Life Sci, 1996, 58(5), PL 81 - 6 The specific binding of the platelet-activating factor (PAF) receptor antagonist WEB 2086 and the benzodiazepine flunitrazepam to rat hepatocytes; Svetlov SI et al.; Thieno-triazolodiazepines WEB 2086 and BN 50739 have been described as the potent PAF receptor antagonists . Binding of radiolabeled {3H}WEB 2086 has been widely employed to characterize PAF receptors in different cells . In a search for a PAF receptor in isolated rat hepatocytes, we discovered that the binding of {3H}WEB to rat hepatocytes was highly specific but had a relatively low affinity with a Kd of 113 nM and Bmax of 0.65 pmol/10(6) cells in freshly isolated cell suspension and Kd of 1.65 muM and Bmax of 2.0 pmol/plate in cultured hepatocytes . No consistent specific binding of {3H}PAF itself was found in the same cell preparations . The binding of {3H}flunitrazepam in the presence of the peripheral type of benzodiazepine receptor antagonist Ro 5-4864 was saturated and exhibited a K(i) of 3.8 nM and Bmax of 3.5 pmol/plate . The central type of benzodiazepine receptor antagonist clonazepam was competed for the {3H}flunitrazepam binding, however with a much lower affinity . Various antagonists inhibited the binding of {3H}WEB 2086 with a rank order BN 50739>>Ro 5-4864 > or = clonazepam . Interestingly, bicuculline, specific antagonist of GABA(A) recognition sites, also significantly reduced the binding of {3H}WEB 2086 . The binding of {3H}flunitrazepam was inhibited with a rank potency BN 50739>>WEB 2086 . Taken together, these findings suggest that the specific binding of PAF receptor antagonists WEB 2086 and BN 50739 in rat hepatocytes does not involve PAF receptors and occurs via peripheral benzodiazepine and, possibly GABA(A) receptor sites. Plant Physiol, 1996 Jan, 110(1), 227 - 32 Study of the intercellular fluid of healthy Lupinus albus organs . Presence of a chitinase and a thaumatin-like protein; Regalado AP et al.; Proteins in the intercellular fluid (IF) of healthy Lupinus albus leaves were characterized . Silver staining of the proteins separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis revealed more than 30 polypeptides, with the major ones having a molecular mass lower than 36 kD . After amino-terminal amino acid sequence analysis, one of the major polypeptides, IF4, was shown to have no identity with any of the proteins present in the data bases . Two others, IF1 and IF3, showed identity with previously reported pathogenesis-related proteins, IF1 with an antifungal protein from Hordeum vulgare that belongs to the thaumatin family (PR-5 family), and IF3 with class III chitinase-lysozymes . IF3 was also present in the IF of stem and root and it represents the major polypeptide in the medium of L . albus cell-suspension cultures . The ubiquitous presence of this enzyme in healthy, nonstressed tissues of L . albus cannot be explained. Anesthesiology, 1996 Jan, 84(1), 103 - 16 Effect of volatile anesthetics on hydrogen peroxide-induced injury in aortic and pulmonary arterial endothelial cells; Johnson ME et al.; BACKGROUND: Oxidant damage to endothelial cells occurs during inflammation and reperfusion after ischemia, mediated in part by endogenously produced hydrogen peroxide (H2O2) . Previous studies have established a role for increased cytosolic calcium in the mechanism of endothelial oxidant injury, and have suggested that volatile anesthetics may exacerbate oxidant injury in pulmonary endothelium . However, the effect of volatile anesthetics on oxidant injury to systemic arterial endothelial cells, and their effect on oxidant-related changes in cytosolic calcium homeostasis, have not been reported previously . METHODS: Primary cultures of human aortic and pulmonary arterial endothelial cells were studied . The rate of cell death after H2O2 exposure was determined in cell suspension by propidium iodide fluorimetry and lactate dehydrogenase release . The final extent of cell death 24 h after H2O2 exposure was determined in monolayer cultures by methyl thiazolyl tetrazolium reduction . Cytosolic calcium and cell death were determined in single cells using fura-2 and propidium iodide imaging with digitized, multiparameter, fluorescent video microscopy . RESULTS: In aortic endothelial cells, clinical concentrations of halothane (1.0%) and isoflurane (1.5%) decreased both the rate of cell death and the final extent of cell death after H2O2 exposure, with halothane being more protective . Supraclinical concentrations of halothane (2.7%) and isoflurane (4.0%) were less protective . In pulmonary arterial endothelial cells, halothane and isoflurane had essentially no effect on H2O2-mediated cell death . The protective effect of anesthetic in aortic endothelial cells was not due to an enhanced removal of H2O2 by endogenous enzymes . Hydrogen peroxide exposure caused a large increase in cytosolic calcium well before cell death, and this was moderated by anesthetic treatment . CONCLUSIONS: The effect of volatile anesthetics on oxidant injury to endothelial cells may differ between cells derived from systemic and pulmonary vascular beds . Halothane, and to a lesser extent, isoflurane, protects against oxidant injury in aortic endothelial cells . The mechanism of protection may involve modulation of the interaction of H2O2 with vital cellular constituents, and/or amelioration of the toxic increase in cytosolic calcium that follows such interaction. Gut, 1996 Jan, 38(1), 104 - 14 Effect of c-kit ligand, stem cell factor, on mediator release by human intestinal mast cells isolated from patients with inflammatory bowel disease and controls; Bischoff SC et al.; The regulation of mediator release in human intestinal mast cells is largely unknown . Apart from IgE receptor crosslinking no secretagogues have been described so far . This study examined the effect of two cytokines (c-kit ligand and interleukin 3) and other agonists on human intestinal mast cell function . Cells were isolated from surgery specimens of 47 patients undergoing intestinal resection because of tumours or inflammatory bowel disease . Cell suspensions contained 3.6% mast cells (mean of 50 experiments) . After preincubation without or with c-kit ligand or interleukin 3, cells were stimulated by IgE receptor crosslinking, C5a or formyl-methionyl-leucyl-phenylalanine (fMLP) . Histamine and sulphidoleukotriene release was measured in supernatants . The sequential stimulation of the cells with c-kit ligand and IgE receptor crosslinking induced the release of high amounts of histamine and leukotrienes, whereas each agonist by itself induced only marginal mediator release . Interleukin 3 induced no release by itself, but enhanced the IgE receptor dependent release, possibly by an indirect mechanism . No significant mediator release was seen in response to C5a and fMLP, even if the cells were pretreated with c-kit ligand . The mediator release, particularly that of leukotrienes, was higher in cells isolated from actively inflamed tissue from patients with inflammatory bowel disease compared with controls . In conclusion, it was found that, apart from IgE receptor crosslinking, c-kit ligand and interleukin 3 regulate mediator release in human intestinal mast cells . The enhancement of mediator release by cytokines may be of particular relevance in the pathogenesis of inflammatory bowel diseases and food intolerance reactions. Scand J Immunol, 1996 Jan, 43(1), 16 - 22 Antibody-dependent cellular cytotoxic activity of cells in the rat metrial gland in pregnancy; Peel S et al.; Antibody-dependent cellular cytotoxic (ADCC) activity of cells in rat metrial glands at days 13 and 20 of pregnancy has been examined . A 51chromium-release cytotoxicity assay was carried out using Yac-1 cells and P815 cells as targets in the presence of the corresponding, heat inactivated rat antibody or non-immune rat serum . No killing was observed when effector metrial gland cells and target cells were cultured in the presence of non-immune rat serum . In the presence of rat antibody to Yac-1 cells, or rat antibody to P815 cells, cells from the metrial glands of rats killed at day 13 of pregnancy demonstrated ADCC activity, but cells from the metrial glands of rats killed at day 20 of pregnancy showed no ADCC activity . Spleen and peritoneal exudate cells displayed ADCC activity . Immunohistological and immunocytological studies showed OX-6, OX-34, OX-35 and OX-42 positive cells were present at days 13 and 20 of pregnancy, in sections of metrial glands and in the cell suspensions prepared for the cytotoxicity assays . It is suggested that macrophages are the cells most likely to be responsible for the ADCC activity in the rat metrial gland. Blood, 1996 Jan 1, 87(1), 73 - 82 Adhesion receptors on bone marrow stromal cells: in vivo expression of vascular cell adhesion molecule-1 by reticular cells and sinusoidal endothelium in normal and gamma-irradiated mice; Jacobsen K et al.; Cell adhesion molecules (CAMs) play a key role in interactions between stromal and hematopoietic cells in bone marrow (BM) and in cell traffic through vascular endothelium . To examine the identity of CAMs involved in these processes in mouse BM, we have investigated the in vivo expression of vascular cell adhesion molecule-1 (VCAM-1) and its counter-receptor, very late antigen-4 (VLA-4) . Radioiodinated monoclonal antibodies (MoAbs) detecting VLA-4 and VCAM-1 were injected intravenously . Antibody binding was detected in BM by light and electron microscope radioautography . VCAM-1 labeling was restricted to stromal reticular cells and endothelial cells lining BM sinusoids . VCAM-1+ reticular cells formed patchy concentrations, especially in subosteal regions, associated with lymphoid, granulocytic, and erythroid cells . After gamma-irradiation to deplete hematopoietic cells, reticular cells and endothelial cells all showed VCAM-1 labeling in apparently increased intensity . VLA-4 labeling was shown by undifferentiated blast cells and lymphohematopoietic cells both in BM cell suspensions and in vivo, especially at reticular cell contact points . The results demonstrate that VCAM-1 is expressed in vivo by certain BM reticular cells, suggesting that the molecule mediates adhesion to multiple lineages of lymphohematopoietic cells . The finding that VCAM-1 is also expressed constitutively by BM sinusoidal endothelium, unlike its inductive expression by endothelia elsewhere, suggests that VCAM-1 and VLA-4 may be involved in regulating the normal cell traffic between BM and the blood stream. Plast Reconstr Surg, 1996 Jan, 97(1), 168 - 78; discussion 179-80 De novo cartilage generation using calcium alginate-chondrocyte constructs; Paige KT et al.; These studies investigated the utility of calcium alginate as a biocompatible polymer matrix within which large numbers of chondrocytes could be held successfully in a three-dimensional structure and implanted . Further, the ability of chondrocyte-calcium alginate constructs to engraft and generate new cartilage was examined . Chondrocytes isolated from calf shoulders were mixed with a 1.5% sodium alginate solution to generate cell suspensions with densities of 0, 1.0, 5.0, and 10.0 x 10(6) chondrocytes/ml . The cell suspensions were gelled to create disks that were placed in subcutaneous pockets on the dorsums of nude mice . The alginate concentration and CaCl2 concentration used to make the disks also were varied . A total of 20 mice were implanted with 67 bovine chondrocyte-calcium alginate constructs . Samples with an initial cellular density of at least 5.0 x 10(6) chondrocytes/ml demonstrated gross cartilage formation 12 weeks after implantation . Cartilage formation was observed microscopically in specimens with a cellular density as low as 1.0 x 10(6) chondrocytes/ml . The histoarchitecture of the new cartilage closely resembled that of native cartilage . Cartilage formation was independent of CaCl2 concentration (15 to 100 mM) or alginate concentration (0.5% to 4.0%) used in gel polymerization. J Biol Chem, 1995 Dec 29, 270(52), 31091 - 6 Acetyl coenzyme A:salutaridinol-7-O-acetyltransferase from papaver somniferum plant cell cultures . The enzyme catalyzing the formation of thebaine in morphine biosynthesis; Lenz R et al.; Acetyl coenzyme A:salutaridinol-7-O-acetyltransferase, a highly substrate-specific enzyme, has been purified nearly 3,000-fold to homogeneity from Papaver somniferum plant cell suspension cultures . Purification was achieved by fractionated ammonium sulfate precipitation, dye-ligand affinity chromatography on matrex red A, gel filtration, ion exchange chromatography on Mono Q and a second dye-ligand affinity chromatography on fractogel TSK AF Blue . The purified enzyme was a single polypeptide with an M(r) = 50,000 displaying an isoelectric point of 4.8, a pH optimum between pH 6 and 9 and a temperature optimum at 47 degrees C . The Km values for the substrate salutaridinol and the co-substrate acetyl co-enzyme A were 7 and 46 microM, respectively . Salutaridinol-7-O-acetyltransferase catalyzes the stoichiometric transfer of the acetyl group from acetyl coenzyme A to the 7-OH group of salutaridinol yielding salutaridinol-7-O-acetate, which is a new intermediate in morphine biosynthesis . Salutaridinol-7-O-acetate undergoes a subsequent spontaneous allylic elimination at pH 8-9, leading to the formation of thebaine (1), the first morphinan alkaloid with the complete pentacyclic ring system, or at pH 7 leading to dibenz{d,f}azonine alkaloids that contain a nine-membered ring . Acetylation and subsequent allylic elimination is a new enzymic mechanism in alkaloid biosynthesis, which in the poppy plant can transform one precursor into alkaloids possessing markedly different ring systems, depending on the reaction pH. Arch Biochem Biophys, 1995 Dec 20, 324(2), 255 - 66 Cloning, expression, and characterization of (+)-delta-cadinene synthase: a catalyst for cotton phytoalexin biosynthesis; Chen XY et al.; In cotton, sesquiterpene phytoalexins are elicited in response to bacterial or fungal infection . A Gossypium arboreum cell suspension culture which produces the sesquiterpene phytoalexin gossypol showed