Cell suspensions

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.; The reactive formation of lymphoid follicles and germinal centres in lymph nodes, induced by subcutaneous transfer of in vitro activated splenic adherent cells into syngeneic mice, were studied . Adherent cells were obtained by incubating spleen cell suspensions for 24 h and activated by incubating for 1 h in the medium containing keyhole limpet haemocyanin (KLH) absorbed onto alumina . Some of the treated adherent cells were irradiated with 10 Gy x-rays, while others were either not stimulated or were stimulated with alumina-KLH but killed by repeated freezing and thawing . Examination of adherent cell smears immunostained with antibodies against, F4/80, Mac-1, Mac-2 and NLDC-145 indicated that many adherent cells displayed macrophage markers but few displayed the interdigitating cell marker . Animals transfused with KLH-treated adherent cells with or without irradiation showed a marked increase in the number of lymphoid follicles and germinal centres in draining lymph nodes, whereas those transfused with adherent cells which had not been KLH-treated or which had been killed after KLH treatment displayed no significant change in the number of follicles . These results were interpreted as indicating that following transfusion, antigen-activated adherent macrophages migrated into the draining lymph nodes and induced the reactive formation of lymphoid follicles and germinal centres outside preexisting follicles.

Ecotoxicol Environ Saf, 1998 Sep, 41(1), 51 - 8
Detection of DNA strand breaks in isolated mussel (Mytilus edulis L . ) digestive gland cells using the "Comet" assay; Mitchelmore CL et al.; Isolated mussel (Mytilus edulis L.) digestive gland cells were analyzed using the single-cell gel electrophoresis or "comet" assay to assess the ability of potential aquatic contaminants to induce DNA strand breaks (SBs) and to investigate the potential application of this technique as part of an aquatic biomonitoring regime . Freshly prepared cell suspensions from digestive gland were exposed in vitro to hydrogen peroxide (H2O2, 0-200 microM), 3-chloro-4-(dichloromethyl)-5-hydroxy-2{5H}-furanone (MX, 0-200 microM), benzo{a}pyrene (BaP, 0-200 microM), 1-nitropyrene (1-NP, 0-250 microM) and nitrofurantoin (NF, 0-1000 microM) for 1 h in the dark at 15 degreesC in the presence of the DNA repair inhibitor cytosine-beta-D-arabinofuranoside (araC) . DNA strand breakage was measured using the comet assay . There were significant concentration-dependent increases in the percentage of DNA in the comet tail (mean values+/-SD) for all doses compared with controls (P<0.05) with H2O2 (up to 61.4+/-5.1% at 100 microM), MX (up to 34 . 3+/-2.2% at 200 microM), BaP (up to 24.7+/-5.1 at 100 microM), 1-NP (up to 54.7+/-5.0% at 200 microM), and NF (up to 68.1+/-4.5% at 500 microM) . There was a decrease (P<0.05) in viability (eosin Y exclusion) of exposed compared with control cells at 200 microM H2O2 and BaP only . This study has demonstrated the potential of the comet assay to detect DNA strand breakage at subcytotoxic concentrations of a range of agents, some of which require metabolic activation . This may provide a sensitive, but nonspecific, molecular biomarker of genotoxicity .

Hepatology, 1998 Oct, 28(4), 1051 - 7
Nitric oxide inactivates rat hepatic methionine adenosyltransferase In vivo by S-nitrosylation; Ruiz F et al.; We investigated the mechanism of nitric oxide (NO) action on hepatic methionine adenosyltransferase (MAT) activity using S-nitrosoglutathione (GSNO) as NO donor . Hepatic MAT plays an essential role in the metabolism of methionine, converting this amino acid into S-adenosylmethionine . Hepatic MAT exists in two oligomeric states: as a tetramer (MAT I) and as a dimer (MAT III) of the same subunit . This subunit contains 10 cysteine residues . In MAT I, S-nitrosylation of 1 thiol residue per subunit was associated with a marked inactivation of the enzyme (about 70%) that was reversed by glutathione (GSH) . In MAT III, S-nitrosylation of 3 thiol residues per subunit led to a similar inactivation of the enzyme, which was also reversed by GSH . Incubation of isolated rat hepatocytes with S-nitrosoglutathione monoethyl ester (EGSNO), a NO donor permeable through the cellular membrane, induced a dose-dependent inactivation of MAT that was reversed by removing the NO donor from the cell suspension . MAT, purified from isolated rat hepatocytes, contained S-nitrosothiol groups and the addition of increasing concentrations of EGSNO to the hepatocyte suspension led to a progressive S-nitrosylation of the enzyme . Removal of the NO donor from the incubation media resulted in loss of most NO groups associated to the enzyme . Finally, induction in rats of the production of NO, by the administration of bacterial lipopolysaccharide (LPS), induced a fivefold increase in the S-nitrosylation of hepatic MAT, which led to a marked inactivation of the enzyme . Thus, the activity of liver MAT appears to be regulated in vivo by S-nitrosylation.

