Proc Natl Acad Sci U S A, 1999 Dec 7, 96(25), 14246 - 51 Structure of the glycosylphosphatidylinositol anchor of an arabinogalactan protein from Pyrus communis suspension-cultured cells; Oxley D et al.; Arabinogalactan proteins (AGPs) are proteoglycans of higher plants, which are implicated in growth and development . We recently have shown that two AGPs, NaAGP1 (from Nicotiana alata styles) and PcAGP1 (from Pyrus communis cell suspension culture), are modified by the addition of a glycosylphosphatidylinositol (GPI) anchor . However, paradoxically, both AGPs were buffer soluble rather than membrane associated . We now show that pear suspension cultured cells also contain membrane-bound GPI-anchored AGPs . This GPI anchor has the minimal core oligosaccharide structure, D-Manalpha(1-2)-D-Manalpha(1-6)-D-Manalpha(1-4)-D-GlcN -inositol, which is consistent with those found in animals, protozoa, and yeast, but with a partial beta(1-4)-galactosyl substitution of the 6-linked Man residue, and has a phosphoceramide lipid composed primarily of phytosphingosine and tetracosanoic acid . The secreted form of PcAGP1 contains a truncated GPI lacking the phosphoceramide moiety, suggesting that it is released from the membrane by the action of a phospholipase D . The implications of these findings are discussed in relation to the potential mechanisms by which GPI-anchored AGPs may be involved in signal transduction pathways. Masui, 1999 Nov, 48(11), 1194 - 201 {Measurements of extracellular water volume by bioelectrical impedance analysis during perioperative period of esophageal resection}; Tatara T et al.; Segmental bioelectrical impedance analysis (BIA) was conducted in five patients who underwent esophageal resections . Resistance values fitted at zero frequency (R0) in each body segment (arm, trunk and leg) were determined before the induction of anesthesia, at the end of surgery and on the second or third postoperative day . Extracellular water volume (ECW) in each body segment was estimated using the equation derived from the cell suspension theory . ECW in whole body was obtained from the sum of each body segment . R0 in trunk and leg significantly decreased at the end of surgery compared to the values before the induction of anesthesia (P < 0.05) . The change ratio of R0 in trunk before the induction of anesthesia was significantly lower at the end of surgery than that in arm (P < 0.05), resulting from the most striking fluid accumulation in the trunk . Postoperatively, R0 in all body segments, however, appeared to decrease similarly compared to the values before the induction of anesthesia, suggesting the redistribution phenomena of extracellular water among body segments . The correlation (r = 0.90, P < 0.001) and good agreement {bias = 0.01 (L)} between net fluid balances and estimates of ECW changes in whole body suggest that BIA allows close monitoring of tissue hydration during perioperative period by providing estimates of ECW in body segments. J Appl Microbiol, 1999 Oct, 87(4), 546 - 56 A study of dextran production from maltodextrin by cell suspensions of Gluconobacter oxydans NCIB 4943; Mountzouris KC et al.; This study investigated dextran synthesis from a commercial maltodextrin substrate using cell suspensions of G . oxydans NCIB 4943 as catalysts . Experiments were arranged according to a central composite statistical design . The effects of substrate concentration (10-100 g l-1), cell concentration (0.32-32.0 g wet weight l-1), time of reaction (8-48 h) and pH (3.5-5.5), each at three levels, on dextran yield and dextran molecular weight (MW), were investigated . Response surface methodology was used to assess factor interactions, and empirical models describing the two responses were fitted . Most of the variance in dextran yield could be explained by the fitted model (R2 = 0.96) . Dextran yield ranged from 1.21 to 41.69% . The presence of significant negative quadratic effects of cell concentration and time indicated that dextran yield reached a plateau and thus, optimum levels of cell concentration and time could be identified to maximize dextran yield . Dextran MW ranged from 6.6 to 38 kDa and was characterized by the significant interactions of reaction time with substrate concentration and cell concentration . The model, however, could account for only 60% of the variance in dextran MW . Possible reasons for this are discussed. Brain Res Bull, 1999 Nov 1, 50(4), 275 - 81 Impact of a preceding striatal excitotoxic lesion and treatment with ciliary neurotrophic factor on striatal graft survival; Petersen A et al.; The survival of grafted embryonic striatal tissue, dissected from the lateral ganglionic eminence, depends on the status of the host striatum . We found significantly larger volumes of surviving graft tissue and of striatal-like tissue (P-zone) within the graft, when the host striatum had been subjected to an excitotoxic lesion prior to transplantation surgery . Concomitantly the numbers of surviving grafted cells, assessed in both cresyl violet-stained sections and in sections stained with an immunohistochemical marker for striatal neurons, increased as compared to when graft tissue was placed in an intact unlesioned striatum . Finally, we examined the impact of treatment of the donor tissue with ciliary neurotrophic factor (CNTF) on graft survival . CNTF has previously been shown to protect striatal neurons against excitotoxic insults both in vitro and in vivo, but it did not improve striatal graft survival when added to the cell suspension prior to implantation. Brain, 1999 Dec, 122 ( Pt 12), 2321 - 35 Primary CA1 and conditionally immortal MHP36 cell grafts restore conditional discrimination learning and recall in marmosets after excitotoxic lesions of the hippocampal CA1 field; Virley D et al.; Common marmosets (Callithrix jacchus, n = 18) were trained to discriminate between rewarded and non-rewarded objects (simple discriminations, SDs) and to make conditional discriminations (CDs) when presented sequentially with two different pairs of identical objects signifying reward either in the right or left food well of the Wisconsin General Test Apparatus . After bilateral N-methyl-D-aspartate (0.12 M) lesions through the cornu ammonis-1 (CA1) field (7 microl in five sites), marmosets showed profound impairment in recall of CDs but not SDs, and were assigned to lesion only, lesion plus CA1 grafts and lesion plus Maudsley hippocampal cell line, clone 36 (MHP36) grafts groups matched for lesion-induced impairment . Cell suspension grafts (4 microl, 15-25 000 cells/microl) of cells dissected from the CA1 region of foetal brain at embryonic day 94-96, or of conditionally immortalized MHP36 cells, derived from the H-2Kb-tsA58 transgenic mouse neuroepithelium and labelled with {3H}thymidine, were infused at the lesion sites . The lesion plus MHP36 grafts group was injected five times per week with cyclosporin A (10 mg/kg) throughout testing . Lesion, grafted and intact control marmosets (n = 4-5/group) were tested on recall of SDs and CDs learned before lesioning and on acquisition of four new CDs over a 6-month period . Lesioned animals were highly impaired in recall and acquisition of CD tasks, but recall of SDs was not significantly disrupted . Both grafted groups of marmosets showed improvement to control level in recall of CDs . They were significantly slower in learning the first new CD task, but mastered the remaining tasks as efficiently as controls and were substantially superior to the lesion-only group . Visualized by Nissl staining, foetal grafts formed clumps of pyramidal-like cells within the denervated CA1 field, or jutted into the lateral ventricles . MHP36 cells, identified by beta-galactosidase staining and autoradiography, showed neuronal and astrocytic morphology, and were distributed evenly throughout the CA1 region . The results indicate that MHP36 cell grafts are as functionally effective as foetal grafts and appear to integrate into the host brain in a structurally appropriate manner, showing the capacity to differentiate into both mature neurons and glia, and to develop morphologies appropriate to the site of migration . These findings, which parallel the facilitative effects of foetal and MHP36 grafts in rats with ischaemic CA1 damage, offer encouragement for the development of conditionally immortal neuroepithelial stem cell lines for grafting in conditions of severe amnesia and hippocampal damage following recovery from cardiac arrest or other global ischaemic episodes. Plant Physiol Biochem, 1999 Nov, 37(11), 869 - 874 Chlorogenic acid in a Nicotiana plumbaginifolia cell suspension; Gillet F et al.; A phenylpropanoid compound has been characterized in a Nicotiana plumbaginifolia cell suspension . This compound has been isolated and purified by semi-preparative reverse phase-high performance liquid chromatography . Its structure has been identified by NMR spectroscopy as 5-O-caffeoylquinic acid, which is chlorogenic acid (CA) . The influence of culture conditions on the accumulation of this metabolite by N . plumbaginifolia cell suspensions has been studied . Darkness strongly inhibits the CA accumulation . Moreover, it has been shown that feeding experiments with caffeic acid had a deleterious effect upon the CA content . This one was not influenced by a supplementation with quinic acid. Bioessays, 1999 Dec, 21(12), 1061 - 8 How I learned to love carrots: the role of the cytoskeleton in shaping plant cells; Lloyd C; The carrot cell suspension was originally used because it provided a model system for studying directional cell expansion - a key process in plant morphogenesis . Early immunofluorescence studies of plant microtubules, using these cells, provided hints that the cortical array of microtubules was dynamic and this was later confirmed by microinjection studies on plant epidermal cells . A nonfixation approach for detecting F-actin was then developed on these cells and showed that, unlike animal cells, actin filaments remained associated with the nucleus throughout division and could have a role in aligning the plane of cell division . Currently, we are using detergent-extracted carrot cytoskeletons for isolating microtubule-associated proteins (MAPs) . I discuss how MAPs may be involved in the oriented deposition of cellulose in the cell wall . Mech Ageing Dev, 1999 Oct 1, 110(1-2), 1 - 12 Age-related alteration of intracellular ATP maintenance in the cell suspensions of mice cerebral cortex; Joo HJ et al.; Neurological alteration in the aging brain has been suggested to be due to, in part, a declined cellular energy metabolism . In order to understand age-related alteration of intracellular ATP maintenance, the present in vitro study measured change of intracellular adenosine triphosphate (ATP) content in cell suspensions of cerebral cortex isolated from male ICR mice aged 2 days (infant), 8 weeks (young adult) and 12 months (aged) under several different conditions, using the chemiluminescence technique . Among the three different ages, significant decrease of intracellular ATP content by oxygen deprivation for 15 min was observed in the cell suspensions of cerebral cortex from 12-month-old mice (P < 0.05) . When cell suspensions of 8-week cerebral cortex were incubated with or without glucose (0-60 min), intracellular ATP content decreased in a time-dependent manner under both conditions, but depletion rate was significantly high in the glucose-free condition . This decrease was maximally restored by adding 1 mM glucose as tested . In addition, the ability for intracellular ATP maintenance in the presence or absence of glucose was age-dependently different . The rank order of difference of intracellular ATP content between with and without glucose was 3 months > 12 months > 2 days . The highest decrease of intracellular ATP content by incubation without glucose was observed in the 12-month samples . Sodium cyanide (100 microM) produced a gradual ATP depletion in cerebral cortex suspended from 2-day-old mice, but rapid change in both 8-week and 12-month samples . Combination of cyanide and iodoacetate (3.5 mM) rapidly depleted the intracellular ATP content in all age groups tested . These results suggest that the aging process in the cerebral cortex of mice is accompanied by alteration of maintenance of intracellular ATP homeostasis under a given condition, and this may be associated with pathological change of overall mechanisms involved in the development of neuronal disease in the senescent brain. Cell Transplant, 1999 Sep-Oct, 8(5), 489 - 99 Lack of effect of short-term depletion of plasma complement C3 on the survival of syngeneic dopaminergic neurons following grafting into the intact rat striatum; Khorooshi MH et al.; Metabolically compromised cells may be subject to complement-mediated cytotoxicity . The aim of this study was to clarify to what extent plasma complement C3 might contribute to the low survival (5-20%) of grafted dopaminergic neurons . The survival of intrastriatal cell suspension grafts of syngeneic dopaminergic, tyrosine hydroxylase (TH)-containing neurons was compared in rats subjected to short-term i.v . treatment with 1) cobra venom factor (CVF), or 2) placebo treatment . Depletion of plasma complement C3 by CVF was confirmed by crossed immunoelectrophoresis . With 159 +/- 37 (mean +/- SEM) TH-immunoreactive and 154 + /- 40 TH mRNA-expressing neurons in the CVF-treated rats (n = 9), and 117 +/- 34 TH-immunoreactive and 160 +/- 49 TH mRNA-expressing neurons in placebo rats (n = 6), the CVF treatment did not increase the survival of the grafted dopaminergic neurons . Similarly, CVF had no apparent effect on the astroglial, microglial, or oligodendroglial cell response within and around the graft . The data indicate that depletion of plasma complement C3 at the time of grafting has no effect on the long-term survival of syngeneic ventral mesencephalic dopaminergic neuronal grafts. Cytometry, 1999 Jun 1, 36(2), 102 - 11 Evaluation of membrane physiology following fluorescence activated or magnetic cell separation; Seidl J et al.; BACKGROUND: Over the past decade, cell separation technology has