Cell suspensions

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 biosynthesis of the newly detected glycoside, methylursubin (4-methoxyphenyl-O-beta-D-primeveroside), from 4-methoxyphenol through the intermediate methylarbutin in cell suspensions of the Indian medical plant, Rauwolfia serpentina . The metabolic dynamics indicate that, within 48 hr, 4-methoxyphenol is almost completely converted into the primeveroside, methylursubin . Because of the higher sensitivity at 150.9 MHz compared to that at 100.6 MHz, measuring times could be reduced to 1.5 hr . This allows detailed monitoring of the conversion of 4-methoxyphenol with an excellent signal-to-noise ratio.

J Nutr, 1995 Oct, 125(10), 2471 - 82
The CD45RA+ (quiescent) cellular phenotype is overabundant relative to the CD45RA- phenotype within the involuted splenic T cell population of weanling mice subjected to wasting protein-energy malnutrition; Woodward BD et al.; The objective of this investigation was to determine whether an imbalance between naive- and memory-phenotype cells occurs within CD4+ and/or CD8+ splenic T cell subsets in models of protein-energy malnutrition (PEM) which produce wasting disease (loss of approximately 1.6% of body weight per day for 14 d) and profound depression in thymus-dependent immunity . Male and female weanling mice of disparate inbred strains, CBA/J and C57BL/6J, were allocated to the following groups: zero-time control (23 d old and 19 d old, respectively), ad libitum intake of a complete purified diet (19% crude protein, 17 kJ/g gross energy), restricted intake of the complete diet, and (C57BL/6J, only) ad libitum intake of an isocaloric low protein diet (0.6% crude protein) . Surface expression of isoforms of CD45, a component of the T cell receptor complex, as well as of the accessory molecule, CD2, were assessed by flow cytometry of splenic mononuclear cell suspensions . Both major T cell subsets in the malnourished groups contained a significantly higher proportion of cells expressing the surface marker, CD45RA, than was found in the spleen cells of the control groups . CD45RA+ (naive-phenotype) T cells represent the extreme of quiescence and stringent activation requirements among thymic lymphocytes . The results provide the first clear evidence of a T cell subset imbalance in PEM which is consistent with depression in acquired immunity and which occurs, apart from antigenic challenge, in a site wherein immune responses take place . The T cell receptor complex may emerge as a focal point of the depressive influence of PEM on the competence of thymic lymphocytes.

J Immunol, 1995 Oct 1, 155(7), 3345 - 52
Human follicular dendritic cells enhance cytokine-dependent growth and differentiation of CD40-activated B cells; Grouard G et al.; Germinal centers constitute microanatomic subunits within secondary follicles where B cells undergo somatic mutations, isotype switch and affinity selection . This allows the generation of memory B cells and plasma cells, whose Igs bind to the eliciting Ag with a high affinity . T cells and follicular dendritic cells (FDCs) are thought to play key roles in the germinal center reaction . To study effects of FDCs on B cell growth and differentiation, we have isolated FDC-lymphocyte clusters from human tonsils by enzymatic digestion and centrifugation of the resulting cell suspension through BSA gradient . Irradiated FDC-lymphocyte clusters induced moderate proliferation of autologous B cells . IL-2 was the only cytokine able to enhance B cell proliferation cocultured with FDCs . When B cells were activated by soluble anti-CD40 Ab with or without IL-2, IL-3, IL-4, IL-10 or IL-13, addition of FDCs increased B cell proliferation . In the presence of FDCs, maximal B cell proliferation was observed in anti-CD40 stimulated cultures supplemented with either IL-4 + IL-10 or IL-2 + IL-10 . Cultures performed in the presence of IL-2 and IL-10 resulted in high levels of Ig production in the presence of FDCs . In conclusion, the present study demonstrates that freshly isolated human FDCs can enhance the growth and differentiation of CD40-activated B cells.

Am J Obstet Gynecol, 1995 Oct, 173(4), 1157 - 60
Fetal liver cell transplantation for the creation of lymphohematopoietic chimerism in fetal baboons; Shields LE et al.; OBJECTIVE: Our purpose was to create xenogeneic lymphohematopoietic chimerism by in utero transplantation of human fetal liver cells in the midgestation fetal baboon . STUDY DESIGN: Human fetal liver cell suspensions obtained from preimmune human fetuses (< 80 days' gestation) were injected into the peritoneal cavity of three fetal baboons (85, 95, and 104 days' gestation) . A total of 9 x 10(6) cells, in a volume of 1 ml, were injected percutaneously into the fetal abdominal cavity under ultrasonographic guidance . The success of the injection was assessed by observing ascites and free loops of fetal bowel after injection . Fetal umbilical cord blood (35 days posttransplantation) and neonatal blood and bone marrow were obtained to be assayed for the presence of donor hematopoietic cells . Chimerism was detected by fluorescence in situ hybridization with a human Y-chromosome specific probe . RESULTS: All the animals survived the in utero procedures . Thirty-five days after transplantation engraftment was noted in one animal . Postnatally the same animal showed engraftment in both the peripheral blood and bone marrow . The rate of chimerism was 1.5% (1.5% of the cells were human) in both the peripheral blood and bone marrow . CONCLUSIONS: This study demonstrates that creation of xenogeneic lymphohematopoietic chimerism is possible in the midgestation fetal baboon . However, the level of chimerism was too low to study the biologic activity of the transplanted cells or to potentially ameliorate lymphohematopoietic disorders . Future studies using allogeneic tissue, evaluating cells obtained from both fetal and adult donors, and comparisons between purified stem cells and fetal liver cells are needed.

Biochem Biophys Res Commun, 1995 Sep 14, 214(2), 348 - 53
Role of interleukin-3 in the regulation of intracellular K+ homeostasis in cultured murine haemopoietic cells; Laskay G et al.; Comparative fluorimetric, flow cytofluorimetric and fluorescence ratiometric determinations of intracellular K+ concentrations in murine haemopoietic cells (FDCP-Mix clone A4) cultured in the presence and absence of the specific growth factor Interleukin-3 were carried out with the fluorescent potassium-binding benzofuran-isophthalate acetoxymethyl ester probe . Cell suspensions kept in the absence of Interleukin-3 for 5 hours exhibited lower fluorescence intensities and smaller fluorescence ratios than their growth-factor-replete counterparts, an effect found to be reversible by readdition of the growth factor . It is concluded that Interleukin-3 deprivation of these cells leads to loss of intracellular K+ . It is tentatively suggested that this deprivation-induced K+ loss might be associated with an early event in apoptotic cell death.

Biochem Biophys Res Commun, 1995 Sep 14, 214(2), 310 - 7
Growth and maturation of small hepatocytes isolated from adult rat liver; Mitaka T et al.; Small hepatocytes existed in the supernatant following low-speed centrifugation of the cell suspension after collagenase liver perfusion . The cells proliferated for more than 2 months and formed colonies in the Dulbecco's modified Eagle's medium supplemented with 10 mM nicotinamide, 10% fetal bovine serum, 1 mM ascorbic 2-phosphate, and 10 ng/ml epidermal growth factor . One small cell finally proliferated to several hundred cells . In addition, some cells in the colonies were shown to differentiate into mature hepatocytes that had a large cytoplasm and sometimes two nuclei . The secretion of albumin in the medium by the hepatocytes increased with time in culture, and the cells possessed connexin 32 in their cell membrane and many peroxisomes . Thus, the small hepatocytes may be "committed progenitor cells" which can further differentiate into mature hepatocytes.

Biochim Biophys Acta, 1995 Sep 14, 1258(2), 135 - 44
Neutrophil release of arachidonic acid, oxidants, and proteinases: causally related or independent; Ely EW et al.; This investigation examined the concept that arachidonic acid (AA) serves as a second messenger in stimulation of the respiratory burst and degranulation of polymorphonuclear neutrophils (PMN) . The main support for this idea is from observations that reagent AA, added to cell suspensions, stimulates the respiratory burst and degranulation and these events are blocked by PLA2 inhibitors . We verified that exogenously-added AA stimulated release of O2-, myeloperoxidase (MPO), and lysozyme (LZ), but this required amounts of AA which approximated the critical micellar concentration . This suggested that such administration of AA might act as an extracellular agonist, similar to particulate stimuli, rather than acting as a second messenger as might occur following mobilization of AA from cellular membranes . To investigate the role of fatty acids released by hydrolysis of cellular phospholipids, exogenously-added group I, II or III PLA2's were used to mobilize fatty acids from cellular membranes . Mole quantities of cell-associated free fatty acids were measured by negative ion chemical ionization gas chromatography/mass spectrometry . AA mobilization in response to exogenous PLA2 was dose- (0.1 to 10 U/ml PLA2) and time-dependent (peak at 1 to 2 min with a reduction by 4 min) . Resting neutrophils contained < 10 pmol free AA/10(7) PMN; the receptor-mediated agonist N-formyl-methionyl-leucyl-phenylalanine (fMLP) alone did not increase these values . Exogenously-added PLA2 generated large quantities of free AA in control and fMLP-treated cells (462 +/- 122 and 2097 +/- 176 pmol/10(7) PMN, respectively); however, this did not induce O2-, nor did it augment the level of O2- stimulated by fMLP . Also, PLA2 caused no degranulation and did not alter degranulation induced by fMLP . PLA2 also did not alter O2- or degranulation responses in primed PMN . The data indicate that mobilization of AA from cellular phospholipids neither stimulates nor modulates the respiratory burst or degranulation of PMN.

Magnes Res, 1995 Sep, 8(3), 215 - 22
Reciprocal changes in intracellular and extracellular magnesium in rat pancreatic acinar cells in response to different secretagogues; Gonzalez A et al.; This study employs the fluorescent bioprobe Mag-fura-2 to measure the mobilisation of magnesium (Mg2+) in rat pancreatic acinar cells at rest and during stimulation with either cholecystokinin-octapeptide (CCK-8; 10(-8) M), acetylcholine (ACh; 10(-5) M) or histamine (10(-4) M) . In Mag-fura-2 loaded acini suspended in normal Krebs-Ringer-Hepes (KRH) solution containing (mMol/litre) NaCL, 130; KCI, 5; HEPES, 20; KH2PO4, 1.2; CaCl2, 1.0; MgSO4, 1.0; glucose, 10; minimum essential medium, 1.0 ml/Litre and pH 7.4, both CCK-8 and ACh evoked a transient rise in intracellular free Mg2+ concentration ({Mg2+}i) followed by a sustained and prolonged decrease . Addition of CCK-8 to acinar cell suspension previously stimulated with ACh resulted in little or no change in {Mg2+}i indicating that the two secretagogues are mobilising magnesium from the same intracellular pool . Histamine had no significant effect on {Mg2+}i or on the CCK-8 evoked response . When acini were challenged in the presence of the cell-impermeant form of Mag-fura-2 (the tetrapotassium salt) in a Ca(2+)-free KRH solution containing 1-mM EGTA, CCK-8 and ACh, stimulation resulted in a gradual release of magnesium reaching a maximum after 300-350 s following application of the secretagogues (magnesium efflux) . Again histamine had no effect on magnesium release . Incubation of acinar cells in a nominally magnesium-free KRH solution had no effect on basal {Mg2+}o . When challenged with CCK-8, {Mg2+}i rose transiently, followed by a sustained decrease . In contrast, ACh stimulation resulted in a triphasic response comprising an initial rise followed by a decrease, superceded by a small rise in {Mg2+}i . Again histamine had no effect on {Mg2+}i in nominally magnesium-free solution . Addition of either ACh or CCK-8 to cells placed in KRH solution with no added Mg2+ caused a small efflux of magnesium, as measured in the presence of the tetrapotassium salt of Mag-fura-2 (cell-impermeant form) . These results, employing two different forms of a fluorescent bioprobe for the measurement of magnesium, indicate that unlike histamine, CCK-8 and ACh can evoke marked changes in intracellular free magnesium concentration during the stimulus-secretion coupling process.

J Recept Signal Transduct Res, 1995 Sep-Dec, 15(7-8), 931 - 49
Characterization of interleukin-13 receptor in carcinoma cell lines and human blood cells and comparison with the interleukin-4 receptor; Feng N et al.; The interleukin-13 receptor is characterized by ligand-binding and crosslinking studies and compared with the interleukin-4 receptor . Crosslinking of radio-labeled hIL-4 and hIL-13 to the receptors on human carcinoma and mast cell lines demonstrated a predominant subunit at 130 kDa with two other minor bands of lower molecular mass (75 kDa and 65 kDa) in autoradiography . All binding of 125I-IL-13 was specifically blocked when the carcinoma cell suspensions were incubated with an excess of unlabeled hIL-4 . However, unlabeled hIL-13 was unable to completely displace 125I-hIL-4 from the 130 kDa protein . In addition, 125I-hIL-13 showed no binding to mouse fibroblast cells transfected with human 130 kDa hIL-4 receptor c-DNA . Using weighted nonlinear computer modeling of the data from several equilibrium binding studies with human mast cells, a model of two binding sites for IL-4 (Kd = 50 and 190 pmol/L) and one site for IL-13 (Kd = 100 pmol/L) fitted better than a one site model with a very high level of significance (F = 10.66, P < 0.0001) . It can be concluded that human IL-4R and hIL-13R are similar but distinct . This conclusion is supported here for the first time by a strong statistical criterion.

Acta Derm Venereol, 1995 Sep, 75(5), 381 - 5
Topical treatment of psoriatic plaques with 1 alpha, 24 dihydroxyvitamin D3: a multiparameter flow cytometrical analysis of epidermal growth, differentiation and inflammation; Glade CP et al.; The clinical efficacy and tolerability of the vitamin D3 analogues calcitriol, calcipotriol and 1 alpha, 24 dihydroxyvitamin D3 in the treatment of psoriasis have been assessed in various clinical studies . In vitro and in vivo investigations have shown interference of these compounds with epidermal growth, keratinisation and inflammation . In this study we quantified the in vivo cell biological effects during treatment of psoriatic plaques with 1 alpha, 24 dihydroxyvitamin D3 . By using a flow cytometric triple labelling procedure, we could discriminate different epidermal subpopulations, permitting precise assessment of epidermal cell cycle kinetics . Twenty patients with plaque-type psoriasis were treated in a double-blind placebo-controlled left-right comparative study with 1 alpha, 24 dihydroxyvitamin D3 ointment (4 micrograms/g applied once daily) for 8 weeks . Epidermal cell suspensions prepared from keratotome biopsies taken before and after treatment were stained with TO-PRO-3 iodide (a new DNA fluorochrome) and monoclonal antibodies against keratin 10 (as a marker for differentiation) and vimentin (as a marker for inflammation), simultaneously . The flow cytometric analyses showed a significant decrease of proliferating basal keratinocytes in verum-treated lesions, whereas such a decrease was not observed in placebo-treated lesions . The amount of keratin 10-positive keratinocytes increased and the presence of vimentin-positive cells decreased in cell suspensions derived from both verum- and placebo-treated lesions, but these effects were not significant . We conclude that multiparameter flow cytometry promises to be an adequate approach to assess the interference of antipsoriatic treatments with cutaneous inflammation, epidermal proliferation and keratinisation . Topical 1 alpha, 24 dihydroxyvitamin D3 seems to exert its in vivo antipsoriatic effect mainly through an inhibition of epidermal growth.

Eur J Gastroenterol Hepatol, 1995 Sep, 7(9), 865 - 9
Arachidonic acid metabolism and intracellular calcium concentration in inflammatory bowel disease; Schmidt C et al.; OBJECTIVE: To study the alteration of prostaglandin E2 (PGE2) and leukotriene B4 (LTB4) synthesis and intracellular Ca2+ concentration in chronic inflammatory bowel disease, and to ascertain the effect of anti-inflammatory drugs and other mediators on eicosanoid synthesis and Ca2+ concentration . METHODS: Biopsies taken from the descending colon were isolated biochemically . The suspension of isolated mucosal cells was incubated for 15 min in the presence and absence of arachidonic acid and the Ca2+ ionophore A23187 . PGE2 and LTB4 concentrations in the incubation medium were measured by radioimmunoassay, and the intracellular Ca2+ concentration was determined using fura-2 . We studied 107 subjects . In addition, the effects of bradykinin, endothelin, cyclosporin A and PGE2 on intracellular Ca2+ concentration were determined in 25 individuals . RESULTS: Untreated patients with active inflammatory bowel disease showed a significant increase in LTB4 synthesis compared with healthy controls . However, in patients receiving steroids, sulphasalazine or 5-aminosalicylic acid, both LTB4 and PGE2 synthesis were markedly decreased . When arachidonic acid was added to the cell suspension, it significantly stimulated LTB4 synthesis, especially in patients with active disease . Patients with active Crohn's disease or ulcerative colitis had moderately higher Ca2+ levels than healthy controls . However, there was a significant decrease in intracellular Ca2+ concentration in patients with quiescent disease who were receiving maintenance therapy . CONCLUSION: We suggest that increased LTB4 synthesis and elevated intracellular Ca2+ concentrations contribute to the pathophysiology of inflammatory bowel disease . Drugs effective in the treatment of these diseases may exert their pharmacological action by normalizing these pathological findings.

Anticancer Res, 1995 Sep-Oct, 15(5B), 2129 - 36
Cytogenetic, molecular and phenotypic characterization of the newly established renal carcinoma cell line KJ29 . Evidence of translocations for chromosomes 1 and 3; Barletta C et al.; The established non papillary human renal carcinoma cell line (RCC) KJ29 was submitted to a multiparametric characterization to evaluate its potential use for in vitro and in vivo studies . The cell line grows in vitro as monolayer as well as cell suspension . Cytogenetic analysis has shown a modal chromosome number of 50 with some marker chromosomes, including rearrangements of chromosomes 1 and 3 . The antigenic phenotype is characterized by co-expression of cytokeratin and vimentin, as well as expression of urothelium differentiation antigens, low levels of class II MHC antigens and no class I antigens . A differential expression of the VLA-3 integrin heterodimer has been detected between the adherent and non adherent cell population . The cell line which is highly tumorigenic in athymic mice displays expression of erb B-2 and c-met oncogenes and high expression of cell-cycle related and Ha-ras 1 genes.

Biorheology, 1995 Sep-Oct, 32(5), 537 - 52
Effect of hematocrit on adenosine diphosphate-induced aggregation of human platelets in tube flow; Goldsmith HL et al.; Both chemical and physical effects of red cells are known to play a role in the adenosine diphosphate (ADP)-induced aggregation of human platelets in sheared blood . Using a previously described double infusion technique (Bell et al., 1989a), we studied the effect of increasing hematocrit from 10 to 60% on the rate and extent of platelet aggregation with 0.2 microM ADP in citrated whole blood undergoing tube flow . Blood and agonist were rapidly mixed in a small chamber and the suspensions flowed through lengths of 1.19 mm-diameter polyethylene tubing at mean transit times <t> from 0.2 to 42.8 s at a mean tube shear rate <G> = 335 s-1 . Effluent was collected into 0.5% glutaraldehyde, the red cells removed by centrifugation through Percoll, and all single platelets and aggregates in the volume range 1-10(5) microns3 counted and sized using an aperture impedance counter . Both the initial rate (over the first 8.6 s) and the extent of aggregation with time increased with increasing mean hematocrit up to 35.8%, being significantly greater than in citrated plasma (cPRP) . However, at 61.5% hematocrit, the extent of aggregation decreased markedly to a level close to that in cPRP . We also studied the effect of washed red cells at 39% hematocrit on the aggregation of washed platelets in Tyrodes-albumin fibrinogen-free suspensions . It had previously been shown that, at <G> > or = 335 s-1, washed platelets in platelet-rich Tyrodes (PRT) aggregated with 0.7 microM ADP . We found that red cells markedly increased the extent of aggregation from that in PRT, and promoted the formation of large aggregates, absent in PRT . Spontaneous aggregation in whole blood or washed cell suspensions in the absence of added ADP at <t> = 42.8 s was < 10% of that in the presence of ADP . The results indicate that a physical effect of red cells, likely manifested as an increase in the efficiency of aggregate formation (Goldsmith et al., 1995), plays an important role at low and normal hematocrits; however, at high hematocrits, particle crowding impedes the formation of aggregates.

Gen Comp Endocrinol, 1995 Sep, 99(3), 364 - 72
Atrial natriuretic peptide stimulates aldosterone production by turkey (Meleagris gallopavo) adrenal steroidogenic cells; Kocsis JF et al.; The inhibitory action of atrial natriuretic peptides (ANPs) on mammalian aldosterone synthesis is well documented . In addition, other work indicates that ANP and an analogue of its second messenger, 8-Br-cGMP, inhibit aldosterone production by chicken adrenal steroidogenic cells . However, the interaction between angiotensin II (AII) and ANP in the regulation of avian aldosterone production is poorly understood because chicken adrenal steroidogenic cells, the commonly used in vitro avian model, are comparatively unresponsive to AII . By contrast, turkey (Meleagris gallopavo) adrenal steroidogenic cells are sensitive to AII . Thus, in the present study, the action of ANPs and related peptides and their interaction with other stimulators of aldosterone production were investigated using freshly isolated and briefly cultured turkey adrenal steroidogenic cells . Surprisingly, several ANPs {rat (r), human (h), chicken (c)}, and rat brain natriuretic peptide (rBNP) were as efficacious as {Ile5}AII for stimulating aldosterone production (2 hr) in freshly isolated cell suspensions but were less potent than {Ile5}AII (ED50 of ANPs approximately 5-10 nM; {Ile5}AII ED50 approximately 0.1 nM) . In addition, chicken ANP enhanced maximal aldosterone production induced by {Ile5}AII (1 nM), K+ (25 mM), and hACTH-(1-39) (ACTH) (1 nM): maximal enhancement of the action of these secretagogues was +49%, +137% and +15%, respectively (P < 0.05; n = 3) . Furthermore, other ANPs and related peptides {rBNP and bovine aldosterone secretion inhibiting factor (bASIF)} enhanced maximal {Ile5}AII-induced aldosterone production: the order of maximal enhancement was rBNP (+180%) > hANP/rANP (+50%) > bASIF (+25%) (P < 0.05; n = 3).(ABSTRACT TRUNCATED AT 250 WORDS)

Phys Med Biol, 1995 Sep, 40(9), 1399 - 409
The influence of membrane permeability for ions on cell behaviour in an electric alternating field; Despa S; The behaviour of a cell in an electric alternating field has been investigated, taking into account the field-induced diffusion flows of ions through the membrane . We computed the difference in ion concentration between the internal and external sides of the membrane and the transmembrane diffusion potential induced by the external field . We also studied the effects of these flows on dielectric properties of a tissue in the radio frequency range . The value of the electric permittivity at low frequencies decreases gradually with the increase of membrane permeability for ions, while the electric permittivity at high frequencies is unchanged . These effects are especially important for analysis of the dielectric spectrum of a tissue or cell suspension which has undergone the influence of various physical or chemical agents, e.g . ionizing radiation or detergents.

Nippon Seikeigeka Gakkai Zasshi, 1995 Sep, 69(9), 721 - 34
{Gamma ray-irradiation in fresh allo-joint transplantation}; Watanabe H; In the first of a series of experiments in rat designed to assess the efficacy of gamma ray irradiation in fresh allo-joint transplantation, it was found that the optimal gamma ray dosage was 4 Gy . At this dosage level, the irradiation rays suppressed the viability of marrow cells which had the highest antigenicity, with no injury to the bone or articular cartilage . In a second experiment, a fresh homologous knee joint was irradiated at 4 Gy and then transplanted while administering the donor's splenic cell suspension (for specific immunosuppression) and the immunosuppressive agent cyclosporine (5 mg/kg) to the recipient rat . All the rats that received a pre-irradiated knee joint graft survived until sacrificed for evaluation without showing any sign of host rejection . In these rats, bone fusion had occurred between the host bone and the graft by the 8th postoperative week . Degeneration of the articular cartilage was similar between the rats that had received a pre-irradiated graft and those that had not . These findings indicated that 4 Gy gamma ray irradiation to a graft before transplantation provided an effective means of immunosuppression.

Cytometry, 1995 Sep 1, 21(1), 101 - 7
Bivariate cytokeratin/DNA flow cytometric analysis of paraffin-embedded samples from colorectal carcinomas; Leers MP et al.; Admixture of normal and neoplastic cells is a serious problem in the evaluation of tumor cell kinetic parameters by flow cytometry, in particular for DNA diploid tumors . The admixture of non-neoplastic cells, such as stromal cells and inflammatory cells, can disturb the estimation of the proliferative tumor fraction . This problem has been addressed in fresh tumor samples by applying bivariate flow cytometric analyses for DNA and cytokeratin . We have adapted this approach for formalin-fixed and paraffin-embedded tissue samples of colorectal carcinomas . After preparation of a single cell suspension from paraffin blocks by means of an enzymatic digestion step, the cells of epithelial origin were selectively stained with a panel of subtype specific cytokeratin antibodies . DNA analysis could thus be performed on the cytokeratin-positive cells . The proliferative fractions of the paraffin-embedded samples could be compared with those of the fresh tissue samples and a very good correlation was seen between DNA indices from fresh and paraffin-embedded material . As expected, after gating on the cytokeratin-positive cells an enrichment of the S-phase fraction was seen compared with the ungated cell population . However, this enrichment was more pronounced in the cell suspensions derived from the paraffin-embedded part of the tumor compared with the fresh disaggregated, ethanol-fixed part of the tumor.

Laryngoscope, 1995 Sep, 105(9 Pt 1), 928 - 33
Characterization of lymphocyte subpopulations in Warthin's tumor; Chin KW et al.; Cell suspensions from six Warthin's tumors (WTs) were characterized with fluorescence-labeled cell cytometry . WT lymphocyte subsets were identified with monoclonal antibodies directed against lymphocyte-associated cell antigens including T lymphocyte subsets, B lymphocytes, and natural killer (NK) cells . Results showed that T cell proportions were 58% and B cell proportions were 39% . The T cell helper:cytotoxic-suppressor ratio was 5.7:1 and the B to T cell ratio was 0.8:1 . NK cells represented 1.3% of cells . When compared to peripheral blood lymphocytes (PBLs) in the same patients, statistically significant differences were noted between PBLs and WT lymphocytes in the percentage of B lymphocytes (P < .01), T cytotoxic-suppressor lymphocytes (P < .02), NK cells (P < .01), and in the ratios of B to T lymphocytes (P < .01) and T helper to T cytotoxic-suppressor lymphocytes (P < .03) . Comparing these data to retrospective data on lymphocyte distribution in normal and reactive lymph nodes, the epithelial component does not appear to exert a local effect on the lymphoid component of WT.

J Invest Dermatol, 1995 Sep, 105(3), 418 - 25
Characterization of an 80-kD membrane glycoprotein (gp80) of human keratinocytes: a marker for commitment to terminal differentiation in vivo and in vitro; Schon MP et al.; We have characterized an 80-kD cell-surface glycoprotein (gp80) identified by monoclonal antibody BT 15, the expression of which is closely associated with a commitment to terminal squamous or follicular differentiation of keratinocytes in normal adult and fetal human epidermis . Maximum expression was found in the suprabasal layers, but basal cells located at the epidermal sulci were also clearly positive, in contrast to the virtually negative basal cells at the epidermal ridges . This protein was also present in benign hyperproliferative disorders of the epidermis (i.e., common warts, keratoacanthoma, psoriasis, and seborrhoic keratoses) with monoclonal antibody BT 15 preferentially staining suprabasal cells and some basal cells at the epidermal sulci . Gp80 was completely lacking in most basal cell carcinomas; the only exceptions were two cases of partially cornifying tumors that were strongly stained around keratotic pearls . In squamous cell carcinomas, gp80 was expressed in keratinized areas of the tumors . In organotypic keratinocyte cultures that resemble the in vivo situation, gp80 was strongly expressed in the suprabasal layers . However, unlike known markers for terminal differentiation, gp80 was weakly expressed by basal cells . Synthesis rates of gp80 were high in keratinocyte cell suspensions freshly prepared from skin, and decreased in primary cultures and first and second subcultures (ratio 10:4:2:1) . Elevated concentrations of the Ca++ that increased stratification of cultured keratinocytes resulted in a two- to threefold increase of gp80 synthesis . GP80 was not synthesized at detectable levels by the immortal keratinocyte cell line HaCaT; however, it was expressed in HaCaT cultures treated with mitomycin C, indicating an association with cessation of growth . Pulse-chase experiments revealed that gp80 is synthesized from a 55-kD precursor molecule, the maturation of which was prevented by treating cells with tunicamycin . Glycosidase digestion of BT 15 immunoprecipitates from untreated cells indicated that the predominant post-translational modification of the protein is N-linked glycosylation . Our data indicate that gp80 is a glycoprotein that is expressed by growth-arrested human keratinocytes or as part of the terminal differentiation program.

J Invest Dermatol, 1995 Sep, 105(3), 383 - 7
Differential extracellular signaling via Fc gamma R and FMLP in functionally distinct antigen-presenting cell subsets: ultraviolet-induced epidermal macrophages versus Langerhans cells; Shibaki A et al.; Sunburned skin is characterized by expanded numbers of macrophages (ultraviolet {UV}-MPH), and these UV-MPH differ from Langerhans cells (LC) in their abilities to initiate T-cell-mediated immune reactions . UV-MPH and LC may themselves be differentially responsive to the surrounding milieu, which may in turn modulate their immunoregulatory activity . We asked whether immunologic signal responsiveness, as assessed by cytosolic calcium mobilization, differed among normal human LC, UV-MPH, and normal blood monocytes . LC from normal skin and UV-MPH from UV-exposed skin were distinguished from keratinocytes in epidermal cell suspensions by labeling with anti-HLA-DR . Intracellular calcium content was monitored in real time with the calcium indicator, indo-1, after cross-linking Fc gamma RI, Fc gamma RII, CD11b, CD11c, or CD18 molecules, or addition of interleukin-1 alpha, IL-1 beta, interferon-gamma, bradykinin, substance P, or FMLP . Using flow cytometric analysis of cell suspensions, UV-MPH and blood monocytes were triggered by cross-linking Fc gamma RII (flux of 6.05 and 12.2, respectively) . UV-MPH could also be triggered by Fc gamma RI crosslinking and FMLP (flux of 6.41 and 15.54, respectively) . By contrast, none of these inflammatory stimuli could cause cytosolic calcium mobilization in normal LC (Flux of -0.2 by FcRII, and 0.18 by FMLP) . Because LC calcium flux may be dependent upon extracellular attachments, LC were anchored onto fibronectin-coated coverslips and then their Fc gamma RII was crosslinked in a continuous flow chamber . However, image analysis also failed to detect calcium flux . Neither population responded to interleukin-1, interferon-gamma, bradykinin, substance P, or beta 2 integrin crosslinking . These results indicate that blood monocytes and infiltrating macrophages differ substantially from LC in their responses to immune complexes and chemoattractants . Differential responsiveness to the inflammatory milieu may influence the antigen presenting or effector capabilities of these populations.

Am J Physiol, 1995 Sep, 269(3 Pt 1), C791 - 6
Whole cell sodium conductance of principal cells freshly isolated from rat cortical collecting duct; Bubien JK; Cortical collecting duct fragments were manually dissected from 6-wk-old Sprague-Dawley rats . The fragments were enzymatically digested (collagenase A) into single cells, washed, and resuspended in serum-free RPMI 1640 . Individual cells were examined electrophysiologically using the whole cell patch-clamp technique . Two morphologically distinct cell types were present in the cell suspension . Small round cells that had a capacitance of 7 pF and larger oval cells with a capacitance of 29 pF were consistently observed . Whole cell electrophysiological examination revealed that the small round cells had virtually no plasma membrane ionic conductance, whereas both inward and outward currents were observed in the larger oval-type cells . Also, superfusion of 250 pM arginine vasopressin specifically increased the inward conductance of only the larger cells . The effect could be completely inhibited by 2 microM amiloride or 100 mumol of the Rp diastereomer of 8-(4-chlorophenylthio)-adenosine 3',5'-cyclic monophosphate (a specific adenosine 3',5'-cyclic monophosphate inhibitor) . These findings are consistent with the hypothesis that the larger cells are principal cells and the smaller cells are intercalated cells and directly demonstrate that an amiloride-sensitive whole cell conductance is readily observable in freshly isolated cortical collecting duct cells . Thus the whole cell configuration of the patch-clamp technique appears to be well suited for assessing cellular mechanisms that regulate the ionic conductances of cortical collecting duct cells.

Dig Dis Sci, 1995 Sep, 40(9), 2022 - 8
Role of calcium in thromboxane B2-mediated injury to rabbit gastric mucosal cells; Wong HM et al.; A sustained increase in cytosolic Ca2+ can damage gastric mucosal cells . The present study has examined the role of Ca2+ in thromboxane B2 (TXB2)-mediated damage of rabbit isolated gastric mucosal cells . Cells were isolated from rabbit oxyntic mucosa by collagenase-EDTA digestion . Cell metabolic activity and cell damage were estimated by alamar blue dye absorbance and trypan blue uptake, respectively . Cellular Ca2+ was monitored by indo-1 dye fluorescence . Addition of TXB2 (10(-6) and 10(-8) M) to the cell suspension resulted in a decrease in metabolic activity, and this effect was reduced when Ca2+ was removed from the incubation medium . TXB2 addition to the incubation medium resulted in an increase in cytosolic Ca2+ and incubation of cells with the intracellular Ca2+ chelator, BAPTA-AM (20 microM), reduced cell injury in response to TXB2 . Incubation of cells with the Ca2+ ionophore A23187 (1-25 microM) resulted in a dose-dependent increase in trypan blue uptake and a reduction in cell metabolism . Cell injury in response to A23187 were exacerbated by addition of TXB2 (10(-8) M) to the cell suspension . TXB2 treatment reduced cellular content of reduced glutathione (GSH), while exogenous GSH addition (10 mM) reduced TXB2-mediated cell injury . These data demonstrate that TXB2 can directly injure gastric mucosal cells . Gastric mucosal cellular damage in response to TXB2 is mediated in part by a disruption of Ca2+ homeostasis as well as a reduction in cellular GSH content.