Indian J Biochem Biophys, 1998 Apr, 35(2), 97 - 102
Poly-ADP-ribosylation of histone proteins of human kidney T1-cells in vitro following gamma-irradiation; Sharan RN et al.; Poly-ADP-ribosylation of cellular proteins is involved with radiation induced damage and its repair . It has been observed that suspension of human kidney T1-cells in vitro attained elevated levels of poly-ADP-ribosylation due to experimental manipulations necessary for preparation of single cell suspension from monolayer cell cultures . These cells in suspension were exposed to various doses of gamma-rays with or without subsequent repair incubation . The PADPR of histones H3, H1 and H2B increased with increasing dose of radiation and decreased after 90 min or repair incubation . Concomitant with these changes, the affinity of histones to DNA in chromatin reduced immediately after irradiation . Normal affinity was reestablished after post-irradiation repair incubation . The results indicate that induction of poly-ADP-ribosylation of histone proteins by radiation and by manipulations to prepare single cell suspension involved different cellular components.

Arthritis Rheum, 1998 Sep, 41(9), 1632 - 8
Linkage of chondrocyte apoptosis and cartilage degradation in human osteoarthritis; Hashimoto S et al.; OBJECTIVE: To examine the occurrence of apoptosis in human osteoarthritis (OA) cartilage, and to determine its relationship to cartilage degradation . METHODS: Knee cartilage was obtained from subjects at autopsy, from a tissue bank, and from OA patients undergoing total joint replacement surgery . Chondrocytes were isolated and the number of apoptotic cells was analyzed by flow cytometry . Apoptotic cells in cartilage sections were identified by the detection of DNA strand breaks . Electron microscopy was applied to demonstrate morphologic changes, and Safranin O staining was performed to analyze the relationship between apoptosis and proteoglycan depletion . RESULTS: Flow cytometry on cell suspensions prepared from collagenase digests of cartilage showed that approximately 22.3% of OA chondrocytes and 4.8% of normal chondrocytes were undergoing apoptosis . Staining of cartilage sections demonstrated the presence of apoptotic cells in the superficial and middle zones . Cartilage areas that contained apoptotic cells showed proteoglycan depletion, and the number of apoptotic cells was significantly correlated with the OA grade . CONCLUSION: These observations demonstrate increased chondrocyte apoptosis in OA cartilage . Chondrocyte apoptosis and proteoglycan depletion are anatomically linked and may be mechanistically related.

Dis Colon Rectum, 1998 Sep, 41(9), 1107 - 11
Excision of trocar sites reduces tumor implantation in an animal model; Wu JS et al.; PURPOSE: The purpose of this study was to determine the effect of excising abdominal trocar wound sites after pneumoperitoneum on the rate of trocar site tumor implantation in a hamster model . This would help determine whether tumor cells seed trocar sites during or after pneumoperitoneum . METHODS: A total of 0.5 ml of GW-39 human colon cancer cell suspension at 2.5 percent v/v (8 x 10(5) cells) was injected into the abdomens of 77 hamsters through a midline incision . Animals were subjected to ten minutes of pneumoperitoneum, after placement of four abdominal trocars, and then randomly assigned to undergo either simple suture closure or 4-mm radius trocar wound site excision at the end of the procedure . Gross and microscopic tumor implants were documented seven weeks later . RESULTS: There were three and four deaths in simple suture closure and wound site excision groups, respectively . Of the remaining 35 hamsters in each group, tumor cells implanted at 89 and 78 percent of trocar sites, respectively (P < 0.03) . There was no significant difference between the two groups in tumor implantation at midline laparotomy sites . Wound site excision also resulted in fewer palpable tumors (44 vs . 61 percent; P < 0.01) and a lower tumor implantation rate (49 vs . 74 percent; P < 0.05) at all four concurrent sites compared with simple suture closure . CONCLUSIONS: Excision of laparoscopic abdominal trocar wound sites can significantly, but not completely, reduce tumor implantation rate compared with simple wound closure.

Phytochemistry, 1998 Sep, 49(2), 403 - 11
Differential regulation and distribution of acridone synthase in Ruta graveolens; Junghanns KT et al.; Cell suspension cultures of Ruta graveolens L . accumulate polyketide metabolites such as acridone alkaloids and flavonoid pigments . Whereas flavonoid synthesis is induced by light, the production of alkaloids can be enhanced in dark-cultured cells by treatment with fungal elicitors . Acridone synthase (ACS) catalyzes the committed condensing reaction of acridone biosynthesis yielding 1,3-dihydroxy-N-methylacridone from N-methylanthraniloyl- and malonyl-CoAs . The reaction proceeds in a manner analogous to that of chalcone synthase (CHS) which catalyzes the first committed step in flavonoid biosynthesis and cDNA and protein sequences of Ruta ACS possess a high degree of sequence homology to heterologous CHSs . ACS transcript abundance and specific activity were monitored in cultured R . graveolens cells irradiated either continuously with white light or treated with fungal elicitor over a period of 24 h and found to increase transiently upon elicitor treatment and to decrease upon light irradiation . Immunodetection with a rabbit polyclonal ACS antiserum revealed that the amounts of ACS polypeptide decreased slightly in light-irradiated cells but increased in elicitor-treated Ruta cells . Fluorescence microscopy and tissue print hybridizations were employed to aid in localizing the sites of storage and biosynthesis of acridone alkaloids in Ruta plants . Yellow fluorescing alkaloids were detected particularly in root tissue adjacent to the rhizodermis, but also in the endodermis and vascular tissue of the hypocotyl . ACS transcript abundance in situ followed the same spatial pattern, indicating that the synthesis of acridones likely proceeds at all sites of deposition rather than exclusively in the root . Expression in planta and the induction response of ACS suggest that the alkaloids serve as phytoanticipins or phytoalexins in the defense of Ruta particularly to soil-borne pathogens or as feeding deterrents.

