Scand J Plast Reconstr Surg Hand Surg, 1998 Dec, 32(4), 359 - 64 Histodifferentiation of hair follicles in grafting of cell aggregates obtained by rotation culture of embryonic rat skin; Takeda A et al.; We have previously reported reconstruction of hair follicles from a single cell suspension of rat fetal upper lip by a two-step culture method consisting of rotation and flotation cultures . Rotation sorted out the cells and flotation facilitated histodifferentiation . In the present study, we added grafting procedures to the previous method to see whether cell aggregates obtained this way were graftable, and whether the grafting promoted histodifferentiation . The aggregates before and after flotation were grafted, and differentiation of hair follicles comparable to those in vivo was confirmed 10 days after grafting . There was no difference in the degree of differentiation between the two kinds of grafts . The grafting procedure therefore resulted in an appreciable increase in histodifferentiation even when aggregates obtained after flotation were grafted. Proc Natl Acad Sci U S A, 1998 Dec 22, 95(26), 15815 - 20 The effect of 4,4'-diisothiocyanato-stilbene-2,2'-disulfonate on CO2 permeability of the red blood cell membrane; Forster RE et al.; It has long been assumed that the red cell membrane is highly permeable to gases because the molecules of gases are small, uncharged, and soluble in lipids, such as those of a bilayer . The disappearance of 12C18O16O from a red cell suspension as the 18O exchanges between labeled CO2 + HCO3- and unlabeled HOH provides a measure of the carbonic anhydrase (CA) activity (acceleration, or A) inside the cell and of the membrane self-exchange permeability to HCO3- (Pm,HCO-3) . To test this technique, we added sufficient 4, 4'-diisothiocyanato-stilbene-2,2'-disulfonate (DIDS) to inhibit all the HCO3-/Cl- transport protein (Band III or capnophorin) in a red cell suspension . We found that DIDS reduced Pm,HCO-3 as expected, but also appeared to reduce intracellular A, although separate experiments showed it has no effect on CA activity in homogenous solution . A decrease in Pm,CO2 would explain this finding . With a more advanced computational model, which solves for CA activity and membrane permeabilities to both CO2 and HCO3-, we found that DIDS inhibited both Pm,HCO-3 and Pm,CO2, whereas intracellular CA activity remained unchanged . The mechanism by which DIDS reduces CO2 permeability may not be through an action on the lipid bilayer itself, but rather on a membrane transport protein, implying that this is a normal route for at least part of red cell CO2 exchange. Anticancer Res, 1998 Sep-Oct, 18(5A), 3481 - 6 Effects of vitamin C on arylamine N-acetyltransferase activity in human liver tumor cells; Wu LT et al.; Arylamine N-acetyltransferase (NAT) activity in a human liver tumor (heptoma) cell line was inhibited by vitamin C . Using high performance liquid chromatography, NAT activity on the acetylation of 2-aminofluorene and p-aminobenzoic acid was examined . Two assay systems were performed, one with cellular cytosols, the other with intact liver tumor cell suspensions . The NAT activity in a human liver tumor cell line was inhibited by vitamin C in a dose-dependent manner in both types of examined system: i.e . the greater the concentration of vitamin C in the reaction, the greater the inhibition of NAT activities in both systems examined . The data also indicated that vitamin C decreased the apparent Km and Vmax of NAT enzymes from human liver tumor cells in both systems examined . This report is the first demonstration which showed vitamin C effect on human liver tumor cell NAT activity. J Exp Med, 1998 Dec 21, 188(12), 2335 - 42 Type I interferon-mediated stimulation of T cells by CpG DNA; Sun S et al.; Immunostimulatory DNA and oligodeoxynucleotides containing unmethylated CpG motifs (CpG DNA) are strongly stimulatory for B cells and antigen-presenting cells (APCs) . We report here that, as manifested by CD69 and B7-2 upregulation, CpG DNA also induces partial activation of T cells, including naive-phenotype T cells, both in vivo and in vitro . Under in vitro conditions, CpG DNA caused activation of T cells in spleen cell suspensions but failed to stimulate highly purified T cells unless these cells were supplemented with APCs . Three lines of evidence suggested that APC-dependent stimulation of T cells by CpG DNA was mediated by type I interferons (IFN-I) . First, T cell activation by CpG DNA was undetectable in IFN-IR-/- mice . Second, in contrast to normal T cells, the failure of purified IFN-IR-/- T cells to respond to CpG DNA could not be overcome by adding normal IFN-IR+ APCs . Third, IFN-I (but not IFN-gamma) caused the same pattern of partial T cell activation as CpG DNA . Significantly, T cell activation by IFN-I was APC independent . Thus, CpG DNA appeared to stimulate T cells by inducing APCs to synthesize IFN-I, which then acted directly on T cells via IFN-IR . Functional studies suggested that activation of T cells by IFN-I was inhibitory . Thus, exposing normal (but not IFN-IR-/-) T cells to CpG DNA in vivo led to reduced T proliferative responses after TCR ligation in vitro. Microsc Res Tech, 1998 Dec 1, 43(5), 456 - 63 Hepatocyte growth factor and Madin-Darby canine kidney cells: in vitro models of epithelial cell movement and morphogenesis; Balkovetz DF; It is becoming increasingly apparent that epithelial cell movement and changes in morphology are central to both development and regeneration of epithelial organs and are involved with pathological processes such as transformation of epithelia to carcinoma and metastasis . Hepatocyte growth factor (HGF) is a mesenchymally derived growth factor with pleiotrophic effects on epithelia depending on culture conditions . In vivo, HGF plays a role in mesenchymal-epithelial interactions . Madin-Darby canine kidney (MDCK) cells, which share many properties with polarized epithelia in vivo, are remarkably sensitive to HGF . In vitro models of HGF-treated MDCK cells have proven to be useful for the study of epithelial cell movement and changes in morphology . When cultured on plastic at low density, MDCK cells scatter in response to HGF . MDCK cells grown as cell suspensions in collagen gels form complex branching tubular structures in response to HGF . When cultivated as a monolayer on permeant supports, MDCK cells are well polarized with established E-cadherin mediated cell-cell junctions and dedifferentiate in response to HGF . Some of the mechanisms responsible for changes in cell movement and morphology that have been characterized using these models are summarized in this review . Models of MDCK cells exposed to HGF will continue to be useful in the study of epithelial cell movement and morphogenesis in vitro and will provide important clues into the cellular mechanisms important during in vivo epithelial processes such as organ development, regeneration, and transformation to carcinoma. Cell Motil Cytoskeleton, 1998, 41(4), 353 - 62 Isolation and characterization of novel Chlamydomonas mutants that display phototaxis but not photophobic response; Matsuda A et al.; The unicellular green alga Chlamydomonas displays two distinct kinds of behavioral response to light: phototaxis, in which cells swim toward or away from the light source under constant illumination; and photophobic responses (also called stop responses or photoshock responses), in which cells transiently convert their flagellar waveform and swim backward upon sudden increase in light intensity . It has been suggested that the two responses partly share a common signal transduction pathway, but exactly how the different responses are produced has not been established . In this study, to help understand the molecular and cellular mechanisms that bring about the photophobic response, we isolated novel mutants (ppr1, ppr2, ppr3, and ppr4) that do not show the photophobic response . Importantly, these mutants retain the ability to display phototaxis, with almost the same sensitivities as in the wild type cell . Demembranated and reactivated flagellar axonemes of the ppr mutants were found to convert the bending patterns depending on the Ca2+ concentration, indicating that the axonemal mechanism for waveform conversion required for the photophobic response was unaffected by the mutations . In addition, measurements of electric currents in cell suspensions showed that these mutants generate normal photoreceptor currents (PRC) upon photostimulation, suggesting that they retain the normal activity of photoreception and the ionic channels that produce PRCs . However, the all-or-none flagellar current (FC), a Ca2+ current generated by PRC-induced depolarization of flagellar membrane, was absent or seriously impaired in the mutants . These findings clearly indicate that the all-or-none FC is necessary for the photophobic response but not for phototaxis . The isolation of the four genetically independent ppr mutants suggests that the generation of the FC is based on multiple components that are not used in the mechanism for phototaxis, and implies that the Chlamydomonas flagellar membrane possesses a voltage-dependent Ca2+-channel specifically used for generation of photophobic responses. J Physiol Biochem, 1998 Jun, 54(2), 77 - 84 Inhibition by estrogens of the oxidant-mediated mobilization of arachidonic acid in hepatocytes; Babenko NA et al.; Oxidative stress is associated with alterations in arachidonic acid (AA) metabolism . The present work was performed to assess the effect of the oxidant tert-butyl hydroperoxide on the release of AA from rat hepatocytes, and the possible preventive actions of estrogens on this effect . The exposure of {14C}-prelabeled cells to tertbutyl hydroperoxide produced the mobilization of {14C}-AA from hepatocyte lipids, both an intracellular {14C}-AA accumulation and an increased release of {14C}-products into the medium being observed . The formation of lysophospholipids was also enhanced significantly in the presence of the oxidant, thus suggesting the involvement of phospholipase A2 (E.C . 3.1.1.4) in the hepatocyte response to tert-butyl hydroperoxide . Estradiol and 2-hydroxyestradiol (25-100 microM) added in vitro to cell suspensions prevented significantly the oxidant- mediated stimulation of AA release, this effect probably being caused by the estrogen inhibitory actions against cellular lipid peroxidation. Scand J Rheumatol, 1998, 27(6), 446 - 53 In vitro synthesis and characterization of a cartilaginous meniscus grown from isolated temporomandibular chondroprogenitor cells; Girdler NM; Internal derangement of the temporomandibularjoint can lead to perforation of the intra-articular meniscus and osteoarthritic degeneration . Current methods of repairing damaged menisci are limited by lack of biological compatibility of graft materials . This project aimed to synthesise and characterise a primate cartilaginous meniscus in vitro from harvested mandibular chondroprogenitor cells . Isolated cells from the mandibular cartilage of 12 young adult marmosets, aged 9-12 months, were grown in monolayer culture . After 21 days confluent colonies were resuspended and dispersed into a unpolymerised solution of type I collagen and fibrinogen . The resultant cell suspension was infiltrated into a resorbable type I collagen sponge carrier and allowed to polymerise . Aliquots of the cell-infiltrated sponge were maintained in organ culture for a further 14 days . Cultures were characterised using histochemical and immunocytochemical localisation of collagen and proteoglycan species . Two-thirds of cells in confluent 21-day monolayers expressed cartilage-specific type II collagen and chondroitin-4-sulphate . After 35 days organ cultures had formed a viable, organised, three-dimensional tissue mass consisting of mature chondrocytic cells interspersed in a dense cartilaginous matrix . The cartilaginous tissue generated in vitro may have potential application in the repair or replacement of damaged menisci in vivo. Int J Mol Med, 1998 Jun, 1(6), 995 - 9 Detection of cytokine gene expression in human monocytes and lymphocytes by fluorescent in situ hybridization in cell suspension and flow cytometry; Wieckiewicz J et al.; The use of digoxigenin (DIG)- and biotin-labelled dsDNA probes to detect TNFalpha-mRNA accumulation in human peripheral blood mononuclear cells (PBMC) and isolated monocytes is described . The fragment of the glyceraldehyde-3-phosphate dehydrogenase GAPDH-cDNA was used as a control probe . The hybridization signals were detected by staining with fluorescein isothiocyanate (FITC)-labelled anti-DIG antibody and avidin-FITC, respectively . The cells were stimulated in vitro with lipopolysaccharide (LPS) for 0.5-6 h . The TNFalpha-mRNA was detected in monocytes 1 h after stimulation with LPS, and the highest accumulation was seen around 2 h . The TNFalpha-mRNA in stimulated PBMC was detected at the lower level peaking around 4 h . The TNFalpha-mRNA accumulation was lower in lymphocytes than in monocytes when PBMC were studied . There was no difference in the level of GAPDH-mRNA between unstimulated and stimulated cells . Finally, an enhanced accumulation of TNFalpha-mRNA was observed in PBMC from some patients with sepsis or cancer . Thus, this study shows that cytokine gene expression may be detected in cells ex vivo . This opens the possibility of studying the level of cytokine gene activation in PBMC of patients with diseases where the role of cytokines in their pathophysiology is implicated. Cell Transplant, 1998 Nov-Dec, 7(6), 573 - 83 The development of intracerebral cell-suspension implants is influenced by the grafting medium; Watts C et al.; The effect of preparing and grafting embryonic striatal and nigral tissue in four different media was evaluated in vitro and in vivo . The proportion of TH-positive and DARPP-32-positive neurons was determined after 2 days in vitro in standard culture medium following preparation in the different media . The effects were more marked for striatal neurons where DARPP-32 expression in tissue prepared in HBSS was poor compared to other media . TH expression was unaffected by the preparation medium . Striatal grafts derived from tissue prepared and grafted in HBSS were smaller, with fewer DARPP-32 cells, compared to other media . Survival of grafts in combined HBSS and DMEM was very poor . Graft volume and TH cell content was enhanced in tissue prepared in DMEM . These results suggest that preparation protocols optimized for one type of embryonic neuronal population do not necessarily transfer to other neuronal populations. Xenobiotica, 1998 Oct, 28(10), 937 - 48 Metabolic activity of fresh and cryopreserved dog hepatocyte suspensions; Swales NJ et al.; 1 . Dog hepatocytes were cryopreserved at 6 x 10(6) viable cells/ml in a suspension buffer containing 10% DMSO and were stored in liquid nitrogen . 