J Periodontal Res, 1995 Sep, 30(5), 360 - 8
Specific cementum attachment protein enhances selectively the attachment and migration of periodontal cells to root surfaces; Pitaru S et al.; A specific cementum attachment protein (CAP) was identified in human cementum and found to bind with high affinity to non-demineralized root surfaces, hydroxyapatite and fibronectin . Attempting to elucidate the biological function of this protein and its possible role in cementogenesis the capacity of CAP to promote selective cell migration towards and attachment of various periodontal derived cell populations to root surfaces in vitro was assessed . Human gingival fibroblasts (HGF), periodontal ligament cells (HPC), and alveolar bone cells (HABC) were labeled with {3H}Thymidine during their exponential growth phase . Root slices, 300 microns thick, were incubated with increasing concentrations of CAP . Untreated and fibronectin (FN) treated root slices served as negative and positive controls, respectively . Migration was assessed by placing root slices on confluent layers of labeled cells maintained in serum free medium and determining the number of cells migrated onto the root surface 3 days thereafter . Attachment was assessed by incubating root slices with labeled cell suspensions for 2 h and determining the number of attached cells . CAP promoted both cell migration and attachment dose dependently . HABC responded better than HPC and HGF to CAP treated root slices, and HPC response was higher than that of HGF . Cell attachment was dose dependently inhibited by synthetic RGD peptides . FN did not affect the migration of HGF, barely enhanced that of HABC, and was less potent than CAP at enhancing the migration of HPC . FN was more effective than CAP in promoting the attachment of HGF to root slices, but it was as potent as CAP in supporting the attachment of HPC and HABC.(ABSTRACT TRUNCATED AT 250 WORDS)

J Clin Pathol, 1995 Sep, 48(9), 815 - 9
Chlamydia trachomatis and ectopic pregnancy: retrospective analysis of salpingectomy specimens, endometrial biopsies, and cervical smears; Lan J et al.; AIMS--To examine the role of Chlamydia trachomatis in ectopic pregnancy by detection of DNA in archival salpingectomy specimens, and in their preceding cervical specimens and endometrial biopsies, by using the polymerase chain reaction (PCR) . METHODS--Archival paraffin embedded salpingectomy tissues (n = 48) from 37 women with ectopic pregnancy were examined for the presence of C trachomatis plasmid and omp1 DNA by PCR . In addition, preceding cervical specimens (n = 58) stored either as cervical cell suspensions or as archival cervical smears, and preceding endometrial biopsies (n = 18), taken 0-5.8 years before the ectopic pregnancy, were examined by PCR for the presence of C trachomatis . RESULTS--C trachomatis DNA was detected in only one of the 48 salpingectomy specimens from 37 women . However, in six of the 37 women, C trachomatis DNA was detected in the genital specimens (cervix and/or endometrial) taken before salpingectomy . C trachomatis infections were mostly found in endometrial or cervical specimens taken more than three years before ectopic pregnancy . No chlamydial DNA was found in endometrial or cervical specimens taken at the same time of the ectopic pregnancy . CONCLUSIONS--Although no C trachomatis DNA was found in salpingectomy specimens, several women with ectopic pregnancy had C trachomatis infections in endometrial and cervical specimens in the past . This suggests that at least in these cases the ectopic pregnancy is a late post-inflammatory complication of an ascending C trachomatis infection resulting in a scarred fallopian tube.

Arzneimittelforschung, 1995 Sep, 45(9), 1002 - 4
Inhibition of leukotriene C4 and B4 release by human eosinophils with the new 5-lipoxygenase inhibitor 6-hydroxy-2(4-sulfamoylbenzylamino)-4,5,7-trimethylbenzothiazo le hydrochloride; Fukuda T et al.; Eosinophils generate and release leukotrienes C4 and B4 (LTC4, LTB4) and platelet activating factor (PAF), all of which have the capacity to cause inflammation and tissue injury in the airways . This study has examined the effects of a new 5-lipoxygenase inhibitor, 6-hydroxy-2-(4-sulfamoylbenzylamino)-4,5,7-trimethylbenzothiazo le hydrochloride (CAS 120164-49-0, E6080) on the release of LTC4, LTB4 and PAF by human eosinophils, Eosinophils stimulated by 1 mumol/l calcium ionophore A23187 for 15 min released 37.5 +/- 2.2 ng, 2.3 +/- 0.3 ng and 4.0 +/- 0.3 pmol per 10(6) cells of immunoreactive LTC4, LTB4 and PAF, respectively (mean +/- SEM, n = 4) . LTC4 and LTB4 releases were inhibited dose-dependently by the addition of E6080 to the cell suspension . The IC50 values were 0.26 mumol/l for LTC4 and 0.23 mumol/l for LTB4 . PAF release was not inhibited . These results suggest that E6080 is a potent inhibitor of LTC4 and LTB4 release from eosinophils and may provide a protective effect against bronchoconstriction during late-phase asthmatic responses.

Biochem Biophys Res Commun, 1995 Aug 24, 213(3), 1017 - 25
Thrombospondin promotes resorption by osteoclasts in vitro; Carron JA et al.; Dentine slices were soaked in TSP-1, FN or buffer before carrying out resorption assays using bone cell suspensions prepared from the long bones of 18-20-day old pre-hatch chick embryos . Soaking dentine slices in FN resulted in a five-fold increase in resorption over controls, while TSP-1 gave a two-fold increase . However, whereas the increase in resorption on FN-soaked slices could be explained by a vast increase in cell adhesion to the slices, TSP-1-soaked wafers showed no increase in cell adhesion . TSP-1, therefore, appeared to promote resorption by a mechanism other than simply increasing adhesion of stromal cells to the dentine slices . In confirmation of this, TSP-1 was able to further increase resorption on FN-soaked slices where cell adhesion was already maximal . We conclude that the effect of TSP-1 is on resorption, and that osteoclast resorption can be regulated by the presence of TSP in the matrix.

J Immunol Methods, 1995 Aug 18, 184(2), 253 - 61
Novel isolation and purification method permitting functional cytotoxicity studies of macrophages from milky spots in the greater omentum; Krist LF et al.; Milky spots in the greater omentum are well organized perivascular infiltrates of leukocytes which are probably involved in the clearance of tumor cells from the peritoneal cavity . In milky spots, macrophages are the predominant cell type forming a distinct population of cells . To investigate whether these macrophages have a function in the control of metastatic spread in the peritoneal cavity, a novel isolation and purification method was developed in order to study the functional cytotoxicity of macrophages from milky spots in the greater omentum against tumor cells in vitro . In order to obtain a cell suspension, greater omenta of unstimulated healthy male WAG/RIJ rats were incubated in collagenase/DNase suspension and filtered . Subsequently, macrophages were isolated and purified using flow cytometry by sorting unstained cells on the basis of size and internal complexity . Macrophages and other cells were identified by routine May-Grunwald-Giemsa staining and by immunophenotyping with the specific macrophage monoclonal antibody ED 1 . Furthermore, macrophage subtypes were characterized by ultrastructural analysis . Functional cytotoxicity of the isolated macrophages was assayed against the syngeneic CC 531 tumor cell line in a colorimetric MTT assay . From three greater omenta of healthy rats 1.16 +/- 0.16 x 10(6) macrophages were isolated with a purity of 83 +/- 2% and a viability of > or = 96% . The macrophages were of the exudate (monocytic), exudate-resident and resident cell type and were in equal proportions . The contaminating cells were mainly mesothelial . A maximum cytotoxicity of approximately 30% was reached with the macrophage fraction at an effector-to-target ratio of 10 . Furthermore, it was established that the mesothelial cells did not exhibit cytotoxicity.

Blood, 1995 Aug 15, 86(4), 1339 - 47
Characterization and purification of a primitive hematopoietic cell type in adult mouse marrow capable of lymphomyeloid differentiation in long-term marrow "switch" cultures; Lemieux ME et al.; In this report, we describe a modification of the assay for long-term culture-initiating cells (LTC-IC) that allows a subset of murine LTC-IC (designated as LTC-ICML) to express both their myeloid (M) and lymphoid (L) differentiative potentials in vitro . The modified assay involves culturing test cells at limiting dilutions on irradiated mouse marrow feeder layers for an initial 4 weeks under conditions that support myelopoiesis and then for an additional week under conditions permissive for B-lymphopoiesis . All of the clonogenic pre-B progenitors (colony-forming unit {CFU} pre-B) detected in such postswitch LTC appear to be the progeny of uncommitted cells present in the original cell suspension because exposure of lymphoid-restricted progenitors to myeloid LTC conditions for > or = 7 days was found to irreversibly terminate CFU-pre-B production and, in cultures initiated with limiting numbers of input cells (no progenitors of any type detected in > 70% of cultures 1 week after the switch), the presence of CFU-pre-B was tightly associated with the presence of myeloid clonogenic cells, regardless of the purity of the input population . Limiting dilution analysis of the proportion of negative cultures measured for different numbers of input cells showed the frequency of LTC-ICML in normal adult mouse marrow to be 1 per 5 x 10(5) cells with an enrichment of approximately 500-fold in the Sca-1+ Lin-WGA+ fraction, as was also found for competitive in vivo repopulating units (CRU) and conventionally defined LTC-IC . LTC-ICML also exhibited the same resistance to treatment in vivo with 5-fluorouracil (5-FU) as CRU and LTC-IC, thereby distinguishing these three populations from the great majority of both in vitro clonogenic cells and day 12 CFU-S . The ability to quantitate cells with dual lymphoid and myeloid differentiation potentials in vitro, without the need for their prior purification, should facilitate studies of totipotent hematopoietic stem cell regulation.

Cancer Res, 1995 Aug 15, 55(16), 3664 - 8
Protection by grafts of embryonal rat tissues (teratomas) against induction and transplantation of malignant tumors; Moroson H et al.; Clinical observations and experimental studies have shown that pregnancy may have inhibitory effects on tumor growth rather than invariably aggravate neoplastic disease as believed previously . It has been suggested that circulating factors of maternal or fetal origin may protect against tumor growth during pregnancy . The previously created experimental model of teratomas provides the means of having an adult animal bearing a permanent graft of embryonal tissues . To investigate the potential effects of embryonal factors on the growth of malignant neoplasms, rats carrying grafts of embryonal tissues were subjected to the induction or transplantation of carcinomas and lymphomas . Finely minced embryo tissues or cell suspensions injected in homologous rat recipients formed permanent benign teratomas composed of a variety of well differentiated tissues . One injection of N-methyl-N-nitrosourea, a potent carcinogen administered to all rats, induced fatal mammary adenocarcinoma in 50-60% of control rats but in none of the rats bearing a grafted teratoma . Transplantation of N-methyl-N-nitrosourea-induced mammary adenocarcinoma or Gross virus-induced lymphoma killed 100% of control rats but resulted in smaller, later appearing tumors in only 25-61% of teratoma-bearing rats . The present experiments showed that rats bearing grafts of embryonal tissues in the form of teratomas were partially or totally protected against the induction and transplantation of malignant tumors that killed 100% of controls . These results suggest that the embryonal tissues are a source of tumor-inhibitory factors, which may be a part of mechanisms controlling the growth and detecting the aberrations of embryonal tissues . Their identification and analysis may provide knowledge about embryonal growth and possibly about new substances with antineoplastic activity.

Plant J, 1995 Aug, 8(2), 269 - 81
Molecular cloning of cDNAs encoding the protein backbones of arabinogalactan-proteins from the filtrate of suspension-cultured cells of Pyrus communis and Nicotiana alata; Mau SL et al.; This paper reports the isolation of cDNAs encoding the protein backbone of two arabinogalactan-proteins (AGPs), one from pear cell suspension cultures (AGPPc2) and the other from suspension cultures of Nicotiana alata (AGPNa2) . The proteins encoded by these cDNAs are quite different from the 'classical' AGP backbones described previously for AGPs isolated from pear suspension cultures and extracts of N . alata styles . The cDNA for AGPPc2 encodes a 294 amino acid protein, of which a relatively short stretch (35 amino acids) is Hyp/Pro rich; this stretch is flanked by sequences which are dominated by Asn residues . Asn residues are not a feature of the 'classical' AGP backbones in which Hyp/Pro, Ser, Ala and Thr account for most of the amino acids . The cDNA for AGPNa2 encodes a 437 amino acid protein, which contains two distinct domains: one rich in Hyp/Pro, Ser, Ala, Thr and the other rich in Asn, Tyr and Ser . The composition and sequence of the Pro-rich domain resembles that of the 'classical' AGP backbone . The Asn-rich domains of the two cDNAs described have no sequence similarity; in both cases they are predicted to be processed to give a mature backbone with a composition similar to that of the 'classical' AGPs . The study shows that different AGPs can differ in the amino acid sequence in the protein backbone, as well as the composition and sequence of the arabinogalactan side-chains . It also shows that differential expression of genes encoding AGP protein backbones, as well as differential glycosylation, can contribute to the tissue specificity of AGPs.

Br J Haematol, 1995 Aug, 90(4), 791 - 6
Platelet-induced neutrophil activation: platelet-expressed fibrinogen induces the oxidative burst in neutrophils by an interaction with CD11C/CD18; Ruf A et al.; Mutual contacts and platelet-expressed fibrinogen seem to be required for the stimulation of neutrophils by activated platelets . The beta 2-integrins CD11b/CD18 and CD11c/CD18 are potential receptors for fibrinogen on neutrophils . In order to investigate whether binding of fibrinogen to these integrins is involved, monoclonal antibodies (MoAbs) and Gly-Pro-Arg-Pro (GPRP) peptide that inhibits fibrinogen binding to CD11c/CD18 were checked for their effects on the interaction of activated platelets and neutrophils . The luminol-amplified chemiluminescence (CL) as a measure for the oxidative burst of neutrophils was recorded simultaneously to the platelet aggregation in mixed cell suspensions . The adhesion of platelets and neutrophils was determined microscopically . The thromboxane A2 mimetic U46619 was used as a potent platelet agonist but that does not stimulate neutrophils . aggregation and a strong CL of neutrophils . The platelet-induced activation of neutrophils required added fibrinogen which fibronectin or thrombospondin could not substitute for . Cytochalasin D (Cyto D) that blocks actin polymerization totally abrogated the platelet-induced Cl of neutrophils . The MoAb OKM1 against CD11b, which blocks fibrinogen binding to CD11b/CD18 as well as the MoAbs IOT16 and IOT18 directed against CD11a and CD18, respectively, had no effect . In contrast, the MoAb LeuM5 which inhibits the binding of fibrinogen to CD11c/CD18 revealed a strong inhibition . Furthermore, GPRP peptide which CD11c/CD18 recognizes on the A alpha-chain of fibrinogen also strongly inhibited the platelet-induced CL of neutrophils, whereas control peptides such as Gly-His-Arg-Pro (GHRP) or Gly-Pro-Gly-Gly (GPGG) had no effect . In contrast to the platelet-induced CL of neutrophils, Cyto D, MoAb against CD11c and GPRP peptide did not inhibit the CL induced by FMLP and PAF in pure neutrophil suspensions . They also did not affect U46619-induced platelet aggregation . The adhesion of platelets and neutrophils was neither dependent on added fibrinogen nor inhibited by Cyto D, MoAb against CD11c and the GPRP-peptide . Therefore fibrinogen and actin polymerization seem not to be required for the adhesion of neutrophils to platelets . However, the activation of neutrophils depends on the interaction of CD11c/CD18 with the A alpha-chain of platelet-expressed fibrinogen and the contractile system of neutrophils.

Eur J Immunol, 1995 Aug, 25(8), 2163 - 9
Establishment of a cell line with features of early dendritic cell precursors from fetal mouse skin; Girolomoni G et al.; During ontogeny, the skin is progressively populated by major histocompatibility complex class II-negative dendritic cell (DC) precursors that then mature into efficient antigen-presenting cells (APC) . To characterize these DC progenitors better, we generated myeloid cell lines from fetal mouse skin by infecting cell suspensions with a retroviral vector carrying an envAKR-mycMH2 fusion gene . These cells, represented by the line FSDC, displayed a dendritic morphology and their proliferation in serum-free medium was promoted by granulocyte/macrophage colony-stimulating factor (GM-CSF), but not macrophage-CSF . FSDC expressed strong surface-membrane ATP/ADPase activity, intracellular staining for 2A1 antigen, and a surface phenotype consistent with a myeloid precursor: H-2d,b+, I-Ad,b+, CD54+, CD11b+, CD11c+, 2.4G2+, F4/80+, CD44+, 2F8+, ER-MP 12-, Sca-1+, Sca-2+, NLDC-145-, B7.2+, B7.1-, J11d-, B220-, Thy-1-, and CD3- . FSDC stimulated poorly allogeneic or syngeneic T cells in the primary mixed-leukocyte reaction, and markedly increased this function after treatment with GM-CSF, GM-CSF and interleukin (IL)-4 or interferon-gamma (IFN-gamma); in contrast, stem cell factor, IL-1 alpha and tumor necrosis factor-alpha had no effect . Preculture with IFN-gamma was required for presentation of haptens to primed T cells in vitro . However, FSDC, even after cytokine activation, were less potent APC than adult epidermal Langerhans cells in both of the above assays . Finally, FSDC derivatized with haptens and injected either intravenously or subcutaneously could efficiently induce contact sensitivity responses in naive syngeneic mice . The results indicate that fetal mouse skin is colonized by myeloid precursors possessing a macrophage/immature DC-like surface phenotype and priming capacity in vivo . These cells need further differentiation and activation signals (e.g . cytokines) to express their antigen presenting potential in vitro.

Am J Physiol, 1995 Aug, 269(2 Pt 2), R339 - 49
Urate transport in Homarus americanus hepatopancreas: studies on membrane vesicles and R cells; Nies AT et al.; {2-14C}urate uptake was studied in hepatopancreatic basolateral membrane vesicles and in R cell suspensions of the American lobster by Millipore filtration techniques . Unspecific binding of urate to the vesicular membrane was 25.5 +/- 3.0% of equilibrium . Vesicular uptake showed a diffusional and a saturable component (Km) 0.37 +/- 0.04 mM and maximal velocity (Vmax) 16.5 +/- 1.2 pmol urate.mg protein-1.s-1) . {2-14C}urate uptake was significantly trans-stimulated by urate . Purine analogues, probenecid, p-aminohippuric acid, pyrazinoic, and oxonic acid cis-inhibited urate transport . Urate uptake was not affected by Na+ or K+ transmembrane gradients but stimulated by 1 mM 2-oxoglutarate at the cis-side in Na(+)-containing media . Cellular urate uptake was inhibited by pyrazinoic acid . Uptake was saturable (Km 0.53 +/- 0.11 mM and Vmax 3.7 +/- 0.4 pmol urate.mg protein-1.s-1) and Na(+)-independent . However, 2-oxoglutarate stimulated uptake in Na(+)-containing media . These results suggest that urate uptake across the basolateral membrane occurs via a specific, Na(+)-independent transport system that may operate in the exchange mode accepting 2-oxoglutarate as countertransported substrate . In vivo, urate uptake thereby would be a tertiary active system driven by a 2-oxoglutarate gradient established across the cell membrane by the operation of a Na(+)-2-oxoglutarate cotransport system.

Exp Cell Res, 1995 Aug, 219(2), 547 - 54
Phagocytic activity of FRTL-5 rat thyroid follicular cells as measured by ingestion of fluorescent latex beads; Ozaki A et al.; A rat thyroid cell line (FRTL-5) was used to study the phagocytic activity of thyroid follicular cells using fluorescent latex beads and flow cytometric analysis . Morphologic studies demonstrated that latex beads were engulfed and located within cytoplasmic vacuoles of thyrocytes . Flow cytometric evaluation of cell suspensions revealed high levels of fluorescence in cells engulfing latex beads . Using thyrotropin (TSH) as a stimulator of thyroid function and human interleukin-1 beta as an inhibitor, protocols were established for measuring the effects of these substances on either basal or TSH-induced phagocytosis . Cells exposed to latex beads over time in basal (0H) or TSH-containing medium had an increase in time-dependent phagocytic activity which was maximal after 24 or 8 h, respectively . Treatment of FRTL-5 cells with either a stimulator or an inhibitor revealed maximal change in phagocytic activity after 72 h as measured by the percentage of phagocytic cells as well as the mean fluorescence intensity . Phagocytic activity and iodide trapping by FRTL-5 cells were qualitatively similar in both sensitivity and magnitude of change in the assays used in this study . Phagocytosis of fluorescent latex beads represents a sensitive nonradioactive assay of thyrocyte function whose regulation is similar to iodide trapping.

J Invest Dermatol, 1995 Aug, 105(2), 215 - 9
Functional intercellular adhesion molecule-3 is expressed by freshly isolated epidermal Langerhans cells and is not regulated during culture; Zambruno G et al.; Activation of T lymphocytes by antigen-presenting cells requires the interaction of major histocompatibility complex/antigen complexes with the T-cell receptor as well as the binding of co-stimulatory molecules to receptors on T cells . Freshly isolated epidermal Langerhans cells (LC) do not display a significant number of co-stimulatory molecules . After short-term culture, LC express and then upregulate intercellular adhesion molecule-1 (ICAM-1) (CD54), leukocyte function-associated antigen (LFA)-3 (CD58), and B7-1 (CD80) accessory molecules and exhibit an enhanced antigen-presenting function . The present study examined the presence on human LC of the LFA-1 ligands ICAM-2 (CD102) and ICAM-3 (CD50) and their functional role in the activation of allogeneic T cells . Immunohistochemistry of skin sections and flow-cytometry analysis of freshly procured epidermal cell suspensions showed that LC (CD1a+ or HLA-DR+) expressed ICAM-3 but not ICAM-2 . After 48-72-h culture in the presence of granulocyte/macrophage colony-stimulating factor, LC did not stain for ICAM-2 but expressed ICAM-3 at the same level as fresh cells . Incubation of both freshly isolated and cultured LC with monoclonal antibodies directed against ICAM-3 reduced T-cell proliferation (25-75% inhibition) in the primary allogeneic mixed leukocyte reaction assay; incubation of cultured LC with anti-ICAM-1 and anti-ICAM-3 synergistically reduced T-cell response . The results indicate that ICAM-3 is constitutively expressed and represents an important costimulatory molecule on freshly isolated LC but, in contrast to other accessory molecules, is not subjected to regulation during LC culture.

J Immunol, 1995 Aug 1, 155(3), 1151 - 64
Immunologic self-tolerance maintained by activated T cells expressing IL-2 receptor alpha-chains (CD25) . Breakdown of a single mechanism of self-tolerance causes various autoimmune diseases; Sakaguchi S et al.; Approximately 10% of peripheral CD4+ cells and less than 1% of CD8+ cells in normal unimmunized adult mice express the IL-2 receptor alpha-chain (CD25) molecules . When CD4+ cell suspensions prepared from BALB/c nu/+ mice lymph nodes and spleens were depleted of CD25+ cells by specific mAb and C, and then inoculated into BALB/c athymic nude (nu/nu) mice, all recipients spontaneously developed histologically and serologically evident autoimmune diseases (such as thyroiditis, gastritis, insulitis, sialoadenitis, adrenalitis, oophoritis, glomerulonephritis, and polyarthritis); some mice also developed graft-vs-host-like wasting disease . Reconstitution of CD4+CD25+ cells within a limited period after transfer of CD4+CD25- cells prevented these autoimmune developments in a dose-dependent fashion, whereas the reconstitution several days later, or inoculation of an equivalent dose of CD8+ cells, was far less efficient for the prevention . When nu/nu mice were transplanted with allogeneic skins or immunized with xenogeneic proteins at the time of CD25- cell inoculation, they showed significantly heightened immune responses to the skins or proteins, and reconstitution of CD4+CD25+ cells normalized the responses . Taken together, these results indicate that CD4+CD25+ cells contribute to maintaining self-tolerance by down-regulating immune response to self and non-self Ags in an Ag-nonspecific manner, presumably at the T cell activation stage; elimination/reduction of CD4+CD25+ cells relieves this general suppression, thereby not only enhancing immune responses to non-self Ags, but also eliciting autoimmune responses to certain self-Ags . Abnormality of this T cell-mediated mechanism of peripheral tolerance can be a possible cause of various autoimmune diseases.

J Parasitol, 1995 Aug, 81(4), 649 - 52
A method for isolation and partial purification of Trichinella spiralis nurse cells; Montgomery J et al.; Invasion of vertebrate muscle cells by larvae of Trichinella spiralis is accompanied by redifferentiation of the host myofiber into a novel structure called the nurse cell . The nurse cell protects and nurtures the enclosed parasite during its long stay in host muscle . It is anatomically independent of the surrounding uninfected muscle cells and can be isolated from host tissue by mechanical or enzymatic means . Current methods employed for this purpose have yielded only small numbers of nurse cells . An apparatus designed to isolate large numbers of nurse cells and a method for removal of all free larvae and most host muscle debris is described . Homogenization and trypsin digestion of muscle tissue was followed by passage of muscle/parasite suspensions maintained at 37 C through a jacketed glass column fitted with a 40-mesh stainless steel screen at the top and a Nitex screen with 150-microns-diameter pores at the bottom . Nurse cells were retained by the Nitex screen . Density gradient centrifugation using Percoll removed all free larvae and most contaminating muscle debris from nurse cell suspensions . The large quantities of nurse cells made available by this method will allow evaluation of the molecular biology, nutrition, biochemistry, and metabolism of the enclosed parasite and of the Trichinella-modified host muscle cell.

Bone Marrow Transplant, 1995 Aug, 16(2), 297 - 301
Long-term storage of human fetal haematopoietic progenitor cells and their subsequent reconstitution . Implications for in utero transplantation; Jones DR et al.; Haematopoietic progenitor cells were isolated from human fetal liver, obtained between 6 and 15 weeks gestation . After preparation of a single cell suspension, the cells were stored using a stepwise freezing protocol; taking the cells from room temperature through -70 degrees C to liquid nitrogen . Viability (trypan blue exclusion), morphology (Leishman stain), identification of cell type (flow cytometry) and growth characteristics in semi-solid culture medium were assessed using the fresh cell suspension . We were able to confirm that the predominant cells in human fetal liver up to about 15 weeks gestation are those of the erythroid lineage . It was established that viability in excess of 75% was required to ensure adequate growth in culture after frozen storage and it was deemed important to ensure morphological integrity of the cell preparations . The colonies formed in culture were observed to be producing haemoglobin between 7 and 9 days after initial seeding . We have determined that cells can be stored in liquid nitrogen for up to 2 years without loss of (1) viability, (2) morphological features and (3) ability to form colonies and produce haemoglobin in culture . These findings offer encouragement for the implementation of a cell bank to support an in utero transplantation programme.

J Pathol, 1995 Aug, 176(4), 391 - 8
The lymphoepithelial organization of the tonsil: an immunohistochemical study in chronic recurrent tonsillitis; Ruco LP et al.; Interactions between leukocytes and crypt epithelium were extensively investigated in 12 cases of chronic recurrent tonsillitis, using immunohistochemistry and cytofluorimetric analysis of cell suspensions . Intraepithelial leukocytes are a mixed cell population composed of 50 per cent CD20-positive B lymphocytes, 40 per cent T lymphocytes with a 2.7 CD4/CD8 ratio, and 10 per cent CD68-positive macrophages . About 4 per cent of intraepithelial leukocytes are proliferating cells, as indicated by Ki-67 staining . Leukocyte infiltration is associated with expression on epithelial cells of the adhesion molecules ICAM-1 and VCAM-1 . Crypt epithelium is supported by a basement membrane showing frequent interruptions and connected with the reticular stroma of the lymphoid tissue, which was stained for fibronectin, tenascin, collagen, and laminin . Extracellular matrix (ECM) distribution was correlated with integrin expression on B and T lymphocytes . It was found that the ECM was arranged differently in the follicles and in the extrafollicular area and that B and T lymphocytes exhibited different patterns of integrin expression.

J Physiol, 1995 Aug 1, 486 ( Pt 3), 679 - 88
Slowing of shortening velocity of rat cardiac myocytes by adenosine receptor stimulation regardless of beta-adrenergic stimulation; Strang KT et al.; 1 . Single ventricular myocytes were enzymatically isolated, incubated with the A1-purinergic and beta-adrenergic receptor-specific agonists N6-cyclopentyladenosine (CPA) and isoprenaline (Iso), and then rapidly skinned . Ca2+ sensitivity of isometric tension and unloaded shortening velocity (Vo) were measured, and protein kinase A (PKA)-specific phosphorylations of troponin I (TnI) and C-protein were assessed by back-phosphorylation of cell suspensions with {gamma-32P}-ATP . 2 . Isoprenaline treatment decreased the Ca2+ sensitivity of isometric tension relative to propranolol-treated controls, as did simultaneous stimulation with Iso and CPA (Iso + CPA) . CPA alone had no effect on Ca2+ sensitivity . Vo was greater in Iso-treated cells than in paired controls, while Vo was significantly less than control in both Iso + CPA-treated and CPA-treated cells . 3 . Phosphorylation of TnI and C-protein was increased by Iso treatment and also when Iso and CPA were simultaneously applied . CPA alone caused a significant decrease in the phosphorylation state of these two proteins . 4 . From these results we conclude that A1-purinergic receptor stimulation does not inhibit beta-adrenergic receptor-mediated phosphorylation of myofilament proteins, nor does it alter the Ca2+ sensitivity of isometric tension at the level of the myofilaments . However, A1-receptor stimulation does decrease Vo at the level of the myofilaments by a mechanism that is independent of beta-adrenergically mediated phosphorylation of TnI and C-protein.

Brain Res, 1995 Jul 31, 687(1-2), 22 - 34
Preservation of fetal ventral mesencephalic cells by cool storage: in-vitro viability and TH-positive neuron survival after microtransplantation to the striatum; Nikkhah G et al.; Preservation of fetal ventral mesencephalic (VM) dopaminergic tissue prior to transplantation has been hampered by the fact that the cells are vulnerable to mechanical and osmotic stress after storage . Previous quantitative studies have shown that cool storage in a so-called 'hibernation medium' prior to grafting, can be used safely for up to 2 days without morphological or functional losses {16,32} using standard transplantation techniques . In the present study on rat fetal VM tissue we have investigated (i) the accuracy of different vital stains (trypan blue exclusion and ethidium bromide stain) to predict in vivo viability of VM cell suspensions after grafting; (ii) the influence of different storage media (glucose-saline, HBSS, DMEM, CO2-independent medium and hibernation medium), temperatures (+4 degrees C or +21 degrees C) and preparations (cell suspension or intact pieces) on the viability scores and total number of cells in vitro; and (iii) the survival and functional effects of intrastriatally grafted VM tissue after preservation by cool storage for up to 12 days using a less traumatic microtransplantation technique . The results show that cool storage at +4 degrees C of intact VM pieces in hibernation medium gives the best in vitro viability scores . Microtransplantation of cell suspensions prepared from cool-stored VM tissue produced good survival of tyrosine hydroxylase (TH)-positive graft neurons for up to 8 days of storage, and functional compensation in the amphetamine-rotation test for up to 12 days of storage . The total yield of surviving TH-positive neurons was unchanged, compared to fresh grafts, after 5 and 8 days of storage, and only reduced by 48% in the grafts stored for 12 days prior to implantation . These findings highlight the potential usefulness of a combination of cool storage and microtransplantation techniques to be able to extend the preservation periods of VM tissue . Such procedures may ultimately help to increase the safety and flexibility in experimental and clinical studies on neural transplantation of dopaminergic neurons.

Transplantation, 1995 Jul 27, 60(2), 158 - 71
Variable chimerism, graft-versus-host disease, and tolerance after different kinds of cell and whole organ transplantation from Lewis to brown Norway rats; Murase N et al.; The bidirectional paradigm of tolerance involving reciprocal host vs . graft and graft vs . host reactions was examined after Lewis (LEW)-->Brown Norway (BN) transplantation of different whole organs (liver, intestine, heart, and kidney) or of 2.5 x 10(8) LEW leukocytes obtained from bone marrow, spleen, lymph nodes, and thymus . The experiments were performed without immunosuppression or under 14 daily doses of postoperative tacrolimus, which were continued in weekly doses to 100 days in a "continuous treatment" subgroup, and to 27 days in a short treatment group . Without immunosuppression, all organs and cell suspensions failed to engraft or were acutely rejected . GVHD (usually fatal) was always caused when either the long or short treatment was used for recipients of intestinal grafts and cell suspensions of spleen and lymph nodes . In contrast, both immunosuppressive protocols allowed engraftment of bone marrow cells, liver, heart, and kidney without clinical GVHD, whereas thymus cell suspensions and small doses of whole blood neither engrafted nor caused GVHD . At 100 days, now drug-free for 73 days, the liver, bone marrow, and heart recipients were tolerant in that they accepted all challenge LEW heart and/or liver grafts for 100 more days despite in vitro evidence of donor-specific reactivity (split tolerance) . At 200 days, histopathologic studies of the challenge livers were normal no matter what the priming graft . However, the still-beating challenge hearts had a spectrum from normal to severe chronic rejection that defined the tolerogenicity of the original primary grafts: liver best-->bone marrow next-->heart least . Both the GVHD propensity and tolerogenicity in these experiments were closely associated with recipient tissue chimerism 30 and 100 days after the experiments began . The tissue chimerism was invariably multilineage, but the GVHD outcome was associated with T cell over-representation . These observations provide guidelines that should be considered in devising leukocyte augmentation protocols for human whole organ recipients . The results are discussed in relation to the historical tolerance studies of Billingham, Brent, and Medawar; Good; Monaco; and Calne.

Biochem Biophys Res Commun, 1995 Jul 26, 212(3), 861 - 7
Loss of calcium responsiveness in cultured bovine parathyroid cells is associated with decreased calcium receptor expression; Brown AJ et al.; Suppression of PTH secretion by extracellular calcium is mediated by a plasma membrane calcium receptor (CaR) . However, primary cultures of bovine parathyroid cells are known to quickly lose their responsiveness to extracellular calcium . The present study was designed to determine if the loss of calcium responsiveness is due to changes in CaR expression . In primary monolayer cultures of parathyroid cells, calcium-mediated suppression of PTH was still evident after 24 hours in culture but was completely absent after 6 days . This was preceded by a 75% drop in CaR mRNA content within 24 hours . CaR mRNA levels remained low for the 6-day culture . Earlier time points, examined in parathyroid cell suspensions, showed a 70% drop in CaR mRNA by 4 hours after collagenase-dispersion of the glands and an 85% drop after 24 hours . The decreased expression of CaR mRNA was not influenced by altering medium serum, calcium, or 1,25-dihydroxyvitamin D3 . Our results indicate that the loss of responsiveness of cultured parathyroid cells to calcium is due to decreased CaR mRNA and, presumably, CaR protein expression.

Int J Radiat Oncol Biol Phys, 1995 Jul 15, 32(4), 1071 - 81
Radiation-induced changes in glomerular and tubular cell kinetics and morphology following irradiation of a single kidney in the pig; Robbins ME et al.; PURPOSE: Radiation-induced changes in glomerular and tubular cell kinetics and morphology following irradiation of a single pig kidney were assessed . METHODS AND MATERIALS: The right kidney of 13 adult female Large White pigs was irradiated with a single dose of 9.8 Gy gamma rays . Animals were serially killed between 2 and 24 weeks postirradiation (PI); 1 h prior to postmortem each pig received 500 mg bromodeoxyuridine (BrdUrd) . At postmortem, both kidneys were removed and tissue taken to prepare cell suspensions . The labeling index (LI) of these suspensions was measured using flow cytometry; in vivo BrdUrd incorporation in glomerular and tubular cells was determined immunohistochemically . The kidneys were also assessed histologically . RESULTS: Irradiation of the right kidney alone resulted in a significant increase in renal cell LI in both the irradiated and the contralateral unirradiated kidney within 2 weeks of irradiation; peak values of 1.57 +/- 0.32% and 1.04 +/- 0.13%, respectively, were seen 4 weeks PI, significantly greater (P < 0.001) than the preirradiation value of 0.18 +/- 0.01% . The LI values then declined with time, but remained greater than those seen prior to irradiation . A similar pattern of response was determined from counts of labeled glomerular and tubular cells identified immunohistochemically . The increase in labeled glomerular cells was seen 2 weeks PI, whereas that for the tubular cells did not occur until 4 weeks PI . The irradiated kidney exhibited diffuse, progressive glomerular alterations . In contrast, tubular damage was focal; the irradiated kidney also exhibited a prominent vasculopathy, involving arteriolar and peripheral interlobular artery thickening . The contralateral unirradiated kidney appeared unchanged . CONCLUSION: These findings confirm the hypothesis that the morphologic and kinetic responses observed after irradiation of a single kidney are similar to those observed after irradiation of both kidneys . Renal irradiation results in significant alterations in glomerular and tubular cell proliferation and morphology within 2-4 weeks of irradiation; glomerular changes appear predominant.