Infect Immun, 1998 Oct, 66(10), 4981 - 8
An ex vivo study of T lymphocytes recovered from the lungs of I/St mice infected with and susceptible to Mycobacterium tuberculosis; Lyadova I et al.; I/St mice, previously characterized as susceptible to Mycobacterium tuberculosis H37Rv, were given 10(3) or 10(5) CFU intravenously . At two time points postinoculation, the cell suspensions that resulted from enzymatic digestion of lungs were enumerated and further characterized phenotypically and functionally . Regarding the T-cell populations recovered at 2 and 5 weeks postinfection, two main results were obtained: (i) the population of CD44(-) CD45RB+ cells disappeared within 2 weeks postinfection, while the number of CD44(+) CD45RB-/low cells slowly increased between weeks 2 and 5; (ii) when cocultured with irradiated syngeneic splenocytes, these lung T cells proliferated in the presence of H37Rv sonicate . Using H37Rv sonicate and irradiated syngeneic splenocytes to reactivate lung T cells, we selected five CD3(+) CD4(+) CD8(-) T-cell clones . In addition to the H37Rv sonicate, the five clones react to both a short-term culture filtrate and an affinity-purified 15- to 18-kDa mycobacterial molecule as assessed by the proliferative assay . However, there was a clear difference between T-cell clones with respect to cytokine (gamma interferon {IFN-gamma} and interleukin-4 {IL-4} and IL-10) profiles: besides one Th1-like (IFN-gamma+ IL-4(-)) clone and one Th0-like (IFN-gamma+ IL-4(+) IL-10(+)) clone, three clones produced predominantly IL-10, with only marginal or no IL-4 and IFN-gamma responses . Inhibition of mycobacterial growth by macrophages in the presence of T cells was studied in a coculture in vitro system . It was found that the capacity to enhance antimycobacterial activity of macrophages fully correlated with INF-gamma production by individual T-cell clones following genetically restricted recognition of infected macrophages . The possible functional significance of cytokine diversity among T-cell clones is discussed.

J Hypertens, 1998 Sep, 16(9), 1261 - 6
Protein carboxyl methylation controls intracellular pH in human platelets; Otsuka K et al.; OBJECTIVES: Carboxyl methylation is a reversible post-translational event which regulates the function of several cellular proteins . Because the human Na+-H+ antiporter (NHE-1) possesses a C-terminal consensus sequence for carboxyl methylation, we examined the role of protein carboxyl methylation in the regulation of intracellular pH homeostasis . DESIGN: Experiments were conducted using human platelets and N-acetyl-S-trans,trans-farnesyl-L cysteine (AFC), a specific prenylcysteine methyltransferase inhibitor . The effect of AFC on both basal intracellular pH (pHi) and on the kinetic properties of the Na+-H+ antiporter was characterized . MATERIALS AND METHODS: pHi was determined in cell suspensions using 2,7-biscarboxyethyl-5(6)-carboxyfluorescein tetraacetoxymethyl ester, a fluorescent pH indicator . The kinetics properties of the Na+-H+ antiporter activity were determined using platelets acidified with nigericin and challenged with varying extracellular concentrations of Na+ . RESULTS: AFC (20 micromol/l) decreased basal pHi significantly (7.047 +/- 0.011 versus 7.133 +/- 0.012 for control, P< 0.001) . The acidification was dose-dependent and reached steady state 3 min after AFC addition . In the absence of extracellular Na+, the platelets were acidified to the same extent with AFC or with ethanol (control): 6.530 +/- 0.031 versus 6.532 +/- 0.031 (P= 0.97) . However, upon addition of Na+, the platelets treated with AFC showed a significant decrease in the maximal value for initial pHi recovery compared with controls: 0.788 +/- 0.041 versus 0.983 +/- 0.047 pH/min (P< 0.02) . AFC also increased the Hill coefficient (2.89 +/- 0.22 versus 2.14 +/- 0.16, P < 0.03), and tended to decrease K0.5, the {Na+} corresponding to half-maximal activation (51.3 +/- 1.8 versus 60.5 +/- 3.9 mmol/l, P = 0.06) of the antiporter . CONCLUSION: Our data indicate that inhibition of carboxyl methylation reduces basal pHi and alters the kinetic properties of the Na+-H+ antiporter in human platelets, suggesting that carboxyl methylation is implicated in the regulation of intracellular pH homeostasis.

