Cell suspensions

Biosci Biotechnol Biochem, 1997 Dec, 61(12), 1995 - 2003
cDNA cloning and gene expression of phenylalanine ammonia-lyase in Lithospermum erythrorhizon; Yazaki K et al.; Two cDNA clones (LEPAL-1 and LEPAL-2) encoding phenylalanine ammonia-lyase were isolated from cell suspension cultures of Lithospermum erythrorhizon . Northern kinetic studies showed that LEPAL-1 mRNA contents markedly increased one day after inoculation of the cells into fresh medium, then decreased to the steady-state level . The course of mRNA accumulation paralleled that of PAL enzyme activity . The rapid induction of PAL activity seems to reflect the induction of dihydroechinofuran biosynthesis, while shikonin was produced at the steady-state level of PAL activity . The course of LEPAL-2 mRNA accumulation seemed to be similar to, but much lower than that of LEPAL-1 . In the intact plant, both genes are expressed mainly in the root, the organ in which shikonin is exclusively produced and accumulated . Genomic Southern blot analyses showed that both genes are present in the genome as single copies.

Arch Immunol Ther Exp (Warsz), 1997, 45(5-6), 471 - 4
Natural and probe fluorescence of lymphocytes of patients with pulmonary tuberculosis exposed to radiation as a result of the Chernobyl nuclear accident; Gurevich GL et al.; Parameters of natural and probe fluorescence of peripheral blood lymphocytes have been studied in 25 normal persons and 72 patients suffering from extensive forms of pulmonary tuberculosis who live in various radioecological conditions . It was found that in cell suspension the intensity of fluorescence of protein and the membrane-bound merocyanine 540 dye was 2.0-3.2 and 3.9-7.1 times higher, respectively, for the lymphocytes of the examined patients . For lymphocytes of the patients who live in radionuclide-contaminated areas fluorescence of reduced pyridine nucleotides and some fluorescent probes (ANS, DPGT, the ratio of eximer to monomer fluorescence of pyrene) and the merocyanine-photosensitized cell death were also found to increase . These can indicate some changes of cell membranes and reduction of the oxidation resistance of lymphocytes . There was not any substantial dependence on the changes in the parameters determined in the patients calculated radiation doses was revealed . It was found that biophysical tests, in particular, photosensitized cell death, are informative for estimation of severity of the tuberculous process and prognosis of its outcome in patients who live in adverse radioecological conditions.

Arch Immunol Ther Exp (Warsz), 1997, 45(5-6), 411 - 7
The use of tissue slices in immunological investigations; Skibinski G et al.; Our knowledge of cellular and molecular events taking place during various immune responses comes mainly from in vitro studies where isolated cells are exposed to defined stimuli . This reductionist approach has greatly advanced our understanding of the immune system . However, these studies do not truly reflect the complexity of the integrative events which take place in both primary and secondary lymphoid tissue in vivo . In order to address this problem we have developed a tissue culture procedure which involves the cultivating of precision cut human spleen slices at gas/liquid interface . We have shown, that cells in this culture system show marked differences in cytokine and immunoglobulin production in comparison with conventional single cell suspension cultures, obtained from the same spleen and run in parallel . In this review article we describe the basic technique, results which we have obtained in this system and discuss the possible basis of the observed differences.

Cancer Immunol Immunother, 1997 Nov-Dec, 45(3-4), 198 - 202
CD3 x CD19 bispecific antibodies and CD28 costimulation for locoregional treatment of low-malignancy non-Hodgkin's lymphoma; Manzke O et al.; In advance of using bispecific antibodies for the treatment of B cell lymphoma in humans, we analysed CD3 x CD19 bispecific antibodies for their capacity to induce T cell activation in cell suspensions from follicular lymphoma lymph nodes . Here, we demonstrate that the lack of costimulatory molecules, such as members of the B7 family, on the tumour cells resulted in insufficient activation of autologous T lymphocytes . However, stimulation and proliferation of T cells could be induced by addition of monospecific CD28 antibodies . Moreover, we show that bispecific CD3 x CD19 antibodies can protect severe combined immunodeficiency (SCID) mice from human Epstein-Barr-virus (EBV)-induced B cell lymphoma growth . In these in vivo studies, CD28 costimulation did not show a significant benefit, possibly because of the high-level expression of CD80 and CD86 on the surface of the lymphoma cells . Furthermore, the treatment of SCID mice with bispecific antibodies, with or without CD28 antibodies, induced tumour-protective effects, as determined by a rechallenging experiment in long-term-surviving animals with the autologous EBV-transformed tumour B cell line . Treatment of a follicular lymphoma patient by intratumoural injection of both antibodies resulted in immunological responses with increases in the T/B ratio of peripheral blood as well as enhanced NK cell activity without toxic systemic side-effects.

Arch Biochem Biophys, 1997 Dec 15, 348(2), 369 - 77
Cloning and heterologous expression of NADPH-cytochrome P450 reductases from the Papaveraceae; Rosco A et al.; Cytochrome P450 reductase was purified to homogeneity from cell suspension cultures of the opium poppy Papaver somniferum, the enzyme was characterized (K(m) cytochrome c, 8.3 microM; K(m) NADPH, 4.2 microM; pH optimum, 8.0; M(r), 80 kDa), and the amino acid sequence of internal peptides was determined . Partial cDNA clones from P . somniferum and from Eschscholzia californica (California poppy) were then generated using the polymerase chain reaction and were used as hybridization probes to isolate full-length cDNAs . The Papaver and Eschscholzia cytochrome P450 reductases are 63% identical at the nucleotide level and 69% identical at the amino acid level . SDS-PAGE of the purified native P . somniferum enzyme as well as genomic DNA gel blot analysis indicate that two cytochrome P450 reductase isoforms are present in each species . This evidence is also supported by translation of nucleotide sequences obtained from the PCR-generated partial cDNAs and the full-length cDNAs isolated from lambda libraries . The Papaver and Eschscholzia cytochrome P450 reductases were functionally expressed in the yeast Saccharomyces cerevisiae and in the insect cell culture Spodoptera frugiperda Sf9 . Coexpression of cytochrome P450 reductase with the C-O phenol coupling cytochrome P450 of bisbenzylisoquinoline alkaloid biosynthesis in Berberis stolonifera, berbamunine synthase (CYP80A1), in insect cell culture resulted in an alteration of the product profile as compared to that obtained by expression of berbamunine synthase in the absence of plant reductase.

Brain Res Dev Brain Res, 1997 Nov 12, 103(2), 185 - 94
GABA(A) receptor subunit expression in intrastriatal striatal grafts comparison between normal developing striatum and developing striatal grafts; Liste I et al.; Expression of the alpha1, alpha2 and beta2/3 GABA(A) receptor subunits in maturing cell-suspension striatal grafts and in normal developing striatum was studied by immunocytochemistry . During normal postnatal development, the alpha1 subunit was present in the striatum only at very low density, while the alpha2 and beta2/3 subunits were present with a patchy distribution, in some patches at high density . Double-staining techniques indicated that DARPP-32 (a marker of striatal projection neurons) was not colocalized with alpha1, but was present in some beta2/3-positive areas and all alpha2-positive areas . In striatal grafts, alpha1 immunoreactivity was first detected 2 weeks post-grafting (p.g.), and by 3-10 weeks p.g . the pattern was similar to that observed in mature grafts (1 year p.g.), in which alpha1-immunopositive patches surrounding DARPP-32-positive (i.e . striatum-like) areas are observed . Alpha2 and beta2/3 immunoreactivity was observed within the first week p.g., and by 3-10 weeks p.g . was similar to that observed in mature grafts (i.e . immunoreactivity throughout the graft but with patches of different intensity) . During graft maturation there was a marked decline in alpha2 immunoreactivity in DARPP-32-negative areas, as is observed during normal development of the globus pallidus and ventral pallidum . Interestingly, alpha1- and beta2/3-positive fibers (perhaps mostly dendrites) entered DARPP-32-positive patches from DARPP-32-negative areas . This study indicates that the time course of expression of GABA(A) receptor subunits in grafted striatal neurons, closely matches that of morphological maturation of the transplant, that of the development of functional synaptic activity and that of GABA(A) receptor subunit immunoreactivity in normal developing striatum . Our results also suggest that there are significant interactions between DARPP-32-positive and DARPP-32-negative areas with respect to the expression of GABA(A) receptors, and support the suggestion that miniature 'striatopallidal systems' may develop within grafts; such interactions may be important for the functional integration of striatal grafts with the host brain.

Neuroreport, 1997 Nov 10, 8(16), 3485 - 8
Effects of lesions of the nigrostriatal pathway and of nigral grafts on striatal serotonergic innervation in adult rats; Guerra MJ et al.; Neonatal destruction of the nigrostriatal dopaminergic system leads to serotonergic hyperinnervation of the striatum . However, it is not clear whether this occurs in adult animals . We investigated whether serotonergic sprouting occurs in adult animals, and also studied the effects of prior or subsequent implantation of dopamine-rich intrastriatal grafts . One group of adult rats received maximal 6-hydroxydopamine lesions . Other rats received maximal lesions and intrastriatal grafts 2 months later, or vice versa . The lesioned non-grafted rats showed clear serotonergic hyperinnervation throughout the striatum ipsilateral to the lesion . Intrastriatal grafts did not prevent or revert this serotonergic hyperinnervation, and were themselves densely innervated by serotonergic fibers . Serotonergic neurons usually present in the grafted cell suspension also contributed to the serotonergic innervation of the graft and the surrounding striatum.

Transfusion, 1997 Nov-Dec, 37(11-12), 1149 - 55
Influence of a recombinant hemoglobin solution on blood rheology; Stetter MN et al.; BACKGROUND: Red cell transfusion is a matter of great concern because of viral infections . Recently, a genetically engineered hemoglobin, rHb 1.1, consisting of two alpha chains and one beta chain, has been developed; it has good oxygen-carrying and -unloading capacity and is devoid of renal toxicity . STUDY DESIGN AND METHODS: An in vitro study of the influence of increasing concentrations of rHb 1.1 on plasma and blood viscosity, red cell aggregation and deformability, and neutrophil deformability was performed . RESULTS: The rHb 1.1 (50 g/L in phosphate-buffered saline) had a viscosity of 0.80 +/- 0.02 mPa-sec at 37 degrees C, which was lower than that of normal Hb solution at the same Hb concentration (0.93 +/- 0.01 mPa-sec, p < 0.001) or of albumin, a protein with similar molecular weight (0.93 +/- 0.01, p < 0.0001) . The admixture of rHb 1.1 to plasma or to red cell suspensions, at constant Hb concentration, led to a dose-dependent decrease in their viscosities . The simulation of replacement therapy during blood loss revealed rheologic properties of rHb 1.1 that were superior to those of all other fluids . The rHb 1.1 did not affect red cell aggregation or the deformability of red cells or white cells, as measured by the cells' transit time through small pores . CONCLUSION: These data indicate that rHb 1.1 has excellent rheologic properties and should hold promise not only as an oxygen-carrying therapeutic agent, but probably also as a hemodilutional agent that simultaneously decreases blood viscosity and provides oxygen-carrying capacity.

Transfusion, 1997 Nov-Dec, 37(11-12), 1143 - 8
The effects of diaspirin-crosslinked hemoglobin on the assessment of immunohematology profiles; Reppucci AJ et al.; BACKGROUND: Extensive studies have been conducted on the in vitro effects of diaspirin-crosslinked hemoglobin (DCLHb) in biochemical, hematologic, hemostatic, and blood banking (immunohematologic) methods . The absence of red cell antigens or plasma and/or serum antibodies allows DCLHb to be used as "universal-donor" material . This study evaluates the effects of DCLHb on the accurate assessment of the immunohematologic profile (ABO and Rh blood grouping, antibody screen, and crossmatching) . STUDY DESIGN AND METHODS: DCLHb, 7.4 g per dL in an electrolyte solution, was mixed in vitro with human whole blood, representing the blood types A Rh-positive . A Rh-negative, B Rh-positive, B Rh-negative, O Rh-positive, O Rh-negative, and AB Rh-positive . Two concentrations of DCLHb were tested: 10-percent (0.74 g/dL) and 30-percent (2.22 g/dL) . Controls were prepared by adding a 5-percent albumin solution to aliquots of whole blood in volumes equivalent to those used in preparing the DCLHb dilutions . Serum and/or red cell suspensions from these admixed samples were analyzed for their ABO and Rh blood groups, the presence of unexpected antibodies (antibody screen), and compatibility in crossmatch testing . RESULTS: DCLHb added to whole blood in vitro had no effect on the accurate interpretation of the immunohematologic profile . CONCLUSION: DCLHb does not appear to inhibit the true response or crossreact in the analysis of blood grouping, antibody screening, or crossmatching . In addition, the red color of DCLHb (up to 2.22 g/dL) did not obscure the visual reading for agglutination.

Biochem Biophys Res Commun, 1997 Dec 18, 241(2), 606 - 10
Barbiturate induced benzophenanthridine alkaloid formation proceeds by gene transcript accumulation in the California poppy; Haider G et al.; Four barbiturates, barbituric acid, butethal, phenobarbital, and 2-thiobarbituric acid, of fourteen tested were found to induce accumulation of benzophenanthridine alkaloids in cell suspension cultures of the California poppy Eschscholzia california . When the plant cell suspension cultures were treated with 1 mM barbiturate, alkaloids accumulated to 100 mg/l within four days . This is a level comparable to that achieved with 300 microM concentration of the established secondary metabolite inducer methyl jasmonate . In contrast to methyl jasmonate, barbituric acid, and 2-thiobarbituric acid, butethal and phenobarbital treatment resulted in a different alkaloid profile, suggesting that only select cytochrome P-450 genes were activated by these latter two barbiturates . RNA gel blot analysis of barbiturate induced cell cultures confirmed that transcripts of at least two benzophenanthridine alkaloid biosynthetic genes cyp80b1 (encoding the cytochrome P-450-dependent monooxygenase (S)-N-methylcoclaurine 3'-hydroxylase) and bbe1 (encoding the covalently flavinylated berberine bridge enzyme) increased up to 5- to 7-fold over control values.

J Physiol, 1997 Dec 1, 505 ( Pt 2), 403 - 10
Apparent Ca2+ dissociation constant of Ca2+ chelators incorporated non-disruptively into intact human red cells; Tiffert T et al.; 1 . A recently developed method of measuring cytoplasmic Ca2+ buffering in intact red cells was applied to re-evaluate the intracellular Ca2+ binding properties of the Ca2+ chelators benz2 and BAPTA . Incorporation of the free chelators was accomplished by incubating the cells with the acetoxymethyl ester forms (benz2 AM or BAPTA AM) . The divalent cation ionophore A23187 was used to induce equilibrium distribution of Ca2+ between cells and medium . 45Ca2+ was added stepwise to cell suspensions in the presence and absence of external BAPTA . To induce full Ca2+ equilibration, the plasma membrane Ca2+ pump was inhibited either by depleting the cells of ATP or by adding vanadate to the cell suspension . 2 . The properties of the incorporated chelators were assessed from the difference in cytoplasmic Ca2+ buffering between chelator-free and chelator-loaded cells, over a wide range of intracellular ionized calcium concentrations ({Ca2+}i), from nanomolar to millimolar . 3 . Under the experimental conditions applied, incorporation of benz2 and BAPTA into the red cells increased their Ca2+ buffering capacity by 300-600 mumol (340 g Hb)-1 . The intracellular apparent Ca2+ dissociation constants (KDi) were about 500 nM for benz2 and 800 nM for BAPTA, values much higher than those reported for standard salt solutions (KD) of about 40 and 130 nM, respectively . These results suggest that, contrary to earlier observations, the intracellular red cell environment may cause large shifts in the apparent Ca2+ binding behaviour of incorporated chelators . 4 . The possibility that the observed KD shifts are due to reversible binding of the chelators to haemoglobin is considered, and the implications of the present results for early estimates of physiological {Ca2+}i levels is discussed.

Diabetes, 1998 Jan, 47(1), 1 - 4
Evidence against a direct effect of leptin on glucose transport in skeletal muscle and adipocytes; Zierath JR et al.; Recently, it has been proposed that leptin, the ob gene product, influences some steps in the insulin-signaling cascade . The purpose of the present study was to determine whether leptin exerts direct effects on glucose transport in insulin target tissues . Epitrochlearis muscles or isolated adipocytes from male SD rats were incubated in the absence or presence of recombinant leptin (3-1,000 ng/ml), and in the absence or presence of submaximal or maximal insulin concentrations . In skeletal muscle, insulin increased 3-O-methylglucose transport (1.88 +/- 0.21, 4.06 +/- 0.59, and 9.35 +/- 1.90 micromol x ml-1 x h-1, for 0, 0.6, and 12.0 nmol/l insulin, respectively) . Leptin exposure (300 ng/ml) for 2 h did not alter the basal, submaximal, or maximal response of glucose transport to insulin in skeletal muscle (1.50 +/- 0.14, 4.76 +/- 0.58, and 9.04 +/- 1.09 micromol x ml-1 x h-1 for 0, 0.6, and 12.0 nmol/l insulin, respectively) . Insulin increased glucose transport in rat adipocytes (0.194 +/- 0.007, 1.059 +/- 0.029, and 3.367 +/- 0.143 pmol {14C}glucose x 0.5 ml-1 cell suspension x min-1 for 0, 0.8, and 80 nmol/l insulin, respectively); in vitro exposure to leptin (300 ng/ml) did not alter glucose transport (0.220 +/- 0.006, 1.269 +/- 0.046, and 3.221 +/- 0.285 pmol {14C}glucose x 0.5 ml-1 cell suspension x min-1 for 0, 0.8, and 80 nmol/l insulin, respectively) . Similar to our findings in the epitrochlearis muscle, leptin had no direct effect on basal or insulin-stimulated glucose uptake in soleus muscle from ob/ob or lean mice or adipocytes from normal mice . In summary, in vitro exposure of skeletal muscle or adipocytes to recombinant leptin did not alter glucose transport in the absence of insulin, nor did it affect the sensitivity or responsiveness of the glucose transport system to insulin.

Biosci Rep, 1997 Oct, 17(5), 487 - 98
Effects of D-glucose on chemokinesis and resting production of reactive oxygen species in neutrophil granulocytes of lean or obese-hyperglycemic mouse; Oldenborg PA et al.; The response to D-glucose (0-21 mM) was studied in neutrophil granulocytes from obese, hyperglycemic and hyperinsulinemic Umea ob/ob mice and their lean, littermate controls in order to further elucidate the effects of in vivo and in vitro hyperglycemia on neutrophil function . Neutrophil random locomotion on glass and neutrophil resting luminol-enhanced chemiluminescence in cell suspension were studied . Random locomotion was stimulated by D-glucose in neutrophils from both Umea ob/ob and control mice but the locomotive activity in Umea ob/ob mouse neutrophils was significantly higher than that found in the controls at 4-21 mM glucose . In both types of mice, the stimulatory effect of D-glucose on random locomotion was diminished at 21 mM glucose (not significantly different from that at 0 mM glucose) . Resting chemiluminescence from mouse neutrophils was also stimulated by glucose but here the magnitude of response was similar in neutrophils from both types of mice . These results indicate that chronic hyperglycemia and hyperinsulinemia in the Umea ob/ob mouse may be associated with an increased neutrophil random locomotive activity but a similar resting production of reactive oxygen species, as compared with neutrophils from control mice at physiological and hyperglycemic glucose concentrations in vitro.

Int J Radiat Biol, 1997 Dec, 72(6), 745 - 50
Hydrogen peroxide protects yeast cells from inactivation by ionizing radiation: a radiobiological paradox; Saran M et al.; PURPOSE: To elucidate mechanisms of the interaction of hydrogen peroxide with chloride-derived cytotoxins under steady-state irradiation conditions and to determine the effects on cell viability . MATERIALS AND METHODS: Yeast cells were suspended in phosphate-buffered saline and exposed to 60Co gamma-irradiation under different conditions . The colony-forming ability was determined . RESULTS: Irradiation of PBS produces H2O2 and HOCl simultaneously . Under slightly acidic conditions and low oxygen tension the yield of HOCl exceeds that of H2O2 while at physiological pH and normoxic conditions H2O2 exceeds HOCl . Both substances react with each other rapidly in a pH-dependent way, even during an irradiation that lasts several seconds . As HOCl is about 1000-fold more toxic than H2O2 to the strain of Saccharomyces cerevisiae used in these experiments, it is evident that in an irradiation that produces more HOCl than H2O2 the radiation-induced damage will be large . If, in contrast, the cells are irradiated under conditions in which H2O2 production predominates, the damage will be small . One would therefore predict that addition of hydrogen peroxide to a cell suspension prior to irradiation should result in protection for suspended cells if H2O2 interferes with the generation of HOCl and thereby inactivates this more powerful toxin . Our data show that addition of H2O2 in sublethal concentration decreases radiation-induced cell death to the level that is found in chloride-free solution, i.e . depending on pH, reduces it by a factor of > or = 3.

J Gastroenterol Hepatol, 1997 Oct, 12(9-10), 678 - 84
Thiolmethyltransferase activity in the human colonic mucosa: implications for ulcerative colitis; Moore JW et al.; Ulcerative colitis is associated with a selective reduction of n-butyrate oxidation by the colonic epithelial cells although the reason for this has been unclear . Colonic epithelial cell n-butyrate oxidation can be inhibited in vitro by incubation with sulphide but the role of mucosal detoxification of sulphide in the metabolic welfare of the colonic mucosa has not been examined . This study aimed to assess the role mucosal detoxification of sulphide by thiolmethyltransferase (TMT)-mediated methylation may play in protecting the healthy colonic mucosa from the adverse effects of luminal sulphide . Colonic epithelial cell suspensions from healthy human proximal (n = 9) and distal colon (n = 10) were incubated in the presence of 14C-labelled n-butyrate (5 mmol/L) alone, butyrate plus sodium hydrogen sulphide (NaHS) (1.5 mmol/L), or butyrate plus NaHS plus S-adenosyl-methionine 1,4 butane disulphonate (SAMe) (5 mmol/L) . Study end points were metabolic performance (14CO2 production) and mucosal TMT activity . Incubation with NaHS induced a significant inhibition of 14CO2 production compared with control incubations (P < 0.001) which was similar for proximal and distal colonic cell suspensions . S-adenosyl-methionine 1,4 butane disulphonate reversed this effect completely in proximal but not in distal cell incubations, suggesting a greater susceptibility of the distal colon to the sulphide effect . Although median whole mucosal TMT values did not differ between proximal and distal colonic mucosa, a non-normal distribution of distal TMT values was observed . However, neither the degree of sulphide inhibition of control 14CO2 production nor the degree to which SAMe reversed this inhibition correlated with whole mucosal TMT activity . The study concluded that regional variation exists in TMT activity in the human colon but whilst methylation appears to protect colonic epithelial cells against sulphide-induced inhibition of n-butyrate oxidation, this cannot be directly correlated with mucosal TMT activity.

Plant Physiol, 1997 Dec, 115(4), 1385 - 95
Identification of proliferation-induced genes in Arabidopsis thaliana . Characterization of a new member of the highly evolutionarily conserved histone H2A.F/Z variant subfamily; Callard D et al.; The changes in gene expression associated with the reinitiation of cell division and subsequent progression through the cell cycle in Arabidopsis thaliana cell-suspension cultures were investigated . Partial synchronization of cells was achieved by a technique combining phosphate starvation and a transient treatment with the DNA replication inhibitor aphidicolin . Six cDNAs corresponding to genes highly induced in proliferating cells and showing cell-cycle-regulated expression were obtained by the mRNA differential display technique . Full-length cDNA clones (cH2BAt and cH2AvAt) corresponding to two of the display products were subsequently isolated . The cH2BAt clone codes for a novel histone H2B protein, whereas the cH2AvAt cDNA corresponds to a gene encoding a new member of the highly conserved histone H2A.F/Z subfamily of chromosomal proteins . Further studies indicated that H2AvAt mRNA expression is tightly correlated with cell proliferation in cell-suspension cultures, and that closely related analogs of the encoded protein exist in Arabidopsis . The implications of the conservation of histone H2A.F/Z variants in plants are discussed.

Anticancer Res, 1997 Sep-Oct, 17(5A), 3671 - 4
5-fluorouracil (5-FU) and 5,10-methylene tetrahydrofolate (5,10-CH2FH4) as adjuvant therapy in an experimental rodent colon carcinoma model; Carlsson G et al.; Eradication of micrometastases is the goal for adjuvant therapy following a radical surgical procedure for cancer . We report an experimental study with 5,10-methylenetetrahydrofolate (5,10-CH2FH4) modulation of 5-fluorouracil (5-FU) cytotoxicity in adjuvant treatment . A colon adenocarcinoma cell suspension was inoculated intrahepatically in a rodent experimental model . Intravenous 5-FU (30 mg/kg) in combination with 5,10-CH2FH4 (15 mg/kg or 30 mg/kg) was administered after 1, 2, 3, 4 and 7 days . 5-FU alone reduced the tumor take to fifty percent compared to one hundred percent tumor take in control animals (p < 0.05), while 5-FU in combination with 5,10-CH2FH4 (regardless of folate-dose) eliminated tumor take (p < 0.0001) . This makes 5,10-CH2FH4 a promising agent for modulation of 5-FU cytotoxicity in adjuvant cancer treatment.

Biofizika, 1997 Jul-Aug, 42(4), 914 - 8
{Differences in osmoregulation of resistant and nonresistant fibroblasts in hypotonic media and effect of verapamil on osmoregulation}; Zatsepina GN et al.; The osmoregulation of resistant and nonresistant striped hairyfooted hamster fibroblasts in hypotonic medium was studied . It was found that the fibroblasts of a resistant population completely regulate their volume 1 min after diluting the cell suspension with water . Verapamil, an inhibitor of Pgp channels, caused equal swelling of both fibroblast populations in isotonic medium . It was shown that the water entering the cells together with verapamil increases equally the volume of both cell types and remains bound to both resistant and nonresistant fibroblasts after the termination of cell volume regulation.

Brain Res Brain Res Protoc, 1997 Feb, 1(1), 91 - 9
Basic neural transplantation techniques . I . Dissociated cell suspension grafts of embryonic ventral mesencephalon in the adult rat brain; Dunnett SB et al.; Lesions and grafts in the nigrostriatal dopamine system in rats is widely used as a model of degeneration and regeneration in the CNS, and the development for alternative strategies for treatment in Parkinson's disease . The methods of preparing a dissociated cell suspension of embryonic rat substantia nigra and its transplantation into the brain of adult rats are described . This is the first of a series of methodological reports on the basic methodology and refinements of neural transplantation techniques in the mammalian central nervous system.

Glia, 1997 Nov, 21(3), 299 - 314
Lack of immune responses to immediate or delayed implanted allogeneic and xenogeneic Schwann cell suspensions; Hermanns S et al.; Previous studies have shown that Schwann cell implantation offers a potential therapeutic approach to a variety of neurodegenerative disorders and traumatic injuries . In a clinically relevant paradigm, however, the implantation of autologous Schwann cells is problematic and the use of heterogenetic Schwann cells will be required . In the present study we addressed this important issue and analysed the immunogenicity and survival of allogeneic and xenogeneic Schwann cell suspension grafts in a prelesioned CNS fiber tract, the transected postcommissural fornix of the adult Wistar rat . Cultured Schwann cells from Wistar rat or human peripheral nerve were injected either immediately or after a delay into the transection site and the spatio-temporal pattern of leukocyte infiltration and of major histocompatibility antigen expression was characterized and semiquantified with immunocytochemical methods . Our main findings are that (1) invasive cerebral lesions induce the expression of MHC class I and II antigens, but only sparse infiltration of T-lymphocytes, (2) both allogeneic and xenogeneic discordant Schwann cell suspension grafts, from either neonatal or adult peripheral nerve, survive without any overt signs of rejection for up to 10 weeks after implantation; and (3) delayed implantation procedures have no effect on immune responses to allogeneic Schwann cell grafts . These results demonstrate that there is no marked ongoing immune reactions to heterogenetic Schwann cell suspension grafts and that long-term survival of cross-species Schwann cell grafts can be achieved in the absence of any immunsuppressive treatment . Thus the conditions for functional transplantation of Schwann cells across immunological barriers seem to be favourable and will have implications for future cross-species studies, and possibly also for clinical application.

Br J Haematol, 1997 Nov, 99(2), 426 - 32
The anti-neoplastic drug 5-fluorouracil produces echinocytosis and affects blood rheology; Baerlocher GM et al.; The anti-neoplastic agent 5-fluorouracil (5-FU) in high therapeutic doses can induce angina pectoris and myocardial infarction . The pathophysiological mechanism of this side-effect has not yet been elucidated . We analysed the influence of 5-FU on blood rheology in vitro . Whole blood, blood cell suspensions and plasma were incubated with increasing concentrations of 5-FU (final concentrations 0, 0.08, 0.4, 2, 10 and 25 mg/ml 5-FU) at 37 degrees C . Erythrocyte morphology was analysed after fixation with glutaraldehyde . Viscosity was measured at high and low shear rates (94 and 0.1 s{-1}) . Erythrocyte aggregation and the cell transit times of erythrocytes through 5 microm pores and polymorphonuclear leucocytes through 8 microm pores were determined . 5-FU induced a dose-dependent formation of echinocytes within minutes and was reversible upon removal of 5-FU, which reflected a preferential intercalation of the drug in the outer hemileaflet of the cell membrane . High shear blood viscosity was increased at the highest 5-FU concentration (148 +/- 12%), and at low shear rate a dose-dependent decrease was found (0 mg/ml: 100%, 0.08 mg/ml: 87 +/- 10%, 0.4 mg/ml: 80 +/- 19%, 2 mg/ml: 70 +/- 15%, 10 mg/ml: 40 +/- 19%, 25 mg/ml: 33 +/- 5%) . Erythrocyte aggregation was decreased by the 5-FU-induced echinocytosis . The transit time of erythrocytes through narrow pores was increased in a dose-dependent manner by 5-FU, whereas the transit time of polymorphonuclear leucocytes was initially decreased at 10 mg/ml and returned to control after 60 min incubation . We conclude that 5-FU interacts with the cell membrane, induces echinocytosis and vesiculation and affects blood rheology in several ways which may contribute to cardiovascular complications.

Am J Respir Crit Care Med, 1997 Nov, 156(5), 1656 - 61
Near-infrared spectroscopic method for assessing the tissue oxygenation state of living lung; Noriyuki T et al.; To quantify changes in tissue oxygenation of pathologic lungs, we applied a novel method using near-infrared spectroscopy (NIRs) . In in vitro experiments, we assayed the effect of photon scattering on the absorption spectra of an in vitro system simulating structures of lung, which consists of test tube containing air in hematocrit tubes and red blood cell suspension with various predetermined hemoglobin concentrations . It was determined that photon scattering of the tissue containing air did not affect the absorption in the NIR region . In in vivo experiments, we tested the applicability of the NIRs technique in rat lungs under the following conditions: (1) hypoxic loading; (2) administration of an inhibitor (NaCN) of the mitochondrial respiratory chain; (3) hemorrhagic shock . We found that: (1) Changes in hemoglobin oxygenation state in the lung measured by NIRs depended on inspired oxygen concentrations; (2) NaCN-induced reduction of cytochrome oxidase a,a3 in the lung was observed; and (3) Total hemoglobin levels in the lung decreased after bleeding . Changes in the hemoglobin oxygenation state and cytochrome oxidase redox state in the lung were determined using the least-square-curve fitting for NIR absorption spectra . Our NIRs technique was capable of assessing the hemoglobin oxygenation and cytochrome oxidase redox state in the lung.

J Histochem Cytochem, 1998 Jan, 46(1), 41 - 8
p53 expression in human carcinomas: could flow cytometry be an alternative to immunohistochemistry?
Benini E, Costa A, Abolafio G, Silvestrini R.
Several studies have shown that p53 expression has important clinical implications as an indicator of prognosis and response to chemotherapy or radiotherapy in different human tumor types . Determination of p53 expression by immunohistochemistry (IHC) has been incorporated into routine practice and its reliability has been consolidated . However, flow cytometric (FCM) analysis might represent an important objective and rapid approach . In the present study we determined p53 expression by IHC and FCM on a series of 118 human solid tumors . IHC determination was performed on histological sections and FCM analysis on cell suspensions . Low correlation coefficients (rs from 0.22 to 0.57) were observed between IHC and FCM data from individual tumors . By considering the IHC approach as the gold standard, high sensitivity and low specificity were found for FCM in detecting p53 expression . The FCM analysis of p53 expression and DNA content showed p53-positive cells in all cell cycle phases . Moreover, in most breast, lung, and colon aneuploid tumors (77%), p53-positive cells were detected only in the subpopulations with abnormal DNA content . In conclusion, FCM-p53 expression cannot be used alternatively to IHC determination, and its clinical relevance remains to be validated . Nevertheless, FCM may provide important information about p53 protein expression in the different subpopulations and cell cycle phases . (J Histochem Cytochem 46:41-47, 1998)

Acta Biol Hung, 1997, 48(2), 209 - 20
Factors affecting transient expression of vector constructs in wheat protoplasts; Ahmed KZ et al.; Direct uptake of reporter gene constructs with the bacterial beta-glucuronidase (GUS) gene fused to various promoters was achieved to embryogenic cell suspension culture-derived protoplasts of GK Sagvari winter wheat (Triticum aestivum L.) with polyethylene glycol (PEG) treatment . Based on GUS specific activity values, it was found that Mg2+ with PEG at 20% final concentration can significantly increase the transient expression in wheat protoplasts in comparison to the Ca(2+)-containing medium . The optimum incubation time in transformation mixture was 5-10 min at 25 degrees C . Transient GUS expression as detected by spectrofluorimetry was positively correlated with the time elapsed after DNA uptake (with maximum activity at 48 h), and the incubation time in GUS reaction mixture . It was also found that the protoplast culture medium plays an important role in the efficiency, and the treated wheat protoplasts cultured in KM medium showed a higher GUS activity than those kept in GM medium . Among the five plasmid constructs 6-16-fold higher promoter activity has been achieved with pKM794 driven by CaMV 35S promoter plus two enhancer elements than with the other constructs tested.

Leuk Res, 1997 Oct, 21(10), 907 - 9
Induction of mixed allogeneic chimerism for leukemia; Seledtsov VI et al.; Hybrid (C56BL/6 x DBA) (BDF1; H-2b/H-2d) mice bearing the P815 leukemia (H-2d) were grafted with a (CBA x C57BL/6)F1 (CBF1; H-2k/H-2b) cell suspension, comprising bone marrow cells (BMC; 25 x 10(6)/mouse) and spleen cells (SC; 55 x 10(6)/mouse) on day-4, then treated with cyclophosphamide (200 mg/kg) on day-2 and finally grafted once more with CBF1 cells (25 x 10(6) BMC + 7 x 10(6) SC) on day 0 . Allogeneic cell graftings performed in this way induced durable mixed hematopoietic chimerism and significantly prolonged the survival of recipients, compared with that of leukemia-bearers grafted with syngeneic cells . The results obtained raise the possibility of using allogeneic hematopoietic tissue transplantation in combination with non-lethal cytoreductive therapy to induce a long-lasting graft-vs-leukemia effect.

J Urol, 1998 Jan, 159(1), 48 - 51
Monoclonal antibodies against renal tumors: the potential application in discrimination of ambiguous adenocarcinoma or transitional cell carcinoma of kidney; Yu DS et al.; PURPOSE: We tested the discrimination ability of 2 monoclonal antibodies, mAB 90 and 2-2, in renal carcinomas, including renal cell carcinoma and transitional cell carcinoma of the kidney . MATERIALS AND METHODS: Two monoclonal antibodies raised in renal adenocarcinoma (mAB 90, IgG3 subclass and mAB 2-2, IgG1 subclass), have been generated in our laboratory by hybridoma technique . The tumor associated antigens recognized by these IgG subclass monoclonal antibodies are located in the cell membrane of tumor cells . Antibodies were purified from mice ascites through protein A-Sepharose 4B affinity column and then conjugated with fluorescein isothiocyanate fluorescence by dimethyl sulfoxide method . The binding activity of these antibodies was measured by direct immunofluorescence method and analyzed in a flow cytometer . Forty-five cases of renal cell carcinoma and 16 cases of transitional cell carcinoma of the renal pelvis were collected, and the recognition power of these 2 antibodies was tested . Frozen tumor tissues were prepared and allocated into single cell suspensions in phosphate buffered saline before adding diluted (1:10) conjugated antibodies . Irrelevant antibody and negative tumor cell lines without any reaction with these antibodies were used as background fluorescence control . RESULTS: Reactivities of mAB 90 and mAB 2-2 for renal cell carcinoma tissues were 80 and 91%, respectively . On the contrary the reactivities of mAB 90 and mAB 2-2 for transitional cell carcinoma of renal pelvis tissues were 0 and 69%, respectively . mAB 90 (+)/mAB 2-2 (-) expression pattern had 76% accurate diagnostic rate for renal cell carcinoma and mAB 90 (+)/mAB 2-2 (+) pattern had 69% accurate diagnostic rate for transitional cell carcinoma of the kidney . CONCLUSIONS: When facing pathologically ambiguous kidney tumors, the binding specificity of mAB 90 and mAB 2-2 may be useful for discrimination between renal cell carcinoma tumors and transitional cell carcinoma of the renal pelvis.

Exp Neurol, 1997 Nov, 148(1), 271 - 80
Effect of embryonic donor age and dissection on the DARPP-32 content of cell suspensions used for intrastriatal transplantation; Watts C et al.; The aim of this study was to determine in vitro the DARPP-32 content of donor cells used for striatal transplantation in vivo . The effect of selective embryonic dissection of the lateral ganglionic eminence (LGE) was compared with the standard dissection of the whole ganglionic eminence (WGE) at each of three embryonic ages (14, 15, and 16 days of gestation) in the rat . The resultant cell suspensions were cultured for up to 7 days and incubated with antibodies against DARPP-32, a marker of striatal medium spiny neurons; beta-tubulin III, a neuronal marker; GFAP, a marker of reactive astrocytes; and Gal-C, a marker of oligodendrocytes . LGE dissection gave rise to more DARPP-32 neurons compared to WGE; but this relationship was only observed in the younger embryos . When older (16 days gestation) embryos are used there is no difference in the yield of DARPP-32 cells obtained from LGE and WGE . LGE dissections were also observed to contain fewer glial cells . There was no beneficial effect of LGE over WGE on survival of striatal neurons in vitro . These results have important implications for the selection and dissection of fetal donor material used in clinical trials of intrastriatal transplantation as a potential treatment for Huntington's disease.

Int J Cancer, 1997 Nov 27, 73(5), 690 - 6
Investigation of mammary epithelial cell-bone marrow stroma interactions using primary human cell culture as a model of metastasis; Brooks B et al.; A model has been established using primary human cell culture to study the cell biology of breast cancer metastasis to bone marrow . Mammary epithelia were obtained in single cell suspension from tumour (macroscopically involved), benign (macroscopically uninvolved) and normal (reduction mammoplasty) breast tissue as well as from locally involved lymph nodes . Stromal layers were generated from long-term cultures of human bone marrow or from mammary fibroblasts derived from normal or malignant tissue . The interaction between epithelia and stroma has been studied in terms of adhesion of the epithelia to the stroma and their subsequent growth in co-culture . Our results show that when assayed up to 9 hr after plating, epithelial cells from malignant tissue (14 primary tumours and 9 metastases in lymph nodes) displayed a significant preference for adhesion to bone marrow stroma compared with mammary fibroblasts . In contrast, epithelial cells from 4 normal and 2 of 4 benign samples showed no significant preferential adherence . Subsequent co-culture of mammary epithelia with each of the 3 stromal layers revealed that under serum-free, in vitro conditions, bone marrow stromal layers did not provide an advantageous environment for colony growth, in contrast to their ability to provide a preferential substratum for adhesion.

Proc Natl Acad Sci U S A, 1997 Nov 25, 94(24), 12904 - 7
Inhibition of NF-kappaB DNA binding and nitric oxide induction in human T cells and lung adenocarcinoma cells by selenite treatment; Kim IY et al.; NF-kappaB is a major transcription factor consisting of 50(p50)- and 65(p65)-kDa proteins that controls the expression of various genes, among which are those encoding cytokines, cell adhesion molecules, and inducible NO synthase (iNOS) . After initial activation of NF-kappaB, which involves release and proteolysis of a bound inhibitor, essential cysteine residues are maintained in the active reduced state through the action of thioredoxin and thioredoxin reductase . In the present study, activation of NF-kappaB in human T cells and lung adenocarcinoma cells was induced by recombinant human tumor necrosis factor alpha or bacterial lipopolysaccharide . After lipopolysaccharide activation, nuclear extracts were treated with increasing concentrations of selenite, and the effects on DNA-binding activity of NF-kappaB were examined . Binding of NF-kappaB to nuclear responsive elements was decreased progressively by increasing selenite levels and, at 7 microM selenite, DNA-binding activity was completely inhibited . Selenite inhibition was reversed by addition of a dithiol, DTT . Proportional inhibition of iNOS activity as measured by decreased NO products in the medium (NO2- and NO3-) resulted from selenite addition to cell suspensions . This loss of iNOS activity was due to decreased synthesis of NO synthase protein . Selenium at low essential levels (nM) is required for synthesis of redox active selenoenzymes such as glutathione peroxidases and thioredoxin reductase, but in higher toxic levels (>5-10 microM) selenite can react with essential thiol groups on enzymes to form RS-Se-SR adducts with resultant inhibition of enzyme activity . Inhibition of NF-kappaB activity by selenite is presumed to be the result of adduct formation with the essential thiols of this transcription factor.

Life Sci, 1997, 61(21), 2103 - 10
Comparative study of radical scavenger and antioxidant properties of phenolic compounds from Vitis vinifera cell cultures using in vitro tests; Fauconneau B et al.; Vitis vinifera cell suspensions were used to isolate and characterize the flavonoids (anthocyanins, catechins) and non-flavonoids (stilbenes) found in red wine . Furthermore, we showed that astringin is produced although this stilbene has not previously been reported to be a constituent of V . vinifera or wine . The ability of these compounds to act as radical scavengers was investigated using 1,1-diphenyl-2-picryl-hydrazyl (DPPH), a stable free radical . Antioxidant activities were assessed by their capacity to prevent Fe2+-induced lipid peroxidation in microsomes and their action on Cu2+-induced lipid peroxidation in low-density lipoproteins . The results showed that astringin has an important antioxidant effect similar to that of trans-resveratrol, and a higher radical scavenger activity than the latter . Astringinin appeared to be more active . These data indicate that phenolic compounds (stilbenes, catechins, anthocyanins) exhibit interesting properties which may account in part for the so-called "French paradox," i.e . that moderate drinking of red wine over a long period of time can protect against coronary heart disease.

J Virol Methods, 1997 Oct, 68(1), 89 - 95
Disinfection of cell-associated and extracellular HIV-1 by PUVA treatment; Deichmann M et al.; To inactivate cell-associated and extracellular HIV-1 while preserving cellular surface antigens, a procedure was used based on PUVA treatment, i.e . addition of psoralen to cell suspensions followed by irradiation with UVA light . T-lymphoid MT-4 cells were infected with HIV-1 strain NL4-3, 4'-aminomethyl-4,5',8-trimethylpsoralen was added, and the cell suspension was irradiated with 20 mW/cm2 UVA light for 3, 4 and 5 min . To evaluate virus inactivation, cells and supernatants were diluted serially and cocultured with uninfected MT-4 cells . Infectious HIV-1 was detected by cytopathic effects, immunofluorescence and p24 antigen ELISA . UVA irradiation at 3.6 J/cm2 (3 min 20 mW/cm2) reduced the amounts of both cell-associated and extracellular infectious HIV-1 by more than five orders of magnitude . Even at more stringent conditions of PUVA treatment (10 min 20 mW/cm2 UVA irradiation), conformational cellular surface epitopes remained detectable by flow cytometry.

Acta Radiol, 1997 Nov, 38(6), 1083 - 6
An in vitro 1H-MRS model of oncogene transfection . The spectral feature of c-erbB-2 and c-Ha-ras transfected NIH3T3 fibroblast cells; Nakai T et al.; PURPOSE: Malignancy is an abnormality of cell division and differentiation based on abnormal expression of oncogenes . This note describes the in vitro 1H-NMR spectral features of oncogene-transfected NIH3T3 fibroblast cells compared to non-transfected cells . MATERIAL AND METHODS: 1H-NMR spectra of cultured NIH3T3 cells and c-erbB-2 or c-Ha-ras gene-transfected cells were obtained by 400 MHz high resolution NMR . The peaks were assigned by 2D HOHAHA spectra of the cell suspension and the spectral changes were evaluated in 1D and 1D differential spectra . RESULTS: The 1H spectra obtained from both transfected cell lines were broadened over all peaks, suggesting reduced mobility in plasma membrane lipid molecules . No other differential spectra for characterizing metabolic change was detected . CONCLUSION: Broadened 1H spectra observed after c-erbB-2 or c-Ha-ras transfection suggest changes of plasma membrane viscosity, which may be related to the oncogene expression.

J Exp Zool, 1997 Dec 1, 279(5), 498 - 503
Potential contribution of epithelial Na+ channel to net secretion of aqueous humor; Civan MM et al.; The aqueous humor of the eye is secreted by the bilayered ciliary epithelium, consisting of the pigmented (PE) cell layer facing the stroma and the nonpigmented (NPE) cell layer facing the aqueous humor . Cells within each layer and between the two layers are linked by gap junctions, forming a ciliary epithelial syncytium . Unidirectional secretion from the stroma to the aqueous proceeds both through the cells (the transcellular pathway) and between the cells (the paracellular pathway) . Net formation of aqueous humor must, however, be the algebraic sum of unidirectional secretion and unidirectional reabsorption from the aqueous humor back into the stoma . The mechanisms potentially underlying reabsorption of aqueous humor by the NPE cells have recently been addressed by studying the regulatory response (RVI) of anisosmotically shrunken NPE cells . The results indicated that epithelial Na+ channels with a high affinity to amiloride likely contribute to reabsorption of solute from the aqueous humor . We have substantiated this possibility by using Northern analysis to identify in human ciliary body RNA a 3.7-kb transcript corresponding to the alpha-subunit of the amiloride-sensitive, alpha beta gamma-ENaC epithelial sodium channel . We have also found that the Na(+)-channel inhibitor benzamil inhibits the RVI without affecting the cell volume of isotonic cell suspensions . This observation supports the hypothesis that the low conductance, highly selective epithelial Na+ channel is activated by shrinkage and contributes to unidirectional reabsorption as aqueous humor . Examples are provided of how the integrative regulation of aqueous humor formation can involve conjugate actions on both unidirectional secretion and reabsorption.

J Med Genet, 1997 Nov, 34(11), 912 - 6
Rapid identification of multiple supernumerary ring chromosomes with a new FISH technique; Mackie-Ogilvie C et al.; Multiple supernumerary ring chromosomes are a rare cytogenetic finding which is poorly understood . With the introduction of FISH techniques, their chromosomal origin can now be defined clearly . The techniques described previously are complicated and time consuming . We report a new rapid technique which has been used to investigate two new cases . Multiple probes were hybridised to a single slide by means of marking the underside with a diamond pen to form a grid of squares, pipetting fixed cell suspension into the centre of each square, forming a rubber solution grid on the denatured, dehydrated slide following the lines on the underside, adding a mixture of probes into each square, and sealing the slide with a silicone rubber rim and a covering slide . The type of probe and the size, dimensions, and number of squares in the grid can be tailored to individual cases . The two new cases examined here are mosaic for three (case 1) and four (case 2) supernumerary ring chromosomes derived from different chromosomes . Normal cell lines were also present . The karyotypes were established as 47,XY,+r(4)/47,XY,+r(17)/.../48,XY,+r(17),+r(20)/ 49,XY,+r(4),+r(17),+r(20)/46,XY for case 1 and 47,XX,+r(4)/47,XX,+r(8)/47,XX,+r (10)/48,XX,+r(X),+r(4)/.. . /49,XX,+r(X),+r (8),+r(10)/46,XX for case 2 . Our findings suggest that the ring chromosomes were formed during meiosis, perhaps involving complex rearrangements, resulting in a germ cell containing all markers, with subsequent loss of markers during cell division . Our second case also shows that the outcome is not invariably mental or physical handicap.