2 . The exclusion of trypan blue dye was 96 +/- 2 and 85 +/- 9% in fresh and cryopreserved (CP) hepatocytes, respectively . Albumin synthesis was unaffected by freezing . 3 . Ethoxycoumarin and ethoxyresorufin O-deethylase activities were equivalent in fresh and CP hepatocytes . 4 . The profile of testosterone metabolism was unaffected by freezing . Total hydroxylase activities were 815 +/- 33 pmol/min/10(6) cells in freshly isolated whole hepatocytes and 463 +/- 24 pmol/min/10(6) CP whole hepatocytes, but they were equivalent in fresh and CP hepatocyte homogenates supplemented with 250 microM NADPH . 5 . Phase 2 enzymes were functional in freshly thawed CP hepatocytes but they required exogenous addition of cofactors (20 microM UDPGA and 1.7 microM PAPS) . 6 . When placed in suspension for longer times, fresh and CP cell viabilities were 88 +/- 6 and 64 +/- 2% after 4 h . ECOD and EROD activities were equivalent in fresh and CP hepatocyte suspensions, over 4 h . Testosterone hydroxylase activities were well maintained in fresh cell suspensions but they declined to 63 +/- 6% of the initial activity after 4 h in CP hepatocytes . 7 . These results indicate that CP dog hepatocytes are a suitable in vitro system for xenobiotic metabolism since enzyme functions in CP hepatocytes were stabilized . Cofactors in freshly thawed CP hepatocytes should be measured and controlled for optimal use. Plant Physiol, 1998 Dec, 118(4), 1317 - 26 Comparison of binding properties and early biological effects of elicitins in tobacco cells Bourque S, Ponchet M, Binet MN, Ricci P, Pugin A, Lebrun-Garcia A. Elicitins are a family of small proteins secreted by Phytophthora species that have a high degree of homology and elicit defense reactions in tobacco (Nicotiana tabacum) . They display acidic or basic characteristics, the acidic elicitins being less efficient in inducing plant necrosis . In this study we compared the binding properties of four elicitins (two basic and two acidic) and early-induced signal transduction events (Ca2+ influx, extracellular medium alkalinization, and active oxygen species production) . The affinity for tobacco plasma membrane-binding sites and the number of binding sites were similar for all four elicitins . Furthermore, elicitins compete with one another for binding sites, suggesting that they interact with the same receptor . The four elicitins induced Ca2+ influx, extracellular medium alkalinization, and the production of active oxygen species in tobacco cell suspensions, but the intensity and kinetics of these effects were different from one elicitin to another . As a general observation the concentrations that induce similar levels of biological activities were lower for basic elicitins (with the exception of cinnamomin-induced Ca2+ uptake) . The qualitative similarity of early events induced by elicitins indicates a common transduction scheme, whereas fine signal transduction tuning is different in each elicitin. Plant Physiol, 1998 Dec, 118(4), 1389 - 93 Biosynthesis of camalexin from tryptophan pathway intermediates in cell-suspension cultures of Arabidopsis; Zook M; Camalexin (3-thiazol-2'-yl-indole) is the principal phytoalexin that accumulates in Arabidopsis after infection by fungi or bacteria . Camalexin accumulation was detectable in Arabidopsis cell-suspension cultures 3 to 5 h after inoculation with Cochliobolus carbonum (Race 1), and then increased rapidly from 7 to 24 h after inoculation . Levels of radioactivity incorporated into camalexin during a 1.5-h pulse labeling with {14C}anthranilate also increased with time after fungal inoculation . The levels of radioactive incorporation into camalexin increased rapidly between 7 and 18 h after inoculation, and then decreased along with camalexin accumulation . Relatively low levels of radioactivity from {14C}anthranilate incorporated into camalexin in the noninoculated controls . Autoradiographic analysis of the accumulation of chloroform-extractable metabolites labeled with {14C}anthranilate revealed a transient increase in the incorporation of radioactivity into indole in fungus-inoculated Arabidopsis cell cultures . The time-course measurement of radioactive incorporation into camalexin during a 1.5-h pulse labeling with {14C}indole was similar to that with {14C}anthranilate . These data suggest that indole destined for camalexin synthesis is produced by a separate enzymatic reaction that does not involve tryptophan synthase. Anat Rec, 1998 Dec, 252(4), 637 - 45 Changes in structure and functions of prostate by long-term administration of an androgen, testosterone enanthate, in rhesus monkey (Macaca mulatta); Udayakumar TS et al.; The increasing use of androgens in clinical trials for developing a safe, effective, and reversible male contraceptive has necessitated a critical evaluation of the effects of their long-term use on the structure and functions of the prostate gland, which is androgen dependent . Combination regimens using progestogens, gonadotropin-releasing hormone antagonists, or antiandrogens along with androgens are undergoing clinical evaluation as antispermatogenic agents . The majority of these regimens have used testosterone enanthate (TE) as the androgen of choice, but very limited information is available on the side effects of long-term androgen use . The present study is the first report that critically evaluates the effects of long-term use of TE on prostate structure and functions . Adult male rhesus monkeys received intramuscular injections of 50 mg of TE once in 14 days for 33 months . The cranial and caudal lobes of the prostate, which were removed under ketamine anesthesia, were processed for the preparation of semithin sections to evaluate histological changes . The DNA distribution in the cells was studied in single cell suspensions of cranial and caudal lobes of the prostate by using flow cytometry . Changes in the levels of testosterone, estradiol, prostate-specific acid phosphatase (PAP), and prostate-specific antigen (PSA) in samples collected during the pretreatment period and at the time of removal of the prostate were estimated by using conventional procedures . Control samples were processed simultaneously . The administration of TE for 33 months caused the following changes: 1) significant increase in the weight of both lobes of the prostate, 2) cellular hypertrophy and increase in secretory material in the cells and in the lumen of the acini in the central and peripheral zones of the two lobes of the prostate, 3) cellular hyperplasia indicated by flow cytometric analysis of DNA content, 4) significant increase in the secretion of PAP and levels of estradiol, and 5) a marked increase in fibromuscular stroma in the central and peripheral zones of both the lobes of the prostate . The present study is the first report to provide evidence that long-term androgen treatment has caused hypertrophy of the prostatic epithelial cells, which showed increased secretory activity . The hyperplastic changes indicate a need for the development of new androgens with a better pharmacokinetic profile for use in male contraceptive regimens. Int J Radiat Oncol Biol Phys, 1998 Nov 1, 42(4), 871 - 6 MIBG inhibits respiration: potential for radio- and hyperthermic sensitization; Biaglow JE et al.; INTRODUCTION: Meta-iodobenzylguanidine (MIBG) in its 131I-labeled form is clinically used as a tumor-targeted radiopharmaceutical in the diagnosis and treatment of adrenergic tumors . This well established drug may have additional clinical applications as a radiosensitizer or hyperthermic agent, ie., MIBG reportedly inhibits mitochondrial respiration in vitro . The mechanism for MIBG inhibition of cellular oxygen consumption is uncertain . Moreover, MIBG reportedly stimulates glycolysis both in vitro and in vivo . Our studies show the effect of MIBG on 9L glioma oxygen consumption and redox status with tumors cells in vitro and in vivo . MATERIALS AND METHODS: The effects on electron transfer were determined by following oxygen consumption with a Clark oxygen electrode . Fluorescence measurements were used to determine effects of MIBG on intracellular electron acceptors, NADPH and flavoproteins, in vitro and in vivo . 31P-NMR was used to determine alterations in tumor cell pH in vivo . RESULTS: Our results show the inhibition of oxygen utilization with MIBG for cell suspensions in vitro . The same results were demonstrated for tumor cell suspensions rapidly isolated from tumors grown in rats . Moreover, NAD(P)H and flavoprotein (Fp) fluorescence changes were observed to rapidly occur following MIBG addition in vitro . Changes in intracellular pH measured with 31P-NMR, in vivo, precede the changes in fluorescence of NAD(P)H and Fp obtained with frozen sections of tumor . CONCLUSIONS: We conclude that 31P-NMR measurements and fluorescence changes, following MIBG injection, can be used as criterion for selecting the proper time to treat tumors with ionizing radiation or hyperthermia. Int J Radiat Oncol Biol Phys, 1998 Nov 1, 42(4), 795 - 8 Redox generation of nitric oxide to radiosensitize hypoxic cells; Mitchell JB et al.; PURPOSE: Previous studies have shown that nitric oxide (NO) delivered from NO donor agents sensitizes hypoxic cells to ionizing radiation . In the present study, nitroxyl (NO-), a potential precursor to endogenous NO production, was evaluated for hypoxic cell radiosensitization, either alone or in combination with electron acceptor agents . METHODS AND MATERIALS: Radiation survival curves of Chinese hamster V79 lung fibroblasts under aerobic and hypoxic conditions were assessed by clonogenic assay . Hypoxia induction was achieved by metabolism-mediated oxygen depletion in dense cell suspensions . Cells were treated with NO- produced from the nitroxyl donor Angeli's salt (AS, Na2N2O3, sodium trioxodinitrate), in the absence or presence of electron acceptor agents, ferricyanide, or tempol . NO concentrations resulting from the combination of AS and ferricyanide or tempol were measured under hypoxic conditions using an NO-sensitive electrode . RESULTS: Treatment of V79 cells under hypoxic conditions with AS alone did not result in radiosensitization; however, the combination of AS with ferricyanide or tempol resulted in significant hypoxic radiosensitization with SERs of 2.5 and 2.1, respectively . Neither AS alone nor AS in combination with ferricyanide or tempol influenced aerobic radiosensitivity . The presence of NO generated under hypoxic conditions from the combination of AS with ferricyanide or tempol was confirmed using an NO-sensitive electrode . CONCLUSION: Combining NO- generated from AS with electron acceptors results in NO generation and substantial hypoxic cell radiosensitization . NO- derived from donor agents or endogenously produced in tumors, combined with electron acceptors, may provide an important strategy for radiosensitizing hypoxic cells and warrants in vivo evaluation. Int J Radiat Oncol Biol Phys, 1998 Nov 1, 42(4), 769 - 73 The measurement of bioreductive capacity of tumor cells using methylene blue; Biaglow JE et al.; PURPOSE: Methylene blue (MB) can be used as an intracellular electron acceptor . The purpose of this study was to demonstrate the usefulness of MB for the determination of total bioreductive capacity of cell suspensions . METHODS AND MATERIALS: We measured oxygen consumption by Clark electrode and pentose cycle activity by release of 14CO2 from 1-14C-glucose . RESULTS: Methylene blue catalyzes the reaction of intracellular reductants NADPH, NADH, and reduced glutathione (GSH) with oxygen, causing the production of hydrogen peroxide . The reaction rate correlates with the negative charge of molecule (NADPH(-4) > NADH(-2) > GSH(-1)), suggesting that reaction with positively charged oxidized MB is the limiting step of the reaction . In a cellular system MB causes the electron flow from cellular endogenous substrates to oxygen . It is activated by the disruption of the NADP+/NADPH ratio due to several processes . These are direct oxidation of NADPH and GSH, the GSH peroxidase catalyzed reaction of GSH with H2O2, followed by NADPH oxidation by oxidized glutathione (GSSG) . This results in increased cellular oxygen consumption and stimulation of the oxidative limb of pentose cycle (PC) in the presence of MB . The cellular effect of MB differs from other electron accepting drugs . Diamide and tert-butylhydroperoxide act as direct oxidants, while MB is an electron carrier to oxygen . Accordingly, MB shows the highest effect on PC activation and oxygen consumption . CONCLUSIONS: Our results indicate that MB may be used for the determination of the total bioreductive capacity of the cells, measured by oxygen consumption and PC activation. Artif Cells Blood Substit Immobil Biotechnol, 1998 Nov, 26(5-6), 449 - 54 Enhanced mitosis in cultured protoplasts: beneficial effects of oxygenated perfluorocarbon and haemoglobin; Anthony P et al.; Cell suspension-derived protoplasts of Petunia hybrida cv . Comanche were cultured for up to 10 d in (1) aqueous medium, (2) aqueous medium overlaying an oxygenated (10 mbar, 15 min) perfluorochemical liquid (perfluorodecalin; PFC), (3) aqueous medium containing 1:50 (v:v) of the commercial haemoglobin (Hb) solution (Erythrogen), (4) aqueous medium supplemented with 1:50 (v:v) of Erythrogen overlaying oxygenated PFC . Treatments 2, 3, and 4 enhanced mean mitotic division by 111%, 80% and 139% respectively, compared to controls . Both fresh-Hb and stored-Hb significantly (P < 0.05) enhanced mitotic division, although this was 45% greater with fresh-Hb compared to stored-Hb . Both oxygenated PFC and Erythrogen provide approaches for enhancing cellular oxygen supply to eukaryotic cells . Commercially, the recoverability and recycleability of PFCs make these compounds a more attractive option in comparison to Erythrogen, despite a high initial investment cost. Schweiz Rundsch Med Prax, 1998 Oct 21, 87(43), 1434 - 40 {Comparison of conventional PAP smears with thin layer specimen (liquid-based PAP test) and correlation with cytopathological findings with HPV status using the hybrid capture system}; Kunz J et al.; Cervical smears of 554 outpatients of a hospital were examined using a blinded, split sample match pair protocol for which a conventional PAP-smear (CS) was first prepared with Cervex brush and the reminder of the sample was used for the thin-layer-preparation (TLP) according to the manual CytoRich System . The preparations of the two methods were compared with respect to quality and to sensitivity for atypias . In addition the HPV status was determined on the same cell suspension in cases with borderline changes (BLC) and dysplasias including carcinoma using the Hybrid Capture System . The use of TLP reduced the proportion of suboptimal preparations by more than 50% (14.6% vs . 