Neurotoxicology, 1998 Aug-Oct, 19(4-5), 605 - 8
Insect (Locusta migratoria migratorioides) test monitoring the toxicity of cyanobacteria; Hiripi L et al.; An insect test was developed to investigate the toxicity of cyanobacteria . The African locust, Locusta migratoria migratorioides R.F . was used as a test animal instead of mouse . The cyanobacteria tested were Aphanizomenon flos-aque, Anabaena aphanizomenoides, Cylindrospermopsis raciborskii, Microcystis aeruginosa . The toxicity of authentic microcystin-LR was also tested . Cyanobacteria producing toxins killed the animals when the homogenized cell suspension was injected into the animals . The locust test proved to be more sensitive than the mouse test . The LD50 values of the different cyanobacteria for locusts and for mice, respectively were the following: 90 microg/animal (60 mg/kg) and 8000 microg/animal (320 mg/kg), for Aphanizomenon flos-aquae; 255 microg/animal (170.2 mg/kg) and 3750 microg/animal (150 mg/kg), for Anabaena aphanizomenoides; 195 microg/animal (131.4 mg/kg) and 5750 microg/animal (230 mg/kg), for Cylindrospermopsis raciborskii; 22.5 microg/animal (15 mg/kg) and 6000 microg/ animal (240 mg/kg), for Microcystis aeruginosa . In locusts the LD50 value for authentic microcystin-LR was 0.2 microg/animal (130 mg/kg) . Since the weight of the mice is 15 to 20 times larger than that of the locusts, hence less toxic cells are needed to kill the locusts . The locust test is cheaper than the mouse test, large number of animals can be used in the experiments and the LD50 values can be estimated more precisely . The toxicity of C . raciborskii was significantly lower when the lyophilized cells were extracted in methanol (LD50 = 767 mg/kg), instead of NaCl solution (LD50 = 131.4 mg/kg).

J Comp Neurol, 1998 Oct 5, 399(4), 530 - 40
Co-grafted embryonic striatum increases the survival of grafted embryonic dopamine neurons; Sortwell CE et al.; To enhance the current therapeutic benefit of dopamine (DA) neuron grafts in Parkinson's disease, strategies must be developed that increase both DA neuron survival and fiber outgrowth into the denervated striatum . Previous work in our laboratory has demonstrated that dopaminergic neurons grow to greater size when co-grafted with striatal cell suspensions and display extensive tyrosine hydroxylase-positive (TH+) projections, but no conclusion could be reached concerning enhancement of survival of grafted DA neurons . The aim of the present study was to characterize further the potential trophic effects of striatal co-grafts on grafted mesencephalic DA neuron survival . Unilaterally lesioned male Fischer 344 rats were grafted with either a suspension of mesencephalic cells or with both mesencephalic and striatal cell suspensions . Co-grafts were either mixed together or placed separately into the striatum . Lesioned rats receiving no graft served as controls . Rotational behavior was assessed following amphetamine challenge at 2 weeks prior to grafting and at 4 and 8 weeks following grafting . Only rats receiving co-grafts of nigral and striatal suspensions separated by a distance of 1 mm showed significant behavioral recovery from baseline rotational asymmetry . Both mixed and separate striatal co-grafts were associated with a doubling of DA neuron survival compared with solo mesencephalic grafts . In the mixed co-graft experiment, DA neurite branching appeared enhanced and TH-rich patches were observed, whereas with co-grafts that were separated, TH+ innervation of the intervening host striatum was increased significantly . These results provide the first evidence suggesting that nigral-striatal co-grafts, particularly those placed separately and in proximity to each other, increase both DA neuron survival and neurite extension from the mesencephalic component of the grafts.

Biochim Biophys Acta, 1998 Sep 16, 1404(3), 475 - 83
Extracellular Ca2+ regulates the respiratory burst of human neutrophils; Bei L et al.; The role of extracellular calcium in the activation of respiratory burst in human neutrophils was studied by using the receptor agonist, N-formyl-methionyl-leucyl-phenylalanine (fMLP), and the activator of protein kinase C phorbol myristate acetate (PMA) . The level of intracellular free calcium was measured by using both cell suspensions and single cells in the presence and absence of extracellular calcium . The Ca2+-ATPase inhibitor, thapsigargin, was used to activate higher Ca2+ influx, while a novel calcium channel blocker, panax notoginseng saponins (PNGS) was used to block the Ca2+ entry from extracellular space during the responding period of cells . It was found that about two-thirds of the activation of respiratory burst initiated by the receptor agonist were attributed to the Ca2+ influx under normal physiological conditions . The higher Ca2+ influx resulted in tremendous enhancement of the intensity of respiratory burst initiated by fMLP and marked acceleration of the onset of the respiratory burst stimulated by PMA . It is evident that both intra- and extracellular Ca2+ are required for full activation of the respiratory burst of human neutrophils, and the Ca2+ influx from extracellular space plays an important role either in generation of reactive oxygen metabolites or in activation of protein kinase C.

Brain Res, 1998 Sep 21, 806(1), 60 - 8
Strands of embryonic mesencephalic tissue show greater dopamine neuron survival and better behavioral improvement than cell suspensions after transplantation in parkinsonian rats; Clarkson ED et al.; The success of embryonic neural transplants as a treatment for patients with Parkinson's disease has been limited by poor survival of transplanted dopamine neurons . To see if a new partially intact tissue preparation method improves survival, we have developed a technique for extruding embryonic tissue into strands . We expected this method to reduce cell damage and improve transplant survival as well as provide improved tissue delivery . We have compared transplants of tissue strands with mechanically dispersed suspensions of embryonic day 15 rat ventral mesencephalon . Tissue from ventral mesencephalon was transplanted into a single site in dopamine denervated striatum of unilateral 6-hydroxydopamine (6-OHDA) lesioned rats . To evaluate the effects of striatal cografts and growth factors on dopamine cell survival, dispersed mesencephalic cells were cotransplanted with dispersed striatal cells . Another group had dispersed mesencephalic cells cotransplanted with striatal cells incubated in the cold for 2 h with glial cell line-derived neurotrophic factor (GDNF, 100 ng/ml), insulin-like growth factor-I (IGF-I, 1500 ng/ml), and basic fibroblast growth factor (bFGF, 150 ng/ml) . Behavioral improvement was assessed monthly by changes in methamphetamine-induced rotational behavior . Animals were sacrificed after 3 months, and dopamine neurons were identified by tyrosine hydroxylase (TH) immunohistochemistry . Transplants of tissue strands produced better dopamine neuron survival and led to more robust behavioral restoration than did cell suspensions even when suspensions were supported with cografts of striatal cells or pretreatment with growth factors .