Biochim Biophys Acta, 1995 Jul 13, 1257(2), 111 - 7
Reversible inhibition by ethanol of Mg(2+)-dependent phosphatidate phosphohydrolase: an in vitro study in the rat reticulocyte; Le Petit-Thevenin J et al.; By using a tracer method, we demonstrate that short-term in vitro exposure of intact rat reticulocytes to ethanol elicits a biphasic response of cell-bound Mg(2+)-dependent phosphatidate phosphohydrolase (PAP) . An initial concentration-dependent (200-750 mM) activity decrease is rapidly (< 10 min) followed by reversal of the inhibition in the presence of ethanol, suggesting the development of a cell resistance to the inhibitory agent . Addition to the cell suspension of propranolol (100 microM), a known PAP inhibitor, does elicit PAP inhibition but unlike ethanol, inhibition is not followed by a return with time to control value . Ethanol-induced inhibition of cell-bound PAP was also demonstrated in cell-free extracts, where the Mg(2+)-dependent activity was decreased both in the particulate and soluble fractions . In the intact cells, the transient PAP inhibition occurs in concomitance with an overall increase in total glycerolipid biosynthesis, which is constant over 60-min incubation . We suggest that the biphasic mode of response to ethanol of Mg(2+)-dependent PAP activity may play a role in the mechanism of membrane adaptation to ethanol, and thereby to the pathogenesis of alcoholism.

Arch Biochem Biophys, 1995 Jul 10, 320(2), 353 - 60
Molecular cloning and expression of alfalfa (Medicago sativa L.) vestitone reductase, the penultimate enzyme in medicarpin biosynthesis; Guo L et al.; Medicarpin, the major phytoalexin in alfalfa, is synthesized by way of the isoflavonoid branch of phenylpropanoid metabolism . One of the final steps of medicarpin biosynthesis, from vestitone to 7,2'-dihydroxy-4'-methoxyisoflavanol, is catalyzed by vestitone reductase . A 1245-bp cDNA clone which encodes vestitone reductase was identified utilizing internal amino acid sequence of purified vestitone reductase . When expressed in Escherichia coli, the cloned enzyme exhibits strict substrate stereospecificity for (3R)-vestitone, as was observed for vestitone reductase purified from alfalfa . The calculated molecular weight of the protein (35,918) is similar to that of purified vestitone reductase from alfalfa (38 kDa by SDS-PAGE) . The levels of vestitone reductase transcript (1.35 kb) greatly increase within 2 h of elicitor addition to alfalfa cell suspension cultures, preceding the rapid increases in vestitione reductase enzyme activity and medicarpin biosynthesis . In healthy alfalfa plants, the highest levels of transcripts were detected in roots and root nodules, consistent with the synthesis of medicarpin and its conjugate in these tissues . The cloning of the vestitone reductase gene provides a specific tool for the study and manipulation of pterocarpan biosynthesis in legumes.

Mol Reprod Dev, 1995 Jul, 41(3), 348 - 54
Separation of round spermatids from the rat using an immunoselection panning technique; Pelengaris SA et al.; A method was devised for the isolation of round spermatids from the rat using a positive immunoselection technique (panning) . A testis suspension was prepared from adult rats by enzymatic digestion of seminiferous tubules with collagenase . Specific mouse monoclonal antibody (97.25) was indirectly attached to Petri dishes and used in a panning protocol to purify spermatids from the testis cell suspension . The quantity and purity of cells isolated were determined by cell counts and histochemical (periodic acid-Schiff stain) or by immunostaining with acrosome-specific antibodies . A mean yield of 1.38 +/- 0.15 x 10(7) cells per dish was obtained with a purity of more than 90% . The viability of the cells was confirmed by epifluorescent microscopy with propidium iodide/carboxyfluorescein acetate probes . Northern blot analysis of RNA extracted directly from the dish indicated good integrity of a spermatid-specific transcript of glyceraldehyde-3-phosphate dehydrogenase (GAPDH).

J Burn Care Rehabil, 1995 Jul-Aug, 16(4), 400 - 6
Immunoglobulin M synthesis after burn injury: the effects of chronic ethanol on postinjury synthesis; Tabata T et al.; The effect of chronic ethanol ingestion on class-specific immunoglobulin (Ig) synthesis after burn injury was investigated in C57BL/6 mice . Animals were divided into four groups: control, burn, ethanol-sham, and ethanol-burn groups . Five days after injury or the last ethanol ingestion, cell suspensions from spleen and mesenteric lymph nodes were prepared . The number of class-specific Ig-bearing cells were counted by flow cytometry . The cell suspensions were cultured with lipopolysaccharide for 4 days . The supernatants from these cultures were tested for class-specific Ig by enzyme-linked immunoassay . No change occurred in the amount of class-specific IgG and IgA produced by 10(5) lymphocytes calculated from both of these data . Both burn and ethanol alone impaired IgM synthesis; splenic IgM was most affected by burn, and mesenteric lymph node IgM was most affected by ethanol . The group receiving ethanol before burn had IgM synthesis significantly impaired in both lymphocyte populations . Because IgM is the most important Ig in resistance to bacterial infection, this consistent suppression of IgM synthesis in both these tissues may contribute to increased incidence and severity of acute infection.

Vopr Med Khim, 1995 Jul-Aug, 41(4), 25 - 8
{Ability of bone marrow cells to generate active forms of oxygen in C57Bl/6 and BALB/C mice and mutagenic effects of dioxidine}; Guseva NV et al.; Phorbol-myristate acetate- and opsonized zymosan-induced statistically significant differences were shown in the intensity of chemiluminescence occurring in the bone marrow cell suspension . The bone marrow cells from male BALB/c mice showed the most pronounced response to stimulation in the two cases . After 5-day administration of dioxidine (300 mg/kg), the number of the injured metaphases in the bone marrow from the examined animals was 43.6 +/- 2.2 and 23.8 +/- 1.9% in BALB/c and C57B1/6 mice, respectively, which provides evidence for marked strain-specific differences in the cytogenetic effect of this proxidative mutagen . The findings indicated that there was a relationship between the capacity of bone marrow cells of producing APA and the severity of cytogenetic lesions in these cells.

J Biolumin Chemilumin, 1995 Jul-Aug, 10(4), 229 - 37
Fast and sensitive chemiluminescence determination of H2O2 concentration in stimulated human neutrophils; Mueller S et al.; A fast and sensitive chemiluminescence assay for the determination of H2O2 in stimulated neutrophils without the use of enzymes was developed . The method is based on the oxidation of luminol by hypochlorous acid . The chemiluminescence of this reaction is highly dependent on the concentration of hydrogen peroxide . Changes in H2O2 concentration in PMA-stimulated neutrophils were followed by injection of NaOCI to cell suspension at different times after cell stimulation . The short integration time of 2 s permits calculation of actual concentrations of H2O2 without influence of H2O2 decomposition by cellular enzymes or newly produced H2O2 due to dismutation of superoxide anion radicals . Concentrations of H2O2 were diminished by catalase and enhanced by sodium azide owing to inhibition of cellular catalase and myeloperoxidase . Changes in H2O2 concentration upon stimulation could be observed at 3000 cells/mL.

In Vitro Cell Dev Biol Anim, 1995 Jul-Aug, 31(7), 508 - 15
Human epidermis reconstructed on synthetic membrane: influence of experimental conditions on terminal differentiation; Noel-Hudson MS et al.; Cell suspensions of human keratinocytes seeded onto cell culture inserts may undergo terminal differentiation in the absence of fibroblasts . Among the parameters that control these morphogenic events, exposure to air and the composition of the culture medium were investigated . In the latter case, three media were considered DMEM:Ham's F12, MCDB 153, and keratinocyte SFM medium at equivalent calcium (1.5 mM) and fetal calf serum (5%) concentrations . Immunochemical methods and transmission electron microscopy show that cells cultured in DMEM:Ham's F12 medium, and then raised at the air-liquid interface, form a basal layer plus suprabasal cell layers corresponding to the stratum spinosum, stratum granulosum, and stratum corneum . The suprabasal keratinocyte layers show morphologies that resemble intact skin in which cells are connected by desmosomes and contain intermediate filaments and keratohyalin-filaggrin granules . When the cultures are kept submerged, the keratinocytes show occasional keratohyalin granules and are connected by fewer desmosomes . Additionally, no proper stratum corneum is formed . In keratinocyte SFM medium and MCDB 153, cultures raised at the air-liquid interface are not able to form an epithelium of normal architecture and do not express terminal differentiation markers . Differentiation is initiated, however, since desmosomes and bundles of keratin filaments appear; on the other hand, filaggrin is not expressed even after 28 d in culture . Membrane-bound transglutaminase is expressed throughout the entire suprabasal compartment in MCDB153 and DMEM:Ham's F12 media but never appears in keratinocyte SFM medium . These studies show the relative independence of epidermal differentiation program to the composition (including the calcium concentration) of the media contacting the dermis and filling the extracellular space.(ABSTRACT TRUNCATED AT 250 WORDS)

Photochem Photobiol, 1995 Jul, 62(1), 184 - 9
Sulfophthalocyanines for photodynamic inactivation of viruses in blood products: effect of structural modifications; Allen CM et al.; Transmission of infectious disease through blood transfusions is well known . Ultraviolet irradiation, solvents, and detergents provide a means of sterilizing noncellular blood components . However, these harsh methods are not applicable to cellular blood products . Recently, attempts have been made to sterilize biological fluids using photodynamic treatment and phthalocyanine (Pc) dyes have been advanced as photosensitizers for this purpose . We have evaluated a series of water-soluble Pc, chelated with different central metal ions, substituted to different degrees with sulfonato and t-butyl groups, for their effectiveness to reduce virus infectivity in red blood cell suspensions . Vaccinia virus cytopathogenicity was determined by endpoint serial dilutions in the CV-1 cell line . Anti-viral activity increased with the central metal ion in the following: Ga(III) < Al(III) < Zn(II), and varied inversely with the degree of sulfonation . Furthermore, addition of a t-butyl group onto the trisulfonated dyes (PcS3{t-Bu}) resulted in a 5-40-fold increase in anti-viral potency, suggesting that amphiphilicity enhances the photodynamic activity of the dye . Strong anti-viral photosensitizing properties cannot be the sole selection criterion . Of equal importance is the preservation of blood component integrity . Accordingly, the photohemolytic activity of the dyes was evaluated using the rate of hemolysis as a parameter and a toxicity index was defined . Among the most active dyes, the AlPcS3(t-Bu) complex exhibited the most favorable anti-viral properties combined with a low toxicity index . Our results suggest that trisulfophthalocyanines, bearing an additional t-butyl group to enhance amphiphilicity, are particularly promising dyes for photodynamic blood sterilization.

Photochem Photobiol, 1995 Jul, 62(1), 162 - 8
Photofrin accumulation in malignant and host cell populations of a murine fibrosarcoma; Korbelik M et al.; Photofrin (25 mg/kg) was administered to the FsaR fibrosarcoma-bearing mice (either syngeneic or severe combined immunodeficient {SCID}) and the tumors were excised 24 h later . The photosensitizer content in the cells dissociated from tumor tissue was analyzed using flow cytometry . Staining the cell suspensions with the monoclonal antibodies against specific membrane markers served to identify the malignant cells and various types of host immune cells infiltrating the tumor . Photofrin content was also examined in the cells from normal tissues of the tumor-bearing mice (spleen, heart muscle, peritoneal macrophages) . The results show a marked heterogeneity in the Photofrin cellular content of FsaR tumor, particularly within the population of tumor-associated macrophages (TAM) . The Photofrin levels in some TAM were lower or similar to those in the malignant cells . In contrast, a subpopulation of TAM accumulated very high levels of the photosensitizer, which exceeded by far the levels found in the other tumor cell populations . This TAM fraction was characterized by particularly high expression of interleukin-2 receptors and increased cell size and granularity when compared to the other TAM, which suggests that these macrophages are in the activated state . Their average Photofrin content was almost 13 times higher than in the malignant cells . The lowest photosensitizer levels in the tumor were found in tumor-infiltrating leukocytes other than TAM . In FsaR tumors growing in SCID mice, the pattern of Photofrin distribution in TAM and other cellular populations was similar to that found in tumors growing in syngeneic mice.(ABSTRACT TRUNCATED AT 250 WORDS)

Am J Physiol, 1995 Jul, 269(1 Pt 2), H203 - 14
Cytoskeleton modulates gating of voltage-dependent sodium channel in heart; Undrovinas AI et al.; To investigate the role of the cytoskeleton in cardiac Na+ channel gating, the action of cytochalasin D (Cyto-D), an agent that interferes with actin polymerization, was studied by whole cell voltage clamp and cell-attached and inside-out patches from rat and rabbit ventricular cardiac myocytes . Cyto-D (20-40 microM) reduced whole cell peak Na+ current by 20% within 12 min and slowed current decay without affecting steady-state voltage-dependent availability or recovery from inactivation . Brief treatments (< 10-15 min) of cell-attached patches by Cyto-D (20 microM) in the bath induced short bursts of Na+ channel openings and prolonged decays of ensemble-averaged currents . Bursting of the Na+ channel was more pronounced when the cell suspension was pretreated with Cyto-D (20 microM) for 1 h before seal formation . Application of Cyto-D on the cytoplasmic side of inside-out patches resulted in more dramatic gating changes . Peak open probability was reduced by > 50% within 20 min, and long bursts of openings occurred . Washout of Cyto-D did not restore ensemble-averaged current amplitude, but burst duration decreased toward control values . Cyto-D also induced an additional slower component to open and closed times . These results suggest that Cyto-D, through effects on cytoskeleton, induced cardiac Na+ channels to enter a mode characterized by a lower peak open probability but a greater persistent activity as if the inactivation rate was slowed . The cytoskeleton, in addition to localizing integral membrane proteins, apparently also plays a role in regulating specific detailed functions of integral membrane proteins such as the gating of Na+ channels.

J Pharmacol Exp Ther, 1995 Jul, 274(1), 540 - 7
Dapsone-induced hemolytic anemia: effect of dapsone hydroxylamine on sulfhydryl status, membrane skeletal proteins and morphology of human and rat erythrocytes; McMillan DC et al.; Dapsone hydroxylamine is a direct-acting hemolytic agent responsible for dapsone-induced hemolytic anemia in the rat . In the present study, we compared the responsiveness of rat and human red cells to dapsone hydroxylamine-induced cellular changes . Dapsone hydroxylamine induced a rapid and concentration-dependent loss of erythrocytic reduced glutathione content with a concomitant increase in protein-glutathione mixed disulfide formation in both human and rat red cell suspensions . However, the rate of mixed disulfide formation in human cells was considerably slower than that in rat cells and was preceded by a transient increase in oxidized glutathione (glutathione disulfide) formation . Sodium dodecylsulfate-polyacrylamide gel electrophoresis and immunoblotting analysis of membrane ghosts from human red cells revealed changes in skeletal proteins that in general were similar to those observed with rat cells, including a loss of protein band 2.1 and the appearance of membrane-bound hemoglobin . Notable differences were the resistance to loss of band 4.2 and a considerably higher amount of protein aggregation in human ghosts . Although the morphology of human red cells was altered, the incidence and degree of change were considerably less than those of rat red cells . Furthermore, the concentration of dapsone hydroxylamine required to induce damage in human red cells (175-750 microM) was significantly higher than that required for rat red cells (50-175 microM), suggesting that human cells are probably less sensitive than rat cells to dapsone hydroxylamine-induced oxidative damage.

Glia, 1995 Jul, 14(3), 237 - 42
Acute dispersion of glial cells following transplantation into the myelin-deficient rat spinal cord; Lipsitz D et al.; Evaluation of glial cell migration following transplantation can be difficult as the force of the injection itself may cause the cells to become immediately dispersed . In this study we evaluated the extent of spread of cells after injection of 1 microliter of a dissociated cell suspension (50,000 cells/microliter) into the dorsal columns of the thoracolumbar spinal cord in the neonatal myelin-deficient (md) rat . Spinal cords were examined at 0, 4, and 24 h after injection to determine the dispersion of cells away from the initial site of deposition . Examination of skip-serial sections collected at 50-microns intervals rostral and caudal to the site of transplantation showed that the injection could result in a spread of transplanted cells up to 1,600 microns . Migration should therefore be defined as the detection of cells beyond the rostral-caudal boundaries defined by the injection deposition . Cell dispersion should be taken into account when evaluating the results of migration in previous and future experiments concerning glial cell transplantation.

Zhongguo Zhong Xi Yi Jie He Za Zhi, 1995 Jul, 15(7), 419 - 21
{Effects of emodin, sennosides and rheum polysaccharides on free calcium in isolated rat liver cells}; Lin XZ et al.; The effects of emodin (EMD), sennosides (SEN) and Rheum polysaccharides (RPP) on the free calcium level in the isolated rat liver cells were studied with Ca2+ level 131.60 +/- 37.79 nmol/L in the liver cells . After adding CaCl2 (2 mmol/L) and KCl (120 mmol/L) to liver cell suspension sequentially, the free Ca2+ levels were significantly elevated compared with that of the resting status (P < 0.01) . When the liver cells were pretreated with EMD (0.037 mmol/L) for 10 min, in the resting status or using the above doses of CaCl2 and KCl, the free Ca2+ levels were significantly increased compared with that of the control groups (P < 0.01) . On the contrary, after administration of SEN (0.046-0.092 mmol/L) and RPP (0.1-0.2 mg/ml), the free Ca2+ levels were obviously decreased compared with that of the control groups (P < 0.01) . Furthermore, the inhibitory effect was dose dependent . The opposite effects of the different active ingredients of rhubarb (EMD,SEN,RPP) on the free Ca2+ levels suggested that rhubarb has many kinds of regulatory function in the liver cells.

J Physiol, 1995 Jul 1, 486 ( Pt 1), 1 - 13
Mitochondrial membrane potential in single living adult rat cardiac myocytes exposed to anoxia or metabolic inhibition; Di Lisa F et al.; 1 . The relation between mitochondrial membrane potential (delta psi m) and cell function was investigated in single adult rat cardiac myocytes during anoxia and reoxygenation . delta psi m was studied by loading myocytes with JC-1 (5,5',6,6'-tetrachloro-1,1',3,3'- tetra-ethylbenzimidazolylcarbocyanine iodide), a fluorescent probe characterized by two emission peaks (539 and 597 nm with excitation at 490 nm) corresponding to monomer and aggregate forms of the dye . 2 . De-energizing conditions applied to mitochondria, cell suspensions or single cells decreased the aggregate emission and increased the monomer emission . This latter result cannot be explained by changes of JC-1 concentration in the aqueous mitochondrial matrix phase indicating that hydrophobic interaction of the probe with membranes has to be taken into account to explain JC-1 fluorescence properties in isolated mitochondria or intact cells . 3 . A different sensitivity of the two JC-1 forms to delta psi m changes was shown in isolated mitochondria by the effects of ADP and FCCP and the calibration with K+ diffusion potentials . The monomer emission was responsive to values of delta psi m below 140 mV, which hardly modified the aggregate emission . Thus JC-1 represents a unique double sensor which can provide semi-quantitative information in both low and high potential ranges . 4 . At the onset of glucose-free anoxia the epifluorescence of individual myocytes studied in the single excitation (490 nm)-double emission (530 and 590 nm) mode showed a gradual decline of the aggregate emission, which reached a plateau while electrically stimulated (0.2 Hz) contraction was still retained . The subsequent failure of contraction was followed by the rise of the emission at 530 nm, corresponding to the monomer form of the dye, concomitantly with the development of rigor contracture . 5 . The onset of the rigor was preceded by the increase in intracellular Mg2+ concentration ({Mg2+}i) monitored by mag-indo-1 epifluorescence . Since under these experimental conditions intracellular {Ca2+} and pH are fairly stable, the increase in {Mg2+}i was likely to be produced by a decrease in ATP content . 6 . The inhibition of mitochondrial ATPase induced by oligomycin during anoxia was associated with a rapid and simultaneous change of both the components of JC-1 fluorescence, suggesting that delta psi m, instead of producing ATP, is generated by glycolytic ATP during anoxia . 7 . The readmission of oxygen induced a rapid decrease of the monomer emission and a slower increase of the aggregate emission . These fluorescence changes were not necessarily associated with the recovery of mechanical function.(ABSTRACT TRUNCATED AT 400 WORDS)

Cell Biol Int, 1995 Jul, 19(7), 585 - 92
F-actin in mitotic spindles of synchronized suspension culture cells of tobacco visualized by confocal laser scanning microscopy; Kengen HM et al.; TRITC-labelled phalloidin was used to visualize F-actin distribution during mitosis in Nicotiana tabacum BY-2 suspension cells . Aphidicolin was used to synchronize cell suspensions, which enabled sufficient numbers of mitotic cells to be obtained . F-actin was present in the spindle, and its orientation seemed to correlate with the known microtubular arrays . The use of confocal microscopy greatly reduced background fluorescence, and therefore fine actin filaments could be observed in spindles previously thought to be devoid of actin.

J Invest Dermatol, 1995 Jul, 105(1), 14 - 21
Proliferative potential of different keratinocytes of plucked human hair follicles; Moll I; We have examined colony-forming ability, localization of colony-forming cells, and in vitro life spans of outer root sheath keratinocytes of different fragments of adult human plucked hair follicles . These were shown by immunohistochemical staining for cytokeratins and integrins to contain a preserved basal cell layer . By microdissection, five fragments of the outer root sheath (B1, B2, B3-1, B3-2, B4) were separated, dispersed by trypsin into single cell suspensions, and grown on human feeder fibroblasts . All fragments gave rise to at least some colonies, but colony-forming ability was mostly marked in the intermediate part (B2) and the lower half of the central part (B3-1); approximately 60% of colony-forming cells of a hair follicle localized to the fragment B3-1 and 28% to the fragment B3-2 (upper half of the central part, including bulge) . To compare the in vitro life spans of cells from the various fragments, we subcultured isolated keratinocytes under identical conditions . The longest was found in the fragment B3-2 and the shortest in the fragment B1 (bulb) . Moreover, the differentiation state of the native cells and the cells of all cultures were studied during their whole life spans by immunocytochemical analysis of various proliferation and differentiation markers . Surprisingly, keratinocytes of all fragments, as shown by expression of high-molecular-weight cytokeratins and filaggrin, were capable of terminal differentiation . These data indicate that cells with long life spans are localized in central parts of the outer root sheath close to the bulge area and that cells with high colony-forming ability are localized in the lower central parts . The latter are usually removed by plucking and may therefore not represent stem cells but rather cells important for hair growth during a single cycle . Cells with long life spans--also included in plucked hair follicles--may be immediate progeny of stem cells that will be segregated in the bulge area . Finally, our results are important for gene transfer and stem cell gene therapy in genodermatoses, because plucked hair follicles are easily available and keratinocytes close to the bulge area should be used selectively.

Mutagenesis, 1995 Jul, 10(4), 365 - 9
Comparison of separated erythrocyte preparations and manual smears of bone marrow in showing micronucleus induction by clastogens and aneuploidogens in mouse; Vigagni F et al.; The removal of nucleated cells from bone marrow cell suspensions by cellulose column separation and further purification in a Percoll gradient, coupled with slide preparation by a cytocentrifuge, produces uniform preparations of pure well-spread erythrocytes . The use of separated erythrocytes has been a considerable improvement in the in vivo micronucleus (MN) assay, because the optimal cell morphology and the possibility of scoring a high number of polychromatic erythrocytes (PCEs) in a short time are factors contributing to the sensitivity of the assay . As the separation procedure may selectively retain cells containing MN, a study comparing the performance of the erythrocyte fractionation technique and standard manual smear preparation was conducted in male NMRI mice treated with a single injection of two clastogens (cyclophosphamide, 25 or 50 mg/kg, or methylmethane sulfonate, 40 or 80 mg/kg) or two aneuploidogens (colchicine, 0.5 or 1 mg/kg, or vincristine sulfate, 0.05 or 0.1 mg/kg) . Both the preparation methods were clearly able to detect the MN-inducing and toxic effects of the treatments . After treatment with cyclophosphamide and methylmethane sulfonate, the frequency of micronucleated PCEs (MNPCEs) was, respectively, 1.6 and 1.8 times higher in the fractionated erythrocytes than in whole bone marrow . Morphological characterization of the MN indicated that this phenomenon was due to improved identification of small micronuclei in the flat cytospun cells of the fractionated erythrocyte preparations . On the other hand, the purified erythrocyte slides contained only about half the number of MNPCEs observed in standard smears, in samples collected from mice treated with colchicine.(ABSTRACT TRUNCATED AT 250 WORDS)

Biochim Biophys Acta, 1995 Jun 9, 1244(2-3), 283 - 90
Microcalorimetric evaluation of the effect of combined chemotherapeutic drugs; Roig T et al.; A study of the effect of combined antineoplastic drugs in vitro was carried out by microcalorimetric monitoring of the metabolic activity of treated cells . Power-time curves of growing T-lymphoma cell suspensions, treated with single or combined drugs, were recorded . The extent of the effect was evaluated by changes in the slopes of the microcalorimetric curves and the kinetics of the drug action were interpreted from the time at which these changes reached their maximum value . The method was validated using two well-established drug combinations, the potentiatory effect of dipyridamole on methotrexate cytotoxicity, and the synergism between methotrexate and 6-thioguanine . In the first case, where one drug is not toxic, the modulation may be evaluated by comparing the inhibition produced by the toxic drug alone and in combination with its modulator . Otherwise, when both drugs are toxic, the combined effect must be evaluated by means of their combination index . The measurement procedure is simple, the electric signal is well suited to automation of data acquisition and the response may be evaluated within 5 to 6 h of drug administration . Moreover, we demonstrate that microcalorimetry is a reliable method for the detection of modulatory effects in combination chemotherapy.

Cryobiology, 1995 Jun, 32(3), 270 - 84
Fracture phenomena in an isotonic salt solution during freezing and their elimination using glycerol; Gao DY et al.; Thermal stress and consequent fracture in frozen organs or cell suspensions have been proposed to be two causes of cell cryoinjury . A specific device was developed to study the thermal stress and the fracture phenomena during a slow cooling process of isotonic NaCl solutions with different concentrations of glycerol (cryoprotectant) in a cylindrical tube . It was shown from the experimental results that glycerol significantly influenced the solidification process of the ternary solutions and reduced the thermal stress . The higher the initial glycerol concentration, the lower the thermal stress in the frozen solutions . Glycerol concentrations over 0.3 M were sufficient to eliminate the fracture of the frozen solutions under the present experimental conditions . To explain the action of glycerol in reducing the thermal stress and preventing the ice fracture, a further study on ice crystal formation and growth of ice in these solutions was undertaken using cryomicroscopy . It is known from previous studies that an increase of initial glycerol concentration reduced frozen fraction of water in the solution at any given low temperature due to colligative properties of solution, which reduced the total ice volume expansion during water solidification . The present cryomicroscopic investigation showed that under a fixed cooling condition the different initial glycerol concentrations induced the different microstructures of the frozen solutions at not only a given low temperature but also a given frozen fraction of water . It has been known that ice volume expansion during solidification is a major factor causing the thermal stress and the interior microstructure is critical for the mechanical strength of a solid . Therefore, functions of glycerol in reducing the total ice volume expansion during water solidification and in influencing interior microstructure of the ice may contribute to reduce the thermal stress and prevent the fracture in the frozen solutions.

Exp Hematol, 1995 Jun, 23(6), 519 - 28
Murine splenic macrophage tumoricidal activation by cytokines; Verstovsek S et al.; Interleukin-2 (IL-2), IL-1 beta, interferon-gamma (IFN-gamma), and tumor necrosis factor-alpha (TNF-alpha), each alone and in all possible combinations, were studied for their capacity to activate murine resident splenic macrophages to a tumoricidal state . Two approaches were used in these studies . The first approach was to add cytokine directly to the adherent macrophages that had been washed free of nonadherent spleen cells . The only agent effective alone was IL-2, inducing significant tumoricidal activity in macrophages obtained after culturing whole spleen cell suspensions for 4, but not 1 to 3, days . Nonadherent splenic populations were required during this 4-day macrophage "culture conditioning." Only combinations of cytokines containing IL-2 were effective, but none more than IL-2 alone . The second approach was to add cytokine to the whole spleen cell suspensions for an activation period before isolation of adherent macrophages . Again, the only agent effective alone was IL-2 . Macrophage tumoricidal activity was highest when IL-2 was added to the whole spleen cell suspensions at the initiation of the 4-day activation culture . In addition, TNF-alpha, but none of the other cytokines, significantly augmented the IL-2-induced effect . The tumoricidal activity was not a consequence of lipopolysaccharide contamination or of lymphokine-activated killer cells . Based on the utilization of neutralizing antibodies, IL-1 alpha, IL-1 beta, and IFN-gamma were not involved as soluble mediators during the activation of tumoricidal splenic macrophages by IL-2 with or without TNF-alpha.

Am J Clin Oncol, 1995 Jun, 18(3), 226 - 30
Resistance to common viruses during intralymphatic injections of tumor cell vaccines . Correlation with circulating cytokines; Juillard G et al.; Patients with advanced malignancies who received intralymphatic injections of irradiated tumor cell suspensions ("vaccines") were unexpectedly found to be resistant to common viral diseases; 17 patients with a documented past history of viral infections who have been observed for 48 to 148 months (median 108 months), were analyzed . The resistance to viruses was found to correlate closely with the presence, in the serum, of certain cytokines . Specifically, the interleukins, -2, -6, -8 and interferon-gamma, at low but sustained levels appeared to be possibly responsible for the nonspecific protection against viral infections obtained by intralymphatic injections of cellular material . These findings suggest that viral infections in normal or immunosuppressed individuals at particular risk might be prevented by treatments aimed at attaining very modest levels of certain cytokines.

Microbiology, 1995 Jun, 141 ( Pt 6), 1385 - 93
Biosynthesis of fluorinated secondary metabolites by Streptomyces cattleya; Reid KA et al.; The biosynthesis of organofluorine compounds by Streptomyces cattleya NRRL 8057 was examined using 19F NMR spectroscopy . The organism produced 1.2 mM fluoroacetate and 0.5 mM 4-fluorothreonine as secondary metabolites when cultured for 28 d on a chemically defined medium containing 2 mM fluoride . Cell suspensions from batch cultures harvested at the growth maximum of 4 d were not capable of fluoride uptake or fluorometabolite biosynthesis, but by 6 d had developed an efficient fluoride-uptake system and biosynthesized the two fluorometabolites in almost equal proportions . As the harvest age increased, the proportion of fluoroacetate to 4-fluorothreonine formed by cell suspensions rose progressively so that 16-d-old cells showed a ratio of 76:26 for the two compounds . Fluoride uptake and fluorometabolite production by cell suspensions were highly dependent on pH, with both processes showing a maximum rate at pH 6.0 but declining rapidly at higher pH values . This decrease was particularly marked in the case of fluoroacetate biosynthesis which was barely detectable at pH 7.5 . Fluoroacetate and 4-fluorothreonine showed only low levels of interconversion by cell suspensions, suggesting that the carbon skeleton of neither was derived by metabolism of the other . The limited interconversion observed is explicable in terms of a small degree of biological defluorination occurring with each compound, followed by reincorporation of the resulting fluoride ion into the organic form by the active fluorinating system, a phenomenon also noted on incubation of cell suspensions with a number of other fluorinated biochemical intermediates.(ABSTRACT TRUNCATED AT 250 WORDS)

J Pathol, 1995 Jun, 176(2), 123 - 35
Numerical aberrations of chromosomes 1 and 7 in renal cell carcinomas as detected by interphase cytogenetics; Beck JL et al.; Alcohol-fixed single cell suspensions of 37 renal cell carcinomas (RCCs) were assessed by both flow cytometry (FCM) and the fluorescence in situ hybridization (FISH) technique, using chromosome 1- and chromosome 7-specific centromere DNA probes . DNA diploidy or near-diploidy was observed in 30 of the 37 RCCs and only 12 of these (near-)diploid tumours were disomic for both chromosomes 1 and 7 . Numerical aberrations of chromosome 1 and/or chromosome 7 were present in 18 of the 30 (near-)diploid RCCs and five of these cases showed monosomy for chromosome 1 in more than 50 per cent of the tumour cells . A double target FISH, with a centromeric and a telomeric specific probe for 1p36, excluded misinterpretation on the basis of clustering of 1q12, and suggested a complete loss of chromosome 1 . All these five (near-)diploid RCCs with monosomy for chromosome 1 were eosinophilic chromophilic cell carcinomas, according to the Thoenes classification of RCC . This observation is of special interest, because it was recently concluded from cytogenetic studies that the diagnosis of chromophilic renal cell carcinoma must be considered as obsolete . Monosomy for chromosome 1 seems to be a non-random numerical aberration of (near-)diploid eosinophilic chromophilic cell carcinomas, and a gain of one or more chromosomes 1 appeared to be a common phenomenon in RCCs, especially in the DNA aneuploid tumours . As these chromosomal abnormalities were not found in the earlier classical cytogenetic studies, we conclude that in situ hybridization techniques are required in addition to chromosome banding techniques to obtain a complete characterization of the chromosome imbalances in RCCs.

Gan To Kagaku Ryoho, 1995 Jun, 22 Suppl 2, 140 - 4
{Evolution of aneuploidy from diploid colorectal carcinoma as revealed by the analyses of ploidy heterogeneity and Ras mutation patterns}; Sugihara H et al.; To analyze ploidy alterations during progression of colorectal tumors, we mapped the ploidy constitutions by cytofluorometry using (measurements of metaphase cells in) tissue sections as well as cell suspensions isolated from the tissue sections . Clonality of the tumor with heterogeneous ploidy constitution was checked by mutation pattern of K-ras codon 12 . To assess the significance of polyploidy detected in the diploid tumor component, the present materials were confined to 23 tumors that contained diploid tumor cells . Results: 1) Eight diploid tumors without polyploidy that invaded the submucosa or deeper were greater than 2 cm in diameter . 2) Aneuploidy was detected in tissue sections from 9 out of 15 tumors that had diploid component with polyploidy, and was occasionally predominant in the extramucosal invasive parts . 3) Near-diploid aneuploidy was detected in the cells isolated from diploid (+ polyploid) regions of 2 tumors with aneuploidy . 4) Three of the 6 tumors with heterogeneous ploidy constituents had ras mutation with the mutation patterns common to diploid and aneuploid parts . These findings suggest that aneuploid cells evolve preferentially from the diploid tumor cell population with polyploidy, which often include near-diploid aneuploidy.

Anal Cell Pathol, 1995 Jun, 8(4), 287 - 95
DNA content in colorectal carcinoma: a flow cytometric study of the epithelial fraction; Bergstrom C et al.; The aim of this study was to estimate the proliferative activity of the epithelial fraction in human colorectal carcinomas . Cell suspensions from 27 human colorectal carcinomas were simultaneously selected for epithelial cells and analyzed for DNA content . The staining procedure we employed, after dispersing the tumour sample into a single-cell suspension, included propidium iodide and a monoclonal antibody to the intermediate filament cytokeratin specific for secretory epithelia of normal human tissue and cells of epithelial origin in adenocarcinomas . This technique made it possible to distinguish the epithelial cell population from non-epithelial cells in the tumour . Flow cytometry was used for DNA analysis and the results obtained after epithelial selection were compared with conventional DNA analysis of crude tumour tissue . No difference in S-phase values of diploid and aneuploid cytokeratin positive cells were seen, whereas analysis of crude tumour cell suspensions showed lower S-phase values in diploid tumours compared to aneuploid ones . When cytokeratin-positive cells in the tumour were selected, we found presence of diploid tumour cells in almost all aneuploid tumours.