become an important tool in various fields of cell biology allowing for the analysis or subsequent cultivation of specific cell subsets . The objective of the present study was to evaluate if the established sorting techniques fluorescence-activated (FACS) and magnetic cell separation (MACS) affect cell membrane physiology in order to define the most non-perturbing application for the separation of tumor and stromal cells . MATERIALS AND METHODS: Membrane physiology was monitored in single cell suspensions of adherently grown BT474 breast tumor cells and N1 normal skin fibroblasts using flow cytometry . Cell membrane integrity was evaluated by propidium iodide (PI) staining . Microviscosity within the lipophilic membrane layer was determined by a monomer/excimer method utilizing pyrene decanoic acid, membrane potential measurements were carried out using the fluorescence indicator DiBAC4(3), and Annexin-V-staining reflected transversal membrane asymmetry, and an altered phospholipid distribution . RESULTS: Not only the number of preparative cycles prior to cell separation but also the sort conditions during FACS resulted in loss of membrane integrity of a certain cell fraction . If these PI-positive cells were excluded from further analysis, neither MACS nor FACS affected membrane microviscosity while a clear hyperpolarization in both cell types after MACS resulting from exposure to the ferromagnetic matrix of the depletion column and the inhomogeneous magnetic field was shown . In addition, cell sorting of BT474 tumor cells by MACS and FACS was accompanied by the generation of an Annexin-V-positive/PI-negative cell fraction with altered phospholipid distribution . Data were discussed with regard to the sort-induced "stress" conditions such as exposure to hydrodynamic forces or magnetic fields . CONCLUSIONS: Both separation procedures modify cell membrane with neither technique being physiologically preferable for subsequent analysis or recultivation of the sorted cells. Blood Cells Mol Dis, 1999 Jun-Aug, 25(3-4), 170 - 9 Adenine nucleotide synthesis in human erythrocytes depends on the mode of supplementation of cell suspension with adenosine; Komarova SV et al.; In suspensions of washed human erythrocytes, adenosine added in a single dose to concentrations of 0.1-10.0 mmol/l suspension was deaminated at rates ranging from 10 to 50 mmol/l cells h . The sum of adenosine, inosine, and hypoxanthine concentrations in the suspension, as well as the intracellular concentration of ATP, remained constant . In the presence of 25-50 mmol/l orthophosphate, addition of a single dose of adenosine into erythrocyte suspension increased the ATP concentration by up to 280% of the initial level . If the initial adenosine concentrations were greater than 5 mmol/l suspension, ATP increased independently of adenosine concentration to the level determined only by the concentration of orthophosphate . After orthophosphate was returned to its initial level, ATP in erythrocytes began to decrease . In the presence of coformycin, erythrocytes utilised adenosine at a rate of 0.2-0.3 mmol/l cells h . Their adenylate pool increased at a rate of 0.10-0.16 mmol/l cells h for several hours, but intracellular ATP increased only slightly . The energy charge of cells decreased significantly from 0.86 +/- 0.05 (control) to 0.82 +/- 0.06 . Adenosine continuously pumped into erythrocyte suspensions at rates of 0.02-5.0 mmol/l cells h for several hours caused the adenylate pool of erythrocytes and intracellular ATP to increase synchronously at a rate of 0.02-0.35 mmol/l cells h . The energy charge of these erythrocytes increased significantly up to 0.91 +/- 0.03 . After pumping of adenosine was stopped, the intracellular ATP and the adenylate pool began to decrease, returning sometimes to the initial level in 2-3 h. J Biol Chem, 1999 Dec 3, 274(49), 34699 - 705 Characterization of the cryptogein binding sites on plant plasma membranes; Bourque S et al.; Cryptogein is a 98-amino acid proteinaceous elicitor of tobacco defense reactions . Specific binding of cryptogein to high affinity binding sites on tobacco plasma membranes has been previously reported (K(d) = 2 nM; number of binding sites: 220 fmol/mg of protein) . In this study, biochemical characterization of cryptogein binding sites reveals that they correspond to a plasma membrane glycoprotein(s) with an N-linked carbohydrate moiety, which is involved in cryptogein binding . Radiation inactivation experiments performed on tobacco plasma membrane preparations indicated that cryptogein bound specifically to a plasma membrane component with an apparent functional molecular mass of 193 kDa . Moreover, using the homobifunctional cross-linking reagent disuccinimidyl suberate and tobacco plasma membranes incubated with (125)I-cryptogein, we identified, after SDS-polyacrylamide gel electrophoresis and autoradiography, two (125)I-cryptogein linked N-glycoproteins of about 162 and 50 kDa . Similar results were obtained using Arabidopsis thaliana and Acer pseudoplatanus plasma membrane preparations, whereas cryptogein did not induce any effects on the corresponding cell suspensions . These results suggest that either cryptogein binds to nonfunctional binding sites, homologues to those present in tobacco plasma membranes, or that a protein involved in signal transduction after cryptogein recognition is absent or inactive in both A . pseudoplatanus and A . thaliana. FEBS Lett, 1999 Sep 24, 458(3), 349 - 53 Indomethacin-induced G1/S phase arrest of the plant cell cycle; Ehsan H et al.; In animal systems, indomethacin inhibits cAMP production via a prostaglandin-adenylyl cyclase pathway . To examine the possibility that a similar mechanism occurs in plants, the effect of indomethacin on the cell cycle of a tobacco bright yellow 2 (TBY-2) cell suspension was studied . Application of indomethacin during mitosis did not interfere with the M/G1 progression in synchronized BY-2 cells but it inhibited cAMP production at the beginning of the G1 phase and arrested the cell cycle progression at G1/S . These observations are discussed in relation to the putative involvement of cAMP biosynthesis in the cell cycle progression in TBY-2 cells. Br J Cancer, 1999 Oct, 81(4), 638 - 46 Characterization of a novel transplantable orthotopic rat bladder transitional cell tumour model; Xiao Z et al.; An animal tumour model that mimics the human counterpart is essential for preclinical evaluation of new treatment modalities . The objective of this study was to develop and characterize such a model . To accomplish this, the established AY-27 rat bladder transitional cell carcinoma (TCC) cell line was transplanted orthotopically into Fischer CDF344 female rats . AY-27 TCC cells were grown in monolayer cell culture and instilled intravesically as single cell suspensions into bladders that had been conditioned with mild acid washing . Tumour growth was assessed weekly by subjecting the rats to magnetic resonance imaging (MRI) . At intervals following implantation and MRI tumour detection, the animals were sacrificed for necropsy, histological examination and immunocytochemical studies . Flow cytometry was also performed for detection of Fas or Fas-ligand expression on AY-27 cells . The overall tumour establishment was 95% (97/102 rats) at 12-50 days, while in a subgroup of animals sacrificed at 16 days, 80 out of 82 animals (97%) developed TCC, the majority of which was superficial . Tumour stage was assessed by gross pathology and light microscopy . Histological examination of the tumour specimens confirmed the presence of grade II-III TCC . Immunocytochemistry confirmed that the tumour model maintained the features of TCC . The changes seen on MRI correlated well with the extent of tumour invasion identified histologically . Patchy carcinoma in situ could be detected histologically 12-13 days post-inoculation, and progressed to papillary tumour or invasive disease thereafter . Neither Fas nor Fas-ligand was expressed on AY-27 cells . The orthotopic AY-27 TCC model is highly reproducible and is ideal for preclinical studies on experimental intravesical therapies. Biomed Mater Eng, 1999, 9(3), 171 - 8 Microprocessor-controlled freezing device for cryopreservation of cell samples; Djuric D et al.; A freezing device for cryopreservation of blood mononuclear cells has been developed . The device is microcontroller operated, allowing cell freezing by a fully automatic, unattended process . To ensure optimum preservation, the temperature in the cell suspension uniformly decreases from room temperature to -100 degrees C and then the samples are transferred to long-term storage . The performance of the device has been tested using both physiological solution and a sample of cell suspension . The control of temperature variation of cell suspension in the entire temperature range has been realised with an accuracy better than +/- 0.1% . The viability of cells recovered from the frozen samples was 95% . The nitrogen consumption for one cycle of cryopreservation was 1.51 . In addition to the fully automatic mode, the manual and semi-automatic modes are available for research purposes . The device has been designed using low cost and widely used electronic components and materials, it is compact and simple to operate. Appl Microbiol Biotechnol, 1999 Oct, 52(4), 516 - 23 Production of functional human alpha 1-antitrypsin by plant cell culture; Terashima M et al.; Recombinant human alpha 1-antitrypsin (rAAT) was expressed and secreted from transgenic rice cell suspension cultures in its biologically active form . This was accomplished by transforming rice callus tissues with an expression vector, p3D-AAT, containing the cDNA for mature human AAT protein . Regulated expression and secretion of rAAT from this vector was achieved using the promoter, signal peptide, and terminator from a rice alpha-amylase gene Amy3D . The Amy3D gene of rice is tightly controlled by simple sugars such as sucrose . It was possible, therefore, to induce the expression of the rAAT by removing sucrose from the cultured media or by allowing the rice suspension cells to deplete sucrose catabolically . Although transgenic rice cell produced a heterogeneous population of the rAAT molecules, they had the same N-terminal amino acids as those found in serum-derived (native) AAT from humans . This result indicates that the rice signal peptidase recognizes and cleaves the novel sequence between the Amy3D signal peptide and the first amino acid of the mature human AAT . The highest molecular weight band seen on Western blots (AAT top band) was found to have the correct C-terminal amino acid sequence and normal elastase binding activity . Staining with biotin-concanavalin A and avidin horseradish peroxidase confirmed the glycosylation of the rAAT, albeit to a lesser extent than that observed with native AAT . The rAAT, purified by immunoaffinity chromatography, had the same association rate constant for porcine pancreatic elastase as the native AAT . Thermostability studies revealed that the rAAT and native AAT decayed at the same rate, suggesting that the rAAT is correctly folded . The productivity of rice suspension cells expressing rAAT was 4.6-5.7 mg/g dry cell . Taken together, these results support the use of rice cell culture as a promising new expression system for production of biologically active recombinant proteins. Biotechnol Bioeng, 1999, 66(2), 114 - 21 Determination of growth and lysis kinetics in plant cell suspension cultures from the measurement of esterase release; Steward N et al.; The death of Medicago sativa L . cells cultivated in a batch culture was investigated by measuring both the appearance of intact dead cells determined on the basis of the trypan blue (TB) dye exclusion, and the release of the cytoplasmic esterase activity into the culture medium upon cell death . Taking into account the strong instability of this released esterase activity, the total dead cell and lysed cell densities have been estimated . A mechanism for cell death and lysis is proposed and the specific rates of cell growth, death and lysis estimated . The specific rate of appearance of TB dead cells was low and essentially constant (0.25 day(-1)) during the first 8 days of the batch culture, and then increased above 1.5 day(-1) after 2 weeks of cultivation . Whereas no lysis occurred during the first seven days, this phenomenon occurred during the second period and accounted for about 20% of the total cell death by the end of the process . Thus, the viability determined by the trypan blue exclusion method appeared to be invalid after 7 days of culture . When lysis of viable cells is taken into consideration, the specific growth rate was significantly increased and growth was shown to continue for a further 8 days . Increased sensitivity of the cells to shear stresses and consequent cell lysis could be the result of a 35% increase in the cell size J Immunol, 1999 Nov 15, 163(10), 5211 - 8 Induction of tumor immunity by removing CD25+CD4+ T cells: a common basis between tumor immunity and autoimmunity; Shimizu J et al.; This study shows that removal of a T cell subpopulation can evoke effective tumor immunity in otherwise nonresponding animals . Elimination of CD25-expressing T cells, which constitute 5-10% of peripheral CD4+ T cells in normal naive mice, elicited potent immune responses to syngeneic tumors in vivo and eradicated them . The responses were mediated by tumor-specific CD8+ CTLs and tumor-nonspecific CD4-8- cytotoxic cells akin to NK cells . Furthermore, in vitro culture of CD25+4+ T cell-depleted splenic cell suspensions prepared from tumor-unsensitized normal mice led to spontaneous generation of similar CD4-8- cytotoxic cells capable of killing a broad spectrum of tumors; reconstitution of CD25+4+ T cells inhibited the generation . In this culture, self-reactive CD25-4+ T cells responding to self peptides/class II MHC complexes on APCs spontaneously proliferated upon removal of CD25+4+ T cells, secreting large amounts of IL-2 . The IL-2 thus produced appeared to be responsible for the generation of CD4-8- NK cells as lymphokine-activated