a time-dependent 10-fold increase in a 1.9-kb mRNA in response to a challenge by a preparation from Verticillium dahliae . The mRNA prepared from these elicited cultures was used to isolated two cDNA clones that contain open frames coding for proteins of 554 amino acids with M(r) 64,096 and 64,118 . The encoded protein shows a significant degree of sequence identity with the other known plant terpene cyclases . Western blot analyses with a cross-reactive monoclonal antibody from a related sesquiterpene synthase in Nicotiana tabacum showed a time-dependent increase of a 65-kDa protein which reached a maximal level 24 h post elicitor treatment . The encoded protein from the pXC1 cDNA was produced in Escherichia coli and purified by affinity column chromatography . The enzymatic properties of this protein were identified by a radiochemical assay for cyclization of farnesyldiphosphate and a product structure was assigned by GC-MS, chiral phase GC, and NMR analyses as (+)-delta-cadinene . The fungal-elicited production of a (+)-delta-cadinene synthase is consistent with a role for this enzyme as the first committed step in the pathways leading to the related phytoalexins gossypol and lacinilene C in cotton. FEBS Lett, 1995 Dec 18, 377(2), 175 - 80 Evidence against specific binding of salicylic acid to plant catalase; Ruffer M et al.; It was demonstrated that salicylic acid (SA) not only binds to catalase from differentiated higher plants and plant cell suspension cultures but also to those of fungi and animals . SA bound specifically to iron-containing enzymes, such as catalase, aconitase, lipoxidase and peroxidase, while not to iron-free plant enzymes . On the grounds of these experiments, the claim is further challenged that SA is a signalling compound and second messenger in plants that activates plant defense-related genes through elevated H2O2 levels by specifically inhibiting catalase activity . SA may just function as a phytoalexin. Biochem J, 1995 Dec 15, 312 ( Pt 3), 879 - 85 Metabolization of iron by plant cells using O-Trensox, a high-affinity abiotic iron-chelating agent; Caris C et al.; A synthetic siderophore, O-Trensox (L), has been designed and synthesized to improve iron nutrition of plants . The affinity for iron of this ligand {pFe(III) = 29.5 and pFe(II) = 17.9} is very high compared with EDTA . In spite of its high and specific affinity for iron, O-Trensox was found to be able to prevent, and to reverse, iron chlorosis in several plant species grown in axenic conditions . It also allows the iron nutrition and growth of Acer pseudoplatanus L . cell suspensions . The rate of iron metabolization was monitored by 59Fe radioiron . Ferritins, the iron storage proteins, are shown to be the first iron-labelled proteins during iron metabolization and to be able to further dispatch the metal . Using Fe(III)-Trensox, the rate of iron incorporation into ferritin was found to be higher than when using Fe-EDTA, but slower than with Fe-citrate, the natural iron carrier in xylem . During a plant cell culture, the extracellular concentrations of iron complex and free ligand were measured; changes in their relative amounts showed that the iron complex is dissociated extracellularly and that only iron is internalized . This suggests a high affinity for iron of a putative carrier on the plasmalemma . In contrast with Fe-citrate and Fe-EDTA complexes, Fe(III)-Trensox is not photoreducible . Its ability to induce radical damage as a Fenton reagent was tested using supercoiled DNA as target molecule . Unlike Fe-citrate and Fe-EDTA, Fe(II)-Trensox and Fe(III)-Trensox were proven to be harmless even during ascorbate-driven reduction, while Fe-EDTA and Fe-citrate generate heavy damage to DNA. J Immunol Methods, 1995 Dec 15, 188(1), 33 - 41 Quantification of natural antibody producing B cells in rats by an improved ELISPOT technique using the polyvinylidene difluoride membrane as the solid support; Schielen P et al.; We describe here a new type of solid support for the ELISPOT assay, the PVDF membrane . In parallel tests, spot yields on this membrane were superior to those obtained with the frequently used nitrocellulose (NC) membrane, coated with the same rat anti-IgM and anti-IgG antibodies, incubated with the same rat spleen cell suspensions, and developed with the same combination of AP-labeled conjugates and substrate . We therefore used the PVDF membrane, coated with anti-rat IgM and IgG antibodies, ssDNA or bromelain-treated mouse erythrocytes (BrMRBC) (exposing phosphatidylcholine (PC) as major autoantigen) to develop ELISPOT assays for the quantification of isotype-specific natural antibody secreting cells (ASC) in rats . We confirmed the isotype specificity of the binding of the anti-rat IgM and anti-rat IgG coating antibodies and conjugates with the secreted rat antibodies in this assay, and, by inhibition of spot formation with soluble antigen, their specificity for ssDNA and BrMRBC . An in-house 18-well culture device for the easy manufacture of PVDF-lined culture wells greatly facilitated coating, blocking, and washing procedures, as compared to the original method in 24 well culture plates . This simple, fast, specific and sensitive ELISPOT assay was used to make an inventory of the numbers of natural splenic ASC in Wistar and Fischer rats. Zhongguo Yi Xue Ke Xue Yuan Xue Bao, 1995 Dec, 17(6), 428 - 33 {Species-specific monoclonal antibodies against the major outer membrane protein (MOMP) of Chlamydia trachomatis}; Ni A et al.; The synthesized one quarter N-terminal MOMP of C . trachomatis was used for primary immunization of three male BALB/c mice (8 weeks of age), and the boost with C . trachomatis L1/440/Bu elementary bodies (EBs) was followed on day 14 . Spleen cells from one mouse with good response of immunization were fused with murine myeloma NS-1 cells on day 24 . The hybrid cell suspension was seeded into the wells of 96-well microtest plates which contained macrophage feeder layers . Anti-chlamydial antibodies in culture fluids were screened by ELISA with 1/4 MOMP & L1 EBs coated 96-well trays . Positive wells were cloned by limiting dilution . Four clones which secreted immunoglobulin G1 & G2a class were obtained after elimination of those clones that produced antibodies to C . psittaci strain EAE, C . pneumoniae strain ATCC VR1310 and uninfected BGMK cells . In micro-IF test, we found that the all four clones of MAbs reacted with our laboratory prepared L1, L2, A, B, C, E EBs, L2 tissue culture inclusions, as well as the EBs of all 15 standard serovars of C . trachomatis . The titers of their ascites were more than 1:12,800 in micro-IF test . It was shown that the four clones of MAbs reacted predominantly with 40,000 MOMP of C . trachomatis L1 in Western blot. Pathol Res Pract, 1995 Dec, 191(12), 1239 - 44 Patterns of bcl-2 expression in placenta; Kim CJ et al.; A total of 39 placentas, whose gestational ages ranged from 8 to 41 weeks, were analyzed for bcl-2 expression using immunohistochemistry and immunoblotting . Immunohistochemically, both intracytoplasmic and nuclear expression of bcl-2 was observed in villous and extravillous trophoblasts, villous mesenchymal cells and capillary endothelial cells, villous macrophages, intermediate trophoblasts, amnionic epithelium, and even in decidua and endometrial glandular epithelium in early gestational periods . The degree of expression significantly decreased in the placentas after the gestational period of 32 weeks which coincides with the declining phase of placental increase . On immunoblotting lysates of 10(4) cells from single cell suspensions of fresh placentas, bcl-2 was detected in the placentas of 22 and 33 gestational weeks, but it was negligible or absent in three term placentas . The results of the present study suggest two possible implications on the role of bcl-2 in placenta: 1) it may be a type of proliferation or maturation-related marker, especially of trophoblasts, which show decreased expression along with terminal differentiation and maturation, and 2) because the primary role of bcl-2 is the inhibition of programmed cell death (PCD), the decrease in placental bcl-2 around term may be a parturition-associated biological change. Neuroscience, 1995 Dec, 69(4), 1169 - 82 Projection neurons in fetal striatal transplants are predominantly derived from the lateral ganglionic eminence; Olsson M et al.; In the present study, we have characterized aspects of integration, growth and phenotypic differentiation of embryonic grafts derived from the selective dissection of either the lateral or medial portion of the ganglionic eminences of the rodent forebrain . Donor tissues were derived from embryonic day 15 rat, or embryonic day 14 mouse embryos, and injected, as single cell suspensions into the striatum or substantia nigra of adult rats previously subjected to an intrastriatal ibotenic acid lesion . Two to six weeks following grafting, immunocytochemical detection of DARPP-32, the 32,000 mol . wt dopamine- and cyclic AMP-regulated phosphoprotein, was used to identify areas with a striatum-like phenotype within both the intrastriatal and the intranigral grafts . It was thus revealed that all the lateral ganglionic eminence grafts, irrespective of their placement, were dominated by striatum-like tissue (up to 90% of the total graft volume), while the medial ganglionic eminence transplants were only sparsely positive (< 10% of the total graft volume) . These striatum-like regions of the grafts were selectively innervated by tyrosine hydroxylase immunopositive fibres from the host substantia nigra . Furthermore, axons derived from the lateral ganglionic eminence mouse grafts placed in the striatum, as detected by the mouse-specific neuronal marker M6, showed a more extensive and directed outgrowth towards the globus pallidus when compared to fibres emanating from the medial ganglionic eminence grafts . Mouse lateral and medial ganglionic eminence grafts placed into the substantia nigra exhibited similar fibre outgrowth patterns; both types of grafts thus innervated the substantia nigra-pars reticulata and extended axons into the cerebral peduncle . These results show that DARPP-32-positive striatal projection neurons are derived, for the most part, from the lateral ganglionic eminence and that the restricted lateral ganglionic eminence dissection provides a more optimal source of striatal tissue for grafting in the rat Huntington model. J Hematother, 1995 Dec, 4(6), 545 - 9 A semiautomated technique for volume reduction of stem cell suspensions for autotransplantation; Ayello J et al.; Infusion of thawed cryopreserved autologous stem cells (SC) is associated with a variety of complications due to the presence of dimethyl sulfoxide (DMSO) and free hemoglobin and to volume overload . Commonly, the DMSO is not removed before infusion for fear that prolonged in vitro exposure of the cells to DMSO leads to loss of clonogenic activity . We describe a simple technique for the substantial reduction in volume and DMSO content of bone marrow (BM) and peripheral blood stem cell (PBSC) suspensions . Sixty-five patients were transplanted with thawed, volume-reduced SC cryopreserved according to Stiff et al . Semiautomated volume reduction was performed on a COBE 2991 cell processor . The median volumes of cryopreserved SC were 1152 ml and 933 ml for the pools of PBSC products and the mixed pools of BM and PBSC, respectively, whereas the volume of SC infused was 153 ml (78% reduction) . There were no differences in cell recoveries between PBSC and BM (98%) . Only 2 patients demonstrated minimal side effects during infusion . A cohort of 16 metastatic breast cancer patients underwent PBSC harvests following chemotherapy/G-CSF priming and subsequent autotransplantation . Median time to an absolute neutrophil count > 500/microliters was 8 days (range 6-14 days), and median time to a platelet count > 20,000/microliters was 11 days (range 6-18 days) . Volume reduction of SC products without the risk of graft failure was performed simply and resulted in few complications during infusion. Diagn Cytopathol, 1995 Dec, 13(5), 486 - 92 Fluorescence in situ hybridization (FISH): a user's guide to optimal preparation of cytologic specimens; Abati A et al.; Fluorescence in situ hybridization (FISH) is a reliable method for tagging centromeric regions of specific chromosomes in interphase nuclei . Not only is FISH useful for chromosome enumeration, but as region-specific chromosome probes are developed, the clinical applications and potentials for use by pathologists are extensive . This technique lends itself particularly to use in cytology preparations because the cells are disaggregated and monolayer preparations yield excellent technical hybridization results . Over a 7-mo period we processed cytologic samples in an attempt to define and outline a method for optimal specimen processing for FISH use in cell suspensions, techniques applicable to all fresh cytology specimens which can also be used for the processing of surgical pathology aspirates and other material . All samples should be promptly processed to ensure specimen viability, and triaged on an individual basis to ensure preparation of moderately cellular monolayered cytospins . Equivalent nuclear probe signals have been obtained with several sample fixation methods: air-drying, 95% ethanol, methanol (Diff-Quik fixative), and Carnoy's solution . No difference was noted in the nuclear probe signals or specimen adhesion on positively charged or noncharged slides . After initial fixation our slides remained at room temperature until FISH was performed, without any adverse effects . A short digestion with proteinase K and subsequent rehybridization yielded positive results on samples that originally yielded poor nuclear probe signals. Neurochem Res, 1995 Dec, 20(12), 1449 - 56 Clearance and metabolism of arachidonic acid by C6 glioma cells and astrocytes; Staub F et al.; Effects of increased levels of arachidonic acid (AA) were analyzed in vitro by employment of C6 glioma cells and astrocytes from primary culture . The cells were suspended in a physiological medium added with