Plant Physiol, 1997 Nov, 115(3), 1039 - 48
Characterization and expression of caffeoyl-coenzyme A 3-O-methyltransferase proposed for the induced resistance response of Vitis vinifera L; Busam G et al.; Cell-suspension cultures of Vitis vinifera L . cv Pinot Noir accumulated resveratrol upon fungal elicitation, and the activity of S-adenosyl-L-methionine:trans-caffeoyl-coenzyme A 3-O-methyl-transferase (CCoAOMT), yielding feruloyl-CoA, increased to a transient maximum at 12 to 15 h . CCoAOMT cDNA was cloned from the elicited cells and was shown to encode a polypeptide highly homologous to CCoAOMTs from cells of Petroselinum species or Zinnia species . The expression of the cDNA in Escherichia coli revealed that grapevine CCoAOMT methylates both caffeoyl- and 5-hydroxyferuloyl-coenzyme A and is probably involved in phenolic esterification and lignification . Commercial plant activators induce the disease-resistance response of test plants and are considered to mimic the action of salicylic acid . Among these chemicals, 2,6-dichloroisonicotinic acid and benzo(1,2,3)-thiadiazole-7-carbothioic acid S-methyl ester provoke systemic acquired resistance (SAR) and were also shown to induce the expression of class III chitinase in grapevine . The SAR response is classified by an unchanged phenotype of tissues, but the mechanistic basis is unknown . Treatment of the cultured V . vinifera cells with either fungal elicitor or low concentrations of salicylic acid and 2,6-dichloroisonicotinic acid, respectively, raised the CCoAOMT or stilbene synthase transcript abundance, suggesting that grapevine is capable of the SAR response, whereas benzo(1,2,3)-thiadiazole-7-carbothioic acid S-methyl ester was ineffective . The data imply for the first time (to our knowledge) that the expression of phenyl-propanoid genes in grapevine is induced by SAR activators without phenotypic consequences and suggest a role for CCoAOMT and stilbene synthase in the disease-resistance response leading beyond the level of pathogenesis-related proteins as markers of the SAR.

Acta Cytol, 1997 Nov-Dec, 41(6), 1757 - 61
Calretinin . A selective marker of normal and neoplastic mesothelial cells in serous effusions; Barberis MC et al.; OBJECTIVE: To document that a polyclonal antiserum to calretinin, a 29-kd calcium-binding protein, consistently decorates normal and tumor mesothelial cells in cytologic preparations . STUDY DESIGN: Thirty-three archival cytologic specimens from eight patients with histologically confirmed malignant mesothelioma and 13 from patients with metastatic serous effusions were destained and then immunostained with anticalretinin antiserum . For investigation of cell suspensions, four pleural fluids were incubated with anticalretinin antiserum . After cytocentrifugation the specimens were stained in accordance with the alkaline phosphatase anti-alkaline phosphatase (APAAP) method . For electron microscopic examination the cell suspensions were then incubated with gold-labeled antirabbit antibody . RESULTS: The diagnostic sensitivity of this new immunocytochemical approach reached 100% for the eight malignant mesotheliomas investigated . Only 3 of the 13 adenocarcinomas metastatic to the serous membranes included in this study were weakly reactive, accounting for 81% specificity . Binding of anticalretinin antiserum to living mesothelial cells was consistently documented in all four cases investigated . CONCLUSION: Calretinin is a very useful marker for positive identification of normal and tumor mesothelial cells in serous effusions.

Endocrinology, 1997 Dec, 138(12), 5238 - 47
Influence of dietary sodium restriction on angiotensin II receptors in rat adrenals; Lehoux JG et al.; We studied the distribution of angiotensin II (AII) receptors type 1 (AT1) and type 2 (AT2) and the effects of a low sodium intake on these two subtypes of receptors in male rat adrenals . Binding studies on adrenal slices, on cell membranes and on cell suspensions were performed using {125I}AII and specific analogs for AT1 (Losartan) and AT2 (PD 123319) receptors . The distribution of AT1 was also studied by immunofluorescence . Complementary approaches were necessary to reach our goal . Indeed, by autoradiography on adrenal slices, {125I}AII was shown to bind to the zona glomerulosa (ZG) and to the medulla (M) . When coincubated with {125I}AII, PD 123319 displaced {125I}AII from the medulla and from the ZG, indicating the presence of AT2 receptors in both zones . Losartan partially displaced {125I}AII from the ZG, indicating the presence of AT1 receptors in that zone . Furthermore, the labeling intensity of the medulla (AT2 receptors) was much stronger in adrenal sections from rats kept on a low sodium regimen than from controls . Immunofluorescence microscopy revealed that AT1 receptors were located mainly in the ZG of control rats . After sodium restriction, AT1 receptors appeared to be uniformly distributed within an enlarged ZG; furthermore AT1 receptor-positive cells were found to a limited degree in the zona fasciculata and possibly in the zona reticularis, and a greater number of these positive cells appeared in these zones under sodium restriction . Cell suspensions from rats fed a low sodium diet showed a 2.7- and 2.1-fold increase in total AII receptors in adrenal ZG and ZFR + M cells when compared with controls . Based on Losartan displacement, we calculated that {125I}AII bound to AT1 and to AT2 receptors was increased in both ZG and ZFR + M cell preparations under sodium restriction . Results of binding studies on cell membranes were also indicative of an increasing effect of sodium restriction on AT1 and AT2 receptors binding capacity . Furthermore, Northern blotting analysis revealed 3.0- and 2.5-fold increases in the level of AT1 receptor mRNA in the ZG and the ZFR + M of rats fed a low sodium diet as compared with those fed a normal diet . The low sodium intake resulted in a weaker increase (1.5-fold) in the level of AT2 receptor messenger RNA in the ZG, with no changes in the ZFR + M preparations . In conclusion, in this study complementary approaches were needed to determine the localization of AT1 and AT2 receptors in the rat adrenal, and to show the increasing effects of a low sodium regimen on the adrenal level of these receptors . Immunofluorescence studies revealed AT1 receptors mainly in the ZG and also in some cells of the inner adrenal cortex zones; in adrenals of rats kept on a low sodium diet the ZG was markedly enlarged, and an increased number of immunoreactive cells with AT1 receptors were observed throughout that zone; also more immunoreactive cells were present in the inner zones of the adrenal cortex . Furthermore in the adrenals of rats kept on a low sodium diet, we observed: 1) an increased number of AT1 and AT2 receptors in cell suspensions from the ZG, and in cell suspensions of the ZFR + M; 2) an increased level of AT1 and AT2 receptor mRNAs in the ZG; 3) an increased level of AT1 receptor mRNA, with no changes in the AT2 mRNA level in the ZFR + M . These results suggest a role for AT1 as well as for AT2 receptors in controlling adrenal function and differentiation under normal as well as under physiological stimulation of AII production following sodium restriction.

J Exp Clin Cancer Res, 1997 Sep, 16(3), 243 - 7
The influence of zymosan and indomethacin on liver and kidney tumor growth . An experimental study in rats; Ohman MG et al.; Zymosan, a non-specific macrophage-stimulating agent, modifies favourably tumour growth in the liver but has minor effect on renal tumours . The mechanism accounting for variation is still to be clarified . The effect of zymosan on liver cancer may be mediated by the macrophage-monocyte system . Kupffer cells are in vitro cytotoxic against colon cancer cell lines . The kidney is sparse in macrophage elements . The prostaglandin synthesis inhibitor, indomethacin, inhibits tumor growth . In Wistar-FU rats inoculated in the liver and the kidney with an adenocarcinoma cell suspension, pretreatment with zymosan (3 mg x 100 g{-1}) significantly reduced both tumour take and liver volume . This effect was attenuated by concomitant administration of indomethacin (0.2 mg x 100 g{-1}) . After 2 weeks there was still reduced liver tumour volume . No significant effects on tumour take or growth were observed when the cells were inoculated into the kidney . There was no significant effect of zymosan on an hepatoma in Lister-Hooded rats . Pretreatment with indomethacin had no effect on tumor take or initial growth.

Eur J Biochem, 1997 Oct 1, 249(1), 161 - 70
Purification and characterization of two isoforms of isopentenyl-diphosphate isomerase from elicitor-treated Cinchona robusta cells; Ramos-Valdivia AC et al.; In Cinchona robusta (Rubiaceae) cell suspension cultures, the activity of the enzyme isopentenyl-diphosphate isomerase (isopentenyl-POP isomerase) is transiently induced after addition of a homogenate of the phytopathogenic fungus Phytophthora cinnamomi . The enzyme catalyses the interconversion of isopentenyl-POP and dimethylallyl diphosphate (dimethylallyl-POP) and may be involved in the biosynthesis of anthraquinone phytoalexins that accumulate rapidly after elicitation of Cinchona cells . From elicitor-treated C . robusta cells, two isoforms of isopentenyl-POP isomerase have been purified to apparent homogeneity in four chromatographic steps . The purified forms are monomeric enzymes of 34 kDa (isoform I) and 29 kDa (isoform II), with Km values for isopentenyl-POP of 5.1 microM and 1.0 microM, respectively . Both isoforms require Mn2+ or Mg2+ as cofactor, isoform II showing a preference for Mn2+ with maximum activity at 1.5-2 mM . Isoform I was most active in the presence of 0.5-1.5 mM Mg2+ or in the presence of 0.5 mM Mn2+ . A pH optimum of 7-7.8 was found for both forms and both were competitively inhibited by geranyl diphosphate (Ki 96 microM for isoform I) and the transition state analogue 2-(dimethylamino)ethyl diphosphate . Rechromatography of purified isoforms did not indicate any interconversion of both forms . Western blot analysis, using antibodies raised against isopentenyl-POP isomerase purified from Capsicum annuum, showed the presence of both isoforms in the crude protein extracts from C . robusta cells . Isoform II was specifically induced by elicitation, non-treated cells contained low activity of this isoform . The possible role of isopentenyl-POP isomerase in the biosynthesis of anthraquinones is discussed.

Brain Res, 1997 Aug 22, 766(1-2), 285 - 8
Swelling and damage of glial cells by lactacidosis and glutamate: effect of alpha-trinositol; Staub F et al.; The therapeutical efficacy of alpha-trinositol (D-myo-inositol-1,2,6-trisphosphate), an isomer of the intracellular messenger IP3, was analyzed on cytotoxic swelling and damage of glial cells in vitro from lactacidosis or glutamate . C6 glioma cells suspended in a physiological medium were either exposed to pH 5.0 by administration of lactic acid, or to 1 mM glutamate . Cell swelling and viability were quantified by flow cytometry . Lactacidosis of pH 5.0 led to an increase in cell volume to 139.7 +/- 1.3% within 20 min whereas alpha-trinositol was reducing the swelling response by approximately 25% (P < 0.01) . In addition, at pH 5.0 the fraction of viable cells was lowered from 94.3 +/- 0.2% (control) to only 53.8 +/- 3.1% after 60 min . Alpha-trinositol was found to protect also cell viability; at 60 min of lactacidosis 70.2 +/- 1.6% of the cells still were viable (P < 0.01) . The addition of glutamate (1 mM) to the cell suspension led to a steady increase in cell size, reaching 110% of control at 120 min, irrespectively of whether alpha-trinositol was added or not.

Spectrochim Acta A Mol Biomol Spectrosc, 1997 Sep, 53A(10), 1645 - 53
Cell activation influences cell staining kinetics; Sunray M et al.; Stimulation of cells has so far been observed, among other methods, by the decrease of the intracellular fluorescein fluorescence polarization (IFFP) . It is shown that the rate constant of leakage of the fluorescent marker out of the cells increases with stimulation much more significantly than the polarization decreases; thus it might provide a more sensitive method to observe cells stimulation . It is also shown that due to negligible leakage of the marker out of the cells shortly after initiation of the staining of the cell suspension, the fluorescein fluorescence polarization (FFP) of the cell suspension, is very close to IFFP.

Mutat Res, 1997 Oct 6, 379(2), 191 - 9
Characterization of a macromolecular matrix isolated from tobacco suspension cell cultures and its role in the activation of promutagenic m-phenylenediamine; Stavreva DA et al.; The medium recovered from the tobacco cell suspension cultures (TX1MX) activated the promutagenic aromatic amine m-phenylenediamine (mPDA) and a macromolecular complex (gel) responsible for the arylamine activation was isolated from the medium . The gel formation and the role of the gel components in the plant activation of mPDA to products mutagenic in S . typhimurium YG1024 were studied . The activation of mPDA was caused by the peroxidases present in TX1MX . We demonstrated an association of the peroxidase activity and gel pectins . Formation of a stable mutagenic association of mPDA with the macromolecular material was observed . The data indicate that the gel isolated from TX1MX is the macromolecular component of the arylamine conjugate proposed in earlier work.

Infect Immun, 1997 Nov, 65(11), 4405 - 10
Interleukin-12 gene expression in human monocyte-derived macrophages stimulated with Mycobacterium bovis BCG: cytokine regulation and effect of NK cells; Matsumoto H et al.; Macrophage-derived interleukin-12 (IL-12) is essential for the activation of a protective immune response against intracellular pathogens . In this study, we examined the regulation of IL-12 mRNA expression by monocyte-derived macrophages (MDM) in response to Mycobacterium bovis BCG stimulation . A reverse transcription-PCR assay detected p40 mRNA of IL-12 at 3 h and showed a peak at 6 to 12 h with a subsequent decline . Semiquantitation of mRNA levels by competitive PCR revealed that pretreatment with gamma interferon (IFN-gamma) amplified the expression approximately 100-fold, while pretreatment with tumor necrosis factor alpha (TNF-alpha) or granulocyte-macrophage colony-stimulating factor augmented this expression about 10-fold . In contrast, pretreatment with IL-10 and IL-4 inhibited IL-12 mRNA expression . These results were further confirmed by measuring the p70 bioactive protein level in each conditioned medium by an enzyme-linked immunosorbent assay . Since IL-12 mRNA expression was weak without cytokine pretreatment and IFN-gamma strongly augmented production, we speculated that IFN-gamma might have a role in BCG stimulation of IL-12 mRNA expression . Unexpectedly, the addition of three different kinds of anti-IFN-gamma antibodies and anti-IFN-gamma receptor antibody and the coaddition of anti-TNF-alpha antibody with anti-IFN-gamma receptor antibody all failed to inhibit IL-12 mRNA expression . However, the MiniMACS method used to remove NK cells from a mononuclear cell suspension inhibited the expression of p40 mRNA but not the expression of mRNA of TNF-alpha or IL-1beta . We concluded that the coexistence of NK cells was essential for the induction of IL-12 in MDM stimulated with BCG rather than through the secretion of IFN-gamma.

Int Immunol, 1997 Oct, 9(10), 1527 - 36
Agonist peptide modulates T cell selection thresholds through qualitative and quantitative shifts in CD8 co-receptor expression; Chidgey A et al.; Engagement of the TCR is a pivotal step in thymocyte development, ultimately resulting in the survival (positive selection) or loss (negative selection) of developing T cells . The roles of peptides and stromal cell interactions necessary for these selection events, however, are still poorly understood . To investigate the effects of agonist peptide in positive selection, we used a novel cell suspension model for in vitro thymic positive selection in adults . Target thymocytes from H-2Db-restricted TCR transgenic mice, specific to the lymphocytic choriomeningitis virus (LCMV) peptide bred on a non-selecting MHC background (H-2d or TAP-1-/-), were co-cultured with freshly isolated H-2b thymic stromal cells . In the presence of selecting stroma the nominal agonist LCMV peptide induced apoptosis at high concentrations and at low concentrations enhanced the efficiency of positive selection both in numbers of cells 'rescued' and kinetics of appearance of selected single-positive cells . We further illustrate down-modulation of CD8 alpha beta or CD8 beta at high but non-deleting concentrations of agonist peptide . This highlights the ability of the T cell, within the window of positive selection, to modify surface co-receptors both qualitatively and quantitatively in response to increasing avidity TCR-peptide-MHC interactions . The direct consequence of this would be to lower the total signaling events below the threshold for apoptosis induction . Hence if self peptide were not presented in sufficient quantities in the thymus, autoreactive cells may escape deletion and may actually be positively selected.

Artif Cells Blood Substit Immobil Biotechnol, 1997 Nov, 25(6), 577 - 83
Haemoglobin-enhanced mitotic division in cultured protoplasts; Anthony P et al.; Protoplasts from cell suspensions of albino Petunia hybrida cv . Comanche were cultured for 9 days in nutrient medium containing Erythrogen, a purified bovine haemoglobin solution (supplied at 10% w/v) at 1:50-1:500 (v/v) . In some assessments, the non-ionic surfactant Pluronic F-68 (Poloxamer 188), was also added to the culture medium at 0.01-1.0% (w/v) . Erythrogen at 1:50 (v/v) increased the mean initial protoplast plating efficiency (IPE; 18.5 +/- 0.8%, n = 5 throughout) by 64% (P < 0.001) above that of controls (11.3 +/- 0.4%) . Supplementation of medium with 1:50 (v:v) Erythrogen and 0.01% (w/v) Pluronic F-68, increased the mean IPE (24.4 +/- 1.4%) by 92% (P < 0.001) over control (12.7 +/- 1.1%) . Similar results were obtained for mesophyll protoplasts of Passiflora suberosa, with 1:50 and 1:100 (v/v) Erythrogen increasing the mean IPEs to 87% and 93% respectively, over controls . This beneficial and synergistic effect of Erythrogen with Pluronic F-68, on mitotic division of cultured Petunia and Passiflora protoplasts, should also facilitate the culture of isolated protoplasts and cells of other, agronomically-important, species.

FEBS Lett, 1997 Sep 29, 415(2), 186 - 91
Identification of the human Lewis(a) carbohydrate motif in a secretory peroxidase from a plant cell suspension culture (Vaccinium myrtillus L.); Melo NS et al.; This paper reports for the first time the presence of the human Lewis(a) type determinant in glycoproteins secreted by plant cells . A single glycopeptide was identified in the tryptic hydrolysis of the peroxidase VMPxC1 from Vaccinium myrtillus L . by HPLC/ESI-MS . The oligosaccharide structures were elucidated by ESI-MS-MS and by methylation analysis before and after removal of fucose by mild acid hydrolysis . The major structure determined is of the biantennary plant complex type containing the outer chain motif Lewis(a) {structure in text} . A corresponding fucosyltransferase activity catalyzing the formation of Lewis(a) type structures in vitro was identified in cellular extracts of the suspension cultures.

Anal Quant Cytol Histol, 1997 Oct, 19(5), 437 - 42
DNA ploidy by image cytometry in urothelial carcinomas . Comparison of touch imprints and paraffin-embedded biopsies from 31 patients; Mainguene C et al.; OBJECTIVE: To compare DNA content measured by image cytometry from touch imprints and formalinfixed, paraffin-embedded samples in bladder carcinomas . STUDY DESIGN: Thirty-one biopsies of urothelial carcinomas were selected for a prospective study . Imprints of fresh specimens were performed . Cell suspensions were obtained from dewaxed samples by the procedure of Hedley . Sections 7 microns thick were used for carcinoma in situ and small biopsies . The DNA ploidy index was measured on Feulgen-stained slides using an image cytometer . RESULTS: From imprint analysis, seven grade 1 carcinomas (n = 9) were found to be diploid (78%) . Nine grade 2 carcinomas (n = 12) exhibited aneuploidy (75%), as did all grade 3 and in situ carcinomas (n = 10) . Multiploidy was demonstrated from imprints in four cases instead of the two detected from dewaxed tissue . In 27 cases (87%), G0/G1 peaks obtained from paraffin blocks showed a shift to the left . In five cases (16%), variations in the DNA index were responsible for discrepancies in the DNA ploidy evaluation between fresh imprints and dewaxed samples of the same tumors . CONCLUSION: Image cytometry on Feulgen-stained imprints of bladder biopsies is a simple and reliable procedure for assessing DNA ploidy in urothelial carcinomas, providing great sensitivity for detecting small aneuploid peaks and multiploid tumors . DNA image analysis of touch preparations is especially useful for carcinoma in situ and small biopsies unsuitable for Hedley's technique.

Gan To Kagaku Ryoho, 1997 Sep, 24(12), 1785 - 8
{Comparison between intraperitoneal and intravenous 5-fluorouracil administration using pancreatic cancer model of nude mouse}; Maruyama M et al.; Carcinoma of the pancreas is a virulent malignancy . The purpose of this study was to evaluate the efficiency of 5-FU intraperitoneal administration for this malignancy . We developed a pancreatic cancer model whereby a human pancreatic cell line, MIA PaCa-2, was orthotopically transplanted to the pancreas of nude mice as cell suspension (1 x 10(6) cells) . IP group (n = 6) received 5 times IP administration (4, 7, 12, 16, 20 days after implantation) of 5-FU 50 mg/kg, 1.5 ml . IV group (n = 6) received 5 times IV therapy (the same dates as IP group) of 5-FU 50 mg/kg, 0.2 ml . Control group (n = 6) received no treatment . The mice were sacrificed 42 days after implantation . The weight of the tumors of IP, IV and Control group was 0.332 +/- 0.143 g, 0.138 +/- 0.047 g and 0.329 +/- 0.085 g . Significant differences were found between IP and IV, and control . There was no difference between IP and control . This experiment demonstrated that 5 FUIP therapy for primary pancreatic cancer showed no effect and 5 FUIV therapy was much more effective.

Int J Obes Relat Metab Disord, 1997 Sep, 21(9), 764 - 8
A simple method to predict cellular density in adipocyte metabolic incubations; Fine JB et al.; OBJECTIVES: The density of isolated adipocyte suspensions, namely the cellular concentration, influences metabolic results when lipolysis and the pattern of glucose metabolism are studied . It is often difficult to obtain reproducible adipocyte concentrations from experiment to experiment, and investigators usually measure the cell concentration at the end rather than at the initiation of metabolic incubations . METHOD: A simple and rapid method to obtain reliable and predictable adipocyte concentration prior to metabolic incubations is described and validated . The method is based on determination of lipocrit, mean adipocyte diameter (by optical sizing), and calculation of volume, in aliquots of isolated adipocyte suspensions . MAIN OUTCOME MEASURES: Lipocrit, mean adipocyte volume, predicted and observed adipocyte number in isolated cell suspensions . RESULTS: In 15 experiments, adipocyte concentration was accurately predicted within 12-18% of actual concentration . This is in contrast to the four or five-fold differences usually encountered in a series of experiments . CONCLUSION: One can rapidly predict the number of adipocytes present in a given cell suspension with the proposed method, and then correct it to a desired adipocyte concentration at the beginning of metabolic incubations . This method will help to eliminate the confounding effects of variable cell concentrations in in vitro metabolic experiments with isolated adipocytes.

Exp Neurol, 1997 Oct, 147(2), 498 - 502
Tirilazad mesylate improves survival of rat and human embryonic mesencephalic neurons in vitro; Othberg A et al.; The survival rate of embryonic dopamine (DA) neurons after transplantation to the striatum is only 5-20% . Therefore, mesencephalic tissue from several donors needs to be implanted in a parkinsonian patient to induce a therapeutic improvement . Lazaroids are a group of neuroprotective compounds which inhibit lipid peroxidation . Previously, two lazaroids (U-74389G and U-83836F) have been found to improve the survival of both cultured and grafted rat DA neurons . The only lazaroid approved for human use is tirilazad mesylate . The objective of the present study was to explore the effects of tirilazad mesylate on DA neuron survival in cultures of rat ventral mesencephalon and its capacity to promote the in vitro cell viability of embryonic rat and human mesencephalic tissue, treated and dissociated in the same way as in clinical trials . After 7 days in vitro, the number of tyrosine hydroxylase-immunopositive, presumed DA neurons was 140% higher in rat cultures treated with 0.3 microM tirilazad mesylate than that in control cultures . Rat and human cell suspensions supplemented with tirilazad mesylate maintained a high degree of viability for several hours longer than control suspensions . These results indicate that tirilazad mesylate promotes the survival of both rat and human embryonic mesencephalic neurons in vitro . Tirilazad mesylate can be administered clinically and may become a useful tool for increasing survival of grafted DA neurons in patients, thereby reducing the needed quantity of human donor tissue.

Anticancer Res, 1997 Jul-Aug, 17(4B), 3179 - 82
Granulocyte-macrophage colony stimulating factor and interleukin-6 enhanced white blood cell synthesis of leukotrienes in chronic myelogenous leukemia; el-Ahmady O et al.; The effect of Granulocyte-Macrophage, Colony Stimulating Factor (GM-CSF) and Interleukin-6 (IL-6) on leukotriene production by CML white blood cells induced by calcium ionophore (A23187) was investigated and the leukotrienes formed were identified and quantified using high performance liquid chromatography (HPLC) . The in vivo levels of IL-6 and LTB4 were determined by enzyme immunoassay reagents, while GM-CSF was measured by enzyme amplified sensitivity immunoassay . Although GM-CSF or IL-6 alone did not stimulate the synthesis of 5-lipoxygenase product, preincubation of the white blood cells of CML with GM-CSF or IL-6 for 30 minutes at 37 degrees C enhanced the ionophore A23187 induced leukotrienes synthesis, thus the CML white blood cell suspension primed with GM-CSF or IL-6 produced 26.6 +/- 2.8 and 18.9 +/- 1.3 pmol LTC4/10(6) cells respectively, and 30.2 +/- 3.6 and 25.5 +/- 2.5 Pmol LTB4/10(6) cells . In contrast minute amount of leukotrienes were produced by the control cells . In vivo levels of GM-CSF, IL-6 and LTB4 were investigated in CML and normal healthy donors, elevated chemotactic B4 was found in plasma from CML (267 +/- 70.4) while the mean value in normal healthy donors was (127 +/- 13.6) pg/ml . The plasma level of GM-CSF was 32.4 +/- 15.7 pg/ml and 10.5 +/- 3.1 pg/ml respectively in CML and normal healthy donors, while the mean value of GM-CSF and IL-6 in normal healthy donors were 6.7 +/- 2.2 and 4.9 +/- 2.4 pg/ml respectively . No significant correlation was observed between the level of LTB4 and the level of GM-CSF or IL-6 in CML.

J Immunol Methods, 1997 Aug 22, 207(1), 33 - 42
A simple method for evaluating the rejection of grafted spleen cells by flow cytometry and tracing adoptively transferred cells by light microscopy; Oehen S et al.; In this report we describe a simple method using the vital dye 5- (and 6-) carboxyfluorescein diacetate succinimidyl ester (CFSE) to follow splenic graft rejection by flow cytometry . CFSE-labelled spleen cell suspensions were injected intravenously into various recipients and blood samples were taken at different time points to follow the transferred cells . We found that the labelled cells could be readily detected by flow cytometry for up to eleven weeks . The loss of these labelled cells in various transfusion experiments with different major and minor histocompatibility differences followed the rejection kinetics previously described for skin transplants . Thus, this method offers a simple tool to test the histocompatibility of donor cells/grafts with the host in adoptive/transplantation experiments in which donor and host are not completely syngeneic . Furthermore, we developed a method to trace adoptively transferred fluorescent CFSE-labelled cells by light microscopy by converting in situ immunofluorescence staining into immunoenzyme staining.

Pharm Res, 1997 Sep, 14(9), 1223 - 7
Enhanced anti-inflammatory effects of Cu, Zn-superoxide dismutase delivered by genetically modified skin fibroblasts in vitro and in vivo; Okumura K et al.; PURPOSE: The purpose of this work was to evaluate the anti-inflammatory effects of secretable human Cu, Zn-superoxide dismutase (hSOD) delivered by genetically modified skin fibroblasts in vitro and in vivo . METHODS: Rat skin fibroblasts were transfected with pRc/CMV-ILSOD including secretable SOD-coding cDNA . The effects of host and transformants on oxidative stress in vitro models using the xanthine/xanthine oxidase (X/XO) system were examined to study the paracrine SOD action . The anti-inflammatory effects by transplantation of host and transformants were evaluated in an acute inflammation model, carrageenin-induced paw edema, in rats . RESULTS: The transformants (ILSOD cells) secreted SOD protein into the extracellular space, and the extracellular SOD activity in ILSOD cells cultures was significantly increased compared with that in host cell cultures . ILSOD cells diminished the cytotoxic activity by X/XO in a paracrine fashion . These protective effects of ILSOD cells against X/XO-induced cytotoxicity correlated well with the decrease in lipid peroxidation in the damaged cells . The in vivo study showed that transplantation of ILSOD cell suspensions into the hind paw in rats inhibited carrageenin-induced paw edema for at least 7 days, and the degree and the durability of these inhibitory effects were dependent on the number of ILSOD cells transplanted . These inhibitory effects of ILSOD cell suspensions were reduced by co-administration of antiserum for hSOD . Furthermore, the healing of paw edema caused by carrageenin was markedly enhanced by transplantation of ILSOD cells into the edemics hind paw . CONCLUSIONS: The findings suggested that genetically modified skin fibroblasts are a suitable delivery system for obtaining an efficient and continuous supply of SOD to the target site, and this strategy may be a useful drug delivery system for therapeutic proteins.

Pharm Res, 1997 Sep, 14(9), 1216 - 22
The absence of accessible vitronectin receptors in differentiated tissue hinders adenoviral-mediated gene transfer to the intestinal epithelium in vitro; Walter E et al.; PURPOSE: Adenoviral (Ad) vectors have been used as efficient tools for gene therapy in various tissues, whereas in some differentiated epithelium transduction efficiency is almost abolished . METHODS: Caco-2 cell monolayers were chosen as an in vitro model for the differentiated intestinal epithelium . Fluorescence-labeled adenoviral particles were used for binding studies to cell surfaces . Internalization receptors for adenoviral uptake were detected by a fluorescence-labeled vitronectin antibody . Gene expression was studied by using the beta-galactosidase reporter gene . All experiments were done on undifferentiated and differentiated Caco-2 cells . Furthermore, adenoviral particles were allowed to bind to differentiated Caco-2 monolayers followed by a trypsinization step that disintegrates the monolayers and result in a cell suspension . Gene expression was tested after reseeding the cells into dishes . RESULTS: The results from adenoviral binding studies, vitronectin immunofluorescence detection and gene expression are in good agreement and indicate that virion binding as well as the expression of internalization receptors almost disappear in fully differentiated cells . Nonetheless, adenoviral binding to differentiated monolayers seems to be sufficient to cause up to 53% gene expression, but only if internalization of the vector can be induced by disintegrating the monolayers and releasing free vitronectin receptors . CONCLUSIONS: These findings indicate that gene transfer to the intestinal epithelium utilizing adenoviral vectors is poor and ineffective, because of the lack of sufficient internalization receptors . If these receptors can be exposed in differentiated epithelium, transduction can be made more efficient . Alternatively, a viral vector must be developed whose uptake mechanism is independent of integrin receptor expression like the enteral virus Ad40, or Ad5 could be conjugated to ligands that trigger viral internalization by receptor-mediated endocytosis.

Ophthalmic Res, 1997, 29(6), 374 - 80
Visualization and flow of platelets and leukocytes in vivo in rat retinal and choroidal vessels; Kim J et al.; PURPOSE: To directly visualize the flow of leukocytes in choroidal vessels and the flow of platelets in retinal vessels in a rat without incision by fluorescein leukocyte angiography (FLA) using a scanning laser ophthalmoscope (SLO) . METHODS: Blood was withdrawn from a tail vein of a Sprague-Dawley rat with a tuberculin syringe traced with sodium heparin and mixed with sodium fluorescein . The fluorescent plasma layer was diluted with saline solution, centrifuged and then the overlying plasma discarded . The remaining cell suspension was diluted with saline to create the original hematocrit, then infused into the vein of the same rat while performing fluorescein angiography with an SLO . The angiographic image was recorded on a videotape using time-lapsed photography . RESULTS: Fluorescent platelets were detected and the flow within the retinal vessels traced over time . Fluorescent leukocytes in the choroidal vessels were also detected and the flow of a leukocyte was traced and its relative velocities were plotted against the time sequence . The relative size and fluorescence intensities of the platelets and leukocytes in the angiographic image corresponded well with the smear of the blood preparation . CONCLUSIONS: FLA using an SLO can be used to detect the flow of platelets in the retinal vessels and the flow of leukocytes in the choroidal vessels in the experimental rat eye model.

Autoimmunity, 1997, 25(4), 193 - 201
Profiles of pulp infiltrating lymphocytes at various times throughout feather regeneration in Smyth line chickens with vitiligo; Shresta S et al.; Smyth line (SL) chickens develop a spontaneous, autoimmune, posthatch loss of pigment cells (vitiligo) in regenerating feather tissue . Smyth line vitiligo (SLV) is associated with lymphocyte infiltrations prior to and throughout the development of the disorder . It was the purpose of this study to determine the type, relative amounts, and proportions of pulp-infiltrating lymphocytes at various times throughout the growth of regenerating feathers . Feathers were plucked from 8-week-old chickens with and without SLV . Feather pulp cell suspensions were prepared when the regenerating feathers were 2, 3, 4, and 6 weeks of age . Cells were fluorescently labeled using a panel of mouse monoclonal antibodies specific for chicken lymphocytes . Both T and B cells infiltrated the feather pulp of chickens with SLV . T cell levels remained elevated throughout the 6 weeks of feather growth, while B cell levels steadily declined to control levels over the same time . The pulp-infiltrating cells were primarily T cells with an alphabeta T cell receptor expressing the Vbeta1 gene (TCR2+) . The ratio between CD4+ and CD8+ cells was 1.42 and 0.75 in 2- and 6-week-old regenerating feathers from chickens with autoimmune SLV, respectively . In non-vitiliginous chickens this ratio was always near 1 . These data suggest that TCR2+ T cells play an important role in SLV . CD4+ cells may play a recruiting/activating role, whereas CD8+ cells may have cytotoxic activity specifically directed against melanocytes . Additionally, this is the first report demonstrating the infiltration of B cells into the feather pulp of vitiliginous chickens . These B cells may directly/indirectly contribute to melanocyte destruction in SLV.

Vet Immunol Immunopathol, 1997 Aug, 58(1), 1 - 16
Immunophenotypic characterization of feline Langerhans cells; Saint-Andre Marchal I et al.; To carry out the characterization of feline Langerhans cells (LC), first described in 1994, we used a panel of monoclonal antibodies (MAb) known to react with human, canine and feline leukocyte membrane antigens (Ag) . The immunolabeling was performed, at light microscope level, on frozen sections of feline skin and labial mucosa using an avidin-biotin-peroxidase technique, and at electron microscope level on epidermal cell suspensions using an immunogold technique . Out of the 52 MAb tested, six labeled basal or suprabasal DC cells in the frozen sections, either in epidermis or lip epithelium: MHM23 (anti-human CD18), CVS20 and vpg3 (respectively anti-canine and feline-major histocompatibility complex class II molecules), vpg5 (anti-feline leukocytes), vpg39 (anti-feline CD4) and Fel5F4 (anti-feline CD1a) . These six MAb were used on suspensions, and labeled cells which showed no desmosomes or melanosomes, but contained 'zipper-like' structures similar to Birbeck granules (BG) in their cytoplasm, revealing they were LC . Consequently, feline LC are CD18-positive (CD18+), major histocompatibility complex class II-positive (Class II+), CD1a-positive (CD1a+), vpg5-positive (vg5+) and CD4-positive (CD4+) . This immunophenotypic and ultrastructural characterization demonstrates that feline LC share many characteristics with their human counterparts, a fact that will allow us to study the role of feline LC in certain feline diseases such as Feline Immunodeficiency Virus (FIV) infection, since it has been shown that human LC cells are HIV-permissive, and to establish an animal model for human AIDS.

Kurume Med J, 1997, 44(3), 185 - 9
Expression of desmogleins in Paget cells of mammary and extramammary Paget's disease; Mori O et al.; Sagebiel (1969) electronmicroscopically observed that desmosomes are present between adjacent Paget cells, as well as between Paget cells and adjacent keratinizing epidermal cells . Desmosomes contain the proteins desmoglein (Dsg) and desmocollin as their transmembrane components, and Dsg has three isotypes . Among the three isotypes of Dsg, Dsg1 and Dsg3 are autoantigens for pemphigus foliaceus and pemphigus vulgaris (PV), respectively . We examined the expression of Dsgs in Paget cells of mammary and extramammary Paget's disease . Skin samples were obtained from one patient with mammary Paget's disease, and from 2 with extramammary Paget's disease . One part of the samples was cut into small pieces and epidermal sheets were separated with dispase, then treated with a mixture of EDTA and trypsin . The resulting cell suspensions were cultured in low Ca++ medium, then the cells were incubated in high Ca++ medium . Paget cells identified with anti-epithelial membrane antigen (EMA) antibody were positive for serum from a PV patient which was confirmed to react with both Dsg1 and Dsg3 . However, the intensity of fluorescence of Paget cells was weaker than that of EMA-negative keratinocytes . In the cryostat sections, Paget cells identified with anti-EMA antibody showed the same staining pattern as cultured cells in high Ca++ medium . The data presented in this study confirm that Paget cells express Dsgs, and are consistent with the electronmicroscopical data by Sagebiel (1969) . However, our data do not support the hypothesis that Paget cells are of keratinocyte origin, because sweat ducts or sweat glands could be positive for sera from PV patients . It is necessary to confirm whether or not sweat ducts or sweat glands express Dsgs, because sera from PV patients exhibit high background in the dermis.

Kurume Med J, 1997, 44(3), 165 - 9
Epidermal cell cultures from involved skin of patients with mammary and extramammary Paget's disease; Mori O et al.; In involved epidermis, Paget cells are completely enclosed by the surrounding keratinocytes, which appear to be intact and unaltered . It is possible that the surrounding keratinocytes inhibit Paget cell proliferation . Accordingly, Paget cells might proliferate differently when cultured as an epidermal cell suspension . In this study, primary monolayer cultures of epithelial cells from involved epidermis of patients with mammary and extramammary Paget's disease were carried out to investigate whether Paget cells proliferate in the same manner as other malignant cells . Skin samples were obtained from one patient with mammary Paget's disease, and from 2 patients with extramammary Paget's disease . The epidermis was separated from the dermis with dispase, and epidermal cell suspensions were obtained with ethylenediamine tetraacetate and trypsin . A commercially available serum-free media, Keratinocyte-SFM, was used . Epithelial monolayers from the involved skin could be maintained for approximately 45 days, while keratinocytes from normal skin were maintained for approximately 35 days . The mechanism for the longer survival of the mixed cell culture of keratinocytes and Paget cells is not known . Permanent cell lines were not developed from these primary cultures . Paget cells could not be distinguished from keratinocytes by phase-contrast microscopy . The proportion of carcinoembryonic antigen (CEA) positive cells in the culture did not increase, but instead decreased . In certain areas of the dish, the CEA positive cells proliferated and accumulated like mushrooms . However, at the periphery of the dish, the Paget cells identified by immunostaining for CEA were dispersed and not clustered . These findings indicate that the influence of keratinocytes on Paget cells also occurs in cultured cells, which may explain why Paget cells survive longer than keratinocytes . In conclusion, the Paget cells in the involved epidermis do not proliferate like other malignant cells.

Br J Cancer, 1997, 76(7), 870 - 7
Menadione-resistant Chinese hamster ovary cells have an increased capacity for glutathione synthesis; Vallis KA et al.; A cell line (MRc40) resistant to the model quinone compound, menadione, has been isolated from a parental Chinese hamster ovary cell line (CHO-K1) . The known relationship between menadione toxicity and glutathione (GSH) depletion led us to investigate whether the mechanism of resistance of MRc40 was related to alteration in GSH homeostasis . Intracellular concentrations of GSH and cysteine (CySH) were twofold and 3.2-fold greater in MRc40 than in CHO-K1 . Following exposure to menadione, GSH and CySH were depleted, but subsequent recovery of thiols was more rapid and of greater magnitude in MRc40 than in CHO-K1 . Twelve hours after exposure to menadione, the concentrations of GSH and CySH were 9.7- and 4.2-fold greater in MRc40 than in CHO-K1 . Using nuclear magnetic resonance (NMR) spectroscopy, we observed the in situ removal of menadione from cell suspensions of CHO-K1 and MRc40 . However, only in CHO-K1 did we observe concomitant depletion of NMR-visible GSH . We conclude that the perturbation of GSH metabolism contributes to the resistant phenotype and is an important characteristic of menadione-resistant CHO cells.

Int Arch Allergy Immunol, 1997 Oct, 114(2), 139 - 43
Secretory response of mast cells contained in monodispersed human choroidal preparations; Miller ST et al.; BACKGROUND: Mast cells have been identified in the choroid of numerous species including man . However, functional studies involving these human mast cells have not been reported . In the current studies, the secretory response of human choroidal mast cells to various stimuli was examined using monodispersed choroidal cell preparations . METHODS: Monodispersed cell suspensions of human choroid were prepared from eye bank globes and the number, histamine content, and secretory response of mast cells in these preparations were determined . Choroids from 27 donors were used for these experiments . RESULTS: Cell suspensions contained an average of 15% mast cells . Mast cells stained positively with toluidine blue and exhibited the classical granular appearance upon electron microscopy . The amount of histamine contained in each mast cell was calculated to be 2.74+/-0.17 pg . Significant histamine release was observed following treatment with anti-human IgE, calcium ionophore A23187, concanavalin A, compound 48/80 and morphine . CONCLUSION: A method has been developed for obtaining monodispersed human choroidal mast cell preparations . The cells were functional as evidenced by their ability to release histamine upon immunological and nonimmunological stimulation . The degranulation noted following compound 48/80 and morphine challenge suggests that these human choroidal mast cells are analogous to connective tissue or chymase/tryptase-positive mast cells.

Int J Cancer, 1997 Oct 9, 73(2), 230 - 5
Foscan-mediated photodynamic therapy for a peritoneal-cancer model: drug distribution and efficacy studies; Veenhuizen RB et al.; Distribution of the photosensitizer Foscan (meta-tetrahydroxyphenylchlorin, mTHPC), after i.v . or i.p . injection, was investigated in Wag/Rij rats bearing i.p . tumours . These results were compared with the efficacy of mTHPC-mediated photodynamic therapy for illumination intervals of 4 hr to 3 days . For the distribution experiments a single tumour (CC53I colon carcinoma) was implanted intra-abdominally in a fat pad, or a cell suspension (1 x 10(6) CC531 cells) was injected into the peritoneal cavity, which results in a dissemination of tumour nodules on the peritoneum . 14C-mTHPC was not selectively taken up in the single-tumour model after i.v . or i.p . injection, but higher concentrations were achieved for i.p . administration . For this tumour model the concentration ratios between tumour and normal tissue never exceeded a value of 3 . In the disseminated-tumour model, an uptake of up to 40% of the injected dose was found per gram tumour at 4 hr after an i.p . injection and this resulted in very high (> 14) concentration ratios of tumour to normal tissues . Low uptake was found after the i.v . injection route (1% of the injected dose per gram tumour) with lower tumour/normal tissue ratios (<8) . The efficacy of i.p . photodynamic therapy (IPPDT) was evaluated using the single-tumour model only . The lower abdomen was illuminated at 4 hr to 3 days after mTHPC, and tumour size was repeatedly measured via a small laparoscopy . Significant delay in tumour regrowth was achieved for 6 J x cm-2 at 1 day after i.v., or at 4 hr after i.p . mTHPC (p values 0.019 and 0.045 respectively) . Response to PDT, of tumours implanted in the fat pad, was not greater for i.p . administration of the photosensitizer and there was a poor correlation between times of maximum drug uptake in tumours and optimal illumination times for PDT efficacy.

Cell Transplant, 1997 Sep-Oct, 6(5), 511 - 3
Evaluation of intracerebral grafting of dopamine-secreting PC12 cells into allogeneic and xenogeneic brain; Ono T et al.; The PC12 phenochromocytoma tumor cell line is derived from a rat adrenal medullary tumor and secretes dopamine . We have previously reported that grafted microencapsulated PC12 cells using agarose and poly (styrene sulfonic acid) survived in the xenogeneic brain without immunosuppression . To investigate whether unencapsulated PC12 cells form a tumor and how they provoke immunological reaction, PC12 cell suspension was implanted into the striatum of Sprague-Dawley rat (allogeneic graft) or guinea pig (xenogeneic graft) and histological analysis using Nissl stain and immunocytochemical analysis using antityrosine hydroxylase (TH) antibody were performed 1, 2, and 4 wk after transplantation . Host animals were not immunosuppressed . PC12 cells formed a mass 1 and 2 wk after transplantation both in allogeneic and xenogeneic brain . These grafted PC12 cells were immunoreactive to anti-TH antibody . Four weeks after transplantation, however, grafted PC12 cells in the allogeneic brain were only found within the restricted area near the site of implantation . In the xenogeneic brain, only the trace of grafted PC12 cells were found around the site of implantation 4 wk after transplantation . In both allogeneic and xenogeneic animals, a number of lymphocytes were found in and around the grafts at all period investigated . These findings indicate that PC12 cells could survive in the allogeneic or xenogeneic brain for 2 wk and were ultimately rejected by immunological reaction by 4 wk after transplantation . Implantation of encapsulated PC12 cells in the allogeneic or xenogeneic brain is considered a safe and effective method for delivering dopamine into the brain because PC12 cells will not form a tumor in the long-term even if capsules are damaged in some reason.

Cell Transplant, 1997 Sep-Oct, 6(5), 479 - 89
Organization and histochemical phenotype of human fetal cerebellar cells following transplantation into the cerebellum of nude mice; Pundt LL et al.; Previous rodent studies have demonstrated the capacity of cerebellar transplants to organize into trilaminar cell layers typically observed in the normal cerebellum . In Purkinje Cell (PC)-deficient animals, PCs will migrate into the host and form synaptic connections . Recently, fetal cerebellar grafts transplanted into the Purkinje cell degeneration (pcd) mutant mouse were shown to result in an improvement of motor behaviors . These studies indicate the potential therapeutic use of neural transplantation in patients with cerebellar degeneration . In the present study, human fetal cerebellar tissue (8.5 wk postconception) was dissociated and transplanted into the normal cerebellum of nude mice . Six months following transplantation, histological analysis revealed donor cells in recipient mice . Immunostaining for the 28 kDa calcium-binding protein (calbindin) revealed the presence of donor PCs that were organized in discrete cellular layers within the transplant neuropil . In most cases the dendritic processes were oriented in a planar fashion perpendicular to the transplant cell layer . Human neurofilament immunostaining revealed bundles of donor fibers within the core of the transplant and/or at the periphery . These bundles were found to be calbindin positive (PC fibers) . Three animals provided evidence of donor PC axon growth ventrally into host white matter, and in one case, this ventral migration reached the deep cerebellar nuclei . Most notable was the development of a pronounced folia-like organization by the implanted cell suspensions . Glial processes within the grafts were aligned perpendicular to the long axis of the transplant folia . These results demonstrate the capacity of human fetal cerebellar cell suspension to reorganize into cell layers typical of the normal cerebellum following transplantation into the rodent cerebellum, and develop an organotypic folia-like organization.

Jpn J Cancer Res, 1997 Aug, 88(8), 770 - 7
Augmentation in chemosensitivity of intratumor quiescent cells by combined treatment with nicotinamide and mild hyperthermia; Masunaga S et al.; C3H/He and Balb/c mice bearing SCC VII and EMT6/KU tumors, respectively, received continuous administration of 5-bromo-2'-deoxyuridine (BrdU) for 5 days using implanted mini-osmotic pumps to label all proliferating (P) cells . Nicotinamide was administered intraperitoneally before cisplatin injection and/or tumors were locally heated at 40 degrees C for 60 min immediately after cisplatin injection . The tumors were then excised, minced and trypsinized . The tumor cell suspensions were incubated with cytochalasin-B (a cytokinesis-blocker), and the micronucleus (MN) frequency in cells without BrdU labeling (quiescent (Q) cells) was determined using immunofluorescence staining for BrdU . The MN frequency in total (P+Q) tumor cells was determined from tumors that had not been pretreated with BrdU labeling . The sensitivity to cisplatin was evaluated in terms of the frequency of induced micronuclei in binuclear tumor cells (MN frequency) . In both tumor systems, the MN frequency in Q cells was lower than that in the total cell population . Nicotinamide treatment elevated the MN frequency in total SCC VII cells . Mild heating raised the MN frequency more markedly in Q cells than in total cells . The combination of nicotinamide and mild heat treatment increased the MN frequency more markedly than either treatment alone . In total SCC VII cells, nicotinamide increased 195mPt-cisplatin uptake . Mild heating elevated 195mPt-cisplatin uptake in total EMT6/KU cells . Cisplatin-sensitivity of Q cells was lower than that of total cells in both tumor systems . Nicotinamide sensitized tumor cells including a large acutely hypoxic fraction, such as those of SCC VII tumors, through inhibition of the fluctuations in tumor blood flow . Tumor cells including a large chronically hypoxic fraction such as Q cells were thought to be sensitized by mild heating through an increase in tumor blood flow.