35%) and eliminated the only inadequate preparation registered in CS . The DSP detected more than twice as many dysplasias of all degrees as CS (3.4% vs . 1.4%) and reduced the proportion of BLC to one third (3.2% vs . 9.6%) . The percentages of cases positive for high- and intermediate-risk HPV in preparations with BLC, LSIL and HSIL were 17, 62.5% and 100% respectively . The TL-method improves significantly the efficiency of PAP-smears and allows the typing of HPV which is of clinical importance for the management of low grade squamous intraepitelial lesions and borderline changes . The findings speak against the further use of CS for cervical screening. J Neurosci Res, 1998 Dec 1, 54(5), 639 - 54 Galectin-3 promotes neural cell adhesion and neurite growth; Pesheva P et al.; Galectin-3 is a member of the galectin family and belongs to a group of soluble beta-galactoside-binding animal lectins . The molecule is expressed by neural and nonneural cells intra- (cytoplasm and nucleus) as well as extra-cellularly (plasma membrane and extracellular space) . By using an in vitro cell-substratum adhesion assay, we have addressed the question whether galectin-3 present in the extracellular milieu may support the adhesion and/or neurite outgrowth of neural cells in a manner analogous to cell adhesion molecules . Galectin-3 was immobilized as a substratum and various cell types, N2A (neuroblastoma), PC12 (pheochromocytoma), and TSC (transformed Schwann cells) cell lines, neural cells from early postnatal mouse cerebellum, and dorsal root ganglion neurons from newborn mice were allowed to adhere to the lectin . Here we show that all cell types studied specifically adhered to galectin-3 by the following criteria: 1) the number of adherent cells was dependent on the galectin-3 concentration used for coating; 2) adhesion of cells to galectin-3, but not to collagen type I or laminin was inhibited by polyclonal antibodies to galectin-3; 3) upon addition of asialofetuin (a polyvalent carrier of terminal beta-galactosides) to the cell suspension prior to the adhesion assay, cell adhesion to galectin-3 was inhibited in a dose-dependent manner; and 4) cell adhesion to galectin-3 was abolished by treatment of cells with endo-beta-galactosidase . In addition, the adhesion of dorsal root ganglion neurons to galectin-3 could be inhibited by lactose . Notably, substratum-bound galectin-3 promoted the outgrowth of neurites from dorsal root ganglia explants and this neurite outgrowth promoting activity could be inhibited by polyclonal antibodies to galectin-3. Phytochemistry, 1998 Nov, 49(5), 1219 - 25 Phytohormone regulation of isoperoxidases in Catharanthus roseus suspension cultures; Limam F et al.; Peroxidase (POD) activity was investigated in Catharanthus roseus cell suspensions cultured under different hormonal conditions . Depletion of 2,4-dichlorophenoxyacetic acid (2,4-D) from the culture medium enhanced POD activity in cells and spent medium . Addition of phytohormones, in particular the auxin 2,4-D, reduced POD activity in medium and cellular compartments and enhanced ionically cell-wall bound POD . The differential modulation of POD is due to hormone effects on synthesis and/or accumulation of POD, rather than on the secretion process . Qualitative analysis showed that 2,4-D, but not cytokinins, regulated the synthesis of a basic isoform . The cytokinin treatment seemed to affect acidic rather than basic isoforms . The presence of basic POD is correlated with the capacity of cells to produce indole alkaloids . The major extracellular basic isoperoxidase was purified to homogeneity from culture medium of Catharanthus roseus cell suspensions . The isolated peroxidase is a haem protein with a M(r) of 33,000 and a pI close to 9 . The effect of pH on peroxidase activity was studied using guaiacol as substrate and the optimum pH determined at 25 degrees was 6.0 . This enzyme acted on guaiacol, 2,2'-azino-bis-(3-ethylbenzthiazoline-6-sulfonic acid) (ABTS), o-dianisidine, o-phenylenediamine (o-PD) and pyrogallol, but had no effect on syringaldazine or coniferyl alcohol substrates. Mediators Inflamm, 1998, 7(1), 35 - 40 Role of the epithelial layer in the generation of superoxide anion by the guinea-pig isolated trachea; Sadeghi-Hashjin G et al.; The lucigenin-dependent chemiluminescence generation by guinea-pig isolated tracheal two rings preparations was studied . Tracheal preparations stimulated with phorbol myristate acetate (PMA) or opsonized zymosan generated chemiluminescence . The total amount of chemiluminescence generated in 120 min was 754+/-63 mV x min for PMA and 4832+/-396 mV x min for zymosan . Generation of chemiluminescence was decreased by more than 50% when the tissues were co-incubated with superoxide dismutase (100 U/ml) . Also, addition of direct donors of nitric oxide diminished chemiluminescence generation by zymosan-activated tracheal rings significantly by about 50% . However, the presence of the precursor or of inhibitors of nitric oxide synthase did not influence zymosan-induced chemiluminescence . Removal of the epithelial layer from tracheal rings caused an approximately 90% decrease in chemiluminescence response . However, isolated epithelial cell suspensions did not generate chemiluminescence . Histologic examination showed that the number of eosinophils in the tracheal tissue was reduced from 56+/-7 to 18+/-8 per mm basal membrane when the epithelial layer was removed . These results indicated that (1) superoxide anion formation can take place in the guinea-pig trachea, (2) eosinophils in the epithelial and submucosal layers of guinea-pig trachea are likely candidates for superoxide generation although other cell types can also be involved, and (3) besides relaxing airway smooth muscle, nitric oxide donors may also affect superoxide in the airways. Biochim Biophys Acta, 1998 Nov 27, 1425(3), 485 - 92 Identification of 7-dehydrocholesterol, vitamin D3, 25(OH)-vitamin D3 and 1,25(OH)2-vitamin D3 in Solanum glaucophyllum cultures grown in absence of light; Curino A et al.; Solanum glaucophyllum contains the calciotropic hormone 1, 25-dihydroxy-vitamin D3 (1,25(OH)2D3) . The metabolic pathway leading to the formation of 1,25(OH)2D3 in the plant is largely unknown . Specifically, there is controversy about the participation of a photolytic reaction in the generation of vitamin D3 and its metabolites . To investigate the requirement for light, S . glaucophyllum tissue (callus) and cell suspension cultures grown under strict conditions of darkness were extracted with chloroform/methanol (1:2, v/v) followed by purification of the lipidic fraction by Sephadex LH-20 and high-performance liquid chromatography . HPLC peaks with elution times similar to those of authentic samples of 7-dehydrocholesterol, vitamin D3, 25(OH)D3 and 1,25(OH)2D3 were detected . The presence of 1,25(OH)2D3 was also evidenced by {3H}1,25(OH)2D3 competitive binding analysis using the chick hormone intestinal receptor . Furthermore, 7-dehydrocholesterol, vitamin D3, 25(OH)D3 and 1,25(OH)2D3 were unequivocally identified by mass spectrometry . Incubation of control samples of 7-dehydrocholesterol under the same conditions as S . glaucophyllum cultures did not result in vitamin D3 formation, excluding the influence of light in these experiments . The results suggest that a synthetic route of vitamin D3 compounds independent of light operates in Solanum glaucophyllum cultured in vitro. Plant Cell, 1998 Dec, 10(12), 2077 - 86 Involvement of plasma membrane redox activity and calcium homeostasis in the UV-B and UV-A/blue light induction of gene expression in Arabidopsis; Long JC et al.; UV and blue light are important regulators of plant gene expression and development . We investigated the signal transduction processes involved in the induction of chalcone synthase (CHS) and phenylalanine ammonia-lyase (PAL) gene expression by UV-B and UV-A/blue light in an Arabidopsis cell suspension culture . Experiments with electron transport inhibitors indicated that plasma membrane redox activity is involved in both signal transduction pathways . Calcium ionophore treatment stimulated expression of the TOUCH3 gene, and this induction was strongly antagonized by UV-A/blue and UV-B light, suggesting that both light qualities may promote calcium efflux from the cytosol . Consistent with this hypothesis, experiments with specific inhibitors indicated that UV-B and UV-A/blue light regulate calcium levels in a cytosolic pool in part via the action of specific Ca2+-ATPases . On the basis of these and previous findings, we propose that plasma membrane redox activity, initiated by photoreception, is coupled to the regulation of calcium release from an intracellular store, generating a calcium signal that is required to induce CHS expression. Neuroreport, 1998 Oct 5, 9(14), 3223 - 7 cAMP included during cell suspension preparation improves survival of dopaminergic neurons in vitro; Branton RL et al.; The physical process of cell suspension preparation from embryonic ventral mesencephala (VM) may be responsible for the low numbers of dopaminergic (DA) neurons that survive following neural transplantation or in vitro culture . In particular, the disruption of cell to extracellular matrix attachment may result in cell death through deactivation of a cAMP-dependent protein kinase involved in cell survival signalling . In an attempt to reduce this death, dibutyryl cAMP was included in all solutions from explant collection to final dissociation . Pretreatment with 700 microM dibutyryl cAMP resulted in 90% survival of the DA neurons originally plated, compared with only 40% in the untreated cultures, after 5 days in vitro . Treatment of VM explants in this manner may result in major improvements in neural transplantation as a technique for the treatment of Parkinson's disease. J Biol Chem, 1998 Dec 4, 273(49), 32602 - 7 Evidence for contribution by increased cytoplasmic Na+ to the insulinotropic action of PACAP38 in HIT-T15 cells; Filipsson K et al.; Pituitary adenylate cyclase-activating polypeptide (PACAP) is localized to pancreatic nerve terminals and stimulates insulin secretion . The insulinotropic effect of PACAP38 in insulin-producing HIT-T15 cells is accompanied by increases in cellular cAMP and cytoplasmic Ca2+ ({Ca2+}cyt) . As also intracellular Na+ is important for insulin secretion after glucose and other cAMP forming peptides, we examined the Na+ dependence of the insulinotropic effect of PACAP38 in HIT-T15 cells . We found that PACAP38 (100 nM)-induced insulin secretion was diminished by approximately 50% by removal of extracellular Na+ (replaced by equimolar N-methyl-D-glucamine) . In contrast, removal of Na+ did not diminish the formation of cellular cAMP (measured by radioimmunoassay) or the increase in {Ca2+}cyt (measured in FURA-2AM-loaded cell suspensions) induced by PACAP38 . Furthermore, PACAP-38 increased the cytoplasmic Na+ ({Na+}cyt) in single HIT-T15 cells as measured by the fluorophore sodium-binding benzofran isophthalate . This increase was reduced by removal of extracellular Na+ and by inhibition of protein kinase A by H-89 . We conclude that the insulinotropic action of PACAP38 is Na+-dependent . We propose that PACAP38 opens plasma membrane Na+ channels by an action partially mediated by cAMP and protein kinase A, and the subsequent raise in {Na+}cyt elicits insulin secretion by an as yet unsolved mechanism. J Hematother, 1998 Oct, 7(5), 437 - 48 Large-scale production of CD4+ T cells from HIV-1-infected donors after CD3/CD28 costimulation; Levine BL et al.; We describe a procedure for large-scale enrichment, growth, and harvesting CD4+ T cells . This method may be effective for HIV-1 immunotherapy, as the mode of stimulation, with anti-CD3 plus anti-CD28 coated beads (CD3/CD28 beads) induces a potent antiviral effect . PBMC were obtained by density gradient centrifugation of an apheresis product . Monocytes/macrophages were removed by incubating PBMC with beads coated with IgG . The cells were then magnetically depleted of B cells and CD8+ cells with mouse anti-CD20 and anti-CD8 MAbs and sheep antimouse coated beads . The remaining cells were >80% CD4+ and were transferred to gas-permeable bags containing CD3/CD28 beads and cultured in a closed system . After 14 days, the cell number increased an average of 37-fold, and cells were nearly 100% CD4+ . Viral load, assessed by DNA PCR for HIV-1 gag, decreased >10-fold during culture in the absence of antiretroviral agents . Removal of CD3/CD28 beads from the cell suspension was accomplished by passing cells plus beads (3-30 x 10(9) cells in 2-12 L) over a MaxSep magnetic separator using gravity-driven flow . The cells were then concentrated to 300 ml in an automated centrifuge . This process allows safe and efficient growth of large numbers