arachidonic acid (AA) in a concentration range from 0.01 to 0.5 mM . The concentration profiles of the fatty acid and AA-metabolites were subsequently followed for 90 min . AA was measured by gas chromatography, whereas the AA-metabolites PGF2 alpha and LTB4 by radioimmunoassay (RIA) . Following administration of AA at 0.05 or 0.1 mM the medium was completely cleared from the fatty acid within 10 to 15 min . However, when 0.5 mM were added, AA concentrations of 0.36 +/- 0.055 mM were found at 20 min, while 0.275 +/- 0.045 mM at 90 min . Addition of AA (0.1 mM) to cell-free medium was also associated with a steady decline of its concentration, although the decrease was markedly delayed as compared to the clearance in the presence of glial cells . AA was subjected to dose-dependent metabolisation in the cell suspension as demonstrated by the production of PGF2 alpha and LTB4 . Following addition of 0.01 or 0.5 mM, concentrations of PGF2 alpha increased to a 1.9- or 4.9-fold level within 10 min, whereas those of LTB4 rose to a 1.3- or 33.7-fold level . This was attenuated or completely blocked, respectively, by the cyclo- and lipoxygenase inhibitor BW 755C . Formation of both metabolites from AA was also observed when studying astrocytes from primary culture . The current findings demonstrate an impressive efficacy of C6 glioma cells and astrocytes to clear arachidonic acid from the suspension medium and to convert the lipid compound into prostaglandins and leukotrienes . Uptake and metabolisation of AA by the glial elements may play an important role in vivo, for example in cerebral ischemia. Int J Biol Macromol, 1995 Dec, 17(6), 381 - 6 Changes in wall-bound polysaccharidase activities during the culture cycle of a Rubus fruticosus cell suspension; Faik A et al.; Several hydrolytic enzyme activities were detected in the wall of developing cells of Rubus fruticosus in suspension culture . The corresponding substrates of the enzymes are mostly polysaccharide wall constituents, except for chitinase activity . The activities measured when the enzymes were in the free state or wall-bound showed the positive influence of the cell wall micro-environment . Changes in the activities during a cell culture cycle demonstrated that those enzymes acting on xyloglucans behaved differently from the others, and suggest that xyloglucans undergo modifications in vivo over a longer period of time during the exponential growth phase . The same activities were identified in the culture medium . Endo-1,4-beta-D-glucanase activities which depolymerized carboxymethylcellulose (CMC) and xyloglucans (XG) were assayed viscosimetrically . It was found that XG oligosaccharides exhibited an inhibitory effect on the depolymerization of xyloglucans but not on that of CMC . This suggests that true xyloglucanases are present in the culture of Rubus cells. J Chromatogr B Biomed Appl, 1995 Dec 1, 674(1), 132 - 7 Rapid radioassay for metabolites of adenosine and deoxyadenosine in erythrocytes; Szabados E et al.; A radioassay has been developed to quantify the uptake and initial metabolism of adenosine (Ado) or deoxyadenosine (dAdo) by human erythrocytes . Cell suspension and {3H}Ado are mixed at 3-s intervals with a novel dual-syringe apparatus, and uptake and metabolism of Ado is stopped by centrifuging the cells through a dibutylphthalate layer into perchloric acid . The neutralized cell extract is analyzed by two-dimensional chromatography on poly(ethyleneimine)-cellulose plates by two procedures using combinations of solvents optimised for the separation of nucleosides and nucleobases, and for nucleotides derived from the exogenous {3H}Ado. Invest Radiol, 1995 Dec, 30(12), 706 - 11 Characterization of reactive versus tumor-bearing lymph nodes with interstitial magnetic resonance lymphography in an animal model; Vassallo P et al.; RATIONALE AND OBJECTIVES: To determine if magnetic resonance lymphography performed with subcutaneously administered AMI-227, a nanoparticulate iron oxide contrast agent, can distinguish reactive from tumor-bearing lymph nodes . MATERIALS AND METHODS: Mature male Copenhagen rats were inoculated with cell suspensions of R3327-MAT-LyLu rat prostate carcinoma (n = 21) or Freund's complete adjuvant (n = 15) in the left footpad to generate ipsilateral popliteal lymph node metastases or lymphadenitis . At 12 to 14 days after inoculation, T1-and T2-weighted magnetic resonance images of bilateral popliteal areas were obtained before and 24 hours after subcutaneous administration of AMI-227 . Contrast-to-noise ratios were calculated in precontrast and postcontrast images . Bilateral popliteal nodes were excised for pathologic assessment . RESULTS: AMI-227 resulted in decreased contrast-to-noise ratios in reactive (T1-W = -7.01 +/- 1.13, T2- W = -31.64 +/- 5.35) and normal (T1 - W = -13.56 +/- 1.97, T2 - W = -21.62 +/- 2.51) nodes . Contrast-to-noise ratios were unchanged (T1 - W = -0.22 +/- 1.71, T2 - W = -2.20 +/- 4.19) in tumor-containing nodes . These differences in contrast-to-noise ratio changes between tumor-bearing versus nontumor-bearing nodes were statistically significant (P < 0.05) . Histologic analysis showed similar distribution of AMI-227 within normal and reactive nodes, but not in tumor-bearing nodes . CONCLUSIONS: Differences in AMI-227-uptake between tumor- and nontumor-bearing nodes detected with magnetic resonance imaging are helpful for distinguishing the two entities. Immunol Cell Biol, 1995 Dec, 73(6), 537 - 43 Dendritic cell presentation of PPD and 19 kDa protein of Mycobacterium tuberculosis and emergent T helper cell phenotype; Baird MA et al.; Protection against infection with Mycobacterium tuberculosis is preferentially associated with the development of the T helper 1 subset, IFN-gamma production and a cell-mediated response, rather than with T helper 2 cells, 4 (IL-4) and antibody production . The type of APC interacting with T cells responsive to mycobacterial peptides may influence which of these responses predominates . This investigation focuses on the role of dendritic cells (DC) because they are the most potent APC in both primary and recall immune responses . Our results show that splenic DC-enriched suspensions prepared from C57BL/6 mice and pulsed with either purified protein derivative (PPD) or the immunodominant 19 kDa protein from M . tuberculosis, can activate antigen-primed T cells in vitro, whereas spleen cell suspensions depleted of DC cannot . DC pulsed with PPD or 19 kDa antigen are able to prime naive T cells in vivo . Supernatants collected from cultures containing T cells from mice injected with PPD-pulsed DC and then challenged in vitro with PPD-pulsed DC were found to contain more IL-2 and IFN-gamma than those from control mice which received either DC or PPD alone . No such antigen-specific IFN-gamma response occurred if DC pulsed with 19 kDa were used in place of PPD-pulsed DC . IL-4 was not detected in any of the culture supernatants . We conclude that DC can induce production of cytokines associated with a protective immune response when presenting peptides derived from heterogeneous mycobacterial antigens but not when exposed to the single 19 kDa immunodominant protein. J Clin Invest, 1995 Dec, 96(6), 2646 - 53 Clusterin promotes the aggregation and adhesion of renal porcine epithelial cells; Silkensen JR et al.; The function of clusterin, a heterodimeric glycoprotein markedly induced in renal and other organ injuries, is unclear . Since renal injury is accompanied by alterations in cell attachment, it is possible that clusterin functions to promote cell-cell and cell-substratum interactions . In this study, a single cell suspension of renal epithelial (LLC-PK1) cells was treated with purified human clusterin, resulting in time- and dose-dependent cell aggregation . Electron microscopy of the cell aggregates demonstrated cell junction and lumen formation . To determine the effect of clusterin on cell adhesion, tissue culture plates were coated with clusterin, fibronectin, PBS, or albumin . Clusterin and fibronectin promoted cell adhesion to the same extent . The adhesion to clusterin was dose dependent and specific, as a monoclonal antibody against clusterin inhibited cell adhesion to clusterin but not fibronectin . Perterbations of the cytoskeleton may underlie the alterations in cell attachment which occur in renal injury . Induction of clusterin mRNA was seen after disruption of both microtubules and microfilaments and after inhibition of cell-substratum interactions . In conclusion, clusterin is a potent renal epithelial cell aggregation and adhesion molecule . We speculate that clusterin functions to promote cell-cell and cell-substratum interactions which are perturbed in the setting of renal injury, thereby preserving the integrity of the renal epithelial barrier. Anal Chem, 1995 Dec 1, 67(23), 4261 - 8 Use of 2,3-naphthalenedicarboxaldehyde derivatization for single-cell analysis of glutathione by capillary electrophoresis and histochemical localization by fluorescence microscopy; Orwar O et al.; We report that 2,3-naphthalenedicarboxaldehyde reacts rapidly with glutathione and its precursor, gamma-glutamylcysteine, to form highly fluorescent derivatives under physiological conditions . In contrast to previous accounts of 2,3-naphthalenedicarboxaldehyde labeling of primary amines, no additional CN- ion or any other additional nucleophile is required . The fluorescence spectral properties of the chromophores (lambda exc max = 472 nm, lambda em max = 528 nm) make these derivatives amenable to excitation and detection by optical instrumentation that is optimized for fluorescein wavelengths . This selective labeling chemistry enabled quantitative determination and histochemical localization of glutathione in neurobiological samples . Intracellular glutathione was labeled by incubating cultured cells or cell suspensions in a 2,3-naphthalenedicarboxaldehyde-supplemented, DMSO-containing physiological buffer (pH = 7.4) for 2-10 min . Applications include imaging of cultured NG 108-15 cells (mouse neuroblastoma x rat glioma) and primary glial and neuronal cell cocultures (rat hippocampus) using epiluminescent and confocal fluorescence microscopy . Quantitative determination of glutathione in single NG 108-15 cells was accomplished using laser-induced fluorescence detection and capillary electrophoresis. Anal Cell Pathol, 1995 Dec, 9(4), 281 - 93 Flow-cytometric analysis of oxidative and proteolytical activities in tissue-associated phagocytes from normal and hypertrophic muscles; Lohrke B et al.; The study was conducted by the known tendencies of increased stress-susceptibility and metabolic disorders in individuals with hypertrophied muscles due to innate factors or intensive exercise which can induce the overtraining syndrome . Using an animal model, muscle-associated cells from normal (N) and hypertrophic (H) skeletal muscle (m.semitendinosus) were examined in their resting and phorbolmyristate acetate (PMA) stimulated oxidation of dihydrorhodamine (DHR) as well as cathepsin B and L activities . Phagocytes were phenotyped by their casein receptors (CR) and fibroblasts by their surface collagens (I and IV) . The portion of CR-cells in single cell suspension was 4-8% and 1-3% in H and N . The CR-cells were enriched by 200 g centrifugation and cultured for 5 days with and without cortisol (C), norepinephrine (NE) and indomethacin (I) . NE suppressed dose-dependently CR-expression in N, with increase in H occurring . C, NE and I elevated cathepsin activities only in N . PMA stimulated DHR oxidation in H and N 5- and 2-fold . Only the oxidative rate in N reacted to C, NE and I significantly . The data suggest that the response of muscle-associated cells from hypertrophied and normal muscles to signals released in stress-coping significantly differs. Plant J, 1995 Dec, 8(6), 865 - 76 Gene activation by UV light, fungal elicitor or fungal infection in Petroselinum crispum is correlated with repression of cell cycle-related genes; Logemann E et al.; The effects of UV light or fungal elicitors on plant cells have so far been studied mostly with respect to defense-related gene activation . Here, an inverse correlation of these stimulatory effects with the activities of several cell cycle-related genes is demonstrated . Concomitant with the induction of flavonoid biosynthetic enzymes in UV-irradiated cell suspension cultures of parsley (Petroselinum crispum), total histone synthesis declined to about half the initial rate . A subclass of the histone H3 gene family was selected to demonstrate the close correlation of its expression with cell division, both in intact plants and cultured cells . Using RNA-blot and run-on transcription assays, it was shown that one arbitrarily selected subclass of each of the histone H2A, H2B, H3 and H4 gene families and of the genes encoding a p34cdc2 protein kinase and a mitotic cyclin were transcriptionally repressed in UV-irradiated as well as fungal elicitor-treated parsley cells . The timing and extent of repression differed between the two stimuli; the response to light was more transient and smaller in magnitude . These differential responses to light and elicitor were inversely correlated with the induction of phenylalanine ammonia-lyase, a key enzyme of phenylpropanoid metabolism . Essentially the same result was obtained with a defined oligopeptide elicitor, indicating that the same signaling pathway is responsible for defense-related gene activation and cell cycle-related gene repression . A temporary (UV light) or long-lasting (fungal elicitor) cessation of cell culture growth is most likely due to an arrest of cell division which may be a prerequisite for full commitment of the cells to transcriptional activation of full commitment of the cells to transcriptional activation of pathways involved in UV protection or pathogen defense . This conclusion is corroborated by the observation that the histone H3 mRNA level greatly declined around fungal infection sites in young parsley leaves. Photochem Photobiol, 1995 Dec, 62(6), 970 - 5 Induction and disappearance of thymine dimers in human skin exposed to UVB