Rhinology, 1995 Jun, 33(2), 66 - 9
Cell suspension cultures and adenoid epithelium: an assessment of the source of material for human ciliary function experiments in vitro; Schuil PJ et al.; The aim of this study was to explore the usefulness of two different in vitro models for studying the function of human upper respiratory cilia, i.e . cell suspension cultures of human upper airway epithelium, and ciliated adenoid epithelium . Ciliary beat frequency (CBF) and signal consistency (SC), as parameters of ciliary function, were measured by a computerized photo-electrical method . Measurements after one week revealed that CBF of ciliated aggregates from cell suspension cultures had deteriorated to a mean of 5.8 Hz . In the subsequent period, it remained at this rather low and non-physiological level . SC decreased too, although not as dramatically . These results indicate that ciliated aggregates from cell suspension cultures cannot be used for human ciliary function experiments in vitro . On the other hand, in ciliated adenoid epithelium, CBF remained constant for a period of 5 h, although SC decreased after 30 min . Because of this result and the fact that ciliated adenoid epithelium is easily obtainable, we regard this material as suitable for studying human ciliary beat in vitro.

Comp Immunol Microbiol Infect Dis, 1995 Jun, 18(3), 215 - 21
Evaluation of different serological tests for detection of antibodies against Serpulina hyodysenteriae in pig sera; Diarra AT et al.; Swine dysentery is a mucohemorrhagic diarrheal disease caused by S . hyodysenteriae . The detection of asymptomatic carriers in herds is possible by serological tests . However, cross-reactions between S . hyodysenteriae and S . innocens pose a major problem in serological diagnosis . Several serological tests were evaluated for detection of antibodies to S . hyodysenteriae such as: indirect hemagglutination, passive hemolysis, conglutination and microagglutination tests . Among the tests used, only the microagglutination test was able to detect antibodies to S . hyodysenteriae . 70 to 95% of the pigs were invariably seropositive in a single dilution of 1:10 in actively infected herds whereas the number of seropositives did not exceed 10% in presumably non-infected herds . The test was found to be simple, and reliable to be used with confidence for detection of herd infection using boiled cell suspension as an antigen.

Infusionsther Transfusionsmed, 1995 Jun, 22(3), 152 - 8
Flow cytometry quantification of CD34+ cells and other leukocyte subpopulations in frozen-thawed blood cell suspensions: investigation of a new teflon container for cryopreservation of hematopoietic progenitor cells; Arseniev L et al.; BACKGROUND: Cryopreservation is the only available method for the long-time maintenance of blood cells . The present study was designed to prove: (i) the reliability of multiparameter flow cytometry (MFC) for estimation of CD34+ cells in frozen-thawed cell suspensions and (ii) the acceptability of a new teflon container for the cryopreservation of hematopoietic progenitor cells . MATERIALS AND METHODS: Each of 15 ABO-compatible buffy coats (BC) were pooled, and mononuclear cells (MNC) were then separated with the Fresenius AS 104 device (n = 10) . MNC harvested by apheresis were then divided into 2 portions and transferred pairwise into either the new Fresenius or into Gambro cryopreservation containers . Paired samples were frozen at controlled rates (9% DMSO final concentration) and stored at -196 degrees C in liquid nitrogen for 2 weeks . Leukocyte, MNC and differential blood counts and proportions of CD3+, CD4+, CD8+, CD14+ and CD34+ cells were assessed from the pooled BC, the apheresis products, and the frozen-thawed samples . Methyl cellulose culture assays as well as trypan blue viability staining were also carried out . RESULTS: The mean content of the divided apheresis products was 4.9 x 10(9) leukocytes with 86% MNC, 6.89 x 10(6) CD34+ cells, 2.1 x 10(5) granulocyte-macrophage colony-forming units (CFU-GM) and 7.1 x 10(5) erythroid burst-forming units (BFU-E) . As expected, there were virtually no granulocytes after freezing in both types of container . The corresponding mean cell content was as follows: 6.3 x 10(6) CD34+ cells, 2.5 x 10(5) CFU-GM, and 8.1 x 10(5) BFU-E in Fresenius containers, and 6.1 x 10(6) CD34+ cells, 1.3 x 10(5) CFU-GM, and 7.7 x 10(5) BFU-E in Gambro containers . The mean MNC viability of the samples frozen in Fresenius was 81.5% and 82.7% in the Gambro containers . MFC was found to compare with stained smear differentials . CD34+ cell counts correlated with CFU-GM (0.69, p = 0.03) and BFU-E (0.63, p = 0.02) colony formation . CONCLUSIONS: The study reported here revealed no significant differences between the 2 types of storage containers . The new Fresenius teflon container could thus be recommended for cryopreservation of hematopoietic progenitor cells . MFC provided reliable data on CD34+ cell content and leukocyte subset composition of the frozen-thawed cell suspension.

Virus Res, 1995 Jun, 37(1), 13 - 22
A helper T-cell epitope of the E7 protein of human papillomavirus type 16 in BALB/c mice; Vandebriel RJ et al.; The helper T-cell response to the E7 protein of human papillomavirus type 16 (HPV16) was studied using BALB/c (H-2d) mice . Twenty-two overlapping synthetic peptides spanning the HPV16 E7 protein were split into 6 groups . Mice were sensitized using mixtures of synthetic peptides corresponding to each of the groups . Lymph node cell suspensions were cultured with the corresponding mixture of synthetic peptides that was used for sensitization . Two mixtures induced a proliferative response . Analysis of the individual peptides from these mixtures showed that two (overlapping) peptides induced a proliferative response . This response was mediated by CD4+ cells . The common region of the two peptides was found to be a single epitope, and a minimal epitope was demonstrated (AHYNIVTFCCK) . In conclusion, in contrast to others, we demonstrated a helper T-cell response in BALB/c mice . This may be due to the fact that we used synthetic peptides as immunizing agent . The helper T-cell epitopes in HPV16 E7 demonstrated previously are partly overlapping with the (minimal) epitope demonstrated here, underlining the 'public' nature of the epitope.

Biochemistry, 1995 May 23, 34(20), 6847 - 56
Involvement of the CP47 protein in stabilization and photoactivation of a functional water-oxidizing complex in the cyanobacterium Synechocystis sp . PCC 6803; Gleiter HM et al.; Oscillation patterns of the oxygen yield per flash induced by a train of single-turnover flashes were measured as a function of dark incubation and different pre-illumination conditions in several autotrophic mutant strains of Synechocystis sp . PCC 6803 carrying short deletions within the large, lumen-exposed hydrophilic region (loop E) of the chlorophyll a-binding photosystem II protein CP47 . A physiological and biochemical characterization of these mutant strains has been presented previously {Eaton-Rye, J . J., & Vermaas, W . F . J . (1991) Plant Mol . Biol . 17, 1165-1177; Haag, E., Eaton-Rye, J . J., Renger, G., & Vermaas, W . F . J . (1993) Biochemistry 32, 4444-4454}, and some functional properties were described recently {Gleiter, H . M., Haag, E., Shen, J.-R., Eaton-Rye, J . J., Inoue, Y., Vermaas, W . F . J., & Renger, G . (1994) Biochemistry 33, 12063-12071} . The present study shows that in several mutants the water-oxidizing complex (WOC) became inactivated during prolonged dark incubation, whereas the WOC of the wild-type strain remained active . The rate and extent of the inactivation in the mutants depend on the domain of loop E, where 3-8 amino acid residues were deleted . The most pronounced effects are observed in mutants delta(A373-D380) and delta(R384-V392) . A competent WOC can be restored from the fully inactivated state by illumination with short saturating flashes . The number of flashes required for this process strongly depends on the site at which a deletion has been introduced into loop E . Again, the most prominent effects were found in mutants delta(A373-D380) and delta(R384-V392) . Interestingly, the number of flashes required for activation was reduced by more than an order of magnitude in both mutants by the addition of 10 mM CaCl2 to the cell suspension . On the basis of a model for photoactivation proposed by Tamura and Cheniae (1987) {Biochim . Biophys . Acta 890, 179-194}, a scheme is presented for the processes of dark inactivation and photoactivation in these mutants . The results presented here corroborate an important role of the large hydrophilic domain (loop E) of CP47 in a functional and stable WOC.

Arch Biochem Biophys, 1995 May 10, 319(1), 10 - 22
Dihydroxy-acid dehydratase, a {4Fe-4S} cluster-containing enzyme in Escherichia coli: effects of intracellular superoxide dismutase on its inactivation by oxidant stress; Brown OR et al.; Dihydroxy-acid dehydratase (DHAD) has a {4Fe-4S} cluster and is reported to be facilely inactivated by oxidant stress . To directly assess the biological effects in vivo of superoxide dismutase (SOD) on the oxidant sensitivity of DHAD, we used an Escherichia coli K-12 parent strain (CGSC5073) and derived strains OB 1, OB 2, and OB 3 that lacked one of or both FeSOD and MnSOD . In the K-12 parent strain half the cellular DHAD activity was lost in 15 min at 0.8 atm oxygen, less than 10 microM aerobic nitrofurantoin, or about 5 microM aerobic paraquat (PQ) and in about 1 min at 10 microM aerobic PQ . Oxygen and metabolism were required for PQ to inactivate DHAD in cells; adding dithiothreitol to cell-free extracts did not restore DHAD activity . The Km was not appreciably changed for DHAD that was 50 and 70% inactivated in cells, respectively, by hyperbaric oxygen (HBO) and PQ, compared to cells in exponential, aerobic growth . Thus, active site oxidative impairment of individual enzyme molecules apparently was all-or-none . DHAD activity was greatly decreased when measured in extracts made from strains that lacked both SODs unless SOD was added to cell suspensions before extracts were made . DHAD was more sensitive in strains lacking both SODs than in the parent strain to inactivation by aerobic PQ and HBO . Anaerobic (compared to aerobic) growth increased DHAD specific activity by 20% or less in the parent strain and in strains OB 1 and OB 2 (lacking MnSOD and FeSOD, respectively); however, in strain OB 3 (lacking both SODs) DHAD was increased 60% . DHAD was partially inactivated by the oxidant stress of aerobic growth, but remained in a form detectable by DHAD antibody, and the ratio of active to inactive DHAD decreased greatly in cells lacking SOD . Thus, SOD helped maintain DHAD as an active holoenzyme and benefitted cells growing aerobically or when exposed to low levels of PQ.

FEBS Lett, 1995 May 8, 364(2), 103 - 8
Characterization of an Arabidopsis thaliana cDNA homologue to animal poly(ADP-ribose) polymerase; Lepiniec L et al.; A full-length Arabidopsis thaliana cDNA (app) encoding a protein with high similarity (about 60%) to the catalytic domain of vertebrate poly(ADP-ribose) polymerase (PARP; EC 2.4.2.30) has been cloned . The N-terminal extension of the Arabidopsis protein shows similarities with domains of different nuclear and DNA binding proteins in agreement with nuclear localization and putative function of a plant PARP . APP is encoded by a single gene mapped at the top of chromosome 4 of the Arabidopsis genome and mRNA is abundant in cell suspension culture compared to its accumulation in whole plant.

Biochem Biophys Res Commun, 1995 May 5, 210(1), 113 - 8
Oxygen consumption rates of free and alginate-entrapped beta TC3 mouse insulinoma cells; Mukundan NE et al.; Oxygen consumption rates of beta TC3 mouse insulinoma cells were measured in three different cell culture systems: monolayer, freshly trypsinized cell suspension, and trypsinized cells entrapped in alginate-poly-L-lysine-alginate (APA) polymer beads . The oxygen consumption rate for cells in the APA beads was similar to the rate for freshly trypsinized cells, indicating that cells can be entrapped without affecting their oxygen consumption rate . The cells in monolayers consumed oxygen at a higher rate than freshly trypsinized cells or cells in APA beads, suggesting that trypsinization may lower oxygen consumption capability . In addition, the oxygen consumption of beta TC3s during glucose-induced insulin secretion was not significantly different from the basal rate, in contrast to the increased oxygen demand of actively secreting islets . Our results suggest that beta TC3s may be better suited than islets for use in a bioartificial pancreas due to their stable oxygen requirements.

Eur Cytokine Netw, 1995 May-Jun, 6(3), 167 - 76
Effect of IFN-gamma administration in virgin and pregnant mice: distribution of lymphoid and myeloid cells in the spleen; Athanassakis I et al.; Administration of Interferon-gamma (IFN gamma) is used in the therapeutic approach for mainly cancer treatment and viral infections in vivo . Recently we observed some important pathologic dysfunctions caused by IFN-gamma administration to pregnant mice . This treatment affected not only the growth and development of the feto-placental unit, but also, among other hematologic disorders, caused splenomegaly to the mother . In an effort to explain the observed hypersplenism, we have analysed the behaviour of macrophages, B and T lymphocytes in the spleen of virgin and pregnant mice after intraperitoneal administration of low IFN-gamma doses . Although the percentage of myeloid Mac-1 and F4/80 positive cells in spleen cell suspensions of virgin and pregnant mice do not change with the IFN-gamma treatment, immunoperoxidase staining of frozen spleen sections shows that in pregnant mice the monocytic cells accumulate at the central white pulp area of the organ, whereas in non-pregnant mice these cells are mainly found at the peripheral red pulp area . In contrast, the same treatment was shown to increase the numbers of Ly5 positive B cells in both virgin and pregnant mice, whereas B cells were found to form clusters only in the case of pregnant animals . We also show that IFN-gamma increases the numbers of Tcyt/sup (Ly2 positive cells) and TH (L3T4 positive cells) in the spleen of virgin mice but not in pregnant mice . Both populations display a physiologic distribution in the white pulp of the organ as assessed by immunoperoxidase staining of frozen spleen sections . Interestingly, the distribution pattern of IL-2- and IL-4-producing cells, which reflects the presence of Th1 and Th2 subpopulations was different in pregnant and virgin mice . Gestating females had IL-2 producing cells dispersed in the white pulp area, whereas IL-4 producing cells formed clusters mainly at the periphery of the organ . Virgin females had almost undetectable levels of IL-4 producing cells, whereas IL-2 producing cells were found at the periphery . Our results indicate that IFN-gamma alters the equilibrium between Th1 and Th2 cells, which in turn is responsible for the redistribution of myeloid and lymphoid cells in the spleen of pregnant mice thereby explaining the development of an active immune/inflammatory reaction.

Braz J Med Biol Res, 1995 May, 28(5), 609 - 13
Atrial natriuretic peptide modulates the effect of angiotensin II on the concentration of free calcium in the cytosol of Mandin-Darby canine kidney cells; Nascimento-Gomes G et al.; The effect of angiotensin II (ANG II) and atrial natriuretic peptide (ANP) on intracellular free calcium concentration {Ca2+}i was investigated in Mandin-Darby canine kidney (MDCK) cells in culture . Changes in {Ca2+}i were monitored fluorometrically with the Ca(2+)-sensitive probe fura-2/AM at 37 degrees C using a Perkin-Elmer LS-5 spectrofluorimeter (excitation 340/380 nm, slit 3 nm; emission 520 nm, slit 10 nm) . MDCK cells exhibited a mean baseline {Ca2+}i of 98 +/- 10 nM . The addition of increasing concentrations of ANG II (1 pM to 1 microM) to the cell suspension led to a progressive increase in {Ca2+}i to 2-3 times basal levels . In contrast, addition of 1 microM ANP to the cell suspension led to a very rapid 60% decrease in {Ca2+}i . The addition of 1 pM to 1 microM ANG II immediately after 1 microM ANP caused an increase in {Ca2+}i which never exceeded the basal level in the absence of ANP . The data indicate that ANG II increases cell {Ca2+}i, as expected, and provide the new observation that ANP reduces {Ca2+}i in these cells . Furthermore, ANP reduces the increase in {Ca2+}i elicited by ANG II, thus modulating the effect of ANG II on {Ca2+}i.

Burns, 1995 May, 21(3), 175 - 80
Early basement membrane formation following the grafting of cultured epidermal sheets detached with thermolysin or Dispase; Germain L et al.; The basement membrane zone is important for graft adhesion and stability . The aim of the present study was to visualize the regeneration of the basement membrane and determine the sequential appearance of its constituents in the early postgrafting period of cultured human epidermal sheets . A keratinocyte single cell suspension, devoid of dermal fibroblast contamination, was obtained from human skin by a two-step tissue digestion method with thermolysin and trypsin . After culturing, epidermal sheets were generated, detached enzymatically by incubating with thermolysin (for 20-30 min) or Dispase (for 45-60 min), and deposited on a muscular graft bed of athymic mice . Immunohistochemistry and ultrastructural analyses were performed on biopsies harvested 2, 4 and 21 days postgrafting . Bullous pemphigoid antigens and laminin were detected at the dermo-epidermal junction, showing an almost continuous line 2 days postgrafting . Type IV collagen was generally absent at this time, but it was detected 4 days postgrafting . Type VII collagen was labelled as a discontinuous line of increasing intensity from 2 to 21 days postgrafting . Ultrastructural analysis revealed hemidesmosomes and a discontinuous lamina densa 2 days postgrafting, and a complete basement membrane with a continuous lamina densa, hemidesmosomes and anchoring fibrils 21 days postgrafting . The sequence of appearance of major basement membrane components was similar for cultured sheets detached with thermolysin or Dispase . However, it differed from that of other wound healing models . Results are discussed in terms of the variable keratinocyte migration requirement between various wound healing models.

Carcinogenesis, 1995 May, 16(5), 1029 - 35
In vivo tumor measurement of DNA damage, DNA repair and NAD pools as indicators of radiosensitization by metoclopramide; Olsson A et al.; Metoclopramide (MCA), a N-substituted benzamide, causes DNA strand breaks and inhibits DNA repair in vitro and sensitizes radiation and chemotherapeutic drugs in human squamous cell carcinomas when xenographed into nude mice or in a rat glioma model . Here we report on the evaluation of the mechanism behind the radiosensitizing effects of MCA . DNA damage was measured in vivo in a CBA-mouse tumor line (A12B3, sarcoma tumor) by using both alkaline elution and nucleoid sedimentation analysis of cell suspensions prepared from either resected tumor, spleen tissues or whole blood samples . The amount of DNA damage caused by radiation alone, measured 30 min after the irradiation was started, was dose dependent up to 18 Gy in all tissues . The radiation-induced DNA damage in tumor tissue was elevated compared to radiation alone in the presence of MCA, but the level was not higher at 18 Gy compared to 6 Gy in the presence of MCA, and it was still not fully repaired 12 h after irradiation . HPLC analysis of the NAD pools in tumor tissue after DNA damage induction showed a delay in the recovery of the NAD pools (presumably due to the presence of still unrepaired DNA) after exposure to MCA (2 mg/kg) + radiation (6 Gy) compared to tumors exposed to radiation (6 Gy) only, which were fully restored after 48 h . These data confirm earlier published in vitro data on MCA as an inducer of DNA damage and an effector of DNA repair . In addition, the in vivo measurement of radiation-induced DNA damage and DNA repair using the nucleoid sedimentation and alkaline elution assays together with NAD pool determinations may prove to be effective intermediate endpoints in the evaluation of drugs as potential radiosensitizers.

Am J Obstet Gynecol, 1995 May, 172(5), 1505 - 10
Interferon gamma inhibits luteinized human granulosa cell steroid production in vitro; Best CL et al.; OBJECTIVE: The purpose of this study was to determine whether interferon gamma affects luteinized human granulosa cell progesterone, estrone, and estradiol production in the presence and absence of associated white blood cells by either cytotoxic or antiproliferative mechanisms . STUDY DESIGN: Luteinized granulosa cells were isolated by Percoll centrifugation from women during in vitro fertilization cycles . Some cell suspensions were further treated with anti-CD45 magnetic immunobeads to remove associated white blood cells . Granulosa cells with and without white blood cells were cultured in the presence of interferon gamma (0.5 to 50 ng/ml) for 48 hours . Medium was changed at 24-hour intervals, and spent medium was assayed for progesterone, estrone, and estradiol . In separate experiments granulosa cell viability was assessed with the tetrazolium salt reduction assay . RESULTS: Interferon gamma significantly inhibited granulosa cell progesterone production in both basal and human chorionic gonadotropin-stimulated cells cocultured with white blood cells in a concentration-dependent manner, whereas cells cultured free of white blood cells demonstrated less inhibition . In the absence of interferon gamma a more profound increase in granulosa cell progesterone synthesis was found in human chorionic gonadotropin-stimulated cultures without associated white blood cells . Interferon gamma inhibited granulosa cell estrone and estradiol production in basal cultures containing white blood cells in both a time- and concentration-dependent manner . Estrone production was not affected by interferon gamma in human chorionic gonadotropin-stimulated granulosa cell cultures containing white blood cells, whereas estradiol secretion was decreased at 48 hours with 50 ng/ml interferon gamma . Both estrone and estradiol synthesis were inhibited by 50 ng/ml interferon gamma in granulosa cell cultures free of white blood cells . In cultures free of interferon gamma, granulosa cell estrone and estradiol secretion was not affected by human chorionic gonadotropin stimulation compared with basal controls regardless of the presence or absence of white blood cells . All concentrations of interferon gamma used had no effect on granulosa cell viability at any time point tested . CONCLUSIONS: Our data suggest that interferon gamma affects granulosa cell steroid production both independently and in synergy with associated white blood cells and further supports the hypothesis that interferon gamma may be an important intraovarian regulator of ovarian steroid production during the luteal phase.

Cell Immunol, 1995 May, 162(2), 265 - 74
Relative roles of T cells and macrophages in cytokine-mediated functional transformation of cultured splenic dendritic cells; Dai R et al.; Splenic dendritic cells resemble epidermal Langerhans cells in the sense that when both cell types are placed in culture for 1 or more days they undergo a functional transformation that equips them to activate unprimed syngeneic T cells . Among Langerhans cells, this transformation has been ascribed to contaminating keratinocytes that are present in cell suspensions prepared from epidermis . Cytokines released from cultured keratinocytes, particularly GM-CSF and IL-1, have been implicated in mediating the functional transformation observed among cultured Langerhans cells . The present experiments have examined the nature of the cytokines responsible for functional conversion of fresh to cultured dendritic cells harvested from spleens of normal mice and have attempted to identify among the cultured cells the cellular source(s) of these factors . The results indicate that splenic dendritic cells acquire unique accessory properties for activation of naive T cells when placed in vitro because factors generated within the culture, especially GM-CSF and IL-1 beta, are present . Our evidence shows that splenic T cells and phagocytic cells, presumably macrophages, contribute to the presence of these conversion-promoting factors . Thus, this evidence suggests that splenic dendritic cells within the spleen of normal mice, like Langerhans cells within the epidermis of normal, unperturbed skin, exist in vivo in a proactive state with respect to the capacity to induce priming among naive T cells . Acquisition of unique accessory cell function depends upon other cell types within spleen, such as T cells and macrophages that secrete GM-CSF and IL-1 beta . These cytokines, as well as other undefined factors, enable dendritic cells to acquire the accessory properties required to activate resting T cells.

Cell Immunol, 1995 May, 162(2), 211 - 6
Human foreskin mast cell viability and functional activity is maintained ex vivo by coculture with fibroblasts; Levi-Schaffer F et al.; The aim of the present study was to develop long-term culture conditions for foreskin-derived mast cells (HSMC) as we have previously done for rodent peritoneal mast cells (MC) and human lung-derived MC . HSMC were obtained by proteolytic treatment of foreskins from 8-day-old babies (4.8 +/- 2.0% purity) and seeded onto a confluent monolayer of human foreskin-derived fibroblasts (HF) in enriched culture medium . HSMC biochemical and functional properties were studied up to 8 days in these cocultures . Twenty-four hours after seeding the cell suspensions from the proteolytic-digested foreskins, all the cells which did not adhere to the HF were washed out . At this time point approximately 30% of the seeded HSMC were found to adhere to the HF monolayer . The only contaminating cells were endothelial cells (< 8%) . Cocultured HSMC maintained their normal resting morphology and histamine content (3.1 +/- 0.7 pg/cell) up to 8 days in these cocultures . When challenged with compound 48/80 on Days 1, 2, 4, and 8 of coculture, HSMC released similar percentages of histamine which were comparable to the one released by freshly isolated HSMC in suspension (approximately 30%) . Similarly, HSMC, sensitized with IgE antibodies and challenged at various time of coculture with anti-IgE antibodies, released throughout the experiment comparable percentages of this mediator (approximately 50%) . Thus, coculture of HSMC with HF fibroblasts provides a suitable in vitro system for long-term studies on HSMC functional and biochemical properties in a microenvironment which mimics the physiologic one.

Scand J Immunol, 1995 May, 41(5), 504 - 8
T lymphocytes bearing the gamma delta T cell receptor are susceptible to steroid-induced programmed cell death; Spinozzi F et al.; The mechanisms by which glucocorticoids suppress immune responses have not yet been clearly defined . In steroid-sensitive pathological conditions, an increase in gamma delta T cells can occur in certain untreated systemic autoimmune disorders and seems to be a peristent feature in most cases of systemic lupus erythematosus (SLE) . Our previously published data demonstrated that immunosuppressive therapy normalized this expanded SLE T cell subset in parallel with clinical remission of the symptoms . To establish how corticosteroid treatment determines the disappearance of peripheral blood gamma delta T lymphocytes, circulating alpha beta and gamma delta T lymphocytes from seven SLE subjects with active disease and seven healthy individuals were cultured in the presence or absence of 10(-7) M Dexamethasone (DEX) . Cell suspensions were then analysed for DNA fragmentation, characteristic of apoptotic cell death, by a new cytofluorimetric method . Conventional agarose-gel electrophoresis on the same T cell populations was carried out for comparison . Regular follow-ups for 6 months revealed in vivo steroid treatment determined a dramatic fall in SLE blood gamma delta T cells, and in vitro experiments seem to indicate that DEX-triggered apoptotic signals are confined to the double negative (CD4-CD8-) gamma delta T cell subpopulation which disappears after in vivo immunosuppressive therapy . Clinical and pathological remission of some autoimmune diseases is often obtained by corticosteroids . Our results offer new insights on the mechanisms through these hormones exert their potent inhibitory activities on immune system cells postulated to play a role in the generation of autoimmune responses.

Radiat Res, 1995 May, 142(2), 204 - 11
The relative significance of repopulation and hypoxic clonogens in the fractionated radiotherapy of a mouse tumor; Urano M et al.; The aim of this study was to analyze the significance of the repopulation of tumor cell clonogens, reoxygenation and hypoxic cells for the outcome of fractionated radiotherapy . Our previous fractionation study using a murine tumor was used for the analysis (Nishimura and Urano, Int . J . Radiat . Oncol . Biol . Phys . 29, 141-148, 1994) . In the previous study, single cell suspensions were prepared from the early-generation isotransplants of a spontaneous fibrosarcoma, FSa-II, in a C3Hf/Sed mouse and transplanted into the foot of C3Hf/Sed mice . Fractionation schedules consisted of equal graded daily doses (1-20 fractions) given in air or under hypoxic conditions with 137Cs . Treatment was initiated when tumors reached an average diameter of 4 mm . A twice-a-day (b.i.d.) irradiation was tested for 20 doses . Tumor control was the end point, and the TCD50 (50% tumor control dose) was obtained . The surviving fractions resulting from these TCD50 doses were calculated using the alpha and beta values for the FSa-II cells, assuming that these values were constant throughout the fractionation period . These surviving fractions were used to calculate the clonogen repopulation and the reoxygenation of the hypoxic cells . Tumor clonogens repopulated rapidly and the hypoxic cells appeared to reoxygenate substantially during 10-20 doses . In the fractionation regimens consisting of more than 5 doses, the repopulation became significant . This rapid repopulation was reflected as a small alpha/beta ratio on the Fe plot . During 20 daily doses given under hypoxic conditions, clonogens (including both oxygenated and hypoxic) repopulated by a factor of 1.9 x 10(6) . The hypoxic clonogens (based on the TCD50 after doses in air) repopulated to a much lesser extent than did the total clonogens . Reoxygenation was not substantial up to 5 daily doses but appeared to become significant during 10 to 20 doses . The magnitude of reoxygenation appeared to be substantially greater during 20 daily doses than during 20 b.i.d . doses . The significance of the hypoxic cells appeared to be less with increasing fractionation for the tumor control, but still remained critical in 20 daily doses . This study also suggested that the inhibition of clonogen repopulation could improve the outcome of radiotherapy, but this inhibition should be accompanied by further reoxygenation as long as hypoxic clonogens remain critical for the tumor control.

J Immunol, 1995 May 1, 154(9), 4851 - 6
Induction of IL-10 gene expression in human keratinocytes by UVB exposure in vivo and in vitro; Enk CD et al.; Numerous studies have demonstrated that ultraviolet B (UVB) irradiation has profound effects on the skin and systemic immune systems . Because many of the effects of UVB result in suppression of contact sensitivity responses and because IL-10 induces a Th2 rather than a Th1 response, we sought to determine whether UVB irradiation induces IL-10 transcription and subsequent protein secretion by human epidermal cells . Skin of nine volunteers was exposed to UVB or sham irradiation, and epidermal cell suspensions were prepared from suction blister roofs 24 h thereafter . mRNA was extracted using oligo dT-coated magnetic beads, and IL-10 cDNA was amplified with a sensitive RT-PCR technique . We found that IL-10 was constitutively expressed by epidermal cells in 5 of 9 volunteers and that IL-10 message was up-regulated by UVB exposure in all experiments . Since epidermis consists of a heterogeneous cell population with distinct cytokine profiles, we determined whether UVB caused enhanced IL-10 transcription and protein secretion in human keratinocyte cultures . In these experiments, IL-10 was constitutively expressed by keratinocytes and UVB up-regulated IL-10 gene expression in a dose-dependent manner 24 h after in vitro irradiation, coinciding with IL-10 protein secretion into the culture supernatants . Taken together, the findings indicate that UVB irradiation induces IL-10 in human keratinocytes and suggest that keratinocyte-derived IL-10 may be an important component of the immunosuppression that results from UVB irradiation.

Hum Reprod, 1995 May, 10(5), 1311 - 8
A mouse monoclonal antibody, S2n8, detects a 140 kDa protein on the surface of human endometrial stromal cells and decidual cells; Imai K et al.; We developed a mouse monoclonal antibody, S2n8, by immunizing mice i.p . with human decidual cells collected in the first trimester of pregnancy . By indirect immunofluorescence staining of frozen sections, S2n8 was found to react with decidual cells and endometrial stromal cells throughout the menstrual cycle, but not with endometrial glandular cells or with the endometrial surface epithelium . Judging from the fluorescence intensity, the antigen expression on stromal cells was weak in the proliferative phase, and became stronger in the secretory phase . Decidual cells in the first trimester of pregnancy and decidual cells at term showed strong expression of this antigen . Indirect immunofluorescence staining of enzymatically dispersed decidual tissue revealed that the S2n8 antigen was expressed on the decidual cell surface . Flow cytometric analysis of 12 freshly prepared stromal cell-enriched cell suspensions showed that 74.8-94.2% (mean +/- SD 86.1 +/- 6.6%) of the cells carried the antigen . The expression of S2n8 antigen on cultured stromal cells was enhanced by the addition of oestradiol and/or progesterone . The antigenic molecule was purified by immunoaffinity chromatography from decidua collected in the first trimester of pregnancy, and the molecular weight was estimated to be approximately 140 kDa . These findings indicate that the S2n8 antigen is a useful cell surface marker for stromal cells/decidual cells and is associated with their differentiation.

Cell Transplant, 1995 May-Jun, 4(3), 323 - 33
Transplantation of mesencephalic cell suspensions from wild-type and heterozygous Weaver mice into the denervated striatum: assessing the role of graft-derived dopaminergic dendrites in the recovery of function; Witt TC et al.; The Weaver (wv) mutation leads to a loss of mesencephalic dopamine cells and nigrostriatal dopamine axons in homozygosity (wv/wv) and to a deficiency of nigral dopaminergic dendrites without a concomitant loss of dopamine cell somata or axons in heterozygosity (wv/+) . Previous studies have shown that grafts of foetal dopamine cells from wild-type (+/+) donors can survive when implanted into the wv/wv striatum, supply both an axonal and a dendritic innervation to the host, establish synaptic connections with host striatal neurons, and bring about a functional recovery evidenced by rotational asymmetry tests . The aims of the present study were to examine whether wv/+ dopamine cells maintain a "dendrite-poor" phenotype after transplantation to the denervated striatum, and to compare their functional effects with those of wild-type (+/+) grafts in reversing amphetamine-induced turning behaviour . To that end, +/+ and wv/+ ventral mesencephalic tissue (dissected out from E10-E12 foetal mice and made into a cell suspension by enzymatic and mechanical dissociation) was stereotactically grafted into the right striatum of either wv/wv hosts or +/+ hosts subjected in advance to 6-OHDA lesions of the right substantia nigra . Viability and morphology of grafted neurons were assessed by tyrosine hydroxylase immunocytochemistry on serial sections of the host forebrains . Dopamine cell bodies survived in comparable numbers in the grafts regardless of donor genotype; however, grafts of either genotype contained fewer dopaminergic cells when they were hosted in the wv/wv striatum as compared to the striatum of +/+ mice with 6-OHDA lesions . Despite the survival of cell somata, the dendritic arborisation of wv/+ cells was strikingly poorer than that of +/+ cells in grafts placed into both host types, most likely reflecting their in situ phenotypic abnormality . Recipient wv/wv mice with +/+ and wv/+ grafts exhibited 88% and 83% left rotations, respectively; 6-OHDA hosts with +/+ and wv/+ grafts showed 178% and 165% reversals of asymmetry, respectively . The differences between the effects of +/+ and wv/+ grafts were not statistically significant.(ABSTRACT TRUNCATED AT 400 WORDS)

Immunology, 1995 May, 85(1), 99 - 105
Ontogeny of rat thymic macrophages . Phenotypic characterization and possible relationships between different cell subsets; Vicente A et al.; In the present study we combined electron microscopy, immunohistology and primary stromal cell cultures to analyse the ontogeny of rat thymic macrophages (M phi) in an attempt to clarify the relationships between the different macrophage cell subsets described in adult rat thymus . Although phagocytic cells were observed in 15-day-old fetal thymus, monoclonal antibodies (mAb) which recognize different adult macrophage types were unable to identify positive cells until the end of embryonic life . However, our in vitro results from primary thymic stromal cell cultures of 16-day-old fetal rats, and the phenotyping of enriched thymic CD2- cell suspensions, demonstrated that monocyte-like cells which strongly expressed major histocompatibility complex (MHC) class II molecules colonized the embryonic thymus early, giving rise later to distinct macrophage subsets . During the process of maturation, macrophage precursors gradually lost their MHC class II expression, acquired other surface markers (CD45, Thy-1, CD25, CD4, etc.) and increased the acid phosphatase activity . In this respect, ED1+ macrophages, which appeared for the first time in the last stages of embryonic life, consisted of a MHC class II molecule-expressing phagocytic cell population, presumably involved in the elimination of non-selected cortical thymocytes, and of non-phagocytic cells which, in the thymic cortex, might differentiate to ED2+ macrophages throughout ED1+ED2lo/med and ED1+ ED2high intermediate cell stages, observed in vitro in 16-day-old fetal thymic stromal cell cultures . At the end of embryonic life and during the postnatal period the numbers of thymic macrophages increased, particularly in the medulla and corticomedullary border (CMZ), and more slowly in the thymic cortex . This increase was presumably due to the arrival, through perivascular spaces, of new macrophage progenitors, rather than in situ proliferation of pre-existent mature macrophages . The possible function of different thymic macrophage subsets, as well as the relationships between themselves and with their presumptive monocyte-like precursors, are discussed.