of CD4+ T cells from HIV-1+ donors. Biol Reprod, 1998 Dec, 59(6), 1360 - 70 Development of germ cell transplants in mice; Parreira GG et al.; Development of spermatogonial transplants was studied by using 5- to 6-wk-old histocompatible mice as cell donors and sterile (W-locus) mice as recipients . Groups of animals transplanted with germ cell suspensions were killed at 10 min, 9 h, 24 h, 1 wk, 1 mo, 2 mo, and 3 mo along with age-matched "start" and "end" W-locus controls . Weight of testes increased significantly at 24 h through 3 mo after germ cell transplantation, suggesting that the infused cells quickly stimulated organ function . Small clones of young spermatocytes were evident at 1 mo and sperm at 2 mo . The percentage of tubular profiles containing active spermatogenesis originating from spermatogonia increased with time (0.8% at 1 mo, 8.9% at 2 mo, and 28.2% at 3 mo) . Most transplanted germ cells were eliminated from the seminiferous epithelium through phagocytosis by Sertoli cells that occurred primarily before 1 wk, although some pachytene cells were able to proceed through meiosis by 1 wk . A variety of abnormal features are described that characterize developing spermatogenesis in the transplanted testis . Spermatogenesis improved quantitatively and qualitatively with time although released sperm were frequently engulfed by intratubular macrophages and Sertoli cells . A quantitative analysis of spermatogenesis from transplanted germ cells will serve as a basis for improving spermatogonial transplant efficiency. Gynecol Oncol, 1998 Nov, 71(2), 308 - 12 In vitro phase II comparison of the cytotoxicity of a novel platinum analog, nedaplatin (254-S), with that of cisplatin and carboplatin against fresh, human cervical cancers; Monk BJ et al.; OBJECTIVE: To compare the in vitro cytotoxicity of nedaplatin, an investigational platinum analog, with that of cisplatin and carboplatin against fresh cervical cancers from untreated patients . METHODS: Specimens were obtained prior to irradiation or radical surgery from 20 patients with locally invasive cervical carcinoma . Cytotoxicity was measured after single cell suspensions were grown in agar using colony counts and incorporation of {3H}thymidine . Nedaplatin and cisplatin were tested at 1 and 10 micrograms/ml dose levels while carboplatin was tested at 10 and 100 micrograms/ml dose levels continuously . When single hour exposures were used, drug doses were increased by 10-fold . RESULTS: The median drug concentrations associated with a 50% inhibition of growth (IC50) for nedaplatin, cisplatin, and carboplatin were 0.435, 0.73, and 18.6 micrograms/ml, respectively . At 10 micrograms/ml for both cisplatin and nedaplatin and 100 micrograms/ml for carboplatin, cisplatin was the most active drug with 70% of tumors sensitive (</=50% survival relative to control plates) to cisplatin and 45 and 50% sensitive to nedaplatin and carboplatin, respectively (P = 0.015, P = 0.074) . Six of 20 (30%) tumors resistant to cisplatin were also resistant to nedaplatin and carboplatin . CONCLUSION: At doses approximating clinically achievable drug concentrations as defined by the mean plasma concentration time product, cisplatin appears more cytotoxic in vitro than either carboplatin or nedaplatin among chemotherapy-naive cervical cancers . However, nedaplatin and carboplatin are also active agents with similar activity . Since differences in drug sensitivity may be related to subtle differences in dose and schedule and the pharmacokinetics and safety profile of nedaplatin are favorable, clinical trials of nedaplatin are indicated . Infect Immun, 1998 Dec, 66(12), 5889 - 96 Specific-antibody-secreting cells in the rectums and genital tracts of nonhuman primates following vaccination; Eriksson K et al.; To determine optimal strategies to induce specific-antibody-secreting cells (specific ASC) in the rectal and vaginal mucosae, we immunized monkeys with a prototype mucosal immunogen, cholera toxin (CT), given locally or via gastric or parenteral administration . Repeated rectal or vaginal CT immunizations induced strong mucosal and systemic ASC responses . The mucosal responses were, however, confined to the immunization sites and comprised high levels of both specific antitoxin immunoglobulin A (IgA) and IgG . Large numbers of specific IgA and IgG ASC were detected in cell suspensions from dissociated genital and rectal tissues, demonstrating local accumulation of effector B cells at these sites . Intragastric immunization with CT did not per se give rise to cervicovaginal or rectal ASC responses but did prime for a rectal IgA ASC response to local booster immunization . Both rectal and vaginal immunizations also induced circulating blood IgG ASC and IgA ASC . In conclusion, these results show that local administration of antigen to the rectal or vaginal mucosa results in higher ASC responses than systemic or distant mucosal delivery . Furthermore, both the vaginal and the rectal mucosae can serve as inductive sites for systemic ASC responses . These observations should be relevant to the development of vaccines against sexually transmitted diseases such as that caused by human immunodeficiency virus. Tumori, 1998 Jul-Aug, 84(4), 493 - 8 Retrospective analysis of ploidy in primary osseous and extraosseous Ewing family tumors in children; Perotti D et al.; AIMS: To retrospectively study the DNA content in a series of childhood Ewing Family Tumors (EFT), and to investigate its prognostic value . METHODS: The study was performed on a series of 27 EFTs (osseous Ewing's sarcoma, 18 cases; extraosseous Ewing's sarcoma, 2; peripheral neuroepithelioma, 4; Askin Rosai tumors, 3) . Ploidy was investigated using both flow cytometry (FCM) and image cytometry (ICM) on tumor cell suspensions from formalin-fixed paraffin-embedded specimens or fresh frozen tissue obtained from the primary tumor at diagnosis . RESULTS: Ploidy was evaluable by FCM in all cases, and by ICM in 23/27 . When fresh frozen tissue and paraffin-embedded samples from the same tumor were available for analysis, they yielded equal results . The rate of agreement between FCM and ICM was 82% . The majority of cases were diploid, and in the present series aneuploidy seemed to be associated with a poor outcome . CONCLUSIONS: These results suggest that aneuploidy could be an indicator of a bad prognosis in EFT; however, the small number of cases precludes any conclusion of statistical value . Larger retrospective studies on ploidy using archival material could be performed and their reliability is supported by the concordance of results from fresh and formalin-fixed tissue. Cytometry, 1998 Nov 1, 33(3), 310 - 7 Evaluation of a flow cytometric method for simultaneous leukocyte phenotyping and quantification by fluorescent microspheres; Schlenke P et al.; We describe a flow cytometric method using a newly designed product, fluorochrome-containing microspheres (Flow Count fluorospheres), which facilitates the precise quantification of cells in whole blood samples or heterogeneous cell suspensions on a single-cell level . These microparticles are easily distinguishable from other events of interest and can be detected by their light-scattering and fluorescence properties . In contrast to the traditional manual or automated cell-counting techniques, this method offers the opportunity to quantify cells simultaneously with flow cytometric immunophenotyping without additional cell loss or other cell preparation steps . We evaluated the accuracy and reproducibility of this flow cytometric method on the determination of CD45+ leukocyte counts and compared the results with those obtained by conventional techniques . Particular interest was focused on the behavior of cells and fluorospheres regarding their sedimentation rate over the period of analysis . The data from 48 blood samples with low, normal, and high leukocyte counts confirmed the reliability and comparability of the flow cytometric method, permitting the determination of white blood cell concentration at least to a limit of 100 cells/microL . A broad field of applications will benefit from this flow cytometric supplement because it is easy to perform and highly accurate . The results appear to be transferable to clinical decision-making monitoring of CD4+ lymphocytes in patients infected with human immunodeficiency virus or of CD34+ hematopoietic cells, optimizing the harvest for peripheral blood stem cell transplantation. Acta Biochim Pol, 1998, 45(2), 621 - 6 Beta-galactosidase in immobilized cells of gherkin Cucumis sativus L; Stano J et al.; Cell suspensions of gherkin (Cucumis sativus L.) were permeabilized by Tween-80, and immobilized by glutaraldehyde . Beta-galactosidase showed pH optimum at 4.9 and temperature optimum at 58 degrees C . The enzyme catalysed hydrolysis was linear for 3 h with 60-68% conversion of the substrate . The cells characterized by high beta-galactosidase activity and stability on long-term storage showed valuable technological properties. Pancreas, 1998 Nov, 17(4), 383 - 9 Involvement of lipid peroxidation in free fatty acid-induced isolated rat pancreatic acinar cell injury; Morita Y et al.; It was reported that free fatty acids degraded from triglycerides by lipase may play a major role in acute necrotizing or hyperlipidemia-induced pancreatitis . We hypothesized that this injury may be related to the peroxidation of cell membrane phospholipids and tested this hypothesis using isolated pancreatic acini . Pancreatic acini were prepared from male Sprague-Dawley rats by collagenase digestion . Linoleic acid was added (0.1-1.0 mM) to the acinar cell suspension to induce cell injury . Acinar cell damage was measured by lactate dehydrogenase release and by trypan blue exclusion . Phosphatidylcholine hydroperoxide and alpha-tocopherol in the acinar cells were measured . Protective effects of alpha-tocopherol (0.5, 5.0 mM) against this type of cell injury were also evaluated . When isolated acinar cells were treated with linoleic acid, a significant decrease in viability was observed in a time- and dose-dependent manner . In addition, the levels of phosphatidylcholine hydroperoxide after treatment of 0.5 mM of linoleic acid were increased and levels of alpha-tocopherol were decreased significantly . alpha-Tocopherol significantly ameliorated both cellular injury (p < 0.01) and increases in phosphatidylcholine hydroperoxide (p < 0.01) . These data suggest that lipid peroxidation of the cellular membrane is an important component of the pancreatic cell injury mediated by free fatty acids. Pflugers Arch, 1998 Dec, 437(1), 86 - 93 Evidence for a Na+/Ca2+ exchange mechanism in rat peritoneal mast cells; Praetorius HA et al.; Mast cells lose their ability to secrete when incubated in nominally Ca2+-free medium, but the Na+/K+ pump inhibitor ouabain prevents this loss, suggesting a Na+ dependence of the Ca2+ gradient in rat mast cells . The present study includes measurements of histamine release from cell suspensions, and fura-2/AM and current-clamp experiments on single cells . KB-R7943, an inhibitor of the reverse mode of the Na+/Ca2+ exchanger, 2,4-dichlorobenzamil and La3+ counteracted the increase in histamine release induced by ouabain in a dose-dependent manner . The Ca2+ response to compound 48/80 was reduced by preincubation of the mast cells for 30 min in nominally Ca2+-free medium . This reduction was partly prevented by ouabain or by a low extracellular Na+ concentration . Superfusion of cells with a medium containing a low Na+concentration resulted in a hyperpolarization of the cells of 38.6+/-8.6 mV, n=8, followed by a repolarization after the superfusion had ceased (45.7+/-5.9 mV, n=4) . KB-R7943 reduced the hyperpolarization and repolarization induced by a low extracellular Na+ concentration to 15.5+/-2.9 mV (n=7) and 0.2+/-3.4 mV (n=3), respectively . These results are consistent with the presence of a Na+/Ca2+ exchanger in rat peritoneal mast cells. Sheng Wu Yi Xue Gong Cheng Xue Za Zhi, 1997 Mar, 14(1), 30 - 2 {Effects of vasoactive intestinal peptide, alpha-chymotrypsin, pancreozymin, lipase, phospholipase A2 and collagenase on the viscoelasticity properties of red blood cell suspension}; Feng X et al.; Using Low Shear-30 Rheometer, we studied the effects of vasoactive intestinal peptide, alpha-chymotrypsin, pancreozymin, lipase, phospholipase A2 and collagenase on the viscoelasticity properties of RBC suspension . The result showed that these drugs could increase the values of eta 0.512 and A . I . It suggests that these drugs could increase the degree of RBC aggregation . Among the drugs and concentrations, there is no significant difference. Clin Cancer Res, 1997 Feb, 3(2), 295 - 9 Improvement of in vitro chemosensitivity assay for human solid tumors by application of a preculture using collagen matrix; Kitaoka A et al.; The use of {3H}thymidine incorporation assay (TIA) to evaluate the drug response of tumor cells has been recognized as a useful chemosensitivity assay for fresh human tumor specimens . However, its low evaluability has been a disadvantage for clinical application . To overcome this drawback, we have applied a preculture stage prior to the TIA . This preculture requires plating the tumor cell suspension onto a collagen matrix for 24 h . In 29 fresh human tumor specimens, a significant increase in both cell viability (P < 0.05) and {3H}thymidine incorporation (P < 0.001) of the cultured cells was observed with preculturing; the composition of cancer cells (epithelial membrane antigen positive) and stromal cells (vimentin positive) did not change . In comparisons between 66 specimens that were precultured and 705 specimens that were not, the evaluability rate increased significantly from 48.5% (342/705) to 75.8% (50/66; P < 0.0001) after preculturing . No significant change in in vitro chemosensitivities was observed . When the clinical responses for cancer chemotherapy were retrospectively compared with the in vitro sensitivities of the corresponding drugs on 16 patients who had measurable lesions, the correlation between the two was satisfactorily strong; the prediction accuracy for sensitivity was 83.3% (5/6), the prediction accuracy for resistance was 95.0% (19/20), and the overall correlation was 92.3% (24/26) . These results indicate that TIA with preculturing yields increased rates of evaluability, preserving in vitro drug responses of cultured tumor cells, and has an improved potential to be used for determining clinical chemotherapy. Clin Cancer Res, 1997 Nov, 3(11), 1923 - 30 Low intercellular adhesion molecule 1 and high 5T4 expression on tumor cells correlate with reduced disease-free survival in colorectal carcinoma patients; Mulder WM et al.; The purpose of this study was to investigate the prognostic value of the expression of intercellular adhesion molecule 1 (ICAM-1), leukocyte function antigen 3 (LFA-3), human leukocyte differentiation antigen (HLA)-ABC, HLA-DR, and 5T4 with regard to disease-free survival in Dukes' B and C colorectal carcinoma patients . Forty-one patients (28 Dukes' B and 13 Dukes' C) were entered into this study . Immunocytochemistry was performed on cytospin preparations of enzymatically digested colorectal carcinoma cell suspensions . The frequency of metastases and the duration of disease-free survival were compared between the 25% lowest expressers and the 75% remaining patients for ICAM-1, LFA-3, HLA-ABC, and HLA-DR, and between the 25% highest expressers and the 75% remaining patients for 5T4 . Low numbers of ICAM-1-expressing tumor cells were associated with a shorter disease-free survival (P < 0 . 