radiation: flow cytometric measurements in replicating and nonreplicating epidermal cells; Berg RJ et al.; We have earlier reported on determining UV-induced DNA damage in murine epidermal cell suspensions by flow cytometric analysis of the fluorescence from a fluorescein isothiocyanate-labeled antibody (H3) directed against thymine dimers (T < > T) . Here we present an optimization of the technique for analysis of epidermal cell suspensions from 4 mm biopsies from human skin . Cells with different DNA contents can easily be distinguished in flow cytometry by the intensity of DNA-specific 7-amino-actinomycin D fluorescence . Genuine G2-M-phase cells can further be distinguished from cell doublets by pulse-shape discrimination . Thus, T < > T levels in individual cells with different DNA contents (i.e . G0-G1, S or G2-M phases) can be determined after in vivo exposure of human skin to environmentally relevant UVB (280-315 nm) doses . The method was applied to measure the decrease of T < > T in nonreplicating cells (G0-G1 phase) and replicating cells (S phase or G2-M phase) from seven volunteers exposed to twice their minimal erythema dose . The reduction in the average T < > T-specific fluorescence at 24 h after exposure was 46% (ranging between 16% and 66%) for the G0-G1 cells and 70% (ranging between 37% and 100%) for the S + G2-M cells . The difference was statistically highly significant . Determination of individual DNA repair capacities with this method can become a convenient diagnostic tool for patients with DNA repair disorders, or it may even be used to identify individuals with low repair proficiencies and increased risk of developing skin cancers. Immunology, 1995 Dec, 86(4), 661 - 7 Sensitizing capacity of Langerhans' cells obtained from ultraviolet-B-exposed murine skin; Dai R et al.; Acute low-dose ultraviolet B (UVB) radiation impairs contact hypersensitivity induction in some strains of mice (called UVB-susceptible, UVB-S), but not in others (called UVB-resistant, UVB-R) . In order to determine whether these UVB-dependent phenotypes are inherent properties of epidermal Langerhans' cells, Ia-enriched epidermal cell suspensions were prepared from normal and UVB-exposed skin of C57BL/6 (UVB-S) and BALB/c (UVB-R) mice . After derivatization with dinitrofluorobenzene (DNFB), the cells were injected into footpads of naive syngeneic mice, and the recipients were evaluated for contact hypersensitivity and for in vitro evidence of hapten-specific T-cell priming . The results indicate that DNFB-conjugated Ia-enriched epidermal cells from normal mice, and from UVB-exposed skin of UVB-R mice induced contact hypersensitivity and primed hapten-specific T cells in the draining lymph node . By contrast, epidermal cells from UVB-exposed skin of UVB-S mice failed to induce contact hypersensitivity, even though hapten-specific T cells were still detectable in the draining lymph node . In addition, UVB radiation impaired the ability of hapten-bearing Langerhans' cells from UVB-S mice to activate hapten-specific, primed T cells in vitro . We conclude the traits of UVB-S and UVB-R can be expressed directly by Langerhans' cells, and that these effects are at least in part responsible for the deleterious consequences of UVB radiation on cutaneous immunity in UVB-S mice. Immunology, 1995 Dec, 86(4), 568 - 74 Hexachlorobenzene treatment increases the number of splenic B-1-like cells and serum autoantibody levels in the rat; Schielen P et al.; In the present study, the role of B-1 cells in hexachlorobenzene (HCB)-induced autoimmune aberrations in the Wistar rat was investigated . To that end, male and female rats were exposed to a semi-synthetic diet containing 0 or 1000 mg HCB/kg food for 3 weeks . After dissection, serum was prepared form coagulated blood to determine (auto)antibody levels, and spleens and lymph nodes were isolated and weighed . Cell suspensions were prepared, counted and analysed for B- and T-cell subsets by flow cytometry . Quantification of antibody-secreting cells (ASC) in spleen cell suspensions was done with an ELISPOT assay . Previous findings that HCB treatment induced an increase of relative lymph node and spleen weights and serum (auto)antibody levels were confirmed, while it appeared that numbers of some lymph nodal, and of the splenic large cell populations, were elevated as well . HCB treatment did not change subsets of lymph nodal T and B cells, but elevated the absolute numbers of large splenic CD4+ T cells by about 70%, IgMdull/IgDbright B cells by about 60%, and IgMbright/IgDdull B cells by about 200% cells of control numbers, and the absolute numbers of splenic IgM and IgG (auto) ASC by 300-400% of the control numbers . As splenic IgMbright/IgDdull numbers and ASC numbers correlated with statistical significance, the results indicate that HCB treatment selectively activates rat splenic B-1 cells, which may underlie the elevation of serum autoantibody levels. Clin Exp Immunol, 1995 Dec, 102(3), 515 - 22 Phenotypic characterization of two cell populations involved in the acquisition of suppressor activity by cultured spleen cells from Mycobacterium lepraemurium-infected mice; Gosselin D et al.; The impairment of cellular immunity in mice infected with Mycobacterium lepraemurium was shown to correlate with the development of suppressor cells . We have previously reported that before suppressor activity is detectable in freshly harvested cell suspensions, suppressor cell precursors accumulate in the spleen of infected mice . Upon overnight culture in the presence of a regulatory cell subset, these precursor cells acquire the capacity to impair the concanavalin A (Con A)-induced proliferation of normal spleen cells . The purpose of this study was to determine the phenotype of the cells involved in this phenomenon . This was done by following the development of suppressor activity in spleen cell suspensions depleted of defined cell subsets of the adherent or the non-adherent cell fractions with selected MoAbs and immunomagnetic beads or by in vivo treatment . Our results indicate that the acquisition of suppressor activity requires the interaction of Ia+CD11b+Fc gamma R+IgG- asialo GM1- adherent cells with Thy1-CD4-CD8-IgG-Ia- asialo GM1-Fc gamma R+CD11b+ non-adherent cells . It is also shown that the development of suppressor activity is impaired by preventing cell-cell contact between these two cell subsets through coculture in 'Transwell chambers' . These observations support the conclusion that the in vitro acquisition of suppressor activity is a consequence of the maturation of suppressor cell precursors of the monocytic lineage induced by a receptor-ligand type interaction with a non-adherent cell subset that is clearly distinct from mature T, B and natural killer (NK) cells. J Pharmacol Exp Ther, 1995 Dec, 275(3), 1077 - 83 The caffeine- and ryanodine-sensitive Ca++ store in porcine myometrial cells: its heterogeneity of all-or-none Ca++ release; Zhuge R et al.; The mechanisms for Ca++ release from caffeine-sensitive stores were investigated in freshly dispersed porcine myometrial cells utilizing the fura-2 method . Because the caffeine-sensitive Ca++ store has not been detected in myometrium of mammals, we first determined the existence of this type of store in porcine myometrial cells . The evidence includes: 1) caffeine (1-33 mM)-induced concentration-dependent increase in the intracellular Ca++ concentration ({Ca++}i) in both the presence and absence of extracellular Ca++ and 2) although ryanodine alone (< or = 10 microM) failed to change {Ca++}i, it inhibited the response to caffeine in a use-, concentration- and time-dependent manner . In the cell suspension study, the amount of Ca++ released by 10 mM caffeine was found to be inversely proportional to the amount released by preadministration of caffeine (1-33 mM) . In the single cell study, about 30% of cells responded to only a certain concentration of caffeine and the others responded to caffeine gradually . Thapsigargin, an inhibitor of Ca(++)-adenosine triphosphatase in sarcoplasmic reticulum, failed to increase {Ca++}i . Pretreatment with thapsigargin inhibited the response to caffeine in a time- and concentration-dependent manner . These results suggest that in porcine myometrial cells: 1) the Ca++ released from the caffeine- and ryanodine-sensitive store is in an all-or-none manner through compartments of stores or the entire store of a cell and 2) the release process is regulated by luminal Ca++ content of the stores. J Exp Med, 1995 Dec 1, 182(6), 1645 - 53 Leukemia treatment in severe combined immunodeficiency mice by antisense oligodeoxynucleotides targeting cooperating oncogenes; Skorski T et al.; Transformation of hematopoietic cells by the p210bcr/abl tyrosine kinase appears to require the expression of a functional MYC protein, suggesting that simultaneous targeting of BCR-ABL and c-myc might be a rational strategy for attempting treatment of Phil-adelphia leukemia . To test this hypothesis, severe combined immunodeficiency mice injected with Philadelphia leukemic cells were treated systemically with equal doses of bcr-abl or c-myc antisense oligodeoxynucleotides (ODNs) or with both ODNs in combination . Compared with the mice treated with individual agents, the disease process was much slower in the group treated with both ODNs, as revealed by flow cytometry, clonogenic assay, and reverse transcriptase-polymerase chain reaction analysis to detect leukemic cells in mouse tissue cell suspensions, and by enumeration of liver metastases . The retardation of the disease process was positively correlated with a markedly increased survival of leukemic mice treated with both ODNs . These data demonstrate the therapeutic potential of targeting multiple cooperating oncogenes. J Invest Dermatol, 1995 Dec, 105(6), 782 - 8 In human dermis, ultraviolet radiation induces expansion of a CD36+ CD11b+ CD1- macrophage subset by infiltration and proliferation; CD1+ Langerhans-like dendritic antigen-presenting cells are concomitantly depleted; Meunier L et al.; Antigen-presenting (APC), suppressor T-cell-inducing macrophages infiltrate both human and murine epidermis after ultraviolet radiation (UVR) exposure . To determine their derivation, we prepared epidermal cell and dermal cell suspensions from human keratome biopsy specimens obtained from nonexposed skin and from UVB-irradiated sites (3 d after four times the minimal erythema dose) . Simultaneous triple-marker flow cytometric analysis established the extended phenotype of macrophages infiltrating sunburned human epidermis (CD1a- CD1c- CD11b+ CD11c+ CD36+ Fc gamma RII+ DR+) . This then enabled us to track dermal cells of this phenotype after UVR in relation to the heterogeneous DR+ populations in normal dermis . By both in situ immunohistology and cell suspension flow cytometry, UVR induced an expansion of bone marrow-derived DR+ cells in the perivasculature and sub-basement membrane zone of the papillary dermis . Despite an overall expansion of DR+ cells, the CD1a+ CD1c+ CD36- DR+ Langerhans-cell-like dendritic APC subset of dermal DR+ cells was depleted (p < 0.05), indicating that UVR-induced epidermal Langerhans cell loss (from 95% to 7% of DR+ epidermal cells) is not accounted for by Langerhans cell accumulation in the dermis . By contrast, UVR exposure induced a selective expansion of the dermal macrophage subset, which is phenotypically identical to the monocytic/macrophagic APCs that appear in the epidermis after UV injury (p < 0.01) . Cell cycle analysis (to determine whether this expansion was accounted for entirely by infiltration) revealed no increase in the percentage of DR+ CD36+ UVR-exposed dermal cells in S/G2/M phase; however, the expanded DR+ CD36+ subset continued its already substantial level of proliferation unabated . Therefore, epidermal macrophages derive not only from transcapillary migration, but also from in situ proliferation of a dermal precursor . Taken together, these findings show that UVR creates an epidermal and dermal APC milieu which is dominated by monocytic/macrophagic cells, through depletion of cells of dentritic APC phenotype, and concomitant selective dermal expansion of a CD1a- CD1c- CD11b+ CD36+ Fc gamma RII+ DR+ (monocyte/macrophage) population. J Am Acad Dermatol, 1995 Dec, 33(6), 947 - 53 Effects of cyclosporine on cytokines and cytokine receptors in psoriasis; Prens EP et al.; BACKGROUND: Oral cyclosporine is effective in the treatment of recalcitrant psoriasis . However, the precise mechanism(s) are not fully understood . A possible mode of action may be via down-modulation of proinflammatory cytokines that are increased in psoriatic lesions . OBJECTIVE: This study was designed to monitor the effects of cyclosporine treatment on the expression of cytokines, cytokine receptors, and other markers of inflammation in psoriatic skin . METHODS: Ten patients with recalcitrant psoriasis were treated with cyclosporine . The in vivo effects of cyclosporine on cytokines and their receptors were studied by the use of cryostat-cut sections and a panel of antibodies . The in vitro effects were studied with flow cytometry of epidermal cell suspensions prepared from psoriatic lesions and control skin . RESULTS: Clinical improvement was noted in all patients after 2 weeks of cyclosporine treatment . The expression of interleukin-1 beta, interleukin-8, CD25(IL-2R), CD36 and E-selectin were significantly decreased, whereas the number of tumor necrosis factor-receptor-positive epidermal cells was significantly increased in psoriatic lesions . CONCLUSION: Clinical improvement of psoriasis with cyclosporine treatment is accompanied by down modulation of proinflammatory epidermal cytokines and decreased dermal inflammation . Thus besides suppressing cytokine production by the inflammatory infiltrate, the beneficial effect of cyclosporine in psoriasis also depends on the inhibition of the epidermal cytokine network. J Immunol Methods, 1995 Nov 16, 187(1), 163 - 9 Isolation of leukocytes infiltrating the islets of Langerhans of diabetes-prone mice for flow cytometric analysis; Faveeuw C et al.; A simple method was developed