Free Radic Res, 1995 May, 22(5), 431 - 40
Inhibition of stimulus-specific neutrophil superoxide generation by alpha-tocopherol; Kanno T et al.; Alpha-tocopherol but not 2-carboxy-2,5,7,8-tetramethyl-6-chromanol (trolox or CTMC) and 2,2,5,7,8 pentamethyl-6-hydroxy chromane (PMC), derivatives of alpha-tocopherol, inhibited the superoxide (O2-.) generation of rat peritoneal neutrophils (RPMN) induced by phorbol 12-myrisate 13-acetate (PMA) . ID50 for neutrophils obtained from the peritoneal cavity of rat and guinea pig was about 1microM . This concentration, however, was much lower than that for the inhibition of PMA-activated phospholipid-dependent protein kinase (PKC) (ID50 = 30 microM) . The alpha-tocopherol sensitive O2- . generation was also observed in neutrophils induced by dioctanoylglycerol (diC8) and calcium ionophore A23187 but not by formylmethionyl-leucyl-phenylalanine (FMLP), opsonized zymosan (OZ) and sodium dodecyl sulfate (SDS) . The pattern of inhibition by alpha-tocopherol was quite similar to that of staurosporine, a specific inhibitor of PKC . The alpha-tocopherol content of RPMN was 12 ng/10(6) cells and a linear increase to 200 ng/10(6) cells by addition of alpha-tocopherol to the cell suspension corresponded with an increased inhibition of O2- . generation . These results indicate that both the chemical structure and the content of alpha-tocopherol might be important factors in O2- . generation by neutrophils.

J Clin Pathol, 1995 May, 48(5), 447 - 55
Oncogene proteins and proliferation antigens in thymomas: increased expression of epidermal growth factor receptor and Ki67 antigen; Gilhus NE et al.; AIMS--To examine thymomas for proteins encoded by oncogenes and to determine whether their presence correlates with tumour growth and associated myasthenia gravis . METHODS--Sections of 24 thymomas were incubated with anti-EGF receptor (EGF-R), anti-Ki67 antigen, anti-p53, and anti-bcl-2 antibodies, and then stained using the alkaline phosphatase/anti-alkaline phosphatase (APAAP) technique . Cell suspensions and epithelial cell cultures from some of the tumours were also studied . RESULTS--Whereas EGF-R expression was not detected in any of the controls (but only in a 20 week old fetus), it was detected in neoplastic epithelial cells of all thymomas, and was most strongly expressed in metastases and in samples from donors with severe myasthenia gravis . Ki67 labelling was also increased, especially in the larger thymomas . Epithelial expression of both of these markers was confirmed in fresh cell suspensions and monolayer cultures from the five available cases . In contrast, p53 and bcl-2 were not detected in the neoplastic cells, but bcl-2 was present in the intermingling thymocytes . CONCLUSIONS--Neoplastic thymoma cells express EGF-R and Ki67, but there is no concomitant increase in the expression of p53 and bcl-2 proteins . Increased EGF-R expression may result in increased proliferation of neoplastic cells and also in myasthenia gravis . Measurement of EGF-R concentrations may be of prognostic value . The bcl-2 staining pattern in T lymphocytes illustrates the broad spectrum of maturational stages in thymoma lymphocytes.

Exp Lung Res, 1995 May-Jun, 21(3), 407 - 21
Purification of murine pulmonary type II cells for flow cytometric cell cycle analysis; Harrison JH Jr et al.; Mice are widely used as animal models for in vivo lung disease . Despite this fact, few methods exist for isolation of type II pneumocytes from mouse lung, limiting the study of alveolar epithelial characteristics in these models . This study investigated several methods for labeling murine lung cell suspensions for flow cytometric identification and sorting of type II pneumocytes . Crude lung cell suspensions were prepared after intratracheal instillation of Dispase and were labeled using phosphine alone or in combination with Helix pomatia lectin, Maclura pomifera lectin, or anti-murine-CD32 . Crude cell suspensions yielded 17.4 million cells per animal with 19.5% type II pneumocytes by Pap staining . Ultrastructural evaluation of the sorted cell pellets (1-1.5 million cells each) demonstrated optimal type II cell purity in preparations labeled with phosphine and anti-CD32 (94.3% type II cells, 0.4% macrophages, 2.8% Clara cells, and 2.5% other) . Nuclear suspensions appropriate for cell cycle analysis were produced by sorting the type II cells directly into hypotonic propidium iodide, and these preparations clearly demonstrated a substantial increase in S-phase type II cells during proliferative repair of BHT-induced acute lung injury.

Diagn Cytopathol, 1995 May, 12(3), 215 - 22
Lacrimal gland lymphoma: a cytomorphologic and immunophenotypic study; Jeffrey PB et al.; The cytomorphology of lacrimal gland lymphoma has not been specifically described . Herein we present six cases of histologically proven lacrimal gland lymphoma which we analyzed using fine-needle aspiration cytology, cell suspension immunophenotype analysis, and immunoglobulin gene rearrangement studies . Fine-needle aspiration cytology revealed atypical populations of cells comprised of either monomorphic small round lymphocytes with or without plasmacytoid features (4 cases), a mixed population of small and large irregular lymphocytes (1 case), or a population of large irregular lymphocytes (1 case) . The initial cytologic diagnosis was malignant lymphoma in all six cases . Cell suspension immunophenotype analysis demonstrated that the lesions were composed predominantly of B-cells that expressed monotypic surface immunoglobulin . Three cases demonstrated an immunoglobulin heavy chain gene rearrangement . The atypical cytologic features and the abnormal immunophenotype were consistently predictive of malignant lymphoma . Given that these lesions are small and biopsy material is often limited, fine-needle aspiration offers the advantage of providing tissue that is ideal for cytologic and cell suspension immunophenotype evaluation, obviating the need to provide surgical biopsy material for this purpose . We conclude that fine-needle aspiration can identify malignant lymphoid lesions of the lacrimal gland and may serve as a valuable adjunct in the assessment of these lesions . Additional study is warranted to determine whether fine-needle aspiration can reliably distinguish between benign and malignant lymphoid proliferations of the lacrimal gland.

Calcif Tissue Int, 1995 May, 56(5), 382 - 9
Volumes of chick and rat osteoclasts cultured on glass; Piper K et al.; We have examined the relationship between the number of nuclei of an osteoclast and its volume . Chick and rat cells were released from long bones by chopping the shafts and flushing the fragments in Eagle's Minimum Essential Medium with added 10% fetal calf serum . The bone cell suspension was seeded onto glass coverslips . In Experiment 1, rat and chick cells were allowed to settle for 15 minutes, more medium was then added, and the cells were cultured in 5% CO2 at 37 degrees C for 4 hours . In Experiment 2, only rat cells were used, and the cells were cultured in the presence or absence of 10(-6) M 3-amino-1-hydroxypropylidene-1,1-bisphosphonate (APD) in the medium for 4 or 6 hours . The coverslips were washed in 37 degrees C phosphate-buffered saline and fixed for 24 hours in 2.5% glutaraldehyde in isotonic cacodylate buffer (initially 37 degrees C) . The chick cells were critical point dried (CPD) or freeze dried (FD); all rat cells were FD . After drying, cells were coated with gold by vacuum evaporation . The volumes and areas of osteoclasts were measured using a video-rate, line-confocal reflection laser scanning microscope and the number of nuclei in each cell was counted . The volumes and volumes per nucleus of the FD cells were larger than those of the CPD cells but there was no significant difference in plan-areas . Rat osteoclasts were larger than chick cells in all the measured parameters except the mean number of nuclei/cell . The correlation coefficients for the areas, volumes, and the numbers of nuclei for rat and chick cells were all high (r > 0.725) . The volumes and volumes per nucleus, but not the areas or areas per nucleus, of the osteoclasts cultured with APD were significantly smaller than control cells . We conclude that FD causes less shrinkage than CPD; chick osteoclasts are about two-thirds the size of rat osteoclasts; and 10(-6) M APD caused a reduction of rat osteoclast volume and volume per nucleus of 21%.

J Comp Pathol, 1995 May, 112(4), 327 - 38
Apoptosis in chicken embryos induced by the infectious bursal disease virus; Vasconcelos AC et al.; Fifteen-day-old fertile eggs (specific pathogen-free) were inoculated with the infectious bursal disease virus (IBDV) by the allantoic route and were opened and examined 2, 4 or 6 days later . The bursas of Fabricius (BFs) were collected and processed for DNA extraction, flow cytometry, and light and electron microscopy . Cellular DNA was subjected to electrophoresis on 1.5% agarose gel and stained with ethidium bromide . Intense internucleosomal DNA fragmentation was detected in IBDV-infected bursas . Cytograms from cell suspensions derived from infected BFs displayed an increased population of cells with either high density and small size (apoptotic cells) or small size and high uptake of ethidium bromide (necrotic cells) . Light and electron microscopical examination of the IBDV-infected BFs revealed death of lymphoid cells without surrounding inflammatory reaction, but with condensation of nuclear chromatin, crescent formation, and nuclear and cellular fragmentation . These data indicated that infection of chicken embryos with IBDV induced apoptosis in bursal lymphoid cells.

Int J Immunopharmacol, 1995 May, 17(5), 453 - 6
Effects of trinitrotoluene (TNT) metabolites on chemiluminescence response of phagocytic cells; Thierfelder W et al.; The effects of TNT metabolites on the generation of activated oxygen species was investigated by a sensitive luminol-dependent chemiluminescence (CL) assay . Spleen cell suspensions containing 2,4-diaminotoluene, 2,4,6-triaminotoluene, 2-amino-6-nitrotoluene, 4-amino-3,5-dinitrotoluene and 2-amino-4,6-dinitrotoluene were stimulated with zymosan . Aminotoluenes and amino-nitrotoluenes induced a dose-dependent inhibition of CL response . The mixed substituted toluenes generally required higher doses than aminotoluenes for the suppression of CL response which was not due to the cytotoxic reduction of cell viability . CL appears to be a well-suited assay for determination of the immunotoxic potential of diverse molecules on phagocytic cells of the immune system.

Reprod Toxicol, 1995 May-Jun, 9(3), 315 - 26
Short-term male reproductive toxicity study with sulfasalazine in the rat; Hoyt JA et al.; Sulfasalazine (2-hydroxy-5-{{4-{(2-pyridinylamino) sulfonyl} phenyl}azo}benzoic acid; SASP) was administered to rats in a short-term male reproductive toxicity study to further examine the utility of this grouping of techniques and to generate reference data with a substance that is known to cause reversible infertility in men . Adult male CD rats (10/group) were orally administered 0, 150, 300, or 600 mg SASP/kg body weight in divided doses for 14 d followed by a 2-week period without treatment . Males were killed on test day (TD) 15 or 29 . At each time point, the reproductive system was evaluated by comparing testicular and epididymal weights, DNA ploidy distributions of testicular cell suspensions, testicular and epididymal histopathology, and epididymal sperm concentrations, motion, morphology, and breakage . Adding time as a factor in the protocol aids in distinguishing testicular from posttesticular effects . Changes in sperm quality after 2 weeks of test article administration (TD 15) predominantly reflect effects that occurred after the sperm entered the epididymis, while testicular effects predominated on TD 29 . Beginning on TD 14, males to be killed on TD 29 were cohabited with untreated females (1:2) . Females were killed at midgestation and examined for pregnancy status . Body weight gain was depressed in all SASP groups during the first 3 d of test article administration . Food consumption was depressed at the 300- and 600-mg/kg dose levels . No changes were seen in testicular weight, but epididymal weight was depressed at the 600-mg/kg dose level . DNA ploidy distributions determined by flow cytometry did not indicate that the kinetics of spermatogenesis were disturbed . However, alterations in sperm release, which have not previously been reported, were seen at all SASP dose levels . On TD 29, the percentage of progressively motile sperm was depressed and beat/cross frequency was increased at the 600-mg/kg dose level . No changes were observed in sperm morphology or breakage . Fertility was slightly depressed at the 600-mg/kg dose level . In this study, testicular histopathology provided the most sensitive endpoint for reproductive toxicity . The impairment of fertility immediately after treatment was stopped, when no changes were apparent in sperm release or sperm motion, suggested that decreased sperm concentrations and altered motility, while contributory, may not be the primary causes of SASP-mediated infertility.

Cell Calcium, 1995 May, 17(5), 375 - 83
Purinoceptor P2U identification and function in human intrahepatic biliary epithelial cell lines; Wolkoff LI et al.; The mechanisms that regulate ion and fluid transport by the human intrahepatic bile duct have not been well defined . Human intrahepatic biliary cell lines that we have developed were used to identify and characterize purinoceptors based on increases in intracellular calcium in response to ATP and other nucleotides . Intracellular free calcium was measured in cell suspensions using the fluorescent probe Fura-2 and a fluorescence spectrophotometer . Halide efflux was measured in single cells using fluorescence microscopy and the fluorescent probe SPQ . Intracellular calcium increases equivalently in response to ATP and UTP, peaking, then diminishing to a new, elevated baseline . The peak elevation of calcium is the result of both the release of intracellular stores of calcium and the influx of extracellular calcium . The purinoceptor P2U-subtype was identified based on the potency rank order of ATP-analogues . Halide efflux increases with P2U-purinoceptor stimulation which is consistent with the opening of a Ca(2+)-sensitive Cl- channel . The physiological significance of P2U-purinoceptor activation and its effect on the ionic content and flow rate of bile remains to be determined.

Int Immunol, 1995 May, 7(5), 843 - 51
Cytokine dependence of V gamma 3 thymocytes: mature but not immature V gamma 3 cells require endogenous IL-2 and IL-7 to survive--evidence for cytokine redundancy; Leclercq G et al.; It has previously been described that V gamma 3 cells can proliferate extensively in vitro in the presence of different cytokines . Here, the role of cytokines in the maintenance of V gamma 3 cells in the thymus has been determined . Culture of fetal thymocytes in cell suspension for 24 h showed that, whereas immature TCRlowHSAhigh V gamma 3 cells remained viable, all mature TCRhighHSAlow V gamma 3 cells died . These cells died by apoptosis since protein synthesis was required and flow cytometric analysis as well as DNA gel electrophoresis showed that the DNA was degraded to oligonucleosomal bands . Addition of IL-2, IL-4 or IL-7 to suspension cultures of fetal thymocytes rescued V gamma 3 cells from dying . Addition of IL-1, IL-3, IL-5, IL-6, IL-9, TNF-alpha or IFN-gamma was without effect . Phenotypic analysis showed that the alpha-chain of the IL-2 receptor (IL-2R alpha) was expressed by part of the immature V gamma 3 thymocytes, all mature V gamma 3 cells expressed the beta-chain of the IL-2 receptor (IL-2R beta) . Addition of anti-IL-2R beta mAb to fetal thymic organ culture (FTOC) resulted in a moderate reduction of the cell number of mature V gamma 3 thymocytes . Addition of anti-IL-2R alpha, anti-IL-4 or anti-IL-7 mAb had no effect . The cell number of mature V gamma 3 cells was highly reduced when both anti-IL-2R beta and anti-IL-7 mAb were added to FTOC.(ABSTRACT TRUNCATED AT 250 WORDS)

Eur J Neurosci, 1995 May 1, 7(5), 907 - 16
The extracellular matrix molecule tenascin: expression in the developing chick retinotectal system and substrate properties for retinal ganglion cell neurites in vitro; Bartsch S et al.; To investigate the molecular mechanisms involved in the outgrowth of retinal ganglion cell axons in the tectum, the expression of the extracellular matrix molecule tenascin was analysed in the tectum and retina of chickens by immunocytochemistry and in situ hybridization . Tissue was analysed between embryonic days 4 and 12, just before and during the period when retinal ganglion cell axons innervate their target region, the optic tectum . In the tectum, tenascin immunoreactivity becomes detectable at the anterior pole at embryonic day 4, 2 days before retinal ganglion cell axons arrive, and spreads caudally with increasing age . At early stages, tenascin is predominantly accumulated in the stratum opticum, the zone of ingrowing retinal ganglion cell axons, and along their prospective pathway . In the stratum opticum, the molecule is associated with radial glial fibres, glial endfeet and retinal ganglion cell axons located in the immediate neighbourhood of radial glial fibres . At all ages investigated, tenascin mRNA is mainly restricted to cells located in the periventricular region, suggesting that the molecule is synthesized by radial glial cells . In the retina, tenascin is expressed by amacrine, displaced amacrine and horizontal cells but not by retinal ganglion cells . To investigate whether the accumulation of tenascin in the developing and prospective pathway of retinal ganglion cell axons may affect their rate of growth we assayed the substrate properties of tenascin for retinal ganglion cell neurites in vitro . When retinal ganglion cell suspensions from 6-day-old chick embryos were maintained on homogeneous mouse or chick tenascin/polyornithine substrates, neurite length was significantly increased when compared to polyornithine substrates at coating concentrations of 10 or 20 micrograms/ml . Higher coating concentrations (35 or 70 micrograms/ml) resulted in neurite lengths comparable to control values . Together, these observations suggest that tenascin in the developing and prospective stratum opticum might serve as a performed pathway to support growth of retinal ganglion cell axons in the tectum.

Thromb Haemost, 1995 May, 73(5), 850 - 6
Platelet accumulation on fibrin-coated polyethylene: role of platelet activation and factor XIII; Rubens FD et al.; Platelet accumulation on small- and medium-calibre vascular grafts plays a significant role in graft occlusion . We examined platelet accumulation on the surface of fibrin-coated polyethylene tubing (internal diameter 0.17 cm) during 10 min flow (10 ml/min) at high wall shear rate (764 s-1) . Washed platelets labelled with 51Cr were resuspended in Tyrode solution containing albumin, apyrase and red blood cells (hematocrit 40%) . When the thrombin that was used to form the fibrin-coated surface was inactivated with FPRCH2Cl before perfusion of the tubes with the platelet: red blood cell suspension, the accumulation of platelets was 59,840 +/- 27,960 platelets per mm2, whereas accumulation on fibrin with residual active thrombin was 316,750 +/- 32,560 platelets per mm2 (n = 4) . When the fibrin on the surface was cross-linked by including recombinant factor XIII (rFXIII) in the fibrinogen solution used to prepare the fibrin-coated surface, platelet accumulation, after thrombin neutralization, was reduced by the cross-linking from 46,974 +/- 9702 to 36,818 +/- 7964 platelets per mm2 (n = 12, p < 0.01) . Platelet accumulation on tubes coated with D-dimer was ten times less than on tubes coated with D-domain; this finding also supports the observation that cross-linking of fibrin with the formation gamma-gamma dimers reduces platelet accumulation on the fibrin-coated surface . Thrombin-activated platelets themselves were shown to cross-link fibrin when they had adhered to it during perfusion, or in a static system in which thrombin was used to form clots from FXIII-free fibrinogen in the presence of platelets.(ABSTRACT TRUNCATED AT 250 WORDS)

Biochim Biophys Acta, 1995 Apr 28, 1266(2), 171 - 8
Control by electrical parameters of short- and long-term cell death resulting from electropermeabilization of Chinese hamster ovary cells; Gabriel B et al.; Chinese hamster ovary (CHO) cells were pulsed by using brief intense square-wave electric field pulses . The electrical treatment induced a transient local permeabilization of the cell membrane . The growth of CHO cells after electropulsation in an iso-osmotic pulsing buffer with low ionic content was measured . Parallel experiments evaluated cell death which took place in the minute range after electropulsation (short-term death) and the cell death upon 24 h (long-term death) . Short-term cell death was defined as the case of cells with membrane still permeable to Direct-blue at 15 min after electropulsation . It was observed only under stringent pulsing conditions where electropermeabilization of the two sides of the cell was triggered . The long-term cell death, i.e., the inability of some pulsed cells to grow was observed as soon as permeabilization had been triggered . The higher the permeabilization level of the cell population was, the higher the long-term cell death level was . The cell death was linearly related to the reciprocal of the electric field intensity, i.e., to the fraction of the membrane area electrically brought to the permeable state . From this work, it appeared that for high levels of permeabilization of a cell suspension, best cell survivals were obtained if limited alterations were triggered over a large area of the plasma membrane (single pulse with high intensity) than if a small area of the membrane was strongly altered (repetitive pulses with small intensity) . The highest yield of viable permeabilized cells was achieved when using one single pulse of duration up to 1 ms.

Brain Res, 1995 Apr 17, 677(1), 1 - 12
Intraspinal noradrenergic-rich implants reverse the increase of alpha 1 adrenoceptors densities caused by complete spinal cord transection or selective chemical denervation: a quantitative autoradiographic study; Roudet C et al.; This study examined, in the adult rat, whether the intraspinal transplantation of a cell suspension of embryonic day (ED)13 rat locus coeruleus primordia was able to normalize the lesion-induced increase of spinal alpha 1-adrenoceptors . Two experimental models of spinal denervation were studied . The first model consisted of a complete spinal cord transection (thoracic vertebrae level T8-T9) and 1 week later, the cell suspension was transplanted below the section; the second one was obtained by a selective chemical lesion of the noradrenergic (NA) system and one month later, the cell suspension was implanted at the same level as in transected rats . Five weeks after grafting, all animals were sacrificed and spinal cord tissue sections were processed for immunohistochemical detection of noradrenaline or for quantification of alpha 1-adrenoceptors binding sites densities using {3H}prazosin as a ligand . After 6-OHDA lesion, as well as caudally to the transection, a significant increase by 21% (P < 0.01) to 68% (P < 0.001) of alpha 1-adrenoceptors densities was detected . The implantation of embryonic NA neurons into the denervated spinal cord led to a reversal of the lesion-induced increase of spinal alpha 1-adrenoceptors, five weeks later . Moreover, this reversal seems to be more effective after mechanical than after chemical denervation.

Blood, 1995 Apr 15, 85(8), 2045 - 51
Interleukin-3 and interleukin-7 are alternative growth factors for the same B-cell precursors in the mouse; Winkler TH et al.; Clones and lines of precursor (pre) B cells can be established by limiting dilutions of unseparated cell suspensions of fetal liver or bone marrow on stromal cells in the presence of interleukin (IL)-7 . When IL-3 is used instead of IL-7, cultures are regularly overgrown by different precursor cells of the myeloid lineage, as well as by adherent cells that inhibit pre-B-cell expansion . However, in the presence of either IL-7 or IL-3, clones of pre-B cells can be established on stroma cells at frequencies near one in one when the cultures are initiated with cell sorter purified CD45RO (B220)+/c-kit+ fetal liver or bone marrow derived pre-B cells . Clones grown on stromal cells in the presence of IL-7 can be regrown in IL-3, and vice versa . Pre-B cells that proliferate on stromal cells in the presence of IL-7 or IL-3 have the same phenotype, ie, are B220+ c-kit+, CD43+, and surrogate light chain+ . Removal of the growth factors (IL-7, respectively IL-3) from the cultures results in differentiation to surface immunoglobulin (slg) positive, c-kit-, CD43-, surrogate light chain- B cells, a fraction of which is lipopolysaccharide (LPS) responsive as shown by IgM secretion . These results show that IL-7 and IL-3 stimulate largely overlapping populations of precursor B cells from bone marrow to proliferate for long periods of time in the presence of stromal cells . Thus, IL-7 and IL-3 are alternative growth factors for the same pre-BI cell.

Toxicology, 1995 Apr 12, 98(1-3), 207 - 14
Structure-activity relationship of phenolic compounds (phenol, pyrocatechol and hydroquinone) on natural lymphocytotoxicity of carp (Cyprinus carpio); Taysse L et al.; We have tested the effects of phenol and two diphenols (pyrocatechol and hydroquinone) on a non-specific immune response, i.e . the natural cytotoxic activity, of the carp . After a 12-day exposure of fish, at a concentration of 0.1 mg/l, hydroquinone appeared to exert the most immunotoxic effect in vivo . In vitro, after a preincubation of 1 h, phenol (10(-2) M), pyrocatechol (4.25 x 10(-4) M) and hydroquinone (4.25 x 10(-5) M) decreased the natural cytotoxic activity of lymphoid cell suspension . In vivo and in vitro experiments show that hydroquinone is the most toxic compound, whereas diphenols are more toxic than phenol . These results demonstrate that the study of immune systems can reveal the presence of toxic substances with varying degrees of toxicity . Also, the position of a second hydroxic group on the benzonic nucleus seems to influence the compound toxicity.

J Immunol, 1995 Apr 1, 154(7), 3383 - 90
Potential involvement of IL-10 in suppressing tumor-associated macrophages . Colon-26-derived prostaglandin E2 inhibits TNF-alpha release via a mechanism involving IL-10; Kambayashi T et al.; The administration of murine TNF-alpha to colon (C)-26 bearing mice, significantly protects the host against the catabolic effects of the tumor . This effect of exogenous TNF-alpha can be primarily attributed to tumor lysis rather than to a direct anticachectic action . Murine peritoneal macrophages cultured with the C-26 line or with C-26 culture supernatant do not release TNF-alpha in response to LPS stimulation . The reduction in TNF-alpha levels is associated with a significant increase in IL-10 levels . Single cell suspension of freshly disaggregated C-26 tumor (which contains host macrophages), do not produce TNF-alpha but contain significant levels of PGE2 and IL-10 . In contrast, PGE2 but not TNF-alpha or IL-10 can be detected in the C-26.IVX cell line that is used to generate tumors in vivo . Neutralizing anti IL-10 Ab but not isotype-matched Ab, significantly reverses the inhibitory effect of the tumor cells and their culture supernatant on macrophage TNF-alpha release . Additional evidence is presented to indicate that the C-26-derived inhibitory activity is related to PGE2 . Taken together, these results support the hypothesis that tumor-derived PGE2 prevents tumor-infiltrating macrophages from producing TNF-alpha, in part by augmenting macrophage IL-10 synthesis.

Immunology, 1995 Apr, 84(4), 669 - 74
Characterization of local antibody responses to the gastrointestinal parasite Haemonchus contortus; Bowles VM et al.; The ability to identify antigens associated with an infection has generally relied on the use of serum antibodies produced by infected or previously exposed individuals . A major drawback with the use of serum is that it does not necessarily reflect the local antibody response at mucosal tissue sites . This study describes an approach that allows the use of antibodies generated close to the infection site to detect the transient expression of stage-specific antigens during infection with the gastrointestinal parasite Haemonchus contortus . This was achieved by infecting immune sheep with H . contortus larvae and removing the abomasal lymph nodes draining the infection site shortly after the challenge infection . Antibody-secreting cell (ASC) probes were generated from these lymph nodes after short-term in vitro culture of cell suspensions, which allowed the accumulation of antibodies secreted by in vivo-induced ASC into the culture supernatant . Lymph node culture supernatants (= ASC probes) from immune sheep challenged 5 days previously were used to probe Western blots of third and fourth stage larval preparations, and revealed distinct reactivity to larval antigens . No antibody reactivity to larval antigen preparations was detected in sheep that were not challenged . The number of antigens identified using ASC probes was significantly restricted compared to either pre- or post-challenge sera . In contrast to the variability of the serum response, the specificity of ASC probes was highly repeatable between different sheep . ASC probes were also used to purify a H . contortus larval antigen by affinity chromatography, which allowed limited biochemical studies to be undertaken . The antigen(s) recognized by the ASC probes were shown to be expressed on the surface of the larvae . These studies illustrate the use of a novel means of studying the local antibody response close to a mucosal infection site in order to identify and isolate stage-specific antigens expressed during infection.

Chin Med J (Engl), 1995 Apr, 108(4), 265 - 8
Effects of daurisoline on cytosolic free calcium in fetal rat cerebral cells; Che J et al.; Cytosolic free Ca2+ ({Ca2+}i) was measured in dissociated cerebral cells isolated from fetal rats with the fluorescent indicater fura-2 . Increase in {Ca2+}i occurred rapidly following exposure of the cells to 50 mmol/L KCl, 10(-7) mol/L Bay K 8644 or 200 mumol/L glutamate (Glu) . {Ca2+}i elevated by K(+)-depolarization was attenuated by pretreatment with 10(-7), 10(-6) mol/L daurisoline (Dau) . The response of {Ca2+}i to K(+)-depolarization did not change when 10(-8) mol/L Dau was added . When 10(-8)-10(-6) mol/L Dau was added to the cell suspensions prior to exposure to Glu, the Glu-stimulated rises in {Ca2+}i were reduced significantly . However, Dau (10(-6), 10(-7) and 10(-8) mol/L) did not alter the response to Bay K 8644 . These results indicate that Dau can inhibit the increase of {Ca2+}i in fetal rat cerebral cells induced by certain Ca2+ agonists, especially Glu, suggesting that this drug may have a protective effect against cerebral cellular injury.

Clin Sci (Lond), 1995 Apr, 88(4), 405 - 12
Metabolic acidosis is a potent stimulus for cellular inorganic phosphate generation in uraemia; Bevington A et al.; 1 . During metabolic acidosis, significant fluxes of inorganic phosphate (Pi) may occur from cellular to extracellular fluid . In this study Pi was measured in erythrocytes of uraemic patients before and after haemodialysis and was related to their plasma pH (acidosis), plasma Pi (hyperphosphataemia) and cellular organic phosphate concentrations . 2 . Before dialysis, the ratio of cellular to extracellular Pi concentration correlated inversely with plasma pH, increasing 2.5-fold as pH fell from 7.4 to 7.2 . 3 . An increase in cellular Pi similar to that seen in the patients was observed within 90 min of adding acid to normal erythrocytes in vitro . 4 . The total Pi content of the cell suspension increased 25% on decreasing plasma pH from 7.4 to 7.2, largely as a result of generation of Pi from 2,3-bisphosphoglycerate in the cells . This was accompanied by net efflux of Pi into plasma . 5 . In addition, the increase in the steady-state cellular Pi concentration on adding a constant extracellular Pi load was 50% greater at pH 7.2 than at 7.4, implying that alterations in the regulation of the transmembrane Pi gradient also contribute to the rise in cellular Pi observed at low pH . 6 . At normal plasma Pi concentration (1 mM), glycolytic flux (lactate production) was inhibited by 20% when pH was lowered from 7.4 to 7.2 . However, this inhibition was blocked when cellular Pi was increased by adding Pi to the plasma in vitro . 7 . Metabolic acidosis is therefore a potent stimulus for Pi generation in erythrocytes, and this Pi may serve to stimulate glycolysis which is normally inhibited by low pH.

Cryobiology, 1995 Apr, 32(2), 114 - 28
DSC measurement of cell suspensions during successive freezing runs: implications for the mechanisms of intracellular ice formation; Bryant G; The formation of intracellular ice in biological cells during freezing is considered almost universally lethal and is the major contributor to cell damage at high cooling rates . Despite its importance, our understanding of the mechanisms of intracellular ice formation (IIF) is still incomplete . In this paper differential scanning calorimetry is used to study IIF in human lymphocytes in the presence of dimethyl sulfoxide (Me2SO) . Under conditions where damage due to IIF on the initial cooling run is 40-60%, the samples are studied as a function of multiple successive cooling runs . This enables the study not only of the cell fraction which undergoes IIF, but also of the fraction which survives . The temperature at which IIF occurs and the fraction of cell volume which undergoes IIF are analyzed as functions of successive cooling runs . Taking advantage of the large number of cells present in the samples (ca . 10(6)), the effect of successive cooling runs on susceptibility to IIF is examined.

Photochem Photobiol, 1995 Apr, 61(4), 410 - 3
Sensitivity of Porphyromonas and Prevotella species in liquid media to argon laser; Henry CA et al.; The phototoxicity of argon laser irradiation was studied in aqueous suspensions of Porphyromonas endodontalis (American Type Culture Collection {ATCC} 35406), Porphyromonas gingivalis (ATCC 33277), Prevotella denticola (ATCC 33184) and two strains of Prevotella intermedia (ATCC 15033 and 49046), all "black-pigmented bacteria," BPB, that accumulate cellular porphyrins . Several of these species have been implicated in the etiology of periodontal disease . Non-black-pigmented bacteria were also studied to test the specificity of irradiation as a potential photodynamic treatment for periodontal infections . Cell suspensions were irradiated with an argon laser at fluences of 20-200 J/cm2 . When cultured in hemin-supplemented media, ATCC 15033 was the most sensitive to irradiation . However, a second strain of the same species (ATCC 49046) was resistant . The photosensitivity of other species ranked ATCC 33277 > 35406 = 33184 = 35496 . When hemin was replaced in media by hemoglobin, ATCC 33277 became resistant to irradiation . Protoporphyrin IX content in BPB cells was shown not to be a major factor determining photosensitivity . Oxygen was required during irradiation for BPB species to be affected . Non-black-pigmented bacteria were much less sensitive to irradiation than BPB.

Carcinogenesis, 1995 Apr, 16(4), 719 - 27
Perturbation of hepatocyte nuclear populations induced by iron and polychlorinated biphenyls in C57BL/10ScSn mice during carcinogenesis; Madra S et al.; The induction of hepatocarcinogenesis by polychlorinated biphenyls (PCBs) in C57BL/10ScSn mice is markedly potentiated by iron . To investigate the effects of iron and PCBs on nuclear populations, C57BL/10ScSn mice received a single dose of iron-dextran (600 mg Fe/kg) and were fed a diet containing 0.01% of the PCBs mixture Aroclor 1254 for up to 6 months . DNA content of isolated nuclei and hepatocytes was estimated by flow cytometry . Cell suspensions and nuclei isolated from Aroclor treated mice after 6 months contained increased diploid (2N) populations compared to controls . In contrast, iron treatment of mice markedly enhanced fractions of octoploid (8N) nuclei by 2 weeks and this effect persisted over the 6 month period . When Aroclor 1254 and iron were administered together there was a synergistic increase in the mononucleated diploid fraction which was significant at 2 weeks and highly significant at 6 months . This became the predominant nuclear effect . At six months, Aroclor 1254 and iron, both alone and in combination, also increased the rate of DNA synthesis in hepatocytes as measured by bromodeoxyuridine (BrdU) incorporation . The chronic polyploidizing effect of iron overload alone was investigated further and shown to be proportional to the dose and was detectable as early as 2 days after 600 mg Fe/kg and 1 week after 150 mg Fe/kg . Polyploidization of nuclei was inhibited by the oral iron chelator CP94 . Iron also induced a prolonged reduction in the incidence of binucleated cells . Histologically, nuclear enlargement due to iron was confined to the midzonal region of the liver lobule, whereas iron deposition was greatest in the periportal region . Iron (600 mg/kg) also caused increased nuclear polyploid states in hepatocytes of adult rats and gerbils . Similarly, weanling mice with a dominantly diploid cell population, when treated with iron (300 mg/kg), exhibited a significant shift to a tetraploid (4N) population and a marked increase in proliferation as measured by BrdU incorporation and proliferative cell nuclear antigen (PCNA) detection . These results indicate that Aroclor 1254 and iron induce changes in the mouse hepatocyte population that involve 2N and 8N nuclei respectively . The combination treatment leads to the emergence and proliferation of a mononucleated, diploid population as observed frequently in chemical hepato-carcinogenesis . The reason for the chronic polyploidizing effect of iron is unknown, but may imply both increased DNA synthesis and impairment of nuclear division with implications in human conditions of iron overload.

Am J Clin Pathol, 1995 Apr, 103(4), 409 - 14
Ploidy analysis of products of conception by image and flow cytometry with cytogenetic correlation; Topalovski M et al.; Ploidy analysis of hydropic placentas is used in conjunction with morphology and clinical data to classify hydatidiform moles and hydropic abortuses . In most studies, ploidy has been assessed by flow cytometry (FCM) . To validate image cytometry (ICM) as a method to determine ploidy in this setting, the authors used both FCM and ICM to study 19 hydropic placentas in which cytogenetic analysis was available . Nuclear suspensions from paraffin-embedded tissue were used for both ICM and FCM . Image cytometry of tissue sections was performed in some cases . Image cytometry and FCM were concordant in all 19 cases, but discordant with cytogenetics in 2 of 19 cases . Two hydropic abortuses (HA) with a diploid karyotype were triploid and tetraploid, respectively, by both ICM and FCM, which suggested that the cultured tissue was not representative . DNA indices were most accurate when an internal diploid control was used as the reference . In ICM, higher resolution was achieved by analyzing cell suspensions rather than tissue sections . This study shows that ICM is a valid method of determining ploidy of hydropic placentas and partial hydatidiform moles in archival tissue.