killer cells, because direct addition of an equivalent amount of IL-2 to the culture of CD4-8- cells generated similar lymphokine-activated killer/NK cells, whereas coculture of normal CD4-8- cells with CD25-4+ T cells from IL-2-deficient mice did not . Thus, removal of immunoregulatory CD25+4+ T cells can abrogate immunological unresponsiveness to syngeneic tumors in vivo and in vitro, leading to spontaneous development of tumor-specific effector cells as well as tumor-nonspecific ones . This novel way of evoking tumor immunity would help to devise effective immunotherapy for cancer in humans. Plant Physiol, 1999 Nov, 121(3), 1025 - 1035 A Re-Evaluation of the Relative Roles of Two Invertases, INCW2 and IVR1, in Developing Maize Kernels and Other Tissues; Carlson SJ et al.; We have examined the relative abundance and distribution of the transcripts and protein products of a cell wall gene (Incw2) and a soluble invertase gene (Ivr1) to better understand their relative roles during maize (Zea mays L.) kernel development . In developing kernels the steady-state levels of Incw2 transcript increased dramatically from 0 to 12 d after pollination, while Ivr1 transcript, in contrast to a previous report, was undetectable . Consistent with the RNA expression data, the IVR1 protein could not be detected in kernel extracts using antisera raised to a synthetic peptide . Fractionation of the soluble form of invertase from developing kernels by isoelectric focusing and protein blots suggested that the enzyme activity was due to contamination of the cell wall invertase protein . A similar observation was made in a maize cell suspension culture in which Ivr1 RNA, but not IVR1 protein, was significantly modulated by sugars in the medium . Protein-blot analyses of the soluble enzyme activity suggested that changes in the enzyme activity are attributable to a cell wall invertase protein in the soluble fraction . Based on the collective evidence, we propose that the cell wall, but not the soluble invertase, is critical to heterotrophic sinks such as cell suspension cultures and developing kernels. Transplantation, 1999 Oct 27, 68(8), 1153 - 60 Discordant neural tissue xenografts survive longer in immunoglobulin deficient mice; Larsson LC et al.; BACKGROUND: The immune response against discordant xenografts in the brain is incompletely understood and remains a major obstacle for future clinical applications of xenogeneic neural tissue transplants in neurodegenerative disorders . To determine the role of antibodies in the rejection process, we compared graft survival and immune reactions between immunoglobulin deficient (IgKO) and normal mice . METHODS: A cell suspension of embryonic porcine ventral mesencephalon was injected into the striatum of adult normal and IgKO mice . Graft sizes and number of infiltrating CD4- and CD8-positive lymphocytes were determined by stereological methods at 4 days and 2, 4, and 6 weeks after the transplants . Microglial accumulation was determined using the optical densitometrical method . Intraparenchymal deposition of IgG was investigated at 4 days and 2 weeks . RESULTS: The majority of IgKO mice had surviving grafts for up to 4 weeks, whereas survival was minimal in control mice beyond 4 days . Graft sizes differed significantly between IgKO and control mice at 2 weeks (P<0.01, Kruskal Wallis ANOVA, followed by Mann Whitney test) . The majority of infiltrating lymphocytes were CD4-positive in control mice but CD8-positive in IgKO mice . Microglial accumulation was strong around surviving grafts in IgKO mice at 4 weeks . Prominent staining of IgG, diffuse in the transplanted hemisphere and specific on grafted neurons, was found in control mice . CONCLUSIONS: Our results suggest that immunoglobulins play an initiating role in rejection of discordant neural xenografts . After a prolonged graft survival of approximately 4 weeks, a cellular response with a large proportion CD8-positive cells leads to rejection in IgKO mice. Arch Toxicol, 1999 Sep, 73(7), 381 - 6 Effect of dental materials on gluconeogenesis in rat kidney tubules; Reichl FX et al.; The effect of dental composite components triethyleneglycoldimethacrylate (TEGDMA) and hydroxyethylmethacrylate (HEMA) as well as mercuric chloride (HgCl(2)) and methylmercury chloride (MeHgCl) on gluconeogenesis was investigated in isolated rat kidney tubules . From starved rats kidney tubules were prepared and isolated by digestion with collagenase . Every 10 min up to 60 min 1-ml samples were drawn from the cell suspension for quantitating the glucose content . Glucose formation in controls was 3.3 +/- 0.2 nmol/mg . per min (mean +/- SEM, n=21) . Relative rates of glucose formation were obtained by expressing individual rates as a percentage of the corresponding control . X-Y concentration curves (effective concentration, EC) of the substances were calculated by fitting a four-parametric sigmoid function to the relative rates of glucose formation at various test concentrations . At the end of the incubation period cell viability was assessed by trypan blue exclusion . Cell viability decreased within the 60 min interval from 90 to approx . 80% (controls), <25 (HEMA), <20 (TEGDMA), <10 (MeHgCl), and <10% (HgCl(2)) . Values of 50% effective concentration (EC(50)) were calculated from fitted curves . EC(50) values were (mmol; mean +/- SEM; n=4): HEMA, 17.7 +/- 2.9; TEGDMA, 1.8 +/- 0.2; MeHgCl, 0.018 +/- 0.0005; and HgCl(2), 0 . 0016 +/- 0.0005 . The toxic effect of HgCl(2) was approximately 1000 or 10 000 higher than that of the dental composite components TEGDMA or HEMA, respectively. C R Acad Sci III, 1999 Sep, 322(9), 743 - 8 Glutamine synthetase activity in Solanaceous cell suspensions accumulating alkaloids or not . 13C NMR and enzymatic assay; Mesnard F et al.; The metabolism of labelled pyruvate followed by 13C NMR and the measure of glutamine synthetase (GS) showed, according to previous results, a high activity of this enzyme in suspension cells of Nicotiana plumbaginifolia . This activity could derive glutamate from the alkaloid synthesizing pathways . However, a recent work showed that the rate of the GS gene transcription was inversely proportional to the Gln/Glu ratio . The measures of Gln and Glu concentrations in Nicotiana plumbaginifolia cells revealed that high GS activity correlates with the weak value of Gln/Glu ratio . Therefore, the hypothesis of GS dysfunctioning for the non-biosynthesis of alkaloids in N . plumbaginifolia suspension cells can be discarded . This conclusion is strengthened by the results obtained when using a GS inhibitor. Appl Environ Microbiol, 1999 Nov, 65(11), 5035 - 41 Oxidation of methyl halides by the facultative methylotroph strain IMB-1; Schaefer JK et al.; Washed cell suspensions of the facultative methylotroph strain IMB-1 grown on methyl bromide (MeBr) were able to consume methyl chloride (MeCl) and methyl iodide (MeI) as well as MeBr . Consumption of >100 microM MeBr by cells grown on glucose, acetate, or monomethylamine required induction . Induction was inhibited by chloramphenicol . However, cells had a constitutive ability to consume low concentrations (<20 nM) of MeBr . Glucose-grown cells were able to readily oxidize {(14)C}formaldehyde to (14)CO(2) but had only a small capacity for oxidation of {(14)C}methanol . Preincubation of cells with MeBr did not affect either activity, but MeBr-induced cells had a greater capacity for {(14)C}MeBr oxidation than did cells without preincubation . Consumption of MeBr was inhibited by MeI, and MeCl consumption was inhibited by MeBr . No inhibition of MeBr consumption occurred with methyl fluoride, propyl iodide, dibromomethane, dichloromethane, or difluoromethane, and in addition cells did not oxidize any of these compounds . Cells displayed Michaelis-Menten kinetics for the various methyl halides, with apparent K(s) values of 190, 280, and 6,100 nM for MeBr, MeI, and MeCl, respectively . These results suggest the presence of a single oxidation enzyme system specific for methyl halides (other than methyl fluoride) which runs through formaldehyde to CO(2) . The ease of induction of methyl halide oxidation in strain IMB-1 should facilitate its mass culture for the purpose of reducing MeBr emissions to the atmosphere from fumigated soils. Biofizika, 1999 Jul-Aug, 44(4), 708 - 13 {Kinetics of decrease in erythrocyte filterability under the effect of ephazol}; Dubniskii VZ et al.; The effect of palladium-containing complex Ephazol on the filtration rate of erythrocyte suspensions through nuclear filters was studied by the constant-pressure filtration method . It was shown that the filterability of red blood cells incubated with ephazol decreased . If the time necessary for a fixed volume of red blood cell suspension to pass through a filter was plotted against the time of incubation with Ephazol or against its initial concentration, the curves typical of autoaccelerated processes were obtained . From analysis of kinetic models, it was concluded that the effects observed are due to the nonlinear dependence of the filtration rate w on the rate at which an erythrocyte passes through a pore and the influence of Ephazol on the distribution of erythrocytes with respect to w . Several models describing changes in the distribution of erythrocytes with respect to w in the presence of Ephazol and possible mechanisms relating the filtration kinetics to the incubation parameters are discussed. J Nat Prod, 1999 Oct, 62(10), 1395 - 8 Isolation of labeled 9-dihydrobaccatin III and related taxoids from cell cultures of taxuscell cultures of taxus canadensis elicited with m; Ketchum RE et al.; Cell suspension cultures of Taxus canadensis rapidly produced paclitaxel (1) and other taxoids in response to elicitation with methyl jasmonate . Three of these taxoids, of potential value in the synthesis of taxoid analogues, have been isolated from cell cultures of Taxus canadensis and identified as 13-acetyl-9-dihydrobaccatin III (2), baccatin VI (3), and 9-dihydrobaccatin III (4) . Of these metabolites, 9-dihydrobaccatin III (4) has not been isolated from any Taxus species, whereas 13-acetyl-9-dihydrobaccatin III (2) and baccatin VI (3) have been isolated from a number of natural sources . 2D NMR techniques, mass spectrometry, and partial synthesis were used to rigorously elucidate the structure and stereochemistry of these natural products. J Cancer Res Clin Oncol, 1999 Nov, 125(11), 609 - 14 Repair of potentially lethal damage by total and quiescent cells in solid tumors following a neutron capture reaction; Masunaga S et al.; Purpose: We analyzed the time-course of changes in the sensitivity of total (proliferating + quiescent and quiescent (Q) cell populations within solid tumors in situ following a neutron capture reaction and compared it with that after gamma-ray irradiation . Methods: After continuous labeling of proliferating cells with BrdU for 5 days, mice bearing SCC VII tumors received thermal neutron irradiation with or without a (10)B-labeled compound (sodium {(10)B}borocaptate, BSH, or DL-p-{(10)B}boronophenylalanine, BPA), or gamma-ray irradiation . From 5 min to 72 h after treatment, tumors were excised, minced, and trypsinized . Cell suspensions were incubated for 48 h with the cytokinesis blocker cytochalasin-B . The micronucleus frequency for BrdU-unlabeled cells, Q cells at treatment, was then determined by immunofluorescence staining for BrdU . The micronucleus frequency for total cells was obtained from tumors that had not been pretreated with BrdU labeling . The sensitivity was evaluated in terms of the frequency of induced micronuclei in binuclear tumor cells (micronucleus frequency) . Results: Overall, Q cells showed greater repair capacities than total cells . gamma-Ray irradiation and neutron irradiation with BPA induced larger repair capacities in each cell population . In contrast, thermal neutron irradiation without a (10)B-labeled compound induced the smallest repair capacity in both cell populations . The use of a (10)B-labeled compound, especially BPA, widened the difference in sensitivity between total and Q cells, resulted in an increase in repair capacity in both cell populations, and made the repair patterns of the two cell populations look like those induced by gamma-ray irradiation . Conclusion: Differences in sensitivity and repair patterns following the neutron capture reaction were thought to depend on differences in the distribution of the (10)B-labeled compound between the proliferating and Q cell populations. Clin Cancer Res, 1999 Oct, 5(10 Suppl), 3232s - 3242s Radioimmunotherapy of small volume disease of colorectal cancer metastatic to the liver: preclinical evaluation in comparison to standard chemotherapy and initial results of a phase I clinical study; Behr TM et al.; At the time of surgery, occult metastases (micrometastases) are present in more than 50% of colorectal cancer patients, and the liver is the most frequent site of apparent metastatic disease . Frequently, adjuvant chemotherapy is unable to prevent tumor recurrence . Thus, novel therapeutic strategies are warranted . The aim of this study was to establish a model of human colon cancer metastatic to the liver of nude mice, to assess, in this setting, the therapeutic efficacy of radioimmunotherapy (RAIT) compared to standard chemotherapy and to evaluate, in a Phase I/II trial, the toxicity and therapeutic efficacy of RAIT in colorectal cancer patients with small volume disease metastatic to the liver . Multiple liver metastases of the human colon cancer cell line GW-39 were induced by intrasplenic injection of a 10% tumor cell suspension . Whereas controls were left untreated, therapy was initiated on day 10 or 20 after tumor inoculation with the 131I-labeled, low affinity anticarcinoembryonic antigen (anti-CEA) monoclonal antibody (MAb), F023C5 (Ka = 10(7) liters/mol), or the high-affinity anti-CEA MAb, MN-14 (Ka = 10(9) liters/mol), or chemotherapy (5-fluorouracil/leucovorin (folinic acid) versus irinotecan) at their respective maximum tolerated doses (MTDs) . Twelve colorectal cancer patients with small volume disease metastatic to