for flow cytometric immunofluorescence analysis of cells infiltrating the islets of Langerhans in diabetes-prone rodents . Pancreases were submitted to collagenase P digestion and, in order to minimize collagenase action on leukocytes, islets were isolated before digestion was completed, that is, when exocrine tissue still remained around the islets . Islets, maintained in medium supplemented with sodium azide to prevent modulation of surface markers, were then pressed through a metal sieve and the cell suspension filtered through two different nylon screens . Cells were washed before immunofluorescence staining . Comparative phenotyping of spleen cells submitted to a similar treatment showed that, provided the collagenase P batch was screened, this procedure did not alter leukocyte surface markers and allowed multicolor analysis of the islet infiltrating cells. Arch Biochem Biophys, 1995 Nov 10, 323(2), 463 - 70 Role of cytochrome P450 in the metabolism and toxicity of hydroperoxides in isolated rat hepatocytes; Thompson JA et al.; The contributions of cytochromes P450 (P450) to the metabolism and toxicity of hydroperoxides in freshly isolated rat hepatocytes were investigated utilizing 2,6-di-tert-butyl-4-hydroperoxy-4-methyl-2,5-cyclohexadienone (BHTOOH) . This hydroperoxide was rapidly degraded in cell suspensions, and the products were identical to those determined previously with subcellular preparations of ferric P450 . With 250 microM BHTOOH, the ratio of glutathione peroxidase-mediated:P450-mediated metabolism was estimated to be about 3:1 . Surprisingly, BHTOOH was found to be a more potent cytotoxin than cumyl hydroperoxide (CuOOH), despite the fact that it caused substantially less lipid peroxidation than the latter . P450 inhibition enhanced the toxicity of BHTOOH, but lowered the toxicity of CuOOH . These data demonstrate that intracellular ferric P450 can compete with glutathione peroxidase to reduce hydroperoxides by 1- and 2-electron processes . If the alkoxy radical from homolytic cleavage of the O-O bond can undergo facile intramolecular reactions to nontoxic products, as with BHTOOH, the role of P450 is detoxification . On the other hand, if the alkoxy radical preferentially attacks membrane lipids, as with CuOOH, P450 contributes to lipid peroxidation and toxicity . It was determined that the levels of glutathione, protein thiols, and ATP decreased in parallel with BHTOOH-induced cell death, but no conclusions are possible concerning mechanisms underlying the relatively potent toxicity of BHTOOH . Toxicity may be related to the high lipophilicity of this hydroperoxide which, presumably, facilitates its passage into cells and distribution to various intracellular sites . BHTOOH appears to be an excellent model compound for investigating mechanisms of hydroperoxide-mediated cytotoxicity which do not involve lipid peroxidation. Eur J Morphol, 1995 Nov, 33(4), 359 - 72 Suitability of different staining methods for the identification of isolated and cultured cells from guinea pig (Cavia aperea porcellus) stomach; Giebel J et al.; Cell suspensions from the guinea pig gastric mucosa were obtained using a pronase/collagenase isolation method, and cultured on Petri dishes in minimum essential medium at 37 degrees C . For proper identification of different gastric cell types in cytospots, cell suspensions or culture, selective staining methods were employed, modified and evaluated . Mucous cells and mucous neck cells were detected by use of lectins . Mucous cells were stained on cytospots and in primary cultures with lectins from peanut, Helix pomatia, Ulex europaeus, wheat germ, and from soybean . Vital chief cells in suspensions but not in culture, were selectively stained by Nile blue sulphate, brilliant cresyl blue or the fluorescence dye dihexyloxacarbocyanine iodide . Pepsinogen granules of isolated and cultured chief cells were detected with a polyclonal antibody against porcine pepsinogen . Isolated parietal cells were identified in cytospots by using acidophilic dyes (aurantia, eosin) . In suspensions and in cultures vital parietal cells were identified by enzymatic detection of succinic dehydrogenase or carboanhydrase activity and by the vital stain Janus green . In cultures exclusively, parietal cells were additionally identified by the vital stain rhodamine . Cytochemically, they were identified with phalloidin by binding to actin filaments . Endocrine cells in the suspension were visualised immunocytochemically with antibodies directed against different amines or peptides . Fibroblasts and endothelial cells were identified after isolation and in primary culture with a vimentin antibody . Mast cells in suspension were either visualised by a histamine antibody or by metachromatic staining behaviour to toluidine blue, respectively . Endothelial cells in suspension or culture were distinguished from fibroblasts by endocytosis of acetylated low-density-lipoprotein . In conclusion, the developed methods are highly suitable to identify guinea pig gastric cells after isolation and follow up their fate in primary culture. Toxicol Pathol, 1995 Nov-Dec, 23(6), 635 - 43 Phagocytosis of fluorescent beads by rat thyroid follicular cells (FRTL-5): comparison with iodide trapping as an index of functional activity of thyrocytes in vitro; Sagartz JE et al.; The ability of FRTL-5 rat thyroid follicular cells to engulf latex beads by phagocytosis was evaluated using flow cytometry and compared to iodide trapping in response to selected growth factors, second messengers, and chemicals . Cell suspensions were analyzed to determine the percentage of fluorescence-positive cells as well as the fluorescence intensity of positive cells . Phagocytosis was stimulated by forskolin, cholera toxin, 8-Br-cAMP, calcitriol, and transforming growth factor-beta . In contrast, phagocytosis was inhibited by insulin, calcium, and aminotriazole, but not by sodium iodide . The results of this study showed that phagocytosis of latex beads was regulated in a manner similar to iodide trapping and could be altered by the addition of numerous compounds . Phagocytic activity was stimulated by both cAMP-dependent and cAMP-independent pathways . Flow cytometric evaluation of phagocytosis of fluorescent latex beads represents a simple, rapid, nonradioactive index of thyroid function in vitro. Braz J Med Biol Res, 1995 Nov-Dec, 28(11-12), 1319 - 25 K+ balance in rainbow trout gill and liver tissue, cell suspensions and cultured cells; Eddy FB et al.; Both intact gill and liver tissue from rainbow trout accumulated K+, as determined by 86Rb+ uptake, a process largely inhibited by ouabain, indicating the presence of functional NaKATPase . Cell suspensions, produced by disaggregation of gill or liver tissues, accumulated very little K+ compared to tissues (less than 10%) . Disaggregation resulted in depolarisation of cells with loss of intracellular K+ and although NaKATPase, as measured by 86Rb+ uptake rate, remained functional and inhibitable by ouabain, the activity was insufficient to replace the rapid K+ loss . While attached, cultured gill and liver cells showed normal K+/Na+ ratios and NaKATPase activity, but release from the substratum resulted in depolarisation and rapid K+ loss as seen in cell suspensions . These results suggest that care is required in interpreting ionic regulatory and other results from cell suspensions and that further research should be directed towards systems where cells can maintain normal ionic balance. Anticancer Res, 1995 Nov-Dec, 15(6B), 2529 - 32 Influence of cyclopropyl antiestrogens on the cell cycle kinetics of MCF-7 human breast cancer cells; Jain PT et al.; Five cyclopropyl compounds, previously shown to exhibit pure antiestrogenic activity in the mouse uterotropic assay and antiproliferative activity of MCF-7 human breast cancer cells in culture, were examined for their influence on the cell cycle kinetics of MCF-7 cells . The DNA-histogram of a single cell suspension was obtained on Coulter Epics V after fixing the cells in 70 % ethyl alcohol and staining in propidium iodide . Tamoxifen increased the percentage of cells in G1-phase with a concomitant decrease in percentage of cells in S-phase, in an estradiol reversible manner . Cyclopropyl compound 7a increased the percentage of cells in G1-phase, in an estradiol-irreversible manner . Further, compounds 5a, 5c, 7a and 7b decreased the percentage of cells in S-phase and increased percentage of cells in the G2M-phase, in an estradiol-irreversible manner . Of the five cyclopropyl compounds tested, only 4d had no influence on the cytokinetic parameters, even though this compound was found to exhibit antiproliferative activity on MCF-7 cells equal to that of tamoxifen . In conclusion, all of the cyclopropyl compounds, except 4d, altered cell cycle parameters of MCF-7 cells in a manner different than that of tamoxifen . Thus, the results of this study indicate that, although these cyclopropyl compounds are antiestrogenic, they produce antiproliferative activity by a distinct mechanism of action in estrogen receptor positive breast cancer cells. Eur J Clin Chem Clin Biochem, 1995 Nov, 33(11), 763 - 74 Nitric oxide regulates peroxisomal enzyme activities; Kremser K et al.; We have previously shown that peroxisomes are involved in the production and detoxification of reactive oxygen species and that peroxisomal functions are damaged by such oxygen species . Since nitric oxide is not only a cellular messenger, but also a free radical, it would be interesting to detect a connection between nitric oxide levels and peroxisomal enzyme activities . To determine if nitric oxide has an effect on the activities of peroxisomal functions and whether this effect is based solely on its chemical properties as reactive oxygen species or its action as a second messenger, effectors of the cellular nitric oxide level were applied to a cell model (human skin fibroblasts in culture) or directly to the enzymatic assays or both . If applied to the monolayer at non-cytotoxic concentrations, N-nitro-L-arginine methyl ester hydrochloride, an inhibitor of nitric oxide synthase (EC 1.14.13.39), increased catalase (EC 1.11.1.6) activity by more than 10% and decreased the activity of the peroxisomal fatty acid oxidation system by more than 10% . The effect was concentration-dependent . L-Arginine had the contrary effect . Combinations of L-arginine and N-nitro-L-arginine methyl ester hydrochloride compensated one another . If applied directly to the assays, S-nitroso-N-acetylpenicillamine and sodium nitroprusside inhibited catalase activity in a concentration-dependent manner . Sodium nitro-prusside had no effect on the peroxisomal beta-oxidation system unless cells were pretreated with N-nitro-L-arginine methyl ester overnight (50% inhibition) . The results show a differential effect for the application of nitric oxide-effectors on fibroblast monolayers, cell suspensions and under assay conditions . Depending on the conditions of the incubation, nitric oxide applied to the cell monolayer at low doses acts as a second messenger in cells rather than as reactive oxygen species . Under assay conditions the effect of nitric oxide is more likely that of a reactive oxygen species because it inhibits all measured enzyme activities. Leuk Lymphoma, 1995 Nov, 19(5-6), 467 - 72 Biological heterogeneity of diffuse mixed small and large cell non-Hodgkin's lymphomas assessed by DNA flow cytometry and Ki67; Palestro G et al.; The cell proliferative activity of the clinico-pathologically heterogeneous non-Hodgkin's lymphomas (NHL) included in the intermediate grade F category of the Working Formulation (WF) was investigated . S-phase fraction with flow cytometry on cell suspensions, and Ki67 on frozen tissue sections were performed in 42 F NHL . An avidin-biotin immunocomplex method was used and 1000 cells from 10 representative fields were counted . DNA content, S-phase and Ki67 were also detected in 194 NHL covering the whole spectrum of the WF . DNA content anomalies were found in 52 of 194 NHL . Their incidence, like that of S-phase fraction and Ki67 positive cells, progressively increased from low- to high-grade . A linear correlation was found between Ki67 and S-phase (r = .59) . Using the median value of proliferating cells obtained with both procedures as a cut off, two very different groups of lymphomas could be distinguished within a series of 42 F-intermediate NHL: with low and high proliferative cell activity (p < .0001) that were termed F(low) and F(high), respectively . A intermediate group was placed between them . It differed significantly from the others if Ki67 was used but only from the F(high) group if the S-phase fraction analysis was applied . No significant differences were seen when comparing F(low) with the single categories of low-grade NHL and F(high) with H high-grade NHL; no significant differences were found between F(high) and G, and between G and H categories . The existence of distinct groups of NHL in the F category, as defined by biological parameters assessing the cell proliferative activity, indicates that this category includes biologically heterogeneous lymphoma subtypes with different grades of aggressiveness . The results also indicate that the G intermediate category displays proliferation indices similar to those of H high grade category. Biophys J, 1995 Nov, 69(5), 1712 - 20 Ligand-receptor interaction rates in the presence of convective mass transport; Model MA et al.; The rate of binding of a ligand to receptors on the cell surface can be diffusion limited . We analyze the kinetics of binding, diffusion-limited in a stationary liquid, in the presence of convective mass transport . We derive a formula that expresses the reaction kinetics in terms of the mass transfer coefficient . A moderately transport-limited kinetics is not readily recognizable from the shape of the binding curve and may lead to erroneous estimates of the rate coefficients . We apply our results to practically important cases: a cell suspension in a stirred volume of liquid and a confluent cell colony under a laminar stream . Using typical numbers