J Cell Sci, 1998 Oct, 111 ( Pt 20), 3091 - 100
A cell cycle regulated MAP kinase with a possible role in cytokinesis in tobacco cells; Calderini O et al.; Mitogen-activated protein (MAP) kinases have been demonstrated to have a role in meiosis but their involvement in mitotic events is less clear . Using a peptide antibody raised against the tobacco MAP kinase p43(Ntf6) and extracts from synchronized tobacco cell suspension cultures, we show that this kinase is activated specifically during mitosis . Entry into mitosis appears to be necessary for the activation of the kinase, which occurs as a post-translational event . The activation of the kinase occurs in late anaphase/early telophase . The p43(Ntf6) protein shows a transient localization to the cell plate in anaphase cells, in the middle of the two microtubule arrays characteristic of the phragmoplast, a plant-specific structure involved in laying down the new cell wall . The combined data support a role for the MAP kinase p43(Ntf6) in cytokinesis in tobacco cells.

Food Chem Toxicol, 1998 Sep-Oct, 36(9-10), 761 - 70
Effects of the garlic components diallyl sulfide and diallyl disulfide on arylamine N-acetyltransferase activity in human colon tumour cells; Chen GW et al.; Diallyl sulfide (DAS) and diallyl disulfide (DADS), major components of garlic, were used to determine inhibition of arylamine N-acetyltransferase (NAT) activity in a human colon tumour (adenocarcinoma) cell line . Two assay systems were performed, one with cellular cytosols (9000g supernatant), the other with intact bacterial cell suspensions . The NAT activity in a human colon tumour cell line was inhibited by DAS and DADS in a dose-dependent manner in both system: that is, the greater the concentration of DAS and DADS in the reaction, the greater the inhibition of NAT activities in both systems . The data also indicated that DAS and DADS decrease the apparent values of Km and Vmax of NAT enzymes from human colon tumour cells in both systems examined . This is the first report to demonstrate that garlic components do affect human colon tumour cell NAT activity.

J Cell Biochem, 1998 Oct 1, 71(1), 127 - 39
Several novel transcripts of glyceraldehyde-3-phosphate dehydrogenase expressed in adult chicken testis; Mezquita J et al.; Glyceraldehyde-3-phosphate dehydrogenase (GAPDH), in addition to being a classic glycolytic enzyme, is a multifunctional protein involved in relevant cell functions such as DNA replication, DNA repair, translational control of gene expression, and apoptosis . Although the multifunctional nature of GAPDH suggests versatility in the mechanisms regulating its expression, no major qualitative changes and few quantitative changes in the GAPDH transcripts have been reported . While studying the expression of GAPDH during spermatogenesis, we detected alternative initiations to TATA box and alternative splicings in the 5' region of the pre-mRNA, resulting in at least six different types of mRNAs . The amount and the polyadenylation of the GAPDH transcripts increased in mature testis in relation to immature testis and further increased when cell suspensions from mature testis were exposed to heat shock . These results suggest that alternative initiation, alternative splicing, and polyadenylation could provide the necessary versatility to the regulation of the expression of this multifunctional protein during spermatogenesis.

Biotech Histochem, 1998 Jul, 73(4), 186 - 97
Chimeric antibodies with specificity for tumor antigens: demonstration of in situ localization to tumors after antibody therapy; Saleh MN et al.; In this study, we compare various methods for the detection of a tumor-associated target antigen and deposition of the bound therapeutic monoclonal antibody in patients enrolled in two separate trials, one involving the administration of two radiolabeled monoclonal antibodies and the other involving an unlabeled antibody . In the first trial, patients with TAG-72 expressing metastatic colon cancer scheduled for surgical intervention received radiolabeled murine and chimeric B72.3 antibody followed by radioimmune imaging and subsequent laparotomy . Normal and tumor tissues obtained at surgery were processed for routine histology, immunohistochemistry, radiometry, and autoradiography . Both anti-TAG-72 antibodies localized to known tumor sites as evidenced by radioimmune imaging . Resected tissue revealed a high tumor-to-normal radiolocalization ratio, and autoradiography demonstrated even deposition of the radiolabeled antibodies throughout the entire tumor deposit with sparing of surrounding normal tissue . In contrast, immunohistochemistry on the same sections revealed comparatively weak antigen expression and patchy antibody localization . In the second trial, patients with GD2 antigen expressing metastatic melanoma received the unlabeled chimeric anti-GD2 antibody C14.18 . Immunologic detection of the GD2 antigen and C14.18 deposition was performed on biopsy section as well as on single cell suspension . FACS analysis of the single cell suspension proved more sensitive for the detection of bound antibody than immunohistochemistry, although both methods yielded comparable results for GD2 antigen expression . Our findings demonstrate that the optimal method for the detection of tumor-associated antigen and bound therapeutic antibody can vary depending upon the nature of the antibody (radiolabeled vs . unlabeled and murine vs . chimeric), fixation stability of the target antigen, and the type of pathologic material available for study.