Southeast Asian J Trop Med Public Health, 1997 Mar, 28(1), 18 - 21
Large scale culture technique for pure Plasmodium falciparum gametocytes; Petmitr P et al.; A large scale technique for pure gametocyte cultures of Plasmodium falciparum was established in culture flasks by using treated erythrocytes and RPMI medium supplemented with 15% human plasma instead of serum . Pure gametocyte cultures were successfully obtained following treatment with 5% sorbitol on day 8 and 9 of cultivation . This method resulted in approximately 97% reduction of asexual parasites and provided pure gametocytes in culture . The highest numbers of gametocytes were obtained from cultures starting with 2% parasitemia and 2% erythrocyte suspension . On day 12 of cultivation, approximately 35 x 10(6) gametocytes per 100 ml of cell suspension could be harvested.

Nervenarzt, 1997 Jun, 68(6), 477 - 84
{Stereotactic treatment of movement disorders}; Ostertag CB et al.; Stereotactic surgery for movement disorders is currently undergoing a re-evaluation . A new understanding of the pathophysiology makes the surgical lesion a logical step for the aleviation of both hyperkinetic symptoms such as tremor and hypokinetic symptoms like bradykinesia . Advances in imaging and electrophysiological control render these procedures more accurate and safer . Indications are medically refractory, Parkinsonean tremor, essential tremor, cerebellar tremor, bradykinesia and L-Dopa induced dyskinesis . The standard procedure is ablative surgery, i.e . thalamotomy for tremors and pallidotomy for bradykinesia, dystonia and L-Dopa induced dyskinesias . Deep brain stimulation is a novel alternative for selected patients which is currently evaluated . Neural transplantation of autologus, fetal or genetically manipulated cell suspensions into the striatum for the time being is experimental.

Mod Pathol, 1997 Sep, 10(9), 859 - 63
Usefulness of a new CD5 antibody for the diagnosis of T-cell and B-cell lymphoproliferative disorders in paraffin sections; Dorfman DM et al.; We used a new antibody (NCL-CD5-4C7) immunoreactive for CD5-positive cells in paraffin-embedded tissue, with the microwave antigen retrieval method, to analyze a number of well-characterized cases of B-cell and T-cell non-Hodgkin's lymphomas whose CD5 immunoreactivity was documented by immunohistochemical analysis performed on frozen sections and/or flow cytometric immunophenotypic analysis of neoplastic cell suspensions . In all of the cases included in the study, small, reactive T lymphocytes within histologic sections were strongly positive for CD5 and served as an internal positive control . All CD5-positive T-cell non-Hodgkin's lymphomas (13 {100%} of 13) from several histologic subgroups, were immunoreactive for CD5 in paraffin sections, although 4 of 13 exhibited weak or limited staining of neoplastic cells . The majority of cases of B-cell small lymphocytic lymphoma/chronic lymphocytic leukemia (SLL/CLL) (13 {81%} of 16) and mantle cell lymphoma (14 {93%} of 15), both B-cell lymphoproliferative disorders that are immunoreactive for CD5, were immunoreactive for CD5 in paraffin sections, although 5 of 13 cases of SLL/CLL and 6 of 14 cases of mantle cell lymphoma exhibited weak and/or limited staining of neoplastic cells . Other B-cell non-Hodgkin's lymphomas of various subtypes, including marginal zone, follicular, diffuse large cell, and lymphoblastic types, were nonreactive for CD5 in paraffin sections, as expected . CD5 antibody NCL-CD5-4C7 should be useful for the routine characterization of suspected T-cell and B-cell lymphoproliferative disorders when only paraffin-embedded tissue is available for immunophenotypic analysis.

Mol Cell Biochem, 1997 Sep, 174(1-2), 121 - 4
Reversibility of thiamine deficiency-induced partial necrosis and mitochondrial uncoupling by addition of thiamine to neuroblastoma cell suspensions; Bettendorff L et al.; Culture of neuroblastoma cells in the presence of low thiamine concentration (16 nM) and of the transport inhibitor amprolium leads to the appearance of signs of necrosis: the chromatin condenses, the oxygen consumption decreases and is uncoupled, the mitochondrial cristae are disorganized, the thiamine diphosphate-dependent dehydrogenase activities are impaired . When 10 microM thiamine are added to these cells, the basal respiration increases, the coupled respiration is restored and mitochondrial morphology is recovered within 1 h . Addition of succinate, which is oxidized via a thiamine diphosphate-independent dehydrogenase, to digitonin-permeabilized cells immediately restores a coupled respiration . Our results suggest that the slowing of the citric acid cycle is the cause of the biochemical lesion induced by severe thiamine deficiency and that part of the mitochondria remain functional.

Neuroscience, 1997 Oct, 80(3), 741 - 52
Specificity of attachment and neurite outgrowth of dissociated basal forebrain cholinergic neurons seeded on to organotypic slice cultures of forebrain; Robertson RT et al.; Development and differentiation of basal forebrain-derived cholinergic neurons were studied using a new technique that combines dissociated cell cultures with organotypic slice cultures . Slices of cerebral cortex or entire forebrain hemispheres were taken from early postnatal rat pups and maintained as organotypic cultures on membranes . Dissociated cell suspensions of basal forebrain tissue, taken from rat or mouse fetuses at gestational day 15-17, were seeded on to the slice cultures . Combined cultures were maintained for two to 14 days in vitro . Cultures processed for acetylcholinesterase histochemical staining demonstrated that stained neurons display regional variation in attachment to the slice, with most attachment occurring on cortex and with no detectable attachment on the caudate-putamen . Regional differences in attachment occur between cortical areas, with medial (cingulate) cortex showing much denser cell attachment than lateral (parietal) cortex, and across cortical layers, with layer I and deep layers showing more attachment than middle cortical layers . Similar patterns were observed on slices from rat brain irrespective of whether rat or mouse dissociated cells were used . Tyrosine hydroxylase-stained dissociated cells from ventral midbrain displayed a different pattern of attachment, with prominent attachment to the caudate putamen and less apparent specificity of regional and cortical laminar attachment . Little evidence of neurite outgrowth occurred during the first two days in vitro, but by four days, acetylcholinesterase-positive basal forebrain cells displayed several short and thick neurites that appeared to be dendrites, and one long process that appeared to be an axon . By seven days in vitro, dendrites are well developed and the presumed axon has extended branches over wide areas of cortex . These studies revealed several different types of cell-tissue interaction . The degree of cell growth and differentiation ranged from robust growth when dissociated cells were seeded on to slice cultures of normal target tissue, to apparently no attachment or growth when cells were seeded on to non-target tissue . This combined technique appears to be a useful method for studies of specificity of cell attachment and patterns of neurite outgrowth.

Biochim Biophys Acta, 1997 Aug 29, 1336(2), 323 - 30
Theophylline metabolism in higher plants; Ito E et al.; Metabolism of {8-(14)C}theophylline was investigated in leaf segments from Camellia sinensis (tea), Camellia irrawadiensis, Ilex paraguariensis (mate) and Avena sativa, root segments of Vigna mungo seedlings and cell suspension cultures of Catharanthus roseus . There was extensive uptake and metabolism of {8-(14)C}theophylline by leaves of tea and Camellia irrawadiensis and, to a lesser extent, mate . These purine alkaloid-containing species converted {8-(14)C}theophylline into 3-methylxanthine, xanthine, the ureides allantoin and allantoic acid, and CO2 . With the other test systems, which were from species that do not produce purine alkaloids, there were low levels of {8-(14)C}theophylline uptake which were accompanied by incorporation of relatively small amounts of label into 3-methylxanthine, xanthine and CO2 . None of the higher plants converted {8-(14)C}theophylline to either 1-methyluric acid or 1,3-dimethyluric acid, which are the main catabolites of theophylline in mammals . The data indicate that the main route of theophylline degradation in higher plants involves a theophylline --> 3-methylxanthine --> xanthine --> uric acid --> allantoin --> allantoic acid --> --> CO2 + NH3 pathway . In tea and mate, large amounts of {8-(14)C}theophylline were also converted to theobromine and caffeine via a theophylline --> 3-methylxanthine --> theobromine --> caffeine salvage pathway . The diversity of theophylline metabolism in higher plants and mammals is discussed.

Leukemia, 1997 Sep, 11(9), 1554 - 64
JURL-MK1 (c-kit(high)/CD30-/CD40-) and JURL-MK2 (c-kit(low)/CD30+/CD40+) cell lines: 'two-sided' model for investigating leukemic megakaryocytopoiesis; Di Noto R et al.; Two novel cell lines (JURL-MK1 and JURL-MK2) have been established from the peripheral blood of a patient in the blastic phase of chronic myelogenous leukemia . The cells grow in a single cell suspension with doubling times of 48 h (JURL-MK1) and 72 h (JURL-MK2) . Cytogenetic analysis has shown that JURL-MK1 is hypodiploid whereas JURL-MK2 is near triploid and that both cell lines retain t(9;22) . Moreover, JURL-MK1 and JURL-MK2 have a bcr/abl-fused gene with the same junction found in the patient's fresh cells, and both cell lines express the b3/a2 type of hybrid bcr/abl mRNA . The morphology and immunophenotype of these cell lines are reminiscent of megakaryoblasts . In both lines, a limited but consistent percentage of cells expresses gpIIbIIIa (CD41a), gpIIIa (CD61) and CD36, with no expression of gplb (CD42b), glycophorin A, hemoglobin and CD34 . Both cell lines are clearly positive for CD33, CD43, CD45RO and CD63, while CD13, CD44, CD54, CD30 and CD40 are specific features of JURL-MK2 . Among cytokine receptors, CD117/SCF-R is strongly displayed by a large fraction of JURL-MK1 cells but is hardly detectable on about 20% JURL-MK2 cells . Both cell lines are clearly positive for CD25/IL2R alpha, while a marked expression of CD116/GM-CSF-R and CDw123/IL3R alpha is restricted to JURL-MK2 . Induction of cell differentiation in vitro has demonstrated that TPA is able to modulate the JURL-MK1 phenotype, causing an increased expression of platelet-associated antigens . The JURL-MK2 phenotype is easily modulated by both TPA and DMSO, which cause an increased expression of CD41a and CD117 accompanied by a decreased expression of CD30 . Proliferation studies demonstrated that JURL-MK1 cell growth is enhanced by stem cell factor, while JURL-MK2 proliferation is unaffected by this cytokine . JURL-MK1 and JURL-MK2 are two novel cell lines with divergent biological features, representing a 'two-sided' model for investigating new aspects of megakaryocytopoiesis.

Br J Cancer, 1997, 76(5), 588 - 93
Reduction of hypoxic cells in solid tumours induced by mild hyperthermia: special reference to differences in changes in the hypoxic fraction between total and quiescent cell populations; Masunaga S et al.; C3H/He mice bearing SCC VII tumours received 5-bromo-2'-deoxyuridine (BrdU) continuously for 5 days via implanted mini-osmotic pumps in order to label all proliferating (P) cells . The tumours were then heated at 40 degrees C for 60 min . At various time points after heating, tumour-bearing mice were irradiated while alive or after being killed . Immediately after irradiation, the tumours were excised, minced and trypsinized . The tumour cell suspensions obtained were incubated with cytochalasin-B (a cytokinesis blocker), and the micronucleus (MN) frequency in cells without BrdU labelling, which could be regarded as quiescent (Q) cells, was determined using immunofluorescence staining for BrdU . The MN frequency in the total (P+Q) tumour cell population was determined from the irradiated tumours that were not pretreated with BrdU . The MN frequency of BrdU unlabelled cells was then used to calculate the surviving fraction of the unlabelled cells from the regression line for the relationship between the MN frequency and the surviving fraction of total (P+Q) tumour cells . In general, Q cells contained a greater hypoxic fraction (HF) than the total tumour cell population . Mild heating decreased the HF of Q cells more markedly than in the total cell population, and the minimum values of HFs of both total and Q cell populations were obtained 6 h after heating . Two days after heating, the HF of total tumour cells returned to almost that of unheated tumours . In contrast, the HF of Q cells did not return to the HF level of unheated tumours until 1 week after heating . It was thought that irradiation within 12 h after mild heating might be a potentially promising therapeutic modality for controlling radioresistant Q tumour cells.

Int J Cancer, 1997 Sep 4, 72(5), 851 - 9
Radiation-induced apoptosis in human ovarian carcinoma cells growing as a monolayer and as multicell spheroids; Filippovich IV et al.; Response to external gamma irradiation was studied in a human ovarian carcinoma cell line (OVCAR 3) growing as a monolayer and as multicell spheroids . Necrosis and apoptosis were documented using Trypan-blue uptake and acridine-orange staining, respectively, and apoptosis was quantified using a terminal deoxynucleotidyl transferase assay . Exposure of OVCAR 3 cells growing as a monolayer to 137Cs gamma radiation at a dose of 10 Gy produced 30-40% apoptosis 72 hr after irradiation . Cell-cycle analysis of irradiated cells showed an accumulation of cells in G2/M phase 24 hr after irradiation and then a decline at 48 hr in conjunction with apoptosis onset . The loss of G0/G1 cells in irradiated cultures suggested a preferential entry into apoptosis . No increase in apoptotic cell number was observed in OVCAR 3 spheroids after irradiation, and the cells probably died as a result of necrosis . When spheroids were disrupted immediately after irradiation to obtain a cell suspension, minor apoptosis was observed in association with a marked increase in TB-positive cell number after 96 hr of incubation following irradiation . Thus, a relationship was found between radiation-induced apoptosis and the cell cycle . Results with spheroids suggested the possible involvement of cell-to-cell interactions in apoptosis regulation.

Immunology, 1997 Jul, 91(3), 473 - 8
Selection of antibodies to cell surface determinants on mouse thymic epithelial cells using a phage display library; Palmer DB et al.; The network of thymic epithelium contributes significantly to the thymic stromal cell environment, which plays a vital role in the generation and maturation of thymocytes . Monoclonal antibodies (mAb) have revealed considerable heterogeneity within this epithelial component of the mouse thymic microenvironment, but many of these antibodies recognize epitopes that are located inside the cell and so cannot be used in functional studies . As an alternative approach to isolate antibodies specific to thymic epithelium, we used a phage display library expressing single chain Fv antibodies . For selection, a thymic cell suspension was incubated with the phage display library, and major histocompatibility complex class II positive cells, the majority of which are epithelial, were then specifically selected . Phage bound to these cells were eluted and the selection procedure was repeated for a further five rounds . Immunohistochemical analysis revealed that these phage antibodies show differential staining of thymic epithelial subsets . Flow cytometric analysis of a thymic epithelial cell line using a panel of these antibodies demonstrated that they recognize epitopes on the cell surface . Furthermore, some of these antibodies also labelled human thymic epithelium, suggesting that the epitopes recognized by these antibodies are conserved between human and rodent thymus . Our approach therefore provides a rapid method to select antibodies specific for thymic epithelial cell surface determinants in their native configuration.

Planta, 1997 Sep, 203(1), 67 - 74
Disruption of the phosphate-starvation response of oilseed rape suspension cells by the fungicide phosphonate; Carswell MC et al.; The influence of the anti-fungal agent phosphonate (Phi) on the response of oilseed rape (Brassica napus L . cv . Jet Neuf) cell suspensions to inorganic phosphate (Pi) starvation was examined . Subculture of the cells for 7 d in the absence of Pi increased acid phosphate (APase; EC 3.1.3.2) and pyrophosphate (PPi)-dependent phosphofructokinase (PFP; EC 2.7.1.90) activities by 4.5- and 2.8-fold, respectively, and led to a 19-fold increase in Vmax and a 14-fold decrease in Km (Pi) and Pi uptake . Addition of 2 mM Pi to the nutrient media caused dramatic reductions in the growth and Pi content of the Pi-starved, but not Pi-sufficient cells, and largely abolished the Pi-starvation-dependent induction of PFP, APase, and the high-affinity plasmalemma Pi translocator . Immunoblotting indicated the cells contain three APase isoforms that are synthesized de novo following Pi stress, and that Pi treatment represses this process . Phosphonate treatment of Pi-starved cells significantly altered the relative extent of in-vivo 32P-labelling of polypeptides having M(rs) of 66, 55, 45 and 40 kDa . However, Phi had no effect on the total adenylate pool of Pi-starved cells which was about 32% lower than that of Pi-sufficient cells by day 7 . Soluble protein levels, and activities of pyruvate kinase (EC 2.7.1.40) and ATP-dependent phosphofructokinase (EC 2.7.1.11) were unaffected by Pi starvation and/or Phi treatment . The effects of Phi on the growth, and APase and PFP activities of Pi-starved B . napus seedlings were similar to those observed in the suspension cells . The results re consistent with the hypothesis that a primary site of Phi action in higher plants is at the level of the signal transduction chain by which plants perceive and respond to Pi stress at the molecular level.

Am J Cardiol, 1997 Aug 4, 80(3A), 17A - 25A
Lactate transport in heart in relation to myocardial ischemia; Halestrap AP et al.; In this article, the importance of lactic acid transport into and out of heart cells is described and the properties of the monocarboxylate transporters (MCTs) responsible are presented . These are monocarboxylate/proton symporters with a broad substrate specificity that includes L-lactate, pyruvate, and the ketone bodies acetate, acetoacetate, and beta-hydroxybutyrate . Although it is unlikely that lactic acid transport constrains heart metabolism under most conditions, it may do so during severe hypoxia or ischemia . The transporter plays a critical role in maintaining intracellular pH because it removes the protons that are produced stoichiometrically with lactate during glycolysis . The kinetics and substrate and inhibitor specificities of the transport process have been determined in cell suspensions using a radiotracer technique and in single cells using a fluorescent measurement of the decrease in intracellular pH that accompanies transport . The results of these experiments suggest the presence of 2 different transporter isoforms in heart cells, at least one of which is different from the cloned MCT1 and MCT2 . Immunofluorescence microscopy shows that MCT1 expression is restricted to the intercalated disk region, yet the rate of lactate transport in this region is slower than in the center of the cell, where there is no MCT1 . New cDNA sequences with strong homology to MCT1 have been found in human cDNA libraries and Northern blots show that the corresponding mRNA is expressed in rat heart . Expressions of these new MCT isoforms have yet to be demonstrated and their properties and cellular distribution defined.

Appl Environ Microbiol, 1997 Sep, 63(9), 3684 - 90
Reduction of azo dyes by redox mediators originating in the naphthalenesulfonic acid degradation pathway of Sphingomonas sp . strain BN6; Keck A et al.; The anaerobic reduction of azo dyes by Sphingomonas sp . strain BN6 was analyzed . Aerobic conversion of 2-naphthalenesulfonate (2NS) by cells of strain BN6 stimulated the subsequent anaerobic reduction of the sulfonated azo dye amaranth at least 10-fold . In contrast, in crude extracts, the azo reductase activity was not stimulated . A mutant of strain BN6 which was not able to metabolize 2NS showed increased amaranth reduction rates only when the cells were resuspended in the culture supernatant of 2NS-grown BN6 wild-type cells . The same increase could be observed with different bacterial strains . This suggested the presence of an extracellular factor which was formed during the degradation of 2NS by strain BN6 . The addition of 1,2-dihydroxynaphthalene, the first intermediate of the degradation pathway of 2NS, or its decomposition products to cell suspensions of the mutant of strain BN6 (2NS-) increased the activity of amaranth reduction . The presence of bacterial cells was needed to maintain the reduction process . Thus, the decomposition products of 1,2-dihydroxynaphthalene are suggested to act as redox mediators which are able to anaerobically shuttle reduction equivalents from the cells to the extracellular azo dye.

Cell Tissue Res, 1997 Oct, 290(1), 89 - 99
Isolation and culture of endocardial endothelial cells from Atlantic salmon (Salmo salar) and Atlantic cod (Gadus morhua); Koren CW et al.; This paper describes a procedure for establishing primary cultures of endocardial endothelial cells and morphologically characterizes the cultivated endothelial cells from Atlantic cod (Gadus morhua L.) and salmon (Salmo salar L.) . Following incubation with collagenase and trypsin, the resulting cell suspension was seeded on substrates of fibronectin, collagen or gelatin . Cod and salmon endothelial cells attached firmly to these substrates within 24 and 5 h respectively, but did not attach to uncoated tissue-culture plastic . The contamination by non-endothelial cells was kept at a minimum by working out optimal combinations of serum concentration and growth substrate . The yield was about 3x10(6) and 1x10(6) cells per kg cod and salmon, respectively . Cultures could be maintained for several weeks but cells did not proliferate . Cultivated cod endocardial endothelial cells differed from salmon endocardial cells in having abundant cytoplasm with many vesicles and avidly taking up labelled collagen . The described method allows, for the first time, functional and morphological studies to be performed under controlled conditions on both a specialized scavenger type of endothelium from cod and a conventional flat endothelium from salmon.

Eur J Biochem, 1997 Aug 1, 247(3), 1127 - 35
Purification, characterization and partial amino acid sequencing of hydroxycinnamoyl-CoA:tyramine N-(hydroxycinnamoyl)transferase from tobacco cell-suspension cultures; Negrel J et al.; We report the purification of hydroxycinnamoyl-CoA:tyramine N-(hydroxycinnamoyl)transferase (THT) to apparent homogeneity in 12% yield from tobacco (Nicotiana tabacum L . cv . Xanthi) cell-suspension cultures elicited with a commercial preparation of pronase . The purification procedure employs only four chromatography steps and takes advantage of the fact that the transferase binds tightly both to phenyl-Sepharose and to hydroxyapatite . The native enzyme has a pI of 5.2 and consists of two identical or very similar subunits of approximately 24 kDa . The purified enzyme can synthesise a wide range of amides due to its relatively low specificity for cinnamoyl-CoA derivatives and hydroxyphenethylamines, but its best substrates are tyramine and feruloyl-CoA . THT follows Michaelis-Menten kinetics in the presence of low concentrations of feruloyl-CoA but negative cooperativity occurs when this concentration increases above 2.5 microM, resulting in a marked decrease of the affinity for tyramine . Large deviations from Michaelis-Menten kinetics are also observed when 3-methoxytyramine is used as acyl acceptor . The activity of tobacco THT is not affected by the addition of CaCl2 or MgCl2 but its maximal velocity is increased up to twofold by addition of ethanol to the assay mixture . It is inhibited in vitro by L-tyrosine benzyl ester, which binds reversibly to the tyramine-binding site . Experiments performed using L-tyrosine benzyl ester and caffeoyl-CoA as inhibitors confirm that feruloyl-CoA is the first substrate to add to the transferase in an ordered bi-bi mechanism . Part of the amino acid sequence of the transferase, elucidated by microsequencing of tryptic peptides, is also described.

Br J Pharmacol, 1997 Aug, 121(8), 1789 - 95
Differential effects of lovastatin on mitogen induced calcium influx in human cultured vascular smooth muscle cells; Clunn GF et al.; 1 . In this study the effect of lovastatin, an inhibitor of cholesterol and isoprenoid synthesis, on the rises in intracellular calcium concentration ({Ca2+}i) induced by platelet derived growth factor BB (PDGF-BB), angiotensin II (AII), low density lipoproteins (LDL) and foetal calf serum (FCS) was examined in human cultured vascular smooth muscle cells (VSMC) from saphenous vein . Changes in {Ca2+}i were measured in cell suspensions by the Ca2+ sensitive probe, fura 2 . 2 . Incubation with lovastatin for 24-26 h markedly reduced the peak rise and sustained phase of {Ca2+}i elevation in response to PDGF-BB but the responses to AII, LDL and FCS were unaffected . Further experiments showed that lovastatin pretreatment inhibited PDGF-BB induced Ca2+ influx but not intracellular Ca2+ release . This inhibition could be overcome by co-incubation with mevalonic acid . 3 . Pretreatment of cells with the heterotrimeric G protein inhibitor pertussis toxin for up to 24 h completely abolished AII-induced {Ca2+}i rises but the response to PDGF-BB was unaffected . 4 . The tyrosine kinase inhibitor genistein largely abolished PDGF-BB-induced {Ca2+}i elevation but had no significant effect on AII-induced responses . 5 . Pre-incubation with lovastatin had no effect on the level of tyrosine phosphorylation of PDGF-beta receptors (as measured by Western blot) in response to the PDGF-BB ligand . 6 . PDGF-BB elicits Ca2+ influx via a tyrosine kinase-dependent mechanism distinct from the heterotrimeric G protein coupled pathway utilized by AII . Lovastatin most likely acts by inhibition of isoprenylation (via blockade of isoprenoid synthesis) of an intermediate molecule involved in PDGF-BB-induced Ca2+ influx.

Neurosci Lett, 1997 Aug 1, 231(1), 5 - 8
Modulation of hippocampal acetylcholine release after fimbria-fornix lesions and septal transplantation in rats; Erb C et al.; Female Long-Evans rats sustained electrolytic lesions of the fimbria and the dorsal fornix causing a partial lesion of the septohippocampal pathway . Two weeks later, the rats received intra-hippocampal grafts of fetal septal cell suspensions . Nine to twelve months later, the release of acetylcholine (ACh) in the hippocampus of sham-operated, lesion-only and grafted rats was measured by microdialysis . The extent of cholinergic (re)innervation was determined by acetylcholinesterase (AChE) staining and densitometry . In both lesion-only and grafted rats, the ratio of ACh release to AChE staining intensity was increased as compared to sham-operated rats, indicating a loss of endogenous inhibitory mechanisms . Scopolamine (0.5 mg/kg i.p.), a muscarinic antagonist, increased ACh release in all treatment groups . 8-OH-DPAT (0.5 mg/kg s.c.), an agonist at serotonergic 5HT1A-receptors, induced an increase of hippocampal ACh release in sham-operated rats . This effect was lost in lesion-only rats, but was fully restored by neuronal grafting . As 8-OH-DPAT influences hippocampal ACh release by a postsynaptic action, this finding indicates that the host brain exerts a serotonergic influence on the grafted cholinergic neurons.

J Physiol, 1997 Aug 1, 502 ( Pt 3), 679 - 91
Mass spectrometric determination of HCO3- permeability and carbonic anhydrase activity in intact guinea-pig colon epithelium; Bollert P et al.; 1 . A mass spectrometric method originally used in red blood cells was applied to suspensions of isolated colonocytes and intact colonic epithelium to measure the exchange of 18O between HCO3-, CO2 and H2O to determine intracellular carbonic anhydrase activity (Ai) and membrane bicarbonate permeability (P) . 2 . In suspensions of isolated guinea-pig colon epithelial cells, colonocytes, we found significantly higher values of Ai and P for cells derived from the proximal colon than for cells from the distal colon . In the case of Ai, this confirms earlier reports . 3 . When the 18O exchange process was observed across the mucosal (apical) side of intact colon mucosa, the estimated values of Ai were identical to those obtained for isolated colonocytes, for both the proximal and the distal part of the colon . This is considered to be strong evidence that this method can be applied to a layer of intact epithelium as well as to cell suspensions . 4 . The values of P obtained from the apical side of intact colon mucosa were 6 times higher than those estimated from measurements with isolated colonocytes . This indicates that the basolateral membrane of colon epithelium, which participates in the 18O exchange process in isolated colonocytes but not in the 18O exchange process across the apical side of intact mucosa, has a markedly lower bicarbonate permeability than the apical membrane . 5 . When the 18O exchange process was observed across the serosal (basolateral) side of intact colon mucosa, the P values, as expected, were low compared with the apical side of intact mucosa . However, rather unexpectedly, the Ai values derived from these measurements were 2-3 times lower than those obtained with isolated colonocytes . It appears possible that the latter finding is an artifact due to the submucosal tissue markedly slowing down CO2 diffusion from the bathing medium into the epithelial cells, thus causing an apparent fall in Ai . 6 . Ai decreased and P increased with increasing temperature, as expected, when studied on the mucosal side of intact colon . This provides additional support for the validity of the method.

Am J Physiol, 1997 Aug, 273(2 Pt 1), C679 - 86
Effects of P2 purinergic receptor stimulation in brown adipocytes; Lee SC et al.; Sympathetic stimulation of brown adipocytes plays a major role in body energy homeostasis by activating energy-wasting pathways . Sympathetic neuronal input initiates a variety of metabolic, developmental, and membrane responses in brown fat cells . Many of these actions are mediated by adrenergic pathways mobilized by released norepinephrine . However, since sympathetic stimulation may also release vesicular ATP, we tested brown fat cells for ATP responses . Micromolar concentrations of extracellular ATP had a number of effects on brown adipocytes . We have shown previously that ATP elicits substantial (average of approximately 30%) increases in cell membrane capacitance (P . A . Pappone and S . C . Lee, J . Gen . Physiol . 108: 393-404, 1996) . Here, we show that cytosolic calcium levels were increased by ATP, both through release from intracellular stores and through influx, as assessed by fura 2 imaging . In addition, ATP indirectly activated a nonselective cation conductance that was independent of cytosolic calcium levels in patch voltage-clamped brown fat cells . Similar calcium, conductance, and capacitance responses could be activated by 2-methylthio-ATP and ADP, consistent with mediation by a P2 type purinergic receptor . Calorimetric measurements from cell suspensions showed that ATP increased basal heat production of isolated brown fat cells by approximately 40% but had no effect on the greater than fivefold increase in heat production seen with maximal adrenergic stimulation . These myriad responses to extracellular ATP suggest that P2 receptor-mediated signaling is important in brown adipocyte physiology and that sympathetic stimulation may normally activate purinergic as well as adrenergic pathways in brown fat.

Anal Quant Cytol Histol, 1997 Aug, 19(4), 338 - 44
Image and flow cytometric analyses of DNA content in human solid tumors . A comparative study; Faranda A et al.; OBJECTIVE: To compare the sensitivity and potentials of flow and image cytometry in assessing DNA content . STUDY DESIGN: The study was performed on 152 tumors (oral cavity, uterine cervix, bladder, colorectum, breast) . Flow cytometry was carried out on cell suspensions from frozen samples, and the results were expressed as the DNA index . Image cytometry was performed on Feulgen-stained sections, and the results were expressed as the rate of cells exceeding 2.5c or 5c . For colorectal and breast cancers, DNA content by image cytometry was also measured on imprints and was expressed as the DNA index or rate of cells exceeding 2.5c and 5c . RESULTS: Among flow cytometric diploid tumors, image cytometric analysis performed on histologic sections showed about 80% diploid tumors from the uterine cervix and breast cancers . The frequency decreased to 36% for oral cavity cancers . Generally satisfactory concordance was observed when flow cytometric aneuploid tumors were analyzed . A highly significant correlation was observed between DNA indices observed by flow and image cytometry on imprints . CONCLUSION: Image cytometry appears more sensitive than flow cytometry in detecting small, aneuploid clones, but its main limitation is the low power in resolving near-diploid cell populations . The results on imprints indicate that image cytometry is a potential alternative approach for small tumor samples.

Microsc Res Tech, 1997 Aug 1, 38(3), 315 - 28
Spray-freezing freeze substitution (SFFS) of cell suspensions for improved preservation of ultrastructure; Fields SD et al.; Some unicellular organisms present challenges to chemical fixations that lead to common, yet obvious, artifacts . These can be avoided in entirety by adapting spray-freezing technology to ultrarapidly freeze specimens for freeze substitution . To freeze specimens, concentrated suspensions of cells ranging in diameter from 0.5-30 pm were sprayed with an airbrush at 140-200 kPa (1.05-1.5 torr; 20.3-29.0 psi) into a nylon mesh transfer basket submerged in liquid propane . After freezing, the mesh basket containing the frozen sample was lifted out of the chamber, drained and transferred through several anhydrous acetone rinses at 188 K (-85 degrees C) . Freeze substitution was conducted in 1% tannic acid/1% anhydrous glutaraldehyde in acetone at 188 K (-85 degrees C), followed by 1% OsO4/acetone at 277 K (4 degrees C) . Freeze substitution was facilitated using a shaking table to provide gentle mixing of the substitution medium on dry ice . High quality freezing was observed in 70% of spray-frozen dinoflagellate cells and in 95% of spray-frozen cyanobacterial cells . These could be infiltrated and observed directly; however, overall ultrastructural appearance and membrane contrast were improved when the freeze-substituted cells were rehydrated and post-fixed in aqueous OSO4, then dehydrated and embedded in either Spurr's or Epon resin . Ultrastructural preservation using this ultrarapid freezing method provided specimens that were consistently superior to those obtainable in even the best comparable chemical fixations.

Exp Cell Res, 1997 Aug 1, 234(2), 486 - 97
Activation of calcium signaling in isolated rat hepatocytes is accompanied by shape changes of microvilli; Lange J et al.; Preceding studies using the hamster insulinoma cell line, HIT, and isolated rat hepatocytes have shown that two essential components of the Ca2+ signaling pathway, the ATP-dependent Ca2+ store and the store-coupled Ca2+ influx pathway, are both located in microvilli covering the surface of these cells . Microvilli-derived vesicles from both cell types exhibited anion and cation pathways which could be inhibited by anion and cation channel-specific inhibitors . These findings suggested that the microvillar tip compartment forms a space which is freely accessible for external Ca2+, ATP, and IP3 . The entry of Ca2+ into the cytoplasm, however, is largely restricted by the microvillar core structure, the dense bundle of actin microfilaments acting as a diffusion barrier between the microvillar tip compartment and the cell body . Moreover, evidence has been presented that F-actin may function as ATP-dependent and IP3-sensitive Ca2+ store that can be emptied by profilin-induced depolymerization or reorganization {K . Lange and U . Brandt (1996) FEBS Lett . 395, 137-142} . Here we demonstrate the tight connection between microvillar shape changes and the activation of the Ca2+ signaling system in isolated rat hepatocytes . Using a combination of scanning electron microscopy (SEM) and fura-2 fluorescence technique, we confirmed a consequence of the "diffusion barrier" concept of Ca2+ signaling: Irrespective of the type of the applied stimulus, activation of the Ca2+ influx pathway is accompanied by changes in the structural organization of microvilli indicative of the loss of their diffusion barrier function . We further show that the cell surfaces of unstimulated hepatocytes isolated by either the collagenase or the EDTA perfusion technique are densely covered with microvilli predominantly of a short and slender type . Beside this rather uniformly shaped type of microvilli, a number of dilated surface protrusions were observed . Under these conditions the cells displayed the well known rather high basal {Ca2+}i of 200-250 nM as repeatedly demonstrated for freshly isolated hepatocytes . However, addition of the serine protease inhibitor, phenylmethanesulfonyl fluoride (PMSF), to the cell suspension immediately after its preparation reduced the basal cytoplasmic Ca2+ level to about 100 nM . Concomitantly, dilated surface protrusions disappeared, and cell surfaces exclusively displayed short, slender microvilli . Activation of the Ca2+ signaling pathway by vasopressin, as well as by the IP3-independent acting Ca2+ store inhibitor, thapsigargin, was accompanied by a conspicuous shortening and dilation of microvilli following the same time courses as the respective increases of {Ca2+}i induced by the effectors . Furthermore, the abundance of the large form of surface protrusions on isolated hepatocytes positively correlated with the size of a cellular Ca2+/Fura-2 compartment which is rapidly depleted from Ca2+ by extracellular EGTA . These findings support the postulated localization of the store-coupled Ca2+ influx pathway in microvilli of HIT cells also for hepatocytes and are in accord with the notion of a cytoskeletal diffusion barrier regulating the flux of external Ca2+ via the microvillar tip region in the cytoplasm.

J Neurosci Res, 1997 Aug 1, 49(3), 355 - 63
NGF and BDNF in the anterior pituitary lobe of adult rats; Hopker VH et al.; Previous studies revealed that NGF-like immunoreactivity is present in cells from the adult rat anterior pituitary lobe, both in vivo and in vitro, and that in both situations NGF colocalizes with the thyroid-stimulating hormone (TSH) . More recently, brain-derived neurotrophic factor (BDNF) was similarly found to occur in the anterior pituitary tissue, again with a general colocalization with TSH . In the present study, we have extended the use of adult rat anterior pituitary cultures to show their content of BDNF-immunoreactive cells and their main colocalization with TSH . We have also explored the question of whether neurotrophins nerve growth factor (NGF) and/or BDNF are actually produced within anterior pituitary cells . Use of the sensitive method reverse transcription-polymerase chain reaction (RT-PCR) has allowed us to confirm the presence of NGF and BDNF mRNAs in the cell suspension freshly derived from adult anterior pituitary . In situ hybridization techniques applied to the cell cultures from such a suspension, however, have revealed only a variable presence of NGF mRNA-positive cells but no recognizable BDNF mRNA . Thus, the question of whether the two neurotrophins are produced within the very cells whose immunoreactive content can be recognized remains an open one.

Magn Reson Med, 1997 Aug, 38(2), 334 - 6
Direct observation of resolved intracellular and extracellular water signals in intact human red blood cells using 1H MAS NMR spectroscopy; Humpfer E et al.; High resolution 400 MHz 1H NMR spectra of red blood cell suspensions when measured using magic angle spinning (MAS) show two water resonances separated by 15 Hz . Based on addition of a paramagnetic Mn-EDTA complex, measurement of relaxation times and variation of extracellular H2O/D2O ratios, these have been assigned as intracellular (linewidth 17.5 Hz) and extracellular water (linewidth 4.6 Hz) . This is the first direct observation of intracellular water using NMR spectroscopy and the 1H MAS NMR spectroscopic approach offers the possibility of studying directly the compartmentation of substances in cells and kinetics of molecular transport.

Biol Reprod, 1997 Aug, 57(2), 428 - 35
Protein kinase C- and Ca2+ ionophore-stimulated production of reactive oxygen species in mechanically dispersed isolated bovine luteal cells; Sakka E et al.; We measured the production of reactive oxygen species (ROS) using luminol-horseradish peroxidase-induced chemiluminescence in mechanically dispersed cell suspensions from bovine corpus luteum (CL) . Since other cell types besides luteal cells were present in crude cell suspensions from CL, cell preparations were purified by centrifugation on Percoll . Only cell suspensions that gave no significant response when stimulated with formyl-methionyl-leucyl-phenylalanine, a potent stimulator of ROS production by phagocytes, were used routinely . Basal ROS production by purified bovine luteal cell preparations was low but could be stimulated rapidly and in a dose-dependent manner by nanomolar concentrations of the protein kinase C (PKC) activator phorbol 12-myristate 13-acetate (PMA), though only in cells purified by fractionation on Percoll . Luteal ROS responses to PMA were quenched by returning bovine erythrocytes to purified luteal cells, or by exogenous catalase or superoxide dismutase . The magnitude of the response to PMA varied markedly from one luteal cell preparation to another but appeared to be unrelated to the stage of the luteal phase of the CL from which the cells were prepared . The luteal ROS response to PMA was blocked by staurosporine, an inhibitor of PKC . Although the inactive phorbol ester (4alpha-phorbol didecanoate; 4alphaPDD) alone had little or no effect on luteal ROS production, 4alphaPDD significantly potentiated the effects of submaximal concentrations of PMA in a dose-dependent manner . ROS production could also be stimulated by the Ca2+ ionophore A23187 . This response was rapidly abolished by treatment with EDTA or EGTA . A23187 also augmented the response to submaximal PMA levels: however, pretreatment with 4alphaPDD did not significantly enhance the ROS response to A23187 . In conclusion, we have shown that isolated bovine luteal cell suspensions are capable of generating a marked acute ROS response triggered by activation of PKC and/or elevation of cytosolic calcium.

Endocrinology, 1997 Aug, 138(8), 3236 - 41
Interaction of mouse placental lactogens and androgens in regulating progesterone release in cultured mouse luteal cells; Thordarson G et al.; Pituitary hormones are essential for the maintenance of the corpus luteum in the pregnant mouse during the first half of gestation . Thereafter, hormones from the placenta take over the luteotropic role of the pituitary hormones . Mouse placental lactogen-I (mPL-I) and mPL-II, two PRL-like hormones produced in the placenta, are probably necessary for the maintenance of the corpus luteum in the latter half of pregnancy . A culture system of luteal cells from pregnant mice was developed to investigate the role of hormones from the placenta that may be important for the function of the corpus luteum . Mice were killed on days 10, 14, and 18 of pregnancy, and the corpora lutea were excised from the ovaries and digested in 0.1% collagenase, 0.002% DNase for 1 h . The resulting luteal cell suspension was plated onto 96-well plates coated with fibronectin (1 x 10(5) cells/well) and cultured for 1-3 days . Medium was changed daily . The cells were treated with various concentrations and combinations of mPL-I, mPL-II, mouse PRL, androstenedione, dihydrotestosterone, 17beta-estradiol (E2), testosterone, hydroxyflutamide, cycloheximide, actinomycin D, and fadrozole to study the effects of these different treatments on progesterone (P4) production . The three lactogens (mPL-I, mPL-II, and mouse PRL) all stimulated the release of P4 from the luteal cells . The potency of the lactogens was similar and did not depend on the stage of pregnancy at which the luteal tissue was obtained . However, the responsiveness of the cells to all hormone-stimulated P4 release was gradually reduced the later in pregnancy the tissue was collected . Androgens also stimulated the release of P4 from the luteal cells, and when administered together, the lactogens and the androgens acted synergistically to stimulate P4 release . The androgens acted directly but not through conversion to E2, as determined by the findings that 1) the effects of the androgens could not be reproduced by E2 administration, 2) nonaromatizable androgen dihydrotestosterone was as effective as aromatizable androgens, and 3) aromatase inhibitor did not prevent the action of the androgens to stimulate the P4 release . The effect of the androgens on the P4 release was rapid, occurring within 15 min of hormone administration . It was not prevented by inhibitors of protein and RNA synthesis, and the intracellular androgen receptor antagonist hydroxyflutamide did not affect the androgen action . Therefore, the androgen effects were not mediated through the intracellular androgen receptor and de novo protein synthesis was not needed for androgen-stimulated P4 release.

Neuroscience, 1997 Aug, 79(4), 963 - 72
The effect of nigral implantation on sensitization to dopamine agonists in 6-hydroxydopamine-lesioned rats; Gancher S et al.; The implantation of fetal nigral tissue into the striatum of patients with Parkinson's disease is a promising approach to treatment which may produce clinical benefit partly by influencing drug responsiveness . The purpose of the present study was to determine the pharmacological mechanisms which drug response changes by measuring to what extent sensitization produced by repeated apomorphine treatment was attenuated by tissue implantation in rats with nigrostriatal lesions . Prior to implantation of nigral cell suspensions, the daily administration of apomorphine to rats with unilateral 6-hydroxydopamine lesions produced a progressive increase in the magnitude and duration of rotational behaviour . After implantation, apomorphine-induced rotational effects were reduced to levels observed upon the initial exposure to drug and did not increase following repeated treatment . Attenuated responses to selective D1 and D2 agonists were also observed after implantation . In vehicle-implanted rats, the initial response to apomorphine was attenuated but then increased following repeated apomorphine administration . No attenuation in responses to selective D1 and D2 agonists was observed in this group . Cell suspensions prepared from fresh and cyropreserved tissue produced similar behavioural effects, even though the volume of transplanted striatum exhibiting tyrosine hydroxylase activity was greater with fresh tissue . The duration of rotational behaviour induced by apomorphine was not affected by cell implantation . These findings suggest that the expression of sensitization in an animal model of parkinsonism may disappear after a period without drug treatment . Implantation of nigral tissue may produce beneficial results in parkinsonism by limiting the development of dopamine agonist-induced sensitization.

Neuroscience, 1997 Aug, 79(3), 711 - 21
The effects of donor stage on the survival and function of embryonic striatal grafts in the adult rat brain . II . Correlation between positron emission tomography and reaching behaviour; Fricker RA et al.; Grafts of embryonic striatal primordia are able to elicit behavioural recovery in rats which have received an excitotoxic lesion to the striatum, and it is believed that the P zones or striatal-like tissue within the transplants play a crucial role in these functional effects . We performed this study to compare the effects of different donor stage of embryonic tissue on both the morphology (see accompanying paper) and function of striatal transplants . Both the medial and lateral ganglionic eminence was dissected from rat embryos of either 10 mm, 15 mm, 19 mm, or 23 mm crown-rump length, and implanted as a cell suspension into adult rats which had received an ibotenic acid lesion 10 days prior to transplantation . After four months the animals were tested on the "staircase task" of skilled forelimb use . At 10-14 months rats from the groups which had received grafts from 10 mm or 15 mm donor embryos were taken for positron emission tomography scanning in a small diameter positron emission tomography scanner, using ligands to the dopamine D1 and D2 receptors, {11C}SCH 23390 and {11C}raclopride, respectively . A lesion-alone group was also scanned with the same ligands for comparison . Animals which had received transplants from the 10 mm donors showed a significant recovery with their contralateral paw on the "staircase test" . No other groups showed recovery on this task . Similarly, the animals with grafts from the youngest donors showed a significant increase in D1 and D2 receptor binding when compared to the lesion-alone group . No increase in signal was observed with either ligand in the group which had received grafts from 15 mm donors . Success in paw reaching showed a strong correlation to both the positron emission tomography signal obtained and the P zone volume of the grafts . These results suggest that striatal grafts from younger donors (10 mm CRL) give greater behavioural recovery than grafts prepared from older embryos . This recovery is due to both the increased proportion of striatal-like tissue within the grafts and an increase in functional D1 and D2 dopamine receptors measured by positron emission tomography, i.e . a more extensive integration of the graft with the host brain.

Eur J Pharmacol, 1997 Jul 23, 331(2-3), 325 - 31
Differential effects of nitrofurans on the production/release of steroid hormones by porcine adrenocortical cells in vitro; Jager LP et al.; Changes in the biogenesis of corticosteroids caused by nitrofurans were studied . The three nitrofurans used: furazolidone, furaltadone and nitrofurantoin, altered the steroid production/release by porcine adrenocortical cells in vitro during 1 h incubations . With pregnenolone as a substrate the nitrofurans inhibited aldosterone production/release . Although the nitrofurans differed in potency (nitrofurantoin > furazolidone > furaltadone) maximum inhibition occurred at 100 microM . In this concentration the nitrofurans changed also the release/production of other corticosteroids . The output of corticosterone and cortisol decreased by 50% . The production/release of deoxycortisol stayed the same . In contrast the output of progesterone and 17alpha-hydroxyprogesterone increased to more than 200% of control . The nitrofurans slightly reduced the output of androstenedione . No significant increases of the production/release of other steroids (testosterone, dehydroepiandrosterone, estradiol-17beta and estrone) by the cell suspension could be observed . The profile of the nitrofuran-induced changes lead to the conclusion that nitrofurans interfere with mitochondrial enzymes . These enzymes, presumably cytochrome P450(11,18) mediate the hydroxylation and the oxidation at C11 and C18, the final steps in the biogenesis of aldosterone, corticosterone and cortisol . The rapid and reversible fall in the output of these steroids occurs in vitro at concentrations which are below therapeutic blood concentrations seen in vivo . At higher concentrations the nitrofurans hinder the biogenesis of androgens . Thus nitrofurans can also affect steps in the steroid biogenesis located in the endoplasmatic reticulum.

Transplantation, 1997 Jul 15, 64(1), 49 - 54
Treatment of graft-versus-host disease by extracorporeal photochemotherapy: a pilot study; Besnier DP et al.; BACKGROUND: Graft-versus-host disease (GVHD) is a major complication after bone marrow transplantation, which may be refractory to immunosuppressive drugs . As preliminary case reports suggested that extracorporeal photochemotherapy (ECP) using a Therakos device might be beneficial, we conducted a pilot study to assess the efficacy and safety of a new ECP method that does not require administration of 8-methoxypsoralen (8-MOP) to the patient . METHODS: ECP was performed three times a week for 3 weeks and then tapered according to the patient's course . Soluble 8-MOP was added ex vivo to an enriched mononuclear cell suspension obtained by a cell separator . This cellular suspension was then ultraviolet A irradiated and reinfused into the patient . Evaluation was performed using specific objective tests depending on clinical conditions . RESULTS: The two patients in the study with acute GVHD and severe liver dysfunction resistant to steroid pulse showed no improvement with ECP treatment . The five patients with chronic GVHD (c-GVHD) had the following clinical features: three patients had myositis and two patients had severe cutaneous c-GVHD, including one patient with sclerodermoid lesions, one with bronchiolitis obliterans, one with bronchitis, and one with liver involvement . Immunosuppressive drugs were either prohibited or ineffective . The number of procedures for each patient ranged from 13 to 30 . Cytapheresis required the use of a double-lumen catheter (4/5) or an arteriovenous fistula (1/5) . No side effects were related to 8-MOP or ultraviolet A irradiation . Four of five patients improved after ECP; one patient with bronchiolitis obliterans, a fibrotic condition, remained stable . CONCLUSIONS: ECP treatment may be helpful for the treatment of severe c-GVHD and the avoidance of increased immunosuppression.