001), independent of Dukes' stage . High numbers of 5T4-expressing tumor cells were associated with shorter disease-free survival in Dukes' B patients (P = 0.04) . Cox proportional hazard analysis indicated that low numbers of ICAM-1(+) and high numbers of 5T4(+) cells were independent prognostic factors with relative risks of 13 . 0 (P = 0.0002) and 4.7 (P = 0.02), respectively . The combination of 5T4 and ICAM-1 marker information identified subgroups of patients with a good (high ICAM-1) or poor (low ICAM-1/high 5T4) prognosis . Neither a lack of HLA-ABC and LFA-3 expression nor the presence of HLA-DR on the tumor cells gave additional prognostic information . These findings demonstrate that low ICAM-1 and high 5T4 expression on tumor cells are prognostic markers, additional to Dukes' stage, for reduced disease-free survival in Dukes' B and C colorectal carcinoma patients. Dev Immunol, 1998, 6(3-4), 157 - 70 An adult thymic stromal-cell suspension model for in vitro positive selection; Chidgey AP et al.; Presented here is a cell-suspension model for positive selection using thymocytes from alphabeta-TCR (H-2Db-restricted) transgenic mice specific to the lymphocytic choriomeningitis virus (LCMV) on a nonselecting MHC background (H-2d or TAP-1 -/-), cocultured with freshly isolated adult thymus stromal cells of the selecting MHC type . The thymic stromal cells alone induced positive selection of functional CD4- CD8+ cells whose kinetics and efficiency were enhanced by nominal peptide . Fibroblasts expressing the selecting MHC alone did not induce positive selection; however, together with nonselecting stroma and nominal peptide, there was inefficient positive . These results suggest multiple signaling in positive selection with selection events able to occur on multiple-cell types . The ease with which this model can be manipulated should greatly facilitate the resolution of the mechanisms of positive selection in normal and pathological states. Brain Res Brain Res Protoc, 1998 Nov, 3(2), 192 - 8 An improved method for culturing cerebellar Purkinje cells with differentiated dendrites under a mixed monolayer setting; Furuya S et al.; We report here a novel cell culture protocol which facilitates in vitro survival and dendritic differentiation of cerebellar Purkinje cells in a monolayer, mixed culture setting . We found that the type of culture medium is a critical factor for the maintenance of these cells . Purkinje cells present in the single cell suspension of embryonic rat cerebellum were best maintained in a medium based on Dulbecco's modified Eagle's medium (DMEM)/F-12 without the addition of known neurotrophic factors . These cells maintained in DMEM/F-12-based media displayed an approximately 2.5-3.5-fold increase in survival compared with cells maintained in the widely used Basal Medium Eagle's (BME)-based serum-free culture medium with the same supplements . This novel protocol permits not only enhanced survival but also accelerated, improved dendritic differentiation of these cells . Purkinje cells developed highly branched spiny dendrites by 14-16 days in vitro, which matches the time course of the dendritic growth of these cells in vivo . The Purkinje cells expressed metabotropic glutamate receptor 1alpha in the cell bodies and branched dendrites, and the intradendritic calcium concentration increased when trans-ACPD, a selective agonist of this receptor, was applied . This novel protocol allows the development of functional branched dendrites and therefore is useful for electrophysiological and ion-imaging studies on dendrites of Purkinje cells grown in vitro . Ultrasound Med Biol, 1998 Sep, 24(7), 1009 - 21 In situ measurements of Doppler power vs . flow turbulence intensity in red cell suspensions; Wu SJ et al.; Whereas previous studies have shown that ultrasonic backscatter and Doppler power from blood are affected by flow turbulence, turbulence level has only been inferred from the flow Reynolds number and not directly measured . In this study, both ultrasonic Doppler power and flow turbulence intensity were measured in situ to quantify the relationship between Doppler power and flow turbulence . Three grid meshes of different geometries were used in a steady-flow mock loop to generate controlled levels of flow turbulence in porcine red blood cell saline suspensions . Doppler power was measured by a 10-MHz PW Doppler flowmeter, and the turbulence intensity by using constant-temperature hot film anemometry . We showed that Doppler power is affected by turbulence and hematocrit in a complex way . At a fixed hematocrit, Doppler power increases nonlinearly with turbulence intensity and, at fixed turbulence intensity, Doppler power peaks at an optimal hematocrit level that increases with turbulence level . The shape factor, introduced by Lucas and Twersky (1987) to take into account effects of shape and orientation of the scatterers in a dense distribution of small and tenuous scatterers, was estimated by fitting the experimental data to the theoretical model . The results indicate that shape factor decreases with increasing turbulence intensity. Exp Nephrol, 1998 Nov-Dec, 6(6), 542 - 50 Highly specific separation of heterogeneous cell populations by lectin-coated beads: application for the isolation of inner medullary collecting duct cells; Grupp C et al.; Conditions for the highly specific selection of a cell type by the use of lectin-coated magnetic beads are reported for the isolation of inner medullary collecting duct (IMCD) cells from a heterogeneous inner medullary cell suspension, containing both single cells and tubular fragments of variable size . The lectin Dolichos Biflorus Agglutinin (DBA), which binds in rat inner medulla exclusively to IMCD cells, was coupled via the avidin-biotin system to beads . By isolating DBA-bead-IMCD cells in a magnetic field (positive selection) from a suspension containing about 50% IMCD, a fraction of 98 +/- 1% purity was obtained; recovery of cells was up to 90% . Suspensions negative on reverse-transcriptase polymerase chain reaction for vimentin as a marker of contaminating interstitial and vascular cells could be received by repeating this procedure and additional trypsinization . On the other hand, it was possible to reduce the portion of IMCD cells in the suspension by one isolation step to 1.5 +/- 0.9% (negative selection) . Performing this step twice resulted in virtually pure suspensions . No significant effects of this isolation technique on cell viability, growth characteristics, and biochemical parameters were observed . Therefore, this method appears to be a powerful tool for the highly specific separation of heterogeneous cell populations. Plant Physiol, 1998 Nov, 118(3), 1005 - 14 The heat-shock element is a functional component of the Arabidopsis APX1 gene promoter; Storozhenko S et al.; Ascorbate peroxidases are important enzymes that detoxify hydrogen peroxide within the cytosol and chloroplasts of plant cells . To better understand their role in oxidative stress tolerance, the transcriptional regulation of the apx1 gene from Arabidopsis was studied . The apx1 gene was expressed in all tested organs of Arabidopsis; mRNA levels were low in roots, leaves, and stems and high in flowers . Steady-state mRNA levels in leaves or cell suspensions increased after treatment with methyl viologen, ethephon, high temperature, and illumination of etiolated seedlings . A putative heat-shock cis element found in the apx1 promoter was shown to be recognized by the tomato (Lycopersicon esculentum) heat-shock factor in vitro and to be responsible for the in vivo heat-shock induction of the gene . The heat-shock cis element also contributed partially to the induction of the gene by oxidative stress . By using in vivo dimethyl sulfate footprinting, we showed that proteins interacted with a G/C-rich element found in the apx1 promoter. Exp Hematol, 1998 Nov, 26(12), 1162 - 71 Candidate hematopoietic stem cells from fetal tissues, umbilical cord blood vs . adult bone marrow and mobilized peripheral blood; Huang S et al.; As part of our ongoing effort to identify a rich source of pluripotent progenitor cells for transplantation and gene therapy, we cultured single-sorted CD34+ subpopulations from different human hematopoietic tissues to assess the relationship between immunophenotype expression and functional characteristics . In combination with index sorting, single cell culture permits precise assessment of the colony efficiency (CE), growth characteristics, and replating potential (RP) of each individual phenotype without interference from other cell types . CD34+ cells from fetal liver (FL), fetal bone marrow (FBM), umbilical cord blood (UCB), adult BM (ABM), and mobilized peripheral blood (MPB) were sorted and cultured as single cells according to the coexpression of CD38 . With the exception of FL, higher CEs were found among CD34+/CD38+ cells vs . CD34+/CD38- cells from the same cell source . However, colonies with dispersed growth pattern (DGP) and mixed growth pattern (MGP) were found predominantly in the CD34+/CD38- subsets . We next examined the functional characteristics of CD34+ subsets defined by three-color analysis and coexpression of CD38 and HLA-DR or CD38 and CDw90 (Thy-1) . Using the combination CD34, CD38, and HLA-DR, the highest CEs, the highest percentages of colonies with DGP/MGP and the maximum RP were found in the CD34+/CD38-/HLA-DR+ subset among samples from FL, FBM, and UCB . The CEs and percentages of colonies with DGP/MGP of single-sorted CD34+/CD38-/HLA-DR+ cells were 72.7+/-11.8% (mean +/- standard deviation) and 20.1+/-10.4%, respectively, in FL; 60.9+/-11.1% and 11.5+/-5.4% in FBM; 57.0+/-16.5% and 24.1+/-7.3% in UCB; 27.2+/-12.8% and 9.0+/-4.9% in MPB, and 9.6+/-7.8% and 4.6+/-3.2% in ABM . Using the combination CD34, CD38, and CDw90(Thy-1), the subset with the highest CEs and highest percentages of colonies with DGP/MGP was found to be CD34+/CD38-/CDw90- in FL and FBM, but colonies with high RP were distributed evenly among CDw90+ and CDw90- subsets derived from FL, FBM, UCB, or MPB . We conclude that the CD34+/CD38-/HLA-DR+ subset contained the highest number of candidate stem cells among the various immunophenotypes, and that FL contained the highest concentration of CD34+ cells (11.4+/-7.5%) and the highest level of CD34+/CD38-/HLA-DR+ subsets (34.7+/-8.2%) among cells from various ontogenic age . Our estimate of candidate stem cells using single cell suspension culture correlated with that obtained by single cell long-term culture-initiating cells . CD34+/CD38-/HLA-DR+ cells from FL appear to represent the best targets for ex vivo stem cell expansion and genetic manipulation. Plant J, 1998 Sep, 15(6), 773 - 81 Activation of MAPK homologues by elicitors in tobacco cells; Lebrun-Garcia A et al.; Elicitors of plant defence reactions (such as cryptogein, an elicitin produced by Phytophthora cryptogea, or oligogalacturonides (OGs)), induced in tobacco cell suspensions (Nicotiana tabacum var Xanthi) a rapid and transient activation of two protein kinases (PKs) with apparent molecular masses of 50 and 46 kDa, respectively . These PKs activated and phosphorylated at tyrosine residues, phosphorylated myelin basic protein (MBP) at serine/threonine residues . Both are recognized by anti-MAPK antibodies . The two MBP kinases possessed the same kinetics of activation, and their activation depended, to the same extent, on different exogenously applied compounds (staurosporine, lanthanum, EGTA) . We demonstrate here that the activation of the MBP kinases is calcium dependent and sensitive to staurosporine, a protein kinase inhibitor which annihilates all known responses of tobacco cells to cryptogein . The activation of MBP kinases appeared to be independent of the production of active oxygen species (AOS) and insensitive to calyculin A, a protein phosphatase type 1 and 2A inhibitor . The activation of MAPKs is discussed in relation to the early responses induced by cryptogein. Neurochem Res, 1998 Oct, 23(10), 1217 - 23 Intrastriatal grafts of fetal mesencephalic cell suspensions in MPP+-lesioned rats: a microdialysis study in vivo; Espejo M et al.; The striatum of rats was lesioned by unilateral administration of MPP+ . Two weeks later, a suspension of fetal mesencephalic cells (FMC), obtained from 14-day rat embryos, was injected into the lesioned striatum . Two weeks after grafting, the success of implantation and recovery of dopamine function were assessed by tyrosine hydroxylase immunocytochemistry (TH) and the measurement of striatal dopamine content . In addition, the extracellular concentrations of dopamine and dopamine metabolites were studied by microdialysis in vivo before and after perfusion of MPP+ to induce dopamine release from vesicular stores . TH+ cell bodies were seen in the lesioned grafted striata, indicating that fetal cells survived in these striata . In addition, there was a marked increase in TH-immunoreactivity in the neuronal fibers and terminals in the area surrounding the cell implant, suggesting a compensatory response of the host tissue which may involve fiber sprouting . Grafting induced a recovery in indices of dopamine function, including recovery in dopamine content, and basal and MPP+-induced dopamine release . Thus, grafts of FMC may provide a significant recovery of dopamine function in MPP+-lesioned striata. Hum Reprod, 1998 Oct, 13(1O), 2791 - 6 Enzymatic digestion of testicular tissue may rescue the intracytoplasmic sperm injection cycle in some patients with non-obstructive azoospermia; Crabbe E et al.; Recovery of testicular spermatozoa from azoospermic patients with testicular failure, followed by intracytoplasmic sperm injection (ICSI) is a recent advance in the treatment of male infertility . In most cases, free spermatozoa are recovered from testicular tissue after mechanical mincing of multiple biopsies . Testicular sperm retrieval, however, remains unsuccessful in 30-50% of male patients suffering from Sertoli cell-only syndrome and maturation arrest . In this study, a strategy was developed in order to maximize the chance of sperm retrieval in difficult cases of testicular failure . The ultimate step was the use of enzymatic procedures (collagenase type IV) to dissociate the testicular tissue completely . Testicular tissue of 41 patients for whom no spermatozoa were found after mechanical mincing of the testicular tissue was investigated . In 14 out of the 41 cases (34%), enough spermatozoa for ICSI were found after fine mincing of multiple biopsies and several hours' search in the cell suspension treated with the erythrocyte-lysing buffer (ELB) . In 27 out of the 41 patients, no spermatozoa were found even after the use of ELB . In seven out of these 27 failures (26%), spermatozoa for ICSI were retrieved after enzymatic dissociation of the residual minced tissue pieces, thus making ICSI possible despite failure to find spermatozoa with conventional mincing . From this study, we may conclude that enzymatic digestion of testicular tissue is easy to perform, is not time-consuming and constitutes a successful method in reducing the sperm recovery failures in patients with non-obstructive azoospermia. Magnes Res, 1998 Sep, 11(3), 161 - 9 Early morphological and immunological alterations in the spleen during magnesium deficiency in the rat; Malpuech-Brugere C et al.; Dietary magnesium deficiency in rodents, and especially in rats, causes inflammation and leads to alterations in the immune response . One of the characteristics of magnesium deficiency in the rat is a marked enlargement of the spleen . Considering the importance of the spleen for the immune response, in this study we have evaluated histological, cytological and immunological changes in this organ of rats in early stages of this deficiency . For this purpose, male weaning Wistar rats were pair-fed with either control or magnesium-deficient diet, for 2, 4 or 8 days . Results indicate that after 8 days on the deficient diet rats presented clinical signs of inflammation, splenomegalia and leukocytosis . As shown by histometrical analysis, both the red and white spleen pulps of deficient rats displayed an increased incidence of polymorphonuclear leukocytes and macrophages in all studied stages of deficiency . Concomitantly, the relative number of lymphocytes decreased . This observation was confirmed by the analysis of the cell suspension obtained from the spleen . The greater number of adherent cells in the cell suspension from deficient rats provides an additional confirmation of the increased number of macrophages in the spleen of these rats . Analysis of lymphocyte populations demonstrated a reduced proportion of CD5+ and CD8+ cells after 8 days of deficiency . The reduction in the number of CD8+ cells in deficient rats could be related to the observed decrease in IFN-gamma concentration in the spleen homogenate . In short, this study shows that magnesium deficiency causes early cytological and immunological modifications in the spleen which appeared before macroscopical changes in this organ and before clinical symptoms of inflammation . These changes could be related to the altered immune response of deficient animals. Anal Biochem, 1998 Oct 15, 263(2), 208 - 13 A perifusion system to control oxygen concentration in cell suspensions; Arthur PG et al.; A system is described for the perifusion of cells with a perifusate containing oxygen at concentrations defined by the user . The metabolic competency of cells in this system was assessed by measuring total ATP turnover for human platelets . Human platelets, which are small (radius of 1-2 micron) anucleate cells involved in blood clotting, maintained ATP turnover over a 4-h period at 37 degrees C . Oxygen concentration in this system was controlled by adjusting the proportions of air-saturated and nitrogen-saturated perifusates . Examples of oxygen consumption and lactate output as a function of oxygen concentration are described . Cell Tissue Res, 1998 Dec, 294(3), 537 - 47 Male-associated polypeptide (MAP) expression in different compartments of the reproductive system of the mussel Mytilus galloprovincialis: immunocytochemical and western blot study; Torrado M et al.; Mytilus mussels are characterized by annually repeated reproduction which is associated with subsequent growth, morphogenesis, breakdown and redevelopment of the gonad and reproductive tract into mantle mesenchyme . We present a description of the expression of the male-associated polypeptide (MAP; see Mikhailov et al . 1995) in different compartments of the male reproductive system as well as in mantle gonad-supporting tissue . MAP is expressed in both gonad and mantle structures in dynamic patterns that show a substantial overlap in terms of dependence on the stage of gonad development/involution . In general, the total MAP concentration directly correlates with the volume of gonad tubule/duct structures but inversely correlates with mantle connective tissue cell fraction . A maximum of MAP expression is reached in the fully ripe male gonad . MAP is localized around gonad tubules/ducts, in the gonoduct epithelium, membranes of follicle-like structures as well as in the extracellular fiber-like structures of the mantle . However, we also demonstrate unique sites of MAP accumulation in the lumen of gonad follicle-like tubules and in ductal fluid . The latter is characterized by a very high MAP concentration . MAP is also detected in sperm-containing cell suspension obtained by gonad biopsy which we interpret as a result of the adsorption of MAP on mature spermatozoa . The results obtained should be taken into consideration in the interpretation of possible MAP functions since they seem to point to MAP as a major component of ductal (seminal) fluid of the male reproductive tract . It is likely that MAP is able to complement the processes of sperm terminal differentiation and maturation . In addition, we demonstrate that the male-predominant character of MAP expression is restricted by gonad-containing tissues (i.e., mantle and visceral mass) only, although the polypeptide is also detected in other somatic organs in both males and females. Ann Rheum Dis, 1998 Aug, 57(8), 487 - 94 Involvement of simultaneous multiple transcription factor expression, including cAMP responsive element binding protein and OCT-1, for synovial cell outgrowth in patients with rheumatoid arthritis; Wakisaka S et al.; OBJECTIVE: To elucidate possible roles of several transcription factors in the pathogenesis of rheumatoid arthritis (RA), the transcription factor expression in RA synovial tissue and their contribution to RA synovial cell functions were studied . METHODS: Single cell suspension of dissociated synovial tissue was cultured to induce in vitro tissue outgrowth of RA synovial cells . Transcription factors were immunohistochemically identified in RA synovial tissue obtained by joint surgery and in the in vitro tissue outgrowth, and confirmed by western blotting and gel shift assays . RESULTS: Immunohistochemical examination of RA synovial tissue revealed simultaneous expression of various transcription factors (NF-kappa B, c-Jun (a component of AP-1), cAMP responsive element binding protein (CREB), and OCT-1) . The same set of transcription factors was expressed in the in vitro tissue outgrowth of RA patients . The early passage RA synovial cells were treated with interleukin 1 beta (IL1 beta) and confirmed translocation of transcription factors into the nucleus by western blotting, and their DNA binding activity by gel shift assays . CONCLUSION: This study emphasises the importance of the simultaneous expression of several transcription factors for the hyperactivity of RA synovial cells that leads to tissue outgrowth. Int Immunol, 1998 Oct, 10(10), 1509 - 17 Homing of transgenic gammadelta T cells into murine vaginal epithelium; Rakasz E et al.; The vaginal epithelium of normal mice contains lymphocytes of fetal thymic origin that express an invariant Vgamma4/Vdelta1 TCR . The apparent lack of other gammadelta TCR species suggests that a selection mechanism might operate to regulate the localization of gammadelta T cells at this anatomical site . Selection might be connected to the Vgamma4/Vdelta1 TCR or to some homing characteristic of the fetal thymic lineage that appears at day 17-18 of embryonic life . In the present studies, we investigated whether transgenic gammadelta cells expressing a TCR species characteristic of the subpopulation of gammadelta T cells found in the blood, spleen and lymph would translocate to the vaginal epithelium . We found that the transgenic Vgamma2 TCR+ cells did accumulate in the vagina of transgenic mice . Furthermore, like normal vaginal gammadelta T cells, the transgenic vaginal gammadelta T cells expressed the phenotype of recently activated memory/effector T cells (CD44(hi), CD62L-, CD45RB(lo), CD69+) . Vaginal gammadelta T cells in normal mice do not express the CD2 and CD28 antigens, but both of these markers are present on transgenic vaginal gammadelta T cells . We observed that a small fraction of splenic transgenic gammadelta T cells had the same surface phenotype as the vaginal transgenic gammadelta T cells, raising the possibility that the gammadelta T cells present in the vaginal epithelium of transgenic mice originated from the peripheral lymphoid organs . Data in support of this possibility came from experiments in which co-incubation of splenic transgenic gammadelta T cells with vaginal epithelial cell suspensions induced the vaginal gammadelta phenotype on the splenic gammadelta T cells . The finding of transgenic gammadelta T cells in the vaginal epithelium suggests that homing of gammadelta T cells to this site is not restricted to gammadelta T cells that express the V4/NS1 invariant TCR . Furthermore, these findings imply that retention of gammadelta T cells in the vaginal epithelium of normal mice is affected by a Vgamma4/Vdelta1-specific mechanism . The finding of a significant level of apoptosis in the transgenic vaginal gammadelta T cells, but not in the normal vaginal gammadelta T cells, could reflect that the mechanism of retention of Vgamma4/Vdelta1 + in the vaginal epithelium involves selective survival at the site. Photochem Photobiol, 1998 Oct, 68(4), 555 - 60 In situ molecular dosimetry and tumor risk: UV-induced DNA damage and tumor latency time; de Gruijl FR et al.; In UV carcinogenesis there is a fundamental chain of causal events from UV-induced DNA damage through mutations up to tumor formation: each of the early events should be predictive of the ultimate tumor risk . Instead of the UV surface exposure, the in situ load of DNA damage should be a more direct measure of the carcinogenicity . To explore this further we measured cyclobutane thymine dimer loads of epidermal cell suspensions from chronically UV-exposed hairless SKH-1 mice; skin samples were taken after various time periods under different daily exposures . Although the average load per cell decreased in the course of time due to dilution of damage in an increasing epidermal hyperplasia, the amount of thymine dimers in a column of epidermis (i.e . per mm2 of skin area) became stationary, and this amount increased with higher daily exposure . The median tumor latency time, t50, is inversely related to this stationary load . Extrapolation of a fitted relationship would imply a t50 between 450 and 1430 days for spontaneous skin carcinomas . The present data suggest that the skin strives to maintain a maximum level of tolerable DNA damage by lowering the average genotoxic load in vital cells in a hyperplastic reaction: pseudo-repair by dilution . This would also explain the strong hyperplastic reactions in DNA repair-deficient mouse strains . An understanding of these short-term adaptive reactions can refine our assessments of skin cancer risks in humans. Neurosci Lett, 1998 May 1, 246(3), 141 - 4 Serotonin increases cytoplasmic Ca2+ concentration in PC12h cells: effect of tachykinin peptides; Takenouchi T et al.; We report here that serotonin (5-hydroxytriptamine, 5-HT) induces an increase in intracellular Ca2+ concentration ({Ca2+}i) in rat pheochromocytoma PC12h cells, a subclone of PC12 cells, which was detected by using Ca2+ sensitive indicator dye fura-2 . The {Ca2+}i increase completely disappeared when extracellular Ca2+ was chelated with excess EGTA and potently suppressed in Na+-free buffer . Nifedipine, a voltage-dependent L-type calcium channel blocker, significantly blocked the 5-HT response . Addition of another 4 mM Ca2+ to the cell suspension attenuated the {Ca2+}i increase induced by 5-HT, whereas the nicotinic action was remarkably potentiated . Furthermore, metoclopramide, a 5-HT3 receptor antagonist, inhibited the 5-HT response in a dose dependent manner . These findings suggest that the 5-HT-induced {Ca2+}i increase involves the mediation of a voltage-dependent