J Surg Res, 1998 Jul 1, 77(2), 137 - 40
5-HT induces cAMP production in crypt colonocytes at a 5-HT4 receptor; Albuquerque FC Jr et al.; Previous studies demonstrate that both 5-hydroxytryptamine (5-HT) and cyclic AMP (cAMP) induce chloride efflux from crypt colonocytes in the rat distal colon; antagonist studies suggest that the 5-HT response is mediated primarily by the 5-HT4 receptor . Since this receptor is known to be positively coupled to adenylate cyclase, we postulated that 5-HT should induce generation of cAMP, which should be inhibited by 5-HT4 antagonists . Method . Mucosal cells from rat distal colon were taken by a sequential calcium chelation technique for enrichment of crypt cells . Cytokeratin stains demonstrated that >99% of cells were colonocytes . {3H}Thymidine uptake studies demonstrate a fivefold increased incorporation in this cell preparation compared to earlier fractions . 3-Isobutyl-l-methylxanthine (IBMX, 100 microM) was added to all cell suspensions in order to prevent cAMP metabolism . Cell suspensions were incubated for 2 min at 37 degreesC with different concentrations of 5-HT (n = 7) . cAMP was measured by enzyme immunoassay . In another series of experiments, 5-HT (0.3 microM) stimulation of cAMP was similarly measured in the presence and absence of 5-HT receptor antagonists: 10 microM 5-HTP-DP (5-HT1P; n = 4), 0.1 microM ketanserin (5-HT2A; n = 4), 0.3 microM ondansetron (5-HT3; n = 4), 3 microM tropisetron (5-HT3 and 5-HT4; n = 4), and 10 nM GR-113808 (5-HT4; n = 5) . Results . 5-HT produced a dose-dependent increase in cAMP . The increase was significant at concentrations >/=0.3 microM when compared to cells incubated with IBMX alone . In the second series of experiment, 5-HT-induced generation of cAMP at a dose of 0.3 microM was significantly inhibited in the presence of GR-113808 and tropisetron . Conclusion . 5-HT acts at a 5-HT4 receptor to induce production of cAMP in rat distal crypt colonocytes .

Plant Physiol, 1998 Sep, 118(1), 209 - 18
Piperonylic acid, a selective, mechanism-based inactivator of the trans-cinnamate 4-hydroxylase: A new tool to control the flux of metabolites in the phenylpropanoid pathway
Schalk M, Cabello-Hurtado F, Pierrel MA, Atanossova R, Saindrenan P, Werck-Reichhart D.
Piperonylic acid (PA) is a natural molecule bearing a methylenedioxy function that closely mimics the structure of trans-cinnamic acid . The CYP73A subfamily of plant P450s catalyzes trans-cinnamic acid 4-hydroxylation, the second step of the general phenylpropanoid pathway . We show that when incubated in vitro with yeast-expressed CYP73A1, PA behaves as a potent mechanism-based and quasi-irreversible inactivator of trans-cinnamate 4-hydroxylase . Inactivation requires NADPH, is time dependent and saturable (KI = 17 &mgr;M, kinact = 0.064 min-1), and results from the formation of a stable metabolite-P450 complex absorbing at 427 nm . The formation of this complex is reversible with substrate or other strong ligands of the enzyme . In plant microsomes PA seems to selectively inactivate the CYP73A P450 subpopulation . It does not form detectable complexes with other recombinant plant P450 enzymes . In vivo PA induces a sharp decrease in 4-coumaric acid concomitant to cinnamic acid accumulation in an elicited tobacco (Nicotiana tabacum) cell suspension . It also strongly decreases the formation of scopoletin in tobacco leaves infected with tobacco mosaic virus.

Haematologia (Budap), 1998, 29(2), 101 - 14
The IL-6 gene expression by leukemic cells from acute lymphoblastic leukemia common and T type and modulation of IL-6 production by TNF; Pituch-Noworolska A et al.; The tumour necrosis factor alpha (TNF-alpha) and interleukin-6 (IL-6) mRNA accumulation and the release of the cytokines TNF-alpha and IL-6 was determined in leukemic cells isolated from bone marrow biopsy from patients with acute lymphoblastic leukemia: ALL-common type (cALL), 11 patients; ALL-T type, nine patients . The non-leukemic bone marrow cells (BMMC) and peripheral blood mononuclear cells (PBMC) from healthy donors were used as a control . The mRNA was assessed by fluorescent in situ hybridization in cell suspension and analyzed with flow cytometry . The accumulation of cytokine mRNA was higher in cALL cells as compared to ALL-T and PBMC (control) and was comparable to cytokines mRNA accumulation in BMMC . The production of IL-6 by leukemic cells from both types of leukemia was significantly lower than in BMMC . The bioactive TNF was not detected in either of the leukemia groups studied . TNF-alpha protein was produced by ALL-T cells and BMMC but not by cALL type of leukemic cells . The synthesis of IL-6 was significantly enhanced by TNF-alpha in BMMC and ALL-T while the presence of TNF-alpha had no effect on IL-6 synthesis in the culture of cALL leukemic cells . It was concluded that despite IL-6 and TNF-alpha mRNA contents, leukemic cells representing early stage of B-cell development (CD10+) showed disregulation of production of these cytokines.