Cell Immunol, 1997 Jul 10, 179(1), 66 - 73
IL-15 mediates anti-tumor effects after cyclophosphamide injection of tumor-bearing mice and enhances adoptive immunotherapy: the potential role of NK cell subpopulations; Evans R et al.; The daily administration of IL-15 to cyclophosphamide (CY)-injected mice bearing the 76-9 rhabdomyosarcoma was shown to prolong the period of remission induced by CY . In addition, IL-15 was shown to enhance the efficacy of adoptive immunotherapy . Cytotoxicity assays using spleens from normal and tumor-bearing mice indicated that IL-15 enhanced NK cell activity but there was no evidence for class I-restricted cytolytic T cell activity . To determine whether IL-15 was likely to induce different cytotoxic effectors at the tumor site compared with the spleen, tumors were removed after CY injection and cell suspensions were incubated with IL-15 in parallel with isolated spleen cells . Both populations were seen to expand to yield predominantly cells coexpressing NK1.1 and B220 antigens . However, tumor-associated NK cells were shown to differ from expanded spleen NK cells in terms of the proportions of LGL-1+ cells and cells expressing early and late NK cell differentiation antigens . Both expanded populations expressed high NK cell cytotoxic activity but only the spleen cells expressed lymphocyte-activated killer cell activity . It was apparent that the expanded tumor-associated NK cells expressed low-level class I-restricted lytic activity . The potential of activated NK cells in the circulation to exert anti-tumor effects was shown by the adoptive transfer of expanded NK cells to tumor-bearing mice after CY injection when significant prolongation of life was seen in all cases . The data indicate that IL-15 may serve as a useful anti-cancer adjuvant by activating initially the NK cell arm of the immune network.

Int J Hyperthermia, 1997 Jul-Aug, 13(4), 401 - 11
Alteration in the hypoxic fraction of quiescent cell populations by hyperthermia at mild temperatures; Masunaga S et al.; We investigated oxygenation of quiescent (Q) tumour cells in vivo by mild heat treatment . C3H/He mice bearing SCC VII tumours received BrdU continuously for 5 days via implanted mini-osmotic pumps, to label all proliferating (P) cells . The tumours were then irradiated after treatment, and were excised, minced and trypsinized . The tumour cell suspension thus obtained were incubated with cytochalasin-B (a cytokinesis blocker), and the micronucleus (MN) frequency in cells without BrdU labelling was determined using immunofluorescence staining for BrdU . This MN frequency was then used to calculate the surviving fraction of unlabelled cells from the regression line for the relationship between the MN frequency and the surviving fraction of total (P + Q) tumour cells . Thus, a cell survival curve could be determined for the cells not labelled with BrdU, which can be regarded as the Q cells in a tumour for all practical purposes . The MN frequency in total tumour cell population was determined from the irradiated tumours that were not pretreated with BrdU . Assays performed immediately after irradiation of both normally aerated and hypoxic tumours showed that Q cells contained higher hypoxic fractions than the total tumour cell population . Mild heat treatment (40.0 degrees C, 60 min) before irradiation decreased the hypoxic fraction, even when is was combined with nicotinamide administration . In contrast, mild heating did not decrease the hypoxic fraction when the mice were placed in a circulating carbogen (95% O2/5% CO2) chamber . Therefore, mild heat treatment was thought to preferentially oxygenate the chronically hypoxic fraction.

Gut, 1997 Jul, 41(1), 77 - 81
Effect of sulphide on short chain acyl-CoA metabolism in rat colonocytes; Moore JW et al.; BACKGROUND: It has been proposed that the diminished n-butyrate oxidation observed in ulcerative colitis may be the result of sulphide induced inhibition of short chain acyl-coenzyme A (acyl-CoA) dehydrogenase activity . AIM: To examine the acyl-CoA ester profiles in isolated rat colonic epithelial cells treated in vitro with sodium hydrogen sulphide (NaHS) . METHODS: Isolated rat colonic epithelial cell suspensions were incubated for 10 minutes in the presence of {1-14C} n-butyrate (5 mM), with and without NaHS (1.5 mM) . Incubations were carried out both in the presence and the absence of exogenous CoA and ATP . Metabolic performance was assessed by 14CO2 production and by acyl-CoA ester production measured by HPLC with ultraviolet detection . RESULTS: Results are given as mean (SEM) . For colonocytes incubated in the presence of exogenous CoA and ATP, treatment with NaHS significantly diminished 14CO2 production (control 0.97 (0.06) mumol/g dry weight cells/min, treated 0.26 (0.09) mumol/g dry weight cells/min, p = 0.0019), was associated with an increase in butyryl-CoA concentrations in the final reaction mixture at 10 minutes (control 2.55 (0.28) mumol/g dry weight cells, treated 3.32 (0.32) mumol/g dry weight cells, p = 0.002), and a reduction in crotonyl-CoA concentrations (control 0.274 (0.02) mumol/g dry weight cells, treated 0.120 (0.04) mumol/g dry weight cells, p = 0.008) . The mean concentration of acetyl-CoA in the reaction mixture at 10 minutes was not significantly different between control and sulphide treated incubations . There were no significant differences in acyl-CoA ester profiles observed when cells were incubated in the absence of exogenous CoA and ATP . CONCLUSIONS: These results support the view that sulphides inhibit n-butyrate oxidation in colonic epithelial cells by inhibiting short chain acyl dehydrogenation of activated fatty acids.

Andrologia, 1997 Jul-Aug, 29(4), 193 - 9
CD 45/67 immunobead preparation of human semen activates granulocytes; Ochsendorf FR et al.; In human semen reactive oxygen species (ROS) produced by spermatozoa or leukocytes can impair spermatozoa functions . The objective of this study was to assess the effects of CD 45- and/or CD 67 immunobead preparation on the chemiluminescence (CL) of seminal plasma free ejaculate cells (= original cell suspension), as well as of the spermatozoa and leukocyte fractions . The original cell suspensions of 68 infertile and 8 fertile men were incubated with CD 45 or CD 67 immunobeads . After separation in a magnetic field the luminol chemiluminescence of the original cell suspensions, the spermatozoa and the leukocyte fractions were recorded on a luminometer . Spermatozoa fractions did not contain any leukocytes as no increase in CL-counts occurred after addition of FMLP . The number of peroxidase-positive cells (per 10(7) spermatozoa) correlated with the CL of the original cell suspensions (r = 0.7; P < 0.0001) as well as the CL of the spermatozoa and the leukocyte fractions after CD 45 or CD 67-preparation (r = 0.64; P < 0.0001) . The CL of the spermatozoa and of the leukocyte fractions after CD 45 immunobead incubation were significantly correlated (r = 0.091; P < 0.0001) . According to these data contaminating leukocytes could be eliminated by immunobead preparation . However, incubation of original cell suspensions with CD 45 or CD 67 immunobeads stimulated leukocytes to release soluble products resulting in elevated CL signals both in the leukocyte and the spermatozoa fractions . These effects have to be taken into account when using immunobeads for the preparation of human semen.

Plant J, 1997 Jul, 12(1), 191 - 202
Roscovitine, a novel cyclin-dependent kinase inhibitor, characterizes restriction point and G2/M transition in tobacco BY-2 cell suspension; Planchais S et al.; Although the developmental programs of plants and animals differ, key regulatory components of their cell cycle have been conserved . Particular attention has been paid to the role of the complexes between highly conserved cyclin and cyclin-dependent kinases in regulating progression through the cell cycle . The recent demonstration that roscovitine is a potent and selective inhibitor of the animal cyclin-dependent kinases cdc2 (CDK1), CDK2 and CDK5 prompted an investigation into its effects on progression through the plant cell cycle . Roscovitine induced arrests both in late G1 and late G2 phase in BY-2 tobacco cell suspensions . Both block were fully reversible when roscovitine was used at concentrations similar to those used in the animal system . Stationary-phase cells subcultured in the presence of roscovitine were arrested at a 2C DNA content . This arrest was more efficient without exogenous addition of plant growth regulator . Roscovitine induced a block in G1 earlier than that induced by aphidicolin . S-phase synchronized cells treated with roscovitine were arrested at a 4C DNA content at the G2/ M transition . The expression analysis of a mitotic cyclin (NTCYC1) indicated that the roscovitine-induced G2 block probably occurs in late G2 . Finally, cells in metaphase were insensitive to roscovitine . The purified CDK/cyclin kinase activities of late G1 and early M arrested cells were inhibited in vitro by roscovitine . The implications of these experimental observations for the requirement for CDK activity during progression through the plant cell cycle are discussed.

Exp Brain Res, 1997 Jul, 115(3), 520 - 30
Behavioral effects of GM1 ganglioside treatment and intrahippocampal septal grafts in rats with fimbria-fornix lesions; Roeser C et al.; The monosialoganglioside GM1 is a compound with neurotrophic properties found to foster functional recovery in various paradigms of brain damage . The present experiment examined whether systemic treatment with GM1 may facilitate behavioral recovery in rats with fimbria-fornix lesions and intrahippocampal grafts rich in cholinergic neurons . Among 68 Long-Evans female rats, 46 sustained a bilateral electrolytic lesion of the fimbria and the dorsal fornix and 22 were sham-operated . Fourteen days later, half the lesioned rats were subjected to intrahippocampal grafts of a fetal septal cell suspension . Starting a few hours after lesion surgery and over a 2-month period, half the rats of each surgical treatment group received a daily injection of GM1 (30 mg/kg i.p.), the other half being injected with saline as a control . All rats were subsequently tested for locomotor activity and radial maze learning . The lesions induced locomotor hyperactivity and impaired learning performances in both an uninterrupted and an interrupted radial maze testing procedure . In all rats with surviving grafts, the grafts had provided the hippocampus with a new and dense organotypic acetylcholinesterase-positive innervation pattern which did not differ between saline- and GM1-treated subjects . The scores/performances of the rats that had received only the grafts or only the GM1 treatment did not differ significantly from those of their respective lesion-only counterparts . However, in the radial-arm maze task, the grafted rats given GM1 showed improved learning performances as compared with their saline-treated counterparts: they used more efficient visit patterns under the uninterrupted testing conditions and made fewer errors under the interrupted ones . The results suggest that GM1 treatment or intrahippocampal grafts used separately do not attenuate the lesion-induced behavioral deficits measured in this experiment . However, when GM1 treatment and grafts are used conjointly, both may interact in a manner allowing part of these deficits to be attenuated.

Biochem Mol Biol Int, 1997 Jul, 42(3), 453 - 67
Cloning, sequence, and expression of a blood group B active recombinant alpha-D-galactosidase from pinto bean (Phaseolus vulgaris); Davis MO et al.; A cDNA encoding pinto bean alpha-D-galactosidase {E.C . 3.2.1.22} was obtained by amplification of cDNA using highly conserved sequences found in eucaryotic alpha-D-galactosidases . Subsequently a full length Phaseolus cDNA clone was obtained that is 1537 nt long and contains untranslated 5' and 3' sequences . The nucleotide sequence of the cDNA has a high degree of homology with other eucaryotic alpha-D-galactosidase genes . The recombinant alpha-D-galactosidase (rGal) was expressed in Escherichia coli and purified by ion exchange and affinity chromatography . Purified rGal was homogeneous by SDS-PAGE and had relative masses of 40.1 and 45.4 kDa under nonreducing and reducing conditions, respectively . The N-terminal sequence of the expressed protein contained the sequence GNGLGQTPPMG corresponding to that deduced from the cDNA sequence . The native molecular weight for rGal was determined to be 32.18 kDa by Sephacryl S-200 chromatography . The specific activity of the rGal was 349 mu moles of PNP-alpha-D-galactopyranoside hydrolyzed per mg of pure rGal per min . rGal was highly specific for alpha-D-galactosyl residues and degraded B oligosaccharide . No detectable hemagglutinin or protease activity was present in the preparations . Furthermore, rGal was active against the blood group B antigen on native human erythrocytes in cell suspension assays . The only detectable RBC phenotypic change was loss of the B and P1 epitopes . Recombinant Phaseolus vulgaris alpha-D-galactosidase may have useful biotechnical applications in the potential mass production of enzymatically converted, universally transfusable type O RBCs . alpha-D-galactosidase {E.C . 3.2.1.22} has been purified from a variety of procaryotic and eucaryotic species . Most alpha-D-galactosidases have similar low molecular weight substrate specificities, but activity against high molecular weight substrates is variable . Terminal alpha-D-galactoside residues are present in glycoproteins and glycolipids . Some alpha-D-galactosidases have activity against alpha-D-galactosyl residues on cell membrane glycoconjugates . Glycosidases with this property are useful for carbohydrate structural studies and biotechnical applications . Enzymes free of other glycosidase activities with activity near neutral pH are particularly useful for membrane modification studies on native cells . Complex sugar chains in glycolipids and glycoproteins have often been implicated in the growth and development of eucaryotes . In particular, complex sugar chains play an important role in the recognition of self in the immune system . Some alpha-D-galactosidases can modify certain carbohydrate membrane epitopes, thereby modulating the immune response . For example, the blood group B epitope expressed on erythrocytes contains a terminal alpha-D-galactosyl residue . Individuals lacking this antigen produce naturally occurring complement fixing antibodies to the B epitope . Hydrolysis of this terminal saccharide destroys the antigenic activity of the B determinant producing H antigen (blood type O) on erythrocytes . Only rare individuals produce clinically significant antibodies to the H antigen, and therefore, type O red blood cells are "universally" compatible and in great demand . Dhar purified alpha-D-galactosidase isozymes from Phaseolus vulgaris and characterized their activity . To our knowledge, our laboratory, in a brief report, is the first to describe the cloning of the gene and the use of recombinant enzyme for seroconverting blood type B to O cells . This paper describes the cloning, sequence, expression, purification, and characterization of recombinant alpha-D-galactosidase . Activity of the recombinant enzyme on the native human erythrocyte blood group B epitope is shown.

Eur J Immunol, 1997 Jul, 27(7), 1691 - 5
Analysis of HLA class Ib gene expression in male gametogenic cells; Fiszer D et al.; We have investigated mRNA expression for nonclassical MHC class I genes (HLA-E,-F,-G) in human gametogenic cells . Testicular tissue was treated by collagenase and the resulting cell suspension was further purified by fractionation on Percoll gradients in a two-step procedure . Three gametogenic cell fractions were analyzed: purified heterogenous suspension of gametogenic cells, fraction of round spermatids and fraction of elongated spermatids . Total RNA isolated from each cell population was subjected to both reverse transcriptase/polymerase chain reaction and Northern blot analysis using oligonucleotides specific for HLA-E, -F and -G . Both method gave similar results . We have found a considerable level of HLA-E mRNA, very low amounts of reamplified cDNA for HLA-F and both a complete lack of mRNA and reamplified cDNA for the HLA-G gene in the analyzed gametogenic cell fractions . Additionally, we have localized HLA-E molecules on the cells of the adluminal compartment within seminiferous tubules using immunostaining with monoclonal antibodies specific for HLA-E heavy chain followed by confocal microscopy analysis . The unique expression pattern of HLA class I antigens in the male gonad could play an important role in an efficient protection against an autoimmunological attack toward germ cells.

ASAIO J, 1997 Jul-Aug, 43(4), 289 - 97
Evaluation of the capabilities of a hemoglobin vesicle as an artificial oxygen carrier in a rat exchange transfusion model; Izumi Y et al.; Encapsulation of hemoglobin within a liposome is one of the strategies in the development of artificial oxygen carriers . It maintains the oxygen transporting properties of hemoglobin and, at the same time, eliminates the side effects of cell free hemoglobin . Hemoglobin vesicles (HbV) are a type of liposome encapsulated hemoglobin . They have a particle size of approximately 250 nm, a hemoglobin concentration of 10 g/dl, and the oxygen affinity, P50, is regulated to 32 Torr . In this study the authors examined the oxygen transporting capability of HbV in vivo, by performing exchange transfusions in rats . Exchange transfusion (90% of the estimated circulatory volume) with HbV suspended in 5% albumin (containing 160 mEq/L, sodium and 107 mEq/L, chloride) was carried out in male Wistar rats . Mean arterial pressure and heart rate were monitored through the arterial catheter . Arterial blood samples for gas analyses were also obtained from the arterial catheter . Abdominal aortic blood flow was measured by an ultrasonic pulsed Doppler flowmeter as an indicator of cardiac output . The oxygen tension of blood withdrawn from the right atrium was measured as an indicator of mixed venous oxygen tension . These values were employed to calculate oxygen delivery and consumption . Renal cortical and skeletal muscle tissue oxygen tensions were monitored as indicators of tissue perfusion . Five percent albumin and washed rat red blood cells suspended in 5% albumin containing 10 g/dl of hemoglobin; were employed as controls . At the completion of a 90% exchange transfusion, renal cortical and skeletal muscle tissue oxygen tensions, along with oxygen delivery and consumption, were sustained almost equally well with the HbV suspension compared to the washed rat red blood cell suspension, but declined significantly with the albumin suspension . The results indicate that the oxygen transporting capability of HbV was almost equivalent to that of rat red blood cells.

Reprod Toxicol, 1997 Jul-Aug, 11(4), 521 - 31
Flow cytometric evaluation of the effects of doxorubicin on rat spermatogenesis; Suter L et al.; Histopathologic examination of testicular tissue allows testicular impairment to be investigated . As an alternative to histopathology, flow cytometry (FCM) using a triple staining technique that combines DNA-ploidy with mitochondria stainability and vimentin immunostaining has also been utilized to evaluate testicular damage . In this article we evaluate the effects on spermatogenesis after acute exposure of rats to doxorubicin . Testicular cell suspensions of treated and control animals were analyzed by FCM . This allows several cell types to be identified and quantified, giving a control pattern . Deviations from this control pattern are considered as an indication of testicular damage . Doxorubicin produced a depletion of spermatogonia as early as 3 d after treatment . This effect could be followed through the temporal evolution of spermatogenesis . Comparable results were obtained by histopathology . The presented results show that FCM is a suitable and sensitive method for the detection of testicular damage . The advantages of FCM over other techniques include its rapidity and objectivity.

Mod Pathol, 1997 Jul, 10(7), 650 - 6
Contribution of flow cytometry to the diagnosis of gastric lymphomas in endoscopic biopsy specimens; Almasri NM et al.; Gastric lymphomas seem to have unique clinical, pathologic, and immunophenotypic features that set them apart from nodal lymphomas . Microscopic examination of endoscopic biopsy specimens is the most frequent procedure used to diagnose gastric tumors, but it is very difficult, and sometimes impossible, to recognize lymphomas in endoscopic samples by histologic or even immunohistologic methods . Because most gastric lymphomas are of B-cell origin, we used flow cytometry to assess B-cell clonality in gastric biopsy specimens containing dense lymphocytic infiltrates thought to represent lymphoma . We prepared viable cell suspensions from unfixed specimens obtained from 29 consecutive patients who had a previous microscopic diagnosis of suspicious gastric lymphoid infiltrates . We performed immunophenotypic studies with multicolor flow cytometry, and we assessed clonality by examination of immunoglobulin (Ig) light-chain expression analyzed exclusively on B cells identified by anti-CD20 or CD19 antibodies . The mean number of cells recovered was 1.04 x 10(6), from an average of 5.5 gastric biopsy fragments per patient . In 26 of the 29 patients, the number of cells was adequate for analysis . We detected B-cell monoclonality in 16 cases, including 5 in which the percentage of clonal B cells was less than 5% . Of the 16 cases, only 8 could be diagnosed as lymphomas on morphologic grounds alone; the remaining 8 patients had either suspicious lymphoid infiltrates or chronic gastritis . The three cases with an insufficient number of cells were considered non-neoplastic either on histologic grounds alone or in conjunction with Southern analysis of Ig genes . We conclude that flow cytometric immunophenotypic analysis of freshly prepared cell suspensions obtained from endoscopic biopsy specimens can be used to evaluate gastric lymphocytic infiltrates . Specifically, the analysis of surface Ig light-chain expression on B cells distinguishes between monoclonal (lymphoma) and polyclonal (nonlymphoma) infiltrates . The rapidity, ease, quantitative properties, and sensitivity of this technique make it a supplement to the morphologic assessment of gastric lymphoid infiltrates.

Lung Cancer, 1997 Jul, 17(2-3), 181 - 95
Detection of K-ras point mutation by in situ PCR in cell suspensions: comparison of the indirect and direct methods; Sagawa M et al.; In situ PCR is a new technique for the localization of low copy number sequences . We report here a method for the in situ visualization of a point mutation in K-ras codon 12 by indirect in situ PCR . Twenty-five primers were examined to select mutant-specific primers . Harvested cell lines were fixed and suspended in PCR mixture . Forty cycles of PCR in cell suspension was performed in a thermal cycler using a hot start method . Cells were cytocentrifuged onto slides, and post-fixation was performed . The specimens on the slides were then hybridized with a digoxigenin-labeled probe, followed by color reaction . Both Calu-1 (mutated: TGT) and NCI-H460 (wild type: GGT) cells had strong hybridization signals in the nuclei with general primers . But with mutant-specific primers, only Calu-1 cells had hybridization signals . No signal was observed without primers or Taq DNA polymerase . Southern blotting of the same preparation confirmed desired amplification . We also applied direct in situ PCR, but this method failed to detect the point mutation . We conclude that our indirect in situ PCR method shows the feasibility of in situ identification of single cells carrying point mutations.

Ann Thorac Surg, 1997 Jul, 64(1), 216 - 9
Establishment of an experimental intrapulmonary tumor nodule model; Wang HY et al.; BACKGROUND: A pulmonary tumor model is necessary to study the biology and therapy of lung cancer . Methods to establish a solitary intrapulmonary nodule are not well defined . Two methods for solitary intrapulmonary tumor nodule development in the Fischer rat are described . METHODS: Methylcholanthrene-induced sarcoma cell suspensions were introduced into lung parenchyma of Fischer rats via limited thoracotomy and lung puncture, or instilled into a distal airway after tracheal puncture and catheterization . Intrapulmonary tumor location, implantation mortality, procedure length, and animal survival were recorded . RESULTS: Single pulmonary nodules developed at the implanted position in 100% (n = 320) and 95% (62/65) of animals after direct injection into the pulmonary parenchyma or via tracheal puncture and instillation . Operative mortality was 2% and 5% via lung or tracheal implantation, respectively . Less than 5 minutes was required for each implantation . Mean survival time was 24 +/- 2 and 26 +/- 6 days after lung or tracheal implantation in animals allowed to survive until tumor-induced death . CONCLUSIONS: These easily performed, reproducible methods of establishing solitary intrapulmonary tumors are useful tools for lung cancer research.

Plant Physiol, 1997 Jul, 114(3), 1103 - 11
Secretion of active recombinant phytase from soybean cell-suspension cultures; Li J et al.; Phytase, an enzyme that degrades the phosphorus storage compound phytate, has the potential to enhance phosphorus availability in animal diets when engineered into soybean (Glycine max) seeds . The phytase gene from Aspergillus niger was inserted into soybean transformation plasmids under control of constitutive and seed-specific promoters, with and without a plant signal sequence . Suspension cultures were used to confirm phytase expression in soybean cells . Phytase mRNA was observed in cultures containing constitutively expressed constructs . Phytase activity was detected in the culture medium from transformants that received constructs containing the plant signal sequence, confirming expectations that the protein would follow the default secretory pathway . Secretion also facilitated characterization of the biochemical properties of recombinant phytase . Soybean-synthesized phytase had a lower molecular mass than did the fungal enzyme . However, deglycosylation of the recombinant and fungal phytase yielded polypeptides of identical molecular mass (49 kD) . Temperature and pH optima of the recombinant phytase were indistinguishable from the commercially available fungal phytase . Thermal inactivation studies of the recombinant phytase suggested that the additional protein stability would be required to withstand the elevated temperatures involved in soybean processing.

Surgery, 1997 Jul, 122(1), 1 - 7
Implantation of colon cancer at trocar sites is increased by low pressure pneumoperitoneum; Wu JS et al.; BACKGROUND: The purpose of this study was to determine the effect of pneumoperitoneum on the implantation of tumor at trocar sites . METHODS: GW-39 human colon cancer cell suspension (0.5 ml of 2.5% v/v) was injected into the peritoneal cavity of golden Syrian hamsters through a 1 cm midline incision . Four 5 mm trocars were inserted through the anterior abdominal wall, and the midline incision was then closed . The animals were randomized to receive pneumoperitoneum (n = 62) or no pneumoperitoneum (n = 60) for 10 minutes . Tumor implantations at trocar sites and midline wound incisions were documented grossly and histologically 8 weeks later . RESULTS: Tumor was identified in 86% (49 of 57) of control animals and 95% (52 of 55) of the experimental group (p = 0.20) . Implants increased with pneumoperitoneum at the midline incision from 44% to 71% (p < 0.01) and at trocar sites from 41% to 64% (p < 0.00001) . CONCLUSIONS: Pneumoperitoneum significantly increased tumor implantation at trocar sites and midline incisions.

Exp Neurol, 1997 Jul, 146(1), 142 - 50
Transplantation of mesencephalic cell suspension in dopamine-denervated striatum of the rat . II . Effects on corticostriatal transmission; Capozzo A et al.; The present study has been designed to investigate whether intrastriatal implantation of mesencephalic dopamine (DA)-synthetizing neurons into the striatum (ST) of rats whose substantia nigra (SN) was previously destroyed by 6-hydroxydopamine (6-OHDA) restores the pattern of corticostriatal transmission from the medial prelimbic and sensorimotor cortices . In 6-month-old normal animals electrical stimulation of these two functionally unrelated cortices evoked a short latency and brief excitation in 81.6% of neurons recorded in the dorsolateral ST . This percentage decreased significantly (70.6%) in age-matched animals whose dopaminergic nigrostriatal pathway was unilaterally destroyed by 6-OHDA 3 months before recording . However a significant increase in neurons (36.9%) which could be simultaneously activated from the two cortices in comparison to intact rats was noted . In addition the lesion caused a significant decrease in the threshold current required to evoke activation of striatal neurons from the sensorimotor cortex . The increase in the number of striatal neurons responding simultaneously to cortical stimulations demonstrates that destruction of the dopaminergic nigrostriatal pathway causes a loss of the focusing action of DA on corticostriatal transmission . Transplantation of embryonic mesencephalic neurons appears to reestablish this action since the number of convergent responses was significantly decreased in grafted animals (23.5%) in comparison to denervated (36.9%) and sham-grafted (35.1%) animals . Furthermore, the grafts showed a trend to increase current intensities required to evoke activation of striatal cells from both cortices . The action of grafted mesencephalic neurons over prelimbic and sensorimotor cortical inputs to the dorsal ST could be involved in recovery of grafted animals in the correct execution of complex sensorimotor tasks requiring integration of different cortical signals within the ST.

Drug Metab Dispos, 1997 Jul, 25(7), 790 - 7
Baculovirus-mediated expression and purification of human FMO3: catalytic, immunochemical, and structural characterization; Haining RL et al.; The baculovirus expression vector system was used to overexpress human FMO3 in insect cells for catalytic, structural, and immunochemical studies . Membranes prepared from infected Trichoplusia ni cell suspensions catalyzed NADPH-dependent metabolism of methyl p-tolyl sulfide at rates 20 times faster than those obtained with detergent-solubilized human liver microsomes . Sulfoxidation of the methyl and ethyl p-tolyl sulfides by recombinant human FMO3 proceeded with little stereochemical preference, whereas sulfoxidation of the n-propyl and n-butyl homologs demonstrated increasing selectivity for formation of the (R)-sulfoxide . This chiral fingerprint recapitulated the metabolite profile obtained when detergent-treated human liver microsomes served as the enzyme source . Catalytically active human FMO3 was purified to apparent homogeneity by cholate solubilization and sequential column chromatography on Octyl-Sepharose, DEAE-Sepharose, and hydroxyapatite . Purified FMO3 exhibited the same electrophoretic mobility as native microsomal enzyme, and immunoquantitation showed that this isoform represents approximately 0.5% of human liver microsomal protein . Therefore, FMO3 is quantitatively a major human liver monooxygenase . LC/electrospray-mass spectrometry analysis of purified FMO3 identified >70% of the tryptic peptides, including fragments containing motifs for N-linked glycosylation and O-linked glycosylation . Although insect cells have the capacity for glycan modification, MS analysis of the tryptic peptides demonstrated that these sites were not modified in the purified, recombinant enzyme . Edman degradation of the recombinant product revealed that posttranslational modification of human FMO3 by insect cells was limited to cleavage at the N-terminal methionine, a process seen in vivo with animal orthologs of FMO3 . These studies demonstrate the suitability of this eukaryotic system for heterologous expression of human FMOs and future detailed analysis of their substrate specificities.

Hum Pathol, 1997 Jul, 28(7), 820 - 6
Rapid polymerase chain reaction-based detection of the causative agent of cat scratch disease (Bartonella henselae) in formalin-fixed, paraffin-embedded samples; Mouritsen CL et al.; Bartonella (formerly Rochalimaea) henselae (Bh) plays a central role in cat scratch disease . A polymerase chain reaction (PCR)-based assay that can detect Bh DNA in formalin-fixed, paraffin-embedded (FF-PE) samples would have utility in the evaluation of processed lymph nodes suggestive of this disorder . Fresh or FF-PE cultures of Bh and related species were analyzed . Thirteen lymph nodes (12 FF-PE and one fresh cell suspension) with necrotizing suppurative granulomatous inflammation and seven FF-PE negative control lymph nodes were also evaluated . PCR was performed using a novel, hemi-nested protocol . Amplified products were analyzed by gel electrophoresis . The fresh and FF-PE Bh cultures showed a specific PCR product with an analytical sensitivity of 0.5 pg bacterial DNA . Seven (54%) of 13 clinical lymph node samples with morphological features suggestive of cat scratch disease also had detectable Bh DNA, whereas none of the seven negative control lymph nodes yielded positive results . We have designed a rapid and sensitive PCR test that can reliably detect Bh DNA in fresh and FF-PE samples . Our findings indicate that this assay has clinical utility in the diagnosis of cat scratch disease.

Glia, 1997 Jul, 20(3), 243 - 53
Microglia in the pineal gland of the neonatal rat: characterization and effects on pinealocyte neurite length and serotonin content; Tsai SY et al.; Microglia in the pineal gland of 1-day-old Sprague-Dawley rats were examined by OX-42 immunocytochemistry and DiI-acetylated-LDL uptake in pineal cell suspension and were found to comprise 3-5% of the total cells in the pineal gland of the neonates . In order to investigate the effects of microglia on pinealocyte structure and function, microglia-depleted and microglia-enriched pineal cell cultures were generated from 1-day-old neonate by fluorescence activated cell sorting (FACS) . After 7 days of culture, tissues were processed for either immunocytochemistry for pinealocyte S-antigen and serotonin or high performance liquid chromatography to measure serotonin . Morphometric analysis of immunoreacted cells revealed that pinealocyte neurite length was enhanced in microglia-depleted cultures and was inhibited in a microglia-enriched environment (ANOVA, P < 0.001) . Serotonin content of pineal cultures decreased in microglia-depleted cultures and was elevated in microglia-enriched cultures (ANOVA, P < 0.001) without any significant change in pinealocyte numbers . These findings are consistent with a working hypothesis that microglia function to mediate neuroendocrine-immune interactions of the gland.

Bone, 1997 Jul, 21(1), 1 - 6
Identification and enrichment of human osteoprogenitor cells by using differentiation stage-specific monoclonal antibodies; Joyner CJ et al.; A major problem in developmental bone biology is the inability to clearly identify early progenitor cells of the osteogenic and related lineages . Identification of these cells is important for the study of their normal development and for determination of potential changes in skeletal diseases . The objective of the present study was to obtain specific markers for early progenitor cells . Monoclonal antibodies were raised against human marrow stromal fibroblastic cell cultures, known to be rich in progenitors for the stromal lineages . Antibodies were selected initially by their reactivity with these marrow cultures and their immunohistochemical localization in human fetal tissues, in progenitor cell regions adjacent to osteoblastic cells . Antibody HOP-26 was strongly reactive with cells in marrow stromal colonies at early stages of differentiation, before the induction of alkaline phosphatase activity, and decreased dramatically after the cells reached confluence . In sections of human fetal limb, binding of HOP-26 was restricted to cells in close proximity to the developing bone, in periosteum, and between the developing bone trabeculae . In adult trabecular bone tissue, HOP-26 was reactive with occasional cells present within the marrow spaces with osteoblasts, adipocytes, and fibrous tissue unreactive . No antibody binding was detected in sections of skin, muscle, appendix, brain, tonsil, or liposarcoma, or cultured SaOS II, MG63, or skin cells . In primary cell suspensions, HOP-26 was unreactive with blood cells but strongly reactive with 0.59 +/- 0.27% of nucleated marrow cells . The antigen associated with these cells was detectable both intracellularly and on the cell surface, and by using immunopanning, HOP-26 selected the marrow stromal fibroblastic colony-forming units (CFU-F) . HOP-26 provides the means to identify osteogenic progenitor cells directly and with high specificity . The present studies demonstrate the value of this antibody in providing enriched populations of progenitor cells for experimental studies of osteogenic differentiation and in histopathology.

IEEE Trans Biomed Eng, 1997 Jul, 44(7), 549 - 54
An approach for measuring ultrasonic backscattering from biological tissues with focused transducers; Wang SH et al.; When the standard substitution method is used with a focused transducer to measure the backscattering coefficient from biological tissues including blood, it yields erroneous results . Extending the backscattering measurements to frequencies beyond 15 MHz necessitates the use of focused transducers because of the worsened signal-to-noise ratio--caused by the increased attenuation and the smaller transducer aperture size--in order to make the measurements close to the transducer . An approach which allows the use of focused transducers in backscattering measurements has been developed . It has been used to measure the backscattering coefficient of red cell suspensions of hematocrit ranging from a few percent to 30% in the frequency range from 5 MHz to 30 MHz . The results at hematocrits below 20% agree well with those obtained with the standard substitution method, although they differ as the hematocrit is increased beyond 20% . The experimental results also show that the fourth-power dependence of backscatter on frequency is in general approximately valid for suspended erythrocytes of hematocrit between 6% and 30%.

Biol Reprod, 1997 Jul, 57(1), 128 - 33
Extra- and intracellular effects of divergent selection for pituitary responsiveness to gonadotropin-releasing hormone in prepubertal ram lambs; Evans NP et al.; Divergent selection based on the response of 10-wk-old male lambs to a GnRH challenge has produced two lines of sheep, referred to as high and low lines, that differ in their ability to release LH in response to pharmacological and physiological doses of GnRH . The aim of this study was to determine whether the between-line differences in pituitary sensitivity were related to differences in GnRH receptor number and/or the transduction of the intracellular signal following GnRH receptor activation . Pituitary glands were collected from fourteen 20-wk-old ram lambs from each line, weighed, and sampled for GnRH receptor analysis . The remaining tissue from 9 lambs from each line was dispersed . Of the resultant cell suspension, a sample was stored for measurement of GnRH receptor content and the remainder was plated and cultured for 24 h . The LH responses of cultured cells were measured after exposure to GnRH, A23187, or the phorbol ester phorbol 12,13 dibutyrate (PDB) . The results indicated that the pituitary glands of the high line contained significantly higher concentrations of GnRH receptors than did those of the low line and released significantly more LH after stimulation with either GnRH or the Ca2(+)-calmodulin or protein kinase C intracellular second messenger systems . Therefore, the between-line difference in the regulation of pituitary LH secretion occurs at a step distal to the stimulatory sites of action of A23187 and PDB.

Blood, 1997 Jul 1, 90(1), 209 - 16
Death of bystander cells by a novel pathway involving early mitochondrial damage in human immunodeficiency virus-related lymphadenopathy; Carbonari M et al.; Destruction of immune cells in peripheral lymphoid tissues plays presumably a pivotal role in acquired immune deficiency syndrome pathogenesis . We found that cell suspensions obtained from lymph nodes of eight human immunodeficiency virus (HIV)-infected individuals contained variable proportions (2.1% to 18.3%, median 11.2%) of dead lymphocytes permeable to supravital dyes, represented by CD4+, CD8+, and B cells . The frequency of dead cells correlated directly (R = 0.847) with the amount of HIV provirus in the cell populations, and HIV provirus was enriched in the dead cell fractions . Similar proportions of dead cells were observed in cell suspensions from lymphadenopathic lymph nodes of HIV- donors, but not from small resting HIV- lymph nodes . Electron microscopic and flow cytometric analyses revealed that most dead cells from HIV+ lymph nodes lacked internucleosomal DNA fragmentation but displayed combined features of apoptosis and necrosis, eg, chromatin condensation and mitochondrial swelling . Cells with similar morphology were readily identified in lymph node tissue sections, and marked mitochondrial swelling could be occasionally observed in cells with otherwise normal morphology . Our findings have two major implications . One is that the in vivo cell death in HIV-infected lymph nodes occurs predominantly through a novel pathway, related to but distinct from classical apoptosis and characterised by early and severe mitochondrial damage . The second implication is that HIV-related lymphadenopathy is accompanied in vivo by massive destruction of uninfected lymph node cells . Comparable levels of cell death were observed in other inflammatory lymphadenopathies not related to HIV; however, the uniquely endless and generalized nature of HIV lymphadenopathy might render this "inflammatory" cell destruction a powerful pathogenetic mechanism, accounting for the progressive disruption and depletion of lymphoid tissues seen in HIV infection.

J Clin Invest, 1997 Jul 1, 100(1), 142 - 8
Immune cell-derived beta-endorphin . Production, release, and control of inflammatory pain in rats; Cabot PJ et al.; Localized inflammation of a rat's hindpaw elicits an accumulation of beta-endorphin-(END) containing immune cells . We investigated the production, release, and antinociceptive effects of lymphocyte-derived END in relation to cell trafficking . In normal animals, END and proopiomelanocortin mRNA were less abundant in circulating lymphocytes than in those residing in lymph nodes (LN), suggesting that a finite cell population produces END and homes to LN . Inflammation increased proopiomelanocortin mRNA in cells from noninflamed and inflamed LN . However, END content was increased only in inflamed paw tissue and noninflamed LN-immune cells . Accordingly, corticotropin-releasing factor and IL-1beta released significantly more END from noninflamed than from inflamed LN-immune cells . This secretion was receptor specific, calcium dependent, and mimicked by potassium, consistent with vesicular release . Finally, both agents, injected into the inflamed paw, induced analgesia which was blocked by the co-administration of antiserum against END . Together, these findings suggest that END-producing lymphocytes home to inflamed tissue where they secrete END to reduce pain . Afterwards they migrate to the regional LN, depleted of the peptide . Consistent with this notion, immunofluorescence studies of cell suspensions revealed that END is contained predominantly within memory-type T cells . Thus, the immune system is important for the control of inflammatory pain . This has implications for the understanding of pain in immunosuppressed conditions like cancer or AIDS.

Neuroscience, 1997 Jul, 79(1), 57 - 78
Extensive migration and target innervation by striatal precursors after grafting into the neonatal striatum; Olsson M et al.; Embryonic striatal precursors grafted into the lesioned adult host striatum show limited integration with little migration and restricted efferent projections . In the present study, the influence of an immature striatal environment on the integrative capacity of grafted neuroblasts was examined after transplantation of striatal progenitors into the striatum at different stages of postnatal development . Mouse progenitors, derived from embryonic day 13.5-14 lateral or medial ganglionic eminence or the cerebellar primordium, were transplanted as a single cell suspension into the developing postnatal day 1, 7 and 21 rat striatum . The grafted cells and their axonal projections were visualized using antibodies raised against the mouse-specific neural markers, M6 and M2 . Cells from the lateral (but not the medial) ganglionic eminence showed a remarkable capacity to innervate selectively the striatal target structures, globus pallidus, entopeduncular nucleus and substantia nigra, reminiscent of endogenous striatal neurons, which is not observed after grafting into adult hosts . M6 and M2-immunopositive cellular profiles from both the lateral and medial ganglionic eminences were observed to have migrated extensively away from the injection site, in contrast to the cerebellar precursors which remained clustered at the implantation site . Cells from the lateral ganglionic eminence were largely confined within the striatal complex where they developed striatal characteristics, displaying expression of DARPP-32, the 32,000 mol . wt dopamine- and cyclic AMP-regulated phosphoprotein, whereas cells from the medial ganglionic eminence had migrated caudally along the internal capsule and were observed predominantly in the globus pallidus and thalamus, in addition to the striatum . The cells located outside the striatum were all DARPP-32 negative . The improved integration and increased projection capacity of the lateral ganglionic eminence precursors grafted into postnatal day 1 hosts gradually declined as the host advanced into later stages of development (postnatal day 7), and in postnatal day 21 hosts the grafted striatal precursors behaved similarly to grafts implanted into adult recipients . These results demonstrate the specific capacity of embryonic striatal progenitors to integrate into the developing basal ganglia circuitry during early postnatal development, and that the extent of neuronal and glial integration and graft host connectivity declines when the host has developed beyond the first postnatal week.

Plant J, 1997 Jun, 11(6), 1167 - 75
Tryptophan decarboxylase is encoded by two autonomously regulated genes in Camptotheca acuminata which are differentially expressed during development and stress; Lopez-Meyer M et al.; Camptothecin (CPT) is a valuable anti-cancer monoterpene alkaloid produced by the Chinese tree Camptotheca acuminata . Tryptophan decarboxylase (TDC) supplies tryptamine for the indole moiety of CPT and its derivatives, and is considered a key step in monoterpene indole alkaloid biosynthesis as it links primary and secondary metabolism . This report describes the isolation and characterization of tdc1 and tdc2, two autonomously regulated TDC genes from Camptotheca . When expressed in Escherichia coli, the products of each gene could decarboxylate tryptophan, but were inactive against tyrosine, phenylalanine and 3,4-dihydroxyphenylalanine (dopa), tdc1 was developmentally regulated, having its highest expression level in the apex, young stem and bark, tissues which also contain the highest levels of CPT . Expression of tdc1 also increased during seedling development and was correlated with alkaloid accumulation during germination . tdc2 expression was induced in Camptotheca leaf discs and cell suspension cultures treated with fungal elicitor or methyl jasmonate, treatments which did not affect tdc1 expression . Unlike tdc1, tdc2 expression was not detected in any unstressed Camptotheca tissues nor in developing seedlings . These data suggest that tdc1 may be part of a developmentally regulated chemical defense system in Camptotheca, while tdc2 serves as part of a defense system induced during pathogen challenge.

Exp Brain Res, 1997 Jun, 115(1), 71 - 82
Effects of graft pooling of foetal rat and mouse tissue and immunosuppression in the 6-hydroxydopamine rat model of Parkinson's disease; Schwarz SC et al.; We employed intracerebral co-transplantation of foetal xenogeneic striatal mouse tissue and allogeneic rat substantia nigra into the adult rat brain to elucidate the effects of xenogeneic mouse graft on the function and survival of an allogeneic rat graft in 6-hydroxydopamine lesioned Sprague-Dawley rats . Foetal mouse striatum (STR) and rat substantia nigra (VM) were transplanted as non-pooled separate deposits or a pooled cell suspension with or without cyclosporin A (Cy A) . Immunosuppressed recipients of pooled rat and mouse co-grafts showed a significantly better compensation of amphetamine-induced rotational behaviour compared with non-immunosuppressed animals with pooled rat and mouse co-grafts 3 and 6 weeks post-grafting.Tyrosine hydroxylase (TH) immunohistochemistry revealed a non-significant reduction in survival in pooled (1806.3+/-367.5 cells) rat and mouse co-transplants without immunosuppression compared with immunosuppressed pooled (3383.3+/-732.7 cells) animals with allo- and xenogeneic tissue and controls (3506.4+/-839.3 cells) . Graft volumes were significantly reduced in pooled transplants without immunosuppression (0.1+/-0.026 mm3; ANOVA post-hoc Scheffe F-test, P<0.0001) compared with immunosuppressed recipients (0.7+/-0.1 mm3) and controls (0.6+/-0.1 mm3) . In non-pooled allo- and xenogeneic grafts without immunosuppression the survival rate of the TH-immunoreactive cells and graft volumes were reduced (2359.3+/-479.5 cells; 0.2+/-0.043 mm3) compared with immunosuppressed animals (2927.3+/-946.6 cells; 0.6+/-0.2 mm3) and controls (2701.1+/-693.8 cells; 0.3+/-0.1 mm3) without reaching a level of significance . Rejection of mouse tissue was observed in all non-immunosuppressed recipients . In summary: (i) continued immunosuppression yielded significant beneficial effects on function and beneficial effects on survival of pooled grafts with an immunogenetic disparity; (ii) the rejection of a xenogeneic graft component may compromise survival and function of other, allogeneic graft components; and (iii) transplantation of non-pooled allo- and xenogeneic tissues may result in a better survival of the graft compared with pooled cell suspensions.

Cell Calcium, 1997 Jun, 21(6), 461 - 7
Okadaic acid induces the release of Ca2+ from intracellular stores in ECV304 endothelial cells; Hepworth TJ et al.; The effects of serine/threonine phosphatase inhibition on endothelial cell cytosolic free Ca2+ ({Ca2+}c) were investigated using okadaic acid and Fura-2-loaded ECV304 endothelial cells . When added to confluent adherent cells, 500 nM okadaic acid induced a transient and oscillatory elevation of {Ca2+}c both in the presence and absence of extracellular Ca2+ . In the absence of extracellular Ca2+, depletion of the intracellular Ca2+ stores with either ATP (1 microM) or thapsigargin (100 nM) prevented any further release of Ca2+ on the subsequent addition of okadaic acid . Likewise (in the absence of extracellular Ca2+), a prior release of Ca2+ induced by okadaic acid reduced the magnitude of the response to ATP (1 microM) . Taken together these observations indicate that okadaic acid induces Ca2+ release from the agonist-sensitive pool . The okadaic acid-induced Ca2+ release was mimicked by another potent phosphatase inhibitor, calyculin A (10 nM), and also the less potent analogue of okadaic acid, 1-nor-okadone (500 nM) . The response to okadaic acid was characterised by a series of asynchronous {Ca2+}c oscillations, which at their peak resulted in 40-100% cells, at any one time, having an elevated {Ca2+}c . The response appeared to propagate between adjacent cells and the elevation of {Ca2+}c appeared initially in the cell periphery . In adherent cells, the release of Ca2+ induced by okadaic acid was found to be dependent upon cell density, as the proportion of cells responding to okadaic acid increased as the cell density increased . The response to okadaic acid was not observed in ECV304 cell suspensions . The data suggest that a kinase activity stimulated either directly or indirectly by cell-cell interactions can lead to the release of Ca2+ from the agonist- and thapsigargin-sensitive intracellular stores.

Exp Neurol, 1997 Jun, 145(2 Pt 1), 434 - 41
Catecholaminergic development of fetal rat ventral mesencephalon: characterization by high-performance liquid chromatography with electrochemical detection and immunohistochemistry; Tomasini R et al.; We determined dopamine (DA), noradrenaline (NA), and adrenaline (A), as well as immunohistochemically stained tyrosine hydroxylase (TH) and DA in dissected rat ventral mesencephalon (VM) tissue from Embryonic Day (ED) 14 to Postnatal Day (P) 17 . Whole VM tissue DA, NA, and A contents increased with advancing age . VM DA/protein increased from ED15 to ED16, whereas NA/protein increased from ED15 to ED16 and from ED20 to P4 . VM DA/NA ratio increased from ED14 to ED15 and decreased from ED18 to P4 . VM cell suspensions exhibited higher DA/NA ratios than whole VM tissue . Washed cell suspensions had higher DA/NA than unwashed counterparts . We conclude that data from both VM immunohistochemistry and catecholamine assays relate to VM development . VM DA is contained mainly in cells, whereas VM NA is located in fibers that channel at the dorsal side of the VM . Determination of tissue catecholamine contents may be helpful for the biochemical characterization of tentatively identified VM grafts.

Eur J Immunol, 1997 Jun, 27(6), 1374 - 80
Interleukin-15-induced maturation of human natural killer cells from early thymic precursors: selective expression of CD94/NKG2-A as the only HLA class I-specific inhibitory receptor; Mingari MC et al.; Immature postnatal thymocytes were shown to contain precursors which, under suitable culture conditions, give rise to phenotypically and functionally mature natural killer (NK) cells . Here, we analyzed the effect of different cytokines for their ability to induce the expression of HLA class I-specific inhibitory receptor(s) during the process of NK cell development from immature thymocytes . From thymocyte cell suspensions depleted of CD2+, CD3+, CD4+, CD8+, CD56+, and CD16+ cells, we further removed cells expressing HLA class I-specific inhibitory receptors including CD94/NKG2-A, p58.1, and p58.2 by immunomagnetic bead separation . The resulting cells did not contain any of the above NK receptors as determined by immunofluorescence and flow cytometric analysis, as well as by reverse transcriptase polymerase chain reaction (RT-PCR) amplification using appropriate sets of primers . Although different cytokines have been used, including interleukin (IL)-7, stem cell factor (SCF), IL-2, and IL-15, only IL-2 or IL-15 induced cell proliferation when used alone . Moreover, maturation towards CD3- CD56+ cells displaying cytolytic activity against the HLA class I- targets K562 or 221 was detectable in cultures containing IL-15 used alone or in combination with IL-7 or SCF . On the other hand, these CD3- CD56+ cell populations did not lyse HLA class I+ target cells, including autologous PHA blasts . Analysis of the expression of the various HLA class I-specific inhibitory NK receptors revealed the presence of high proportions of CD94/ NKG2-A+ cells, while the NK receptors belonging to the Ig superfamily were undetectable both by immunofluorescence and by RT-PCR analysis . The expression of CD94/NKG2-A appeared to be responsible for the inability of cells to lyse HLA class I+ target cells . Thus, addition of anti-CD94 monoclonal antibodies of IgM isotype resulted in lysis of autologous target cells . The use of 221 cells transfected with different HLA class I alleles as target cells confirmed the broad class I specificity of CD94/NKG2-A receptor . Our experiments indicate that IL-15 provides an appropriate stimulus to the expression of CD94/NKG2-A, but not of other class I-specific NK receptors in the process of maturation of NK cells from thymocyte precursors.