the liver (all lesions < or = 2.5 cm) were entered into a mCi/m2-based Phase I dose escalation study with 131I-labeled humanized version of MN-14, hMN-14 . The patients were given single injections, starting at 50 mCi/m2 and escalating in 10-mCi/m2 increments . The MTD was defined as the dose level at which < or = 1 of 6 patients develop grade 4 myelotoxicity . In the mice, untreated controls died from rapidly progressing hepatic metastases at 6-8 weeks after tumor inoculation . The life span of mice treated with 5-fluorouracil/leucovorin was prolonged for only 1-3 weeks, whereas irinotecan led to a 5-8-week prolongation . In contrast, at their respective MTDs, the 131I-labeled low-affinity anti-CEA MAb, F023C5, led to a 20% permanent cure rate, and the high affinity MAb, MN-14, led to an 80% permanent cure rate, when therapy was initiated at 10 days after tumor inoculation . In the 20-day-old tumor stage, although it prolonged life, 131I-F023C5 was unable to achieve cures, whereas 131I-MN-14 was still successful in 20% . Histologically, no remaining viable tumor cells could be demonstrated in these animals surviving > 6 months . In patients, the MTD was reached at 60 mCi/m2 of hMN-14 (at 70 mCi/m2, two of three grade 4 myelotoxicities) . Of 11 assessable patients, 2 had partial remissions (corresponding to an objective response rate of 18%), and 5 (45%) had minor/mixed responses or experienced stabilization of previously rapidly progressing disease . These data suggest that in small volume disease, RAIT may be superior to conventional chemotherapy . Antibodies of higher affinity seem to be clearly superior . The clinical response rates in patients with small volume disease are encouraging, being comparable to the response rates of conventional chemotherapeutic regimens but with fewer side effects . Ongoing studies will show whether treatment at the MTD will further improve therapeutic results. J Investig Dermatol Symp Proc, 1999 Sep, 4(2), 169 - 72 Molecular mechanisms involved in the migration of epidermal dendritic cells in the skin; Nakamura K et al.; The murine epidermis contains two types of dendritic cells (DC), Thy-1+ dendritic epidermal T cells (DETC) and Langerhans cells . In this review, we introduce our data obtained using a skin organ culture system to examine the migratory capacity of DETC and Langerhans cells into the epidermis . DETC or Langerhans cells were depleted by topical application ofclobetazole propionate (CP) solution onto the murine ears . CP-treated or untreated ear skin was co-cultured with syngeneic (semi-syngeneic, or allogenic, in experiments with Langerhans cells) epidermal cell suspension . We found (i) that donor DETC or Langerhans cells migrated into the CP-treated epidermis as well as into untreated epidermis, (ii) that leukosialin Ly48 recognized by monoclonal antibody S11 and TNF-alpha strongly inhibited donor Langerhans cell migration into the epidermis . We mention other molecules that may participate in the migration of Langerhans cells such as chemotactic cytokines, monocyte chemoattractant protein (MCP)-1, TGF-beta and skin-homing molecule, cutaneous lymphocyte-associated antigen (CLA) on Langerhans cells. Glia, 1999 Nov, 28(2), 156 - 65 Effects of schwann cell suspension grafts on axon regeneration in subacute and chronic CNS traumatic injuries; Stichel CC et al.; In a previous study, we have shown that microtransplanted Schwann cell suspensions foster structural recovery of the acutely transected postcommissural fornix . The emphasis of the present study was to examine whether subacutely and chronically injured axons also demonstrate significant responsiveness to implanted Schwann cells . Microinjected suspensions of cultured Schwann cells i) elicited a growth response and attracted axons in a subacute and chronic traumatic lesion but ii) failed to stimulate regrowth of the postcommissural fornix projection at any nonacute postlesion stage . In conclusion, the single intervention strategy of Schwann cell microimplantation is not sufficient to ensure regeneration of the subacutally or chronically transected postcommissural fornix . The use of Schwann cells as stimulators of axon regrowth depends on the neuronal cell type and the appropriate postinjury time point . Jpn J Physiol, 1999 Aug, 49(4), 379 - 87 Automatic measurement of the red cell oxygen dissociation curve identical with the whole blood curve; Mawjood AH et al.; Automatic measurement of the entire oxygen dissociation curve (ODC) of blood and hemoglobin provides a useful means for evaluating their gas-transport function . The automatic oxygenation apparatus previously developed by Imai et al . (1970, 1981), which uses a polarographic determination of partial pressure of oxygen and a spectrophotometric determination of oxygen saturation of hemoglobin, has mostly been used for the measurement of accurate ODCs of hemoglobin solution . However, it was not suitable for red cell suspension because a significant noise was superimposed on the absorbance signal due to light-scattering by red cells . In the present study, we have overcome this problem by using an integrating sphere for the photometric system . Through extensive tests we found the optimal experimental conditions for obtaining the red cell oxygenation data that were identical with the whole blood data with respect to the position (oxygen affinity) and shape (sigmoid character) of the ODC and its pH-dependence (the Bohr effect) . The accuracy was higher than that of commercially available automatic apparatuses such as the "Hemox-Analyzer" (Technical Consulting Service) and "Hem-O-Scan" (Aminco) . Thus, our method provides an easy and convenient means for obtaining accurate ODCs mimicking the whole blood ODCs from one drop of whole blood . An application of our method to the effect of blood storage on ODC is presented, demonstrating the usefulness of our method. J Neurosci Methods, 1999 Sep 15, 91(1-2), 101 - 7 A chemiluminescent catecholamine assay: its application for monitoring adrenergic transmitter release; Israel M et al.; A chemiluminescent procedure for measuring catecholamines (dopamine, norepinephrine, epinephrine) is described . It is based on the observation that lactoperoxidase catalyses both the oxidation of catecholamines, and the chemiluminescent reaction of luminol with their oxidation product . The assay has been adapted for continuously monitoring the release of catecholamines from adrenergic tissues, from cell suspensions and from cells loaded in culture with dopamine. Br J Haematol, 1999 Sep, 106(4), 1033 - 6 Kaposi's sarcoma-associated herpesvirus (KSHV/HHV-8) DNA sequences are absent in leukapheresis products and ex vivo expanded CD34+ cells from multiple myeloma patients; De Greef C et al.; Recently it was reported that Kaposi's sarcoma-associated herpesvirus (KSHV/HHV-8) infects bone marrow (BM) dendritic cells (DC) in multiple myeloma (MM) patients and therefore might play a role in MM development . Because of the use of myeloid growth factors like GM-CSF and G-CSF for the mobilization of peripheral blood progenitor cells (PBPC), the subsequent increase of DC precursors might imply a risk for KSHV contamination in PBPC grafts . Therefore, in this study leukapheresis products and ex vivo cultured CD34+ cell suspensions were analysed . KSHV DNA could not be amplified in any of them. Plant Physiol, 1999 Oct, 121(2), 535 - 43 Ascorbate biosynthesis in Arabidopsis cell suspension culture; Davey MW et al.; The biosynthesis of L-ascorbic acid (L-AA) in an Arabidopsis (L.) Heynh . cell suspension culture was studied by quantifying the effects of incubation with a range of potential biosynthetic precursors, analogs, and inhibitors on the intracellular levels of reduced and oxidized forms of L-AA . Our results support the recently published biosynthetic pathway of L-AA from L-galactose (G.L . Wheeler, M.A . Jones, N . Smirnoff {1998} Nature 393: 365-369), but suggest that Arabidopsis cell suspension culture simultaneously contains two other routes leading to L-AA . The possible physiological significance of these alternate routes is discussed. Mol Gen Genet, 1999 Sep, 262(2), 239 - 49 Four mismatch repair paralogues coexist in Arabidopsis thaliana: AtMSH2, AtMSH3, AtMSH6-1 and AtMSH6-2; Ade J et al.; By using degenerate oligonucleotides based on the sequence homology between known MutS homologues, three MSH cDNAs belonging to the MSH2, MSH3 and MSH6 families, as defined in eukaryotes, have been isolated from Arabhidopsis thaliana (ecotype Columbia) . Genomic sequences for two of these genes (AtMSH2 and AtMSH6-2) were also isolated and determined, whereas the genomic sequence of AtMSH3 was obtained through the Arabidopsis sequencing project, as was the sequence of a second, distinct AtMSH6 homologue (AtMSH6-1) . Comparative analysis of the AtMSH2 Landsberg erecta genomic sequence (reported here) and the previously described AtMSH2 Columbia allele revealed several polymorphisms, including the presence of a small, transposon-like element in the 3' untranscribed region of the former allele . Arabidopsis is the first organism to show such divergence of two AtMSH6 genes; the divergence is strongly supported by sequence data and phylogenetic analysis . Southern analysis revealed that the three genes we have isolated exist as single copies, and genetic mapping indicated that AtMSH2 and AtMSH6-2 both reside on chromosome III . Finally, expression of these three genes could only be observed in suspensions of A . thaliana cells . Such a cell suspension divides actively after subculture, and the AtMSH genes are most strongly expressed at this stage. Blood, 1999 Oct 15, 94(8), 2819 - 26 Early maturation of T-cell progenitors in the absence of glucocorticoids; Sacedon R et al.; In the present work, we demonstrated that both fetal liver and thymic T-cell precursors express glucocorticoid receptors (GRs) indirectly suggesting a role for glucocorticoids (GCs) in the earliest events of T-cell differentiation . To evaluate this issue, we analyzed the thymic ontogeny in the progeny of adrenalectomized pregnant rats (Adx fetuses), an in vivo experimental model, which ensures the absence of circulating GCs until the establishment of the fetal hypothalamus-pituitary-adrenal (HPA) axis . In the absence of maternal GCs, T-cell development was significantly accelerated, the process being reversed by in vivo GC replacement . Mature single positive thymocytes (both CD4 and CD8) appeared in 16-day old fetal Adx thymus when in the control fetuses, most thymocytes still remained in the double-negative (DN) CD4(-)CD8(-) cell compartment . In addition, emigration of T-cell receptor (TcR)alphabeta positive cells to the spleen also occurred earlier in Adx fetuses than in control ones . In vitro recolonization of cultured deoxiguanosine-treated mouse fetal thymus lobes with 13-day-old fetal liver cell suspensions from both Adx and control fetuses demonstrated changes in the developmental capabilities of fetal liver T-cell precursors from embryos grown in the absence of GCs . Furthermore, a precocious lymphoid colonization of the thymic primordium from Adx fetuses was evidenced by ultrastructural analysis of both Adx and Sham early thymus . Both findings accounted for the accelerated T-cell differentiation observed in Adx fetuses . Together, these results support a role for GCs not only in the thymic cell death, but also in the early steps of T-cell differentiation. Eur J Oral Sci, 1999 Oct, 107(5), 338 - 43 Chemosensitivity testing of oral cancer cells treated with a p185neu-specific agent; Werkmeister R et al.; The amplification and overexpression of the erbB-2 oncogene and its involvement in tumorigenesis makes this receptor an appropriate target for specific agents directed towards tumor cells . The purpose of this study was to evaluate the in vitro effect of the bacterially produced recombinant immunotoxin scFv(FRP5)-ETA on the protein synthesis and adenosine triphosphate (ATP) reduction in oral squamous cell carcinoma (OSCC) cells . This agent recognizes the erbB-2 receptor and inhibits protein synthesis in receptor-overexpressing cells . OSCC cells were selected for this study, and amplification and expression levels of the erbB-2 receptor were determined . Cell suspensions were cultured for 6 d with various concentrations of scFv(FRP5)-ETA (1-1000 ng/ml) . A431 and MDA-MB468 cell lines were used as controls . Chemosensibility of tumor cells was measured by {3H}leucine incorporation assay and by an ATP luminescence assay . In OSCC cells with amplification and overexpression of erbB-2 inhibition, up to 92% of protein synthesis and 90% of ATP reduction was observed when cells were exposed to 1,000 ng/ml immunotoxin . In OSCC cells showing a deletion of erbB-2 and in erbB-2-negative MDA-MB468 cells, protein synthesis was inhibited by 22% and 8%, respectively . These results indicate that the effectiveness of a recombinant immunotoxin targeting erbB-2 receptors in OSCC cells depends on the level of erbB-2 amplification and expression, that it is highly specific for tumor cells expressing these receptors, and that a dose-dependency can be observed. Eur J Gastroenterol Hepatol, 1999 Aug, 11(8), 839 - 43 Increased release of interleukin-6 by oesophageal mucosa in children with reflux oesophagitis; Corrado G et al.; OBJECTIVE: To evaluate the release of interleukin-6 (IL-6) by oesophageal mucosa and to establish the serum levels of IL-6 and C-reactive protein (CRP), and plasma fibrinogen in children with reflux oesophagitis . DESIGN: In a prospective study, IL-6 release by tissue fragments obtained from oesophageal biopsies was determined and serum IL-6 and CRP as well as plasma fibrinogen were analysed . METHODS: The study population comprised ten children with reflux oesophagitis, diagnosed on the basis of 24 h oesophageal pH monitoring and endoscopy with biopsies . Ten children with recurrent abdominal pain were studied for comparative purposes . Biopsy tissue fragments were processed to obtain a cell suspension and the release of IL-6 was determined in culture medium . Serum IL-6 levels were measured by ELISA, serum CRP by turbidimetry, and