characterizing the ligand-receptor interactions, we show that stirring and perfusion can be important factors determining the reaction rates . With the confluent colony, the early reaction kinetics requires a different treatment, and we provide it for the case of low receptor occupancy . We show that, even with a fast perfusion, a cell monolayer can transiently generate a zone of depletion of the ligand, and that would affect the early stages of the reaction . Our results are expressed in a simple analytical form and can be used for the design and interpretation of experimental data. Anat Rec, 1995 Nov, 243(3), 347 - 56 Culture of bovine oviduct epithelial cells (BOEC); Walter I; BACKGROUND: Bovine oviduct epithelial cells are widely used in co-culture experiments to improve early embryonic development and in vitro fertilization in embryo transfer programmes for domestic animals . METHODS: The present study compares different methods for harvesting and culture of bovine oviduct epithelial cells in order to optimize handling . Bovine oviduct epithelial cells were mechanically or enzymatically isolated and cultured on glass, on permeable membranes, or in suspension . Growth of the cells and their state of differentiation was examined by means of classical staining methods, immunohistochemistry and electron microscopy . RESULTS: Initial cell suspensions contained sheets of ciliated and nonciliated (secretory) cells; 24 h after seeding, free floating epithelial cells formed vesicles with cilia on their external surface . First adhesion of cells was seen 72 h after seeding . Later on, cells grew continuously and confluent monolayers were formed after 7 days . Results were identical after mechanical or enzymatical cell harvesting and were identical on both substrata tested, i.e., on glass and on permeable membranes . Light and electron microscopy proved the monolayers to resemble a polarized, simple, cuboidal to columnar epithelial membrane with intact junctional complexes and numerous apical microvilli . Their epithelial nature was established by immunostaining for cytokeratins . Cilia were missing and secretory granules were scarce . A layer of acidic glycoprotein material was demonstrated on the apical surface . Monolayers of bovine oviduct epithelial cells stored lipid droplets and large quantities of glycogen . About 50% of the seeded cells did not adhere but survived in the culture medium as free floating cells . These suspended cells maintained morphological criteria of differentiation (cilia and secretory granules) until day 12 of culture . Proliferation rates of cultivated cells were determined by counting mitoses and by immunostaining with MIB1 antibody . Results showed coincidence of rapid proliferation and morphological dedifferentiation of monolayers . Suspended cells, by contrast, did not proliferate but retained cellular differentiation under identical culture conditions . CONCLUSIONS: The results strongly suggest that monolayers of bovine oviduct epithelial cells will not fully substitute for original oviduct epithelium when used in co-culture experiments after in vitro fertilization. Cell Prolif, 1995 Nov, 28(11), 609 - 15 In vitro bromodeoxyuridine-labelling of single cell suspensions: effects of time and temperature of sample storage; Carbajo-Perez E et al.; The present study was aimed to explore how the in vitro BrdUrd-labelling of rat thymocytes might be affected by both the time elapsed between obtaining the sample and the beginning of the labelling (0, 15, 30 or 60 min) and the effect of the temperature of storage (4 degrees C versus room temperature) . Single cell suspensions obtained after in vivo labelling with BrdUrd were used as controls . The S phase fraction was calculated by flow cytometry both according to BrdUrd-immunolabelling and DNA content . Immediate incubation with BrdUrd after the sample was obtained resulted in a slight decrease of the proportion of S phase cells analysed either according to DNA content or to BrdUrd-immunolabelling . Regardless of storage-temperature, the S phase fraction decreased in samples kept for 15 min or more before BrdUrd incubation . No BrdUrd-positive cells were detected in samples stored for 60 min at room temperature . This effect was related to temperature since positive cells were found when the samples were kept at 4 degrees C during the same time period . Our results suggest that during in vitro incubation a relative loss of S phase cells exists and that a delay beyond 15 min between obtaining the sample and the in vitro labelling seriously compromises the results of this technique. Br J Haematol, 1995 Nov, 91(3), 551 - 61 Haemopoietic growth factor production by normal and aplastic anaemia stroma in long-term bone marrow culture; Gibson FM et al.; Defective marrow stroma, or microenvironment, have been proposed as one of several mechanisms to account for bone marrow failure in aplastic anaemia (AA) . This could involve defects in positive- or negative-acting haemopoietic regulator expression by AA stroma, or alteration of normal stroma-stem cell interactions . We have used a sensitive bioassay to investigate production of granulocyte-colony stimulating factor (G-CSF), granulocyte-macrophage-colony stimulating factor (GM-CSF), interleukin (IL)-3, IL-6 and stem cell growth factor (SCF), by normal and AA stroma in long-term bone marrow culture (LTBMC) . LTBMC were grown to confluence, irradiated and harvested to yield a single cell suspension . These cells were cocultured with normal target bone marrow mononuclear cells (BMMC), or CD34+ cells, in clonogenic assays, in the absence of exogenous cytokines . Cytokines responsible for the colony-stimulating activity (CSA) and burst-promoting activity (BPA) produced by stromal cells were identified by neutralizing antibodies to specific cytokines . All normal stroma populations produced G-CSF and GM-CSF, 93% produced IL-3, 80% produced IL-6, and 70% produced SCF . Similarly, all AA stroma produced G-CSF and GM-CSF, and 71% produced SCF . In contrast, only 71% of AA stroma produced IL-3 and 36% produced IL-6 . Target cell stimulation was not dependent on direct stroma-target cell contact, suggesting production of soluble cytokines . However, although both IL-6 and G-CSF were detected in LTBMC supernatants by enzyme-linked immunoassay (ELISA), IL-3 and GM-CSF were undetectable, perhaps indicating low-level local production of these factors. Nippon Ganka Gakkai Zasshi, 1995 Nov, 99(11), 1277 - 82 {Analysis of infiltrating lymphocytes in choroidal melanoma}; Ohta K et al.; We performed immunohistochemistry on tumor infiltrating lymphocytes (TILs) in a 78-year-old man with choroidal malignant melanoma . Cell suspensions of TILs from fresh specimens and peripheral blood lymphocytes (PBLs) were stained with anti-CD3, anti-CD4, anti-CD8, anti-CD29, anti-CD45RA, and anti-human leukocyte antigen (HLA)-DR monoclonal antibodies and analyzed using three-color flow cytometry . In light microscopy, the number of infiltrating lymphocytes around the tumor was very small . Immunohistochemically, T lymphocytes were more numerous than B lymphocytes . Flow cytometric analysis showed that CD8+ cells were more numerous than CD4+ cells in CD3+ cells in TILs, and most of these cells also expressed HLA-DR antigen . CD29+ (memory) cells were increased and CD45RA+ (naive) cells were decreased in CD4+ cells in TILs as compared with PBLs . We concluded that the increase in the percentage of activated memory T lymphocytes and the decrease of naive T lymphocytes may reflect a localized antigen-specific immunological response in choroidal malignant melanoma. Biol Reprod, 1995 Nov, 53(5), 985 - 95 Effect of cryoprotectant solutes on water permeability of human spermatozoa; Gilmore JA et al.; Osmotic permeability characteristics and the effects of cryoprotectants are important determinants of recovery and function of spermatozoa after cryopreservation . The primary purpose of this study was to determine the osmotic permeability parameters of human spermatozoa in the presence of cryoprotectants . A series of experiments was done to: 1) validate the use of an electronic particle counter for determining both static and kinetic changes in sperm cell volume; 2) determine the permeability of the cells to various cryoprotectants; and 3) test the hypothesis that human sperm water permeability is affected by the presence of cryoprotectant solutes . The isosmotic volume of human sperm was 28.2 +/- 0.2 microns3 (mean +/- SEM), 29.0 +/- 0.3 microns3, and 28.2 +/- 0.4 microns3 at 22, 11, and 0 degrees C, respectively, measured at 285 mOsm/kg via an electronic particle counter . The osmotically inactive fraction of human sperm was determined from Boyle van't Hoff (BVH) plots of samples exposed to four different osmolalities (900, 600, 285, and 145 mOsm/kg) . Over this range, cells behaved as linear osmometers with osmotically inactive cell percentages at 22, 11, and 0 degrees C of 50 +/- 1%, 41 +/- 2%, and 52 +/- 3%, respectively . Permeability of human sperm to water was determined from the kinetics of volume change in a hyposmotic solution (145 mOsm/kg) at the three experimental temperatures . The hydraulic conductivity (Lp) was 1.84 +/- 0.06 microns.min-1.atm-1, 1.45 +/- 0.04 microns.min-1.atm-1, and 1.14 +/- 0.07 microns.min-1.atm-1 at 22, 11, and 0 degrees C, respectively, yielding an Arrhenius activation energy (Ea) of 3.48 kcal/mol . These biophysical characteristics of human spermatozoa are consistent with findings in previous reports, validating the use of an electronic particle counter for determining osmotic permeability parameters of human sperm . This validated system was then used to investigate the permeability of human sperm to four different cryoprotectant solutes, i.e., glycerol (Gly), dimethylsulfoxide (DMSO), propylene glycol (PG), and ethylene glycol (EG), and their effects on water permeability . A preloaded, osmotically equilibrated cell suspension was returned to an isosmotic medium while cell volume was measured over time . A Kedem-Katchalsky model was used to determine the permeability of the cells to each solute and the resulting water permeability . The permeabilities of human sperm at 22 degrees C to Gly, DMSO, PG, and EG were 2.07 +/- 0.13 x 10(-3) cm/min, 0.80 +/- 0.02 x 10(-3) cm/min, 2.3 +/- 0.1 x 10(-3) cm/min, and 7.94 +/- 0.67 x 10(-3) cm/min, respectively . The resulting Lp values at 22 degrees C were reduced to 0.77 +/- 0.08 micron.min-1.atm-1, 0.84 +/- 0.07 micron.min-1.atm-1, 1.23 +/- 0.09 microns.min-1.atm-1, and 0.74 +/- 0.06 micron.min-1.atm-1, respectively . These data support the hypothesis that low-molecular-weight, nonionic cryoprotectant solutes affect (decrease) human sperm water permeability. Toxicol Lett, 1995 Nov, 81(1), 73 - 8 The localization of DMPO spin adducts of OH in endothelial cells exposed to hydrogen peroxide; Kaneko M et al.; Examination by electron spin resonance (ESR) spectroscopy revealed the localization of 5,5-dimethyl-l-pyrroline-N-oxide (DMPO) spin adducts of hydroxyl radicals (.OH) produced by bovine endothelial cells exposed to hydrogen peroxide . Addition of 10 mM chromium oxalate, a line-broadening agent, to the reaction mixture virtually abolished the signal of DMPO-OH spin adducts . Moreover, the spin adducts were recovered in the filtrated fraction of the cell suspension . We, therefore, concluded that the location of DMPO-OH due to .OH radicals produced by endothelial cells was extracellular . Contrastingly, the site of formation of DMPO-OH was confirmed to be intracellular by the effect of Desferal, an iron chelator, and the effect of poly(ethylene glycol), an extracellular scavenger of OH radicals, as previously reported . The DMPO-OH adducts in the cell suspension mixture were degraded by a cyanide sensitive pathway and they were apparently more unstable than in the extracellular fraction . The initial amount of DMPO-OH adducts formed in endothelial cells could potentially be monitored by the DMPO-OH signals in the extracellular reaction mixture better than those in the cell suspension mixture. J Allergy Clin Immunol, 1995 Nov, 96(5 Pt 1), 677 - 86 One of the rubber latex allergens is a lysozyme; Yagami T et al.; BACKGROUND: Type I hypersensitivity reactions caused by latex products are ascribed to proteins eluted from them, but little is known about the properties of these allergenic proteins . The reason for the cross-reaction between rubber latex and fruits is also not known . We have speculated that a series of defense-related proteins in plants is a cause of latex allergy and the cross reaction . OBJECTIVE: To verify our hypothesis, we selected a lysozyme as a representative defense-related protein and examined its relationship to latex allergy . METHODS: Lysozymes eluted from latex gloves were detected with a cell-suspension clearing test . A chromatographically separated lysozyme was investigated for its physicochemical and enzymatic properties and allergenicity . RESULTS: Lysozyme activity was detected in extracts from ammoniated latex and latex gloves . We separated a lysozyme (27 kd; isoelectric point, 9.5) using cation-exchange and gel filtration chromatography . This lysozyme was enzymatically very similar to fruit lysozymes and was demonstrated to be an allergen . CONCLUSIONS: One of the rubber latex allergens is a lysozyme that has similarities to fruit lysozymes . This suggests the relevance of defense-related proteins to latex allergy and the cross reaction. Mol Cell Biochem, 1995 Oct 18, 151(2), 91 - 8 Indo-1 can simultaneously detect Ba2+ entry and Ca2+ blockade at a plasma membrane calcium channel; Owen CS et al.; The fluorescent chelator Indo-1 can make simultaneous determinations of two intracellular ion concentrations, such as {Ca2+} and {Cd2+}, or {Ca2+} and {Ba2+}, in a normal cell suspension . The second ion can be detected even if its spectrum when bound to Indo-1 is same as for the calcium-bound or the ion-free Indo-1, as long as there is a change in height . This is because the mathematical analysis uses not only the spectral shape, but also takes into account increases in total signal intensity . For maximum