J Parasitol, 1997 Jun, 83(3), 434 - 9
Temperature modulation of the response of Ig-positive cells to Goussia carpelli (Protozoa: Apicomplexa) infections in carp, Cyprinus Carpio L; Steinhagen D; The influence of temperature on the kinetics of immunoglobulin-positive (Ig+) leukocytes in cell suspensions prepared from the pronephros and the intestine of common carp Cyprinus carpio during the development of Goussia carpelli, a gut-dwelling coccidian parasite, was examined . The development of the parasite was temperature dependent . At 20 C, oocysts were formed 2-3 wk postexposure (PB), at 15 C for 3-4 wk PE, and at 12 C for 5-6 wk PE . At all 3 temperatures, changes of relative numbers of Ig+ cells were observed in cell suspensions . During merogony and gamogony, the proportion of Ig+ cells increased, peaked during oocyst formation of the parasite, and then remained elevated . Serum collected from infected carp 8-20 days PE contained immunoglobulins binding to G . carpelli merozoites . The development of the parasite and the increase of Ig+ cells in intestine and pronephros of infected carp were temperature dependent . The peak level of Ig+ cells appeared not to be influenced by temperature . These findings indicate that even at low temperatures local and systemic immune responses are induced in carp with enteritic parasite infection.

Trends Biotechnol, 1997 Jun, 15(6), 230 - 5
Expanded-bed adsorption in industrial bioprocessing: recent developments; Hjorth R; Expanded-bed adsorption allows the capture of proteins from particle-containing feedstocks without prior removal of particulates, thus enabling clarification of a cell suspension or cell homogenate and the concentration of the desired product in a single operation . This usually results in higher product recovery in a shorter time period . Process development and scale-up of an expanded-bed operation is convenient because both the adsorbent and the equipment are similar to those in conventional packed-bed chromatography . This article reviews the recent developments in expanded-bed adsorption technology and the range of applications that are now being achieved.

Cancer, 1997 Jun 1, 79(11), 2073 - 86
Prognostic significance of DNA ploidy and proliferation in 309 colorectal carcinomas as determined by two-color multiparametric DNA flow cytometry; Zarbo RJ et al.; BACKGROUND: Although DNA flow cytometry has been shown to be of independent value in determining the prognosis of colorectal carcinoma, a number of well-designed studies with contradictory findings have left unresolved the clinical significance of DNA ploidy and proliferation in biologically meaningful subsets of colorectal carcinoma cases . METHODS: To evaluate the prognostic significance of DNA ploidy and proliferation as determined by flow cytometry in a prospective series of 309 human colorectal carcinomas with 4-6 years of follow-up, fresh tumors were mechanically dissociated into whole cell suspensions and dual fluorescence-labeled to allow gated analysis of subpopulations with phenotypic markers . Software programs with histogram-dependent algorithms employing background, aggregate, and debris correction were used in DNA and cell cycle quantitation . Data were analyzed according to recommendations of the 1992 DNA Flow Cytometry Consensus Conference . RESULTS: None of the clinical, site, or staging parameters, including TNM stage variables, correlated with any flow cytometric DNA ploidy or proliferation measurement . Tumors classified as DNA aneuploid or tetraploid, by any definition, did not differ in prognosis or correlate with stage or any pathologic parameter . Univariate Kaplan-Meier survival analysis showed prognostic significance of the following: Dukes staging, individual components of TNM stage (tumor depth, lymph node status, and metastasis), vascular invasion, histologic pattern of tumor infiltration, and peritumoral lymphocytic inflammation . DNA ploidy status and proliferation measurements were not predictive of survival for the overall group or within any particular stage . Only Dukes Stage D (metastasis), vascular invasion, and depth of invasion (T classification) were significant independent predictors of survival in multivariate Cox regression models . CONCLUSIONS: In this analysis, DNA ploidy and proliferation measurements were not predictive of survival in any stage of colorectal carcinoma . However, clinical and pathologic documentation of staging and select histopathologic observations were significant predictors of survival in univariate and multivariate analyses.

Virology, 1997 May 26, 232(1), 187 - 97
Mutations that alter a conserved element upstream of the potato virus X triple block and coat protein genes affect subgenomic RNA accumulation; Kim KH et al.; The putative subgenomic RNA (sgRNA) promoter regions upstream of the potato virus X (PVX) triple block and coat protein (CP) genes contain sequences common to other potexviruses . The importance of these sequences to PVX sgRNA accumulation was determined by inoculation of Nicotiana tabacum NT1 cell suspension protoplasts with transcripts derived from wild-type and modified PVX cDNA clones . Analyses of RNA accumulation by S1 nuclease digestion and primer extension indicated that a conserved octanucleotide sequence element and the spacing between this element and the start-site for sgRNA synthesis are critical for accumulation of the two major sgRNA species . The impact of mutations on CP sgRNA levels was also reflected in the accumulation of CP . In contrast, genomic minus- and plus-strand RNA accumulation were not significantly affected by mutations in these regions . Studies involving inoculation of tobacco plants with the modified transcripts suggested that the conserved octanucleotide element functions in sgRNA accumulation and some other aspect of the infection process.

Cancer Lett, 1997 May 19, 115(2), 243 - 8
Extensive DNA damage induced by monochloramine in gastric cells; Suzuki H et al.; Colonization of Helicobacter pylori (Hp) to gastric mucosa plays an important role for the pathogenesis of gastric mucosal lesions . We previously reported the importance of monochloramine (NH2Cl), which was derived from the interaction between Hp-urease and infiltrated leukocytes, in the course of Hp-associated gastric mucosal injury . While the long-term infection of Hp in the gastric mucosa is known to be one of the virulent factors which closely link to the gastric carcinogenesis, the details of its pathogenetic mechanisms remain speculative . The present study is designed to examine whether a NH2Cl could damage the DNA of gastric cells . Rabbit gastric mucosal cells (RGMC) or KATO III cells were cultured and suspended . Cell suspensions were exposed to HOCl, NH3 or NH2Cl for 15 min to give a final concentration of 0.1 mM . The magnitude of a double strand break of DNA was quantified by measuring the remnant double strand stained by ethidium bromide (EB), and the fluorescence intensity of EB was analyzed by spectrophotometer . Separately, cell nuclei were stained by fluorescent dye (Hoechst No . 33258) in order to evaluate the levels of chromatin condensation evoked by DNA fragmentation . The number of cells with chromatin condensation was counted . During the entire experimental period, more than 85% of cells were persistently viable in all groups . NH2Cl significantly induced the DNA double strand break as well as chromatin condensation in RGMC and KATO III cells (P < 0.05) . However, NH3 or HOCl did not induce the DNA double strand break as well as chromatin condensation in both cells . NH2Cl, but not its precursors (NH3 or HOCl), enhanced the levels of DNA injury, suggesting the possible involvement in the carcinogenesis of Hp-associated gastric mucosa.

Biochem Biophys Res Commun, 1997 May 8, 234(1), 64 - 7
Selective clinical ultrasound signals mediate differential gene transfer and expression in two human prostate cancer cell lines: LnCap and PC-3; Tata DB et al.; Low intensity ultrasound signals, similar to that employed in clinical therapy, are found to mediate differential gene transfer and expression of the Green Fluorescence Protein (GFP) reporter in two human prostate cancer cell lines, LnCap and PC-3 . Cell suspensions in the presence or in the absence of GFP (44.5nM) were treated at 37 degrees C under a standing wave condition . Cells were exposed to either continuous wave, 932.7kHz ultrasound, or to several independent bursts, each burst comprising a 20% duty cycle (932.7kHz) sine wave . The burst "repetition" frequency was varied from 10Hz to 10kHz in several different experiments and each treatment received a net identical ultrasound energy exposure . Transient GFP expression levels in viable cells were monitored by flow cytometry . The findings revealed a strong ultrasound tone-burst frequency dependence on the transfection efficiencies . Interestingly, the ultrasound signal parameters which are routinely employed in clinical therapy did not yield any statistically significant enhancement in transfection efficiency relative to their sham counterparts.

FEBS Lett, 1997 May 5, 407(3), 357 - 60
Expression of a highly basic peroxidase gene in NaCl-adapted tomato cell suspensions; Medina MI et al.; A tomato peroxidase gene, TPX2, that is only weakly expressed in the roots of young tomato seedlings is highly expressed in tomato suspension cells adapted to high external NaCl concentration . The protein encoded by this gene, with an isolectric point value of approximately 9.6, is found in the culture medium of the growing cells . Our data suggest that the expression of TPX2 in the salt-adapted cells is not the result of the elicitation imposed by the in vitro culture or the presence of high NaCl concentration in the medium.

Mol Hum Reprod, 1997 May, 3(5), 445 - 50
Direct assessment of triploid cells in mosaic human fetuses by fluorescence in-situ hybridization; Horiuchi I et al.; Villous tissues from 30 spontaneous abortions and the same number of artificial abortions were obtained and analysed for the frequency of polyploid cells . Single cell suspensions were made from these tissues without culture and the ploidy of > 100 cells was analysed . Trisomies of chromosomes 17 and 4 have rarely been reported in villous cells of spontaneous abortions, suggesting that the presence of more than three copies of chromosomes 17 and 4 per cell indicates polyploidy . The number of chromosomes 17 and 4 was detected by fluorescence in-situ hybridization analysis using centromeric probes D17Z1 and D4Z1 . Most villous cells from cases of spontaneous and artificial abortions had two D17Z1 or D4Z1 signals per cell, with very small percentages of cells (0.5 +/- 0.4%) showing three signals per cell . However, in four cases of spontaneous abortions, 2-12% of cells had three D17Z1 or D4Z1 signals per cell . This indicates the presence of triploid cells in these cases of spontaneous abortion, at a significantly higher frequency compared to artificial or the remaining 26 cases of spontaneous abortion . In addition, three cases contained 0.2-0.4% of cells showing six signals, indicating that these cells were dividing triploid cells . The low frequency of mosaicism reported here would not be detectable by conventional chromosomal analysis.

J Pathol, 1997 May, 182(1), 86 - 91
Terminal deoxynucleotidyl transferase staining of malignant lymphomas in paraffin sections: a useful method for the diagnosis of lymphoblastic lymphoma; Suzumiya J et al.; Terminal deoxynucleotidyl transferase (TdT) is a DNA polymerase located in the cell nucleus which catalyses the polymerization of deoxynucleotides at the 3'hydroxyl ends of oligo- or polydeoxynucleotide initiators without a template . TdT is known as a useful marker for the diagnosis of acute lymphoblastic leukaemia/lymphoma, but its detection usually requires fresh tissue specimens or cell suspensions, using either an enzyme analysis or immuno-fluorescence or -peroxidase staining . Until the recent development of the use of microwave-treated paraffin sections for immunoperoxidase staining, detection of TdT in paraffin sections required rather complicated processes . This new simple technique was applied to paraffin sections from the tumour tissue specimens of 16 patients with lymphoblastic lymphoma and of seven patients with non-endemic Burkitt's lymphoma, which is sometimes difficult to differentiate from lymphoblastic lymphoma because of their similar clinicopathological characteristics . In addition, as a control, ten cases each were examined of adult T-cell leukaemia/lymphoma (ATLL) and angioimmunoblastic lymphoma (AILD), which are both peripheral T-cell lymphomas . The tumour cells from 15 of the 16 (94 per cent) patients with lymphoblastic lymphoma were found to be TdT-positive . The specificity of the anti-TdT antibody used was confirmed by immunoblot and the specific 60 kD band was detected only in a specimen of lymphoblastic lymphoma . These results show that the immunostaining of TdT on paraffin-embedded sections is a useful method for differentiating lymphoblastic lymphoma from other lymphomas . This method is applicable to a routine diagnostic service.

Plant Mol Biol, 1997 May, 34(2), 233 - 42
Molecular characterization of an Arabidopsis thaliana cDNA coding for a monofunctional aspartate kinase; Frankard V et al.; A cDNA clone encoding a monofunctional aspartate kinase (AK, ATP:L-aspartate 4-phosphotransferase, EC 2.7 . 2.4) has been isolated from an Arabidopsis thaliana cell suspension cDNA library using a homologous PCR fragment as hybridizing probe . Amplification of the PCR fragment was done using a degenerate primer designed from a conserved region between bacterial monofunctional AK sequences and a primer identical to a region of the A . thaliana bifunctional aspartate kinase-homoserine dehydrogenase (AK-HSDH) . By comparing the deduced amino acid sequence of the fragment with the bacterial and yeast corresponding gene products, the highest identity score was found with the Escherichia coli AKIII enzyme that is feedback-inhibited by lysine (encoded by lysC) . The absence of HSDH-encoding sequence at the COOH end of the peptide further implies that this new cDNA is a plant lysC homologue . The presence of two homologous genes in A . thaliana is supported by PCR product sequences, Southern blot analysis and by the independent cloning of the corresponding second cDNA (see Tang et al., Plant Molecular Biology 34, pp . 287-294 {this issue}) . This work is the first report of cloning a plant putative lysine-sensitive monofunctional AK cDNA . The presence of at least two genes is discussed in relation to possible different physiological roles of their respective product.

Eur J Endocrinol, 1997 May, 136(5), 499 - 507
Assessment of thyroid growth stimulating activity of immunoglobulins from patients with autoimmune thyroid disease by cytokinesis arrest assay; Miyamoto S et al.; OBJECTIVE: To develop a novel bioassay for the assessment of thyroid cell growth stimulating activity using cytochalasin B (CB) and to test immunoglobulins (IgGs) from patients with autoimmune thyroid diseases . DESIGN: The assay is based on the principle that growing cells during incubation with CB show an increased number of nuclei in a cell (N/C index), since CB, at appropriate concentrations, is known to inhibit cytoplasmic cleavage without affecting nuclear mitosis . The N/C index represents potential DNA production while cells are incubated with CB . METHODS: FRTL-5 thyroid cells were incubated with various thyroid stimulators in TSH-free medium containing 2 mg/J CB for 3 days . After the incubation, the cells were harvested in trypsin/EDTA to obtain single cell suspension, fixed, dropped onto a glass slide, stained and observed under a microscope to determine the N/C index . RESULTS: Bovine TSH at 10(-3)-1.0 U/I, forskolin at 1x10(-7)-10(-5) mol/l, cholera toxin at 10x10(-5)-10(-3) mg/l, or (Bu)2cAMP at 1 x 10(-5)-10(-3) mol/l increased the N/C index up to approximately 2.0 in a dose-dependent manner . IgGs not only from 27 patients with untreated goitrous Graves' disease but also from 14 patients with goitrous Hashimoto's thyroiditis elicited an increase in the N/C index, which exceeded the mean + 2 S.D . of the values for 17 normal subjects (mean +/- S.D., 1.063 +/- 0.014) . Four patients with primary myxedema displayed a normal N/C index . In Graves' disease, the N/C index did not correlate significantly with thyroid stimulating antibodies (TSAb) activities but did correlate significantly with estimated goiter size (P < 0.05) . IgGs containing blocking-type TSH-receptor antibodies inhibited the TSH- or Graves IgG-stimulated increase in N/C index almost completely, but did not influence the stimulatory effect of IgG from two patients with Hashimoto's thyroiditis . CONCLUSIONS: We have developed a sensitive and simple assay for thyroid growth stimulating activity by using CB, and found that all tested patients with goitrous Graves' disease and goitrous Hashimoto's thyroiditis have thyroid growth stimulating immunoglobulins whose activity does not correlate with TSAb.

Cell Transplant, 1997 May-Jun, 6(3), 339 - 46
MAP2 expression in the developing human fetal spinal cord and following xenotransplantation; Giovanini MA et al.; Human fetal spinal cord (FSC) tissue was obtained from elective abortions at 6-14 wk gestational age (GA) . The specimens were then either immediately processed for immunohistochemical analysis or xenotransplantation . In the latter case, donor tissue was prepared as a dissociated cell suspension and then introduced either subpially or intraspinally into contusion lesions of the adult rat midthoracic spinal cord . The xenografts were subsequently examined by conventional histological and immunohistochemical methods at 2-3 mo postgrafting . Immunostaining showed that MAP2 was expressed heavily in cells residing in the mantle layer of the human fetal spinal cord in situ as early as 6 wk GA . Subpial and intraparenchymal xenografts also were intensely immunoreactive for MAP2, but no staining of surrounding host neural tissue was detected . We conclude that the differential expression of MAP2 can be used to distinguish human graft tissue from the surrounding rat spinal cord in this xenograft paradigm . Under appropriate staining conditions, MAP2 can thus serve to facilitate analyses of host-graft integration, donor cell migration, and neuritic outgrowth.

Cell Transplant, 1997 May-Jun, 6(3), 221 - 30
Colonization of neural allografts by host microglial cells: relationship to graft neovascularization; Pennell NA et al.; In order to illuminate functional roles of microglial cells within neural allografts, we have transplanted both whole and microglial and endothelial cell-depleted E14 neural cell suspensions into the intact striatum of Sprague-Dawley rats . Following posttransplantation times of up to 30 days, the intrastrial allografts were analyzed histochemically using the Griffonia simplicifolia B4 isolectin, a marker for both microglia and blood vessels . Our results indicate that both whole and depleted suspension grafts develop identically in terms of neovascularization and microglial colonization . In both types of transplants microglial cells appeared before any blood vessels were apparent . The main phase of graft vascularization occurred between days 7 and 10 posttransplantation and neovascularization was complete by day 21, as revealed by quantitative image analysis . Microglial cells, which were present as ameboid cells during early posttransplantation times, underwent continuing cell differentiation with time that paralleled graft vascular development . By 30 days posttransplantation microglia within the grafts had assumed the fully ramified phenotype characteristic of resting adult microglia . During graft development and vascularization, microglia were often seen in close proximity to ingrowing blood vessels and vascular sprouts . In conclusion, our study has shown that microglial colonization of grafts and graft vascularization occurs independent of donor-derived microglial and endothelial cells, and suggests that the great majority of microglia and vessels within the graft are host derived . We hypothesize that the host microglia invading the allografts play an active role in promoting graft neovascularization.

Lab Invest, 1997 May, 76(5), 703 - 16
Cell-surface levels of human carcinoembryonic antigen are inversely correlated with colonocyte differentiation in colon carcinogenesis; Ilantzis C et al.; Human carcinoembryonic antigen (CEA) is overexpressed in a wide variety of epithelial malignancies including colon cancer . CEA can function in vitro as a homotypic intercellular adhesion molecule and can block the terminal differentiation of rodent myoblasts, thus raising the possibility that deregulated expression of CEA might directly contribute to malignant progression . To address this question, the expression pattern and cell-surface levels of CEA were studied during malignant transformation of the colonic epithelium in sporadic and familial adenomatous polyposis-related neoplasms . The level of immunohistochemically detected CEA was higher in 30% to 62% of microadenomas and small adenomas from familial adenomatous polyposis patients compared with adjacent normal mucosa, and this proportion was positively correlated with lesion size and degree of dysplasia . Cytofluorometric analysis of highly purified single epithelial cell suspensions from freshly excised carcinomas versus adjacent normal tissue demonstrated up to a 20-fold increase of mean cell-surface CEA in a group of colon carcinomas representative of the overall majority of such tumors--from Dukes' stages A to D and ranging mainly from well to moderately differentiated, the degree of overproduction was inversely correlated with tumor differentiation and directly correlated with stage . A marked tendency toward nonpolarized versus apical cell-surface expression with progression was also noted . Nonspecific cross-reacting antigen (NCA), a CEA family member, is also a homotypic adhesion molecule and blocks terminal myogenic differentiation, whereas biliary glycoprotein is a CEA family adhesion molecule that does not . Cell-surface NCA showed even greater overexpression (up to 70-told) in dedifferentiated tumors, whereas total-cell biliary glycoprotein showed approximately 2-fold lower levels than was normal in more differentiated tumors and approximately 2-fold higher levels than in further progressed tumors . These results therefore support the suggestion that CEA and NCA can directly contribute to colon carcinogenesis by inhibiting colonocyte differentiation.

Poult Sci, 1997 May, 76(5), 753 - 60
Production of chicken chimeras from injection of frozen-thawed blastodermal cells; Kino K et al.; To execute a strategy for reconstituting genetic resources from cryopreserved blastodermal cells, experiments were conducted to optimize conditions for producing chimeric chickens from frozen-thawed blastodermal cells . Stage X blastodermal cells were collected from Barred Plymouth Rock embryos and dispersed . Cells were resuspended in 10% dimethyl sulfoxide in Dulbecco's modified Eagle's medium (DMEM) containing 20% fetal bovine serum, and distributed into plastic ampules . Cell suspensions were seeded to induce ice formation at -7 C, cooled from -7 to -35 C at 1 C/min and then ampules were plunged into liquid nitrogen . Thawing was done by plunging the ampules into warm water (37 C) for 3 min . After centrifugation, the supernatant was replaced with DMEM, and dead or broken cells were removed by density gradient centrifugation . Approximately 500 cells were injected into irradiated Stage X White Leghorn recipient embryos . Following incubation, several somatic chimeras were produced . The frequency of somatic chimerism when fresh (unfrozen) cells, or cells that were frozen and selected by density gradient centrifugation on Percoll or Nycoprep were injected into recipient embryos was 84, 79, and 85%, respectively . The percentage of donor-derived pigmentation in the down of these chimeric chickens was 79, 50, and 58%, respectively . Germline chimerism was determined by mating the chimeras that survived to sexual maturity to Barred Plymouth Rocks . Nine of 16 birds (56.2%) injected with fresh cells, 2 of 26 birds (7.7%) injected with cells that were frozen and selected by density gradient centrifugation on a Percoll gradient, and 3 of 26 birds (11.5%) injected with cells that were frozen and selected on a Nycoprep gradient showed germline transmission; the percentage of donor-derived progeny in these chimeras were 29.5, 5.2, and 6.8%, respectively . The Barred Plymouth Rock donor stock was "reconstituted" by inter se mating of germline male and female chimeras . These data demonstrate that the strategy described here for reconstituting genetic resources from cryopreserved blastodermal cells via chimeric intermediates can be performed successfully.

Aust N Z J Surg, 1997 May, 67(5), 289 - 92
Adverse impact of pneumoperitoneum on intraperitoneal implantation and growth of tumour cell suspension in an experimental model; Mathew G et al.; BACKGROUND: An investigation of the effect of laparoscopy and CO2 pneumoperitoneum on the pattern of tumour implantation and growth in the peritoneal cavity was carried out . METHODS: A suspension of viable adenocarcinoma cells was introduced into the left upper quadrant of the peritoneal cavity of 36 syngeneic immune-competent rats at laparotomy, laparoscopy with CO2 insufflation, and gasless laparoscopy (12 rats in each group) . Six days later the peritoneal cavity and surgical wounds were examined for macroscopic evidence of implanted tumour . The abdominal cavity was divided into sectors and macroscopic tumour implantation was determined for each sector and wound . This was confirmed by histological examination . RESULTS: While tumour implantation occurred in the vicinity of the tumour suspension introduction site in the laparotomy and gasless laparoscopy groups, implantation occurred throughout the peritoneal cavity, including areas remote to the introduction site, in the laparoscopy with CO2 insufflation group . Tumour growth was more likely in the port wounds of rats undergoing laparoscopy with insufflation than without . CONCLUSIONS: In this model, CO2 insufflation during laparoscopy resulted in widespread tumour dissemination and implantation, when compared to laparotomy and gasless laparoscopy, supporting the postulate that wound metastasis and tumour spread may be more likely following laparoscopic cancer surgery in humans when CO2 insufflation is used.

Aust N Z J Surg, 1997 May, 67(5), 245 - 9
Hydrogen sulphide produces diminished fatty acid oxidation in the rat colon in vivo: implications for ulcerative colitis; Moore JW et al.; BACKGROUND: Several lines of evidence suggest a possible role for reduced forms of sulphur (including sulphide) in ulcerative colitis . The aims of this study were to assess the metabolic profile of colonic epithelial cells after treatment in vivo with hydrogen sulphide and correlate this with mucosal histological appearances . METHODS: Adult Sprague-Dawley rats had antegrade Roux-en-Y colostomies fashioned to allow access to the 'in-flow' bowel . Animals were treated with 2 mL sodium hydrosulphide (10, 20, 30 mmol/L) or saline control twice daily via the stoma for four (acute experiments) and 90 (chronic experiments) days . Isolated colonic epithelial cell suspensions prepared from such animals were incubated in the presence of {1-14C}-labelled n-butyrate (5 mmol/L) or {6-14C}glucose (5 mmol/L) . Metabolic performance was measured radiometrically (14CO2 production) and enzymatically (ketone body production and lactogenesis) . The histological appearances of treated mucosa were scored for acute inflammatory changes . RESULTS: There was a highly significant reduction in 14CO2 production from both n-butyrate and glucose in all groups compared to the control in both acute and chronic experiments . There was no difference between groups with respect to histological appearance and no evidence of acute inflammation in any specimen . CONCLUSIONS: Sodium hydrosulphide impairs rat colonic epithelial metabolic performance in vivo, but does not produce mucosal inflammation.

J Neuropathol Exp Neurol, 1997 May, 56(5), 490 - 8
Xenogeneic adrenal medulla graft rejection rather than survival leads to increased rat striatal tyrosine hydroxylase immunoreactivity; Bresjanac M et al.; Adrenal medulla has often been used as a donor tissue for transplantation into damaged central nervous system, with functional effects ranging from very good to nonexistent . The grafts have often been associated with morphological evidence of stimulated recipient dopaminergic fiber plasticity . The interpretation of these results has been difficult due to variable but mostly poor graft survival . The present study combines two experiments which evaluated the effects of intrastriatal xenogeneic adrenal medullary cell suspension grafts on rat recipients . First, bovine adrenal medulla cell suspension grafts of various compositions were tested for their functional and morphologic effects on immunosuppressed hemiparkinsonian rats . In the second experiment, graft rejection was allowed to occur in half of the rats in order to determine a possible contribution of the inflammatory/immune response to increased dopaminergic fiber plasticity of the recipient . At 28 days, grafts of all cell types survived well in immunosuppressed rats, but none of the grafted cell types was associated with either an amelioration of amphetamine-induced rotation or an increase in striatal tyrosine hydroxylase immunoreactivity around the graft site . The latter phenomenon was observed only in the nonimmunosuppressed rats with rejected grafts . Our findings strongly support the role of inflammatory/immune response to grafting in stimulating dopaminergic fiber plasticity and in the appearance of sprouting.

Eur J Nucl Med, 1997 May, 24(5), 488 - 96
Indium-111 labelled lymphocytes: isotope distribution and cell division; Kuyama J et al.; Since lymphocytes continue to proliferate and divide in vivo, it is important to determine the fate of a radionulide following lymphocyte labelling . Using the mixed lymphocyte reaction (MLR), we induced indium-111 labelled lymphocytes from a specific in-bred rat strain (AS) to divide and then observed the subsequent 111In distribution between cells and supernatant . L10 and L12.4 cells, which are allospecific CD4+ T lymphocytes from the AS rat, were stimulated in the MLR by antigen-presenting cells from the August rat, a different strain . We labelled L10 or L12.4 lymphocytes on day 0, the first day of the stimulation cycle, and continued to culture the lymphocytes in vitro . The proliferation of the cells was estimated according to their increase in number . The distribution of 111In between cell and supernatant fractions and between viable and dead (but intact) cells was measured in the cell suspension each day after labelling . The metabolic activity of 111In-labelled lymphocytes was compared with control cells by measuring their uptake of fluorine-18 fluorodeoxyglucose ({18F}FDG) . 111In-labelled lymphocytes showed a poor proliferative response compared with control cells 24-48 h after labelling but increased in number after this time . From 24 to 72 h, about 70% of 111In was in the supernatant but only about 5%-10% was associated with intact dead cells . These dead cells tended to retain their 111In, losing less than 30% per day, suggesting that 111In in the supernatant was the result of active elimination from viable cells . Moreover, 24 h after culture, considerably more 111In was associated with viable than with dead lymphocytes, although over the next few days this distribution reversed . 111In-labelled lymphocytes took up more {18F}FDG than control cells at 24 h but not at 0 or 72-96 h; the maximum {18F}FDG uptake coincided with the greatest reduction in cell number . Furthermore, {18F}FDG uptake correlated with the initial 111In burden in lymphocytes labelled with 111In 24 h previously . The results are consistent with active elimination of 111In by 111In-labelled lymphocytes . The energy requirements for this are diverted away from cell division, thereby increasing the probability of cell death . As lymphocytes become 111In deplete, they recover their capacity to proliferate and their risk of death decreases . These findings have important implications for 111In-labelled lymphocyte scintigraphy, suggesting that cells remaining viable immediately after labelling will either subsequently die or alternatively eliminate the label.

Cytometry, 1997 May 1, 28(1), 74 - 80
Mitochondrial membrane potential measurement in rat cerebellar neurons by flow cytometry; Sureda FX et al.; Mitochondrial membrane potential (MMP) in dissociated rat cerebellar neurons was measured using rhodamine 123 (Rh 123) as fluorescent dye, and flow cytometry . Dye distribution was studied by confocal scanning microscopy . Propidium iodide (PI)-marked cells (dead cells) were not stained by Rh 123, while the green fluorescence of living cells was restricted to mitochondria . Incubation of cells with different ionophores resulted in a maximal inhibition of Rh 123 fluorescence of 27.0 +/- 5.9% (valinomycin), 55.6 +/- 7.2% (ionomycin), and 37.3 +/- 5.1% (gramicidin) . Ionophores decreased cell viability at high concentrations, measured as the number of propidium iodide-marked cells . Exposure of cell suspensions to the mitochondrial specific uncoupling agent CCCP caused a decrease in Rh 123 fluorescence (40 +/- 6.1%) . Conversely, oxidative stress induced by H2O2 did not affect Rh 123 fluorescence . Impairment of glucose bioavailability reduced Rh 123 fluorescence . 2-Deoxy-D-glucose decreased the MMP with a maximal inhibition of 24.0 +/- 4.4% . Lack of glucose in the incubation medium also resulted in a decrease in MMP . Moreover, application of L-glutamate and N-methyl-D-aspartate (NMDA) (the excitatory amino acids) decreased Rh 123 uptake in a dose-dependent manner, which suggests that the measurement of MMP in dissociated cerebellar neurons by flow cytometry is a suitable method to detect the activity of drugs acting on glutamate receptors.

Cytometry, 1997 May 1, 28(1), 11 - 24
Specificity of seven monoclonal antibodies against p53 evaluated with Western blotting, immunohistochemistry, confocal laser scanning microscopy, and flow cytometry; Bonsing BA et al.; p53 immunostaining of histological sections shows inter- and intratumor variability in distribution and staining intensity which are usually scored semiquantitatively . In order to investigate the variation in p53 expression more accurately and its possible relation to other cellular parameters (e.g., DNA content), we have studied the possibility to measure p53 accumulation by multiparameter flow cytometry . To this end we have evaluated seven, commercially available, monoclonal antibodies (MAbs) against p53 (MAbs 1801, 240, 246, 421, 1620, Do1, and Do7) on five tumor cell lines with known p53 gene status: MCF-7 (wild-type p53 gene), COV362.cl4 and T47d (mutated p53 genes), and SAOS-2 and HL60 (no p53 mRNA) . Localization of immunofluorescence was investigated with confocal laser scanning microscopy, immunofluorescence signal intensity with flow cytometry, and antibody specificity with Western blotting . Subsequently, single cell suspensions from two breast carcinomas were flow cytometrically analyzed after triple staining for p53, cytokeratin 8/18, and DNA, and compared to immunohistochemical staining . MAbs Do1 and Do7, and to a lesser extent MAb 421, accurately discriminated p53 positive from p53 negative cell lines . Even at high concentrations these MAbs yielded nuclear immunofluorescence, whereas with MAbs 1801, 240, and 246 strong cytoplasmic signals in both the p53 accumulating and p53 negative cell lines were seen . By using lower antibody concentrations the cytoplasmic immunofluorescence disappeared, but simultaneously the nuclear p53 immunostaining intensity in p53 accumulating cell lines decreased, resulting in false negative nuclei . With MAb 1620 only weak intranuclear spots were obtained in all cell lines tested . Western blotting yielded results with MAbs 1801, Do1, and Do7 in the 53 kD region of the p53 accumulating cell lines . The signal intensity obtained with MAb 1801 was much less compared to MAbs Do1 and Do7 . Although all three MAbs are also described as wild-type p53 specific, only MAbs, Do1 and Do7 showed bands in the 53 kD region of cell line MCF-7 . With MAb 1801 ascites and MAb 1801 supernatant an additional approximately 80 kD band was present in all cell lines tested, including SAOS-2, indicating cross reactivity of this MAb . Immunohistochemical staining of two clinical breast carcinomas confirmed the results obtained in the cell lines . Multiparameter flow cytometric analysis of these breast carcinomas with MAbs Do1 and Do7 showed intratumor heterogeneity for p53 accumulation, which was independent of DNA index heterogeneity . We conclude that MAbs Do1 and Do7 enable quantitative analysis of p53 accumulation in a multiparameter flow cytometric analysis.

Cancer, 1997 May 1, 79(9), 1686 - 97
Endothelial-selectin ligands sialyl Lewis(x) and sialyl Lewis(a) are differentiation antigens immunogenic in human melanoma; Ravindranath MH et al.; BACKGROUND: Sialyl Lewis(x) (sLe(x)) and sialyl Lewis(a) (sLe(a)), the endothelial-selectin ligands involved in extravasation of neutrophils and carcinomas, have been identified in human melanoma . This study explored the following issue: If these ligands are immunogenic tumor-differentiation antigens, they would be potential targets for immunotherapy because of their putative roles in extravasation and metastasis . METHODS: Using a cell-suspension enzyme-linked immunosorbent assay (ELISA), the expression of sLe(x) and sLe(a) on the surface of normal melanocytes, melanoma cells from biopsies, and cell lines (M10-v, M24, and M101) constituting melanoma cell vaccine (MCV) were quantitated . Melanoma patients were immunized with the MCV expressing these antigens . Sera of normal individuals, sera of patients, and sera that adsorbed to sLe(x) and sLe(a) were titrated for anti-sLe antibodies by ELISA to verify the immunogenicity of the ligands . RESULTS: The normal melanocytes did not express sLe(x) and poorly expressed sLe(a) . Melanoma cells from tumor biopsies and MCV lines expressed both sLe(x) and sLe(a) . Sialyl Le(x) was associated with glycoprotein(s) in M10-v, and sLe(a) occurred as a glycolipid moiety in M24 . MCV recipients developed high titers for immunoglobulin (Ig)M but not IgG to both ligands . IgM titers to these ligands were low in normal subjects . In some of the preimmune sera of patients, the titers were threefold above normal . Six of 13 MCV recipients developed at least a twofold increase in anti-sLe titers above preimmune level after the second or third immunization . Adsorption studies suggested that both ligands were immunogenic . CONCLUSIONS: The melanoma-associated sLe(x) and sLe(a) are immunogenic neoplasm-differentiation antigens and are therefore potential targets for passive and active specific immunotherapy in the treatment of melanoma.

J Immunol Methods, 1997 Apr 25, 203(2), 171 - 80
Flow-cytometric screening for the modulation of receptor-mediated endocytosis in human dendritic cells: implications for the development of an in vitro technique for predictive testing of contact sensitizers; Becker D et al.; The aim of this study was to explore the usefulness of human blood dendritic cells (DC) in the development of an in vitro model for predictive testing of contact sensitizers . A method was established to monitor the influence of chemicals on the intracellular targeting of antibody-crosslinked MHC class II molecules after their uptake by human DC . Using a three-colour flow-cytometric technique, freshly prepared DC were distinguished from other MHC class II-bearing cell types such as B-cells and monocytes in unseparated mononuclear cell suspensions of healthy volunteers . The assay is based on the pH-sensitivity of internalized fluorescein-coupled MHC class II specific antibodies . Quenching of fluorescence intensity due to internalization into acidic intracellular compartments was observed with untreated DC whereas internalization into less acidic structures following stimulation with strong contact sensitizers ensured that the fluorescence intensity was conserved . The usefulness of this approach for predictive testing of the preservatives MI/MCI, imidazolidinyl urea, methyl-4-hydroxy-benzoate and 2-phenoxyethanol in comparison to the strong allergen DNFB and the irritants sodium lauryl sulphate and dithranol was explored . Whereas low concentrations of MI/MCI resembled the strong allergen DNFB, high concentrations of imidazolidinyl urea were required for a moderate response . Methyl-4-hydroxy-benzoate and 2-phenoxyethanol as well as the irritants SLS and dithranol failed to induce a significant effect in this assay . The non-responsiveness to the latter compounds reflected their minor or absent capacity to induce contact hypersensitivity in humans, whereas DNFB, MI/MCI and imidazolidinyl urea are well established contact sensitizers . These data suggest that the capacity of a chemical to modulate endocytotic mechanisms in dendritic cells in vitro seems to reflect the probability of that substance acting as a hapten in vivo.

Eur J Biochem, 1997 Apr 15, 245(2), 294 - 9
Enzymes of octadecanoid biosynthesis in plants--12-oxo-phytodienoate 10,11-reductase; Schaller F et al.; Octadecanoids, potent cyclic plant signaling molecules derived from alpha-linolenic acid, are involved in the regulation of a multitude of physiological processes such as senescence, herbivore and pathogen defense, mechanoperception and morphogenesis . The first cyclic intermediate in the Vick-Zimmerman pathway of octadecanoid biosynthesis is 12-oxo-phytodienoic acid . Its conversion to the end product of the pathway, jasmonic acid, a C12 compound, first proceeds through reduction to 3-oxo-2-(pent-2'-enyl)-cyclopentane-1-octanoic acid, which is then converted to jasmonic acid by three cycles of beta-oxidation . The first of these conversions is a decisive point in the biosynthetic sequence, in that it channels the octadecanoid into the pathway of beta-oxidation . 12-Oxo-phytodienoate reductase was purified to apparent homogeneity from a cell suspension culture of Corydalis sempervirens . The enzyme is soluble and a monomer of apparent molecular mass 41 kDa which prefers NADPH over NADH to reduce the 10,11-double bond of 12-oxo-phytodienoic acid . The structure of the reaction product was proved by derivatization, GC/MS and NMR analysis . The enzyme accepts both the cis and the trans isomer of 12-oxo-phydodienoic acid, with a preference for the cis-isomer (6:1) . 12-Oxo-phytodienoate reductase will also convert the synthetic substrate 2-cyclohexenone to cyclohexanone, but the enzyme did not reduce some other cyclic alpha,beta-unsaturated ketones tested (the plant hormone abscisic acid or the steroids testosterone and progesterone) . Characteristic parameters of 12-oxo-phytodienoate reductase were determined.

Braz J Med Biol Res, 1997 Apr, 30(4), 471 - 7
Interactions of ANP and ANG II in tubular nephron acidification; Mello-Aires M et al.; In order to examine the effects and the interaction of angiotensin II (ANG II, 1 pM) and atrial natriuretic peptide (ANP, 1 microM) on the kinetics of bicarbonate reabsorption in the rat middle proximal tubule, we performed in vivo experiments using a stopped-flow microperfusion technique with the determination of lumen pH by Sb microelectrodes . These studies confirmed that ANG II added to the luminal or peritubular capillary perfusion fluid stimulates proximal bicarbonate reabsorption and showed that ANP alone does not affect this process, but impairs the stimulation caused by ANG II . We also studied the effects and the interaction of these hormones in cortical distal nephron acidification . Bicarbonate reabsorption was evaluated by the acidification kinetic technique in early (ED) and late (LD) distal tubules in rats during in vivo stopped-flow microperfusion experiments . The intratubular pH was measured with a double-barreled microelectrode with H(+)-sensitive resin . The results indicate that ANG II acted by stimulating Na+/H+ exchange in ED (81%) and LD (54%) segments via activation of AT1 receptors, as well as vacuolar H(+)-ATPase in LD segments (33%) . ANP did not affect bicarbonate reabsorption in either segment and, as opposed to what was seen in the proximal tubule, did not impair the stimulation caused by ANG II . To investigate the mechanism of action of these hormones in more detail, we studied cell pH dependence on ANG II and ANP in MDCK cells using the fluorescent probe BCECF . We showed that the velocity of cell pH recovery was almost abolished in the absence of Na+, indicating that it is dependent on Na+/H+ exchange . ANP (1 microM) alone had no effect on this recovery but reversed both the acceleration of H+ extrusion at low ANG II levels (1 pM and 1 nM), and inhibition of H+ extrusion at higher ANG II levels (100 nM) . To obtain more information on the mechanism of interaction of these hormones, we also studied their effects on the regulation of intracellular free calcium concentration, {Ca2+}i, monitored with the fluorescent probe Fura-2 in MDCK cells in suspension . The data indicate that the addition of increasing concentrations of ANG II (1 pM to 1 microM) to the cell suspension led to a progressive increase in {Ca2+}i to 2-3 times the basal level . In contrast, the addition of ANP (1 microM) to the cell suspension led to a very rapid 60% decrease in {Ca2+}i and reduced the increase elicited by ANG II, thus modulating the effect of ANG II on {Ca2+}i . These results may indicate a role of {Ca2+}i in the regulation of the H+ extrusion process mediated by Na+/H+ exchange and stimulated/impaired by ANG II . The data are compatible with stimulation of Na+/H+ exchange by increases of {Ca2+}i in the lower range, and inhibition at high {Ca2+}i levels.

Cardiovasc Res, 1997 Apr, 34(1), 169 - 78
Glucose elevations alter bradykinin-stimulated intracellular calcium accumulation in cultured endothelial cells; Pieper GM et al.; OBJECTIVE: Diabetes selectively injures receptor-mediated endothelium-dependent relaxation . In this study, we investigated the effect of elevated glucose concentrations on intracellular calcium (Ca2+i) signal transduction in response to stimulants of EDRF/nitric oxide release in cultured bovine aortic endothelial cells . METHODS: {Ca2+i} was measured in cell suspensions using Fura-2 and fluorescence spectroscopy while nitric oxide production was evaluated using radioimmunoassay of cGMP production . RESULTS: After 24 h exposure to 25 mM glucose in Ham's F-12 media, the increase in endothelial cell {Ca2+i} in response to 100 nM bradykinin was attenuated by 40% while the response to ionomycin was unaltered . When RMPI medium was used, no reduction in response to bradykinin was observed at 25 mM glucose, but a significant reduction in {Ca2+i} signal was observed after exposure to 35 mM glucose for a similar time period . Defective {Ca2+i} signaling was also seen in cells using MEM medium . {Ca2+i} signal responses to ionomycin and NaF, a G-protein activator of extracellular calcium entry via calcium channels, were unaltered by elevated glucose exposure . The defect in {Ca2+i} signal was not mimicked by either mannose or sucrose, but was prevented by co-incubation with cytochalasin B to inhibit glucose uptake . Neither superoxide dismutase nor catalase nor the extracellular hydroxyl radical scavenger, mannitol, blocked the reduction in the bradykinin-induced increase of {Ca2+i} in elevated glucose-exposed cells; however, the reduction was completely blocked by the cell-permeable hydroxyl radical scavenger, dimethylthiourea . Bradykinin-stimulated (but not ionomycin-stimulated) cGMP production within endothelial cells or in RFL-6 detector cells was attenuated by elevated glucose exposure . CONCLUSIONS: Hyperglycemia may contribute to defective endothelium-dependent relaxation in diabetes via an attenuated increase in Ca2+i signal transduction for the release of nitric oxide by endothelial cells . This defect possibly arises as a consequence of hydroxyl radicals formed intracellularly.

Allergy, 1997 Apr, 52(4), 465 - 9
Rapid expression of the CD69 antigen on T cells and natural killer cells upon antigenic stimulation of peripheral blood mononuclear cell suspensions; Werfel T et al.; The CD69 antigen has been identified as the earliest activation marker on the surface of cytokine- or mitogen-activated lymphocytes . The expression of this molecule may be a useful early marker of antigen- or allergen-specific activation of lymphocytes in vitro . We evaluated the expression of the CD69 and CD25 antigens on antigen- or allergen-stimulated lymphocytes and the proliferative responses as detected by thymidine incorporation . Peripheral blood mononuclear cells (PBMC) of allergic patients sensitized to Dermatophagoides pteronyssinus, bovine casein, or nickel sulfate were cultured in the absence or presence of clinically relevant allergens, tetanus toxoid, or recombinant interleukin (IL)-2 . The respective binding of CD69 or CD25 antibodies to PBMC and thymidine incorporation were measured . An early expression of CD69, but not of CD25, antigen was detectable after 24-72 h of stimulation on up to 80% of natural killer (NK) cells and up to 10% of CD4+ T cells in PBMC cultures . Anti-IL-2 antibodies inhibited these increases of CD69 on NK cells and T cells by up to 60% . After 6 days of antigenic stimulation, the rates of both CD25+ and CD69+ lymphocytes were higher . Seventy-four percent of the CD25+ PBMC but only 55% of the CD69+ cells were CD3+ T lymphocytes at this time . No qualitative differences were detectable in allergen- or tetanus-toxoid-stimulated PBMC from allergic patients . The high expression of CD69 on NK cells in antigen-stimulated cultures suggests that these cells are easily activated by cytokines from antigen-stimulated T cells . CD69+ NK cells may serve as early-indicator cells in cultures with antigen- or allergen-stimulated mononuclear cells.

Inflammation, 1997 Apr, 21(2), 251 - 67
Chemotactic factors released in culture by intact developing and healing skin lesions produced in rabbits by the irritant sulfur mustard; Tanaka F et al.; Development, peak and healing lesions were induced in the skin of rabbits by topical applications (on different days) of the chemical irritant sulfur mustard (SM) . Immediately after the rabbits were euthanized, the intact lesions were excised and organ-cultured for 17 to 20 hours . The culture fluids from early, peak and healing SM lesions all showed high chemotactic activity for both PMN and MN . This finding suggests that the PMN and MN, seen microscopically in tissue sections of the lesions, were entering continuously, even during the healing process . The chemotaxins identified were the eicosanoid LTB4, the chemokine IL-8, and proteases producing the complement fragment C5a . Other studies from our laboratory showed that the number of cells containing IL-1, IL-8, MCP-1, and GRO mRNAs was increased in SM lesions . Chemotactic activity was released by both live and dead (frozen and thawed) cell suspensions of PMN, MN, and fibroblasts, suggesting that these cells were major sources of the chemotaxins produced by the SM lesion explants . Explants of normal skin produced considerable chemotactic activity for MN, but not for PMN . Chemotactic activity for PMN, and the release of LTB4, IL-8 and proteases cleaving C5 to C5a, occurred only in explants infiltrated by leukocytes.

Vaccine, 1997 Apr-May, 15(6-7), 624 - 30
Total and isotype humoral responses in cattle vaccinated with foot and mouth disease virus (FMDV) immunogen produced either in bovine tongue tissue or in BHK-21 cell suspension cultures; Capozzo AV et al.; The anti-foot and mouth disease virus (FMDV) serum antibody activity of protected and non protected animals immunized with inactivated FMDV originated in either bovine tongue tissue (BTTV vaccines) or BHK-21 cell suspension cultures (BHKV vaccines) was evaluated . The results show that 80-100% of the BTTV immunized and only 40-60% of the BHKV immunized animals with liquid-phase blocking sandwich ELISA (lp ELISA) serum titres of 1.5-1.7 U, were protected against the challenge with any of the four infectious FMDV argentine reference strains . This difference becomes almost marginal among BTTV and BHKV vaccinated animals with a strong anti-FMDV humoral response (i.e . lp ELISA titres > or = 1.95 U) . Isotyping of the anti-FMDV response in immunized cattle with low lp ELISA titres revealed that BTTV vaccines were able to induce remarkably higher anti-FMDV IgG1 titres than their BHKV counterparts (i.e . mean titres of 1.95 and 1.35 U . respectively) . This difference in specific IgG1 serum levels induced by BTTV and BHKV vaccines seems to be also limited to those animals with low anti-FMDV lp ELISA titres . These results together with the fact that the specific serum IgG1, but not the IgG2, isotype response of 219 vaccinated animals correlates almost linearly with their capacity to pass the challenge, suggests that the superior performance of BTTV vaccines is close related to their ability to raise a stronger anti-FMDV IgG1 response than BHKV vaccines.