Ca2+ channel, evoked by membrane depolarization via the activation of cation channel-type receptors, 5-HT3 receptors . We also noted the inhibitory action of tachykinin peptides on the 5-HT response, suggesting that the cell line is useful to investigate these neuromodulatory actions in the nervous system. Cell Immunol, 1998 Nov 1, 189(2), 149 - 59 Sca1(+)/Mac1(+) nitric oxide-producing cells in the spleens of recipients early following bone marrow transplant suppress T cell responses in vitro; Johnson BD et al.; Spleen cells collected from allogeneic chimeras early after bone marrow transplantation (BMT) consistently showed suppressed proliferative responses to interleukin-2 in vitro and failed to proliferate in mixed lymphocyte reaction (MLR) assays . However, isolation of Thy 1.2(+) T cells from the heterogeneous spleen cell suspension prior to culture resulted in heightened proliferation, suggesting the presence of cells capable of suppressing T cell responses in vitro . When separated into subpopulations by negative and positive selection with specific monoclonal antibodies, a non-T, non-B population with immunosuppressive properties was identified . The suppressive cells were found in the spleens of both allogeneic and syngeneic chimeras, but not normal donor mice . Suppressor activity was transient and typically declined by 3 weeks post-BMT . The cells suppressed the response of alloactivated T cells isolated from BMT chimeras as well as naive donor T cells in MLR assays in a dose-dependent manner . To explore the mechanism(s) involved in the suppression, the effects of interferon-gamma (IFN-gamma)-specific mAb and the nitric oxide (NO) synthase inhibitor NG-methyl-l-arginine were examined . The results support a role for both IFN-gamma and NO in the suppressive activity . Separation of cells based on Mac-1 expression indicated that there were both Mac-1-enriched and Mac-1-depleted cells capable of producing NO, but that the Mac-1-depleted cells were the most potent suppressors in MLR assays . The Mac-1-depleted cells still contained a residual population of Mac-1(dim) cells which showed increased levels of Mac-1 expression after overnight culture . Intracellular staining with an inducible nitric oxide synthase (iNOS)-specific mAb indicated that the NO-producing cells expressed the cell surface markers Mac-1 and Sca-1 . When iNOS knockout transgenic mice were used as transplant donors, in vitro suppression of T cell responses was reduced but not eliminated, suggesting that other mechanism(s) could contribute to the suppression . Collectively, these results demonstrate that Sca-1(+)/Mac-1(+) cells capable of producing NO are present in the spleens of recipients early after BMT and suggest that these cells may have immunoregulatory roles in vivo . Mol Gen Genet, 1998 Sep, 259(5), 511 - 5 A tobacco gene encoding a novel basic class II chitinase: a putative ancestor of basic class I and acidic class II chitinase genes; Ohme-Takagi M et al.; Various chitinases have been identified in plants and categorized into several groups based on the analysis of their sequences and domains . We have isolated a tobacco gene that encodes a predicted polypeptide consisting of a 20-amino acid N-terminal signal peptide, followed by a 245-amino acid chitinolytic domain . Although the predicted mature protein is basic and shows greater sequence identity to basic class I chitinases (75%) than to acidic class II chitinases (67%), it lacks the N-terminal cysteine-rich domain and the C-terminal vacuolar targeting signal that is diagnostic for class I chitinases . Therefore, this gene appears to encode a novel, basic, class II chitinase, which we have designated NtChia2;B1 . Accumulation of Chia2;B1 mRNA was induced in leaves in association with the local-lesion response to tobacco mosaic virus (TMV) infection, and in response to treatment with salicylic acid, but was only slightly induced by treatment with ethephon . Little or no Chia2;B1 mRNA was detected in roots, flowers, and cell-suspension cultures, in which class I chitinase mRNAs accumulate to high concentrations . Sequence comparisons of Chia2;B1 with known tobacco class I and class II chitinase genes suggest that Chia2;B1 might encode an ancestral prototype of the present-day class I and class II isoforms . Possible mechanisms for chitinase gene evolution are discussed. Biophys J, 1998 Nov, 75(5), 2587 - 96 Electrooptics studies of Escherichia coli electropulsation: orientation, permeabilization, and gene transfer; Eynard N et al.; Fast optical transient signals are suitable approaches to the investigation of the behavior of bacteria during an electric pulse . In a previous work, by a dual approach taking advantage of a video method and a fast kinetic study of the light transmitted across a cell suspension, we showed that a field-induced orientation phenomenon was affecting the rod-shaped bacteria during the pulse (Eynard et al., 1992 . Eur . J . Biochem . 209:431-436) . In the present work, time courses of electro-induced responses of bacteria during a single square-wave pulse are analyzed . Observations of both the orientation step and the permeabilization process are relevant . These two steps are affected by the addition of DNA . They both obey to a first-order kinetic . The conclusion of this work is that Escherichia coli permeabilization and transformation are multistep processes: orientation (step 1) is followed by an envelope alteration (step 2), all steps being affected by plasmid addition . In the case of E . coli, a rod-shaped bacteria, the orientation process (step 1) brings the cell parallel to the field direction . The pulse duration must be longer than the orientation characteristic time (approximately 1 ms) to trigger an effective permeabilization and its associated events . The permeabilization process (step 2) is associated with a field-induced dipole effect. Surg Endosc, 1998 Nov, 12(11), 1300 - 2 Tumor implantation following laparoscopy using different insufflation gases; Neuhaus SJ et al.; BACKGROUND: Laparoscopic manipulation of malignancies is associated with an increased incidence of metastasis to port sites in experimental models . This study investigated the effect of different insufflation gases on the implantation of a tumor cell suspension following laparoscopic surgery in an established small animal model . METHODS: Forty Dark Agouti rats underwent laparoscopy and the introduction into the peritoneal cavity of a tumor cell suspension . The insufflating gas used for each procedure was one of the following gases (10 rats in each group): carbon dioxide (CO2), nitrous oxide (N2O), helium, and air . The rats were killed 7 days after surgery, and the peritoneal cavity and port sites were examined for the presence of tumor . RESULTS: Although no significant differences were seen between air, CO2, and N2O insufflation groups, tumor involvement of peritoneal surfaces was less likely following helium insufflation . CONCLUSION: The results of this study suggest that tumor metastasis to port sites following laparoscopic surgery may be influenced by the choice of insufflation gas . In this study, helium was associated with reduced tumor growth. Br J Pharmacol, 1998 Sep, 125(2), 357 - 64 Characterization of the P2 receptors on the human umbilical vein endothelial cell line ECV304; Conant AR et al.; 1 . To characterize the P2 receptors present on the human umbilical vein endothelial-derived cell line, ECV304, cytosolic Ca2+, ({Ca2+}c), responses were recorded in single cells and in cell suspensions to a series of nucleotides and nucleotide agonists . 2 . Concentration response curves were obtained in fura-2-loaded ECV304 cell suspensions, with EC50 values of 4.2 microM for ATP, 2.5 microM for UTP and 14 microM for adenosine-5'-O-(3-thio)triphosphate (ATPgammaS) . EC50 values for 2-methylthioATP, ADP, adenosine-5'-O-(2-thio)diphosphate (ADPbetaS) and AMP were 0.5 microM, 3.5 microM, 15 microM and 4.7 microM respectively, but maximal {Ca2+}c responses were less than those produced by a maximal addition of ATP/UTP . ECV304 cells were unresponsive to UDP and beta,gamma,methyleneATP . 3 . Cross-desensitization studies on ECV304 cells suggested that ATP and UTP recognized the same receptor . However, ADP recognized a receptor distinct from the UTP-sensitive receptor and AMP recognized a third distinct receptor . 4 . ECV304 {Ca2+}c responses to 2-methylthioATP were inhibited in the presence of 30 microM pyridoxalphosphate-6-azophenyl-2',4'-disulphonic acid (PPADS), whereas {Ca2+}c responses to UTP were unaffected by this treatment . 5 . ECV304 cells responded to the diadenosine polyphosphate Ap3A with rises in {Ca2+}c . Apparent responses to Ap4A, Ap5A and Ap6A, were shown to be due to a minor nucleotide contaminant that could be removed by pre-treatment of the diadenosine samples with either alkaline phosphatase or apyrase . 6 . ECV304 cells display a pharmacology consistent with the presence of at least two P2 receptors; a P2Y2 receptor insensitive to the diadenosine polyphosphates and a P2Y1 receptor sensitive to Ap3A . In addition, ECV304 cells respond to AMP with increases in {Ca2+}c via an as yet uncharacterized receptor. Am J Reprod Immunol, 1998 Oct, 40(4), 295 - 302 Effects of pregnancy on lymphocytes within sheep uterine interplacentomal epithelium; Fox A et al.; PROBLEM: Previous studies demonstrate increases in the number and granularity of gamma delta T cells within the sheep uterine interplacentomal epithelium during pregnancy . To further characterize their activation and function, gamma delta T-cell receptor (TCR)+ intraepithelial lymphocytes (IELs) from nonpregnant and pregnant uteri were phenotyped extensively . Cytokine mRNA expression in the epithelium and by gamma delta TCR+ IELs isolated from pregnant uteri was also examined . METHOD OF STUDY: Cell suspensions were prepared from the uterine interplacentomal epithelium and from the peripheral blood of nonpregnant and pregnant ewes (120-140 days of gestation) . Surface marker expression was determined by two-color flow cytometry and cytokine expression determined by reverse transcriptase--polymerase chain reaction . RESULTS: Uterine gamma delta TCR+ IELs exhibited increased beta 1-integrin expression but decreased leukocyte function associated antigen (LFA)-1 and major histocompatibility complex class I expression during pregnancy . Major histocompatibility complex class II, CD44, CD2, and LFA-3 expression was unchanged during pregnancy, whereas CD25, VLA-4 and L-selectin were never expressed . The same cytokines were expressed in the pregnant and nonpregnant uterine interplacentomal epithelium with detectable mRNA for interferon (IFN)-gamma, tumor necrosis factor (TNF)-alpha, and interleukin (IL)-1 alpha, but not for IL-2 or IL-4 . gamma delta TCR+ and CD8+ IEL purified from pregnant uteri expressed mRNA for IFN-gamma, TNF-alpha, transforming growth factor-beta, and IL-10 . CONCLUSIONS: gamma delta TCR+ IELs from pregnant uteri have cytoplasmic granules, and express CD8 and cytokines indicative of cytotoxic potential . Phenotypic changes induced during pregnancy differed from those observed after activation of circulating naive cells and may represent further stimulation of fully differentiated effectors . gamma delta TCR+ IELs are present only in interplacentomal areas of pregnant uteri and may control trophoblast invasion within these areas. Radiother Oncol, 1998 Aug, 48(2), 221 - 8 Are hypoxic cells critical for the outcome of fractionated radiotherapy in a slow-growing mouse tumor? Urano M, Nishimura Y, Kuroda M, Reynolds R. PURPOSE: To investigate the significance of hypoxic cells, reoxygenation and repopulation for the outcome of fractionated radiotherapy of a slow-growing subline of a murine fibrosarcoma and to compare the results with those previously obtained from the original fast-growing tumor . MATERIALS AND METHODS: A slow-growing subline, 457-O, was obtained among the tumors that recurred after a single irradiation to the third generation isotransplants of a mouse fibrosarcoma, FSa-II . The single cell suspensions were transplanted into the mouse foot and when the tumors reached an average diameter of 4 mm, they were subjected to one to 20 equal daily y-ray doses given in air (A) or under hypoxic conditions (H) . The TCD50 (50% tumor control radiation dose) was calculated according to the tumor control frequency within 180 days . The linear-quadratic plus time model was fitted to these data by logistic regression analysis . RESULTS: The volume doubling time of the 457-O tumors was approximately 2.2 times slower than that of the original FSa-II tumors . The TCD50(H) (single dose) was 52.3 Gy and increased with an increasing number of fractions to a TCD50(H) (20 doses) of 90.8 Gy . This increase of 38.5 Gy was much smaller than that of 149 Gy for the original FSa-II . The TCD50(A) (single dose) and TCD50(A) (20 doses) were 41.3 and 50.6 Gy, respectively . This small difference of 9.3 Gy contrasted with a significant increase of 52.9 Gy for the FSa-II . DISCUSSION: These results suggested no repopulation of 457-O tumor clonogens during the course of up to 20 daily doses, while the original FSa-II tumor cells repopulated substantially . Hypoxic clonogens in the slow-growing tumor reoxygenated but some fractions remained critical . CONCLUSION: The present data together with those obtained from the fast-growing FSa-II suggested that hypoxic clonogens were critical for the outcome of fractionated radiotherapy . Repopulation was insignificant in this slow-growing tumor during five to 20 daily doses. J Gen Virol, 1998 Oct, 79 ( Pt 10), 2301 - 11 Promoter activity associated with the intergenic regions of banana bunchy top virus DNA-1 to -6 in transgenic tobacco and banana cells; Dugdale B et al.; Promoter regions associated with each of the six ssDNA components of banana bunchy top virus (BBTV) have been characterized . DNA segments incorporating the intergenic regions of BBTV DNA-1 to -6 were isolated and fused to the uidA (beta-glucuronidase) reporter gene to assess promoter activity . In tobacco cell suspensions, the BBTV DNA-2 and -6 promoters generated levels of GUS expression 2-fold