accuracy, whole spectra were analyzed . When 3 mM {Ba2+} was added to a B cell line that had been stimulated with antiimmunoglobulin to open receptor operated calcium channels, there was a sudden drop in 400 nm Indo-1 fluorescence . Spectral analysis showed that this was due to a drop in intracellular {Ca2+}, which was consistent with blockage of the receptor-operated calcium current by extracellular Ba2+ . The conductance for Ba2+ was also observable as a slow rise in total fluorescence . There was also a slow increase in intracellular {Ca2+} as barium accumulated in the cell, which was tentatively attributed to blockage of the plasma membrane calcium pump by intracellular Ba2+. Eur J Pharmacol, 1995 Oct 15, 291(2), 153 - 8 Ca(2+)-activated outward-rectifier K+ channels and histamine release by rat gastric enterochromaffin-like cells; Sakai H et al.; Gastric enterochromaffin-like (ECL) cells were isolated from rat gastric fundic mucosa by Percoll density-gradient centrifugation and counter-flow elutriation . About 67% of cells in the purified cell suspension were ECL cells, which were reacted with anti-histidine decarboxylase antibody . A23187, a calcium ionophore, at 0.1-10 microM induced histamine release from ECL cell-rich suspension, indicating that the Ca2+ pathway is involved in the mechanism of histamine release from the ECL cells . A23187 at 5 microM significantly increased outward-rectifier cationic current in 62% of cells in the ECL cell-rich factions . A23187-sensitive cells showed acridine orange uptake . In single-channel recordings, a Ca(2+)-dependent outward-rectifier K+ channel of large conductance (146 +/- 22 picosiemens) was found in the cell that showed acridine orange uptake . The channel opened in a voltage-dependent manner at 0.1 microM of intracellular free Ca2+ concentration . These results may suggest that opening of the Ca(2+)-activated K+ channel is one of the steps involved in the mechanism of histamine release in ECL cells. Eur J Biochem, 1995 Oct 15, 233(2), 531 - 7 New inhibitors of the ubiquinol oxidase of higher plant mitochondria; Hoefnagel MH et al.; A screen has been performed of possible inhibitors of the ubiquinol oxidase of higher plant mitochondria by assaying their effects on cyanide-insensitive NADH oxidase of mitochondria of Arum maculatum . A number of compounds which have powerful inhibitory effects have been identified . Potent inhibition was found with compounds related to the previously described n-propyl gallate, but with the n-propyl sidechain replaced with alkyl chains of greater hydrophobicity . Titration of a range of partial reactions showed that the inhibitors act specifically on the ubiquinol oxidase . The concentrations of inhibitor required are dependent on the respiratory substrate and on the amount of mitochondria used in the assay . Octyl gallate also proved to be a potent inhibitor of the ubiquinol oxidase in tobacco cell suspensions . A second class of compounds which strongly inhibit cyanide-insensitive NADH oxidation is aurachin C and its analogues . Compounds related to aurachin D are much less effective . Titrations of a range of partial reactions indicate that inhibition is caused by a direct action on the ubiquinol oxidase . However, both types of aurachins also act strongly at the Qi site of the cytochrome bc1 complex, as already known to be the case in other systems, and so they are of more limited value for studies of the ubiquinol oxidase . Titration of the oxidation of NADH via the ubiquinol oxidase in a purified mitochondrial fraction from the spadices of Arum maculatum with octyl gallate gave a half-maximal effect at a concentration of around 6 nM when the protein concentration was 14 micrograms ml-1 . A similar titre was obtained with a decyl derivative of aurachin C . This allowed us to estimate an upper limit for the concentration of ubiquinol oxidase in these mitochondria of 0.72 +/- 0.15 nmol mg-1 protein, or a ratio of ubiquinol oxidase/cytochrome oxidase of about 15 +/- 7:1 . The measurements also provide a minimal turnover number for the ubiquinol oxidase of 186 +/- 42 electrons.s-1 . Titration of the ubiquinol oxidase in soybean cotyledon mitochondria with these compounds gave the concentration of inhibitor required to elicit 50% of the maximum observed effect (I50) values about one order of magnitude higher than those found with Arum mitochondria, and again the values depended on the respiratory substrate . An explanation for the variation in I50 values may be found in terms of differences in oxidase concentrations in the different mitochondrial membranes and in the differences in rate-controlling steps with substrates of different activities. Eur J Pharmacol, 1995 Oct 6, 293(3), 259 - 66 Ca(2+)-mediated damage to rabbit gastric mucosal cells: modulation by nitric oxide; Tepperman BL et al.; Pertubations in cellular Ca2+ homeostasis can lead to oxidative stress whereas nitric oxide has been shown to inactivate oxygen radicals . Therefore the effects of inhibition of nitric oxide (NO) synthase activity on Ca2+-mediated disruption to rabbit dispersed gastric mucosal cells have been examined . Addition of the Ca2+ ionophore A23187 (3-25 microM) to the incubation medium induced a concentration-dependent increase in cell damage is assessed by trypan blue dye uptake and decreased cellular metabolic activity as estimated by alamar blue absorbance . These responses were exacerbated by inhibition of NO synthase activity with NG-monomethyl-L-arginine (300 microM) . The deleterious effects of ionophore A23187 and NG-monomethyl-L-arginine were ameliorated by addition of the NO donor S-nitroso-acetyl-penicillamine to the cell suspension . An increase in cellular Ca2+ in response to ionophore A23187 (12.5 microM) resulted in enhanced 2'7'-dichlorofluorescein fluorescence suggesting an elevation in oxidative stress . Ca2+-mediated cell injury was abolished by the oxygen radical scavengers, catalase and 2',2'-dipyridyl . However, the cytotoxic effect of combined treatment with A23187 and NG-monomethyl-L-arginine was not reduced by administration of oxygen radical scavengers . NG-monomethyl-L-arginine treatment exacerbated the increase in cytosolic Ca2+ in response to ionophore A23187 as assessed by indo-1 fluorescence . Furthermore this increase in cytosolic Ca2+ was reduced by addition of S-nitroso-acetyl-penicillamine to the incubation medium . These data suggest that NO synthase inhibition in gastric mucosal cells exacerbates the damaging actions of the Ca2+ ionophore A23187 . The increase in cell damage in response to the NO synthase inhibitor NG-monomethyl-L-arginine does not appear to be mediated by an increase in oxidative stress and may be associated in part with changes in cellular Ca2+ flux. Neuroreport, 1995 Oct 2, 6(14), 1827 - 32 Graft-induced restoration of function in hereditary cerebellar ataxia; Triarhou LC et al.; Fetal cerebellar cell suspensions, prepared from wild-type (+/+) mice, were implanted bilaterally into the cerebellum of Purkinje cell degeneration' (pcd) mutant mice, a model of adult-onset recessively inherited cerebellar ataxia, to study the functional effects of the grafts on motor coordination and fatigue resistance in a rotating rod treadmill paradigm . The viability of transplanted Purkinje cells was verified with immunocytochemistry for calbindin-D28k and for glutamate receptor 2/3 subunits and with in situ hybridisation histochemistry for insulin-like growth factor I mRNA, biochemical markers normally expressed by Purkinje cells in the cerebellum . Sham injections of vehicle did not appreciably modify the performance of pcd mutants in the rota-rod tests . On the other hand, bilateral cerebellar grafts led to a 3.5-fold increase in the time period that recipient pcd mice were able to stay on the rotating drum based on the comparison of mean scores (of three trials) or a 5.5-fold increase based on the comparison of maximum scores (of the three trials) . These findings provide evidence for a motor enhancement in the pcd mouse model of hereditary cerebellar ataxia following intracerebellar transplantation of primordial Purkinje cells. Endod Dent Traumatol, 1995 Oct, 11(5), 245 - 9 In vitro evaluation of the cytotoxicity of calcium hydroxide-based root canal sealers; Beltes P et al.; The cytotoxicity of three calcium hydroxide-containing root canal sealers (Sealapex, CRCS and Apexit) was tested by using L929 and BHK 21/C13 cells . After setting for 24 h, the sealers were covered with cell suspension . Cytotoxicity was determined by a quantitative technique at 24 h, 48 h and 72 h . All the sealers were found to be cytotoxic . Sealapex showed the highest cytotoxicity, causing a significant decrease in cell density . CRCS was less toxic than Sealapex and more toxic than Apexit . Apexit proved to be the least toxic material. Biochem Mol Med, 1995 Oct, 56(1), 52 - 62 Cholesterol modulation of transmembrane cation transport systems in human erythrocytes; Lijnen P et al.; The aim of this study was to investigate whether in vitro cholesterol enrichment of human erythrocytes affects transmembrane cation transport systems by changes induced in the membrane microviscosity of these cells . Human erythrocytes in suspension were incubated with cholesterol-lecithin dispersions to obtain an enrichment of their membrane cholesterol . The ouabain-sensitive Na+ efflux, the Na+, Li+(-)countertransport activity, the Na+, K+(-)cotransport activity, the basal transmembrane leakage of Na+ and K+, and the enzymatic activity of ATPases were determined in these cholesterol-rich cells and compared with control cells . Membrane core and surface microviscosity was also measured in the control and cholesterol-enriched cells, using the fluorescent probes, 1,6-diphenyl-1,3,5-hexatriene (DPH) and trimethylammonium (TMA)-DPH, respectively . The cholesterol content of the erythrocytes incubated in the presence of cholesterol-rich dispersions increased gradually over time . A 47% increase membrane cholesterol content was obtained after 16 h of incubation, while no change in the erythrocyte phospholipid content was found . High membrane cholesterol in the human erythrocyte phospholipid content was found . High membrane cholesterol in the human erythrocyte, obtained by in vitro enrichment of the cells with cholesterol-lecithin dispersion, inhibited in intact cell suspensions the ouabain-sensitive Na+ efflux, an estimate of the Na+(-)pump activity, and in isolated erythrocyte membranes the enzymatic activity of Na+, K+(-)ATPase, and Mg2+(-)ATPase . The dissociation constant for internal sodium and the maximal rate of ouabain-sensitive Na+ efflux is decreased in cholesterol-rich erythrocytes compared to control cells . The elevated erythrocyte membrane cholesterol content was also accompanied by a decrease in the Na+,K+(-)cotransport activity, the Na+, Li+(-)countertransport activity, and the transmembrane basal leakage of Na+ and K+ . Microviscosity, measured in the erythrocyte membrane core with the fluorescence probe DPH, was increased in the cholesterol-rich cells compared to the control cells . However, the membrane surface microviscosity, measured with the probe TMA-DPH, was not different between the control cell and the cholesterol-rich cells . The present data show that enrichment of the human erythrocyte membrane with cholesterol results in an increase of membrane core microviscosity, resulting in an inhibition of transmembrane cation transport systems in erythrocytes in suspensions and of erythrocyte membrane Na+,K+(-)ATPase, Ca2+(-)ATPase, and Mg2+(-)ATPase. J Magn Reson B, 1995 Oct, 109(1), 39 - 43 1H NMR investigations of tumor spheroids grown from a human glioma biopsy or from a human malignant glioma cell line; Kotitschke K et al.; Three-dimensional spherical aggregates of cells, grown from a permanent human malignant glioma cell line (multicellular GaMG spheroids) and from a human glioma biopsy (fragment spheroids), were investigated by 1H NMR spectroscopy . In addition, 1H NMR spectra of biopsy specimens immediately after explantation and of cell monolayers from primary passage and passage 5 were acquired and compared to those of fragment spheroids . By allowing tumor cells to grow in a three-dimensional arrangement, many biological characteristics of the original tumor in vivo are preserved . A technical procedure was established, indicating that spheroids are particularly suited for NMR spectroscopic studies . Well-resolved proton NMR spectra were obtained from homogeneously reaggregated as well as from fragment spheroids which were immobilized in agar . We found that fragment spheroids closely resemble characteristic spectral patterns of the corresponding tumor tissue in vitro, thus making such tissue available for 1H NMR spectroscopic measurements under easy to standardize tissue-culture conditions . Furthermore, the effect of altering glucose supply on metabolism and growth was studied with multicellular GaMG spheroids . The spectroscopic differences found between cell suspensions and multicellular spheroids indicate that GaMG spheroids produce large amounts of lactate and that they can adapt their metabolism depending on glucose supply similar to tumors in vivo. Neuroscience, 1995 Oct, 68(3), 737 - 49 Intrathalamic striatal grafts survive and affect circling behaviour in adult rats with excitotoxically lesioned striatum; Labandeira-Garcia JL et al.; Current models of basal ganglia disorders suggest that choreoathetosis is the end result of reduced GABAergic inhibition of the motor thalamus . Graft-derived release of GABA from intrastriatal striatal grafts has also been reported . In the present work, cell suspension grafts from embryonic day 14-15 rat striatal primordia were implanted close to the ventromedial thalamic nucleus to investigate whether they can develop and survive in this ectopic location, and whether they induce changes in the circling behaviour of the host . The grafts were implanted either in normal rats or in rats whose striatum had been lesioned with ibotenic acid . These grafts were implanted either ipsilateral or contralateral to the lesioned striatum . Additionally, some rats