Br J Radiol, 1997 Apr, 70(832), 391 - 8
Modification of the response of a quiescent cell population within a murine solid tumour to boron neutron capture irradiation: studies with nicotinamide and hyperthermia; Masunaga S et al.; C3H/He mice bearing SCC VII tumours received 5-bromo-2'-deoxyuridine (BrdU) continuously for 5 days via implanted mini-osmotic pumps, to label all proliferating (P) cells . 20 min after intraperitoneal injection of sodium borocaptate-10B (BSH), or 3 h after oral administration of dl-p-boronophenylalanine-10B (BPA), the tumours were irradiated with thermal neutrons . To modify the uptake dose of 10B, nicotinamide (NA) was intraperitoneally injected 60 min before the administration of 10B-compounds and/or the tumours were heated to 41.5 degrees C for 20 min immediately before irradiation . After irradiation, the tumours were excised, minced and trypsinized . The tumour cell suspensions were then incubated with cytochalasin-B (a cytokinesis-blocker) . The micronucleus (MN) frequency in cells not BrdU-labelled (quiescent (Q) cells) was determined using immunofluorescence staining for BrdU . With or without the administration of 10B-compounds, the sensitivity of Q cells was lower than that of total (P + Q) tumour cells . With thermal neutron irradiation in the presence of either BPA or BSH, the MN frequency in each cell population was increased . A greater increase in the MN frequency of total tumour cells was observed after thermal neutron irradiation in the presence of BPA than in the presence of BSH . The distribution of 10B from BPA into tumour cells was thought to be more dependent on the uptake ability of the tumour cells than that from BSH . Sufficient quantity of 10B from these two 10B-compounds to cause a highly lethal event inside the cancer cell with thermal neutron irradiation could not be delivered to Q cells . When NA and/or heat treatment were combined with 10B-compound administration, NA increased MN frequency in the BSH treated total cells, and heat treatment elevated MN frequency in Q cells . From the viewpoint of cell kill effect, the combined treatment with nicotinamide and heat treatment was more useful than treatment with either nicotinamide or heat treatment alone, not only in the total tumour cells but also in the Q cells.

Arch Dermatol Res, 1997 Apr, 289(5), 285 - 91
Binding and in vitro modulation of human epidermal Langerhans cell functions by substance P; Staniek V et al.; Substance P (SP) is distributed in both the central and peripheral nervous system . It has various effects on immunocompetent cells, such as macrophages and lymphocytes . The aim of our study was to search for the presence of SP receptors (SP-R) on human cutaneous Langerhans cells (LC), and to determine the effects of SP on LC immunological functions in a model of mixed epidermal cell-lymphocyte reaction (MELR) . Radioligand binding studies showed that LC-enriched epidermal cell suspensions reversibly bound SP, and that the specific binding increased with the percentage of LC . Functional assays showed that SP had no effect when added at concentrations from 10(-6) M to 10(-12) M to the MELR . The addition of SP at concentrations of 10(-4) M and 10(-5) M was able to inhibit the allogeneic T-cell response (98.3 +/- 1.8% and 92.8 +/- 8.9% inhibition, respectively) without modifying the cell viability . This inhibition was through an effect of SP on both T-cell and LC function . We conclude that SP has receptors on LC and may inhibit antigen presentation.

Plant Mol Biol, 1997 Apr, 33(6), 979 - 87
Molecular cloning, structure and expression of an elicitor-inducible chitinase gene from pine trees; Wu H et al.; We have cloned, sequenced, and examined the expression of genes from pine trees that appear to encode extracellular class II chitinase . Nucleotide sequence analysis indicates a coding sequence composed of three exons interrupted by two introns at locations identical to those found in other chitinase genes that possess introns . One of the genes, Pschi4, potentially encodes a protein that shares 62% amino acid sequence identity through the catalytic domain with class II chitinase from tobacco . In contrast, Pschi1 contains a stop codon in the first exon and may be a pseudogene . Pschi4 genes are conserved in several species of pine, and appear to comprise a small multigene family . Treatment of pine cell suspension cultures with the general elicitor chitosan induced Pschi4 expression . The regulatory sequences associated with the Pschi4 gene were sufficient to direct chitosan-inducible expression of Pschi4 in transgenic tobacco plants, which indicates that Pschi4 is an actively expressed member of the multigene family . The observation that the Pschi4 gene from pine (a gymnosperm) was appropriately regulated by chitosan in tobacco (an angiosperm) suggests that the signaling pathways that mediate chitosan-induced transcription are highly conserved in the plant kingdom.

Neurosci Res, 1997 Apr, 27(4), 305 - 15
Localization of dopamine receptors and associated mRNA in transplants of human fetal striatal tissue in rodents with experimental Huntington's disease; Pundt LL et al.; Huntington's Disease (HD) is characterized by deficits in motor and cognitive functions . This neurodegenerative disease shows an extensive loss of medium-sized spiny projection neurons (GABAergic) within the neostriatum . With the loss of these neurons, there is a concomitant loss of associated receptors, such as those for GABA, glutamate, and dopamine . In the present study, we have addressed the question of whether dopamine receptors are re-established in the lesioned rodent striatum following the transplantation of human striatal cells . Human striatal cell suspension or saline (transplant controls) was injected into the striatum of rats previously lesioned with quinolinic acid (QA) . Three nine months following transplantation, the animals were sacrificed and the brains were processed for receptor autoradiography and in situ hybridization of dopamine D1 and D2 receptor subtypes . Our results demonstrate that animals transplanted with human striatal cells show a significant increase in D1 receptors following transplantation when compared to the lesion area in control animals, while D1 receptor mRNA remains unchanged . In contrast to D1 receptor binding, D2 receptor levels are not increased in the lesioned and transplanted area of the striatum when compared to controls; however, D2 receptor mRNA levels are significantly increased . These results demonstrate that at the times the animals were examined, D1 and D2 receptors were differentially regulated . Our results further indicate that human striatal primordium will survive following transplantation and will express D1 receptors and D2 receptor mRNA that are depleted in the QA lesioned rodent striatum . This study compliments and extends previous findings on human striatal cell transplantation in rodent models of HD.

Biosci Biotechnol Biochem, 1997 Apr, 61(4), 737 - 9
Spectroscopic analysis of the cytoagglutinating activity of abrin-b isolated from Abrus precatorius seeds against leukemic cells; Ohba H et al.; The cytoagglutinating activity of abrin-b, a toxic lectin isolated from Abrus precatorius seeds, against cultured cell strains derived from acute lymphoblast leukemia (ALL) was investigated by visible (VIS) spectroscopy . Upon addition of abrin-b, the turbidity at 600 nm of cell suspension decreased and this change could be recorded as the cytoagglutination curve . From this curve, the cytoagglutination velocity (CV) and cytoagglutination intensity (CI) of each cell strain was measured . Each cell strain showed the respective CV and CI values and the cell strains derived from the T cell line were strongly agglutinated by abrin-b compared with those derived from the B cell line . Further, it has become apparent that the cytoagglutinating activity increased with an increase in the order of the differentiation of cell strains.

Hum Immunol, 1997 Apr 1, 53(2), 216 - 23
Generation of dendritic cells expressing bcr-abl from CD34-positive chronic myeloid leukemia precursor cells; Smit WM et al.; Patients with a relapse of chronic myeloid leukemia (CML) after allogeneic bone marrow transplantation can be successfully treated with blood mononuclear cells from the original bone marrow donor . However, the antileukemic effect of this treatment is often accompanied by graft-versus-host disease (GVHD) . Treatment with cytotoxic T-lymphocyte (CTL) lines or clones that are specifically generated against leukemic antigen-presenting cells from the patient, may separate antileukemic effects from GVHD . In this report we demonstrate that after culturing CD34-positive cells purified from bone marrow of patients with chronic phase CML in medium containing human serum, GM-CSF, TNF alpha, and IL-4 up to 28% of the cultured cells were dendritic cells, characterized by morphology, phenotypic analysis, and their efficient capacity to stimulate allogeneic T lymphocytes . The expression of HLA and costimulatory molecules and the stimulatory capacity of the dendritic cell-enriched cell suspensions were optimal between days 7 and 10 after onset of the cultures . Fluorescence in situ hybridization revealed that all cultured dendritic cells contained the CML specific t(9;22) translocation . PCR analysis showed expression of the translocation specific bcr-abl mRNA . These leukemic dendritic cells may enhance the induction and proliferation of CTL lines and clones with more specificity for the leukemic cells.

Photochem Photobiol, 1997 Apr, 65(4), 622 - 9
UVA II exposure of human skin results in decreased immunization capacity, increased induction of tolerance and a unique pattern of epidermal antigen-presenting cell alteration; LeVee GJ et al.; The risks incurred from increased exposure to UVA II (320-340 nm) (i.e . during sunscreen use and extended outdoor exposure, tanning parlors) are not well understood . Therefore, we explored the effects of UVA II on skin immune responses in humans . After a single local exposure (4 minimum erythemal dose {MED}) using a xenon are lamp filtered with a narrow bandpass filter (335 +/- 5 nm full width at half maximum), individuals were contact-sensitized with dinitrochlorobenzene (DNCB) through a UVA II exposure site or through normal skin . UVA II induced a marked decrease in the magnitude of skin immune responses (P < 0.0001) . The UVA II group had only 29% successful sensitizations, as compared to 83% in the control group . The percentage of individuals who remained tolerant to DNCB after two sensitizations was 23.6% for the UVA II-exposed group, as compared to 3.8% in the controls (P = 0.006) . UVA II also uniquely altered the type of antigen-presenting cells present in the epidermis . Human leukocyte antigen (HLA)-DR+ cells in control epidermal cell suspensions (C-EC) comprised a single, homogeneous population of Langerhans cells (LC) with the phenotype: CD1ahi DRmid CD11b CD36 (1.5 +/- 0.3% of EC) . UVA II irradiation reduced the number of such LC to 0.6 +/- 0.2% of EC . Although cells expressing the macrophage phenotype: CD1a- DRhi CD11b+ CD36+ were increased in UVA II skin, relative to C-EC, these comprised only 10.1 +/- 6.1% of the DR+ cells, which is less than that after UVB exposure . Also distinct from UVB, a third population was found in UVA II-EC, which exhibited a novel phenotype: CD1a+ DR+ CD36+ CD11b+; these comprised 11.1 +/- 6.9% of the DR+ UVA II-EC . In conclusion, despite the above differences in infiltrating DR+ cells, both UVB and UVA II reduce the skin's ability to support contact sensitization, induce active suppression (tolerance) and induce a reduction in LC.

J Physiol, 1997 Apr 1, 500 ( Pt 1), 139 - 54
Cytoplasmic calcium buffers in intact human red cells; Tiffert T et al.; 1 . Precise knowledge of the cytoplasmic Ca2+ buffering behaviour in intact human red cells is essential for the characterization of their {Ca2+}i-dependent functions . This was investigated by using a refined method and experimental protocols which allowed continuity in the estimates of {Ca2+}i, from nanomolar to millimolar concentrations, in the presence and absence of external Ca2+ chelators . 2 . The study was carried out in human red cells whose plasma membrane Ca2+ pump was inhibited either by depleting the cells of ATP or by adding vanadate to the cell suspension . Cytoplasmic Ca2+ buffering was analysed from plots of total cell calcium content vs . ionized cytoplasmic Ca2+ concentration ({CaT}i vs . {Ca2+}i) obtained from measurements of the equilibrium distribution of 45Ca2+ at different external Ca2+ concentrations ({Ca2+}o), in conditions known to clamp cell volume and pH . The equilibrium distribution of 45Ca2+ was induced by the divalent cation ionophore A23187 . 3 . The results showed the following . (i) The known red cell Ca2+ buffer represented by alpha, with a large capacity and low Ca2+ affinity, was the main cytoplasmic Ca2+ binding agent . (ii) The value of alpha was remarkably constant; the means for each of four donors ranged from 0.33 to 0.35, with a combined value of all independent measurements of 0.34 +/- 0.01 (mean +/- S.E.M., n = 16) . This contrasts with the variability previously reported . (iii) There was an additional Ca2+ buffering complex with a low capacity (approximately 80 micromol (340 g Hb)(-1)) and intermediate Ca2+ affinity (apparent dissociation constant, K(D,app) approximately 4-50 microM) whose possible identity is discussed . (iv) The cell content of putative Ca2+ buffers with submicromolar Ca2+ dissociation constants was below the detection limit of the methods used here (less than 2 micromol (340 g Hb)(-1)) . 4 . Vanadate (1 mM) inhibited the Vmax of the Ca2+ pump in inosine-fed cells by 99.7% . The cytoplasmic Ca2+ buffering behaviour in these cells was similar to that found in ATP-depleted cells.

Anal Biochem, 1997 Mar 15, 246(2), 218 - 24
Flow cytometric detection of mitochondrial dysfunction in subpopulations of human mononuclear cells; Kunz D et al.; At 488 nm argon-ion laser excitation human mononuclear cells emit flavoprotein-related autofluorescence signals . Approximately 60% of these are caused by the mitochondrial flavoproteins alpha-lipoamide dehydrogenase and electron transfer flavoprotein, having differences in their fluorescence emission spectra . At the emission wavelength of 530 nm the redox changes of alpha-lipoamide dehydrogenase fluorescence in human mononuclear cells can be monitored by flow cytometry . This allows the estimation of the steady-state reduction level of this flavoprotein being in redox equilibrium with the mitochondrial NAD-system . We applied this method to elucidate the possible impairment of mitochondrial function in subpopulations of mononuclear cells of patients harboring deletions of the mitochondrial DNA in skeletal muscle . In the monocyte fraction of three patients and in the lymphocyte fraction of one patient we observed in the presence of the mitochondrial substrate octanoate elevated steady-state reduction levels of alpha-lipoamide dehydrogenase . This is an indication for the presence of respiratory chain-inhibited mitochondria in mononuclear cell subpopulations of the described patients . These data were confirmed by conventional determinations of maximal oxygen consumption rates of digitonin-permeabilized cells . Therefore, the flow cytometric determination of flavoprotein-caused autofluorescence changes is a useful and sensitive method for the detection of an impairment of mitochondrial respiratory chain in subpopulations of heterogeneous cell suspensions.

Braz J Med Biol Res, 1997 Mar, 30(3), 347 - 58
Assessment of the degree of contamination of rat germ cell preparations using specific cDNA probes; Savaris RF; Recent reports showing a decrease in sperm count in men have brought new concerns about male infertility . Animal models have been widely used to provide some relevant information about the human male gamete, and extrapolations are made to men and to the clinical context . The present-study assesses one of the methods used for separation of germ cells of the adult rat testis, namely centrifugal elutriation followed by density gradients (Percoll) . This method was chosen since it presents the best results for cell purity in separating germ cells from the rat testis . A comparison between continuous and discontinuous Percoll gradients was performed in order to identify the best type of gradient to separate the cells . Maximal cell purity was obtained for spermatocytes (81 +/- 8.2%, mean +/- SEM) and spermatids (84 +/- 2.6%) using centrifugal elutriation followed by continuous Percoll gradients . A significant difference in purity was observed between elongating spermatids harvested from continuous Percoll gradients and from discontinuous gradients . Molecular analysis was used to assess cell contamination by employing specific probes, namely transition protein 2 (TP2), mitochondrial cytochrome C oxidase II (COX II), and sulfated glycoprotein 1 (SGP1) . Molecular analysis of the samples demonstrated that morphological criteria are efficient in characterizing the main composition of the cell suspension, but are not reliable for identifying minimal contamination from other cells . Reliable cell purity data should be established using molecular analysis.

Food Chem Toxicol, 1997 Mar-Apr, 35(3-4), 409 - 16
Cutaneous xenobiotic metabolism: glycine conjugation in human and rat keratinocytes; Nasseri-Sina P et al.; Glycine conjugation is an important route of metabolism and detoxication of carboxylic acids in the liver . In this paper the in vitro cutaneous metabolism of {carboxyl-14C}benzoic acid to its glycine conjugate hippuric acid in rat and human skin is reported . Cutaneous glycine conjugation was studied in F344 rat and human epidermal keratinocytes using two systems: (1) freshly isolated keratinocytes in suspension and (2) primary keratinocyte cultures . For comparative purposes, studies were also carried out in freshly isolated and cultured F344 rat hepatocytes . After incubation of 5 x 10(6) cells with 1 microM benzoic acid at 37 degrees C for 8 hr, no glycine conjugation was observed in rat and human keratinocyte suspensions, with greater than 98% of the radioactivity recovered as the parent compound . In contrast, cultured keratinocytes exhibited glycine conjugation, with 10.9 +/- 1.0% (mean SEM, n = 3) and 2.1 +/- 0.6% (mean SEM, n = 3) conversion to hippuric acid at 8 hr in rat and human cells, respectively . Tissue-specific differences in metabolism were observed, with conjugation in hepatocytes significantly greater (P < 0.05) than in keratinocytes at all times up to 8 hr . After incubation of benzoic acid with cultured hepatocytes for 8 hr, more than 98% of the of the radioactivity was recovered as the glycine conjugate . These studies indicate that rat and human skin possesses low, but demonstrable, glycine-conjugating activity, and that keratinocytes in primary culture may provide a better system than freshly isolated cell suspensions for studying such activity.

Bull Cancer, 1997 Mar, 84(3), 29 - 34
{Oncogen N-myc expression and measurement of DNA ploidy in neuroblastoma: a double staining flow cytometric analysis}; Chassevent A et al.; When N-myc copy number was assessed by molecular biology, it has been proven that an amplification of the oncogene was a bad prognosis in childhood neuroblastoma, and the same goes for DNA-diploidy . This study concerns the development of a biparametric flow cytometric analysis of 2 neuroblastoma cell lines (SK-N-SH and IGR-N-91), which exhibited respectively 1 and 60 copies of the N-myc oncogene . An indirect immunofluorescence technique allowed N-myc oncoprotein staining and an isotypic control was used to assess the threshold of specific fluorescence . Simultaneously, a double staining with propidium iodide gave the nuclear DNA content . For both types of cells, the level of N-myc expression was calculated as a fluorescence index (IF) . IF for IGR-N-91 appeared 2.5 times higher than those of SK-N-SH . This fluorescence index increases significantly during the exponential growth of N-myc amplified cells, whereas it does not vary for SK-N-SH . During IGR-N-91 tumoral evolution, the cell line which derived from murine heart metastasis was the only one to show an increased IF . When applied to 10 neuroblastoma cell suspensions, this double staining showed an high IF for only 1 N-myc amplified case . A poor cell yield after tumoral dissociation and too much debris did not allowed the calculation of IF for half of them, which hampered a routine development of this technique.

Immunology, 1997 Mar, 90(3), 427 - 34
Demonstration of cytoplasmic CD32 (Fc gamma RII) within human lymphocytes following microwave treatment; Sandilands GP et al.; We have recently described a cytoplasmic from of CD32 (Fc gamma RII) within the vast majority of normal human peripheral blood lymphocytes (PBL) including T cells . The function of cytoplasmic CD32 is not known . These flow cytometric studies were conducted using single cell suspensions of PBL that had been pre-fixed and permeabilized using methanol/triton-X-100 . In this study we have attempted to visualize cytoplasmic CD32 by immunocytochemistry using normal PBL processed in various ways and have also looked for CD32 within tissue lymphocytes . Weak cytoplasmic CD32 staining was observed in paraffin sections of normal lymphocytes but only when sections were microwave treated . The intensity of staining for CD32 did however, appear to be much stronger within infiltrating lymphocytes found in autoimmune diseases or in rejecting allografts: an observation that suggests that up-regulation of cytoplasmic CD32 may occur when T cells become activated in vivo . Microwave treatment of PBL suspensions was shown to disrupt the outer cell membrane, thus effectively permeabilizing the cell and allowing for the detection of cytoplasmic components, like CD32, by flow cytometry . Microwave treatment may, therefore, afford an alternative method for cell permeabilization and may prove to be a useful method for the study of cytoplasmic molecules in cell suspensions and in paraffin-embedded tissues.

Plant Mol Biol, 1997 Mar, 33(4), 699 - 708
Sequence and RT-PCR expression analysis of two peroxidases from Arabidopsis thaliana belonging to a novel evolutionary branch of plant peroxidases; Kjaersgard IV et al.; cDNA clones encoding two new Arabidopsis thaliana peroxidases, ATP 1a and ATP 2a, have been identified by searching the Arabidopsis database of expressed sequence tags (dbEST) . They represent a novel branch of hitherto uncharacterized plant peroxidases which is only 35% identical in amino acid sequence to the well characterized group of basic plant peroxidases represented by the horseradish (Armoracia rusticana) isoperoxidases HRP C, HRP E5 and the similar Arabidopsis isoperoxidases ATP Ca, ATP Cb, and ATP Ea . However ATP 1a is 87% identical in amino acid sequence to a peroxidase encoded by an mRNA isolated from cotton (Gossypium hirsutum) . As cotton and Arabidopsis belong to rather diverse families (Malvaceae and Crucifereae, respectively), in contrast with Arabidopsis and horseradish (both Crucifereae), the high degree of sequence identity indicates that this novel type of peroxidase, albeit of unknown function, is likely to be widespread in plant species . The atp 1 and atp 2 types of cDNA sequences were the most redundant among the 28 different isoperoxidases identified among about 200 peroxidase encoding ESTs . Interestingly, 8 out of totally 38 EST sequences coding for ATP 1 showed three identical nucleotide substitutions . This variant form is designated ATP 1b . Similarly, six out of totally 16 EST sequences coding for ATP 2 showed a number of deletions and nucleotide changes . This variant form is designated ATP 2b . The selected EST clones are full-length and contain coding regions of 993 nucleotides for atp 1a, and 984 nucleotides for atp 2a . These regions show 61% DNA sequence identity . The predicted mature proteins ATP 1a, and ATP 2a are 57% identical in sequence and contain the structurally and functionally important residues, characteristic of the plant peroxidase superfamily . However, they do show two differences of importance to peroxidase catalysis: (1) the asparagine residue linked with the active site distal histidine via hydrogen bonding is absent; (2) an N-glycosylation site is located right at the entrance to the heme channel . The reverse transcriptase polymerase chain reaction (RT-PCR) was used to identify mRNAs coding for ATP 1a/b and ATP 2a/b in germinating seeds, seedlings, roots, leaves, stems, flowers and cell suspension culture using elongation factor 1alpha (EF-1alpha) for the first time as a positive control . Both mRNAs were transcribed at levels comparable to EF-1alpha in all plant tissues investigated which were more than two days old, and in cell suspension culture . In addition, the mRNA coding for ATP 1a/b was found in two day old germinating seeds . The abundant transcription of ATP 1a/b and ATP 2a/b is in line with their many entries in dbEST, and indicates essential roles for these novel peroxidases.

DNA Cell Biol, 1997 Mar, 16(3), 357 - 68
Characterization of the adrenal cytochrome P450C17 in the hamster, a small animal model for the study of adrenal dehydroepiandrosterone biosynthesis; Cloutier M et al.; The hamster, like the human produces cortisol as its major glucocorticoid, rather than corticosterone, typical of most enzyme rodents . It is not known, however, if the hamster cytochrome P450C17 (P450C17), a key enzyme for cortisol formation, also exhibits 17,20-lyase activity and if it catalyzes the formation of dehydroepiandrosterone (DHEA) at the adrenal level . To study this, we isolated the cDNA of P450C17 from a hamster adrenal library . This cDNA was sequenced and was found to have an open reading frame for a protein of 511 amino acids, as compared to the human P450C17, which contains 508 amino acids . The hamster P450C17 cDNA, in the coding region, is 76% homologous with the human P450C17 cDNA . The cDNA was then cloned in the expression vector pSV-SPORT 1, which was transiently transfected into COS 1 cells . The transfected cells were used for temporal studies on the transformation of radiolabeled C21-delta5- and C21-delta4-precursors . When transfected cells were incubated with {14C}pregnenolone, rapid formation of {14C}DHEA occurred . The intermediate 17alpha-hydroxypregnenolone accumulated initially with subsequent metabolism to DHEA . Likewise, when incubated with C21-delta4-steroids, {14C}progesterone and {3H}17alpha-hydroxyprogesterone, the 17,20-lyase product androstenedione was produced efficiently . In these studies, with respect to the delta5 pathway, the expressed hamster P450C17 gave similar results to bovine P450C17 cDNA inserted in the same expression vector . However, in contrast to the bovine enzyme, which converted low amounts of progesterone to androstenedione, the expressed hamster P450C17 enzyme showed an active metabolism via the delta4 pathway . Northern blot analysis, using the complete alpha-32P labeled hamster P450C17 cDNA as the probe, demonstrated a strong presence of P450C17 mRNA in hamster adrenals, a weaker presence in testes and ovaries, and no detectable species in brain, mesentery, and kidney . Immunoblotting analysis using an anti-rat P450C17 antibody demonstrated the presence of P450C17 protein in hamster adrenals, testes, and ovaries . Hamster adrenal cell suspensions and microsomal preparations were used to demonstrate the biosynthesis of {14C}17alpha-hydroxypregnenolone and {14C}DHEA from {14C}pregnenolone; both metabolites were formed during incubations . However, the ratio of {14C}DHEA/{14C}17alpha-hydroxypregnenolone was much lower in adrenal cells than in transfected COS 1 cells, indicating the presence of putative factors in hamster adrenal cells, favoring the 17alpha-hydroxylase activity rather than that of the 17,20-lyase . In conclusion, these studies demonstrate that the hamster adrenal is both a DHEA and a cortisol producer, and, therefore, this animal could be a suitable small animal model for the study of the role of DHEA in relation to human biochemistry and physiology.

Neuroendocrinology, 1997 Mar, 65(3), 200 - 9
Hormonal status and the neuroendocrine response to a novel heterotypic stressor involving subchronic noise exposure; van Raaij MT et al.; Despite a number of studies on noise-induced health effects, it is still unclear to what extent different neuroendocrine pathways are affected by noise exposure . Male Wistar rats were housed in sound-attenuated rooms isolated for noise from outside . Three groups of chronically cannulated rats were exposed to either background noise (+/-64 dB) only or irregular experimental white noise (90 dB, 2-22 kHz) . Two protocols, with approximately the same total amount of noise but with different densities, were used: protocol N1 (180 min random noise per day for 18 days) or protocol N2 (540 min random noise per day for 8 days) . Basal levels of circulating hormones (ACTH, corticosterone, prolactin and catecholamines) and plasma glucose were measured . In control animals, no significant changes in any of these parameters were observed over 18 days . Except for plasma prolactin, N1 did not induce a significant elevation in basal hormonal levels . N2 however induced significant elevation in basal prolactin, corticosterone and noradrenaline levels . At the end of the exposure period, all animals were subjected to a novel heterotypic stressor (restraint stress) to monitor differences in neuroendocrine activation (ACTH, corticosterone and prolactin) . Compared to nonexposed control animals, N1 animals showed a normal ACTH and an enhanced corticosterone response, whereas N2 animals showed an increased ACTH but a normal corticosterone response . The prolactin response of both N1 and N2 animals was significantly decreased . Adrenal cell suspension experiments revealed that in noise-exposed rats both basal- and ACTH-stimulated corticosterone production were significantly increased as compared to control animals . These results indicate that chronic noise exposure at mild intensities induces subtle but significant changes in hormonal regulation.

Photochem Photobiol, 1997 Mar, 65(3), 451 - 5
Comparison of methylene blue and methylene violet for photoinactivation of intracellular and extracellular virus in red cell suspensions; Skripchenko A et al.; Previous studies with methylene blue (MB) in red cell suspensions have demonstrated that extracellular, but not intracellular, virus can be readily photoinactivated . To test if the resistance of intracellular virus to inactivation is related to the permanent positive charge of the phenothiazine, a series of uncharged phenothiazine dyes, methylene violet (MV), monodemethylated MV and didemethylated MV, were studied . Values of the sensitivity of intracellular relative to extracellular vesicular stomatitis virus (VSV) inactivation for the three dyes (D10 extracellular/D10 intracellular) in buffer were 1.0, 0.60 and 0.33, respectively . In contrast, intracellular virus was resistant to inactivation with MB, with a D10 extracellular/D10 intracellular of 0.05 in buffer . Because virucidal activity of MV was inhibited by the presence of plasma, the red cells (30% hematocrit) were repeatedly washed prior to photoinactivation and storage . Under conditions where MB and MV inactivated approximately 5 log10 of extracellular VSV, intracellular VSV was inactivated by more than 4 log10 with MV compared to 0.88 log10 with MB . These phototreatment conditions did not significantly affect red cell morphology, extracellular pH, ATP or 2,3-diphosphoglycerol levels during 42 days of 1-6 degrees C storage . There was enhanced potassium efflux and hemolysis over values obtained from untreated control; the extent of change from controls was comparable for each phototreatment . These results indicate that the uncharged phenothiazine dye, MV, can inactivate both intracellular and extracellular virus yet exhibit similar in vitro red cell storage properties as MB phototreatment.

Blood, 1997 Mar 1, 89(5), 1708 - 15
Expression of matrix metalloproteinases and tissue inhibitors of metalloproteinases in reactive and neoplastic lymphoid cells; Stetler-Stevenson M et al.; We have studied the expression of gelatinase A, gelatinase B, interstitial collagenase, tissue inhibitor of metalloproteinase (TIMP)-1 and TIMP-2 in reactive lymphoid cells, as well as in a series of cell lines derived from neoplasms of B- and T-cell lineage . Using both Northern blot analysis and zymography, gelatinase B activity was detected by zymography in two Burkitt cell lines and in a tonsillar cell suspension, while gelatinase A and interstitial collagenase activities were not detected by either method . TIMP-1 expression was demonstrated by Northern blot analysis in the multipotential neoplastic K-562 cell line, the high grade Burkitt's B-cell lymphoma lines, isolated tonsillar B cells and at low levels in peripheral blood T cells, but was not expressed in any of the neoplastic T-cell lines or isolated peripheral blood B cells . In contrast, TIMP-2 expression was restricted to tissues containing cells of T-cell lineage with high levels being observed in the neoplastic T-cell lines and lower levels in normal peripheral blood T cells and hyperplastic tonsil . Expression of TIMP-1 and TIMP-2 was confirmed at the protein level by reverse zymography and immunofluorescence assays using antihuman TIMP polyclonal antibodies . Expression of gelatinase B by the high grade B-cell Burkitt's lymphoma cell lines is consistent with previous findings in large cell immunoblastic lymphomas and indicates that this enzyme may play an important role in high grade non-Hodgkin's lymphomas . TIMP expression correlated with cell lineage in that TIMP-1 was primarily observed in B cells and TIMP-2 was restricted to T cells.

J Neurooncol, 1997 Mar, 32(1), 29 - 38
Ex vivo expansion of tumor-draining lymph node cells using compounds which activate intracellular signal transduction . II . Cytokine production and in vivo efficacy of glioma-sensitized lymphocytes; Rice CD et al.; We have investigated the anti-tumor activity of ex vivo activated and expanded T cells which had been sensitized in vivo to one of two different syngeneic rat glioma cell lines; D74 or RT-2 . Rats were sensitized by inoculation of irradiated tumor cells into each hind foot pad . After 10 days, the tumor-draining lymph node (DLN) from each popliteal region was excised and prepared as a single cell suspension . Tumor-DLN lymphocytes were next activated overnight in RPMI-1640 medium containing 10% fetal bovine serum (FBS), Bryostatin-1 (5 nM), ionomycin (1 microM), and 20 U human recombinant interleukin-2 (IL-2) per ml . Culture for seven days in RPMI-1640 supplemented with FBS and IL-2 resulted in approximately 100-fold expansion of the lymphocyte population . Both D74- and RT-2-sensitized T cells constitutively secreted tumor necrosis factor-alpha, and both lymphocyte populations produced comparable amounts of the cytokine when co-cultured with either glioma cell line . Neither D74- and RT-2-sensitized effectors constitutively secreted gamma-interferon (gamma-IFN), but both populations produced gamma-IFN when exposed to either glioma cell line in vitro . D74-sensitized T cells released significantly more gamma-IFN than the RT-2 DLN lymphocytes . In vitro Chromium-release assays indicated that RT-2-sensitized T cells were more cytotoxic for RT-2 targets than for the D74 line and that D74-sensitized effectors were also more cytotoxic for RT-2 targets . To assess in vivo therapeutic efficacy, rats who had been inoculated intradermally with RT-2 cells three days earlier received an intravenous injection of RT-2- or D74-sensitized DLN cells (10(6) cells/gram body weight) expanded after activation with Bryostatin-1 and ionomycin or an equal number of lymphokine-activated killer (LAK) cells . Tumor diameters were measured daily and revealed that injection of glioma-sensitized lymphocytes led to the elimination of tumor while treatment with LAK cells had no therapeutic benefit . These results indicate, that at least for these two glioma lines, gamma-IFN release, rather than in vitro cytotoxicity, was a better predictor for in vivo immunotherapeutic efficacy of the glioma-sensitized, expanded T cells.

Cytometry, 1997 Mar 1, 27(3), 283 - 9
Multi-parameter flow cytometric analysis with detection of the Ki67-Ag in paraffin embedded mammary carcinomas; Leers MP et al.; In the present study we describe a novel multiparameter flow cytometric (FCM) assay to estimate the fraction of cycling cells in epithelial tumors derived from fresh frozen as well as archival material . To this end, MCF-7 cells as well as a series of breast carcinomas (n = 10; fresh frozen as well as formalin fixed and paraffin embedded) were stained using a panel of different antibodies directed against the Ki67-Ag (DAKO/PC, MIB-1, Ki-S5, and poly-Ki67) for a 3-parameter cytokeratin/Ki67-Ag/DNA FCM analysis . Whereas all Ki67-Ag antibodies work equally well in the methanol fixed cell line, MIB-1 and Ki-S5 epitopes are retained in cell suspensions mechanically derived from fresh frozen tissue . Only antibody Ki-S5 shows specific nucleolar staining patterns in cell suspensions prepared by trypsin digestion of formalin fixed, paraffin embedded tissue sections . A good correlation was found between the fractions of Ki67-Ag-positive epithelial cells measured in cell suspensions derived from fresh frozen and the corresponding formalin fixed and paraffin embedded tumor samples . Furthermore, the fraction of Ki67-Ag-positive epithelial cells as determined by 3-parameter FCM correlated very well with the Ki67-Ag-labeling index in paraffin embedded tissue sections.

Cytometry, 1997 Mar 1, 27(3), 262 - 8
Flow cytometric analysis of glucose transport by rat brain cells; Aller CB et al.; The fluorescent, non-metabolizable glucose analog 6-{N-(7-nitrobenz-2-oxa-1,3-diazol-4-yl)amino}-6-deoxyglucose (NBDG) was used to measure rates of hexose transport by dissociated brain cells from developing and adult rats . Flow cytometric analysis of glucose uptake and expression of glucose transporters was performed by mapping on size by granularity, which discriminated between neurons and astrocytes in a suspension of mixed brain cells . These mapped cell populations were identified by immunofluorescent staining with antisera to neuron-specific enolase (NSE) and glial fibrillary acidic protein (GFAP) . Specific uptake of the analog by membrane glucose transporters was confirmed by its inhibition by D-glucose and by cytochalasin B . Both neurons and astrocytes expressed the GLUT1 and GLUT3 transporter isoforms . This was confirmed by the additive inhibition of NBDG uptake by antibodies to these transporter isoforms in both cell types . The advantages of flow cytometric analysis of glucose transport include continuous monitoring over extremely short periods of time, increased precision of cell-by-cell flow cytometric measurements versus average uptake rates obtained with radioisotopes, and simultaneous analysis of uptake by different cell populations . Moreover, both uptake rates and the abundance of specific transporters can be determined directly and rapidly on the same cell suspension.

Arch Ital Urol Androl, 1997 Feb, 69 Suppl 1, 33 - 7
{Comparison of flow-cytometry and immunohistochemistry with proliferating monoclonal antibody . Cell nuclear antigen (PCNA) in the study of proliferative kinetics of renal carcinoma}; Villari D et al.; Tumor Proliferative Fraction (TPF) has been shown to correlate with prognosis in some malignancies . A reliable, accurate method for application in a clinical practice is still being sought . The aim of this study is to compare TPF as determined by Proliferating Cell Nuclear Antigen (PCNA) and Flow Cytometry (FC) in 36 consecutive patients affected by Renal Cell Carcinoma (RCC) . Proliferating cells were identified in paraffined sections using a anti-PCNA monoclonal antibody (PC 10 Dako) . Cell suspension for FC were prepared from fresh/frozen samples DNA index and S phase were evaluated using a computerized program (Multicycle, Phoenix) . 16 samples (47.1%) were found to be aneuploid by FC (DI range 0.72-2.40) . Aneuploid vs diploid tumors had significantly higher mean FC-S phase (p = 0.049) and PCNA LI (p = 0.034) . Weak correlation (r-Spearman 0.416 p = 0.01) was found between PCNA LI and grading and near to significativity between PCNA LI and tumor size (r = 0.335 p = 0.0061) . When patients are classified according to nuclear grading, is evident that all PCNA G4 are aneuploid and that 62.5% of PCNA G1 are diploid . A week correlation near to significativity is found between PCNA LI and S phase only in the aneuploid tumors . A more reliable measurement of TPF in RCC could be provided by combining the two methods . Further research on larger series is needed.

Am J Physiol, 1997 Feb, 272(2 Pt 2), H1020 - 32
Contribution of red blood cell aggregation to venous vascular resistance in skeletal muscle; Cabel M et al.; The effects of red blood cell aggregation on venous vascular resistance and conductance were examined in the cat lateral gastrocnemius muscle . During perfusion with blood of normal hematocrit, venous conductance fell linearly by 41% when blood flow was reduced from 5 to 1 ml x min(-1) x 100 g tissue(-1) and increased linearly by 155% when flow was increased from 5 to 20 ml x min(-1) x 100 g tissue(-1) . This effect was not seen when the muscle was perfused with an acellular solution of 12% Dextran 40 in Ringer solution and was greatly reduced or absent with a nonaggregating suspension of red blood cells in Ringer solution + Dextran 40 . Also, the venous vascular conductance at a control flow of 5 ml x min(-1) x 100 g tissue(-1) during perfusion with the nonaggregating red blood cell suspension was twice that with normal blood of the same hematocrit . The effect of flow on venous conductance was significantly reduced when red blood cell aggregation was increased by addition of Dextran 250 to the blood (200 mg/kg body wt) and was also reduced in animals with systemic hematocrit >50% . These findings suggest that red blood cell aggregation contributes importantly to venous vascular resistance in resting muscle.

Mech Ageing Dev, 1997 Feb, 93(1-3), 15 - 24
IL-6, DHEA and the ageing process; James K et al.; The age-related increase in circulating IL-6 levels in humans which has been attributed to a decline in DHEA production by the adrenal gland is currently attracting attention because of its possible relevance to the aetiology and management of a number of age-related clinical disorders . The potential importance of these observations and suggestions has prompted us to perform more detailed studies on the relationship between IL-6 and DHEA . Using immunoassay techniques we have found in normal healthy individuals over the age of 40 an inverse relationship between plasma DHEA levels and the presence of detectable levels of IL-6 (more than 1 pg/ml) . In vitro, studies also revealed that low dose (10(-6)-10(-8) M) of DHEA and DHEAS inhibited the production of IL-6 in unstimulated human spleen cell suspension cultures whilst enhancing its release by explant cultures of the same tissue . In contrast they had no effect on immunoglobulin production . These studies suggest that there is a real, but complex relationship between IL-6 production and DHEA levels which warrants further investigation.

Kansenshogaku Zasshi, 1997 Feb, 71(2), 116 - 24
{Determination of cutoff value of serum anti-Legionella antibody titer--microplate agglutination test (MPAT)}; Yabuuchi E et al.; In order to promote the serological diagnosis of legionellosis in the clinical laboratory, the cutoff values of serum anti-Legionella antibody titers for microplate agglutination (MPAT) test were determined . Antibody levels were tested for 178 serum specimens including 98 healthy persons, 22 ordinally workers having either metabolic or renal failure, definitely diagnosed patients of 17 mycoplasmal and 9 chlamydial pneumonia, 32 patients of other bacterial pneumonia . Heat killed unstained cell suspension of each strain of Legionella pneumophila serogroup (SG) 1a, 1b, SGs 2 to 6, L . bozemanii, L . dumoffii, L . gormanii, and L . micdadei were used as antigens . Strains of L . pneumophila SG 1b were mainly isolated from environmental specimens . However, in some Legionella pneumonia cases, etiologic agents were determined as L . pneumophila SG 1 b . Thus the representative strain of SG 1b was used as an antigen for the determination of the patient's antibody titer . Quantitative agglutination was performed by using a 96-well U-bottom microplate for each antigen . Test sera were diluted from 1:16 to 1:256 . Results were read after 20 h at 25 degrees C . Cutoff values for 11 antigens were determined, at this moment, as 4-fold or greater increase in level to > or = 1:128 in paired sera, and > or = 1:256 in single serum . However, final diagnosis should be given by over-all coordination of serological results and clinical symptoms together with other laboratory findings . Two culture-positive Legionella pneumonia cases due to either SG3 or 6 in which significant rise of serum antibody titers against organisms of corresponding SG estimated by MPAT method were discussed.

Int J Dev Biol, 1997 Feb, 41(1), 111 - 22
Transplantation of testis germinal cells into mouse seminiferous tubules; Ogawa T et al.; In the adult male, germ cell differentiation takes place in the seminiferous tubules of the testis by a complex, highly organized and very efficient process . A population of diploid stem-cell spermatogonia that lie on the basement membrane of the tubule continuously undergoes self-renewal and produces progeny cells, which initiate the process of cellular differentiation to generate mature spermatozoa . Each testis contains many seminiferous tubules, which are connected at both ends to a collecting system called the rete testis . The mature spermatozoa pass from the tubules into the rete and are then carried through efferent ducts to the epididymis for final maturation before they are ready to fertilize an egg . In previous studies, we have demonstrated that donor testis cells collected from a fertile mouse are able to generate spermatogenesis when transplanted to the seminiferous tubules of an infertile male . The spermatozoa produced by the recipient from the donor-derived spermatogonial stem cells are able to fertilize eggs and produce progeny carrying the donor male haplotype . Furthermore, donor testis stem cells from a rat will generate normal rat spermatozoa following transplantation to a mouse testis . The spermatogonial transplantation technique is clearly valuable and applicable to many species, but it is difficult . Therefore, several procedures to introduce donor cells into the seminiferous tubules of a recipient have been developed using the mouse as a model, and they are described here in detail . The results indicate that microinjection of cell suspensions into the seminiferous tubules, efferent ducts or rete testis are equally effective in generating donor cell-derived spermatogenesis in recipients . Each approach is likely to be useful for different experimental purposes in a variety of species.

Diagn Cytopathol, 1997 Feb, 16(2), 126 - 31
Ex vivo fine-needle aspiration cytology and flow cytometric phenotyping in the diagnosis of lymphoproliferative disorders: a proposed algorithm for maximum resource utilization; Saddik M et al.; Surface marker characterization of lymphoproliferative disorders is an essential component in the diagnostic work-up of these lesions . Immunohistochemical surface marker analysis (SMA) is somewhat costly, fixation-dependent, and difficult to objectively quantitate . Two-color flow cytometric (TCFCM) SMA allows for more quantitative dual marker analysis of a wide range of surface antigens, and is less expensive . Ex vivo fine-needle aspiration (xvFNA) has been reliably used for FCM DNA analysis . The procedure has also been used to harvest tumor cells for xenotransplantation . In this study, we attempted to test the reliability of material obtained by xvFNA for SMA . We also designed an algorithm initiated by cytological assessment of the xvFNA smears in order to tailor the panel of antibodies required for TCFCM SMA of the aspirates . We performed 20 xvFNAs on freshly resected specimens from 19 patients with suspected lymphoproliferative disorders . The specimens included 12 lymph node biopsies, seven splenectomies, and one breast biopsy . There were 10 male and nine female patients with a median age of 58 yr . The aspirate cell suspensions were examined by FCM within 24 hr of harvesting . The number of markers used ranged from four to 14 with an average of eight . The diagnoses included non-Hodgkin's lymphoma (n = 5), lymphocytic leukemia (n = 5), reactive lymphoid hyperplasia (n = 8), and Hodgkin's disease (n = 1) . Combining cytological assessment of the xvFNA smears and TCFCM SMA, the diagnosis was reached prior to histopathologic examination in 17 cases (90%) . The two remaining cases showed a reactive pattern on cytology and a polyclonal FCM SMA profile, and the diagnosis of sarcoidosis and toxoplasmosis was made on histological examination . Our study suggests that xvFNA provides adequate material for TCFCM SMA . An algorithm combining xvFNA cytology, FCM SMA, and histological examination is appropriate for the diagnosis of lymphoproliferative disorders in most instances with maximal resource utilization and minimal expense.

Antonie Van Leeuwenhoek, 1997 Feb, 71(1-2), 137 - 41
Molecular and biochemical basis of the interaction between tomato and its fungal pathogen Cladosporium fulvum; de Wit PJ et al.; The interaction between the biotrophic fungal pathogen Cladosporium fulvum and tomato complies with the gene-for-gene model . Resistance, expressed as a hypersensitive response (HR) followed by other defence responses, is based on recognition of products of avirulence genes from C . fulvum (race-specific elicitors) by receptors (putative products of resistance genes) in the host plant tomato . The AVR9 elicitor is a 28 amino acid (aa) peptide and the AVR4 elicitor a 106 aa peptide which both induce HR in tomato plants carrying the complementary resistance genes Cf9 and Cf4, respectively . The 3-D structure of the AVR9 peptide, as determined by 1H NMR, revealed that AVR9 belongs to a family of peptides with a cystine knot motif . This motif occurs in channel blockers, peptidase inhibitors and growth factors . The Cf9 resistance gene encodes a membrane-anchored extracellular glycoprotein which contains leucine-rich repeats (LRRs) . 125I labeled AVR9 peptide shows the same affinity for plasma membranes of Cf9+ and Cf9- tomato leaves . Membranes of solanaceous plants tested so far all contain homologs of the Cf9 gene and show similar affinities for AVR9 . It is assumed that for induction of HR, at least two plant proteins (presumably CF9 and one of his homologs) interact directly or indirectly with the AVR9 peptide which possibly initiates modulation and dimerisation of the receptor, and activation of various other proteins involved in downstream events eventually leading to HR . We have created several mutants of the Avr9 gene, expressed them in the potato virus X (PVX) expression system and tested their biological activity on Cf9 genotypes of tomato . A positive correlation was observed between the biological activity of the mutant AVR9 peptides and their affinity for tomato plasma membranes . Recent results on structure and biological activity of AVR4 peptides encoded by avirulent and virulent alleles of the Avr4 gene (based on expression studies in PVX) are also discussed as well as early defence responses induced by elicitors in tomato leaves and tomato cell suspensions.

Eur J Immunol, 1997 Feb, 27(2), 442 - 8
High and low doses of haptens dictate whether dermal or epidermal antigen-presenting cells promote contact hypersensitivity; Bacci S et al.; In the induction of contact hypersensitivity (CH) to an epicutaneously applied hapten, we have previously proposed that low doses of hapten sensitize primarily through epidermal Langerhans' cells (LC), whereas high doses rely largely on dermal antigen-presenting cells (APC) . To examine this issue further, we applied either high or low doses of dinitrofluorobenzene (DNFB) epicutaneously to mice . We observed reduced LC density at the site after 12 h (nadir), which returned to normal levels at 24 h only after a low dose of hapten . When a low dose of an unrelated hapten, oxazolone, was painted on skin that had been painted 12 h previously with high dose of DNFB, oxazolone-specific CH was impaired . When grafts of whole skin, dermis alone, and epidermis alone prepared from skin painted 2 h previously with low or high doses of DNFB were placed onto naive, syngeneic mice, CH was induced by whole skin after both types of doses, by epidermis only after a low dose, and by dermis only after high dose . When epidermal cell suspensions were derivatized in vitro with low or high doses of DNFB, only cells exposed to a low dose induced proliferation of hapten-specific Tcells . Thus, only a low dose of hapten reveals the APC functions of LC without the participation of dermal APC.