Steroids, 1998 Sep, 63(9), 459 - 63
Non-ACTH POMC fragments stimulate aldosterone production by human adrenal cells in vitro; Molloy ES et al.; The possibility that non-ACTH proopiomelanocortin-derived fragments may stimulate aldosterone production has previously been studied using nonhuman cells with inconsistent results . We have examined the response of aldosterone to beta-endorphin (beta-End) and joining peptide (JP) and compared these with the response to ACTH using eight cell suspensions prepared from human adrenal glands . ACTH, 10(-6), 10(-8), and 10(-10) M, consistently stimulated aldosterone accumulation above that occurring in unstimulated cells (150 +/- 83, 120 +/- 62, and 77 +/- 32 fmol/10(4) cells above basal, respectively; mean +/- SE; p < 0.05) . beta-End significantly stimulated aldosterone production at 10(-6) and 10(-8) M (114 +/- 84 and 50 +/- 24 fmol/10(4) cells above basal; p < 0.05); 10(-10) M beta-End did not provide significant stimulation . Furthermore, JP stimulated aldosterone biosynthesis (41 +/- 16 fmol/10(4) cells above basal; p < 0.05), only at the highest concentration used, 10(-6) M . The addition of 10(-8) M ACTH plus 10(-6) and 10(-10) M beta-End to human adrenal cells yielded values significantly greater than those achieved with either agent alone (267 +/- 152 and 183 +/- 89 fmol/10(4) cells above basal; p < 0.05) . These data indicate for the first time that beta-End and JP have the capacity to stimulate aldosterone production in human adrenal cells in vitro . The physiological and potential clinical significance of these observations has yet to be elucidated.

Am J Physiol, 1998 Sep, 275(3 Pt 2), H776 - 82
Depression of endothelial and smooth muscle cell oxygen consumption by endotoxin; Motterlini R et al.; An optical method based on the oxygen-dependent quenching of a phosphorescent probe (palladium-porphyrin) was used to investigate the effect of bacterial endotoxin {lipopolysaccharide (LPS)} on oxygen consumption (VO2) by vascular cells . Endothelial (EC) and smooth muscle (SMC) cells from pig aorta were suspended in culture medium in the presence of palladium-porphyrin and transferred to glass capillary tubes that were sealed to create a hypoxic environment . Measured PO2 changed as a function of time in a highly predictable fashion when cell suspensions were exposed to agents or treatment known to affect cellular metabolism . Both EC and SMC showed a significant decrease in VO2 as cell density increased, and SMC VO2 was significantly higher than EC (1.94 +/- 0.09 vs . 1.0 +/- 0.15 nmol . min-1 . 10(6) cells-1) . Exposure to LPS (1 microg/ml) caused a decrease in VO2 of 46% and 15% for EC and SMC, respectively . Pretreatment of cells with N-acetyl-L-cysteine, a substrate for glutathione synthesis with antioxidant properties, restored VO2 to normal values after exposure to LPS . These data suggest that endotoxin impairs VO2 in cells derived from the vascular wall and indicate the importance of EC and SMC respiration in maintaining vascular homeostasis under conditions of sepsis.

Mech Ageing Dev, 1998 May 15, 102(2-3), 263 - 77
Relation between exploratory activity and immune function in aged mice: a preliminary study; De la Fuente M et al.; Previous studies show that fast exploration of a T-shaped maze by mature mice may predict an above average longevity . Since the nervous and the immune systems work in a coordinated fashion, and it seems that these two homeostatic systems both influence organismic aging and suffer a senescent decline, we have performed a comparative study of the above behavioral parameter and different functions of three representative immune cells: lymphocytes, macrophages and natural killer (NK) cells obtained from old (76 +/- 1 weeks of age) female OF1-Swiss mice . At 70 weeks of age the mice were divided into a 'fast' and a 'slow' group, containing 100 and 0%, respectively, of animals able to explore the 50 cm-long first arm of the maze in 20 s or less . At 76 +/- 1 weeks of age the animals were sacrificed, the peritoneal cell suspensions were obtained and the immune organs (axillary nodes, spleen and thymus) were isolated . The following leukocyte functions were studied in peritoneal macrophages: adherence to substrate, mobility (spontaneous and chemotaxis), ingestion of particles and superoxide anion production whereas mobility, lymphoproliferative response to the mitogen Con A and NK activity were studied in the immune-organ leukocyte suspensions . The results show that the aged fast mice have better immune functions than the aged slow mice.