Anesthesiology, 1995 Apr, 82(4), 1004 - 12
Triiodothyronine increases contractility independent of beta-adrenergic receptors or stimulation of cyclic-3',5'-adenosine monophosphate; Ririe DG et al.; BACKGROUND: Triiodothyronine regulates cardiac contractility; however, the mechanisms by which it produces its acute contractile effects remains unknown . We compared the acute effects of thyroid hormones (triiodothyronine {T3} and thyroxine {T4}) and of isoproterenol on the contractility of isolated rat hearts . In addition, we sought to determine whether the acute inotropic effects of thyroid hormones were mediated by beta-adrenergic receptors or by increased production of cyclic-3',5'-adenosine monophosphate (cAMP) . METHODS: A Langendorff heart preparation harvested from euthyroid male Sprague-Dawley rats was used . Drugs were administered through an aortic perfusion catheter . A pressure-transduced left-ventricular balloon catheter measured pressure and heart rate changes . Changes in the maximum positive rate of change in pressure (dP/dT) and maximum negative dP/dT were determined . Responses to varying doses of T3, T4, and isoproterenol were assessed in the presence and absence of beta-adrenergic receptor blockade with propranolol . cAMP production, measured by radioimmunoassay, was determined in myocardial cell suspensions after incubation with T3 or isoproterenol . RESULTS: T3 0.74 nmol rapidly and significantly increased maximum dP/dT by 335 +/- 38 mmHg/s within 30 s after bolus injection; however, contractility was unchanged after as much as 12.9 nmol T4 . The maximal increase in dP/dT after 0.8 nmol isoproterenol was comparable to that produced by T3 . However, the cardiotonic actions of isoproterenol were significantly slower to develop (peaking at 60 vs . 15 s) and lasted longer than those of T3 . Pretreatment with propranolol 1 mumol diminished the contractile effects of isoproterenol but had no effect on those of T3 . Concentrations of isoproterenol that increase contractility also significantly increased cAMP production in isolated rat myocardial cells . However, T3 failed to increase cAMP production . CONCLUSIONS: These results demonstrate that the acute inotropic effects of T3 are not shared by T4 and appear unrelated to beta-adrenergic receptor mechanisms or to generation of cAMP . Thus, T3 acutely stimulates cardiac contraction by mechanisms that differ from those of the more commonly used beta-adrenergic receptor agonists and phosphodiesterase inhibitors . Further studies are needed to identify the mechanisms underlying the acute contractile effects of T3 and to determine whether T3 will prove useful for increasing ventricular function in patients.

Eur Respir J, 1995 Apr, 8(4), 559 - 65
Some technical factors influencing the induction of sputum for cell analysis; Popov TA et al.; Inhalation of hypertonic saline aerosol is a relatively noninvasive method to obtain sputum for examination of inflammatory processes in the airways . We investigated some technical factors which might influence the success of induction and sputum cell counts . In total, twenty six asthmatic and 13 healthy subjects, unable to raise sputum spontaneously, inhaled nebulized saline for three 7 min intervals . In three randomized, cross-over studies we repeated sputum induction on separate days with two ultrasonic nebulizers (De Vilbiss Ultraneb 99 and Fisoneb) and one jet nebulizer (Pari LL with Master Compressor) (Study 1, n = 15), with different saline concentrations (normal saline 0.9%; hypertonic saline 3% on 2 days; and hypertonic saline 3, 4 and 5%, sequentially) (Study 2, n = 14) and with pretreatment with either salbutamol or placebo (Study 3, n = 10) . The latter two studies were double-blind . Sputum cells were dispersed with dithiothreitol, and the cell suspension was used to perform total cell counts and to prepare cytospins for differential cell counts . We compared success rate, cell counts, subject discomfort and percentage fall in forced expiratory volume in one second (FEV1) during the procedures . All sputum examinations were performed blind to the clinical procedures . The success rates and the cell counts of the specimens obtained with the two ultrasonic nebulizers were not different, whilst general discomfort was proportional to the saline output of the nebulizer . Induction of sputum by hypertonic saline was more successful than normal saline, but more disagreeable to the subjects . Induction with saline 3% on two days was only successful in 6 of 14 subjects.(ABSTRACT TRUNCATED AT 250 WORDS)

Clin Orthop, 1995 Apr, (313), 103 - 14
Interleukin-10 stimulates hematopoiesis in murine osteogenic stroma; Van Vlasselaer P et al.; Bone marrow from 5-fluorouracil-treated mice support osteogenesis when cultured in the presence of beta-glycerophosphate and vitamin C . These cultures are unable to support the growth of granulocyte/macrophage colony-forming units for longer than 2 weeks . In contrast, granulocyte/macrophage colony-forming units were detected for more than 6 weeks in interleukin-10 (IL-10)-treated cultures . In addition, IL-10-treated cultures contain long-term culture initiating cells, suggesting the presence of pluripotent hematopoietic cells . Apparently, IL-10 does not directly stimulate the proliferation of granulocyte/macrophage colony-forming units . Interleukin-10 is unable to stimulate {3H}-thymidine incorporation or to increase the number of granulocyte/macrophage colony-forming units in cell suspensions harvested from untreated or interleukin-10-treated bone marrow cultures . Interleukin-10 acts via an indirect pathway . Because exogenous transforming growth factor-beta (TGF-beta) reverses IL-10's stimulatory activity on myeloid progenitors, IL-10 most likely works by blocking TGF-beta synthesis, which acts as an endogenous suppressor of hematopoiesis in osteogenic marrow cultures . This is shown further by the increased numbers of granulocyte/macrophage colony-forming units in cultures treated with neutralizing anti TGF-beta antibodies (1D11.16) . Interleukin-10 and 1D11.16 change the cultured bone marrow stroma from an osteogenic into a hematopoietic morphology . It may be that by blocking endogenous TGF-beta production, IL-10 drives marrow mesenchymal cells away from osteogenic differentiation toward hematopoietic support.

Int J Dev Neurosci, 1995 Apr, 13(2), 105 - 11
Neurobehavioral, neurochemical and electrophysiological studies in 6-hydroxydopamine lesioned and neural transplanted rats; Agrawal AK et al.; Unilateral injection of 6-hydroxydopamine (6-OHDA) into the caudate nucleus of rat caused degeneration of dopaminergic terminals, evidenced by significant (P < 0.05) elevation of spontaneous and drug-induced motor behaviour, enhanced DA receptor binding and significant increase in the neuronal firing rate of caudate neurons, suggesting supersensitivity of dopaminergic receptors . Eight weeks following the transplantation of embryonic cell suspensions from caudate at the lesioned site, a significant restoration of the enhanced 3H spiperone binding and neuronal activity of caudate neurons was observed in comparison with lesioned rats . These results clearly demonstrate that transplanted embryonic neuronal tissue at the lesioned site is capable of restoring the neuronal deficits caused by 6-OHDA as evidenced by significant amelioration in neurochemical, behavioral and electrophysiological alterations.

Mod Pathol, 1995 Apr, 8(3), 275 - 81
Quantitative DNA analysis of fresh solid tumors by flow and image cytometric methods: a comparison using the Roche Pathology Workstation Image Analyzer; Ellison DA et al.; The clinical utility of DNA ploidy and cell cycle parameters as prognostic indicators has been demonstrated for selected malignant tumors . Previous quantitative DNA analysis studies have used various tumor sample preparation methods and analyzers . We undertook a pilot study to compare the results of DNA analysis of fresh solid tumors by flow cytometry with the new Roche Pathology Workstation Image Analyzer . Flow cytometric DNA analysis was done on cell suspensions of fine needle aspirates from fresh tumor specimens and analyzed for ploidy and cell cycle statistics with a Becton-Dickinson FACScan Analyzer, using a rectangular model . Small aliquots from these same aspirates were prepared as direct cytologic smears and Feulgen stained for DNA analysis with the Roche Image Analyzer . Additional smears were stained with Diff-Quik for morphologic correlation with DNA histograms . The study group consisted of 40 malignant neoplasms . There was a high correlation between the flow and image DNA indices (R = 0.93, slope = 1.0036, P < 0.001) but a weaker relationship between the flow and image estimated S-phase fractions (R = 0.57, slope = 0.5401, P < 0.01) . DNA ploidy categorization for the two methods was concordant in 30 (75%) cases, discordant in seven (17.5%) cases, and equivocal in three (7.5%) cases . In our experience, quantitative DNA analysis of fresh tumor aspirates by flow and image cytometric methods yielded comparable and/or complementary results, with each method having certain advantages and disadvantages . Proposed reasons for false and true discordances and an approach for evaluation are discussed.

Clin Exp Allergy, 1995 Apr, 25(4), 364 - 70
Antiallergic activity of loratadine: inhibition of leukotriene C4 release from human leucocytes; Miadonna A et al.; The H1 antagonist loratadine has the capacity to inhibit histamine release from human basophils . The aim of this study was to investigate whether loratadine can also inhibit leukotriene C4 (LTC4) release from human leucocytes . Basophil-enriched mononuclear cell suspensions were prepared by centrifugation of peripheral venous blood (n = 10) on discontinuous Percoll gradients . Leucocytes were stimulated with anti-IgE, N-formylmethionyl-leucyl-phenylalanine (FMLP) and Ca2+ ionophore A23187; immunoreactive (i) LTC4 release in the cell supernatant was measured by a competitive radioimmunoassay and histamine release was evaluated by an automated fluorometric technique . Loratadine, in the concentration range of 1-50 microM, exerted a dose-dependent inhibitory effect on IgE-mediated and IgE-independent histamine and iLTC4 release . The concentrations inhibiting 50% of histamine release were 30 microM (anti-IgE), 27 microM (FMLP) and 19 microM (Ca2+ ionophore A23187) . The concentrations inhibiting 50% of iLTC4 release were 2.3 microM (anti-IgE) . 11 microM (FMLP) and 1.7 microM (Ca2+ ionophore A23187) . The inhibitory activity on iLTC4 release was optimal after preincubation for 2 h at 37 degrees C, and was no longer evident when leucocytes were stimulated 2 h after cell washing . Increased extracellular Ca2+ concentrations reduced the inhibitory activity of loratadine . These results indicate that loratadine has the capacity to inhibit the release of preformed and newly generated mediators from human basophil-enriched mononuclear cell suspensions.

Proc Natl Acad Sci U S A, 1995 Mar 14, 92(6), 2071 - 5
Molecular cloning and heterologous expression of a cDNA encoding berbamunine synthase, a C--O phenol-coupling cytochrome P450 from the higher plant Berberis stolonifera; Kraus PF et al.; A cDNA encoding a cytochrome P450-dependent oxidase, berbamunine synthase (EC 1.1.3.34; CYP80), from cell suspension cultures of the higher plant Berberis stolonifera Koehne and Wolf (barberry) has been isolated and heterologously expressed in functional form in insect cell culture using a baculovirus-based expression system . This cytochrome P450-dependent enzyme is unusual in that it catalyzes the regio- and stereoselective formation of a C--O phenol couple in bisbenzylisoquinoline alkaloid biosynthesis without concomitant incorporation of activated oxygen into the product . Consistent with the function of an oxidase rather than a monooxygenase, an essential glycine residue in the distal helix, which forms the oxygen-binding pocket in the well-studied bacterial enzyme P-450cam, is replaced by proline at the equivalent position in berbamunine synthase . This oxidase was accumulated in an active form in insect cell microsomes and accepted electrons from the endogenous NADPH-cytochrome P450 reductase . The heterologously expressed enzyme oxidatively couples either two molecules of (R)-N-methylcoclaurine to form the (R,R) dimer guattegaumerine or one molecule each of (R)- and (S)-N-methylcoclaurine to form the (R,S) dimer berbamunine . The ratio of the two bisbenzylisoquinolines formed could be altered by reductase source or by varying the enantiomer composition of the substrates.

J Immunol Methods, 1995 Mar 13, 180(1), 93 - 100
Altered expression of CD11b/CD18 and CD62L on human monocytes after cell preparation procedures; Lundahl J et al.; We have investigated the effect of different cell preparation procedures on the surface expression of CD11b/CD18 and CD62L on human monocytes . Both EDTA and heparin anticoagulated blood were used as sources for leukocytes . The monocytes were analysed by flow cytometry in a mixed leukocyte suspensions obtained after ammonium chloride mediated lysis and in mononuclear cell suspension prepared by density gradient centrifugation (Ficoll-Paque) performed both at 4 degrees C and at 20 degrees C . Monocytes from heparinized blood had a higher expression of CD11b/CD18 and a more pronounced inter-individual variation than monocytes from EDTA blood . Monocytes isolated by Ficoll-Paque had a higher degree of ex vivo activation by means of altered expression of CD11b/CD18 and CD62L compared to monocytes prepared by ammonium chloride mediated lysis . This was more pronounced when the isolation procedure was performed at 20 degrees C . Our findings indicate that monocytes prepared by ammonium chloride mediated lysis of EDTA blood and with the cell handling temperature kept at 4 degrees C are exposed to the smallest ex vivo modulation by means of receptor alteration . An awareness of ex vivo modulation by different cell preparation procedures is of importance especially when comparing the expression of functional receptors on leukocytes of disparate origin.

Neurosci Lett, 1995 Mar 10, 187(3), 153 - 6
An avian model for the reversal of 6-hydroxydopamine induced rotating behaviour by neural grafting; Yanai J et al.; A new animal model of parkinsonism was established in 'Black Silkie' chickens by means of unilateral injections of 6-hydroxy-dopamine into the substantia nigra . Apomorphine produced a strong contralateral turning pattern in the lesioned chickens, amphetamine had no effect . 6-OHDA treated animals received embryonic transplants of substantia nigra cell suspensions which caused them to cease rotating (P < 0.01) . This finding allows us to add an avian model, which offers unique methodological advantages, for reversal of 6-OHDA-induced rotating behavior by transplantation.

Yan Ke Xue Bao, 1995 Mar, 11(1), 16 - 21
Characteristics of an established retinoblastoma cell line HXO-Rb44; Xu H et al.; PURPOSE: To study the retinoblastoma cell culture and to establish a new retinoblastoma cell line . METHODS: 22 retinoblastomas were cultured by using the method of single cell suspension . Characteristics of the cultured cells were studied in the following programs: tumor cell morphology in vitro, electron microscopic, growth curve, cloning in soft agar, immunohistochemistry, karyotype and tumorigenicity . RESULTS: 22 retinoblastomas were cultured successfully in vitro, only a continued cell line HXO-Rb44 was established (more than 3 years) . The characteristics of this cell line are that it grew as a suspension of round cell in graps like clusters in vitro, its population doubling time was 44 hours, and it could be cloned in soft agar . Histopathologic and ultrastructured pictures showed the characteristics of Rb . HXO-Rb44 cell was positive to NSE and negative to GFAP in immunohistochemical staining . A subcutaneous injection of HXO-Rb44 cells produced a retinoblastoma in BALB/C athumic nude mice . CONCLUSIONS: HXO-Rb44 has the characteristics of retinoblastoma and is a new retinoblastoma cell line . It is a useful material for study this tumor both in basic and clinical fields.

Biochem J, 1995 Mar 1, 306 ( Pt 2), 571 - 80
Strictosidine synthase from Catharanthus roseus: purification and characterization of multiple forms; de Waal A et al.; Multiple (six) forms of strictosidine synthase from Catharanthus roseus cell suspension cultures were purified and characterized . A purification protocol is presented composed of hydrophobic-interaction, gel-permeation and ion-exchange chromatography and chromatofocusing . Four of six isoforms were purified to apparent homogeneity, whereas two others were nearly homogeneous . All strictosidine synthase isoforms were found to be glycoproteins . The isoforms were also found in leaves and roots of the plant, in seedlings and in hairy root cultures . The ratio of the different isoforms differed slightly between these sources . The kinetic parameters of the isoforms showed no significant differences . The maximal velocity (300-400 nkat/mg of protein) is the highest reported so far . It was demonstrated that the apparent Michaelis constant for tryptamine (approx . 9 microM) is much lower than values reported previously . The presence of weak product inhibition (Kp approx . 35 times Km) was established, whereas substrate inhibition was not detected.

J Leukoc Biol, 1995 Mar, 57(3), 484 - 90
Differential regulation of fyn-associated protein tyrosine kinase activity by macrophage colony-stimulating factor (M-CSF) and granulocyte-macrophage colony-stimulating factor (GM-CSF); Li Y et al.; The proliferation and differentiation of macrophages are regulated by, among others, GM-CSF and M-CSF . Treatment of bone marrow nonadherent (NA) cells with M-CSF induced a greater percentage of NA cells into adherent cells, recognized as monocyte/macrophages, than did GM-CSF . The effect of GM-CSF and M-CSF on the activation of fyn kinase, a 59-kDa src family-related protein tyrosine kinase (PTK), was studied . Control cultures of bone marrow NA cells expressed only minimal levels of fyn kinase activity . Treatment of bone marrow NA cells with M-CSF, but not GM-CSF, for 12 to 24 h greatly enhanced the levels of fyn kinase activity . The effect of M-CSF on the activation of fyn kinase was further investigated using bipotential adherent bone marrow-derived macrophages (BMDMs) that coexpress receptors for both GM-CSF and M-CSF . BMDMs can be induced by either growth factor to undergo extensive proliferation in vitro . Compared to bone marrow NA cells, BMDMs displayed higher levels of basal fyn kinase activity, which were similarly elevated by M-CSF but not GM-CSF treatment . The role of fyn kinase in regulating cell adhesion was investigated by growing BMDMs in both tissue culture and Teflon flasks . The growth of BMDMs was anchorage independent; the majority of them continued to proliferate as cell suspension in Teflon flasks . Whereas the levels of fyn kinase activity in adherent BMDMs grown in tissue culture flasks increased steadily, those in BMDMs grown in Teflon flasks diminished . To study the role of fyn kinase in growth regulation, BMDMs were treated with c-fyn sense and antisense s-oligos . In the presence of c-fyn antisense s-oligos, the proliferative response of BMDMs to M-CSF but not GM-CSF was inhibited . In contrast, the proliferation of BMDM in response to either GM-CSF or M-CSF was not influenced by c-fyn sense s-oligos . Collectively, our data suggest that the activation of fyn kinase is closely associated with the acquisition of adherent capacity in maturing macrophages.

Infect Immun, 1995 Mar, 63(3), 853 - 7
Induction of compartmentalized B-cell responses in human tonsils; Quiding-Jarbrink M et al.; The capacity of tonsillar and nasal mucosal lymphoid tissues to serve as induction sites of local and/or distant B-cell responses in humans has been examined . The frequencies of vaccine-specific antibody-secreting cells (ASC) in cell suspensions from palatine tonsils (PT) and adenoids were determined after local (intra-tonsillar {i.t.}) and regional (intranasal {i.n.}) immunizations as well as peroral and parenteral immunizations with cholera and tetanus toxoids . While peroral and parenteral immunizations evoked negligible ASC responses in PT, i.t . vaccination induced a substantial ASC response which consisted of immunoglobulin G (IgG) and IgA ASC . Responses were highly restricted to immunized tonsils . Primary immunization in one PT followed by a second immunization of both PT evoked a larger ASC response in the primed tonsil . The latter ASC response was associated with higher frequencies of ASC precursors in primed tonsils . Furthermore, two i.n . immunizations induced only modest ASC responses in PT, although such immunizations evoked high ASC responses in adenoids . However, both i.t . and i.n . routes of immunization induced specific peripheral blood ASC responses, suggesting that a fraction of B cells activated in tonsils or in nasal mucosa may enter the circulation and disseminate to distant organs . These blood ASC responses preceded increases in both IgA and IgG antibody titers in nasal washes and serum samples . However, vaccine-specific ASC were not detected in duodenal cell suspensions from volunteers who had received i.t . or i.n . immunizations . Collectively, these results indicate that tonsils can serve as expression sites of locally induced antibody responses and support the development of immunological memory . Furthermore, tonsils may serve as powerful inductive sites for immune responses expressed in the upper aerodigestive tract.

J Invest Dermatol, 1995 Mar, 104(3), 329 - 34
Regulation of transgenic class II major histocompatibility genes in murine Langerhans cells; Longley J et al.; I-E is a class II major histocompatibility complex molecule normally expressed by Langerhans cells . A series of transgenic mice were developed previously that carry E alpha d gene constructs with promoter-region deletions that cause expression of I-E by different cell types when maintained on a B6 (I-E{-}) genetic background . To study cis-acting gene sequences that regulate expression of class II proteins by Langerhans cells, we identified transgenic I-E expression by tissue immunoperoxidase staining and by epidermal cell suspension immunofluorescence cytometry . Mice with a transgene containing 1.4 kilobase pairs (kb) of flanking sequence 5' to the E alpha initiation site expressed barely detectable levels of I-E on a tiny percentage of Langerhans cells, indicating that sequences promoting Langerhans cell expression of E alpha exist between 2.0 and 1.4 kb 5' of the E alpha initiation site . Removal of an additional 170 bp of 5' flanking sequence caused near-normal levels of expression by approximately one third of epidermal Langerhans cells, which contrasts with studies that showed minimal transgene expression by splenic dendritic cells in these animals . Thus, sequences between 1.4 and 1.23 kb 5' of the E alpha initiation site decrease expression of I-E by epidermal Langerhans cells, but enable I-E expression by splenic dendritic cells . These studies identify Langerhans cell-specific regulatory sequences and genetic regions controlling major histocompatibility complex class II gene expression in Langerhans cells and splenic dendritic cells . The genetic regions identified may be particularly important because differential regulation of class II major histocompatibility complex protein synthesis by Langerhans cells and dendritic cells may be crucial to immune functions of intact animals.

Shock, 1995 Mar, 3(3), 184 - 8
Acinar cell respiration in experimental acute pancreatitis; Schulz HU et al.; The early pathogenetic steps that finally lead to acinar cell necrosis in acute pancreatitis have been characterized only scarcely as yet . Among a lot of hypotheses, one concept favors disturbances of cellular energy metabolism as a major factor that contributes to preterm cell decline . To investigate, whether an experimental acute pancreatitis alters cell respiration, the respiratory capacities of acinar cells isolated from rats with acute pancreatitis were measured . Acute pancreatitis was induced using Popper's model, i.e., a combination of duct obstruction, secretory stimulation, and mesenteric short-term ischemia with subsequent reperfusion . Acinar cells were isolated using a collagenase digestion technique . The respiratory rates of the isolated cells in suspension were measured at 37 degrees C in 100% oxygen-saturated N-(2-hydroxyethyl)piperazine-N'-2-ethanesulfonic acid-buffered Eagle's-minimal essential medium . Resting respiration of the acinar cells uniformly amounted to about 60 pmol of O2/s x 10(6) cells in both the control and the pancreatitis group . Cellular respiration could significantly be stimulated by stepwise uncoupling of oxidative phosphorylation by means of 2,4-dinitrophenol in all cell suspensions investigated . The maximum rate of stimulated respiration was diminished in the cells isolated from rats with acute pancreatitis as compared with the controls (79.3 +/- 5.0 vs . 160.2 +/- 15.5 pmol of O2/s x 10(6) cells, p < .05), however . This reduced respiratory load capacity of the acinar cells in acute pancreatitis reflects the restricted ability of the cells to increase respiration on enhanced cellular demand . Since mitochondrial respiration is coupled to oxidative phosphorylation, an altered energy-transforming potential of the acinar cells in acute pancreatitis becomes evident.(ABSTRACT TRUNCATED AT 250 WORDS)

Plant Mol Biol, 1995 Mar, 27(5), 901 - 10
Purification, immunological characterization and cDNA cloning of a 47 kDa glycoprotein secreted by carrot suspension cells; van Engelen FA et al.; A 47 kDa glycoprotein, termed EP4, was purified from carrot cell suspension culture medium . An antiserum raised against EP4 also recognized a protein of 45 kDa that was ionically bound to the cell wall . EP4 was detected in culture media from both embryogenic and non-embryogenic cell lines and was found to be secreted by a specific subset of non-embryogenic cells . Secretion of the 47 kDa glycoprotein by embryogenic cells was not evident . The 45 kDa cell wall-bound EP4 protein was specific for non-embryogenic cells and was shown by immunolocalization to occur in the walls of clustered cells, with the highest levels in the walls separating adjacent cells . In seedlings, EP4 proteins were mainly found in roots . EP4 cDNA was cloned by screening a cDNA library with an oligonucleotide derived from an EP4 peptide sequence . The EP4 cDNA sequence was found to be 55% homologous to ENOD8, an early nodulin gene from alfalfa.

Anal Biochem, 1995 Mar 1, 225(2), 346 - 8
DNA preparation from cryopreserved bone marrow cell samples; Jorgensen H et al.; 1 . Quickly thaw cryopreserved MNCs (1 ml) in 9 ml PBS supplemented with DNase I immediately before use (final concentration: 0.1 mg/ml) . 2 . Incubate at 37 degrees C for 10 min . 3 . Wash twice in PBS supplemented with 2% human AB serum at 4 degrees C . 4 . Incubate with saturating amounts of selected MoAbs for 20 min at 4 degrees C . 5 . Wash twice in PBS supplemented with 2% human AB serum at 4 degrees C . 6 . Immunomagnetic bead separation of viable cells as described by Lea et al . (1) . 7 . DNA extraction . 8 . Measure quality and yield of DNA by spectrophotometry . In conclusion, the DNA extraction method presented here, based on DNase I pretreatment of the cryopreserved cells followed by an immunological selection for viable cells, provides cell suspensions with close to 100% viability; thus, high-quality DNA can be extracted even from cryopreserved cell samples of low initial viability . Furthermore, using this method DNA analyses can be performed on selected cellular subsets if desired . We thus recommend this method for DNA analyses in hematological research when the only materials available are cryopreserved bone marrow samples of low viability.

Breast Cancer Res Treat, 1995 Mar, 33(3), 225 - 35
Characterization of platelet aggregation induced by human breast carcinoma and its inhibition by snake venom peptides, trigramin and rhodostomin; Chiang HS et al.; MCF-7 cells, a metastatic human breast carcinoma line, caused dose-dependent platelet aggregation in heparinized human platelet-rich plasma (PRP) . MCF-7 tumor cell-induced platelet aggregation (TCIPA) was almost blocked by apyrase (0.5 U/ml) and completely inhibited by hirudin (5 U/ml) . This TCIPA was unaffected by cysteine proteinase inhibition with E-64 (10 microM), but was limited by cell pretreatment with phospholipase A2 . MCF-7 cell suspension caused marked, dose-dependent decrease in plasma recalcification times using normal, Factor VIII-deficient, and Factor IX-deficient human plasma . This effect was potentiated in cell lysates but was inhibited in intact cells preincubated with sphingosine . MCF-7 cell suspension did not affect the recalcification time of Factor VII-deficient plasma . Taken together, these data suggest that MCF-7 TCIPA arises from MCF-7 tissue factor activity expression . Trigramim and rhodostomin, RGD-containing snake venom peptides which antagonized the binding of fibrinogen to platelet membrane glycoprotein IIb/IIIa, prevented MCF-7 TCIPA . Likewise, synthetic peptide GRGDS as well as monoclonal antibodies against human tissue factor, platelet membrane glycoprotein IIb/IIIa and Ib prevented MCF-7 TCIPA, which was unaffected by control peptide GRGES . On a molar basis, trigramin (IC50, 0.1 microM) and rhodostomin (IC50, 0.03 microM), were about 5,000 and 18,000 times, respectively, more potent than GRGDS (IC50, 0.54 mM).

Cytometry, 1995 Mar 1, 19(3), 267 - 72
Limited loss of nine tumor-associated surface antigenic determinants after tryptic cell dissociation; Corver WE et al.; Disodium ethylenediaminetetraacetic acid (EDTA) or trypsin/EDTA are frequently used for the dispersion of monolayer cells into single cell suspensions allowing flow cytometric analysis of surface antigenic determinants . A disadvantage of EDTA is the slow action of this agent, whereas trypsin might affect the antigenic determinants under focus . We studied the possible deleterious effect of trypsin on three different ovarian carcinoma cell lines, COV413b, COV362.c14, and NIH:OVCAR-3, on cell surface antigenic determinants by flow cytometry . Either EDTA or trypsin/EDTA was used for detachment and dissociation of monolayer ovarian cancer cell lines, followed by indirect immunofluorescence with a panel of monoclonal antibodies directed against nine different surface antigenic determinants, including six markers directed against widely distributed antigens . Compared to EDTA, trypsin/EDTA resulted in higher total cell yields and rapid detachment and dissociation into single cell suspensions with significantly lower amounts of dead cells detected by both trypan blue and propidium iodide (PI) . Large differences in antigen expression were observed for the different cell lines . However, all antigenic determinants tested could still be detected after tryptic proteolysis . Three antigenic determinants were significantly decreased after trypsin/EDTA compared to EDTA detachment . CA 125 was decreased on COV362.c14 and NIH: OVCAR-3 cells, respectively . BMA 180 and ICAM-1 were decreased on COV413b cells . This cell line-dependent decrease might be caused by differences in glycosylation . We conclude that trypsin/EDTA can be used for rapid monolayer cell detachment with high cell yields and limited loss of antigenic determinants tested.

Cytometry, 1995 Mar 1, 19(3), 263 - 6
Usefulness of the scraping method for DNA flow cytometry in breast tumors; Cornacchiari A et al.; Flow cytometric DNA analysis is an important prognostic tool in breast cancer . We evaluated the possibility of performing DNA analysis on cell suspensions obtained by scraping the cut surface of breast tumors; 31 breast tumor nodules, including six benign and 25 malignant lesions, were studied . From each case, cell suspensions acquired by mechanical mincing of a fresh frozen tissue fragment and by two different scrapings (central and peripheral) from the cut surface of the tumor were analyzed via flow cytometry . In all cases, comparison of the DNA histograms for three samples showed no significant differences in the appearance of debris or in the value of coefficient of variation of the G0-G1 peak . All benign nodules showed a normal DNA stemline in all specimens . In 23 of 25 cases of breast carcinoma, the ploidy of the three preparations was similar, with a concordance in 12/14 (85, 71%) cases in DNA nondiploid tumors . Linear regression analysis showed a good correlation in DNA index between either scraping sample and the tissue fragment (r = .955 and r = .905) . The results indicate that the scraping technique provides excellent cell suspensions and DNA histograms comparable to those obtained from mechanical mincing of tissue fragments . The technique minimizes preparation time and avoids consuming much tissue and, thus, is the method of choice when very small cancers have to be analyzed.

Cell Biol Int, 1995 Mar, 19(3), 215 - 22
EPR study of the sea anemone cytolysin, equinatoxin II, cytotoxicity on V-79 cells; Batista U et al.; Electron paramagnetic resonance (EPR) was used to study the effect of equinatoxin II (EqT II), a cytolytic protein isolated from the sea anemone Actinia equina L., on membrane fluidity and cell metabolism of V-79 cells; the reduction of the spin probe incorporated into the cell membranes as well as the oxygen consumption in the cell suspension were measured . The results were compared with the results obtained by the cell viability study . Under the influence of EqT II (less than 37.5 micrograms/10(6) cells) no significant changes in cell membrane fluidity were observed, while reduction kinetics of the spin probe and the oxygen consumption decreased when the cells were kept in Tris buffer solution . However, in the presence of 10% fetal calf serum, which prevented cell lysis, the effects of EqT II were diminished . The oxygen consumption corresponds to the cell viability changes but the reduction kinetics alterations indicate that some oxidation-reduction processes other than cell respiration are affected by EqT II in the absence of serum . The effect seems to be indirect, probably due to the formation of pores which are associated with changed permeability of plasmalemma for metabolites and ions.

Vet Immunol Immunopathol, 1995 Mar, 45(1-2), 73 - 84
Response of the regional lymph node to bluetongue virus infection in calves; Barratt-Boyes SM et al.; Bluetongue virus (BTV) infection of cattle is characterized by prolonged cell-associated viremia . In an effort to further evaluate the antiviral response of BTV-infected cattle, the role of the regional lymph node (LN) in the immune response of calves to BTV was characterized . Calves were inoculated with BTV in the skin of the neck in an area drained by the superficial cervical LN . Calves were euthanized at regular intervals after inoculation and both BTV-challenged and contralateral (control) superficial cervical LNs were harvested . In addition, some calves had cannulation of the superficial cervical efferent lymphatics prior to inoculation . Lymphocyte subpopulation analysis was done on LN cell suspensions and lymph cells using a panel of cell-specific monoclonal antibodies . There was a significant increase in the proportion of B cells in the challenged LN after inoculation as compared with the control LN . In addition, BTV-specific antibodies were detected in efferent lymph plasma from the challenged LN in one cannulated calf by 9 days after inoculation (DAI), as determined by competitive enzyme-linked immunosorbent assay, whereas BTV-specific antibodies were not detected in serum from this calf through 12 DAI . Analysis of lymph cells revealed a sustained increase in cell output from the challenged LN due to an increase in lymphoblasts and CD8+ T cells . In contrast, the cell output from the control LN dropped markedly by 8 DAI and there was no significant increase in any specific cell population . Double label analysis characterized lymphoblasts as activated CD8+ cells, as determined by expression of MHC Class II antigens (CD8+ MHC II+) . These cells were only transiently present as CD8+ MHC II+ cells were not identified in lymph from the challenged LN at 14 DAI . Few CD8+ MHC II+ cells were identified at any time in lymph from the control LN or in lymph from a mock infected calf . The data indicate that B cell proliferation in the challenged LN and release of activated CD8+ cells from this LN were specific responses to BTV infection . The rapid expansion of activated CD8+ T cells indicates that these cells may limit early viral spread . It is concluded that the regional LN draining inoculated skin is critical to the immune response of calves to BTV infection.

In Vivo, 1995 Mar-Apr, 9(2), 149 - 54
Ex-vivo fine needle aspiration . A new method of xenografting non-small cell carcinoma of the lung; Mourad WA et al.; Ex-vivo needle aspiration (xvFNA) has been rarely used to obtain viable tumor cells . It has been occasionally employed for short-term cultures . Xenografting of lung carcinoma in athymic nude mice provides a good animal model for the study of this neoplasm . Successful engraftment using conventional methods has been disappointing) low (d 40%) . Enzymatic digestion of the tumor fragments to obtain cell suspension lowers viability . We postulated that xvFNA might provide readily available tumor cell suspensions for xenografting lung carcinoma and that it would provide a higher success rate of engraftment than the conventional techniques . We aseptically performed xnFNA in 35 cases of freshly resected non-small cell carcinoma of the lung . These included 15 adenocarcinomas, 17 squamous carcinomas and 3 undifferentiated non-small cell carcinoma (UNSCC) . Tumor cell suspensions were injected subcutaneously in athymic nude mice . Tumor necrosis in the aspirates ranged from 20-90% (median 60%) . Gross evidence of engraftment was seen in 30 of 35 cases (85.7%) 1-19 weeks postimplantation (median 2 weeks) . This was seen in UNSCC (3/3), squamous carcinomas (13/17) and adenocarcinomas (14/15) . Xenograft sizes ranged from 5-34 mm (median 19 mm) . They showed similar morphology to the primary tumors . Ex-vivo FNA used for harvesting lung carcinoma cells and their xenografting is an effective method for obtaining viable material for studying this neoplasm.