Br J Haematol, 1997 Feb, 96(2), 403 - 11
Persistence of residual tumour cells after cytokine-mediated ex vivo expansion of mobilized CD34+ blood cells in multiple myeloma; Van Riet I et al.; Mobilized CD34+ blood cells were immunomagnetically enriched from leukapheresis products in five multiple myeloma (MM) patients . Thawed samples of selected CD34+ cells were cultured for up to 21 d in a liquid and stroma-free culture system with different combinations of recombinant cytokines . The most successful cell expansion was obtained when a combination of rh-IL-1beta, rh-IL-3, rh-IL-6, rh-SCF, rh-G-CSF and rh-GM-CSF was used . After 14 d this mixture gave a 120-187-fold overall increase of total nuclear cells and a 4-8-fold overall increase of early CFU-GM numbers . In four patients a very sensitive patient-specific PCR analysis showed the presence of monoclonal cells in the initial leukapheresis products . After immunomagnetic separation a tumour cell depletion of 2-4 logs was observed, although all samples still contained malignant cells . Cell suspensions that were cultured with the most potent cytokine combination showed tumour contamination in two-thirds of evaluable cases at the moment of maximal CFU-GM output . Serial cDNA dilution experiments indicated that the positive PCR results at day 14 reflected the persistence of pre-culture tumour cells rather than in vitro expansion of tumour cells in two cases . This study demonstrates that ex vivo expansion of myeloid precursor cells from mobilized CD34+ cells in MM patients does not always result in an effective purging of residual tumour cells . On the other hand, our culture conditions do not seem to favour in vitro expansion of malignant cells, despite the use of a cytokine cocktail that includes potential myeloma growth factors.

Anal Biochem, 1997 Feb 1, 245(1), 55 - 60
Determination of catalase activity at physiological hydrogen peroxide concentrations; Mueller S et al.; A method for the determination of catalase activity (EC 1.11.1.6.) in homogenates and cell suspensions is described by following the decomposition of H2O2 at physiological H2O2 levels . This first chemiluminescence assay for catalase activity is based on the reaction of luminol (5-amino-2,3-dihydro-1,4-phthalazinedione) and NaOCl . The chemiluminescence of this reaction specifically depends on the H2O2 concentration and shows fast kinetics of less than 2 s . Using a flow technique, the exponential decay of H2O2 in the presence of catalase is followed down to 10(-8) M H2O2 at pH 7.4 over three orders of magnitude . At these very low H2O2 concentrations neither oxygen is liberated in gaseous form nor enzyme inactivation or loss of cell viability is observed . Addition of the catalase inhibitor NaN3 completely inhibits H2O2 decomposition . Since the method is not influenced by sulfhydryl and amino group containing compounds, it is especially suited for crude tissue homogenates and suspensions of intact cells . Interestingly, application to cell suspensions shows that intact human erythrocytes and rat hepatocytes exhibit only 5.8 and 1.9% of catalase activity when compared to homogenized cells . These data suggest that the diffusion of H2O2 through membranes is lower than that assumed so far.

Exp Cell Res, 1997 Feb 1, 230(2), 337 - 41
Hepoxilin-evoked intracellular reorganization of calcium in human neutrophils: a confocal microscopy study; Mills L et al.; Hepoxilin A3 has previously been shown to cause a rapid dose-dependent rise in intracellular calcium in intact human neutrophils in suspension . Two components have been observed, an initial rapid phase of intracellular calcium rise, followed by a slow decline to plateau levels that remain above the original baseline calcium levels . These changes have been suggested to involve the release of calcium from intracellular stores in the ER (initial rapid phase), while the slower rate of decline (plateau phase) was presumed to be due to calcium influx as it was abolished in zero calcium extracellular medium . The present study used confocal microscopy to examine the response to hepoxilin A3 at the subcellular level . Our results show that calcium dynamics in response to hepoxilin A3 varies in different subcellular compartments within the cell and that hepoxilin A3 evoked a persistent accumulation of calcium in organelles . The hepoxilin-evoked calcium sequestration was eliminated by prior exposure to CCCP, a mitochondrial uncoupler . CCCP also eliminated the plateau phase of the calcium response in cell suspension, suggesting that this phase was due to mitochondrial accumulation of calcium rather than calcium influx . Experiments with DiI-loaded cells, a membrane marker, showed that the nuclear calcium was not elevated by hepoxilin addition to the cells . These results demonstrate that hepoxilins evoke the release of calcium from the ER which is taken up by the mitochondria where it is tightly sequestered . These results offer an explanation of observations previously made with cell suspensions in which hepoxilin A3 was shown to inhibit the calcium mobilizing effects of chemotactic agents.

Gastroenterology, 1997 Feb, 112(2), 429 - 36
Parenteral nutrition modifies glucose and glutamine metabolism in rat isolated enterocytes; Colomb V et al.; BACKGROUND & AIMS: After small bowel resection, parenteral nutrition is often required to provide energy and nitrogen supplies and also to stimulate intestinal adaptation, despite the absence of glutamine in formulas . The aim of this study was to investigate the effect of nutrient supply route on fuel utilization by enterocytes . METHODS: Rats received an intravenous or intragastric continuous infusion of an all-in-one glutamine-free formula . Sham-operated control rats were orally fed and received the same protein-caloric supplies as the other two groups . On day 7, the rats were killed in the fed state, blood samples were collected, and the jejunoileum was removed . Enterocytes were isolated . Aliquots of cell suspensions were incubated (30 minutes at 37 degrees C) in the presence of {14C}glucose and {14C}glutamine (2 mmol/L) . Substrate utilization was determined by measuring metabolites and CO2 generated . RESULTS: Intravenously fed rats showed mild hyperglycemia and marked hyperinsulinemia . Plasma glutamine levels were similar in the three groups . Intravenously fed rats showed a simultaneous increase in glutamine utilization and a decrease in glucose utilization compared with intragastrically fed and control rats, without parallel changes in glutaminase and hexokinase activities . The basolateral glucose transporter protein concentration was reduced in intravenously fed rat enterocytes . CONCLUSIONS: The route of nutrient delivery influences fuel utilization by enterocytes.

Cytometry, 1997 Feb 1, 27(2), 179 - 88
Trivariate flow cytometric analysis of paraffin-embedded lung cancer specimens: application of cytokeratin subtype specific antibodies to distinguish between differentiation pathways; Leers MP et al.; The aim of the present study was to investigate whether trivariate FCM analysis, for the simultaneous detection of two different CK subtypes in combination with DNA content, can be applied to paraffin embedded samples of different types of non-small cell lung cancer in order to evaluate the cell cycle of individual sublines . Single cell suspensions were prepared from 50 microm thick paraffin sections of 22 lung carcinomas by pepsin digestion and immunostained with CK-antibodies which were chosen to distinguish glandular differentiation (adenocarcinomas) and squamous differentiation . There was a good correlation between the immunocytochemical results of the different CK antibodies in tissue sections and in the corresponding single cell suspensions . Gating for CK-positivity revealed a higher S-phase fraction as compared to the ungated cell population . The tumor cells in adenocarcinoma cases were specifically recognized by CK7 antibodies, while well-differentiated squamous cell carcinomas were specifically stained for CK14 and/or CK17 . In poorly differentiated squamous cell carcinomas simultaneous expression of CK7 and CK17 was detected in a subpopulation of the tumor cells, next to cells positive for CK7 or CK17 alone . The trivariate FCM analysis allowed the separate estimation of ploidy status and cell cycle parameters in the three different cell populations of these, apparently (phenotypically) heterogeneous, malignancies.

Cytometry, 1997 Feb 1, 27(2), 161 - 8
Three-parameter flow cytometric analysis of rat spermatogenesis; Suter L et al.; Mammalian spermatogenesis is a complex process which is not yet fully understood . In this paper we describe the analysis of rat spermatogenesis by means of 3-parameter flow cytometry . Since the analysis of DNA content only provides sufficient information for the identification of 4 cell populations, additional parameters were combined with propidium iodide (PI) staining . Immunostaining of the intermediate filament vimentin allowed the identification of somatic (vimentin positive) and germ (vimentin negative) cells . Utilizing the combination of DNA and vimentin staining, we have been able to quantitate the somatic cells present in a testicular cell suspension and to analyze somatic and germinal cells separately . Furthermore, the addition of mitochondrial staining with the fluorochrome nonyl acridine orange (NAO) allowed several cell subpopulations within each ploidy group to be distinguished . After 3-color staining and subsequent cell sorting, 11 testicular cell subpopulations could be identified: somatic cells, and 10 subtypes of germinal cells . The method described in this paper represents a valuable tool for the evaluation of spermatogenesis in both normal and perturbed situations.

J Virol, 1997 Feb, 71(2), 1567 - 75
Multiple widely spaced elements determine the efficiency with which a distal cistron is expressed from the polycistronic pregenomic RNA of figwort mosaic caulimovirus; Edskes HK et al.; The polycistronic expression mechanism of the plant pararetrovirus figwort mosaic caulimovirus (FMV) depends upon cis-acting elements present in its pregenomic RNA and a trans-acting protein (P6) which is expressed from a monocistronic subgenomic RNA . Using transient expression of FMV-derived polycistronic reporter constructs in Nicotiana edwardsonii cell suspension protoplasts, we further analyzed the cis-acting elements involved in polycistronic expression . A cis-acting element located within the first 74 nucleotides of the 7,954-nucleotide pregenomic RNA appears to be essential for P6 to transactivate expression of an internal cistron . Expression of this internal cistron, in the presence of P6, is greatly enhanced by the combined presence of two cis-acting elements located at the 3' end of the polycistronic RNA . Surprisingly, deletion of the most upstream of these two 3' cis-acting elements exposed a negative-acting element located internally on the polycistronic RNA, at the 3' end of open reading frame I . The action of both this negative-acting internal element and the positive-acting 3' elements is more pronounced when the large 5' untranslated leader region is present . This indicates that the 5' untranslated leader region is central to regulation of the FMV gene expression mechanism . Although a limited set of elements suffices to direct polycistronic expression in this eukaryotic system, a complex interplay between elements is involved in the spatial regulation of the genes present on the pregenomic RNA of FMV.

FEBS Lett, 1997 Jan 27, 402(1), 21 - 4
Expression of the cell-adhesion molecule VCAM-1 by stromal cells is necessary for osteoclastogenesis; Feuerbach D et al.; Osteoblastic cells have been shown to be involved in osteoclast formation through cell to cell contacts . This study was designed to examine the possible function of vascular cell adhesion molecule 1 (VCAM-1) during osteoclastogenesis . As a source for stromal cells we used the recently established mouse bone marrow stromal cell line mBMS-B1 which has the ability to support osteoclastogenesis when used in co-culture with a crude spleen cell suspension . mBMS-B1 cells express a single approximately 3.9 kb VCAM-1 mRNA species . Expression was low under basal culture conditions and a 5-10-fold increase was observed in the presence of 1,25(OH)2D3 . Cell surface expression of VCAM-1 examined by FACS analysis was increased about 2-fold after 1,25(OH)2D3 treatment . Immunoprecipitation of cell surface expressed VCAM-1 or total VCAM-1 protein using the anti-VCAM-1 monoclonal antibody MK2.7 resulted in a single approximately 110 kDa protein on SDS-PAGE . Induction by 1,25(OH)2D3 was about 2-5-fold on day 3 . The stromal cell-osteoclast precursor cell interaction was investigated in a co-culture of the mBMS-B1 and mouse spleen cells in the presence of 1,25(OH)2D3 . The monoclonal antibody MK2.7 which is known to block hemopoietic-stromal cell recognition inhibited the formation of osteoclasts when added to the co-culture at day 2 but not day 4 . These data suggest that VCAM-1 is involved in the interaction between stromal cells and osteoclastic precursor cells during osteoclastogenesis presumably most important during early stages of the formation of osteoclasts.

Int J Cancer, 1997 Jan 17, 70(2), 201 - 7
Platelets mediate tumor cell adhesion to the subendothelium under flow conditions: involvement of platelet GPIIb-IIIa and tumor cell alpha(v) integrins; Dardik R et al.; The aim of our study was to explore the role of platelets and their specific integrin receptors in mediating the interaction of 4 human tumor cell lines (3 melanoma and 1 carcinoma) with the extracellular matrix (ECM) under static and arterial flow conditions . Under static conditions, all 4 cell lines adhered to the ECM . The adhesion capacity of all 4 cell lines was virtually abolished by application of flow during incubation with the ECM . Under static conditions, tumor cell adhesion was not affected by adding platelets to the cell suspension and was slightly reduced by pre-coating the ECM with platelets prior to the addition of tumor cells . In contrast, under flow conditions, platelets significantly increased tumor cell adhesion to the ECM, the enhancing effect being more pronounced when platelets were pre-incubated with the ECM prior to the addition of tumor cells than when incubated simultaneously with the cells . Platelet-mediated tumor cell adhesion under flow was markedly inhibited by blockade of the platelet GPIIb-IIIa or of the tumor cell alpha(v) integrins . Platelets of a Glanzmann thrombastenia (GT) patient were unable to support tumor cell adhesion to the ECM under flow . Our results suggest that the interaction of tumor cells with subendothelium-bound platelets under flow conditions is mediated by platelet GPIIb-IIIa and by tumor cell alpha(v) integrins independently of the nature of the beta subunit.

Toxicol Lett, 1997 Jan 15, 90(1), 61 - 6
Effect of ozone on metabolic activities of Escherichia coli K-12; Komanapalli IR et al.; Escherichia coli K-12 cell suspensions in buffer were exposed to ozone at a concentration of 600 ppm . Measurements were made of cell viability, glyceraldehyde-3-phosphate dehydrogenase, malate dehydrogenase, lactate dehydrogenase, glutathione disulfide reductase, nonprotein sulfhydryl and total sulfhydryl compounds . Cell viability was not affected when E . coli K-12 was exposed to ozone for less than 10 minutes . The most sensitive parameter was glyceraldehyde-3-phosphate dehydrogenase followed by nonprotein sulfhydryl and total sulfhydryl compounds . Effects on malate dehydrogenase, lactate dehydrogenase and glutathione disulfide reductase were negligible . Cell survival and induction of lipid oxidation were also determined using two strains of E . coli K-12 (rec A, deficient in DNA repair and wild-type) . The extent of membrane lipid oxidation correlated with cell viability in a dose-dependent manner and the survival curves of both strains showed similar sensitivity to ozone . The data suggest that the sulfhydryl group in the membrane is the primary target of ozone attack . Rec A DNA repair system does not appear to play a role in ozone resistance.

Arch Biochem Biophys, 1997 Jan 15, 337(2), 185 - 90
Taxol production and taxadiene synthase activity in Taxus canadensis cell suspension cultures; Hezari M et al.; The cyclization of geranylgeranyl diphosphate to taxa-4(5),11(12)-diene represents the first committed, and a slow, step in the complex biosynthetic pathway leading to the anticancer drug Taxol . The cyclization enzyme, taxadiene synthase, has been previously purified from Pacific yew (Taxus brevifolia) stem and characterized, and the corresponding cDNA has been isolated . To better assess the role of taxadiene synthase in the control of pathway flux in Canadian yew (T . canadensis) cells, a reliable system for production of Taxol in suspension culture, the enzyme from this source was isolated and shown to be chromatographically, electrophoretically, and kinetically identical to that of T . brevifolia stem . Results from the analysis of enzyme activity levels during the time course of Taxol accumulation in developing cell cultures of T . canadensis indicate that rate-limiting transformations lay farther down the pathway than the cyclization step in this system.

Folia Neuropathol, 1997, 35(2), 87 - 93
Development of human fetal substantia nigra grafts in the brain of non-immunosuppressed rats; Bystron I et al.; The survival of xenogenous tissue after transplantation to the brain of Wistar rats without immunosuppression was studied . Cell suspension was prepared from the ventral mesencephalon of human embryos 7-8 weeks of gestation, and injected either into the striatum or motor cortex of adult rats . After 1, 3, 7, 14 days and 1, 3, 6, 8 months the rats were sacrificed and the brains were processed for tyrosine hydroxylase (TH), glial fibrillary acidic protein (GFAP), ferritin immunocytochemistry and electron microscopy study . The intensity of inflammatory reaction of the host brain strongly affected the viability of grafted cells, the extend of GFAP and ferritin reactions in the tissue around the graft . Grafted human TH-positive neurons were found for 3 month survival only . After transplantation, we observed the grafted cell differentiation similar to that in normally developing mesencephalon . Six and eight months after transplantation glial cells prevailed in the grafts, while most neurons looked abnormal . An extensive bundles of myelinated fibers transpassing the intracortical transplants were seen . We are concluding that human mesencephalic cells can survive and develop in the brain of non-immunosuppressed rats.

Eur Surg Res, 1997, 29(4), 292 - 302
Processing of tumor tissues for vaccination with autologous tumor cells; Lahn M et al.; Vaccination with gene-transfected tumor cells has recently been proposed as a new strategy in the immunotherapy of cancer . Since autologous tumor cells provide an optimal antigen profile, the possibility of generating single cell suspensions from renal cell carcinoma (RCC), malignant melanoma (MM), colon carcinoma (CC), and non-small-cell lung cancer (NSCLC) biopsies was investigated . One hundred and seventy-four tumor biopsies were processed by mechanic and enzymatic dissociation, yielding 1-2 x 10(6) cells/g tumor (median), irrespective of tumor type . Primary tumor cell cultures (PTCC) of > or = 10(7) cells were established from 29 of 86 (34%) RCC, 14 of 38 (37%) MM, 11 of 23 (48%) NSCLC and 4 of 27 (15%) CC specimens . The amount of non-tumor cells, as assessed by morphology and immunocytology, was generally low (< 30%) in RCC (35 of 41) and MM (11 of 17), while it exceeded 60% in 8 of 11 PTCC from NSCLC and 3 of 11 CC . A high tumor cell yield was obtained in biopsies with a high degree of vascularization and in the virtual absence of necrosis . Thus, PTCC > or = 10(7) cells were obtained in 73% of MM with a high degree of vascularization and in 22% of MM with a low degree of vascularization (p < 0.007) . Long-term tumor cell cultures exceeding 20 passages were established in 24 of 86 (18%) RCC, 7 of 38 (18%) MM and 3 of 27 (11%) CC, while successful implantation in nude mice was achieved in 8 of 20 RCC and 5 of 10 MM . Thus, under the conditions described, > or = 10(7) primary tumor cells of high purity could be generated from about one third of RCC and MM biopsies, while the success rate increased to > 50 and > 70%, respectively, in samples with a high degree of vascularization generated by an optimized biopsy technique excluding necrotic parts.

Ultrasound Med Biol, 1997, 23(5), 793 - 6
Acoustic cavitation nuclei survive the apparent ultrasonic destruction of Albunex microspheres; Brayman AA et al.; The hypothesis tested was that gas bodies capable of nucleating violent cavitation activity in vitro would survive the rapid disruption of Albunex microspheres by 1-MHz ultrasound . Human erythrocyte hemolysis was used as a proxy measure of cavitation . Fluid (5% human serum albumin {HSA}) with or without Albunex (ALX) was exposed or sham-exposed to 1-MHz ultrasound (P+ = 1.25 +/- 0.01 MPa, P- = 0.81 +/- 0.01 MPa; ISPTP approximately 35 W/cm2) for 60 s using 10-microseconds pulses and a duty factor of 0.5 . An equal volume of whole human blood was then added to the fluid, followed by a second 60-s treatment . Insonation of cell suspensions prepared in previously sham-exposed HSA + ALX fluid produced about 4% hemolysis, a level significantly greater than in the controls . Insonation of cell suspensions prepared in previously insonated HSA + ALX fluid produced about 0.4% hemolysis; this also differed significantly from the controls . The data thus support the hypothesis.

Brain Res Bull, 1997, 43(4), 381 - 92
Effects of combined ventral forebrain grafts to neocortex and amygdala on behavior of rats with damage to the nucleus basalis magnocellularis; Shoham S et al.; In rats with damage to the nucleus basalis magnocellularis, transplantation of the embryonic ventral forebrain to the neocortex improves behavioral performance in some behavioral tasks . The present investigation focuses on improvement of behavioral performance by combined graft placement to both neocortex and amygdala . Male rats received unilateral microinjections of quisqualate to the nucleus basalis magnocellularis to produce cell damage . Embryonic ventral forebrain cell suspensions were placed in one group of rats in the frontal and parietal neocortex, in a second group in the amygdala, and in a third group in the frontal and parietal neocortex and in the amygdala . These groups were compared to a group of nonoperated rats and a group of rats with damage but with no grafts . Quisqualate-induced damage to the nucleus basalis magnocellularis reduced cholinergic innervation in the ipsilateral cortical hemisphere, impaired performance in the one-trial training version of passive avoidance, an increased motility and time spent in the open arms of the elevated plus maze . Combined graft placement to neocortex and amygdala normalized performance of passive avoidance and restored the normal time spent in the open arms of an elevated plus maze . These results suggest that after damage to the nucleus basalis magnocellularis, modulation of function in multiple brain regions may be necessary for optimization of adaptive behavior in situations involving induction of fear.

Biofizika, 1997 Jan-Feb, 42(1), 191 - 5
{Death of cells from mouse peritoneal exudate induced by products of reaction of Fe2+ ions with previously photo-oxidized psoralen}; Kiagova AA et al.; Toxic effect of previously photooxidized in water psoralen on survival of mouse peritoneal exudate cells was investigated . Cell survival was determined by trypan exclusion test . It was shown that photooxidized psoralen itself did not induce trypan-positive cells while decrease in cell survival was observed in the case of simultaneous addition of photooxidized psoralen and Fe2+ ions to cell suspension . To evaluate the quantity of psoralen photoproducts which react with Fe2+ photooxidized psoralen Fe2+ mixtures were titrated by different cell concentrations . Then parameter c50 (cell concentration such that 50% trypan-positive cells were observed) was determined . It was shown that c50 increased with the increase in preirradiation fluence of psoralen from 4.5 to 45 kJ/m2 . But further increase in preirradiation fluence resulted in decrease of c50 . The photodestruction of psoralen photoproducts which react with Fe2+ ions was proposed . Simultaneous addition of photooxidized psoralen, Fe2+ ions and antioxidant butylated hydroxytoluene significantly increased cell survival . That indicated free radical nature of reaction products of photooxidized psoralen with Fe2+ ions.

Biomed Mater Eng, 1997, 7(1), 49 - 58
Viable bone formation in porous hydroxyapatite: marrow cell-derived in vitro bone on the surface of ceramics; Yoshikawa T et al.; With the aim of in vitro production of bone fragments more closely resembling autogenous bone, rat cultured bone marrow cells were combined with porous hydroxyapatite (HA) discs and cultured in the presence of dexamethasone (Dex) . Bone marrow cells were collected from the femoral diaphyses of a 7-week-old male Wistar rat, and primary culture was performed for six days . Then, cell suspensions were prepared by trypsin treatment, and combined with the porous HA discs . After a 2-hour incubation, the composites were additionally cultured for up to 4 weeks (subculture) in the presence of Dex . In a control group, the subculture was performed without Dex . After 1, 2, 3 and 4 weeks of subculturing, the HA discs were removed, and alkaline phosphatase (ALP) activity, bone Gla protein (BGP), and DNA were quantitated . A portion of the disc was prepared for scanning electron microscopy (SEM), from which bone formation was evaluated morphologically . ALP activity peaked at 2-3 weeks and decreased at 4 weeks . BGP levels began to increase at 3 weeks . In the SEM study, mineralized collagenous extracellular matrix was noted at 3 and 4 weeks . In the control group, neither significant ALP activity nor increased BGP was detected . These biochemical and morphological results suggest that with the culture technique, active bone formation in the pore regions of HA can be fabricated in vitro . It is anticipated that when composites are subcultured in this way they will function as a bone graft with properties similar to those of autogenous bone.

Radiat Med, 1997 Jan-Feb, 15(1), 37 - 43
Determination and drug modification assessed by micronucleus frequency assay of potentially lethal damage repair in quiescent cell populations within murine solid tumors; Masunaga S et al.; We investigated potentially lethal damage repair (PLDR) by quiescent (Q) tumor cells in vivo . SCC VII tumor-bearing C3H/He mice were irradiated with 60Co gamma-rays after being given 10 injections of 5-bromo-2'-deoxyuridine (BrdU) to label all proliferating (P) cells in their tumors, and the tumors were than excised and trypsinized . The tumor cell suspensions thus obtained were incubated with cytochalasin-B (Cyt-B, a cytokinesis blocker), and the micronucleus (MN) frequency in cells without BrdU labeling was determined using immunofluorescence staining for BrdU . Thus, the MN frequency was determined for cells not labeled by BrdU; for all practical purposes, such cells can be regarded as the Q cells in a tumor . The MN frequency in the total (P + Q) tumor cell population was determined from irradiated tumors that were not pretreated with BrdU . Assays were performed immediately after irradiation alone, 24 hours after the injection of cis-diaminedichloroplatinum(II) (CDDP), mitomycin C (MMC), misonidazole {1-(2-nitro-1-imidazolyl)-3-methoxy-2-propanol} (MISO), 3-aminobenzamide (3-AB), camptothecin (CPT) or caffeine (CAF) following irradiation, and 24 hours after irradiation alone . Q cells were more radioresistant and had a greater capacity for PLDR than the tumor cell population as a whole . CDDP and MISO (especially the latter) inhibited PLDR more strongly in Q cells than in the tumor cell population as a whole . However, CPT and CAF exerted similar inhibition of PLDR in Q cells and in the tumor cell population as a whole . This assay method appears to be useful for detecting the responses of Q tumor cells to various chemical agents.

Ann Biomed Eng, 1997 Jan-Feb, 25(1), 135 - 53
Stratified multiphase model for blood flow in a venular bifurcation; Das B et al.; Available in vitro and in vivo experimental observations suggest that red cell aggregation and blood vessel geometry are important determinants of the flow characteristics of blood in venules . However, no consistent relationship has been observed between red blood cell aggregation and vascular resistance . The present work attempts to understand this relationship by evaluating computationally the effect of red cell aggregation on the flow characteristics of blood in a converging vessel bifurcation . The proposed mathematical model considers blood as a two-phase continuum, with a central core region of concentrated red cell suspension that is surrounded by a layer of plasma adjacent to the vessel wall . In the central core region, blood is described by Quemada's non-Newtonian rheological model, in which local viscosity is a function of both the local hematocrit and a structural parameter that is related to the size of red blood cell aggregates . Fluids from the two feeding branches are immiscible, which results in a stratified multiphase flow in the collecting venule . Calculations predict a complex, three-dimensional pattern of blood flow and generally nonaxisymmetric distribution of velocity, hematocrit, and shear stress in the collecting venule . The calculations are a first step toward a realistic model of blood flow in the venous microcirculation.

J Dairy Sci, 1997 Jan, 80(1), 86 - 93
Expression of immunoglobulin G1 receptors by bovine mammary epithelial cells and mammary leukocytes; Barrington GM et al.; The objective of this study was to identify and evaluate expression of IgG1 receptors by different cell types in mammary tissue sections and digest-dispersed cells from the bovine mammary gland . An immunohistochemical system utilizing avidin-biotin-peroxidase complex demonstrated epithelial expression of IgG1 receptors in mammary tissue sections from cows producing colostrum but not from cows in lactation . Fluorescence flow cytometry demonstrated that cells dispersed in digests from both tissues producing colostrum and lactating tissues selectively bound IgG1 . Fluorescence flow cytometry, using monoclonal antibodies to cell surface molecules, cytokeratin, and IgG1, revealed that leukocytes constituted the largest percentage of cells and were the predominant cell type binding IgG1 in mammary tissue digests . Although IgG1 binding to epithelial cells predominated in the gland during colostrum production in situ, digestion and filtration to produce single cell suspensions resulted in the loss of large numbers of epithelial cells . Studies of Ig binding of cells produced by enzymatic digestion must account for the types of cells surviving the digestion process.

AIDS, 1997 Jan, 11(1), 27 - 32
gp120 is present on the plasma membrane of apoptotic CD4 cells prepared from lymph nodes of HIV-1-infected individuals: an immunoelectron microscopic study; Sunila I et al.; OBJECTIVE: To study whether free gp120 can be detected on the plasma membranes of apoptotic CD4+ T lymphocytes in lymph nodes from HIV-positive patients . METHODS: Lymph-node cell suspensions prepared from three HIV-positive patients were studied by pre-embedding, double-immunogold-labeling to identify cell type, determine cell morphology, and detect the presence of bound gp120 molecules . Cells were classified by their surface antigens as helper/inducer T lymphocytes (CD4+), cytotoxic/suppressor T lymphocytes (CD8+), B cells (CD20+), and total lymphocytes {CD45+, leukocyte common antigen (LCA)+} . RESULTS: gp120 colabelled with both apoptotic and normal CD4+ T lymphocytes and LCA+ cells, but not with either apoptotic or normal CD8+ T lymphocytes or B cells . gp120 was more often identified on apoptotic than on normal CD4+ T lymphocytes . The gp120 and CD45 label were often colocalized . HIV particles were not identified to be associated with or budding from either normal or apoptotic lymphocytes . CONCLUSIONS: Free gp120 is found bound to CD4+ T cells in lymph nodes of HIV-infected individuals and potentially mark them for premature death by apoptosis.

Haematologica, 1997 Jan-Feb, 82(1), 5 - 10
Characterization of the biophysical properties of human erythroblasts as a preliminary step to the isolation of fetal erythroblasts from maternal peripheral blood for non invasive prenatal genetic investigation; Sitar G et al.; BACKGROUND AND OBJECTIVE: Fetal erythroblasts in maternal circulation represent a valuable source of fetal cell material which can be obtained with non-invasive procedures that do not endanger the fetus . Physical separation techniques have been invaluable in the isolation and characterization of different cells . There are basically two principles that have been used most successfully: separation according to density and separation according to size . In order to determine whether physical separation procedures are capable of purifying human erythroblasts, the biophysical characteristics of these cells were determined . METHODS: Bone marrow particles were obtained from formal adults and peripheral blood buffy coats from blood banks . A single cell suspension was initially fractionated by buoyant density gradient centrifugation . Fractions enriched in erythroblasts were pooled and further processed by velocity sedimentation in order to take advantage of the differences in size of erythroblasts and other cells . RESULTS: Density distribution curves were drawn after density gradient centrifugation for the different cell types present in the starting cell samples . Separation of the erythroblast-enriched density fractions by velocity sedimentation was successful and a highly purified population of erythroblasts was obtained . Cell size distribution of the different cell types was determined . INTERPRETATION AND CONCLUSIONS: This initial study defines the biophysical properties (size and density) of human erythroblasts in bone marrow and peripheral blood and is a necessary preliminary step in setting up the optimal procedure for the isolation of fetal erythroblasts from maternal peripheral blood in sufficient amounts and purity for prenatal non-invasive genetic investigation.

Magn Reson Imaging, 1997, 15(2), 193 - 202
Low-molecular weight lanthanide contrast agents: evaluation of susceptibility and dipolar effects in red blood cell suspensions; Fossheim S et al.; Red blood cell (RBC) suspensions, containing low-molecular weight (LMW) dysprosium (Dy) and gadolinium (Gd) chelates, were selected as a two-compartment system for the evaluation of the magnetic dipolar and susceptibility contributions to the transverse (T2) relaxation of solvent water protons . The influence of RBC geometry and degree of metal chelate compartmentalization on T2 was investigated by variation of the osmolality and hematocrit (HC), respectively . The T2-relaxation ability of Dy-chelates was markedly improved in RBC suspensions, in comparison to aqueous solutions, due to the presence of susceptibility effects that more than compensated for the low dipolar relaxation efficacy . Despite a smaller susceptibility effect, the Gd-chelates were still the most efficacious in shortening T2 due to their comparatively larger dipolar relaxation contribution . The results obtained with the Dy-chelates allowed the evaluation of the relative contributions of susceptibility and dipolar mediated relaxation for the Gd-chelates . The RBC geometry and degree of compartmentalization influenced strongly the T2 relaxation efficacy of Dy-chelates, as opposed to the Gd-chelates . Hemolysis eliminated the susceptibility effect, essentially removing the T2 relaxation ability of Dy-chelates . The T2 relaxation efficacy of Gd-chelates was improved by hemolysis due to enhancement of the dipolar relaxation . As a conclusion, RBC suspensions have clearly been shown to be a suitable ex vivo model with which to distinguish the different contrast mechanisms of LMW Dy- and Gd-based MRI contrast agents.

Free Radic Biol Med, 1997, 22(7), 1183 - 93
Formation of free radicals and protein mixed disulfides in rat red cells exposed to dapsone hydroxylamine; Bradshaw TP et al.; The hemolytic activity of dapsone is well known to reside in its N-hydroxylamine metabolites . Addition of dapsone hydroxylamine (DDS-NOH) to red cell suspensions causes damage such that when reintroduced into the circulation of isologous rats, the injured cells are rapidly removed by the spleen . Hemolytic activity is associated with the extensive formation of disulfide-linked hemoglobin adducts on red cell membrane skeletal proteins . To determine if free radicals could be involved in this process, rat red cells were incubated with DDS-NOH in the presence of the spin trap, 5,5'-dimethyl-1-pyrroline-N-oxide (DMPO) and subjected to EPR analysis . Addition of DDS-NOH (25-50 microM) to a red cell suspension gave rise to a four-line (1:2:2:1) EPR spectrum with coupling constants identical to those of a DMPO-hydroxyl radical adduct (DMPO-OH) standard . No other radicals were detected; however, preincubation of red cells with cysteamine caused the DDS-NOH-generated DMPO-OH signal to be replaced by a cysteamine thiyl radical adduct signal . DDS-NOH-treated red cells were also found to contain ferrylhemoglobin, indicating the presence of hydrogen peroxide . Furthermore, DDS-NOH was found to stimulate salicylate hydroxylation in red cell suspensions, confirming the presence of oxygen radicals . These data support the hypothesis that oxygen radicals are involved in the mechanism underlying dapsone-induced hemolytic anemia.

Arch Immunol Ther Exp (Warsz), 1997, 45(1), 43 - 7
Stimulatory effect of ovine colostrinine (a proline-rich polypeptide) on interferons and tumor necrosis factor production by murine resident peritoneal cells; Blach-Olszewska Z et al.; We describe effects of ovine colostrinine (proline-rich polypeptide--PRP) isolated from ovine colostrum and nonapeptide fragment of PRP on interferon (IFN) and tumor necrosis factor (TNF) production by murine resident peritoneal cells (RPC) . The cells from several mouse strains have been found to produce small amounts of IFN-beta and TNF-alpha constitutively . The colostrinine at concentrations of 1-100 micrograms per one ml of cell suspension containing 1 x 10(6) RPC isolated from BALB/c mice, enhanced the IFN and TNF production by 3-30 folds . Upregulation of TNF and IFN production has been observed in the RPC cultures that produced spontaneously less than 16 units of the cytokines only . Synthetic nonapeptide fragment of the colostrinine (Val-Glu-Ser-Tyr-Val-Pro-Leu-Phe-Pro) at concentration of 1-100 micrograms/ml stimulated TNF synthesis but not IFN production . In 1996 Inglot et al . suggested that the colostrinines may be classified as cytokines produced by the mammary gland of mammals . In this paper we have found that the ovine colostrinine at low concentrations modulate the production of other cytokines (IFN-beta and TNF-alpha) in mouse cells that means that it may function in the cytokine network.

Cancer Immunol Immunother, 1997 Jan, 43(6), 317 - 23
Human ex vivo carcinoma cells produce transforming growth factor beta and thereby can inhibit lymphocyte functions in vitro; Vanky F et al.; We tested 20 human carcinoma samples for the production of transforming growth factor beta (TGFbeta) in vitro . Tumour cell suspensions without obvious contamination with non-malignant cells were kept in culture conditions for 16 h and their supernatants were added to CCL-64 cells . The proliferation of these cells is inhibited by TGFbeta . According to this assay, the supernatants contained both active and latent TGFbeta . In addition, the supernatants were found to suppress the spontaneous cytotoxic function and activation of T-cell-enriched lymphocyte populations . A specific monoclonal antibody (mAb) counteracted these effects and therefore we concluded that they were mediated to a large extent by TGFbeta . In line with the results obtained with the supernatants, activation of lymphocytes could also be inhibited by tumour cells and their inhibitory effect was weaker in the presence of the TGFbeta-specific mAb . It is important to note that, when TGFbeta-specific mAb was added to autologous mixed lymphocyte/tumour cell cultures, lymphocyte activation occurred more often . These results thus substantiate the assumption that production of TGFbeta may help the survival of potentially immunogenic tumour cells in immunocompetent patients.

Eur J Neurosci, 1997 Jan, 9(1), 140 - 50
Localization of voltage-sensitive Ca2+ fluxes and neuropeptide Y immunoreactivity to varicosities in SH-SY5Y human neuroblastoma cells differentiated by treatment with the protein kinase inhibitor staurosporine; Kukkonen JP et al.; The distribution of voltage-sensitive elevations of the level of Ca2+ in untreated SH-SY5Y cells and cells that had been induced to differentiate with staurosporine was investigated by monitoring fura-2 fluorescence in cell suspensions, and by using microfluorometry and quantitative fluorescence imaging on cell bodies and on cellular processes . Cell bodies of both types of cells displayed small Ca2+ elevations, which were composed of transient and sustained components . Elevations were partially sensitive to the L- and N-channel blockers nifedipine (1 microM) and omega-conotoxin GVIA (100 nM) respectively . Up to ten times Ca2+ elevations were observed in varicosities of treated cells than in cell bodies of treated and cells . These elevations were insensitive to compounds known to release Ca2+ from intracellular stores . Elevations of Ca2+ were sustained, and they were insensitive to 5 microM nifedipine, 100 nM omega-agatoxin IVA and 100 nM omega-conotoxin GVIA, and partially sensitive to 2 microM omega-conotoxin GVIA, indicating predominance of non-L-type, non-N-type, non-P-type channel activity . The intracellular localization of neuropeptide Y, a marker of differentiation in these cells, was also investigated by fluorescence immunocytochemistry . Varicosities of treated cells displayed marked fluorescence when viewed in a confocal microscope . These findings show that the varicosities of staurosporine-treated cells exhibit some of the functional properties of nerve terminals . The varicosities resemble boutons en passant nerve endings and they seem to express Ca2+ channels different from those in the cell body.

J Magn Reson Imaging, 1997 Jan-Feb, 7(1), 251 - 7
Low molecular weight lanthanide contrast agents: in vitro studies of mechanisms of action; Fossheim S et al.; The MR contrast properties of a series of structurally dissimilar low molecular weight (LMW) gadolinium (Gd) and dysprosium (Dy) chelates have been investigated under controlled experimental conditions in various in vitro test systems . Relaxation analysis (water, pH = 5.8, 37 degrees C, .47 T) demonstrated the high dipolar relaxation efficacy of the tested Gd chelates . The T1 and T2 relaxivities of both metal chelate series decreased with decreasing hydration number, confirming the strong correlation between metal chelate structure and dipolar relaxivity . Susceptibility-induced T2 relaxation, commonly known as the susceptibility effect, is modulated primarily by the magnetic susceptibility and compartmentalization of the contrast agent . The influence of these parameters on the susceptibility effect of Dy diethylenetriamine penta-acetic acid bis-methylamide (DTPA-BMA) and GdDTPA-BMA was investigated in two-compartment in vitro models . In red blood cell suspensions (45% hematocrit, 37 degrees C, .47 T, 2 and 3 mM metal ion concentration), the T2 relaxation efficacy of DyDTPA-BMA was markedly improved due to susceptibility effects that were shown to depend on compartmentalization . As the relaxation ability of GdDTPA-BMA was modulated by the dipolar interactions, compartmentalization was not a prerequisite for its T2 relaxation efficacy . In a coaxial glass system with no intercompartmental water exchange, which eliminated the dipolar relaxation mechanism, DyDTPA-BMA was shown to be the most efficient susceptibility agent because of its higher magnetic susceptibility . The reported one- and two-compartment model studies have demonstrated the different mechanism of action of LMW Gd- and Dy-based contrast agents . Gd chelates are predominantly dipolar relaxation enhancers, whereas Dy chelates are efficient susceptibility agents only in compartmentalized systems.

Immunology, 1997 Jan, 90(1), 14 - 22
Expression of HIS50 Ag: a rat homologue of mouse heat-stable antigen and human CD24 on B lymphoid cells in the rat; Hermans MH et al.; Recently, a cDNA encoding a newly identified rat antigen (HIS50 Ag) that binds to monoclonal antibody (mAb) HIS50 was cloned and shown to be homologous to cDNA encoding murine heat-stable antigen (HSA) and human CD24 . Here we show that, like CD24 and HSA, at least part of HIS50 Ag is inserted into the plasma membrane by a glycosylphosphatidylinosito: (GPI)-lipid linkage and we describe its expression in rat haemolymphopoietic tissues . HIS50 Ag expression was almost exclusively confined to B lymphoid cells, the vast majority of T lymphoid cells, erythroid and myeloid cells were HIS50+ . Cell suspension analysis indicated that in bone marrow (BM) almost all Thy-1+ cells, HIS24+ cells {HIS24 recognizes the B-cell form of leucocyte common antigen (LCA)}, terminal deoxynucleotidyl transferase-positive (TdT+) cells and (c + s)kappa cells expressed HIS50 Ag, and all (c + s)mu 1 cells . A presumably early population of B lymphoid cells, expressing HIS24 Ag without HIS50 Ag, TdT or immunoglobulin HIS24+HIS50+ TdT Ig+), constituted 1.6% of BM nucleated cells . In blood, one-fifth of mononuclear cells were HIS50+, and about 85% of these expressed mu and/or kappa chains . In spleen, flow cytometry analysis and immunohistology demonstrated heterogeneous expression of HIS50 Ag: immunoglobulin M (IgM)bright cells (as found largely in red pulp and marginal zone) were HIS50bright, while IgMdull cells expressed low or undetectable levels of HIS50 Ag . Germinal centre B cells expressed high levels of HIS50 Ag . Germinal centres of lymph nodes and tonsil of man also bound HIS50 . We conclude that HIS50 Ag expression in the haemolymphopoietic system of rat is virtually restricted to the B lineage.

Virchows Arch, 1997 Jan, 430(1), 47 - 51
Nonrandom gain of chromosome 7 in central neurocytoma: a chromosomal analysis and fluorescence in situ hybridization study; Taruscio D et al.; Central neurocytoma is a benign, slow-growing neoplasm with favourable prognosis . Biomolecular analysis has failed to demonstrate significant alterations, and no cytogenetic alterations have been reported . In this study we demonstrate chromosome 7 gain in three of nine neurocytomas (33%) . Traditional cytogenetic analysis performed in four of the nine cases identified trisomy 7 as the sole chromosomal abnormality in one case . Interphase cytogenetics utilizing fluorescent in situ hybridization (FISH) on cell suspensions from formalin-fixed paraffin-embedded tumour tissue performed in all nine cases detected trisomy 7 in two more cases and tetrasomy in another . Our results suggest that chromosome 7 gain is a feature of neuroectodermal tumorigenesis, possibly conferring growth advantage on the neoplastic cells . FISH on interphase nuclei is a valuable adjunct in the genetic evaluation of rare central nervous system neoplasms with low baseline proliferative activity.

Plant Mol Biol, 1997 Jan, 33(2), 279 - 89
Characterization of a tobacco extensin gene and regulation of its gene family in healthy plants and under various stress conditions; Hirsinger C et al.; A genomic clone (Ext 1.4) encoding an extensin was isolated from a Nicotiana tabacum genomic library . The encoded polypeptide showed features characteristic of extensins such as Ser-(Pro)4 repeats and a high content in Tyr and Lys residues . The presence of one Tyr-Leu-Tyr-Lys motif suggests the possibility for one intramolecular isodityrosine cross-link whereas numerous Val-Tyr-Lys motifs may participate in intermolecular cross-links . This extensin appears to be close to an extensin already characterized in N . tabacum but very different from the Ext 1.2 extensin of N . sylvestris . The analysis of genomic DNA gel blots using probes spanning different parts of the gene showed that the Ext 1.4 gene belongs to a complex multigene family having one member originating from N . sylvestris and three members from N . tomentosiformis . The Ext 1.4 specific probe found a 1.4 kb mRNA in stems, roots, ovaries and germinating seeds of healthy plants . The Ext 1.4 gene family is strongly induced in actively dividing cell suspension cultures and after wounding of leaves or stems in conditions where root formation occurs . On the contrary, it is not induced in leaves in response to a hypersensitive reaction to a viral infection or after elicitor treatment.

Pharmazie, 1997 Jan, 52(1), 60 - 4
Antioxidant activities of polyphenolic extracts from flowers, in vitro callus and cell suspension cultures of Crataegus monogyna; Rakotoarison DA et al.; Numerous plants synthesize among their secondary metabolites phenolic compounds which possess antioxidant effects . The aim of the present work was to assay the antioxidant activities of phenolics from Crataegus monogyna Jacq . flowers and in vitro tissue culture (calli and cell suspensions) extracts . In the case of tissue culture extracts, the phenolic production is studied at three different stages of one subculture period (initial growth period, increasing and maximal phenolic synthesis phases) . Attention was paid to the main categories: flavonoids and proanthocyanidins, and to the principal individual components . Total phenolic amounts decrease in the order: fresh flowers > cell suspension cultures > callus cultures . The antioxidant activities of these different extracts against H2O2 and HOCl, have been determined in vitro . All the extracts are efficient and the scavenging capacity is clearly related to the total phenol content . The scavenging effects of the cell suspension extracts are similar to those of the flowers . Among individual compounds, the flavanol-type derivatives, specially the proanthocyanidin B2, are more efficient . Thus, in vitro plant tissues could be an interesting source of bioactive molecules.

Int J Hyperthermia, 1997 Jan-Feb, 13(1), 125 - 31
Thermal enhancement of the effect of ifosfamide against a spontaneous murine fibrosarcoma, FSa-II; Kuroda M et al.; The effect of hyperthermia on the cytotoxicity of 3-(2-chloroethyl)-2-{(2-chloroethyl)amino}-tetrahydro-2H-,1,3, 2-oxazaphosphorine-2-oxide, ifosfamide (IFO) was investigated in vivo . Tumours were early generation isotransplants of a spontaneous C3Hf/Sed mouse fibrosarcoma, FSa-II . The tumour cell suspensions containing approximately 2 x 10(5) cells were transplanted into the dorsal site of the C3Hf/Sed mouse foot . Hyperthermia was given by immersing the tumour-bearing foot into a constant temperature water bath set at 41.5 degrees C for 0-90 min when tumours reached 34 mm3 . IFO was administered i.p . immediately before hyperthermia . Tumour response was studied by the tumour growth (TG) time assay; namely, the TG time or the time for one-half of the treated tumours to reach 700 mm3 from the initial treatment day was determined and the dose-response curves was fitted between the TG time and IFO dose . The anti-tumour effect of IFO was enhanced at this elevated temperature . The thermal enhancement ratio (TER) or the ratio of the slope of dose-response curve at 41.5 degrees C to that of dose-response curve without hyperthermia was relatively small for a short treatment time of 30 min . This TER was smaller for IFO than the TERs for cyclophosphamide (CY) and BCNU which had been studied in our laboratory . However, the TER for IFO increased greatly with a prolongation of treatment time from 30 to 90 min, and exceeded the TER for CY . The TERs were 1.5, 2.6 and 3.6 for heating time of 30, 60, and 90 min, respectively, indicating that a long treatment time such as 90 min at moderately elevated temperatures could result in a substantial enhancement of the antitumour effect of IFO.

Eur J Cell Biol, 1997 Jan, 72(1), 39 - 45
Histone H1(0) expression is restricted to progenitor cells during human hematopoiesis; Valiron O et al.; The histone H1(0) accumulates in cells with little or no proliferative activity during the terminal phase of differentiation in adult tissues . The hematopoietic cell system is an interesting in vivo model to study the relationship between H1(0) and both the proliferative capacity and differentiation state of cells . Using immunofluorescence techniques, we have analyzed the distribution of histone H1(0) during human hematopoietic differentiation, in normal bone marrow cells and in cell lines representative of cells blocked at early stages of differentiation . In enriched bone marrow cell suspensions, H1(0) was not expressed in any cell population highly engaged into a differentiation pathway . However, more than 50% of cells from blastic population (CD34-positive cells) were expressing H1(0), whereas only 5% of CD34-negative cell population expressed H1(0) . We show that H1(0) was also detected in almost all the cell lines studied . These results indicate that histone H1(0) is expressed in immature cells which, although committed, still retain several differentiation potentialities . For normal human hematopoiesis, cells expressing H1(0) belong to a population of cells that are largely quiescent, although having a high proliferative capacity . H1(0) is no longer present in terminally differentiating or differentiated cells with limited or no proliferative potential . Thus, we suggest that H1(0) accumulates in cells with little or no proliferative activity but which are able to resume cell proliferation if required . These results are in keeping with the hypothesis that H1(0) contributes to stabilize a chromatin structure in cells for which integrity and/or longevity are essential.