plasma fibrinogen by spectrophotometry . RESULTS: Oesophageal cells obtained from reflux oesophagitis patients synthesize and release in vitro a significantly higher amount of IL-6 than controls (71.26+/-19.5 versus 31.67+/-8.02 pg/10(6) cells; P<0.01) . Serum IL-6, serum CRP and plasma fibrinogen levels were not statistically different between patients with reflux oesophagitis and controls . CONCLUSIONS: These results suggest a short-term action of IL-6 since its effects could be exerted only in the microenvironment of the oesophageal mucosa. Radiat Med, 1999 Jul-Aug, 17(4), 259 - 64 Potentially lethal damage repair by total and quiescent tumor cells following various DNA-damaging treatments; Masunaga S et al.; After continuous labeling of proliferating (P) cells with 5-bromo-2'-deoxyuridine (BrdU) for 5 days, SCC VII tumor-bearing mice received various kinds of DNA-damaging treatments: gamma-ray irradiation, tirapazamine (TPZ, hypoxia-specific cytotoxin) administration, or cisplatin injection . From 0.5 to 72 hr after treatment, tumors were excised, minced, and trypsinized . Single tumor cell suspensions were incubated for 48 hr with a cytokinesis-blocker, cytochalasin-B . Then, the micronucleus (MN) frequency for BrdU-unlabeled cells, quiescent (Q) cells at treatment, was determined using immunofluorescence staining for BrdU . The MN frequency for total (P+Q) cells was obtained from tumors that were not pretreated with BrdU labeling . The sensitivity to each DNA-damaging treatment was evaluated in terms of the frequency of induced micronuclei in binuclear tumor cells (MN frequency) . Treatment with gamma-rays or cisplatin resulted in a larger MN frequency in total cells than in Q cells . In contrast, TPZ treatment produced a smaller MN frequency in total cells than in Q cells . Regardless of the treatment used, Q cells showed greater repair capacities than total cells . However, TPZ caused much smaller repair capacity in both total and Q cells, compared with gamma-rays or cisplatin . Gamma-rays and cisplatin produced similar repair patterns . Differences in sensitivity between total and Q cells and repair patterns of the two cell populations were thought to depend on differences between the two cell populations in the toxicity of the DNA-damaging treatment and distribution pattern of the anticancer agent. Br J Pharmacol, 1999 Sep, 128(2), 472 - 8 The effects of endomorphin-1 and endomorphin-2 in CHO cells expressing recombinant mu-opioid receptors and SH-SY5Y cells; Harrison C et al.; 1 Endomorphin-1 and -2 (E-1/E-2) have been proposed as endogenous ligands for the mu-opioid receptor . The aims of this study are to characterize the binding of E-1/E-2 and the subsequent effects on cyclic AMP formation and {Ca2+}i levels in SH-SY5Y and Chinese hamster ovary (CHO) cells expressing endogenous and recombinant mu-opioid receptors . 2 E-1 displaced {3H}-diprenorphine ({3H}-DPN) binding in CHO micro and SH-SY5Y membranes with pKi values of 8.02+/-0.09 and 8.54+/-0.13 respectively . E-2 displaced {3H}-DPN binding in CHOmu and SH-SY5Y cells with pKi values of 7.82+/-0.11 and 8.43+/-0.13 respectively . E-1/E-2 bound weakly to CHOdelta and CHOkappa membranes, with IC50 values of greater than 10 microM . 3 In CHOmu cells, E-1/E-2 inhibited forskolin (1 microM) stimulated cyclic AMP formation with pIC50 values of 8.03+/-0.16 (Imax = 53.0+/-9 . 3%) and 8.15+/-0.24 (Imax = 56.3+/-3.8%) respectively . In SH-SY5Y cells E1/E2 inhibited forskolin stimulated cyclic AMP formation with pIC50 values of 7.72+/-0.13 (Imax=46.9+/-5.6%) and 8.11+/-0.31 (Imax = 40.2+/-2.8%) respectively . 4 E-1/E-2 (1 microM) increased {Ca2+}i in fura-2 loaded CHOmu cell suspensions in a thapsigargin sensitive and naloxone reversible manner . Mean increases observed were 106+/-28 and 69+/-6.7 nM respectively . In single adherent cells E-1/E-2 (1 microM) increased {Ca2+}i with a mean 340/380 ratio change of 0.81+/-0.09 and 0.40+/-0.08 ratio units respectively . E-1/E-2 failed to increase intracellular calcium in CHOdelta, CHOkappa and SH-SY5Y cells . 5 These data show that E-1/E-2 bind with high affinity and selectivity to mu-opioid receptors and modulate signal transduction pathways typical of opioids . This provides further evidence that these two peptides may be endogenous ligands at the mu-opioid receptor. Eur J Neurosci, 1999 Sep, 11(9), 3073 - 81 Differential effects of Bcl-2 overexpression on fibre outgrowth and survival of embryonic dopaminergic neurons in intracerebral transplants; Schierle GS et al.; The causes of death of transplanted neurons are not known in detail, but apoptotic mechanisms involving caspase activation are likely to play a role . We examined whether overexpression of the anti-apoptotic protein Bcl-2 may enhance the survival of dopaminergic {tyrosine hydroxylase (TH)-immunoreactive} grafted neurons . For this purpose, we prepared cells from embryonic day 13 ventral mesencephalon (VM) of mice overexpressing human Bcl-2, or from their wild-type littermates . The bcl-2 transgene was strongly expressed in these cells, and resulted in protection of neuronal cultures from death triggered by serum deprivation or exposure to staurosporine . To model pretransplantation stress more closely in vitro, we stored dissociated embryonic mesencephalic cells for 8 h in the same type of medium used for intracerebral transplantation . This resulted in massive cell death as quantified by lactate dehydrogenase (LDH) release, and increased DNA fragmentation . Although this cell loss was strongly reduced by a caspase inhibitor, Bcl-2 had no significant protective effect . Finally, mesencephalic cell suspensions were xenografted into the striatum of immunosuppressed hemiparkinsonian rats . Neither the survival of TH-immunopositive transplanted neurons nor the functional recovery of the rats was improved by Bcl-2, although the Bcl-2 protein was strongly expressed in transgenic grafts 5 weeks after implantation, and dopaminergic fibre outgrowth from the grafts was significantly improved . These data suggest that cell death in neuronal transplants involves apoptotic mechanisms that can bypass negative regulation by Bcl-2. J Pediatr Surg, 1999 Sep, 34(9), 1378 - 84 Immunoscintigraphy of xenotransplanted hepatoblastoma with iodine 131-labeled anti-alpha-fetoprotein monoclonal antibody; Fuchs J et al.; BACKGROUND/PURPOSE: Hepatoblastoma (HB) is the most common primary malignant liver tumor affecting infants and young children . The alpha-fetoprotein level is elevated in 95% of all children with hepatoblastoma . Therefore, it is of interest to assess targeting of the HB marker alpha-fetoprotein by antibody imaging . In this pilot study, the authors investigated the radioimmunoscintigraphy of xenotransplanted HB in nude mice utilizing an anti-alpha-fetoprotein antibody . METHODS: HB cell suspensions from tumors of 3 children were transplanted subcutaneously into nude mice NMRI (nu/nu) . A total of 200 microg of intact anti-alpha-fetoprotein antibody was injected intravenously into 8 animals from each HB . Before injection, the monoclonal antibody was labeled with iodine (I) 131 (specific activity of 75 MBq/mg, labeling yield of 95%) using the conventional iodogen method . Planar scintigraphic images of anesthetized mice in posterior views were acquired with a gamma camera immediately after injection, and after 1, 2, 3, 7, and 14 days . The biodistribution data were obtained by killing and dissecting animals, and the activity in the tissues was measured in a gamma counter . The alpha-fetoprotein levels in the animals' sera were recorded 15 days after imaging and were compared with the control group . RESULTS: A total of 66% of the hepatoblastomas could be detected by scintigraphy . Within 24 hours, the mean specific tumor uptake in nude mice hepatoblastomas with a volume of over 1,000 mm3, was 14% per injected dose (+/-3.9%) . The biological half-life of the labeled antibody complex in the tumor was 3.86 (+/-0.84) days . Thyroid uptake of free I-131 was 2.85% per injected dose (+/-1.5%) reflecting the deiodination of the labeled antibody complex . CONCLUSIONS: The results show the possibility of imaging xenotransplanted hepatoblastoma with 131I-labeled anti-alpha-fetoprotein and may, in the future, determine tumor recurrence and extension, and thereby improve the prognosis of advanced HBs. AIDS Res Hum Retroviruses, 1999 Sep 20, 15(14), 1305 - 14 The dendritic cell-T cell milieu of the lymphoid tissue of the tonsil provides a locale in which SIV can reside and propagate at chronic stages of infection; Hu J et al.; Previous studies described the presence of numerous human immunodeficiency virus (HIV)-positive cells within and just beneath the mucosal surfaces of the tonsillar tissue of HIV-1-infected individuals . The virus-positive cells were most abundant in the dendritic cell (DC)-T cell rich areas of the lymphoepithelia lining the crypts, and consisted of multinucleated syncytia that contained DCs . This suggested that such cells within the tonsillar tissue might represent a site for chronic virus replication in infected individuals . Using the simian immunodeficiency virus (SIV)-macaque system, we chose to study further the viral distribution within the tonsillar tissue of animals infected via the vaginal route 8-10 months earlier . Our initial studies demonstrated that in situ hybridization (ISH)-positive DCs and T cells could be identified within the genital mucosa and draining lymph nodes of these infected animals even at this chronic stage of infection . Here we specifically examined the distal mucosa-associated lymphoid tissues of the tonsil . ISH-positive cells were mostly restricted to the DC-rich T cell areas of the underlying lymphoid tissue . However, T cells were the most commonly infected cell type and virus-positive cells were rarely found within the epithelia . In isolated cell suspensions, ISH-positive lymphocytes were often tightly associated with ISH-negative DCs, although few ISH-positive lymphocytes were often tightly associated with ISH-negative DCs, although few ISH-positive DCs could be identified within these clusters . Therefore, the naturally occurring DC-T cell milieu of the lymphoid tissue of the tonsil provides a locale in which SIV can reside and propagate on a chronic basis, even many months after the animals were infected by virus crossing the genital mucosa. Plant J, 1999 Sep, 19(5), 509 - 19 Cloning and expression of a cDNA encoding betanidin 5-O-glucosyltransferase, a betanidin- and flavonoid-specific enzyme with high homology to inducible glucosyltransferases from the Solanaceae; Vogt T et al.; Based on protein sequence data and RT-PCR, a full length cDNA encoding betanidin 5-O-glucosyltransferase (5-GT) was obtained from a cDNA library of Dorotheanthus bellidiformis (Burm.f.) N.E.Br . (Aizoaceae) . 5-GT catalyses the transfer of glucose from UDP-glucose to the 5-hydroxyl group of the chromogenic betanidin . Betanidin and its conjugates, referred to as betacyanins, are characteristic fruit and flower pigments in most members of the Caryophyllales, which fail to synthesise anthocyanins . The 5-GT cDNA displayed homology to previously published glucosyltransferase sequences and exhibited high identity to sequences of several inducible glucosyltransferases of tobacco and tomato (Solanaceae) . The open reading frame encodes a polypeptide of 489 amino acids with a calculated molecular mass of 55.24 kDa . The corresponding cDNA was expressed in Escherichia coli . The recombinant protein displayed identical substrate specificity compared to the native enzyme purified from D . bellidiformis cell suspension cultures . In addition to the natural substrate betanidin, ortho-dihydroxylated flavonols and flavones were glycosylated preferentially at the B-ring 4'-hydroxyl group . 5-GT is the first enzyme of betalain biosynthesis in plants, of which the corresponding cDNA has been cloned and expressed . The results are discussed in relation to molecular evolution of plant glucosyl- transferases. Br J Surg, 1999 Sep, 86(9), 1180 - 4 Impact of laparoscopic colonic resection on tumour growth and spread in an experimental model; Gutt CN et al.; BACKGROUND: The influence of surgical manipulation and carbon dioxide pneumoperitoneum on intraperitoneal tumour growth and port-site metastasis during laparoscopic colon resection is still unknown . METHODS: Some 33 male WAG/Rij rats were randomized into three experimental groups: a laparoscopy group with carbon dioxide pneumoperitoneum (n = 11), a gasless laparoscopy group (n = 11) and a laparotomy group (n = 11) . After transanal injection of a tumour cell suspension (1 x 106 CC 531 cells) into the distal colon, a colon segment resection and an end-to-end anastomosis (laparoscopy; intra-abdominal technique) were performed . Tumour growth was scored semiquantitatively 24 days after the operation . Data were analysed by the Kruskal-Wallis test . RESULTS: The tumour indices from the four locations with the greatest tumour growth were significantly decreased in the laparoscopy group with carbon dioxide pneumoperitoneum compared with the gasless laparoscopy and laparotomy groups (P < 0.01) . Port-site metastases were significantly decreased in the carbon dioxide pneumoperitoneum group compared with the gasless laparoscopy group (P = 0.05) . CONCLUSION: A full laparotomy incision promotes greater tumour growth than does carbon dioxide pneumoperitoneum . Surgical manipulation stimulates local tumour spread more than the establishment of a carbon dioxide pneumoperitoneum. Eur J Pharmacol, 1999 Aug 27, 379(2-3), 237 - 42 The effect of C-terminal truncation of the recombinant delta-opioid receptor on Ca2+i signaling; Harrison C et al.; We have previously shown a stimulatory coupling of the recombinant delta-opioid receptor to phospholipase C leading to production of inositol (1,4,5) triphosphate {Ins(1,4,5)P3} that is affected by truncation of the C-terminus of the receptor . Using a C-terminal mutant of the delta-opioid receptor lacking the final 37 amino acids (CHOdelta37), we examined its coupling to intracellular calcium ion concentration ({Ca2+}i) compared