received intrastriatal grafts, and lesioned but non-grafted rats and lesioned rats that had received injections of saline or of cell suspensions from fetal spinal cord in the thalamus were used as control . Four to eight months after transplantation, circling behaviour after amphetamine or apomorphine injection was evaluated . Serial sections were stained with Cresyl Violet and studied immunohistochemically with antibodies against DARPP-32 (dopamine- and adenosine 3',5'-monophosphate-regulated phosphoprotein, as striatal marker), Fos protein, glutamate decarboxylase (67,000 mol . wt), glutamate decarboxylase (65,000 mol . wt) and GABA . Cresyl Violet sections showed that the intrathalamic striatal grafts developed into tissue masses resembling those observed in intrastriatal striatal grafts . DARPP-32 immunohistochemistry revealed that the grafts were composed of DARPP-32 immunoreactive (striatum-like) and DARPP-32-negative patches . The intrathalamic grafts of rats which had received a low dose of apomorphine (0.25 mg/kg) 2 h before perfusion showed clusters of intensely Fos-immunoreactive nuclei throughout the transplant, indicating that these cells had developed dopamine receptors and supersensitivity to dopamine agonists . Double Fos and DARPP-32 immunohistochemistry revealed that the Fos-positive nuclei were located in the striatum-like areas . Finally, the intrathalamic grafts also contained neurons immunoreactive to GABA and glutamate decarboxylase (65,000 and 67,000 mol . wt) . Rats that had received intrathalamic grafts contralateral to the lesioned striatum (i.e . contralateral to the lesion-induced turning direction) showed a significant reduction of circling both after amphetamine (78% reduction) or apomorphine (77% reduction) injection . Rats that had received grafts ipsilateral to the lesioned striatum showed a 75% decrease in amphetamine-induced circling, but no significant change in apomorphine-induced circling . No significant drug-induced circling was observed in non-lesioned and grafted rats . Sham grafting (saline) or grafting of weakly GABAergic tissue (fetal spinal cord) had no significant effects on lesion-induced circling behaviour.(ABSTRACT TRUNCATED AT 400 WORDS) J Physiol, 1995 Oct 1, 488 ( Pt 1), 65 - 75 A maxi Cl- channel coupled to endothelin B receptors in the basolateral membrane of guinea-pig parietal cells; Kajita H et al.; 1 . To study endothelin (ET) receptors in guinea-pig stomach, ET-binding assays and in vitro autoradiography were performed on fundic cell suspensions and on sections of the fundus, respectively . ETA and ETB receptor subtypes were found to coexist in the parietal cells . 2 . Endothelin 1 (ET-1) added to the (basolateral) bathing solution was found to activate noisy whole-cell Cl- currents within about 1 min in both single, isolated parietal cells and those within gastric glands obtained from the fundus . 3 . ET-1-induced Cl- currents were rapidly blocked by a Cl- channel blocker (NPPB) added to the (basolateral) bathing solution in a concentration-dependent manner with a half-maximum inhibition concentration of 33 microM . 4 . The anion selectivity sequence of the ET-1-induced conductance was I- > Br- > Cl- > F-, corresponding to Eisenman's sequence I . 5 . Changes in extracellular pH between 5 and 8 did not affect the ET-1-induced activation of Cl- currents . 6 . Similar activating effects were also observed with ET-3 and a specific ETB receptor agonist (IRL1620) . An ETB receptor antagonist (IRL1720) prevented the ET-1 effect, whereas an ETA-selective antagonist (FR139317 or BQ123) failed to antagonize the ET-1 effect . 7 . In the whole-cell mode, unitary Cl- channel events could be observed in association with ET-1-activated macroscopic currents . The single-channel conductances were around 200 and 350 pS at negative and positive membrane potentials, respectively . 8 . It is concluded that gastric parietal cells of guinea-pig possess pH-insensitive 'maxi' Cl- channels coupled to ETB receptors in the basolateral membrane. Plant Cell Physiol, 1995 Oct, 36(7), 1319 - 29 Isolation and characterization of two cDNAs encoding 4-coumarate:CoA ligase in Lithospermum cell cultures; Yazaki K et al.; Two near full-length cDNAs (LE4CL-1, LE4CL-2), which encode 4-coumarate:CoA ligase (4CL), were cloned from a library of Lithospermum erythrorhizon cell suspension cultures by the use of heterologous probe of potato 4CL . These cDNAs are 2.1 kb and 2.2 kb in length, respectively . LE4CL-1 encodes 636 amino acids, whose homologies to the 4CL protein sequences known to potato, parsley, pine and rice, were found to be 68%, 66%, 56% and 50% (identities on amino acid level), respectively, whereas those of the predicted translation product of LE4CL-2 (594 amino acids) to the above 4CL proteins were 49 approximately 54% . The similarity of the deduced amino acid sequences between the two 4CLs from Lithospermum cell cultures was 49% in identity . Northern analyses showed that the mRNA levels of both LE4CL-1 and LE4CL-2 were much higher under illumination than in the dark, as reported for the 4CL genes of such plants as parsley . In comparison of mRNA levels of LE4CL-1 and LE4CL-2, the former was demonstrated to be generally higher than the latter by means of an application of RT-PCR . The genomic southern blot experiments suggested that there are probably three copies of LE4CL-1 in the Lithospermum genome DNA, whereas only one copy was detected for LE4CL-2. Plant Cell Physiol, 1995 Oct, 36(7), 1213 - 20 Callose synthesis in spirostanol treated carrot cells is not triggered by cytosolic calcium, cytosolic pH or membrane potential changes; Messiaen J et al.; Carrot (Daucus carota L.) cell suspensions were treated with a spirostanol saponin from Yucca . This saponin is an elicitor of callose synthesis . Irrespectively of the mode of action of spirostanol on the callose synthase activity itself, the spirostanol-induced callose synthesis in carrot is not preceded by changes in membrane potential, cytosolic free calcium or cytosolic pH . The inability of modulators of cytosolic free calcium content (verapamil, nifedipine and Br-A23187), EGTA and a proton pump inhibitor (vandate) to inhibit or induce callose formation is consistent with a calcium- and pH-independent mechanism for callose deposition. Biophys J, 1995 Oct, 69(4), 1584 - 95 Physical and chemical effects of red cells in the shear-induced aggregation of human platelets; Goldsmith HL et al.; Both chemical and physical effects of red cells have been implicated in the spontaneous aggregation of platelets in sheared whole blood (WB) . To determine whether the chemical effect is due to ADP leaking from the red cells, a previously described technique for measuring the concentration and size of single platelets and aggregates was used to study the shear-induced aggregation of platelets in WB flowing through 1.19-mm-diameter polyethylene tubing in the presence and absence of the ADP scavenger enzyme system phosphocreatine-creatine phosphokinase (CP-CPK) . Significant spontaneous aggregation was observed at mean tube shear rates, (G) = 41.9 and 335 s-1 (42% and 13% decrease in single platelets after a mean transit time (t) = 43 s, compared to 89 and 95% decrease with 0.2 microM ADP) . The addition of CP-CPK, either at the time of, or 30 min before each run, completely abolished aggregation . In the presence of 0.2 microM ADP, CP-CPK caused a reversal of aggregation at (t) = 17 s after 30% of single cells had aggregated . To determine whether red cells exert a physical effect by increasing the time of interaction of two colliding platelets (thereby increasing the proportion of collisions resulting in the formation of aggregates), an optically transparent suspension of 40% reconstituted red cell ghosts in serum containing 2.5-micron-diameter latex spheres (3 x 10(5)/microliters) flowing through 100-microns-diameter tubes was used as a model of platelets in blood, and the results were compared with those obtained in a control suspension of latex spheres in serum alone . Two-body collisions between microspheres in the interior of the flowing ghost cell or serum suspensions at shear rates from 5 to 90 s-1 were recorded on cine film . The films were subsequently analyzed, and the measured doublet lifetime, tau meas, was compared with that predicted by theory in the absence of interactions with other particles, tau theor . The mean (tau meas/tau theor) for doublets in ghost cell suspensions was 1.614 +/- 1.795 (SD; n = 320), compared to a value of 1.001 +/- 0.312 (n = 90) for doublets in serum . Whereas 11% of doublets in ghost cell suspensions had lifetimes from 2.5 to 5 times greater than predicted, in serum, no doublets had lifetimes greater than 1.91 times that predicted . There was no statistically significant correlation between tau meas/tau theor and shear rate, but the values of tau meas/tau theor for low-angle collisions in ghost cell suspensions were significantly greater than for high-angle collisions. Biomed Tech (Berl), 1995 Oct, 40(10), 272 - 5 {Computer-controlled laser irradiation unit for studies of light-induced processes in cell cultures}; Knappe A et al.; For the photodynamic treatment of tumours, synergistic effects of photosensitizing substances and light (today usually laser light) are used . With the aim of optimizing photosensitizing drugs and therapy, the effects of light and drug dose were studied in cell experiments . To automate and standardize such in vitro experiments, a laser irradiation chamber was developed . Cells cultured from tumour cell lines are placed on micro-titre plates or in petri dishes, together with the photosensitizer, and subsequently irradiated in the irradiation chamber with a well-defined dose of laser light of a wavelength corresponding to the absorbance of the photosensitizing agent . The plates or dishes are irradiated from below . In this way, light dose errors due to refraction from the meniscus of the cell suspension as occurs with irradiation from above, are avoided . During irradiation, speckle effects on the underside of the plates or petri dishes lead to variation in irradiation . A vibrator keeps the light transmission fibre and thus speckle pattern in motion, guaranteeing a homogeneous irradiation of the cells. Carcinogenesis, 1995 Oct, 16(10), 2455 - 9 Substitution of equally carcinogenic UV-A for UV-B irradiations lowers epidermal thymine dimer levels during skin cancer induction in hairless mice; Berg RJ et al.; Cyclobutane pyrimidine dimers (CPD) are the predominant DNA lesions induced by UV-B radiation, among these lesions thymine dimers are most frequent . Although UV-A radiation may also induce CPD, it has been found that equally cytotoxic or equally mutagenic UV-A and UV-B doses do not induce equal amounts of CPD, indicating that other DNA adducts contribute to the UV-A effects . Thus far it has not been established whether this finding can be extrapolated and also holds true for the more complex biological endpoint of skin cancer . Therefore, we compared thymine dimer levels during skin cancer induction by combined UV-A and UV-B daily exposures with the levels from equally carcinogenic daily UV-B exposures . From control experiments it was known that both groups would react similarly regarding the occurrences of carcinomas, with a median latency time of 170 +/- 10 days . After 50, 106 and 151 days of irradiation eight hairless mice (SKH:HR1) from both groups were euthanized and thymine dimers in epidermal cell suspensions were quantified by flow cytometry . Staining on DNA content enabled us to quantify thymine dimers in G0/G1-phase, in S-phase and in G2M-phase subpopulations . Both in total epidermal cell populations and in subpopulations of replicating epidermal cells thymine dimer levels were significantly lower in the UV-A/B combination group than in the UV-B group (0.010 < P < 0.025 and P < 0.005 respectively) . This indicates that the carcinogenicity of UV-A relative to that of UV-B is not properly measured by thymine dimers and that other DNA lesions than CPD, for example, from reactive oxygen species, are likely to contribute to UV-A carcinogenicity. Plant Cell, 1995 Oct, 7(10), 1681 - 9 TATA box and initiator functions in the accurate transcription of a plant minimal promoter in vitro; Zhu Q et al.; The functional architecture of the proximal region of a rice phenylalanine ammonia-lyase (PAL) promoter was analyzed by transcription of PAL-beta-glucuronidase (GUS) templates by whole-cell extracts of rice cell suspension cultures . The promoter 5' truncated to position -35 was sufficient for accurate initiation of basal transcription . Substitution of the TATTTAA sequence between positions -35 and -28 with GCGGGTT or 2-bp substitutions to give TCGTTAA and TATGGAA inactivated the minimal promoter . Moreover, the function of the TATTTAA sequence was dependent on its position relative to the initiation site; hence, this element is an authentic TATA box essential for RNA polymerase II-dependent transcription . Substitutions in the TCCAAG initiator cis element (-3 to +3) at the -1 (C to A or G) and +1 (A to C or T) residues caused inaccurate initiation, whereas mutations at the other residues of this conserved element or sequence substitutions between the TATA box and initiator had little effect . TATA box and initiator functions were confirmed by analysis of the effects of promoter mutations on expression in stably transformed rice cell suspensions and plants . We concluded that the proximal region of the PAL promoter has a simple functional architecture involving a TATA box appropriately positioned upstream of the initiator . Transcription of derivatives of such minimal promoters by highly active cell extracts should allow molecular analysis of functional interactions between specific cis elements and cognate trans factors. Phytochemistry, 1995 Oct, 40(3), 801 - 6 Dynamics of the biosynthesis of methylursubin in plant cells employing in vivo 13CNMR without labelling; Lutterbach R et al.; In vivo NMR experiments with a digital 600 MHz instrument, exploiting the natural abundance of 13C, allowed us for the first time to follow the biosy