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 metabo