to the full length wild type receptor (CHOdeltaWT) in transfected Chinese hamster ovary (CHO) cells . D-{Pen2,5}enkephalin (DPDPE) mediated increases in {Ca2+}i were measured fluorimetrically in fura-2 loaded whole cell suspensions . DPDPE produced time- and concentration-dependent increases in {Ca2+}i in CHOdeltaWT and CHOdelta37 . In both cell types the DPDPE simulated increase in {Ca2+}i was naloxone reversible and pertussis toxin and thapsigargin sensitive . Removal of the C-terminus resulted in a rightward shift of the Ca2+ release concentration-response curve {pEC50 = 8.43 +/- 0.13 and 6.08 +/- 0.25 for CHOdeltaWT and CHOdelta37, respectively} . These data indicate that the C-terminus of the recombinant delta-opioid receptor is important in {Ca2+}i coupling and may be attributed to the effect of C-terminus truncation on phospholipase C coupling reported previously. J Am Soc Nephrol, 1997 Apr, 8(4), 530 - 4 Inhibition of IMCD 11 beta-hydroxysteroid dehydrogenase type 2 by low pH and acute acid loading; Nolan PJ et al.; Mineralocorticoid receptors in the inner medullary collecting duct (IMCD) are protected from glucocorticoid binding by an enzyme, 11 beta-hydroxysteroid dehydrogenase type 2 (11 beta-HSD2) . To study the role of 11 beta-HSD2 in acid-base homeostasis, 11 beta-HSD2 activity was measured in rat IMCD-enriched cell suspensions . Homogenates of cell suspensions were incubated in buffers ranging in pH from 6.00 to 8.15 in the presence of 1 microCi of 3H-corticosterone (CS) and 400 microM NAD+ . Enzyme activity was expressed as the amount of 3H-CS converted to 3H-11-dehydrocorticosterone (DHCS) . IMCD 11 beta-HSD2 activity at pH 6.5 was 49% of activity at pH 7.5; 22.5 versus 11.0 fmol/microgram of protein per h . Experiments also were performed on intact cell suspensions at pH 7.5 and 6.5 . There was a 42% inhibition in the IMCD cell suspension conversion rate of 3H-CS to 3H-11-DHCS at pH 6.5; 13.1 versus 7.6 fmol/microgram per h (P < 0.005) . In cell suspensions at pH 7.5, 1-day acid loading caused a 26% inhibition in conversion rate, 13.2 versus 9.9 fmol/microgram per h (P < 0.05), when compared with controls . These results suggest that during acute metabolic acidosis, IMCD 11 beta-HSD2 is inhibited and may allow access to the mineralocorticoid receptors by glucocorticoids. Oral Microbiol Immunol, 1999 Jun, 14(3), 143 - 52 Cloning of Prevotella intermedia loci demonstrating multiple hemolytic domains; Beem JE et al.; A gene bank was created from Prevotella intermedia strain 27 chromosomal DNA, and a clone was isolated that conferred the expression of two separate modes of hemolytic activity in recombinant Escherichia coli . The original recombinant hemolytic strain (EB34) contained plasmid, pEB34, with a 5.6-kb insert from Sau 3 AI-digested P . intermedia strain 27 chromosomal DNA cloned into the Bam HI site of pUC18 . EB34 and deletion subclones were tested for expression of hemolytic activity in a standard tube assay, measuring lysis of erythrocytes spectrophotometrically as a function of hemoglobin release . Cell suspensions of EB34 demonstrated a dose-dependent hemolytic activity, inhibitable by proteases, and heat treatment but not dependent on calcium ions, and not inhibitable by osmoprotectants . Cell-free lysates also demonstrated a heat inhibitable, dose dependent hemolytic activity . Sub-cloning experiments localized the hemolytic region of the insert to a 3.9-kb fragment under direction of the lac promoter . Sequence analysis of the entire insert revealed the presence of multiple open reading frames (1 to 3) in this region which correlated to different forms of hemolytic expression, such that subclones containing all open reading frames 1 to 3 demonstrated strong hemolytic phenotype on blood plates and in the tube assay . Subclones containing only ORF1 demonstrated hemolysis on plates, but not in the tube assay . Subclones containing only open reading frames 2 and 3, but not ORF1 demonstrated hemolysis in the tube assay but not on plates . Homology searches of DNA and protein databases have not revealed significant homologies with reported hemolysins or proteins in any of the open reading frames. Nat Toxins, 1999, 7(2), 71 - 9 Mechanisms underlying the hemolytic and ichthyotoxic activities of maitotoxin; Igarashi T et al.; Maitotoxin (MTX), a putative Ca(2+) channel activator produced by the dinoflagellate Gambierdiscus toxicus showed extremely potent hemolytic and ichthyotoxic activities . Hemolysis of 1% mouse blood cell suspension in saline occurred at 15 nM of MTX . The activity was enhanced six-fold in the presence of 10 microM of Ca(2+) and completely blocked by EDTA2Na, indicating its dependency on external Ca(2+) . The MTX-induced hemolysis was little affected by L-type Ca(2+) channel blockers (diltiazem, nifedipine, verapamil) but was strongly inhibited by calmodulin blockers (prenylamine and chlorpromazine) or a phospholipase A2 inhibitor (quinacrine) . MTX was mimicked by a calcium ionophore, calcimycin . Based on these results, a series of cellular events triggered by MTX were presumed to occur in the following sequence: increased Ca(2+) entry in cells, activation of calmodulin, promotion of phospholipase A2 activity, and finally destruction of cell membrane resulting from hydrolysis of membrane lipids . The sensitivity of blood cells to MTX varied significantly, dependent on the animal sources . Nucleated blood cells of carps and chickens were 100 times more resistant than those of mammals . LC(50) of MTX to freshwater fish Tanichthys albonubes in Ca(2+) free media (pH 8) was 5 nM but was markedly lowered to 3 pM by raising pH to 8 and increasing Ca(2+) concentration to 2 mM . In a marine environment MTX was 2000 times more toxic to fish than 42-di-hydrobrevetoxin-B (PbTx-3), one of the best known ichthyotoxins of red-tide origins . Int J Cancer, 1999 Oct 29, 83(3), 393 - 400 Targeting HER-2/neu for active-specific immunotherapy in a mouse model of spontaneous breast cancer; Cefai D et al.; The identification of tumor-associated antigens has led to increased interest in vaccination strategies to treat and/or prevent cancer . This study examined the feasibility of active-specific immunotherapy against the breast-tumor antigen HER-2/neu using a HER-2/neu transgenic (rNeu-TG) mouse model . rNeu-TG mice develop spontaneous breast tumors after pregnancy, indicating that they fail to mount an effective immune response against rNeu . Allogeneic fibroblasts expressing HER-2/neu were used as a cell-based vaccine . Vaccination induced a rNeu-specific anti-tumor immune response that prevented tumor formation of transplanted breast-tumor cells, and also protected mice from spontaneous tumor formation . Both T-cell-mediated and humoral immune responses were detectable in vaccinated mice . Vaccination also protected tumor-bearing mice from a challenge with cell suspensions isolated from spontaneous tumors, indicating that rNeu-TG mice are not tolerant to rNeu, even after spontaneous tumor formation . However, established spontaneous tumors themselves were never affected . This observation correlated with T-cell infiltrations in the injected but not in the established spontaneous tumor . Thus, allogeneic fibroblasts are efficient vaccine vectors to prime a specific immune response against an over-expressed tumor antigen . Moreover, our results suggest striking differences in the immunological requirements for the rejection of an established vs . a transplanted tumor . Haemostasis, 1999 Sep, 29(1), 41 - 9 Recent advances in platelet-polymorphonuclear leukocyte interaction; de Gaetano G et al.; Epidemiological evidence suggests a positive correlation between the number of PMN and the risk of ischemic vascular disease . The observation that activated PMN induce platelet activation my provide some biological plausibility to the role of PMN in thrombogenesis . Between other PMN products, cathepsin G, a protease released during PMN activation, is a potent platelet agonist . However, the antiproteinases present in plasma could virtually abolish its activity . Indeed it was shown that, when PMN were stimulated after interaction with platelets in mixed cell population, P-selectin-mediated platelet-PMN adhesion may result in the formation of a sequestered microenvironment in which cathepsin G activity is protected by antiproteases . P-selectin-mediated adhesion was also shown to facilitate the transcellular metabolism of arachidonic acid, resulting in increased production of both thromboxane B2 and leukotriene C4 . PMN adhesion to activated platelets in mixed cell suspensions subjected to high shear rate can be modeled as an adhesion cascade involving a P-selectin-dependent recognition step followed by an adhesion-strengthening interaction mediated by the beta(2)-integrin Mac-1 . Moreover, an intermediate tyrosine-kinase-dependent signal regulating beta(2)-integrin adhesiveness is required . Indeeed activated platelets express not only P-selectin but also different beta(2)-integrin ligands including fibrinogen and ICAM-2 . Some of the functional responses elicited by P-selectin on PMN could be prevented by specific antibody to the P-selectin glycoprotein ligand-1, indicating that this adhesive receptor is able to transduce an 'outside-in' signal when engaged by the ligand . By using activated platelets, P-selectin-expressing CHO cells and soluble recombinant P-selectin, P-selectin was shown to trigger protein tyrosine phosphorylation in PMN and the tyrosine kinase-dependent function of Mac-1 . In conclusion, adherence of activated platelets to PMN may be a key event in the sequence of thrombus formation . The recognition of the essential contribution of PMN beta(2)-integrins in addition to P-selectin in platelet-PMN adhesion provides an additional evidence to the broad range of function and mechanisms in which PMN integrins are involved and may be potential targets for pharmacological intervention. J Rheumatol, 1999 Sep, 26(9), 1869 - 76 Increased HLA-DR and CD44 antigen expression in the gut: evidence of extraarticular immunological activity in rheumatoid arthritis; Abuzakouk M et al.; OBJECTIVE: To examine the gastrointestinal (GI) immune system in rheumatoid arthritis (RA) for evidence of activation . METHODS: Duodenal biopsies from 25 patients with RA were obtained by endoscopy . Single cell suspensions from the epithelial layer and lamina propria were prepared . Flow cytometry was used to examine the expression of CD4, CD8, T cell receptor-gammadelta (TCR-gammadelta), TCR-alphabeta, HLA-DR, CD44, and interleukin 2 receptor on gut T lymphocytes . Fifteen disease control (DC) individuals and 6 patients with osteoarthritis (OA) taking longterm nonsteroidal antiinflammatory drug (NSAID) therapy were also investigated . Peripheral blood T lymphocytes from all individuals were examined for the expression of these surface molecules . RESULTS: HLA-DR expression was significantly increased on intraepithelial lymphocytes (IEL) and enterocytes from patients with RA (n = 13) compared with the 2 control groups (p<0.01) . Immunohistochemistry also revealed increased expression of HLA-DR on enterocytes from patients with RA . RA IEL (n = 6) expressed significantly higher levels of CD44 (p<0.02) . In the lamina propria, a small but significant gammadelta T lymphocyte population (mean 5.5%, range 2-12%) was detected in rheumatoid factor positive RA patients (n = 8) compared with RF negative RA patients (n = 8, mean 2%, range 0.4-6%; p<0.01) and the disease control group (n = 15, mean 2%, range 0.5-5%; p<0.01) . None of these changes were detectable in peripheral blood lymphocytes from patients with RA . CONCLUSION: This study demonstrates evidence of activation of specific components of the GI immune system in RA . Peripheral blood T lymphocytes from patients with RA did not show increased expression of activation markers, suggesting that changes in the RA GI tract are not systemic but localized . Moreover, these changes appear to be independent of NSAID therapy. Vision Res, 1999 Aug, 39(17), 2817 - 32 Enhanced retinal longwave sensitivity using a chlorophyll-derived photosensitiser in Malacosteus niger, a deep-sea dragon fish with far red bioluminescence; Douglas RH et al.; Through partial bleaching of both visual pigment extracts and cell suspensions we show that the deep-sea stomiid Malacosteus niger, which produces far red bioluminescence, has two visual pigments within its retina which form a rhodopsin/porphyropsin pigment pair with lambda max values around 520 and 540 nm, but lacks the very longwave sensitive visual pigments (lambda max > 550 nm) observed in two other red light producing stomiids . The presence of only a single opsin gene in the M . niger genome was confirmed by molecular and cladistic analysis . To compensate for its apparently reduced longwave sensitivity compared to related species, the outer segments of M . niger contain additional pigments, which we identify as a mixture of defarnesylated and demetallated derivatives of bacteriochlorophylls c and d, that are used as a photosensitiser to enhance its sensitivity to longwave radiation. Biol Reprod, 1999 Oct, 61(4), 927 - 34 A comparative morphological study of human germ cells in vitro or in situ within seminiferous tubules; Johnson L et al.; For many infertile couples, intracytoplasmic germ cell/spermatozoon injection into unfertilized eggs may be their only hope for producing their own biological children . Thus far, success with injection of pre-spermatozoan germ cells such as round spermatids has not been as great as that of spermatozoon injection . This could be due in part to the difficulty of identifying younger (less mature) male germ cells in testicular biopsy dispersions . To improve the identification of various types of live, dispersed, human testicular cells in vitro, a comparative study of the morphological characteristics of human spermatogenic germ cells in vitro or in situ within seminiferous tubules was conducted . Live human testicular tissue was obtained from an organ-donating, brain-dead person with a high density of various germ cells . A cell suspension was obtained by