greater and similar to the 800 bp CaMV 35S promoter, respectively . Deletion analysis of the BBTV DNA-6 promoter suggested all the necessary promoter elements required for strong expression were located within 239 nucleotides upstream of the translational start codon . In transgenic tobacco plants, the BBTV-derived promoters generally provided a weak, tissue-specific GUS expression pattern restricted to phloem-associated cells . However, in callus derived from tobacco leaf tissue, GUS expression directed by the BBTV DNA-6 promoter was strong and, in some lines, comparable to the CaMV 35S promoter . Detectable promoter activity associated with the BBTV promoters in banana embryogenic cells was only observed using a sensitive green fluorescent protein (GFP) reporter . Promoters derived from BBTV DNA-4 and -5 generated the highest levels of transient activity, which were greater than that of the maize ubi-1 promoter . In transgenic banana plants, the activity of the BBTV DNA-6 promoter was restricted to the phloem of leaves and roots, stomata and root meristems. Acta Neurochir Suppl, 1998, 71, 203 - 5 Relevance of calcium homeostasis in glial cell swelling from acidosis; Plesnila N et al.; Tissue acidosis from trauma or ischemia induces cytotoxic brain edema, mainly affecting astrocytes . In vitro, lactacidosis induces a dose-dependent swelling of glial cells . Activation of membrane transporters and channels, also involved in regulation of intracellular pH (pHi), has been identified as underlying mechanism, although details are poorly understood . We have currently studied whether Ca(2+)-ions play a role in acidosis-induced glial swelling and the associated intracellular acidification . The medium pH of a cell suspension (C6 glioma) was lowered from control (7.4) to 6.2 by lactic acid . Cell volume (CV) and pHi were assessed by flow cytometry . During acidosis in normal medium (2.2 mM Ca2+) CV reached a maximum of 125.1% . In a calcium-free medium swelling from acidosis was inhibited by 74%, while additional buffering of intracellular calcium (Ca2+i) by BAPTA-AM had no further effect . Buffering of Ca2+i alone did not affect the CV increase from acidosis at all . pHi which is decreasing during acidosis was not influenced by the above modifications . The present experiments indicate that lactacidosis-induced glial swelling depends on the presence of extracellular Ca(2+)-ions, while alterations of Ca2+i do not seem to be involved. Wound Repair Regen, 1998 Jan-Feb, 6(1), 28 - 37 p53 and apoptosis alterations in keloids and keloid fibroblasts; Ladin DA et al.; Keloids are the result of a dysregulated wound-healing process and are characterized by formation of excess scar tissue that proliferates beyond the boundaries of the inciting wound . In this study, we investigated the expression of key proteins involved in regulating apoptosis in keloids . Twenty archival paraffin-embedded keloid samples were randomly selected for an immunoperoxidase assay with antibodies against fas, p53, bcl-2, and bcl-x proteins using the target antigen-retrieval technique . Apoptosis was assessed in keloids and normal skin and in keloid and normal fibroblasts by the TdT-mediated dUTP nick-end labeling (tunel) assay on tissue sections, fibroblast cultures, and by flow cytometry for cell suspensions . We found that 18 of 20 keloids expressed p53 protein; bcl-2 was expressed by keloid fibroblasts in 19 of 20 keloids, and all specimens had prominent fas expression throughout the tissue . The distribution of these three antigens was regional within each lesion and followed a consistent pattern of p53 and bcl-2 expression colocalized to the hypercellular, peripheral areas of each keloid in a perinuclear pattern (p < .001) . In contrast, an inverse distribution of fas expression was shown, with staining being more diffuse across the cell surfaces and limited to the central, more hypocellular regions in16 of 17 keloids (p < .001) . There was no specific staining pattern in these keloids with antihuman bcl-x . In vitro studies on cultured keloid fibroblasts (derived from six patients) revealed maintenance of the p53+, bcl-2+ phenotype up to passage 10 . Neither neonatal nor normal adult skin fibroblasts expressed either antigen but could be induced to express p53 by exposure to adriamycin . Keloid lesions and keloid fibroblasts were found to have lower rates of apoptosis than normal controls . Keloid fibroblasts displayed enhanced apoptosis rates in response to hydrocortisone, gamma interferon, and hypoxia treatment as compared with normal adult fibroblasts . Focal dysregulation of p53 combined with upregulation of bcl-2 may help produce a combination of increased cell proliferation and decreased cell death in the younger, hypercellular areas of the keloid . This phenotype is reversed in the older areas of the keloid and may prevent malignant degeneration, thus favoring normal apoptosis as evidenced by prominent fas expression. Eye, 1998, 12 ( Pt 3a), 431 - 9 Simultaneous cell cycle and phenotypic analysis of primary uveal melanoma by flow cytometry; Lawry J et al.; BACKGROUND AND PURPOSE: DNA ploidy and cell cycle measurements of uveal melanoma tissue are regarded as having limited prognostic significance . In contrast, dual-parameter (DNA monoclonal antibody) flow cytometry offers a convenient and rapid way to screen tumour samples for a variety of phenotypic markers, whilst simultaneously measuring DNA ploidy and cell cycle, and therefore has the increased potential to identify clinically relevant indicators of disease progression . The aim of the present study was to identify a simple yet robust method for isolating, preserving and staining cells that could be analysed by flow cytometry . METHODS: Using a simple preparation procedure, a panel of membrane-associated antibodies (ICAM-1, W632, HLA-DR) and nuclear or cytoplasmic oncoprotein antibodies (c-erbB-2, c-myc, bcl-2, p53), together with positive (PHM-5) and negative (FITC F(ab')2) controls, were assayed . It was considered important to test the protocol with markers expressed on the cell surface, and in the cytoplasm and nucleus, so as not to be restrictive and thereby exclude an antigen of potential clinical interest . In addition, such panels would also enable the generation of a 'phenotypic profile' for each specimen that may reveal clinically significant trends . RESULTS: Our results indicate that tissue dissociation followed by brief fixation in 1% paraformaldehyde and permeabilisation in 70% methanol produces a stable single cell suspension, which can subsequently be stained with a wide range of antibodies for the accurate identification of cells in a potentially heterogeneous tumour population . CONCLUSION: This technology can rapidly identify sub-populations of cells expressing differing levels of proteins, which may prove to be indicative of disease progression for this aggressive disease. Immunology, 1998 Sep, 95(1), 132 - 40 A long-lasting interferon-gamma response is induced to a single inoculation of antigen-pulsed dendritic cells; Dillon SM et al.; Vaccines against infectious organisms must produce not only long-lasting immunity but also the appropriate immune response to clear the infection . Obligate intracellular parasites, such as mycobacteria, require a predominantly cell-mediated immune response rather than antibody . Presentation of antigen by dendritic cells (DC) has been associated with the development of strong cell-mediated responses generating the production of interferon-gamma (IFN-gamma) . This cytokine has an essential role in the elimination of mycobacteria . Therefore, we investigated both the duration and the nature of the immune response after priming with DC pulsed with mycobacterial antigen and compared this with priming using a conventional adjuvant . We used two strains of mice: C57BL/6, which inherently produces a T-helper 1 (Th1)-type response to mycobacterial antigen, and BALB/c, which does not . DC-enriched cell suspensions, purified DC or cultured bone marrow cells resembling DC (BMAPC) were prepared, pulsed overnight with PPD and injected intravenously (i.v.) into naive mice . Six and 12 weeks later, splenic T lymphocytes from these mice were challenged in vitro with antigen and their proliferative response and cytokine production was determined . Significant antigen-specific proliferation was observed in all assays on rechallenge with antigen in vitro 6 and 12 weeks after the initial priming with DC . IFN-gamma was detected in both strains but was only antigen specific in the C57BL/6 strain . Purified protein derivative (PPD)-pulsed BMAPC generated similar responses 6 weeks after priming . Thus, long-term T-lymphocyte responses and the production of IFN-gamma can be generated using a single inoculation of PPD-pulsed DC. Br J Dermatol, 1998 Sep, 139(3), 468 - 71 The effect of long-term treatment with tacalcitol on the psoriatic epidermis . A flow cytometric analysis; Mommers JM et al.; During the last decade, novel analogues of 1alpha,25-dihydroxy vitamin D3 have been developed for the treatment of psoriasis . Recently, the efficacy of short-term treatment with the novel derivative tacalcitol (1alpha,24-dihydroxy vitamin D3) has been documented . However, data on the long-term effect of tacalcitol on psoriatic skin are sparse . In this study, we assessed the cell characteristics of the psoriatic epidermis after treatment with tacalcitol for up to 24 weeks . We investigated how long-term treatment with tacalcitol modulates the percentages of differentiated keratinocytes, inflammation cells and basal keratinocytes, and the percentage of cells in the SG2M phase in the basal cell population . From 11 patients who were treated with tacalcitol for up to 18 months, we obtained single-cell suspensions of a representative psoriatic lesion after 0, 8, 12, 18 and 24 weeks of treatment . A Psoriasis Area and Severity Index was performed at each visit as well . Cell suspensions were stained with markers for inflammation (Vim3B4), differentiation (RKSE60) and proliferation (TO-PRO-3 iodide) and analysed flow cytometrically . Clinically, patients improved significantly after 8 weeks of treatment . This clinical effect was preserved for the rest of the period of treatment with no further significant improvement . Proliferative activity also decreased significantly after 8 weeks of treatment . Proliferation did not show further significant decreases or habituation after 12, 18 and 24 weeks . For inflammation, no statistically reliable trends could be seen . Differentiation improved significantly after 8 weeks of treatment, but decreased again significantly after 12 weeks . In the period from 12 to 24 weeks, no further significant change was observed . We conclude that tacalcitol is an effective antipsoriatic drug . Prolonged treatment with tacalcitol will generally maintain improvement at the level reached after 8 weeks . Owing to the beneficial effect on both clinical state and proliferation, tacalcitol is likely to be an adequate maintenance therapy. J Hepatol, 1998 Sep, 29(3), 450 - 4 Isolation from human fetal liver of cells co-expressing CD34 haematopoietic stem cell and CAM 5.2 pancytokeratin markers; Lemmer ER et al.; BACKGROUND/AIMS: Ductal plate and bile duct cells in developing human liver express haematopoietic stem cell markers, such as c-kit and CD34, in association with cytokeratin markers CAM 5.2 and CK 18 . The identification of such ductal plate cells as likely progenitors for both bile duct epithelial cells and hepatocytes and their possible reappearance as oval cells in the regenerating liver have generated much interest in their pluripotential capacities . This study aimed to isolate cells from human fetal liver that co-express haematopoietic stem cell and epithelial cell markers . METHODS: Human fetal liver was harvested following legal termination of pregnancy at week 14-22 . CD34+ mononuclear cells were isolated from liver cell suspensions with immunomagnetic beads . Immunofluorescent staining, using anticytokeratin CAM 5.2 against CK 8 and 18, was performed on permeabilised CD34+ cells for flow cytometry and fluorescent microscopy . CD34+ cells were also stained for other stem cell markers (HLA-DR, c-kit) and committed haematopoietic cell markers (CD33, CD38) . RESULTS: Approximately 0.9% (range 0.07-4.0%) of the mononuclear cells isolated were CD34+ cells . The number of mononuclear cells isolated correlated with fetal liver weight (r=0.508) . About 3-8% of these CD34+ cells stained positively for CAM 5.2 . In addition, CD34+ cells were positive for HLA-DR, but only a small percentage was positive for c-kit . Staining for the committed haematological markers, CD33 and CD38, was consistently negative . CONCLUSIONS: This study describes an immunoaffinity method for the enrichment from human fetal liver of cells that co-express haematopoietic stem cell and epithelial cell markers . Such cellular subsets may correspond to pluripotential ductal plate and bile duct cells. Biotechnol Prog, 1998 Sep, 14(5), 797 - 9 Intermittent light irradiation with a second-scale interval enhances caffeine production by coffea arabica cells Kurata H, Achioku T, Okuda N, Furusaki S. We developed novel equipment that intermittently illuminates Coffea arabica cell suspensions at a second-scale interval and investigated how intermittent irradiation enhances caffeine biosynthesis by C . arabica cells . The light/dark cycles consisting of 2 s of illumination and 18 s of darkness enhanced caffeine production, reaching the same level as for continuous light . The intermittent illumination increased the production efficiency regarding light consumption by a factor of 10 . Caffeine production was determined by light intensity regardless of intermittent or continuous light irradiation . We propose a new concept for designing a photobioreactor that is applicable to secondary metabolite production by plant cell culture. J Anat, 1998 Jul, 193 ( Pt 1), 49 - 59 Splenic adherent cells, stimulated in vitro, induce the reactive formation of lymphoid follicles and germinal centres in draining lymph nodes after subcutaneous transfusion into syngeneic mice; Tanaka K et al.; Th