Cell Transplant, 1995 Mar-Apr, 4(2), 173 - 200
A comparative study of preparation techniques for improving the viability of nigral grafts using vital stains, in vitro cultures, and in vivo grafts; Barker RA et al.; The intracerebral transplantation of embryonic dopaminergic nigral neurons, although relatively successful, leads to a fairly low yield of surviving cells . Many factors may influence the viability of dopaminergic grafts and one of these is the preparation of the tissue prior to transplantation . We have investigated the effects of different steps during the preparation and storage of embryonic rat nigral cell suspensions on their subsequent survival at a variety of different time points using a combination of techniques and studies . For studies concerned with the first 24 h we employed vital stains, in the period covering the next 7 days we used in vitro cultures, and in the long term experiment we used in vivo grafts . The results suggest that nigral cell suspensions may remain sufficiently viable for grafting for much longer periods than previously reported . In addition a number of parameters which affect cell survival have been characterised, including the age of the embryonic donor tissue, the use of proteolytic enzymes and the trituration procedure used during the preparation of the suspension . The optimal preparation technique, therefore, uses E13-E14 embryos with the dissected ventral mesencephalon being incubated in purified 0.1% trypsin solutions for 60 min and triturated using a flame polished Pasteur pipette . This may have important implications in improving intracerebral transplantation for Parkinson's disease.

Toxicology, 1995 Feb 27, 96(3), 225 - 38
Possible incorporation of an immunotoxicological functional assay for assessing humoral immunity for hazard identification purposes in rats on standard toxicology study; Ladics GS et al.; The objective of this study was to examine the feasibility of conducting an immunotoxicological assay for assessing humoral immunity in rats on standard toxicology study . Male CD rats were untreated or dosed intraperitoneally daily for 30 or 90 days, excluding weekends, with vehicle or 2 mg/kg cyclophosphamide (CY) . Six days prior to sacrifice, selected rats were injected intravenously with sheep red blood cells (SRBC) . One day prior to necropsy, blood samples for hematological and clinical chemical measurements were collected from each rat . On the day of necropsy standard protocol tissues were collected, weighed, processed to slides, and examined microscopically . One-half of each spleen was used to prepare a single cell suspension in order to assess spleen cell numbers . Serum was analyzed for anti-SRBC IgM antibody using an enzyme-linked immunosorbent assay . A second set of studies was performed to examine further the effect of SRBC administration on lymphoid organ weights using 30- and 90-day study age-equivalent naive male CD rats . Exposure of animals to 2 mg/kg CY for 30 or 90 days resulted in a 28% and 61% decrease, respectively, in SRBC-specific serum IgM levels . CY treatment also caused mild alterations in some leukocytic parameters, with significant decreases of 35% and 33% in white blood cell and lymphocyte counts, respectively, observed in 30-day CY-treated animals receiving SRBC . Injection of SRBC alone did not alter hematological or clinical chemistry parameters . With the expected exception of the spleen (increased number and size of germinal centers), administration of SRBC did not significantly alter the weights or morphology of routine protocol tissues . Furthermore, administration of SRBC did not mask the immunosuppressive effects of CY treatment under the conditions of this study . Based on our preliminary findings, a functional assay for assessing humoral immunity may be conducted in animals on standard toxicology study.

FEBS Lett, 1995 Feb 20, 360(1), 57 - 61
Changes in protein methylation associated with the elicitation response in cell cultures of alfalfa (Medicago sativa L.); Daniell T et al.; The methylation of endogenous proteins increased in alfalfa cell suspension cultures following treatment with a fungal elicitor . Carboxyl methylation, a post-translational modification associated with controlling the localisation and longevity of proteins, was the dominant form of protein methylation in both elicited and unelicited cells . Protein methylation was restricted to a limited number of peptides prior to elicitor treatment but as elicitation progressed the number of endogenous substrates increased . Increases resulted from a combination of an elicitor-dependent increase in the activity of a protein carboxyl methyltransferase and the accumulation of preferred endogenous substrates in the latter stages of elicitation.

Int J Radiat Oncol Biol Phys, 1995 Feb 15, 31(4), 905 - 10
Thermal response and hyperthermic radiosensitization of scid mouse bone marrow CFU-C; O'Hara MD et al.; PURPOSE: Scid mice are severely immunodeficient as a result of a defective recombinase system . Mice with the scid mutation have been shown to have an increased sensitivity to ionizing radiation, presumably as a result of an inability to repair DNA damage . Little is known of the impact of this mutation on the thermal response and on hyperthermic radiosensitization . This investigation established the thermal response (42-44 degrees C), patterns of thermotolerance development, and the impact of hyperthermia (60 min at 40 degrees C or 42 degrees C) on the radiation response of bone marrow colony forming unit-culture cells (CFU-C) in scid mice . METHODS AND MATERIALS: Anesthetized scid mice (pentobarbital, 90 mg/kg) were killed by cervical dislocation and the nucleated marrow obtained from both tibia and femora by passing 2 ml of cold McCoy's 5A medium supplemented with 15% fetal bovine serum through each bone . Single cell suspensions of nucleated marrow were heated in 12 x 75 mm sterile tissue culture tubes at a concentration of approximately 5 x 10(6) cells/ml . Radiation, when used, was delivered immediately prior to hyperthermia by a 137Cs irradiator (dose rate of 1.20 Gy/min) . Colony forming unit-culture were cultured in semisolid agar in the presence of colony stimulating factor (conditioned medium from L929 cells) for 7 days . RESULTS: The slope of the radiation dose-response curve for CFU-C in scid mice was biphasic, the Dos (+/- SE) were 0.29 +/- 0.03 Gy and 1.09 +/- 0.20 Gy, respectively . The Dos of the radiation dose-response curve for wild type marrow from CB-17 and Balb/c mice were 1.28 +/- 0.05 Gy and 1.47 +/- 0.15 Gy, respectively . The Dos of the hyperthermia dose-response curves for scid mice were 75 +/- 5, 10 +/- 1.4, and 4 +/- 0.2 min, respectively, for temperatures of 42 degrees, 43 degrees, and 44 degrees C . Thermotolerance development at 37 degrees C increased to a maximum at approximately 240 min after acute hyperthermia (15 min at 44 degrees C) and thereafter, decreased to control levels within 15 h . Thermotolerance did not develop in scid CFU-C during chronic hyperthermia at temperatures < 42.5 degrees C . Hyperthermia (60 min at 40 degrees or 42 degrees C) immediately after ionizing radiation did not significantly alter the terminal slope of the radiation dose-response curve of scid CFU-C (Do = 1.28 +/- 0.08 Gy) . By contrast, hyperthermia following radiation of wild type CFU-C resulted in a decrease in the Do from 1.47 +/- 0.05 Gy (Balb/c, rad only) to 1.31 +/- 0.08 or 1.06 +/- 0.18 Gy for 60 min at 40 degrees or 42 degrees C, respectively . CONCLUSION: These results show that the thermal response and the pattern of thermotolerance development of scid CFU-C were similar to that of wild type Balb/c CFU-C, but that hyperthermia given immediately after ionizing radiation did not alter the radiation response of scid CFU-C . The scid mutation does not increase hyperthermic sensitivity or change the pattern of thermotolerance development of scid mouse CFU-C, implying that the scid mutation is not involved with thermal response, but does render the already radiation-sensitive scid cells incapable of thermal radiosensitization.

Biochem Pharmacol, 1995 Feb 14, 49(4), 453 - 60
Ethylmorphine O-deethylation in isolated rat hepatocytes . Involvement of codeine O-demethylation enzyme systems; Xu BQ et al.; The O-dealkylation of ethylmorphine (EM) and codeine (CD) to morphine (M) co-segregates with debrisoquine/sparteine genetic polymorphism in man . CD O-demethylation is catalysed by cytochrome P450 2D1 (CYP2D1) in rats . In the present study, the O-deethylation of EM was examined and compared with that of CD in suspensions of freshly-isolated hepatocytes prepared by a collagenase method from Wistar rats with and without CYP2D1 inhibitors . Isolated hepatocytes were also prepared from Dark Agouti (DA) rats deficient in CYP2D1, and were incubated with EM or CD . EM, CD and their metabolites were quantified by HPLC with UV detection . EM had a similar pattern of metabolism to that of CD in suspensions of hepatocytes from Wistar rats . Both EM and CD were O-dealkylated to form M plus morphine-3-glucuronide (M3G) and N-demethylated to form norethylmorphine (NEM) or norcodeine (NCD), respectively, which were further metabolized to normorphine (NM) and finally glucuronidated to normorphine-3-glucuronide (NM3G) . As compared to hepatocytes from Wistar rats, DA rats were characterized by a markedly decreased formation (70 approximately 75% reduction) of M plus M3G from both EM and CD . Quinine, quinidine, propafenone and sparteine all inhibited EM O-deethylation as well as CD O-demethylation . Quinine was the most potent inhibitor of both these O-dealkylations (Ki = 0.2 microM for both EM and CD, respectively) . Quinine as well as the other inhibitors inhibited both EM and CD O-dealkylation competitively and with small differences in Ki versus EM and CD, respectively . The metabolism of EM to M plus M3G and that of CD to M plus M3G was highly correlated when results from the various separate cell suspensions were plotted . In conclusion all findings indicated that the enzyme responsible for O-demethylation of CD, CYP2D1 was also responsible for the O-deethylation of EM to M.

J Immunol Methods, 1995 Feb 13, 179(1), 37 - 49
Organ culture of human lymphoid tissue . I . Characteristics of the system; Hoffmann P et al.; The major aim of three-dimensional tissue culture is to preserve the natural architecture of the tissue and thereby allow the cells to retain their original functions during in vitro cultivation . Here we describe a method for the rapid preparation of three-dimensional tissue explants from human lymphoid organs . The precision-cut tissue slices are of uniform size and thickness and can be cryopreserved and stored in liquid nitrogen without substantial loss of viability or functionality of the cells . Upon in vitro culture, cells within the explants survived as well as their counterparts cultured in single cell suspension . However, spontaneous immunoglobulin (Ig) production in explants started more promptly and often reached considerably higher levels than that in suspension cultures run in parallel . Lymphocytes within the slices could be activated by polyclonal stimuli such as PHA, as shown by the upregulation of the activation markers CD23 and CD25 on B and T cells, respectively . However, approximately five-fold higher concentrations of mitogen than those used for suspension cultures were needed . Taken together, the system presented here constitutes a potent tool for the investigation of the complex interactions leading to activation and differentiation of B and T cells in lymphoid organs.

Photochem Photobiol, 1995 Feb, 61(2), 196 - 9
Efficacy of photodynamic therapy on original and recurrent rat mammary tumors; Gibson SL et al.; Photodynamic therapy has demonstrated efficacy toward primary, metastatic and recurrent human tumors . Here, we investigated the ability of photodynamic therapy, using Photofrin, to inhibit growth of R3230AC mammary adenocarcinomas when tumors were treated as original implants and again as lesions recurring at the initial treatment site . The results demonstrate that both initial implants and lesions recurring after the first photodynamic treatment respond similarly to the same photodynamic therapy protocol, with mean tumor volume doubling times of approximately 11 days in both cases . Cells cultured from original tumor implants or tumors that recurred after photodynamic treatment accumulate equivalent amounts of {14C}polyhematoporphyrin . Single cell suspensions prepared from either original or recurrent tumors from animals administered 5 mg/kg Photofrin and exposed to light in vitro displayed comparable phototoxicity . Additionally, examination of tumors by light microscopy revealed no morphological differences between the original tumor implants and the recurrent lesions . Taken together, these data indicate that lesions which recurred at the site of the initial photodynamic treatment were not resistant to a second identical course of photodynamic therapy.

Plant Mol Biol, 1995 Feb, 27(3), 619 - 22
Drought, heat and salt stress induce the expression of a citrus homologue of an atypical late-embryogenesis Lea5 gene; Naot D et al.; In a search for genes that are induced in citrus cell suspension in response to salt stress, a cDNA clone with high homology to cotton Lea5 gene was isolated . Data base analysis of the protein deduced from the nucleotide sequence indicates that, like in cotton, the protein from citrus contains regions with significant hydropathic character . The gene, designated C-Lea5, is expressed in citrus leaves as well as cell suspension . The steady-state level of C-Lea5 is increased in cell suspension that is grown in the presence of 0.2 M NaCl . This phenomenon is also observed in leaves of citrus plants irrigated with NaCl and in citrus seedlings which are exposed to drought and heat stress . We suggest that the osmotic stress resulted from elevated level of salt is responsible for the increase in the level of C-Lea5.

Cancer Immunol Immunother, 1995 Feb, 40(2), 125 - 31
Specific tumor memory induced by polyethylene-glycol-modified interleukin-2 requires both helper and cytotoxic T cells; Balemans LT et al.; Local polyethylene-glycol (PEG)-modified interleukin-2 (IL-2) immunotherapy of the guinea pig Line 10 (L10) tumor was previously demonstrated to evoke long-lasting systemic immunity after cure of the tumor and metastases . T cells, most likely the helper T cell subpopulation, were demonstrated to be crucial to the antitumor effects . Here we show that systemic immunity is induced within 7 days after the start of PEG-IL-2 therapy, indicating a rapid systemic priming of L10-specific T cells . No in vitro cytotoxic activity was detected in cell suspensions obtained from the primary tumor site, the regional lymph node or the spleen when isolated during (days 21 and 28) intratumoral treatment with 200,000 IU PEG-IL-2 . These data confirm our earlier results obtained with 60,000 IU PEG-IL-2 . Moreover, no cytolytic activity was observed in the chromium-release assay after in vitro restimulation with irradiated tumor cells . Specific L10 immunity can be transferred using spleen cell suspensions . Depletion of such a suspension of helper T cells resulted in rejection of the primary tumor in two out of four animals, but all the guinea pigs developed lymph node metastases . Removal of the cytotoxic/suppressor phenotype caused rejection of the dermal tumor in four of eight guinea pigs, but the capacity to prevent lymph node metastases was retained in all animals . Thus, depletion of either subtype reduces, but does not abrogate, the capacity to transfer L10 immunity with spleen cells . In conclusion, our data suggest that tumor cell killing through direct T cell cytotoxicity is not the main mode of action in PEG-IL-2-induced L10 tumor regression . PEG-IL-2 therapy induces early systemic immunity, resulting in rejection of a distant tumor, and the transfer of this immunity depends mainly on the presence of helper T cells, although cytotoxic T cells may also play a role.

Br J Haematol, 1995 Feb, 89(2), 380 - 5
A flow cytometric assay using mepacrine for study of uptake and release of platelet dense granule contents; Wall JE et al.; Diagnosis of platelet dense granule storage pool disease and release defects at present requires a combination of studies including lumiaggregometry, conventional platelet aggregation, radioactive serotonin uptake and release, and electron microscopy . Flow cytometric methods have been developed to study platelet activation, aggregation, and alpha-granule protein release . Here, we have investigated the use of flow cytometry for analysis of platelet dense granule content uptake and release using mepacrine as a fluorescent marker . Mepacrine (quinacrine) is rapidly taken up and localized in dense granules of platelets . For the assay, as little as 20 microliters of blood from a fingerstick collected without anticoagulant or venous blood collected in 3.8% sodium citrate were diluted 1:40 with 2 ml Hanks balanced salt solution (BSS) . 300 microliters of this cell suspension were incubated with mepacrine alone, or simultaneously with a mouse monoclonal antibody to human platelet glycoprotein IIb (Tab), used as a platelet-specific marker . The bound monoclonal antibody was then indirectly labelled with the fluorochrome, RED670 . 100 microliters of the sample were further diluted with Hanks BSS for one- or two-colour flow cytometric analysis . To verify that mepacrine uptake was related to platelet dense granule content, platelets of beige mice, a strain with dense granule deficiency, were examined . Their mepacrine uptake was substantially decreased compared to that of normal mice . Decreased mepacrine uptake also was demonstrated in platelets of a patient with Hermansky-Pudlak syndrome in which a deficiency of platelet dense granules is characteristic . In both human and mouse platelets, mepacrine uptake was proportional to platelet size . Thrombin induced mepacrine release in a dose-dependent manner from 0.003 to 0.4 U/ml . Therefore both platelet uptake and release of mepacrine can be readily detected by flow cytometry . Flow cytometry provides an attractive alternative to aggregation and radioactive serotonin as methods to study defects in platelet dense granule function.

Arthritis Rheum, 1995 Feb, 38(2), 248 - 53
Reduced red blood cell deformability in patients with rheumatoid vasculitis . Improvement after in vitro treatment with dipyridamole; Lau CS et al.; OBJECTIVE . To assess red blood cell deformability (RCD) in patients with rheumatoid arthritis (RA) without extraarticular manifestations and in RA with vasculitic complications (RV), and to assess whether in vitro dipyridamole improves RCD . METHODS . An improved filtration technique was used to measure RCD in 15 patients with RA, 18 patients with RV, and 20 age- and sex-matched normal control subjects . Washed erythrocytes suspended in buffer, at 5% hematocrit, were filtered through 4.7 mu Nuclepore Hemafil PC membranes . The initial steady-state relative filtration pressure (iRFP) was used as an index to assess RCD . A lower iRFP value reflects increased deformability, a higher value reflects a decrease . For each sample, 2 cell suspensions were prepared, one blank (control) and one containing 5 microM dipyridamole . RESULTS . The mean iRFP values of cells obtained from patients with RV were significantly higher than those of cells obtained from normal controls . There were no appreciable differences in iRFP between RA patients and normal controls . When the erythrocytes were pretreated in vitro with 5 microM dipyridamole before filtration, their deformability improved markedly (iRFP values were reduced) in all study subjects, compared with untreated cells . CONCLUSION . RCD is reduced in patients with RV, and treatment with dipyridamole may be beneficial if reduced RCD contributes to impaired microvascular perfusion.

Br J Cancer, 1995 Feb, 71(2), 265 - 70
The Arg-Gly-Asp-containing peptide, rhodostomin, inhibits in vitro cell adhesion to extracellular matrices and platelet aggregation caused by saos-2 human osteosarcoma cells; Chiang HS et al.; Saos-2 cells, derived from a primary human osteosarcoma, caused dose-dependent platelet aggregation in heparinised human platelet-rich plasma . Saos-2 tumour cell-induced platelet aggregation (TCIPA) was completely inhibited by hirudin but unaffected by apyrase . The cell suspension shortened the plasma recalcification times of normal, factor VIII-deficient and factor IX-deficient human plasmas in a dose-dependent manner . However, the cell suspension did not affect the recalcification time of factor VII-deficient plasma . Moreover, a monoclonal antibody (MAb) against human tissue factor completely abolished TCIPA . Flow cytometric analysis using anti-integrin MAbs as the primary binding ligands demonstrated that the integrin receptors alpha v beta 3, alpha 5 beta 1 and alpha 6 beta 1 were present of Saos-2 cells, which might mediate tumour cell adhesion to extracellular matrix . Rhodostomin, an Arg-Gly-Asp (RGD)-containing snake venom peptide which antagonises the binding of fibrinogen to platelet membrane glycoprotein IIb/IIIa, prevented Saos-2 TCIPA as well as tumour cell adhesion to vitronectin, fibronectin and collagen type I . Likewise, the synthetic peptide Gly-Arg-Gly-Asp-Ser (GRGDS) showed a similar effect . On a molar basis, rhodostomin was about 18,000 and 1000 times, respectively, more potent than GRGDS in inhibiting TCIPA and tumour cell adhesion.

Int J Dev Neurosci, 1995 Feb, 13(1), 41 - 9
Enhanced survival and differentiation in vitro of different neuronal populations by some interleukins; Moroni SC et al.; Data from the literature demonstrate the existence of a growing family of neuropoietic cytokines; members of this group have structural motifs in common with other members and with neurotrophic factors . In this research we studied the responses elicited in vitro by some of these molecules in two different neuronal populations: murine neuroblastoma N18TG2 and neurons from chicken dorsal root ganglia . Both IL-2 and IL-6 improve the survival of murine neuroblastoma cells in clonal density plating experiments; in addition IL-2 significantly inhibits thymidine incorporation by single cell suspension . The survival of sensory neurons, on the other hand, non-responsive to IL-2 and IL-6, was significantly supported by IL-3, which also stimulates their morphological differentiation, inducing the formation of a well-developed neural net . In conclusion, results reported here confirm the neurotrophic activity of some ILs and provide additional neuronal models for future investigations.

Mod Pathol, 1995 Feb, 8(2), 183 - 6
Interphase cytogenetic analysis of single cell suspensions prepared from previously formalin-fixed and paraffin-embedded tissues; Kuchinka BD et al.; Fluorescence in situ hybridization (FISH) provides a rapid and accurate method for the detection of chromosomal aneuploidy . We have developed a technique for the use of FISH on single cell suspensions produced from either formalin-fixed or paraffin-embedded tissues . Preparation of such tissues involves sequential rehydration, enzymatic digestion to release single nuclei, and hybridization with a fluorescently labeled chromosome-specific centromeric probe . In a clinical setting formalin-fixed tissue from many tissue types is readily available for additional retrospective study . FISH on formalin-fixed tissues is especially beneficial in follow-up studies of cases involving termination after prenatal diagnosis or patients with a malignant disease where previous routine cytogenetics established the chromosomal aneuploidy . The use of this technique eliminates the biases of cytogenetic analysis due to clonal selection in tissue culture, the low number of cells analyzed, and the restriction to only dividing cell populations . We have demonstrated that this application of interphase cytogenetics to the study of various formalin-fixed tissues is amenable to the detection of chromosomal aneuploidies and has specific advantages over cytogenetic analysis.

Eur J Nucl Med, 1995 Feb, 22(2), 101 - 7
Labelling of leucocytes with colloidal technetium-99m-SnF2: an investigation of the labelling process by autoradiography; Puncher MR et al.; Autoradiography of smears and frozen sections of labelled cell suspensions was used to study the distribution of radioactivity in and among blood cells labelled in either whole blood or leucocyte-rich plasma (LRP) with technetium-99m-SnF2 colloid . The tracer proved selective for neutrophils: the labelling probability (relative to that for erythrocytes) for each cell type in LRP (mean of five samples) was: neutrophils, 9.4; lymphocytes, 3.7; monocytes, 3.0; eosinophils 1.4; erythrocytes, 1.0 . When labelling was carried out in whole blood (five samples), 74.5% +/- 8.3% of the cell-bound radioactivity was bound to erythrocytes, 13.6% +/- 6.5% to neutrophils, and 11.9% +/- 2.1% to lymphocytes, whereas in LRP (in which the leucocytes were only slightly outnumbered by erythrocytes), 76.5% +/- 14.9% of radioactivity was neutrophil bound . Labelled cells in smear autoradiographs exhibited two distinct silver grain patterns, "diffuse", consistent with an intracellular radioactive particle (in neutrophils), and "focal", consistent with a cell surface-adhering particle in direct contact with the emulsion (in other leucocyte types and erythrocytes) . The phagocytic inhibitor cytochalasin B neither reduced the proportion of labelled neutrophils nor altered the labelling pattern . Neutrophils were able to scavenge radioactivity from the surface of erythrocytes . It is concluded that neutrophils bind 99mTc-SnF2 intracellularly by phagocytosis, with high affinity; other cells become labelled at the cell surface reversibly and with lower affinity . This selectivity is high enough to permit predominantly leucocyte labelling in LRP but not in whole blood.

Biomaterials, 1995 Feb, 16(3), 201 - 7
Adhesion of leucocytes to microscope slides as influenced by electrostatic interaction; Seyfert S et al.; The adhesion of leucocytes to standard microscope slides is low, which severely limits the cell recovery in cytological preparations on such slides . We coated the slides with various polycations with the aim of achieving an electrostatic surface charge which would increase the cell adhesion but would still avoid cell disruption . The adhesion of leucocytes from a K562 myeloid cell suspension and from cerebrospinal fluid was measured on standard slides made of glass and of oxidized polystyrene, and on slides coated with several chemically different cationic polymers . The electrostatic surface charge and the surface tension of these matrices were determined . We found the cell adhesion to be significantly higher on the matrices with a more attractive zeta potential and not to be measurably influenced by the surface tension of the matrices . The results help to improve the cytological preparation technique, especially for suspensions with a low cell content.

Cytometry, 1995 Feb 1, 19(2), 146 - 53
Time-related effects of enzymatic disaggregation on model human lung carcinomas; Howard RB et al.; Enzyme cocktails used to prepare tumor cell suspensions may influence yield, viability, and cytology, thus time-related cocktail effects on model human lung carcinomas were examined . A549, NCI-H125, and NCI-H460 carcinomas were completely disaggregated at 25 degrees C over 2 h with either (mg/ml) collagenase/DNAase (C/D, 1/0.1), collagenase/hyaluronidase/DNAse (C/H/D, 1/0, 1/0.1), or polymyxa protease/DNAse (PP/D, 3/0.1) . Trypan blue viabilities, total yields, viable yields, and flow cytometric percent tumor cells (TC) were measured every 20-30 min (n = 4-7 per tumor type) . The final percentages of TC, mononuclear cells (MN), polymorphonuclear cells (PMN), lymphocytes, and necrotic cells were determined by cytology (n = 4-5 per tumor type) . The time-dependent measurements showed that 1) disaggregation was progressive and complete with all cocktails; 2) viability was stable or increasing with all cocktails; 3) percent TC was stable for all cocktails, but lower for PP/D than C/D in final suspensions; and 4) PP/D gave lower final total yields, higher final viabilities, but the same final viable yields as the C cocktails, suggesting selective elimination of dead cells by PP/D . Final cytology measurements showed that PP/D gave a lower percent MN and a higher percent PMN than C cocktails . Cocktail effects may importantly influence cell suspension properties.

Plant Mol Biol, 1995 Feb, 27(4), 681 - 92
Molecular cloning and heterologous expression of acridone synthase from elicited Ruta graveolens L . cell suspension cultures; Junghanns KT et al.; Cell suspension cultures of Ruta graveolens L . produce a variety of acridone alkaloids, and the accumulation can be stimulated by the addition of fungal elicitors . Acridone synthase, the enzyme catalyzing the synthesis of 1,3-dihydroxy-N-methylacridone from N-methylanthraniloyl-CoA and malonyl-CoA, had been isolated from these cells, and the partial enzyme polypeptide sequence, elucidated from six tryptic fragments, revealed homology to heterologous chalcone synthases . Poly(A)+ RNA was isolated from Ruta cells that had been treated for 6 h with a crude cell wall elicitor from Phytophthora megasperma f . sp . glycinea, and a cDNA library was constructed in lambda 2AP . Clones harboring acridone synthase cDNA were isolated from the library by screening with a synthetic oligonucleotide probe complementary to a short stretch of sequence of the enzyme peptide with negligible homology to chalcone synthases . The identity of the clones was substantiated by DNA sequencing and by recognition of five additional peptides, determined previously from tryptic acridone synthase digests, in the translated sequence . An insert of roughly 1.4 kb encoded the complete acridone synthase, and alignments at both DNA and protein levels corroborated the high degree of homology to chalcone synthases . Expression of the enzyme in vector pET-11c in the Escherichia coli pLysS host strain proved the identity of the cloned cDNA . The heterologous enzyme in the crude E . coli extract exhibit high acridone but no chalcone synthase activity . The results were fully supported by northern blot hybridizations which revealed that the specific transcript abundance did not increase but rather decreased upon white light irradiation of cultured Ruta graveolens L . cells, a condition that commonly induces the abundance of chalcone synthase transcripts.

J Microsc, 1995 Feb, 177 ( Pt 2), 162 - 70
PHOEBE, a prototype scanning laser-feedback microscope for imaging biological cells in aqueous media; Wong TL et al.; Based on the principle of laser-feedback interferometry (LFI), a laser-feedback microscope (LFM) has been constructed capable of providing an axial (z) resolution of a target surface topography of approximately 1 nm and a lateral (x,y) resolution of approximately 200 nm when used with a high-numerical-aperture oil-immersion microscope objective . LFI is a form of interferometry in which a laser's intensity is modulated by light re-entering the illuminating laser . Interfering with the light circulating in the laser resonant cavity, this back-reflected light gives information about an object's position and reflectivity . Using a 1-mW He-Ne (lambda = 632.8 nm) laser, this microscope (PHOEBE) is capable of obtaining 256 x 256-pixel images over fields from (10 microns x 10 microns) to (120 microns x 120 microns) in approximately 30 s . An electromechanical feedback circuit holds the optical pathlength between the laser output mirror and a point on the scanned object constant; this allows two types of images (surface topography and surface reflectivity) to be obtained simultaneously . For biological cells, imaging can be accomplished using back-reflected light originating from small refractive-index changes (> 0.02) at cell membrane/water interfaces; alternatively, the optical pathlength through the cell interior can be measured point-by-point by growing or placing a cell suspension on a higher-reflecting substrate (glass or a silicon wafer).(ABSTRACT TRUNCATED AT 250 WORDS)

Glia, 1995 Feb, 13(2), 141 - 6
Binding and action of glucagon in cultured mouse astrocytes; Cockram CS et al.; This study investigates glucagon binding in primary cultures of differentiated mouse astrocytes and the effect of glucagon on intracellular cAMP accumulation . Binding of 125I-glucagon (0.53 nM) to mouse astrocyte suspensions reached equilibrium after 10 min at 22 degrees C . Equilibrium binding corresponded to 46 +/- 15 pmol/mg protein (n = 3) representing approximately 10,000 occupied sites per cell at the tracer concentration used . Dissociation occurred with a half-time of 2.5 min at 22 degrees C and was not accelerated in the presence of unlabelled glucagon (1 microM) . Scatchard analysis suggested the presence of more than one class of binding site . The Ka for the higher affinity sites was 5.7-7.4 x 10(6) M-1 . The Ka for the lower affinity sites was 3.6-5.3 x 10(4) M-1 . The results suggest the presence of approximately 43,000 high affinity sites per cell . Binding was inhibited by unlabelled glucagon with an IC50 of 50 nM but unaffected by insulin and somatostatin . However, no 125I-glucagon binding could be detected when intact monolayer cells attached to culture dishes were used . Glucagon stimulated cyclic-AMP accumulation in both cell suspensions and intact monolayer cells in a dose-dependent fashion . However high concentrations were required when compared to the receptor-binding studies . Marked degradation of 125I-glucagon by astrocytes during binding experiments was observed and this was inhibited by unlabelled glucagon but also by insulin and desoctapeptide insulin.

J Clin Endocrinol Metab, 1995 Feb, 80(2), 648 - 53
Decidual histamine release and amplification of prostaglandin F2 alpha production by histamine in interleukin-1 beta-primed decidual cells: potential interactive role for inflammatory mediators in uterine function at term; Schrey MP et al.; Inflammatory cytokines such as interleukin-1 (IL-1) have been implicated as paracrine mediators of decidual prostaglandin (PG) production during preterm labor . The aim of the present in vitro study was to investigate a similar potential role for the inflammatory autacoid histamine . Histamine action on human primary decidual cell cultures was monitored by measuring both PGF2 alpha production and PG precursor release . Histamine release from decidual cell suspensions was measured in response to a variety of putative mast cell secretagogues . Histamine stimulated a dose-dependent increase in PGF2 alpha production and {14C}arachidonate release from prelabeled decidual cells, with ED50 values around 1 and 2 mumol/L, respectively . Pretreatment of cells with IL-1 beta enhanced maximal PGF2 alpha production in response to histamine by approximately 4-fold . Mepyramine, an H1 receptor antagonist, completely blocked this PGF2 alpha response . The mean histamine content of unpurified decidual cells was approximately 81 pmol/10(6) cells . Upon challenge with antihuman immunoglobulin E, these cells exhibited a dose-dependent release of histamine . Basal release from decidual cell suspensions and release in response to antiimmunoglobulin E (1:400) or A23187 (1 mumol/L) were 7.6 +/- 2.7%, 25.5 +/- 0.9%, and 63.5 +/- 5.6% of the total histamine content, respectively . No significant changes in histamine secretion were seen in response to compound 48/80, substance-P, phorbol ester, or bacterial endotoxin . These in vitro findings support a potential role for histamine as a local regulator of phospholipase-A2 and PGF2 alpha production in human term decidua . The interaction of this autacoid with other inflammatory mediators, such as IL-1, could play a role in the control of PG production during preterm labor.

FEBS Lett, 1995 Jan 16, 358(1), 67 - 70
Molecular characterization and cell cycle-regulated expression of a cDNA clone from Arabidopsis thaliana homologous to the small subunit of ribonucleotide reductase; Philipps G et al.; A cDNA clone isolated from an Arabidopsis thaliana cell suspension library showed highly significant homology to the small subunit of ribonucleotide reductase (R2) from different species . The 340 amino acid-long deduced putative protein contains all the residues that are important for the enzyme activity and structure . In A . thaliana this enzyme is encoded by a single-copy gene . In synchronized tobacco BY2 cells the corresponding mRNAs specifically accumulate during the S phase of the cell cycle.

J Biotechnol, 1995 Jan 15, 38(2), 165 - 72
Measurement of yeast intracellular pH by image processing and the change it undergoes during growth phase; Imai T et al.; The intracellular pH of the yeast Saccharomyces cerevisiae was determined by a fluorescence microscopic image processing technique . Image processing was carried out using a modification of the ratio imaging method for measurement of yeast intracellular pH . Care was necessary when taking fluorescence images in order to obtain accurate measurement of yeast intracellular pH . Until now it has been difficult to measure the intracellular pH of cells in actual cultivation conditions . This method enabled us not only to measure the intracellular pH of dilute cell suspensions, but also to obtain two-dimensional information . In the case of resting cells, the intracellular pH was dependent upon the extracellular pH, and this value was constant when the extracellular pH was constant . On the other hand, in the case of actively growing cells, intracellular pH was found to change, even if the extracellular pH was constant: the values observed were intracellular pH 5.7 during lag phase, intracellular pH 6.8 during exponential phase and intracellular pH 5.5 during stationary phase . These results for intracellular pH indicate that the yeast proton pump was activated during growth from the point of view of pH in vivo.

Exp Clin Endocrinol Diabetes, 1995, 103 Suppl 2, 118 - 22
Comparison of the survival of fresh or cultured pancreatic islets, pseudoislets and single cells following allotransplantation beneath the kidney capsule in non-immunosuppressed diabetic rats; Wolf-Jochim M et al.; The purpose of the present study was to examine the effect of culture pretreatment and islet structure on transplantation survival time . Donors for islet isolation were highly inbred male Lewis rats (RT 1(1)) . Production of single cells was performed by using EDTA and Trypsin . Pseudo-islets were produced by culturing single cell suspensions at 37 degrees C for 12-14 days . Recipients were BDE rats (RT 1u), made diabetic by streptozotocin injection . 1000-1200 islets (or corresponding amount of single cells or pseudoislets) were transplanted to the subcapsular renal space . Five groups were transplanted . Group 1 (n = 5) received freshly isolated islets of Langerhans . Group 2 (n = 7) received single cells, produced from freshly isolated islets . In group III (n = 7) pseudoislets were transplanted . The animals of group IV received 37 degrees C cultured islets (12-14 days), while group V received single cells consisting of 12-14 day cultured islets at 37 degrees C . The median survival times were: gr . I 7 d.; gr . II 5 d.; gr . III 120 d.; gr . IV 11 d.; gr . V 9 d. . Group III showed a prolongation of allograft survival that was statistically significant compared to all other groups . 4 from 7 animals showed a long-term acceptance . It can be concluded that neither culturing islets at 37 degrees C nor producing single cells achieves long-term acceptance . Transplanting pseudoislets resulted in a long-term acceptance of allograft, without immunosuppression of the host . Three factors may be responsible for this success: Firstly, a reduced number of class-II-antigen positive cells, secondly, metabolic state of rest, and thirdly, the transplantation site.