enzymatic digestion, and cells were cultured for 3 days in an excessive volume (100-fold medium:cells; v:v) of HEPES-TC 199 medium at 5 degrees C and observed live with Nomarski optics (interference-contrast microscopy) . For comparative purposes, testes from ten men obtained at autopsy were fixed, embedded in epoxy resin, sectioned at 20 microm, and observed unstained by Nomarski optics . This approach allowed comparison of morphological characteristics of individual germ cells seen in vitro or in situ in the human testis . In both live and fixed preparations from control men with varied daily sperm production rates, Sertoli cells have oval to pear-shaped nuclei with indented nuclear envelopes and large nucleoli, which makes their appearance distinctly different from germ cells . The size, shape, and chromatin pattern of nuclei, and the presence of meiotic metaphase figures, acrosomic vesicles/structures, tails, and/or mitochondria in the middle piece of germ cells are characteristically seen in live cells in vitro and in those cells observed in the fixed seminiferous tubules . Hence, this comparative approach allows verification of the identity of individual germ cells seen in vitro and provides a checklist of distinguishing characteristics of live human germ cells, to be used by scientists and technical staff in infertility clinics when selecting specific germ cells from a testicular aspirate or enzymatically digested biopsy. Br J Cancer, 1999 Sep, 81(1), 114 - 21 A quantitative polymerase chain reaction-enzyme immunoassay for accurate measurements of human papillomavirus type 16 DNA levels in cervical scrapings; Jacobs MV et al.; A quantitative polymerase chain reaction-enzyme immunoassay (Q-PCR-EIA) was developed to measure the amount of human papillomavirus (HPV) 16 DNA per genome equivalent in cervical scrapings . The quantitative approach was based on a combined competitive PCR for both HPV 16, using the general primer GP5+/6+ PCR, and beta-globin DNA . The two competitive PCRs involve co-amplification of target sequences and exogenously added DNA constructs carrying a rearranged 30 bp sequence in the probe-binding region . The accuracy of quantification by combining the two competitive PCR assays was validated on mixtures of HPV 16 containing cervical cancer cells of CaSki and SiHa cell lines . Comparison of this fully quantitative PCR assay with two semi-quantitative HPV PCR assays on a series of crude cell suspensions from HPV 16 containing cervical scrapings revealed remarkable differences in the calculated relative HPV load between samples . We found evidence that correction for both intertube variations in PCR efficiency and number of input cells/integrity of DNA significantly influence the outcome of studies on viral DNA load in crude cell suspensions of cervical scrapings . Therefore, accurate measurements on viral DNA load in cervical scrapings require corrections for these phenomena, which can be achieved by application of this fully quantitative approach. J Chromatogr A, 1999 Aug 20, 853(1-2), 381 - 9 Direct measurement of ascorbic acid biosynthesis in Arabidopsis cell suspension culture using capillary electrophoresis; Davey MW et al.; We describe procedures to directly measure the biosynthesis of vitamin C (L-ascorbic acid, L-AA) in crude extracts of an Arabidopsis thaliana cell suspension culture by capillary electrophoresis . Optimal conditions have been established for the quantitation of L-AA formed by the oxidation of three different substrates: L-galactose, L-galactono-1,4-lactone, and L-gulono-1,4-lactone . We also demonstrate that L-galactono-1,4-lactone dehydrogenase activity does not require exogenous cofactor . The minimal sample handling requirements, the high selectivity, and short analysis times represent significant advantages over existing protocols. Cytometry, 1999 Oct 1, 37(2), 147 - 55 Phenotyping of epidermal dendritic cells: clinical applications of a flow cytometric micromethod; Wollenberg A et al.; BACKGROUND: The differential diagnosis of inflammatory skin diseases is largely based on the patient's history and the morphological analysis of the skin lesion . Laboratory data, such as serum IgE-level and prick and patch tests, may be helpful but do not assess individual lesions . The assumption of our approach is that each individual lesion is associated with a specific microenvironment and that the immunophenotype of the two epidermal dendritic cell populations, Langerhans cells (LC) and inflammatory dendritic epidermal cells (IDEC), reflects this environment in a disease-specific manner . METHODS: A flow cytometric micromethod was developed to directly analyze individual inflammatory human skin lesions . Crude epidermal single cell suspensions were prepared by trypsinization, stained for three-color analysis with different monoclonal antibodies and the vital stain 7-amino-actinomycin-D, and finally analyzed on a single laser equipped FACScan flow cytometer . RESULTS: With a limited set of cell surface markers, such as FcepsilonRI, FcgammaRII/CD32, CD1b and CD36, highly specific diagnostic criteria for atopic dermatitis and inflamed human skin could be established . CONCLUSIONS: Phenotyping of epidermal dendritic cells is a useful procedure helpful in differential diagnosis of inflammatory skin diseases . Biotechnol Bioeng, 1999 Nov 5, 65(3), 247 - 57 A new coiled hollow-fiber module design for enhanced microfiltration performance in biotechnology; Luque S et al.; The microfiltration performance of a novel membrane module design with helically wound hollow fibers is compared with that obtained with a standard commercial-type crossflow module containing linear hollow fibers . Cell suspensions (yeast, E . coli, and mammalian cell cultures) commonly clarified in the biotechnology industry are used for this comparison . The effect of variables such as transmembrane pressure, particle suspension concentration, and feed flow rate on membrane performance is evaluated . Normalized permeation fluxes versus flow rate or Dean number behave according to a heat transfer correlation obtained with centrifugal instabilities of the Taylor type . The microfiltration performance of this new module design, which uses secondary flows in helical tubes, is significantly better than an equivalent current commercial crossflow module when filtering suspensions relevant to the biotechnology industry . Flux and capacity improvements of up to 3.2-fold (constant transmembrane pressure operation) and 3.9-fold (constant flux operation), respectively, were obtained with the helical module over those for the linear module . Am J Physiol, 1999 Sep, 277(3 Pt 1), C572 - 9 Regulation of intestinal tyrosine phosphorylation and programmed cell death by peroxovanadate; Scheving LA et al.; Cell suspensions of ileal mucosa undergo a rapid and synchronized form of programmed cell death when cultured in a simple medium at 37 degrees C . Because tyrosine phosphorylation of proteins plays a crucial role in the signal transduction of many cellular processes, we examined its role in intestinal programmed cell death by use of immunoblot and immunohistochemical methods . We observed a 50-70% reduction in tyrosine phosphorylation during the initial 10 min of intestinal epithelial cell culture . We hypothesized that the inhibition of protein tyrosine phosphatases would increase protein tyrosine phosphorylation in these suspensions and decrease programmed cell death . A strong inhibitor of these phosphatases (peroxovanadate) but not a weaker one (sodium orthovanadate) abolished the DNA fragmentation/laddering normally seen in dying enterocytes . Peroxovanadate enhanced protein tyrosine phosphorylation of many intestinal proteins, dramatically increasing the dually phosphorylated and active form of mitogen-activated protein kinase . Immunohistochemistry revealed a particularly high level of increased tyrosine phosphorylation in the intestinal crypts in peroxovanadate-treated mucosa . Kinetic studies indicated that the pivotal time for protein tyrosine phosphatase inhibition occurred within 5 min of ex vivo culture, precisely when protein tyrosine phosphorylation declined . Our data suggest that tyrosine kinase inactivation or tyrosine phosphatase activation may initiate intestinal epithelial cell death. J Virol, 1999 Oct, 73(10), 8571 - 7 Prevalence of varicella-zoster virus DNA in dissociated human trigeminal ganglion neurons and nonneuronal cells; LaGuardia JJ et al.; Previous analyses using in situ hybridization alone or together with PCR have yielded conflicting results regarding the cell type in which latent varicella-zoster virus (VZV) resides . We separated human trigeminal ganglia (TG) into neuronal and nonneuronal fractions, followed by primary and nested PCR to quantitate VZV DNA at the single cell level . Both TG from each of eight cadavers were dissociated and separated into neuronal and nonneuronal cell suspensions by differential filtration . Analysis of the neuron fraction (5,000 neurons per sample) revealed VZV DNA in 9 of 16 samples, with copy numbers ranging from 1 to 12, whereas only 2 of 16 nonneuronal cell samples were positive for VZV DNA, with 1 copy each . Further analysis of 10 samples of 100 neurons and the corresponding nonneuronal cell fractions from each TG of a single subject revealed VZV DNA in 3 of 10 samples of the left TG (range, 2 to 5 copies) and in 1 of 10 samples of the right TG (2 copies) but in none of the 20 nonneuronal cell fractions . These data indicate that latent VZV DNA is present primarily, if not exclusively, in neurons, at a frequency of two to five copies per latently infected neuron. FEBS Lett, 1999 Sep 17, 458(2), 204 - 8 An early salicylic acid-, pathogen- and elicitor-inducible tobacco glucosyltransferase: role in compartmentalization of phenolics and H2O2 metabolism; Chong J et al.; Treatment of tobacco cell suspension cultures with a fungal elicitor of defense responses resulted in an early accumulation of the phenylpropanoid glucosyltransferase TOGT, along with the rapid synthesis and secretion of scopolin, the glucoside of scopoletin . Elicitor-triggered extracellular accumulation of the aglycone scopoletin and of free caffeic and ferulic acids could only be revealed in the presence of diphenylene iodonium, an inhibitor of extracellular H2O2 production . Our results strongly support a role for TOGT in the elicitor-stimulated production of transportable phenylpropanoid glucosides, followed by the release of free antioxidant phenolics into the extracellular medium and subsequent H2O2 scavenging. J Cancer Res Clin Oncol, 1999 Aug-Sep, 125(8-9), 461 - 74 Galectins-1 and -3 and their ligands in tumor biology . Non-uniform properties in cell-surface presentation and modulation of adhesion to matrix glycoproteins for various tumor cell lines, in biodistribution of free and liposome-bound galectins and in their expression by breast and colorectal carcinomas with/without metastatic propensity; Andre S et al.; Protein (lectin)-carbohydrate (cellular glycoconjugate) recognition is operative in biochemical information transfer . Galectins constitute a family of endogenous galactoside-binding lectins with conserved features in the binding site . The members of this lectin category are assumed to be involved in cell adhesion and growth regulation . To assess to what extent the different modes of binding-site presentation and/or carbohydrate fine-specificities will affect aspects of galectin behavior, homodimeric cross-linking galectin-1 and monomeric chimeric galectin-3, with its collagenase-sensitive stalk linked to the carbohydrate-recognition domain, were investigated . Cell-surface expression of the two galectins and accessible galectin-binding sites on various tumor cell lines was ascertained by FACScan analysis . In particular, ligand accessibility for the two galectins differed for the tested cell line types . Binding of tumor cells to laminin and plasma or placental fibronectin was generally reduced by treatment of cells or matrix with galectins . Galectin-3 was more efficient than galectin 1 at impairing laminin's potency as matrix . Cell binding of galectin-1, on the other hand, proved on average more effective for blocking cell association to fibronectins after its preincubation with cell suspensions . Differences were also apparent in the biodistribution of the galectins, where an avian homolog of galectin- served as the control to distinguish effects of spatial and sugar-binding features . Histopathological analysis of lymph-node-negative and -positive breast and colorectal carcinomas (n = 180 including 60 metastatic lesions) indicated a correlation of either increased galectin-1 binding and reduced galectin-3 expression or reduced binding of both galectins with the occurrence of lymph node lesions . Together with data on the heparin-binding lectin, revealing reduced expression to be associated with a positive lymph-node status in the breast cancer group, these results can be interpreted to reflect cell-type-dependent requirements of galectin ligand presentation during the metastatic cascade . By introducing mammalian lectins to lectin-histochemical studies, the detection of quantitative differences in glycosylation brings an understanding of its cell biological significance one step closer. Bull Cancer, 1999 Jul-Aug, 86(7-8), 685 - 91 {Standardization and quality control in the evaluation of proliferation parameters in T1T2, N0N1, M0 breast cancer: multicentric retrospective study II . DNA-ploidy and S-phase fraction}; Chassevent A et al.; As part of a clinical research project, proliferative parameters were studied in primary breast cancer: standardization and technical validation of thymidine kinase (TK), thymidylate synthase (TS) and protein tyrosine kinase (PTK) are described . A total of 633 frozen tumor specimens, available in four institutions, was analyzed in three flow cytometry laboratories for DNA content and percentage of S-phase cells (%S) measurement . 1) The standardization step consisted in developing a common protocol for sample preparation; then, common cell suspensions were analyzed in order to perform an inter-laboratory control . Objective guidelines were elaborated to interpret DNA histograms in breast carcinoma . 