Arch Ophthalmol, 1997 Jan, 115(1), 89 - 94
Isolation and culture of iris pigment epithelium from iridectomy specimens of eyes with and without exfoliation syndrome; Hu DN et al.; OBJECTIVE: To culture iris pigment epithelium (IPE) from surgical iridectomy specimens of eyes with and without exfoliation syndrome . METHODS: The IPE was treated to obtain a single cell suspension . Cells were cultured in Ham F12 nutrient mixture, which was supplemented with 30% fetal bovine serum, 50-micrograms/mL {corrected} gentamicin, and 2-mmol/L glutamine . After confluence, the cells were detached using a 0.125% trypsin-0.01% edetic acid solution, resuspended, diluted, and subcultured . The IPE from primary cultures and subcultures was studied by transmission electron microscopy . Immunocytochemical staining was performed . RESULTS: In the primary cultures of IPE from patients with exfoliation syndrome, curved, cross-banded, fine fibrils (diameter, 10-15 nm; periodicity, 10-14 nm) were found on the cell surface . Thicker fibrils (diameter, 24-48 nm; periodicity, 24-36 nm) were found external to the fine fibrils . Subcultures contained mainly fine fibrils . The IPE cells stained positively with anticytokeratin, S100 protein, and vimentin antibodies . CONCLUSION: Iris pigment epithelium can be successfully cultured from eyes with exfoliation syndrome . Studying the production of exfoliation material in vitro should provide information about the pathogenesis of exfoliation syndrome and about the nature of the exfoliation material . The cultivation of normal IPE from surgical specimens provides a source for the study of the growth regulation and pharmacophysiology of IPE in vitro.

Am J Pathol, 1997 Jan, 150(1), 99 - 106
Immunobead filtration: a novel approach for the isolation and propagation of tumor cells; Rye PD et al.; We have developed a method to facilitate the isolation and expansion of tumor cells from body fluids and tissue biopsies . Antibody-conjugated magnetic beads (immunobeads) were used to isolate tumor cells from blood, bone marrow, ascitic/pleural fluids, and enzyme-digested tissue biopsies . Filtration of the resulting cell suspension through a 20-micron nylon monofilament filter secured to the base of polystyrene 96-well strips purged the bead-rosetting cell fraction of contaminating normal cells and unbound beads . Tumor cells that bound the magnetic beads were retained on the membrane due to their increased size and concentrated into a small area (0.332 cm2), thus maintaining a high cell density . The filters provided a stable and uniform three-dimensional matrix for cell growth, with a total surface area of 1.42 cm2 available for cell attachment . The filters could be easily removed from the base of the 96-well strips to facilitate handling and transfer between culture vessels . Tumor cells grown on the filters could subsequently be harvested using trypsin/EDTA or left in situ for immunostaining with conventional immunohistochemical procedures . Filter-grown cells have shown extended passage in conventional cell culture in six cases . In two of five cases, the orthotopic implantation of confluent filters that contained approximately 10(4) cells/8 x 8 mm filter successfully produced tumors in nude mice after only 4 weeks . Our new approach may be of value in improving the success rate of generating long-term cultures from previously unproductive sources of tumor cells and thus may yield a greater variety of cell lines/strains for the study of malignant disease.

Exp Hematol, 1997 Jan, 25(1), 57 - 65
Reverse transcriptase-polymerase chain reaction (RT-PCR)-controlled immunomagnetic purging of breast cancer cells using the magnetic cell separation (MACS) system: a sensitive method for monitoring purging efficiency; Hildebrandt M et al.; A modified reverse transcriptase-polymerase chain reaction (RT-PCR) technique was established with the aim of monitoring the tumor cell contamination in peripheral blood stem cells harvested from breast cancer patients . In an experimental approach, single cell suspensions of different breast cancer cell lines were mixed to normal peripheral blood mononuclear cells in order to 1) determine the sensitivity of tumor cell detection within PBMC and 2) compare polymerase chain reaction in its capacity of monitoring the efficiency of immunomagnetic purging using the magnetic cell separation (MACS) system to immunocytochemical staining . Several target sequences were assessed for their indicative potential and specificity allowing the detection of breast cancer cells by RT-PCR . Among the sequences evaluated, epithelial growth factor receptor (EGF-R) mRNA and Cytokeratin 19 mRNA were shown to be highly specific and sensitive markers for the detection of breast cancer cells within normal peripheral blood mononuclear cells and for the evaluation of the efficiency in immunomagnetic purging . In addition, we were able to show that the MACS is a potent and efficient tool for the selection of tumor cells from peripheral blood mononuclear cells, thus establishing its value for clinical scale immunomagnetic purging.

Radiat Res, 1997 Jan, 147(1), 35 - 40
An assay for quantifying DNA double-strand break repair that is suitable for small numbers of unlabeled cells; Longo JA et al.; A system based on pulsed-field gel electrophoresis (PFGE) is described which measures the induction and repair of DNA double-strand breaks (DSBs) in a biologically relevant X-ray dose range (below 10 Gy) using as few as 125 cells per time . This system was used to measure repair in cells of a freshly obtained human glioblastoma multiforme tumor . No prelabeling of the cells is required, and many different cell types can be studied using this system . Under the pulsed-field conditions used, DNA in the range of 2 to 6 Mb enters the PFGE gel and forms an upper compression zone directly under each well . To quantify the DSBs after electrophoresis, the DNA was transferred to nylon membranes and hybridized with 32P-labeled chromosomal DNA . Phosphor screens were exposed to the membranes and scanned on a phosphor imager . The kinetics of induction and repair was determine by measuring the amount of DNA in the compression zones compared to the amount in the wells . EMT-6 cells were used to demonstrate this method . Induction of DSBs by doses of 0-7.5 Gy X rays was assayed using approximately 12,500 cells per dose and was shown to be linear . Double-strand breaks from 1 Gy were detected above background . To determine a lower limit of the number of cells that could be used to measure DSB repair, cells were embedded in agarose at decreasing concentrations per plug, exposed to 7.5 Gy X irradiation and allowed to repair at 37 degrees C for up to 60 min . DNA from approximately 12,500, 1,250 and 125 cells per time was loaded and subjected to PFGE . The average fast-repair half-time was 3 min and the slow-repair half-time was 35 min . The kinetics of DSB repair in glioblastoma multiforme cells was also determined using this system . Agarose plugs were prepared from a cell suspension, irradiated with 7.5 Gy X rays and allowed to repair for up to 90 min . DNA from approximately 1,250 tumor cells was electrophoresed and analyzed as described above for EMT-6 cells . For this particular tumor, approximately 75% of the induced DSBs were repaired after 90 min . Data presented show that this PFGE-based system is an extremely sensitive method for measuring DSB induction and repair after low doses of X rays using very few cells.

Neurosci Lett, 1996 Dec 27, 221(1), 5 - 8
Early effects of basic fibroblast growth factor on foetal rat mesencephalic cell suspensions; Espejo M et al.; Mesencephalic cell suspensions are used, experimentally but also clinically, to compensate for neurological deficiencies, by implantation into the striatum . Here, we have studied the metabolism of mesencephalic cell suspensions obtained from rat embryos by measuring heat dissipation, oxygen consumption, ATP and lactate production . The effect of basic fibroblast growth factor (bFGF) at a 50 ng/ml concentration on these parameters was studied in order to assess the effect of in vitro exposure of cell suspensions to this trophic factor . Heat production and oxygen consumption were low, as could be expected from an immature nervous tissue, and they further decreased after addition of bFGF . This trophic factor decreased the total ATP concentration and increased the lactate production . The viability of the cell suspensions was reduced by nearly a half, 2 h after the addition of bFGF, and numerous fragmented nuclei were observed . It seems that, in contrast to the neuroprotective effect of bFGF on mesencephalic cultures and nigrostriatal neurons, this factor could have an initial sorting effect in the development of mesencephalic structures.

EMBO J, 1996 Dec 16, 15(24), 6869 - 76
Primitive lymphohematopoietic precursor cell lines generated in culture from day 7 early-mid-primitive streak stage mouse embryo; Palacios R et al.; During mouse development, the first lymphohematopoietic precursor cells and myeloid or erythroid cell lineage-determined cells can be detected in the yolk sac at days 8-8.5 of gestation . The characteristics of the cells that give rise to these yolk sac primitive lymphohematopoietic cells and the molecular events controlling this process remain poorly defined . We show here that cell suspensions from day 7 early-mid-primitive streak stage embryo proper generated early immature PgP-1+ Joro 177+ Lin- hematopoietic cells and some Mac-1+ myeloid and TER 119+ erythroid cells after co-culture with the yolk sac-derived stromal cell line YS6 without addition of exogenous cytokines . Purified Lin- hematopoietic cells generated in these cultures did not express genes known to be transcribed at early stages of lymphoid, myeloid or erythroid cell differentiation and were able to give rise to T and B lymphocytes, myeloid cells and erythroid cells after appropriate further induction in vitro . Several cell lines were established in culture with a mixture of four cytokines from the PgP-1+ Joro 177+ Lin- cell population . The cell lines shared phenotypic and genotypic characteristics with the PgP-1+ Joro 177+ Lin- cell population generated in culture from day 7 embryo proper and they were able to reconstitute the lymphohematopoietic system of irradiated mice . Taken together these results support a model of lymphohematopoiesis in which cells from day 7 early-mid-primitive streak mouse embryo proper migrate and colonize the visceral yolk sac . There they generate primitive lymphohematopoietic precursor cells and the first erythroid and myeloid hematopoietic cells under the influence of yolk sac stromal cells like the YS6 cells described here.

J Immunol, 1996 Dec 15, 157(12), 5249 - 53
Thymic expression of myelin basic protein (MBP) . Activation of MBP-specific T cells by thymic cells in the absence of exogenous MBP; Fritz RB et al.; Previous studies have shown golli-myelin basic protein (MBP) mRNA to be expressed in the thymus of normal SJL mice, but translation of the mRNA was not assessed . To test for the presence of immunoreactive protein, single cell suspensions were prepared from adult SJL thymus and cultured with syngeneic MBP-specific T cells . After 48 h {3H}thymidine was added to the microcultures to assess T cell proliferation . MBP-specific T cell lines proliferated strongly (stimulation index range, 13-31) . T cell lines specific for MBP exon 2, MBP peptide 89-101, proteolipid protein peptide 139-151, and OVA gave stimulation indices of 10-13, 5-6, 2-3, and 2-3, respectively . Stimulatory activity could be abrogated by irradiation of either the thymic cells or the MBP-specific T cells . Stimulatory activity was a property of a minor population of plastic-adherent thymic cells . Monoclonal anti-I-As Ab added to the microcultures inhibited the reaction by 77% . MBP-specific T cells cultured with syngeneic nonirradiated thymus cells in the absence of added MBP transferred experimental autoimmune encephalomyelitis adoptively to syngeneic recipients . These findings indicate that golli-MBP mRNA is translated in normal SJL thymus, and that peptides reactive with MBP-specific T cells in the context of class II MHC molecules are expressed.

Int J Cancer, 1996 Dec 11, 68(6), 788 - 94
Change in morphological and functional cytodifferentiation induced by seminal vesicle mesenchyme in cell suspensions of rat Dunning prostatic adenocarcinoma cells; Hayashi N et al.; Previous experiments have shown that seminal vesicle mesenchyme (SVM) can induce small 0.5 mm fragments of the rat Dunning tumor (DT) to undergo secretory differentiation with a concomitant reduction in tumorigenesis . In the present experiments Dunning tumor epithelial cells (DTE) were purified from DT cell suspensions by Percoll gradient centrifugation and recombined with neonatal rat SVM . The resultant tissue recombinants (SVM + DTE) were grafted under the renal capsule of male athymic mice and grown for 2 months . Under these conditions SVM induced the DTE to exhibit a highly differentiated secretory phenotype by forming ducts lined with tall columnar epithelial cells or large clear cells with pale cytoplasm . Undifferentiated epithelial cells of the parental DT were rarely observed in these tissue recombinants . The loss of tumorigenicity in SVM + DTE recombinants was associated with a striking reduction of epithelial 3H-thymidine labeling index in SVM + DTE recombinants (DT = 8.31%; SVM + DTE recombinants = 1.10%) . Differences in putative secretory proteins were also observed by SDS-PAGE in SVM + DTE recombinants in comparison with DT . Testosterone metabolism was examined in epithelial cells recovered from grafts of DT vs . SVM + DTE tissue recombinants by thin layer chromatography and revealed that the major metabolite produced by DTE was androstenedione, whereas in epithelium isolated from SVM + DTE tissue recombinants the major androgen metabolite was 5alpha-DHT . Thus, after induction by SVM the DTE metabolized androgens in a pattern similar to the normal rat dorsal prostate . The SVM-induced changes in DTE suggest the possibility that emerging or established carcinomas might be regulated at least in part by their connective tissue microenvironment.

FEBS Lett, 1996 Dec 2, 398(2-3), 243 - 7
Structure and organ specificity of an anionic peroxidase from Arabidopsis thaliana cell suspension culture; Ostergaard L et al.; The predominant peroxidase (pI 3.5) (E.C . 1.11.1.7) of an Arabidopsis thaliana cell suspension culture was purified and partially sequenced . Oligonucleotides were designed and a specific probe was obtained . A cDNA clone was isolated from an Arabidopsis cell suspension cDNA library and completely sequenced . The cDNA clone comprised 1194 bp and encodes a 30 residue signal peptide and a 305 residue mature protein (Mr 31,966) . The sequence of the mature protein is 95% identical to the well-characterized horseradish peroxidase HRP A2 and has therefore been designated ATP A2 . Three introns at positions identical to those found in Arabidopsis and horseradish genes encoding cationic peroxidases were identified . RT-PCR analysis revealed root-specific expression.

Toxicology, 1996 Dec 2, 114(2), 135 - 45
Relationship between mitochondrial dysfunction and toxicity of propyl gallate in isolated rat hepatocytes; Nakagawa Y et al.; The relationship between cytotoxicity and mitochondrial dysfunction caused by propyl gallate (PG) has been studied in hepatocytes freshly prepared from fasted rats . Hepatocytes isolated from fasted (18 h) rats were significantly more susceptible to the toxicity of PG than hepatocytes from fed rats . The addition of fructose (15 mM), an alternative carbohydrate source, to hepatocyte suspensions resulted in the prevention of PG (1 mM)-induced cell killing accompanied by decrease in intracellular ATP loss during a 3 h-incubation period . Despite this, fructose did not completely prevent an abrupt loss of intracellular glutathione caused by PG, but effectively inhibited the loss of protein thiol levels . Fructose elicited a concentration (0.5-20mM)-dependent protection against the cytotoxicity of 1.5 mM PG . The incubation of hepatocytes with sodium azide (4 mM), an inhibitor of oxidative phosphorylation, enhanced the toxicity induced by PG (1 mM), but coincubation with fructose delayed the onset of toxicity . Neither azide alone nor fructose plus azide did affect the cell viability during the incubation period . Furthermore, the addition of 2 mM salicylamide, nontoxic to hepatocytes during the incubation period, enhanced PG (1 mM)-induced cytotoxicity and decreased the loss of free PG . These results indicate that the onset of cytotoxicity caused by PG may depend on the intracellular energy status and that mitochondria are critical target for the compound . In addition, the toxicity caused by the inhibition of mitochondrial ATP synthesis is related to the concentration of PG remaining in cell suspensions.

Radiother Oncol, 1996 Dec, 41(3), 237 - 48
Enrichment of tumor cells for cell kinetic analysis in human tumor biopsies using cytokeratin gating; Haustermans K et al.; PURPOSE: To determine the feasibility of using cytokeratin antibodies to distinguish normal and malignant cells in human tumors using flow cytometry . The goal was ultimately to increase the accuracy of cell kinetic measurements on human tumor biopsies . MATERIAL AND METHODS: A panel of four antibodies was screened on a series of 48 tumors from two centres; 22 head and neck tumors (Amsterdam) and 26 esophagus carcinomas (Leuven) . First, screening was carried out by immunohistochemistry on frozen sections to test intensity of staining and the fraction of cytokeratin-positive tumor cells . The antibody showing the most positive staining was then used for flow cytometry on the same tumor . RESULTS: The two broadest spectrum antibodies (AE1/AE3, E3/C4) showed overall the best results with immunohistochemical staining, being positive in over 95% of tumors . Good cell suspensions for DNA flow cytometry could be made from frozen material by a mechanical method, whereas enzymatic methods with trypsin or collagenase were judged failures in almost all cases . From fresh material, both collagenase and trypsin produced good suspensions for flow cytometry, although the fraction of tumor cells, judged by proportion aneuploid cells, was markedly higher for trypsin . Using the best cytokeratin antibody for each tumor, two parameter flow cytometry was done (cytokeratin versus DNA content) . Enrichment of tumor cells was then tested by measuring the fraction of aneuploid cells (the presumed malignant population) of cytokeratin-positive cells versus all cells . An enrichment factor ranging between 0 (no enrichment) and 1 (perfect enrichment, tumor cells only) was then calculated . The average enrichment was 0.60 for head and neck tumors and 0.59 for esophagus tumors . CONCLUSIONS: We conclude that this method can substantially enrich the proportion of tumor cells in biopsies from carcinomas . Application of this method could significantly enhance accuracy of tumor cell kinetic measurements.

Lung Cancer, 1996 Dec, 16(1), 13 - 9
Interleukin-2 receptors in pulmonary adenocarcinoma tissue; Yano T et al.; We previously reported that the serum soluble interleukin-2 receptor (sIL-2R) level increased with the advance of disease stage in non-small cell lung cancer . The present study was thus conducted to investigate the origin of serum sIL-2R in patients with pulmonary adenocarcinoma . Fresh tumor cell suspensions were prepared from surgically resected specimens of pulmonary adenocarcinoma . They were adjusted to a cell density of 5 x 10(5)/ml and then cultured for 24 h at 37 degrees C . The culture supernatants were collected and assayed to determine the sIL-2R levels using an enzyme immunoassay . The resultant cells were thereafter cytocentrifuged onto glass slides and immunochemically stained with anti-human IL-2R alpha (CD25) monoclonal antibody . In three of six cases examined, a substantial level of sIL-2R was identified in the culture supernatants . In four cases, including those three cases with the presence of sIL-2R in the culture supernatants, various proportions of tumor cells were positively stained with the anti-IL-2R alpha antibody . Further examinations revealed that tumor cells expressed IL-2R alpha (CD25) in seven of 16 cases with pulmonary adenocarcinoma . These results thus suggested that the tumor cells did express IL-2R alpha and release sIL-2R in some cases with pulmonary adenocarcinoma.

Plant Mol Biol, 1996 Dec, 32(6), 1093 - 101
G2-and early-M-specific expression of the NTCYC1 cyclin gene in Nicotiana tabacum cells; Qin LX et al.; We have previously reported the isolation of a cDNA encoding a mitotic cyclin, NTCYC1, from a tobacco cell suspension library . Here we describe the expression patterns of NTCYC1 and of Ntsuc1, a suc 1 plant homologue, in synchronized tobacco cell suspensions . Furthermore, the expression pattern of this cyclin is compared to that of Ntcdc2-1, a Nicotiana tabacum homologue of cdc2 . While no NTCYC1 transcript was detected in cells synchronized in the G1 and S phases, NTCYC1 expression was observed in late G2 and early M phases, disappearing in the G1' of a new cell cycle . On the other hand, Ntsuc1 and Ntcdc2-1 exhibited a constitutive expression during the cell cycle . A functional analysis performed by microinjecting NTCYC1 mRNA into immature Xenopus oocytes, indicates that NTCYC1 could participate in the control of the G2/M transition in plant cells . Subsequently NTCYC1 expression was used to assess the status of mesophyll cells in expanded leaves of N . tabacum . Depending on leaf position along the shoot axis, a large population of mesophyll cells appeared with a 4C DNA content, suggesting a G2 arrest . It was found that leaves with such a population also contained high levels of NTCYC1 transcripts . With respect to these results concerning a naturally occurring G2-arrested cell population, the regulation of NTCYC1 expression in planta is discussed.

Int J Hematol, 1996 Dec, 65(1), 49 - 59
Regulation of neutrophil O2- production by neutrophil-endothelial cell interaction via CD11b: its modulation by tumor necrosis factor-alpha (TNF-alpha) and lipopolysaccharide (LPS); Miyabayashi M et al.; Polymorphonuclear leukocyte-endothelial cell (PMN-EC) adhesion and the O2- production by subsequently triggered polymorphonuclear leukocytes (PMN) must be involved in the development of multiple organ failure at septic inflammatory sites . In this study, the adhesion and O2- production of PMN treated with LPS and serum, were markedly enhanced on the EC monolayer by treatment with TNF-alpha or LPS . However, in the intact EC monolayer, neither adhesion nor O2- production was increased . Monoclonal antibodies (mAb) against CD18, CD11b, and ICAM-1 inhibited PMN-EC adhesion . All antibodies except for anti-CD11b mAb had no effect on O2- production by adhered PMN . Anti-CD11b mAb stimulated O2- production in a PMN cell suspension . The pertussis toxin, an inhibitor of some G-proteins, inhibited this reaction . These findings indicate that the adhesion mediated by CD11b provides the signal for O2- production by PMN . This O2- production may involve a signal transduction mechanism mediated by pertussis toxin-sensitive G-proteins.

Biosci Biotechnol Biochem, 1996 Dec, 60(12), 1956 - 61
Three extracellular chitinases in suspension-cultured rice cells elicited by N-acetylchitooligosaccharides; Inui H et al.; In rice suspension culture, a large part (about 90% of total activity in the culture) of the chitinase activity was found in the medium . Two extracellular chitinases (which we named RCH-A and -B) were separated from the cell suspension by DEAE-cellulofine column chromatography . When cells were treated with N-acetylchitooligosaccharides (chitin oligosaccharides) for 3 days, extracellular chitinase activity increased about 3-fold over the control culture . After the treatment, another extracellular chitinase (named RCH-C) appeared in addition to increases in the levels of RCH-A and -B . Partial amino acid sequences of these enzymes indicated that RCH-A (33.5 kDa) and -B (34 kDa) were class Ib chitinases but RCH-C (27 kDa) was a class III chitinase . RCH-A and -B were capable of actively degrading water-insoluble chitin with high affinities, while RCH-C had less affinity for the substrate . However, when a water-soluble chitin derivative, 6-O-hydroxyethylchitin (glycolchitin) was used, RCH-C as well as RCH-A and -B degraded actively with a high affinity . A synergistic effect was observed when these three chitinases acted simultaneously in the hydrolysis of chitin.

Phytochemistry, 1996 Dec, 43(6), 1235 - 7
Structural characterization of 15-hydroxytrichodiene, a sesquiterpenoid produced by transformed tobacco cell suspension cultures expressing a trichodiene synthase gene from Fusarium sporotrichioides; Zook M et al.; Tobacco (Nicotiana tabaccum) cell suspension cultures transformed with a gene encoding trichodiene synthase, a sesquiterpene synthase from the fungus Fusarium sporotrichioides, produced a novel sesquiterpenoid derived from the in vivo production of trichodiene . Mass and nuclear magnetic resonance spectroscopic analyses identified the new compound as 15-hydroxytrichodiene . The in vivo hydroxylation of trichodiene by transformant tobacco cell suspension cultures demonstrates that the introduction of a foreign sesquiterpene synthase gene can result in the production of novel sesquiterpenoid metabolites.

Phytochemistry, 1996 Dec, 43(6), 1141 - 4
Elicitation of dihydrobenzophenanthridine oxidase in Sanguinaria canadensis cell cultures; Ignatov A et al.; Dihydrobenzophenanthridine (DHBP) oxidase catalyses the last step in the biogenesis of the benzo{c}phenanthridine alkaloid sanguinarine . Addition of autoclaved fungal preparations or putative plant defence signalling intermediates (jasmonic acid (JA), methyl jasmonate (MeJ), acetylsalicylic acid (ASA)) to Sanguinaria canadensis cell suspension cultures elicited an increase in the activity of DHBP oxidase . MeJ and ASA were better inducers of oxidase activity than were the fungal elicitor and JA . Enzyme-specific activity could be induced in a dose- and time-dependent manner up to 4- to 14-fold, respectively, when cells were treated with MeJ or with ASA . A change in total enzyme activity in cultured cells was observed only at the highest concentration of MeJ and not at any level of ASA tested . The results suggest that MeJ and ASA may play a role in the S . canadensis defence against pathogens by eliciting the enzymes involved in the synthesis of the phytoalexin benzophenanthridine alkaloids.

Genome, 1996 Dec, 39(6), 1185 - 93
Negative regulation of gene expression of a novel proline-, threonine-, and glycine-rich protein by water stress in Lycopersicon chilense; Yu LX et al.; We have isolated a full length cDNA clone (designated PTGRP) encoding a proline-rich protein from leaves of Lycopersicon chilense . Sequence analysis of the 552-bp insert revealed that the open reading frame encodes a 12.6-kDa protein . The deduced amino acid sequence of PTGRP consists of a C-terminal proline-rich domain with two identical repeat motifs Phe-Pro-Met-Pro-Thr-Thr-Pro-Ser-Thr-Gly-Gly-Gly-Phe-Pro-Ser . The N terminus lacks proline and is hydrophobic . Unlike other proline-rich proteins this protein contains five glycine-rich repeat motifs (Gly-X)n representative of glycine-rich proteins . Southern blot analysis showed that PTGRP is a member of a small gene family within the L . chilense genome . Northern blot experiments revealed that the PTGRP gene is significantly down regulated by water stress . PTGRP mRNA transcription decreased 5- to 10-fold in leaves and stems after 4-8 days of water stress . The mRNA reaccumulated when the drought-stressed plants were rewatered . The in situ hybridization experiments also revealed that PTGRP mRNAs were more abundant in leaf sections of plants watered regularly compared with those of plants submitted to water stress . Down regulation of the PTGRP gene was also observed in desiccated cell suspensions of L . chilense and in those treated with abscisic acid, mannitol, and NaCl . Based on the common features of proline-rich proteins (high proline content, repeated motifs, and a putative signal peptide) and their involvement in the cell wall, it is likely that the PTGRP protein is targeted to the cell wall . Its down regulation by drought could be correlated with the remodeling of the plant cell wall in response to water stress.

Eur J Cell Biol, 1996 Dec, 71(4), 387 - 94
Hydrocortisone is involved in regulating the proliferation and differentiation of mouse epidermal melanoblasts in serum-free culture in the presence of keratinocytes; Hirobe T; Mouse epidermal melanoblasts preferentially proliferated from disaggregated epidermal cell suspensions derived from newborn mouse skin in serum-free medium (MPM) supplemented with dibutyryl adenosine 3', 5' cyclic monophosphate (DBcAMP) and basic fibroblast growth factor (bFGF) . After 12 days, almost all keratinocytes died, and pure and enriched cultures of melanoblasts (approximately 90%) and melanocytes (approximately 10%) could be obtained . In order to clarify the role of hydrocortisone (HC) which is thought to be important for the regulation of melanocyte proliferation and differentiation, the hormone was added to MPM from the initiation of primary culture . The proliferation of melanoblasts was inhibited by HC in a dose-dependent manner . Instead, most of the proliferating melanoblasts were induced to differentiate into melanocytes by HC in a dose-dependent manner . A small number of pure melanoblasts derived from primary cultures at 12 days with MPM depleted of DBcAMP and bFGF were cultured with MPM with or without HC in the presence of secondary keratinocytes that were subcultured from a pure population of primary keratinocytes in a serum-free medium . The inhibition of the proliferation of melanoblasts by HC as well as the stimulation of the differentiation of melanoblasts into melanocytes by HC was observed in the presence of keratinocytes, but not in the absence of keratinocytes . Conditioned media or extracts prepared from pure keratinocytes in serum-free primary culture failed to replace the role of keratinocytes . These results suggest that HC plays an important role in the regulation of the proliferation and differentiation of mouse epidermal melanoblasts in culture in cooperation with factors supplied by keratinocytes.

Eur J Immunol, 1996 Dec, 26(12), 3114 - 8
RAG1, RAG2 and pre-T cell receptor alpha chain expression by adult human hepatic T cells: evidence for extrathymic T cell maturation; Collins C et al.; Flow cytometric analysis of cell suspensions obtained from normal adult liver tissue at the time of transplantation revealed significant populations of T lymphocytes . These were examined for molecular evidence of local T cell maturation using reverse transcription-polymerase chain reaction to detect expression of recombination activation gene 1 (RAG1), RAG2 and pre-T cell receptor alpha chain (pTalpha), which occurs only in early thymocyte development . Four specimens of whole liver were positive for RAG1 and RAG2 expression, whereas peripheral blood mononuclear cells from the same individuals were negative . To localize RAG expression, immature (CD2+CD7+) and mature (CD45R0+) T cell subpopulations were isolated by magnetic separation from hepatic and peripheral blood mononuclear cell preparations . We detected the expression of RAG1, RAG2 and pre-TCRalpha in five specimens of hepatic CD2+CD7+ but not in CD45RO+ hepatic lymphocytes . Four out of six specimens of CD2+CD7+ cells from the peripheral blood were negative for RAG1 and RAG2 while all six specimens were positive for pTalpha expression . These results suggest that pre-T cells are trafficking from the bone marrow or the thymus to other tissues to continue differentiation and selection in the context of an appropriate cellular and molecular environment . The presence of immature populations of T cells in the adult liver and high levels of RAG expression suggests that the adult liver provides such an environment for extrathymic T cell maturation . These findings may have important implications for tolerance induction after liver transplantation and offer help in understanding the etiology of autoimmune liver disease.

Cancer Genet Cytogenet, 1996 Dec, 92(2), 147 - 9
Trisomies 8 and 20 in desmoid tumors; Qi H et al.; A nonrandom occurrence of trisomy 8 and of trisomy 20 in desmoid tumors has been recently reported . The finding of trisomy 8 in nondividing desmoid tumor cells by in situ hybridization prompted us to evaluate, in a similar way, the occurrence of trisomy 20 and the possible occurrence of both trisomies together because their co-existence was cytogenetically observed in a few cases . Double fluorescence in situ hybridization (FISH) with centromeric probes for chromosomes 8 and 20 was performed on 16 single cell suspensions of desmoid tumors . FISH confirmed the occurrence of trisomy 8 or 20 in a single cell suspension of desmoid tumors . Both individual trisomies, and even more their association in the same cells, are rare to extremely rare in solid tumors in general and in mesenchymal tumors in particular, and are only known to occur in infantile fibrosarcoma.

Cancer Genet Cytogenet, 1996 Dec, 92(2), 144 - 6
Cytogenetic analysis in a case of intraocular medulloepithelioma; Betts DR et al.; We report the cytogenetic analysis of a medulloepithelioma, a rare neuroectodermally-derived tumor of childhood, which to our knowledge is the first reported karyotype of this tumor . The tumor sample was received at diagnosis and was mechanically dissociated to create cell suspension cultures . The primary cytogenetic abnormality was a der(16)t(1;16) chromosome, which was typical of that reported in a wide range of other tumor types . The other abnormalities seen were a del(6q) and monosomy 15.

J Exp Med, 1996 Dec 1, 184(6), 2417 - 22
A role for endogenous transforming growth factor beta 1 in Langerhans cell biology: the skin of transforming growth factor beta 1 null mice is devoid of epidermal Langerhans cells; Borkowski TA et al.; Transforming growth factor beta 1 (TGF-beta 1) regulates leukocytes and epithelial cells . To determine whether the pleiotropic effects of TGF-beta 1, a cytokine that is produced by both keratinocytes and Langerhans cells (LC), extend to epidermal leukocytes, we characterized LC (the epidermal contingent of the dendritic cell {DC} lineage) and dendritic epidermal T cells (DETC) in TGF-beta 1 null (TGF-beta 1 -/-) mice . I-A+ LC were not detected in epidermal cell suspensions or epidermal sheets prepared from TGF-beta 1 -/- mice, and epidermal cell suspensions were devoid of allostimulatory activity . In contrast, TCR-gamma delta + DETC were normal in number and appearance in TGF-beta 1 -/- mice and, importantly, DETC represented the only leukocytes in the epidermis . Immunolocalization studies revealed CD11c+ DC in lymph nodes from TGF-beta 1 -/- mice, although gp40+ DC were absent . Treatment of TGF-beta 1 -/- mice with rapamycin abrogated the characteristic inflammatory wasting syndrome and prolonged survival indefinitely, but did not result in population of the epidermis with LC . Thus, the LC abnormality in TGF-beta 1 -/- mice is not a consequence of inflammation in skin or other organs, and LC development is not simply delayed in these animals . We conclude that endogenous TGF-beta 1 is essential for normal murine LC development or epidermal localization.

Biochem J, 1996 Dec 1, 320 ( Pt 2), 563 - 70
Activation effects of a prion protein fragment {PrP-(106-126)} on human leucocytes; Diomede L et al.; Prion-related encephalopathies are characterized by the intracerebral accumulation of an abnormal isoform of the cellular prion protein (PrPC) named scrapie prion protein (PrPSc) . The pathological forms of this protein and its cellular precursor are not only expressed in the brain but also, at lower concentrations, in peripheral tissues . We recently showed that a synthetic peptide corresponding to residues 106-126 {PrP-(106-126)} of the human PrP is toxic to neurons and trophic to astrocytes in vitro . Our experiments were aimed at verifying whether PrP-(106-126) and other peptides corresponding to fragments of the amyloid protein purified from brains of patients with Gerstmann-Straussler-Scheinker disease-namely PrP-(89-106), PrP-(106-114), PrP-(127-147)-were capable of stimulating circulating leucocytes . Native PrP expression in human lymphocytes, monocytes and neutrophils was first confirmed using PCR amplification of total RNA, after reverse transcription, and immunoblot analysis of cell extracts with anti-PrP antibodies . PrP-(106-126), but not the other peptides, increased membrane microviscosity, intracellular Ca2+ concentration and cell migration in circulating leucocytes, and O2- . production in monocytes and neutrophils . Membrane microviscosity was determined by the fluorescence polarization technique, using diphenylhexatriene as a probe, 300 s after the addition of PrP-(106-126) to the cell suspension in the concentration range 5-50 microM . The increase in intracellular Ca2+ elicited by PrP-(106-126) was dose-dependent in the range 5-500 microM . PrP-(106-126) stimulated O2- . production in monocytes and neutrophils in a dose- (10-300 microM) and time-(5-30 min) dependent manner in the presence of 10 microM dihydrocytochalasin B . Both the increase in Ca2+ concentration and the O2- . production were partially sensitive to pertussis toxin . PrP-(106-126) stimulated leucocyte migration in a dose-dependent (30-300 microM) manner and, at the highest concentration used, this migration was comparable with that elicited by 2.5 nM interleukin 8 or 10 nM fMet-Leu-Phe peptide.

Plant Physiol, 1996 Dec, 112(4), 1499 - 508
Induced new mutation of D1 serine-268 in soybean photosynthetic cell cultures produced atrazine resistance, increased stability of S2QB- and S3QB- states, and increased sensitivity to light stress; Alfonso M et al.; We have isolated several herbicide-resistant cell lines from photosynthetic cell suspensions of soybean (Glycine max) that possessed different levels of herbicide resistance, photosystem II activity, and chlorophyll a/b ratio . We have further studied the STR7 mutant, which showed the highest level of resistance to atrazine as well as a cross-resistance to 3-(3,4-dichlorophenyl)-1, 1-dimethylurea (50- and 3-fold, respectively, compared with the wild type) . Sequencing of the psbA gene (coding for the D1 polypeptide of photosystem II) from this mutant revealed a single change, serine-268 to proline, in the D1 protein . To our knowledge, this substitution has not previously been described in any photosynthetic organism . In addition to affecting atrazine resistance, this single amino acid change caused a decrease in the electron transfer rate between the secondary acceptors QA and QB and a stabilization of the S2QB- and S3QB- states . The mutant also showed a larger antenna size, an increase in non-QB-reducing centers, and a higher sensitivity to light stress . The unusual stability of the S2QB- and S3QB- states indicates that STR7 belongs to a new class of QB-site mutants.

Br J Cancer, 1996 Dec, 74(11), 1734 - 42
Intrinsic radiosensitivity of human pancreatic tumour cells and the radiosensitising potency of the nitric oxide donor sodium nitroprusside; Verovski VN et al.; A panel of eight human pancreatic tumour cell lines displayed high intrinsic radioresistance, with mean inactivation doses between 2.4 and 6.5 Gy, similar to those reported for melanoma and glioblastoma . The radiosensitising potency of sodium nitroprusside, a bioreductive nitric oxide donor, was assessed in a model of metabolism-induced hypoxia in a cell micropellet . Sodium nitroprusside at 0.1 mM revealed a radiosensitising effect with an overall enhancement ratio of 1.9 compared with 2.5 for oxygen . Radiosensitising activity correlated with the enhancement of single-strand DNA breakage caused by radiation . In suspensions with cell densities of between 3% and 30% (v/v), the half-life of sodium nitroprusside decreased from 31 to 3.2 min, suggesting a value of around 1 min for micropellets . Despite this variation, the radiosensitising activity was similar in micropellets and in diluted cell suspensions . S-nitroso-L-glutathione was found to possess radiosensitising activity, consistent with a possible role of natural thiols in the storing of radiobiologically active nitric oxide adducts derived from sodium nitroprusside . As measured by a nitric oxide-specific microsensor, activation of sodium nitroprusside occurred by bioreduction, whereas S-nitroso-L-glutathione showed substantial spontaneous decomposition . Both agents appear to exert radiosensitising action through nitric oxide as its scavenging by carboxy phenyltetramethylimidazolineoxyl N-oxide (carboxy-PTI0) and oxyhaemoglobin resulted in attenuated radiosensitisation . Sodium nitroprusside was at least 10-fold more potent than etanidazole, a 2-nitroimidazole used as a reference . Our data suggest that sodium nitroprusside, a drug currently used for the treatment of hypertension, is a potential tumour radioresponse modifier.

Biol Reprod, 1996 Dec, 55(6), 1397 - 404
Isolation and culture of microvascular endothelial cells from the primate corpus luteum; Christenson LK et al.; Endothelial cells have common as well as specialized roles in different tissues and organs; as such, the abundant endothelial cells in the corpus luteum (> 50% of total cell population) could have unique activities necessary for luteal function . Our objective was to establish a method for isolating a pure population of endothelial cells from the primate corpus luteum and to determine the basic conditions for in vitro culture . Corpora lutea collected from rhesus monkeys throughout the luteal phase of the menstrual cycle were minced and enzymatically dispersed into single cell suspensions . Endothelial cells were isolated from the remaining cells (i.e., steroidogenic, fibroblastic, etc.) utilizing magnetic beads labeled with a lectin, Ulex europaeus agglutinin-1 (UEA-1), which binds a sugar found only on primate endothelial cells . After exposure to a magnetic field, UEA-1-negative (-) cells were decanted from the pelleted UEA-1-positive (+) cells; to remove beads from the UEA-1 (+) cells, excess sugar was applied . After optimization of the bead-to-cell ratio, the UEA-1 (+) group contained a population of cells 8-12 microns in diameter (typical endothelial cell size) and < 1% steroidogenic cells . UEA-1 (-) cells were larger (15-35 microns) and stained histochemically for the steroidogenic marker, 3 beta-hydroxysteroid dehydrogenase . Immunocytochemical analysis demonstrated that > 93% of all cells in the UEA-1 (+) group stained positive for the specific endothelial cell marker, platelet/endothelial cell adhesion molecule-1 . Cultured UEA-1 (+) cells produced low levels of progesterone and were unresponsive to hCG (100 ng/ml) . In contrast, cultured UEA-1 (-) and mixed (unsorted) cells produced high basal levels of progesterone and exhibited a > 3-fold increase in response to hCG treatment . Preliminary experiments comparing different culture media and matrices demonstrated that 1) cell proliferation was unaffected by type of medium (i.e., Dulbecco's Modified Eagle Medium {DMEM}/ F12 or McCoy's 5A); 2) the presence of serum was essential in the absence of added growth factors, and 3) extracellular matrix had a profound effect on proliferation . UEA-1 (+) cells exhibited a dose-dependent increase in cell proliferation in response to vascular endothelial growth factor (VEGF) in the presence and absence of fetal calf serum . In the absence of serum, VEGF stimulated proliferation of UEA-1 (+) cells plated on fibronectin but not collagen I, whereas in the presence of 10% fetal calf serum, both matrices supported VEGF-induced mitogenesis . These studies provide for the first time an efficient, reliable method for isolating primate luteal endothelial cells and describe in vitro culture conditions for subsequent studies examining luteal endothelial cell function and regulation.

J Cell Biol, 1996 Dec, 135(5), 1377 - 82
A role for Jun-N-terminal kinase in anoikis; suppression by bcl-2 and crmA; Frisch SM et al.; The disruption of interactions between extracellular matrix and specific cognate integrins triggers apoptosis in epithelial cells, in a process termed "anoikis." To understand anoikis, the connections between epithelial cell integrin signaling and the apoptosis-regulatory proteins are being explored . We report herein that early after detachment from matrix, epithelial cells activate Jun-N-Terminal Kinases (JNKs; alternatively known as Stress-activated Protein Kinases), which are also activated by other apoptotic stimuli . The activity of this pathway was required for anoikis . Another early response to cell suspension was the activation of the ICE-related cysteine protease, ICE/LAP3; this activation and anoikis were suppressed by the ICE-protease inhibitor, crmA . The overexpression of bcl-2 suppressed ICE/LAP3 activation as well . Surprisingly, bcl-2 and crmA attenuated the activation of JNKs following cell suspension, suggesting that the JNK pathway is regulated directly or indirectly by proteolysis . In addition, the blockage of the JNK pathway attenuated the activation of ICE/LAP3, suggesting a positive feedback loop between the ICE and JNK systems . These results indicate the following sequence of information flow in anoikis: integrins-->bcl-2/bax-->(ICE-proteases<-->JNK)-->apopt osis . Cell-cell interactions, which were previously shown to sensitize cells to anoikis, caused bcl-2 mRNA to be downregulated, a permissive event for downstream apoptotic signaling.

J Invest Dermatol, 1996 Dec, 107(6), 815 - 21
Langerhans cells express inducible nitric oxide synthase and produce nitric oxide; Qureshi AA et al.; The importance of nitric oxide (NO) in mediating macrophage functions has been demonstrated, but production of this potent gas has not been examined in Langerhans cells (LC) . Using murine LC purified from epidermal cell suspensions and the recently established LC-like cell line derived from newborn BALB/c epidermis (XS-52), it was shown with reverse transcriptase (RT)-PCR that inducible nitric oxide synthase (iNOS) message is present in these cells . Murine keratinocytes did not contain iNOS message . iNOS mRNA was increased in a concentration-dependent manner by lipopolysaccharide (LPS) in purified murine LC and XS-52 cells, and immunofluorescence using an antibody to iNOS revealed bright cytoplasmic staining in LPS-treated XS-52 cells . Anti-iNOS antibody brightly stained LC on human neonatal foreskin cryosections . An increase in NO production by LPS-treated XS-52 cells over 16 h, as measured by the determination of nitrite levels in culture supernatants using the Griess Reaction, was observed . Interferon-gamma (IFNgamma) did not affect NO production on its own . In the presence of LPS and IFNgamma, NO production was 3 times more than observed with LPS alone . NO production was inhibited by the NOS inhibitor L-NAME . Western blots with anti-iNOS antibody demonstrated an increase in iNOS expression in LPS-treated XS-52 cells that was suppressed by IL-10 . NO produced in LC may affect LC functions such as microbicidal activity, antigen presentation, and cytotoxicity and may affect adjacent keratinocytes and melanocytes.

J Immunol Methods, 1996 Nov 29, 199(1), 1 - 4
A new and simple method to study the role of soluble factors in antibody-mediated cell modulation; Nizet Y et al.; This paper describes an in vitro method to study how interactions between cell suspensions incubated in two culture compartments can occur by diffusion of small molecules through a dialysis membrane . This consists of in vitro cultures of a human PBMC suspension, divided into two parts separated by a dialysis membrane . In one of the cultures, a mitogenic monoclonal antibody (mAb) is added . The porosity of the membrane permits cytokines secreted by activated cells to pass while blocking the antibodies . As a model, PBMC from the same blood donor were divided into two parts and one portion was incubated with an anti-CD3 monoclonal antibody (OKT3 Ortho-Cilag), known to induce IL-2 secretion whereas the other was enclosed in a dialysis bag without antibody . After a 4 day incubation, the cells incubated outside the bag proliferate and secrete cytokines which pass through the membrane and induce cell proliferation inside the bag . This method could complement the currently used methods for the analysis of antibody-mediated cell activation.

Early Hum Dev, 1996 Nov 21, 46(3), 217 - 27
Assessment of the contribution of the spleen to granulocytopoiesis and erythropoiesis of the mid-gestation human fetus; Calhoun DA et al.; Several current textbooks of hematology describe the spleen of the mid-gestation human fetus as a granulocytopoietic and an erythropoietic organ . Although studies in fetal rodents support this view, a convincing demonstration of such in normal human fetuses is lacking . We tested the hypothesis that the human mid-gestation fetal spleen is normally (1) a site of active granulocytopoiesis and erythropoiesis, or (2) a site of production of specific granulocytopoietic or erythropoietic growth factors . This was accomplished using the spleens, livers, and long-bones of 18 human fetuses, 13-22 weeks gestation, immediately following elective, induced, pregnancy terminations . Organs of some of the abortuses were placed directly into formalin for histologic evaluation . Organs from others were subjected to RNA extraction for subsequent probing for specific hematopoietic growth factor mRNA . Cell suspensions were created from the organs of other abortuses for quantification of the absolute number of neutrophils and erythrocytes and their precursors and progenitors . Evidence of active hematopoiesis was present in marrow and liver but not spleen . Transcripts for granulocyte colony-stimulating factor (G-CSF) were detected in the marrow but not the spleen or liver, and transcripts for erythropoietin (Epo) were detected in the liver but not the spleen or marrow . The populations of hematopoietic progenitor cells and normoblasts in the fetal spleen cell were similar to those in fetal blood . Thus, it is likely that the hematopoietic progenitors recovered from fetal spleen cell suspensions are the result of blood within the spleen, rather than from hematopoiesis within the organ . The spleen of mid-gestation human fetuses, unlike the spleen of fetal rats, does not normally function as an active site of granulocytopoiesis or erythropoiesis, nor is it an active site for production of G-CSF or Epo.

J Neurosci, 1996 Nov 15, 16(22), 7240 - 52
A role in migration for the alpha V beta 1 integrin expressed on oligodendrocyte precursors; Milner R et al.; Myelination of the CNS requires the migration of oligodendrocyte precursors throughout the CNS from restricted regions within the ventricular and subventricular zones . In light of the significant effects of cell-extracellular matrix (ECM) interactions on cell migration in other developing systems, we have analyzed the role of integrins in oligodendrocyte precursor migration . We have shown previously that oligodendrocyte precursors in vitro express a limited repertoire of integrins, including alpha 6 beta 1, alpha v beta 3, and that differentiation is associated with downregulation of alpha v beta 1 and upregulation of alpha v beta 5 . Using a migration assay based on the movement of cells away from an agarose drop containing a high-density cell suspension, we find that RGD peptides (that block alpha v but not alpha 6 integrins) and anti-beta 1 antibodies block migration on an astrocyte-derived ECM, whereas anti-beta 3 antibodies have little effect . These results suggest that alpha v beta 1 but not alpha 6 beta 1 plays a role in oligodendrocyte precursor migration, and this is confirmed by the use of blocking monoclonal antibodies that distinguish these two integrins . In keeping with the results of others, we find that differentiated oligodendrocytes lose migratory potential and that the timing of this loss correlates with downregulation of alpha v beta 1 . Taken together with the work of others showing that ECM ligands for alpha v beta 1 are expressed within the CNS, we propose that this integrin plays a significant role in the migration of oligodendrocyte precursors in vivo and that its downregulation during differentiation could be an important factor regulating the migratory phenotype of these cells.

Transfus Sci, 1996 Dec, 17(4), 643 - 9
Development of tumor B-cell lymphocyte hybridoma (TBH) autovaccination . Results of a phase I-II clinical trial; Moviglia GA; To improve patient immune recognition of autologous tumor cells, we have developed a tumor B-cell lymphocyte hybridoma (TBH) autovaccination protocol . This approach is based on immunization of a cancer patient with a hybridoma cell suspension derived from the fusion of autologous activated B-cells and autologous cancer cells . This hybrid allows the host immune system to recognize and destroy oncocytes with low toxicity and high specificity . Of 21 treated patients with very advanced diseases, six complete responses and four partial responses were achieved . Overall, survival was prolonged . Side-effects and combination therapies with IL2, IL6 and gamma I/FN are discussed in this paper . Breast and colon cancer seem to be sensitive to this therapy.

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