Adv Exp Med Biol, 1995, 380, 387 - 90
Identification of 120 kD and 30 kD receptors for human coronavirus OC43 in cell membrane preparations from newborn mouse brain; Collins AR; A biotinylated virus overlay was used to identify a 120kD virus-binding molecule in dissociated newborn mouse brain (nmb) cell suspensions after separation of the proteins by polyacrylamide gel electrophoresis, electroblotting, and blockage of non-specific binding sites . The virus-binding molecule was not detected in adult mouse brain cell suspensions . Mannose- and glucose-rich glycoproteins from nmb cell membranes were selected by ConA-Sepharose (Pharmacia) chromatography . A 30kD virus-binding molecule was eluted by 0.2 M alpha-methyl-D-mannoside . O-linked sialic acid, a receptor component, was identified in the eluate.

Scanning Microsc, 1995, 9(4), 1223 - 30; discussion 1230-2
The formation of filopodia-like protrusions during preparation of cell suspensions for scanning electron microscopy; Rovensky YA; The standard preparation of cell suspensions (e.g., blood cells, cell suspensions derived from monolayer cultures or from various tissues, etc.) for scanning electron microscopy (SEM) includes fixation of the cells in suspension and subsequent dehydration and critical point drying (CPD) of the cells after their preliminary attachment to the special substrata . In the course of the SEM examination of cell suspensions of various origins, unusual morphological cell surface structures, filopodia-like protrusions (FLP), were consistently detected in 3-20% of the cells in the populations . FLP can be effectively observed only by using a stage tilt angle of no less than 30 degrees . FLP were single or multiple thin threads extending from basal parts of a spherical cell and attaching to the substratum surface used . FLP strikingly resembled substratum-attached filopodia formed by a viable cell at its earliest stages of spreading . The percentages of the cells with FLP were not significantly affected by the character of cell fixation (primary aldehyde fixation alone or primary aldehyde with subsequent OsO4 post-fixation) or the raising of the temperature and pressure inside the CPD bomb . It seems that the protrusions imitating the natural cell surface structures can probably be formed at later stages of the preparation of cell suspensions for SEM, namely during dehydration and (or) CPD when the cells undergo substantial shrinkage . One of the possible mechanisms by which the cell shrinkage could induce FLP formation follows . A prefixed spherical cell which settles down to the substratum sticks to it at some discrete points on the cell surface . As a result of the subsequent cell shrinkage, FLP could be formed and then stretched, connecting the same points on the cell surface with the points of the initial cell-substratum adhesion.

Int Urol Nephrol, 1995, 27(5), 603 - 13
Impact of the preparation technique on DNA content measured by image analysis in early stage human testis cancer; de Riese C et al.; Current clinical staging which includes serum tumour markers and imaging techniques fails to identify 30-40% of clinical stage I nonseminomatous germ cell testicular tumour (NSGCT) patients who have occult metastatic disease at time of orchiectomy and who will, therefore, develop clinically evident metastases, usually within two years of follow-up . Therefore, there is a real clinical need to evaluate new biological parameters of the primary tumour which might be useful as predictors for occult metastatic disease . Some investigators have described that DNA content measured by image cytometry is of prognostic value in early stage NSGCT to detect patients at risk for occult metastatic disease . However, optimal preparatory techniques are mandatory in establishing new tumour biological markers in order to obtain reliable and reproducible results . This study has analyzed the impact of the sedimentation technique in comparison to the cytocentrifugation technique on DNA measurement in early stage NSGCT obtained by image cytometry . Different tissue blocks of formalin fixed, paraffin embedded primary testicular tumours (NSGCT) of 50 clinical stage I patients, who underwent retroperitoneal lymph node dissection between 1985 and 1989, were analyzed . Thirty (60%) patients had histologically proven lymph node involvement (pathological stage B), whereas 20 (40%) patients (pathological stage A) had neither lymph node metastases nor tumour recurrence during follow-up . The samples were prepared according to a modified Hedley technique: Individual tissue digestion times were monitored closely to avoid overdigestion . The times varied from 30 to 60 min depending on the constituents of the tissue section . Prolonged digestion times did not correlate with poor quality of the preparations and brief digestion times did not always yield optimal specimens . The impact of two different techniques (cytocentrifugation and gravity sedimentation) on the Feulgen staining results were compared . Cytocentrifuged samples consistently provided larger and paler nuclei with less well defined borders compared to slides from the same cell suspension processed by the sedimentation technique . Nuclei from pathologic stage II samples were more vulnerable to cytocentrifuge alteration than those of stage I . According to the results obtained in this study, the sedimentation slide preparation technique should be preferred for DNA ICM in NSGCT, and possibly in other human malignancies as well.

Exp Brain Res, 1995, 107(1), 52 - 8
Repeated administration of a selective dopamine D2 receptor agonist to 6-OHDA-lesioned rats does not affect the survival and outgrowth of intrastriatal fetal mesencephalic grafts; Van Muiswinkel FL et al.; The objective of the present study was to investigate the effects of chronic activation of dopamine D2 receptors on the development of grafted fetal rat mesencephalic dopaminergic neurons . Therefore, unilaterally 6-hydroxydopamine - lesioned rats received intrastriatal mesencephalic cell suspension grafts and were subsequently chronically treated with the selective dopamine D2 receptor agonist LY 171555 (Quinpirole) . After treatment for 6 consecutive weeks, the rats were processed for tyrosine-hydroxylase immunocytochemistry to assess the survival and outgrowth from grafted dopaminergic neurons . morphological analysis revealed that, like the volume and morphology of the graft, neither the number nor the cell area of grafted dopaminergic neurons was significantly different between vehicle- and LY 171555-treated animals . To obtain a quantitative estimate of the graft-derived dopaminergic reinnervation, a computerized image analysis system was used . Using this procedure, which was based on the densitometric measurement of tyrosine hydroxylase immunoreactivity in the area adjacent to the grafted tissue, it was found that the extent of graft-derived outgrowth also appeared to be unaffected upon chronic treatment with LY 171555 . It is concluded that long-term concurrent administration of a dopamine D2 receptor agonist for 6 consecutive weeks does not impair the survival and outgrowth of grafted rat fetal mesencephalic dopaminergic neurons.

Epithelial Cell Biol, 1995, 4(2), 52 - 62
Epidermal growth factor receptor expression on human breast luminal and basal cells in vitro; Monaghan P et al.; The expression of EGF receptors has been studied on luminal and basal cells of human breast in vitro . Primary cultures of normal adult human breast epithelium were prepared as single cell suspensions containing a mixture of luminal and basal cells . The cells were simultaneously immunolabelled with antibodies recognising EMA (luminal epithelial cells), CALLA/CD10 (basal cells) and the epidermal growth factor receptor (EGFR) . Flow cytometric analysis of these triple labelled cells detected low levels of EGFR on both cell types, with proportionally more EGFR on basal cells compared with luminal cells . Separated populations of basal and luminal cells were prepared from single cell suspensions by flow sorting or by immunomagnetic methods and cultured with and without EGF . Increased proliferation was detected in both cell types in the presence of EGF . To determine the localisation of the EGF receptor, purified cell populations were immunolabelled with anti-EGFR antibody and an FITC-labelled second antibody for fluorescence light microscopy and colloidal gold-labelled antibody for scanning electron microscopy (SEM) . Low levels of EGFR were detected by indirect immunofluorescence on both cell types with higher levels on basal cells compared with luminal cells . The detailed subcellular distribution of the receptor was examined by SEM, with gold-labelling of EGFR detected using a field emission scanning electron microscope with a YAG crystal backscattered electron detector . Both luminal and basal cells expressed EGFR over the upper surface of individual cells when these were growing in isolation, but when cells formed part of a confluent island, levels of EGFR on the upper surface of cells were obviously reduced . Observations made by SEM on cells at the edges of such confluent islands showed that cultured basal cells expressed much higher levels of EGFR on their basal, as compared with their upper surfaces.

Acta Haematol Pol, 1995, 26(4), 367 - 75
Activated lymphocytes in the marrow cell suspension decrease the mafosfamide-induced CFU-GM cytotoxicity; Wozny T et al.; The aim of the study was to assess whether other cells, besides erythrocytes, may influence the cytotoxic effect of mafosfamide (maf) during ex vivo bone marrow purging from residual tumor cells before autologous transplantation . It was shown that the presence of normal granulocytes, blast cells from acute myeloid leukemia-patients (AML) and lymphoma cells from patients with chronic lymphocytic leukemia (CLL) during maf incubation did not change the maf-induced growth inhibition of CFU-GM . Similar observation was made in experiments with resting lymphocytes . However, when phytohaemagglutinin- and pokeweed mitogen-preincubated lymphocytes were present in the marrow cell suspension, significant decline of the maf-related CFU-GM cytotoxicity was observed . These results suggest that besides erythrocytes also the activated lymphocytes in the marrow mononuclear suspension may change the final effect of maf purging.

Ultrasound Med Biol, 1995, 21(6), 827 - 32
Roles of hematocrit and fibrinogen in red cell aggregation determined by ultrasonic scattering properties; Kitamura H et al.; Parameters of the power spectrum of backscattered echoes were applied to quantitatively evaluate red cell aggregation in vitro . Human red cell suspensions were circulated in a closed loop of tubing, and ultrasonic, radiofrequency, echo-signal data were obtained using a 10-MHz transducer . Data acquisition was performed at 30-s to 1-min intervals for 5 min after flow stoppage . Two parameters of the normalized power spectrum of the echo signals, spectral slope and Y-intercept, were computed, and estimates of two scattering properties, the scatterer size and acoustic concentration were calculated from these parameters using equations based on scattering theory . Size and acoustic concentration were observed as they changed over time after the stoppage of flow . The key findings were that hematocrit affected the rate of cell aggregation while fibrinogen controlled aggregate size and acoustic concentration.

Exp Clin Endocrinol Diabetes, 1995, 103(5), 308 - 16
Efficiency of various dissociation methods for the preparation of thyroid single cell suspensions; Frohlich E et al.; For comparison of the physiological potential of single thyroid cells versus cells integrated into follicles it would be ideal to work with suspensions consisting exclusively of single cells instead of a mixture of single cells and follicle fragments . In this study, various techniques for the isolation of single cells have been tested for their effect on cell viability, the ultrastructure of the isolated cells, the percentage of single cells and the ability of these cells to form follicles in culture . In addition, the cells were characterized for the preservation of their morphology and the ability to respond to TSH by comparing their immunocytochemical staining pattern with anti-vimentin and anti-ras p21 antibody to that of the intact thyroid tissue . Dispase treatment of thyroid tissues alone produced suspensions with a relatively small proportion of single cells . These cells stained with anti-vimentin and anti-ras p21 antibody to a similar percentage as thyroid cells in the intact gland . A combination of dispase treatment with either filtration or trypsin treatment severely compromised the viability of the cells . A high proportion of single cells with a good viability could be obtained either by centrifugation of dispase treated tissues or by culturing of dispase treated tissues as monolayers and subsequent detachment from the culture vessels with trypsin . Whereas the immunological staining with anti-vimentin and anti-ras oncogene antibody in the centrifuged cells resembled that of intact tissue, cells cultured as monolayers reacted differently . The differences in the immunological staining were still observed when the cells which had been grown as monolayers were stimulated with TSH . Differential centrifugation appeared to be the ideal method for the isolation of unaltered and viable single cells but is a rather laborious method to obtain larger amounts of single thyroid cells.

Brain Res Bull, 1995, 38(4), 383 - 91
Microglial chimaerism in human xenografts to the rat brain; Geny C et al.; Neural tissue from human fetuses is currently used for intracerebral transplantation to treat patients with Parkinson's disease . The development of the human fetal tissue following grafting has been considered mostly, up to now, from the neuronal point of view in xenografts . Very little is known, in contrast, about nonneuronal, glial, or vascular cells in the grafts . Comparison of the data gathered on the development of grafted human neurons with those obtained in comparable studies using rat transplants has demonstrated species-specific features . We have therefore undertaken a series of studies dealing with nonneuronal cells in human-to-rat transplants to reveal other possible species-specificity of the human tissue . This study has, accordingly, been devoted to the immunohistochemical analysis of microglia of host and donor origins in a human to rat xenograft paradigm allowing clear distinction of the origin of the cells . Human neural tissue was transplanted as a cell suspension into the thalamus of adult rats . Amoeboid human microglia were observed in 1-, 2-, and 3-month-old transplants, but their density, already relatively low at the first stage, decreased further over time . Ramified human microglia were only occasional . In sharp contrast, host rat microglia rapidly invaded the transplant in the absence of any sign of necrosis . The rat cells exhibited first an amoeboid morphology but progressed at the later stages toward a more mature, ramified morphology . These results indicate that donor microglia are quite few in number at first and, at least, do not proliferate actively after transplantation.(ABSTRACT TRUNCATED AT 250 WORDS)

J Immunol, 1995 Jan 1, 154(1), 281 - 9
Antitumor activities of subsets of human IL-2-activated natural killer cells in solid tissues; Vujanovic NL et al.; Human NK cells can be separated into two functionally distinct subpopulations based on the ability to rapidly respond to IL-2 by adherence to solid surfaces . To determine functions of the NK cell subsets in solid tumor tissues, adherent (A) and nonadherent (NA) NK cells were evaluated for their ability to infiltrate multicellular tumor spheroids in vitro, to kill carcinoma (CA) cell targets in these spheroids, and to mediate antitumor activity in vivo . A-NK cells were less cytolytic than NA-NK cells against CA targets in single cell suspensions or in monolayers . However, A-NK cells showed a significantly better ability than NA-NK cells to infiltrate tumor tissues and kill tumor cells in spheroids of human squamous cell CA of the head and neck or breast CA . Perilesional delivery of human A-NK cells and IL-2 resulted in regression of established human squamous cell carcinoma of the head and neck tumors growing subcutaneously in immunosuppressed nude mice . Similarly, in a xenograft model of human gastric CA metastatic to liver of nude mice, a single intrasplenic injection of A-NK cells in combination with i.p . infusions of IL-2 significantly reduced the number of established hepatic metastases (p < 0.007) and prolonged survival of the mice (p < 0.003) . In contrast, NA-NK cells were ineffective in either of the in vivo xenograft tumor models . These findings demonstrate that A-NK cells represent a biologically unique and important subset of NK cells that, in contrast to the rest of NK cells, function as effector cells in solid tumor tissues and, consequently, have a great antitumor therapeutic potential.

Laryngorhinootologie, 1995 Jan, 74(1), 36 - 8
{Surgical treatment of the obliterated tympanic cavity with autologous mucous membrane cell suspension}; Bohm K et al.; 5 patients suffering from high-grade sound conduction deafness due to extensive adhesion process on both ears underwent tympanoplasty on one of the ears . The missing tympanic mucosa was replaced by an autologous cell suspension prepared from mucosa of the maxillary sinus . 6-12 months after re-epithelialisation of the tympanic cavity, reconstruction of the ossicular chain was performed using glasionomer cement prostheses (IONOS) . 3 patients showed an improvement of hearing after transplantation of the isolated mucosal cells and following replacement of auditory ossicles . One patient had good improvement of hearing already after reconstruction of the tympanic cavity . In one case reobliteration of the tympanic cavity occurred . The clinical results confirm that using mucosal cell suspensions for reepithelisation of an obliterated tympanic cavity is yet another successful step forward in reconstructive surgery of the middle ear.

Acta Oncol, 1995, 34(1), 105 - 9
Dosimetry of irradiation models . The 96-well clonogenic assay for testing radiosensitivity of cell lines; Kulmala J et al.; Radiation experiments with cells in single cell suspension in test tubes and on 96-well plates were carried out and compared . The cells originated from cell lines established from carcinomas of the floor of the mouth and from endometrial carcinoma . Two irradiation models were constructed . Both models allowed the absorbed doses to the cells to be administered with a high accuracy in both experimental settings (better than 5.0%) . These irradiation models were compared on cancer cell lines with dissimilar inherent radiation sensitivity and histologic type (UM-SCC-1 resistant, UM-SCC-14A sensitive, and UT-EC-2B highly sensitive); various radiation doses were used . The fractions of surviving cells as a function of radiation dose were compared: there was no significant difference between cells irradiated in test tubes and cells irradiated in 96-well plates . Thus, if the absorbed doses in cells suspended in a tube and in a plate were the same, the survival was similar regardless of the type of irradiation model.

J Exp Zool, 1995 Jan 1, 271(1), 18 - 26
Characterization of plasma membrane Na+/H+ exchange in eel (Anguilla anguilla) intestinal epithelial cells; Vilella S et al.; The ability of eel intestinal epithelial cells to recover from an acute acid load was analysed using the fluorescent dye 2',7'-bis-carboxy-ethyl-5,6-carboxyfluorescein (BCECF) and cell suspensions . Under these experimental conditions (bicarbonate-free solutions) the resting pHi in cells prepared from sea-water (7.52 +/- 0.031) and fresh-water (7.50 +/- 0.094) adapted animals proved to be similar . The recovery rate (following an acid load) increases by increasing the Na ion concentration in the extracellular medium . This pHi recovery is competitively inhibited by the specific inhibitor dimethylamiloride (DMA) with a low Ki in sea- (1.2 microM) as well as in fresh-water (1.3 microM) adapted animals, indicating the presence of a specific Na/H exchange activity in these cells . Using basolateral membrane vesicles it could be demonstrated that this activity is located on the basolateral side of the enterocyte membrane . The kinetic parameters (Kapp and Jmax) of this exchanger are similar in fresh-water and sea-water adapted animals suggesting that no salinity adaptation occurs, thus excluding the involvement of the antiporter in the osmoregulatory processes . These results are in agreement with the presence in the plasma membrane of the eel enterocytes of a Na/H-1 (housekeeper) form of the antiporter.

Am J Physiol, 1995 Jan, 268(1 Pt 2), H260 - 4
Effect of erythrocyte deformability on myocardial hematocrit gradient; Baskurt OK et al.; Myocardial hematocrit gradient was determined between epicardium and endocardium of the left ventricular wall in rat heart under the influence of erythrocyte deformability alterations . Hematocrit determinations were performed by measuring two different radionuclides labeling plasma (125I-labeled albumin) and erythrocytes (99mTc) in 100-microns-thick left ventricular myocardium slices . Myocardial hematocrit gradient calculated after exchange transfusions with partially hardened red blood cell suspensions was compared with the results of the control group, in which the exchange transfusions were done using normal, hematocrit-matched blood . In the control group, the hematocrit value in the myocardium adjacent to epicardium was 0.331 +/- 0.076 l/l and decreased to 0.232 +/- 0.054 l/l near the endocardium . Myocardial hematocrit between these two was represented by a linear gradient . In the group with impaired erythrocyte deformability, the hematocrit value was 0.359 +/- 0.074 l/l in the epicardial myocardium and remained at 0.341 +/- 0.082 l/l in the endocardial layer . These results indicate that tissue hematocrit gradient in the left ventricular myocardium may be disturbed if erythrocyte deformability is altered.

Am J Physiol, 1995 Jan, 268(1 Pt 1), C210 - 7
Regulation of intracellular pH in J774 murine macrophage cells: H+ extrusion processes; McKinney LC et al.; Mechanisms of intracellular pH (pHi) regulation were characterized in the murine macrophage cell line J774.1, using 2',7'-bis(carboxyethyl)-5(6)-carboxyfluorescein to measure pHi . Under nominally HCO3(-)-free conditions, resting pHi of nonadherent J774.1 cells was 7.53 +/- 0.02 (n = 86), and of adherent cells was 7.59 +/- 0.02 (n = 97) . In the presence of HCO3-/CO2, pHi values were reduced to 7.41 +/- 0.02 (n = 12) and 7.40 +/- 0.01 (n = 28), respectively . Amiloride, an inhibitor of Na+/H+ exchange, did not affect resting pHi . Inhibitors of a vacuolar type H(+)-ATPase {bafilomycin A1, N-ethylmaleimide (NEM), 7-chloro-4-nitrobenz-2-oxa-1,3-diazide (NBD), and p-chloromercuriphenylsulfonic acid (pCMBS)} reduced pHi by at least 0.2 pH units . Inhibitors of other classes of H(+)-ATPases (oligomycin, azide, vanadate, and ouabain) were without effect . Inhibition of H+ efflux, measured by the change in extracellular pH of a weakly buffered cell suspension, followed the same pharmacological profile, indicating that the reduction of pHi was due to inhibition of H+ extrusion . Mechanisms of recovery from an imposed intracellular acid load were also investigated . In NaCl-Hanks' solution, pHi recovered exponentially to normal within 2 min . The initial rate of recovery was inhibited > 90% by amiloride or by replacement of extracellular Na+ concentration by N-methyl-glucamine . Inhibitors of the vacuolar H(+)-ATPase also inhibited recovery . NEM and NBD nonspecifically inhibited all recovery . Bafilomycin A1 and pCMBS did not inhibit the initial amiloride-sensitive portion of recovery, but they did inhibit a late component of recovery when pHi was above 7.0 . We conclude that the Na+/H+ exchanger is primarily responsible for recovery from an acid load but does not regulate resting pHi . Conversely, a vacuolar H(+)-ATPase regulates the resting pHi of J774 cells but contributes little to recovery from acidification.

Biochem J, 1995 Jan 1, 305 ( Pt 1), 263 - 8
Distribution of albumin, alpha 1-inhibitor 3 and their respective mRNAs in periportal and perivenous rat hepatocytes isolated by the digitonin-collagenase technique; Racine L et al.; The expression of albumin and alpha 1-inhibitor 3 genes was investigated in rat cell suspensions enriched in periportal (n = 10) and perivenous (n = 10) hepatocytes obtained by the digitonin-collagenase technique . The degree of enrichment of the cell suspensions was assessed: (1) by enzymic assays for the periportal marker alanine aminotransferase and for the perivenous marker glutamine synthetase; and (2) by their content of mRNAs for the periportal marker hepatic glutaminase and for glutamine synthetase . The existence of an antegrade intra-lobular gradient for albumin and alpha 1-inhibitor 3 mRNAs was demonstrated, with periportal:perivenous ratios of 2.33 and 3.80, respectively . However, no gradient was demonstrated for the respective protein contents with corresponding ratios of 0.98 and 1.21 . A certain degree of overlap existed between periportal and perivenous suspensions for their content in albumin and alpha 1-inhibitor 3 mRNAs . A morphometrical analysis of the surface of digitonin-permeabilized hepatic tissue revealed that this overlap could be explained by a variable extent of permeabilization of the mediolobular zone from one rat to another and from one lobule to another in a given animal . These results suggest that while the digitonin-collagenase technique is well suited for studies in vitro of proteins expressed in sharp intra-lobular gradients or restricted to an intra-lobular compartment, it is not completely reliable for proteins distributed in continuous moderate intra-lobular gradients, such as albumin and alpha 1-inhibitor 3.

Am J Clin Pathol, 1995 Jan, 103(1), 8 - 13
Comparison of two methods of mechanical disaggregation of scirrhous breast adenocarcinomas for DNA flow cytometric analysis of whole cells; Torres FX et al.; Mechanical release of single cell suspensions from solid tumors for DNA analysis by flow cytometry has been shown to be optimal for adenocarcinomas in general . In the breast, many adenocarcinomas are fibrotic or scirrhous, creating difficulty in separating the neoplastic cells from the stroma . The authors have conducted a parallel study in 20 consecutive cases of scirrhous adenocarcinomas of the breast using two mechanical methods of cell disaggregation by: (1) mincing tumor tissue in culture media (MINCE); and (2) scraping the surface of the solid tumor with a scalpel blade and rinsing the blade in culture media (SCRAPE) . Both methods were compared with PI staining and quantitation of percent debris and coefficient of variation (CV), as an index of resolution of the G0/G1 tumor peak . Debris was quantitated using Cell-FIT (Becton Dickinson Immunocytometry Systems, San Jose, CA) and Multicycle (Phoenix Flow Systems, San Diego, CA) software programs . With Multicycle, in addition to debris and aggregate determination, an index that included background, aggregates and debris (%BAD) occurring between the boundaries of the tumor G1 and G2 region was evaluated . In the 4 DNA diploid and 16 DNA aneuploid tumors, there was no significant difference in histogram quality measured by CV or amount of debris, aggregates, and %BAD between MINCE and SCRAPE methods . The authors conclude that either method of mechanical disaggregation will produce DNA histograms of comparable quality and degree of resolution . However, the scrape method may be advantageous in the mechanical disaggregation in scirrhous tumors . The scrape method also may be useful in small tumors, where tissue preservation for histology is paramount, and there is insufficient material to separately submit for flow cytometry . Combination of both MINCE and SCRAPE may provide higher cell yields, than using only one of these dissociation techniques . In addition, DNA analysis methods using intact cells obtained with the SCRAPE method result in percent CV values of similar resolution to those reported for methods producing bare nuclear suspensions from fresh tumors.

Exp Brain Res, 1995, 103(3), 355 - 71
A comparison of the behavioural effects of embryonic nigral grafts in the caudate nucleus and in the putamen of marmosets with unilateral 6-OHDA lesions; Annett LE et al.; The behaviour of marmosets with unilateral 6-hydroxydopamine lesions of the nigrostriatal bundle and grafts of embryonic mesencephalon in either the caudate nucleus or the putamen was compared with that of lesion-alone and unoperated controls . The grafts comprised injections of cell suspensions prepared from marmoset ventral mesencephalon (i.e . allografts) targeted at four sites either entirely within the caudate nucleus or entirely within the putamen . Behavioural tests, including measures of amphetamine-induced rotation, neglect and use of each arm to retrieve food from inside tubes, were given before and after the 6-hydroxydopamine lesion and at regular intervals for 6 months after transplantation surgery . Grafts in the caudate nucleus reduced the ipsilateral rotation induced by amphetamine, whereas grafts in the putamen did not . Despite the absence of an effect on rotation, the putamen grafts were effective in reducing lesion-induced deficits on the task in which the marmosets were required to reach into tubes . In this latter task, the caudate grafts were also effective when the monkeys were given a free choice of which hand to use . However, when constrained to use the hand contralateral to the lesion and graft, the performance of the marmosets with caudate grafts was not significantly improved compared with that of lesion-alone controls . Neither the grafts in the caudate nucleus nor the grafts in the putamen abolished the contralateral somatosensory neglect induced by the lesion, although there was a trend for the marmosets with putamen grafts to contact the label on the contralateral side more quickly than those with caudate grafts or the lesion-alone controls . These results demonstrate that the location of embryonic nigral grafts within the primate striatum influences the profile of functional recovery.

J Mol Cell Cardiol, 1995 Jan, 27(1), 371 - 81
Phospholipid hydroperoxides are precursors of lipid alkoxyl radicals produced from anoxia/reoxygenated endothelial cells; Kramer JH et al.; Endothelial cells have been shown to generate primary oxygen-centered free radicals (hydroxyl, superoxide anion) during post-anoxic reoxygenation, but little evidence is available concerning subsequent initiation of lipid peroxidative injury in this model . Electron spin resonance (ESR) spectroscopy with alpha-phenyl-N-tert-butylnitrone (PBN) spin trapping was used to monitor lipid peroxidation (LPO)-derived free radicals formed by cultured bovine aortic endothelial cell suspensions exposed (37 degrees C) to anoxia (A, 45 min, N2 gas) and reoxygenation (R, 15 min, 95% O2/5% CO2) . In some studies, superoxide dismutase (SOD, 10 micrograms/ml) was introduced just prior to R to assess the effects of this primary free radical scavenger on LPO-derived free radical production . At various times, aliquots were removed and PBN was introduced to either the cell suspension aliquot (8 mM PBN final, 1 min), or to the corresponding cell-free filtrate (60 mM PBN final), prior to extraction with toluene and ESR spectroscopy . A LPO-derived alkoxyl radical adduct of PBN (PBN/RO., hyperfine splitting alpha N = 13.63 G and alpha H = 1.94-1.98 G) was observed during R using both trapping procedures, with maximal production at 4-5 min and a second minor peak at 10 min . SOD effectively reduced PBN/RO . production and improved viability of A/R cells . In parallel studies, lipid hydroperoxide production was assessed in lipid extracts of A/R cells by high-performance liquid chromatography . Their separation profiles revealed a peak of oxidized lipid occurring between phosphatidylethanolamine (PE) and phosphatidylcholine (PC) in samples taken at 4-5 min and 10 min of R . Resolubilizing cell lipid extracts in oxygen-free benzene containing cobalt (II) acetylacetonate and PBN led to alkoxyl radical production, but only in the oxidized lipid samples, confirming the presence of hydroperoxides . These results suggest that A/R leads to primary free radical induced-lipid peroxidative injury to endothelial cells, as indicated by alkoxyl radical production originating from oxidized membrane phospholipids.

Virchows Arch, 1995, 426(2), 155 - 61
Determining factors which predict response to primary medical therapy in breast cancer using a single fine needle aspirate with immunocytochemical staining and flow cytometry; Fernando IN et al.; The increasing use of neoadjuvant chemotherapy and endocrine therapy in the management of breast cancer has lead us to evaluate and optimise the standard technique of cytocentrifugation of a single fine needle aspirate (FNA) taken from a breast tumour in-vivo, to determine a range of both immunocytochemical and flow cytometric factors which are predictive of response to primary medical therapy . Some of these factors are also of prognostic significance in early stage disease . An analysis of the cellularity and immunocytochemical staining characteristics of FNAs obtained from a series of 206 patients with palpable breast cancers indicate that in a sample of 46 cases it is possible to measure oestrogen receptor, progesterone receptor and c-erbB-2 providing over 400 cells per slide are obtained, with material obtained in a single FNA prepared by cytocentrifugation, using standard immunocytochemical methods . The staining results obtained were comparable to those obtained using frozen or paraffin embedded tissue sections taken from the same tumour . In addition an estimate of the proliferation indices could be made by flow cytometric analysis of the residual cell suspension fluid with measurement of DNA index and S-phase fraction in 131/164 (80%) and 110/164 (67%) of cases respectively . Providing all FNAs obtained for cytocentrifugation were taken at first presentation rather than immediately following a standard FNA, then it was possible to obtain adequately cellular (> 400 cells/slide) samples in 96 out of 126 (75%) of the last cohort of breast aspirates . These effects may be independent of T stage but not histological type as patients with lobular tumours only produced cellular aspirates in 1/7 (14%) of cases.(ABSTRACT TRUNCATED AT 250 WORDS)

Neurosci Res, 1995 Jan, 21(3), 223 - 33
Development of human fetal ventral mesencephalic grafts in rats with 6-OHDA lesions of the nigrostriatal pathway; Kondoh T et al.; Neuronal transplantation is an approach that can be exploited to study the development of the human central nervous system as well as being used in attempts to restore neurological function . In the present study, we have examined cellular events that appear to precede the development of dopamine nerve fiber extension by neurons from the human fetal ventral mesencephalon . These cellular events were examined using neuronal cell suspensions from human fetal ventral mesencephalic tissue (gestational ages 7-10 weeks) transplanted into the striatum of unilaterally lesioned 6-hydroxydopamine (6-OHDA) rats . Animals were sacrificed for immunohistochemistry 9-10 weeks after the transplantation prior to the manifestation of behavioral recovery . Histological analysis revealed tyrosine hydroxylase (TH) immunoreactive neurons in the grafts . The majority of these neurons had very short TH positive processes (60-70 microns), indicating that the maturation of grafted dopaminergic neurons was still incomplete . Immunostaining for the human specific intermediate neurofilament (hNF, clone: BF-10) showed dense neuronal fibers in the grafts . These fibers extended deeper into the host brain than the TH positive neuronal processes . The whole striatum, particularly the medial part of the striatum, exhibited long NF positive processes . Glial fibrillary acidic protein (GFAP) immunohistochemistry revealed fine astrocytic processes inside the grafts, which were clearly different from host reactive glial cells surrounding the grafts . These graft-derived glial processes tended to extend into the host brain deeper than the TH positive neuronal processes from the grafts . These early histological findings of the grafted human fetal ventral mesencephalon suggest that the graft-derived NF positive neuronal processes, as well as the glial processes, radiate from the grafted tissue and extend into the host brain prior to the extension of TH positive processes . These results further suggest that human-to-rat xenografts can be used to study the neural development of human fetal brain tissue.

Biomed Pharmacother, 1995, 49(1), 27 - 31
Naloxone behaves as opioid agonist/antagonist in clonal cultures of mouse bone marrow cells; Krizanac-Bengez L et al.; The opioid peptide methionine (Met)-enkephalin and the opioid-receptor blocking agent naloxone were added to unseparated or to progenitor-enriched cell suspensions of mouse bone marrow before assay in clonal cultures . Bone marrow samples harvested at 18:00 hours produced more granulocyte-macrophage (GM) colonies than the 06:00 hour samples, and were more sensitive to the proliferation inhibition by both agents . Additive inhibitory effects of naloxone with the enkephalin were occasionally seen . Thus, in this experimental system, naloxone could behave as an opioid agonist . However, there were examples of naloxone diminishing (blocking) the suppressive effect of the enkephalin, as a true opioid antagonist . Significant naloxone/enkephalin interactions occurred in opioid-sensitive (18:00 h) samples of unseparated bone marrow . The interactions were virtually absent in progenitor cell-enriched populations, indicating a significant role of accessory cells in opioidergic regulation of hematopoietic progenitors.

J Appl Toxicol, 1995 Jan-Feb, 15(1), 27 - 31
Red blood cell damage by wollastonite: in vitro study; Aslam M et al.; Asbestos is known to cause lung diseases in occupationally exposed workers . These properties have restricted its use . Industries have been exploring the possibility of other mineral fibres to replace the asbestos . In this direction, wollastonite has gained great attention owing to its high thermal resistance . In the present paper, the toxicity of three samples of Indian wollastonite was compared to that of chrysotile . Dust suspensions were added to the red blood cell suspensions to obtain a final dust concentration of 1.0-5.0 mg ml-1 in the system . The wollastonite varieties were found to have smaller haemolytic potential in human red blood cells than that of chrysotile in vitro . Chrysotile also was more effective in inducting peroxidative damage of polyunsaturated fatty acid (PUFA) than wollastonites in the present system . The peroxidative damage of PUFA and the haemolysis were both time and dose dependent . A higher value of malonaldehyde (a lipid peroxidation product) formation in low-speed supernatant of haemolysate was observed than in the intact cells . As the free-radical scavengers vitamin E and reduced glutathione prevent haemolysis and lipid peroxidation, these data are consistent with the involvement of lipid peroxidation in the haemolytic process.

Electrophoresis, 1995 Jan, 16(1), 92 - 7
Sodium chloride in separation medium enhances cell compatibility of free flow electrophoresis; Bondy B et al.; Free flow electrophoresis of cell suspensions in buffers containing sodium chloride was investigated using a modified procedure and the new apparatus Octopus PZE . The major methodical innovations are upward fluid flow, margin buffers flowing through the electrophoresis chamber at both sides of a central cell suspension buffer, adjacent to the electrode membranes, and a sample injection device which focuses the cells hydrodynamically to the middle of the chamber thickness . Mononuclear leukocytes, suspended in a buffer containing 35 mM NaCl, could be fractionated with the same accuracy as by conventional free flow electrophoresis, operated with a single NaCl-free chamber buffer . However, testing the vitality of separated cells with the help of the calcium indicator FURA2-AM clearly demonstrated the biological importance of the presence of a minimum amount of sodium chloride during cell electrophoresis . Only if at least 35 mM NaCl were present could an undisturbed cytosolic Ca2+ metabolism be maintained for the time of a free flow electrophoresis cell separation experiment.

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