2) DNA-aneuploidy was observed in 61% of cases of the retrospective series . Compared with DNA-aneuploid tumors, mean %S was significantly lower in case of DNA-diploidy (respectively: 6.4% and 2.2%, p < 0.001) . When compared between the four institutions, %S distributions did not differ significantly . 3) %S is strongly correlated with TK, TS and PTK and high percentages were also observed in high grade tumors or tumor without hormone receptors . These results show that a standardization in using flow cytometers and DNA software allows multicenter studies. J Neurosci Methods, 1999 Jul 1, 89(1), 17 - 24 In vivo predegeneration of peripheral nerves: an effective technique to obtain activated Schwann cells for nerve conduits; Keilhoff G et al.; In vivo predegeneration of peripheral nerves is presented as a convenient and effective method to obtain activated Schwann cells and an enhanced cell yield following in vitro cultivation . The experiments conducted in rats were aimed at clinical use in gaining Schwann cell suspensions for filling artificial conduits in order to bridge peripheral nerve gaps . The rat sciatic nerve used as a model was transected distally to the spinal ganglia . Predegeneration in vivo was allowed to take place for 1, 2, 3 and 4 days and up to 1, 2 and 3 weeks . The nerve was then resected and prepared for cell cultivation . Schwann cells cultivated from the contralateral untreated nerve served as control . Immunostaining for S100, nerve growth factor receptor and the adhesion molecules N-cadherin and L1 was used to characterize the general state of the cultures . Viability was assessed by fluorescein fluorescence staining, and the proliferation index was determined by bromodeoxyuridine-DNA incorporation . The Schwann cells from predegenerated nerves revealed an increased proliferation rate compared to the control, whereas fibroblast contamination was decreased . Best results were obtained 1 week after predegeneration. J Neurosci Methods, 1999 Jul 1, 89(1), 1 - 8 Separation of dorsal and ventral dopaminergic neurons from embryonic rat mesencephalon by buoyant density fractionation: disassembling pattern in the ventral midbrain; Silverman WF et al.; The dopaminergic neurons of the ventral mesencephalon, though physically mixed with non-dopamine neurons, are organized into dorsal and ventral 'tiers' with regard to their ontogeny, efferent projections and their relative position in the various mesencephalic sub-nuclei . We have employed buoyant density fractionation to separate the dopaminergic neurons of the two compartments and compare their subsequent phenotype development with respect to their expression of the gene encoding tyrosine hydroxylase, the rate-limiting enzyme in the catecholamine biosynthetic pathway . Using immunocytochemistry, separately and combined with in situ hybridization, we demonstrate here that sedimentation of cell suspensions from E19 rat ventral mesencephalon on 5-step Percoll gradients produces cell fractions enriched in ventral and dorsal tier DA neurons, respectively. Plant J, 1999 Aug, 19(3), 321 - 31 Yariv reagent treatment induces programmed cell death in Arabidopsis cell cultures and implicates arabinogalactan protein involvement; Gao M et al.; Arabinogalactan proteins (AGPs) are a family of highly glycosylated, hydroxyproline-rich glycoproteins implicated in various aspects of plant growth and development . (beta-D-glucosyl)3 and (beta-D-galactosyl)3 Yariv phenylglycosides, commonly known as Yariv reagents, specifically bind AGPs in a non-covalent manner . Here (beta-D-galactosyl)3 Yariv reagent was added to Arabidopsis thaliana cell suspension cultures and determined to induce programmed cell death (PCD) by three criteria: (i) DNA fragmentation as detected by terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling (TUNEL) of DNA 3'-OH groups; (ii) inter- nucleosomal DNA fragmentation as visualized by genomic Southern blotting; and (iii) structural changes characteristic of PCD including cytoplasmic shrinkage and condensation, chromatin condensation and nuclear membrane blebbing . These findings implicate AGP involvement in PCD in plants, presumably by perturbation of AGPs located at the plasma membrane-cell wall interface. Plant J, 1999 Aug, 19(3), 297 - 307 MAP kinase activation by hypoosmotic stress of tobacco cell suspensions: towards the oxidative burst response? Cazale AC, Droillard MJ, Wilson C, Heberle-Bors E, Barbier-Brygoo H, Lauriere C. Hypoosmotic stress activates a phosphorylation-dependent oxidative burst . In-gel kinase assays were performed to characterize the protein kinases that could be implicated in osmoregulation and in the activation of the oxidative burst . Hypoosmotic stress activated several kinases among which 50 and 46 kDa proteins displayed mitogen-activated protein kinase (MAP kinase) properties . They phosphorylated myelin basic protein in the absence of calcium, were recognized by antibodies directed against human MAP kinases, and were phosphorylated on tyrosine . Immunoprecipitation with an antibody directed against the tobacco MAP kinase Ntf4 showed that at least one of the activated kinases would be Ntf4-like . Apigenin, a MAP kinase and cyclin-dependent kinase inhibitor which prevents the hypoosmotically induced oxidative burst (Cazale et al . 1998; Plant Physiol . 116, 659-669), inhibited these kinases in vitro suggesting that they may play a role in the activation of the oxidative burst . Like the oxidative response, activation of the kinases depended on extracellular calcium influx and protein kinases sensitive to staurosporine and 6-DMAP . However, kinase activation did not depend on effluxes through anion channels or on the oxidative burst . Two-dimensional in-gel kinase assays revealed the presence of three protein kinases with an apparent molecular mass of 50 kDa and one of 46 kDa, all four being activated by hypoosmotic stress . The same kinases were also activated by oligogalacturonides and salicylic acid, underlying the importance of these MAP kinases as common components of different signaling pathways triggered by different extracellular stimuli. Biorheology, 1998 Jul-Oct, 35(4-5), 263 - 79 Wall shear stress in backward-facing step flow of a red blood cell suspension; Gijsen FJ et al.; An experimental investigation of the wall shear stress distribution downstream of a backward-facing step is carried out . The wall shear stress distribution was determined by measuring the deformation of a gel layer, attached to the wall downstream of the step . Speckle pattern interferometry was applied to measure the deformation of the gel layer . The measured deformation, combined with the properties of the gel layer, served as an input for a finite element solid mechanics computation to determine the stress distribution in the gel layer . The wall shear stress, required to generate the measured deformation of the gel layer, was determined from these computations . A Newtonian buffer solution and a non-Newtonian red blood cell suspension were used as measuring fluids . The deformation of the gel layer was determined for a Newtonian buffer solution to evaluate the method and to obtain the properties of the gel layer . Subsequently, the wall shear stress distribution for the non-Newtonian red blood cell suspension was determined for three different flow rates . The inelastic non-Newtonian Carreau-Yasuda model served as constitutive model for the red blood cell suspension . Using this model, the velocity and wall shear stress distribution were computed by means of a finite element fluid mechanics computation . From the comparison between the numerical and the experimental results, it can be concluded that wall shear stresses, induced by the red blood cell suspension, can be modeled accurately by employing a Carreau-Yasuda model. Cytobios, 1999, 98(388), 77 - 94 Influence of noradrenaline on the respiratory status of Rana balcanica red cell suspension under normoxia, hypoxia and hypercapnia: alpha 1-receptor involvement; Kaloyianni M et al.; The effect of normoxia, hypoxia and hypercapnia on the extracellular pH, partial pressure carbon dioxide (pCO2), partial pressure oxygen (pO2) and HCO3- levels after noradrenaline treatment of Rana balcanica erythrocytes, was investigated . Noradrenaline caused a significant reduction of the extracellular pH which may have been due to the activation of red blood cell Na+/H+ exchange . Significant falls in the partial extracellular pressure of CO2 and O2 were evident . The initial reduction in extracellular pCO2 and pO2 was followed by a rise reflecting the desensitization of the Na+/H+ exchange after 15 min of hormone stimulation . Both hypercapnia and hypoxia increased the magnitude of these changes in relation to normoxia, although the greatest changes were observed under hypercapnic conditions . The involvement of alpha 1 receptors in regulating the concentration of respiratory gases after catecholamine stimulation was demonstrated . It is suggested that these responses increased the effectiveness of gas transfer over the respiratory surfaces. Arch Immunol Ther Exp (Warsz), 1999, 47(3), 161 - 8 Tumor infiltrating lymphocytes in HLA+ and HLA- laryngeal cancer--quantitative approach; Dworacki G et al.; In search of factors governing the accumulation of tumor infiltrating lymphocytes (TIL), frozen sections from fresh surgical specimens of laryngeal carcinoma (n = 36) were tested by alkaline phosphatase-anti-alkaline phosphatase (APAAP) immunohistochemistry for monomorphic determinants of HLA class I and class II expression on tumor cells and for the distribution of lymphoid cells bearing CD differentiation antigens . Cell subsets were quantitated in two tumor compartments, tumor mass and tumor stroma, by computer-assisted image analysis . In a portion of examined samples lymphoid cell suspension was isolated from cancerous tissues and assessed by flow cytometry . It has been found that T cells, localized mostly in tumor stroma, were predominant cell population in the tumor microenvironment . Their ability to penetrate tumor mass but not tumor stroma, by CD8+ T cells in particular, but also by natural killer (NK) cells, was associated with HLA class I antigen expression on tumor cells . In flow cytometric analysis activated T lymphocytes (CD3+DR+) were abundant in HLA+ tumors as compared to HLA- ones . In 4 year follow up of 20 patients the mortality was higher in HLA- group but the data were not statistically significant . These results show that HLA class I expression on tumor cells favor penetration of cytotoxic lymphoid cells into tumor mass, at least in the laryngeal cancer. J Bone Miner Res, 1999 Sep, 14(9), 1562 - 9 Development and characterization of a human in vitro resorption assay: demonstration of utility using novel antiresorptive agents; James IE et al.; A human in vitro resorption assay has been developed using osteoclastoma-derived osteoclasts and used to evaluate novel antiresorptive agents including antagonists of the alphavbeta3 integrin, and inhibitors of cathepsin K and the osteoclast ATPase . The potency of novel compounds in the in vitro resorption assay correlates with functional assays for each class of inhibitor: the human alphavbeta3-mediated cell adhesion assay for the vitronectin receptor antagonists (r2 = 0.82), the chick osteoclast vacuolar ATPase enzyme assay for the H+-ATPase inhibitors (r2 = 0.77) and the recombinant human cathepsin K enzyme assay for the cathepsin K inhibitors (r2 = 0.80) . Cell suspensions, rich in osteoclasts, are prepared by collagenase digestion of the tumor tissue . These cells can be stored long-term in liquid nitrogen and upon thawing maintain their bone-resorbing phenotype . The cryopreserved cells can be cultured on bovine cortical bone for 24-48 h and resorption can be measured by either confocal microscopy or biochemical assays . The resorptive activity of osteoclasts derived from a number of tumors can be inhibited reproducibly using a number of mechanistically unique antiresorptive compounds . In addition, the measurement of resorption pits by laser confocal microscopy correlates with the release of type I collagen C-telopeptides or N-telopeptides, as measured by enzyme-linked immunosorbent assay . Resorption can be measured reproducibly using a 48-h incubation of osteoclasts on bone slices, or a 24-h incubation with bone particles . This in vitro human osteoclast resorption assay provides a robust system for the evaluation of inhibitors of osteoclastic function that may be developed for the treatment of metabolic bone diseases such as osteoporosis. Eur J Biochem, 1999 Aug, 263(3), 686 - 94 Identification and characterization of cDNA clones encoding hydroxycinnamoyl-CoA:tyramine N-hydroxycinnamoyltransferase from tobacco; Farmer MJ et al.; The sequences of three cDNA clones that include the complete coding region of hydroxycinnamoyl-CoA:tyramine N-hydroxycinnamoyltransferase (THT) from tobacco are reported . The three cDNAs were isolated by antibody screening of a cDNA expression library produced from poly(A)+RNA purified from tobacco leaves (Nicotiana tabacum cv . Bottom Special), previously infiltrated with an incompatible strain of Ralstonia solanacearum . The identity of these clones was confirmed by the detection of THT activity in extracts of transformed Escherichia coli and by matching the translated polypeptides with tryptic enzyme sequences . cDNA clones tht4 and tht11 differ only by their 5' leader and 3' UTRs and therefore encode the same protein, whereas tht10 and tht11 exhibit 95 and 99% sequence identity at the DNA and deduced amino acid levels, respectively . The three clones encode proteins of 226 amino acids with calculated molecular masses of 26 kDa . The deduced amino acid sequences show no similarity with the sequence of anthranilate hydroxycinnamoyl/benzoyltransferase from Dianthus caryophyllus, the only enzyme exhibiting hydroxycinnamoyltransferase activity to be cloned so far in plants . In contrast, comparison of the THT amino acid sequence with protein sequ