Microbiology, 1998 Aug, 144 ( Pt 8), 2271 - 80
Redox poise and oxygenation of cytochrome bd in the diazotroph Azotobacter vinelandii assessed in vivo using diode-array reflectance spectrophotometry; Kavanagh EP et al.; A ferrous oxygenated form of cytochrome d is characteristic of all cytochrome bd-type oxidases so far examined, but its participation in enzyme turnover is unclear . It is relatively stable, occurs in aerated cell suspensions and predominates during enzyme preparation . In this study, diode-array reflectance spectrophotometry was used to assess the redox poise and oxygenation of cytochrome bd in vivo, in the aerobic diazotroph Azotobacter vinelandii . Mutants either lacking or overproducing the cytochrome bd oxidase were used to confirm the reliability of the optical configuration . Changes in absorbance attributed to cytochromes b, c and d were followed as the O2 supply was altered either in suspensions of harvested cells or during steady-state growth . In washed cell suspensions, three states of cytochrome d, which differed in absorbance characteristics, were seen: (1) an oxygenated form that absorbs at 650 nm, (2) a form which has little absorbance at either 650 or 630 nm and (3) the reduced form that absorbs at 630 nm . The transition between states 2 and 3, but not 1 and 2, correlated with the changes in the redox states of cytochromes b595 and b560 . The dissolved O2 concentration at which this transition occurred coincided approximately with the apparent O2 affinity for the oxidase in vivo (approx . 5 microM) . During steady-state growth, the cytochromes were partially reduced and the oxygenated form of cytochrome d was undetected . These in situ measurements support the view that an oxygenated form of cytochrome d (absorbing at 650 nm) in the one-electron-reduced cytochrome bd-type oxidase does not take part in enzyme turnover.

Zentralbl Veterinarmed B, 1998 Aug, 45(6), 353 - 61
Assessment of phagocytic capacity and opsonic activity in blood and mammary secretions during lactation in sows; Magnusson U et al.; The objective of this study was to determine whether phagocytic capacity and opsonic activity in blood and mammary secretions of sows are impaired at parturition compared with later on during lactation . The study comprised eight primiparous sows (Landrace x Yorkshire) free from clinical signs of disease . Blood and mammary secretion samples were collected within 48 h of parturition and 7 and 16 days after parturition . Numbers and proportion of polymorphonuclear neutrophils (PMN) were determined in blood and mammary secretions . Phagocytic capacity was assessed in whole blood and in a cell suspension derived from mammary secretions . Opsonic activity was assessed in serum and i cell-depleted, skimmed mammary secretions . The two assays were based on chemiluminescence, both having zymosan and Escherichia coli as target particles . Numbers and proportion of PMN in mammary secretions were higher (P < 0.05) at parturition than later on during lactation . A parturition, phagocytic capacity in cell suspensions derived from mammary secretions was higher for both (P < 0.05) and E . coli (P < 0.1) . However, when phagocytic capacity was related to the number of PMN in the suspension no such difference was observed . The opsonic activity in cell-depleted, skimmed mammary secretions at parturition was lower (P < 0.05) for zymosan but not for E . coli . None of the described variations were reflected in blood or serum . The findings of this study do not unequivocally support the theory that an immune suppression at parturition in the sow can help explain the increased incidence of coliform mastitis at that time.

Int J Radiat Oncol Biol Phys, 1998 Jul 15, 41(5), 1163 - 70
Response of quiescent and total tumor cells in solid tumors to neutrons with various cadmium ratios; Masunaga S et al.; PURPOSE: Response of quiescent (Q) and total tumor cells in solid tumors to neutron irradiation with three different cadmium (Cd) ratios was examined . The role of Q cells in tumor control was also discussed . METHODS AND MATERIALS: C3H/He mice bearing SCC VII tumors received continuous administration of 5-bromo-2'-deoxyuridine (BrdU) for 5 days using implanted mini-osmotic pumps to label all proliferating (P) cells . Thirty minutes after intraperitoneal injection of sodium borocaptate-10B (BSH), or 3 h after oral administration of dl-p-boronophenylalanine-10B (BPA), the tumors were irradiated with neutrons, or those without 10B-compounds were irradiated with gamma rays . This neutron irradiation was performed using neutrons with three different cadmium (Cd) ratios . The tumors were then excised, minced, and trypsinized . The tumor cell suspensions were incubated with cytochalasin-B (a cytokinesis-blocker), and the micronucleus (MN) frequency in cells without BrdU labeling (Q cells) was determined using immunofluorescence staining for BrdU . The MN frequency in total (P + Q) tumor cells was determined from tumors that were not pretreated with BrdU . The sensitivity to neutrons was evaluated in terms of the frequency of induced micronuclei in binuclear tumor cells (MN frequency) . RESULTS: Without 10B-compounds, the MN frequency in Q cells was lower than that in the total cell population . The sensitivity difference between total and Q cells was reduced by neutron irradiation . Relative biological effectiveness (RBE) of neutrons compared with gamma rays was larger in Q cells than in total cells, and the RBE values for low-Cd-ratio neutrons tended to be larger than those for high-Cd-ratio neutrons . With 10B-compounds, MN frequency for each cell population was increased, especially for total cells . This increase in MN frequency was marked when high-Cd-ratio neutrons were used . BPA increased the MN frequency for total tumor cells more than BSH . Nevertheless, the sensitivity of Q cells treated with BPA was lower than that in BSH-treated Q cells . This tendency was clearly observed in high-Cd-ratio neutrons . CONCLUSION: From the viewpoint of enhancing the Q-cell sensitivity, tumors should be irradiated with high-Cd-ratio neutrons after BSH administration . However, normal tissue reaction remains to be examined because of its low tumor-to-normal tissue and tumor-to-blood biodistribution ratios.

Pflugers Arch, 1998 Oct, 436(5), 782 - 7
Adenovirus-mediated gene expression in isolated rat pancreatic acini and individual pancreatic acinar cells; Padfield PJ et al.; In this study we have examined the feasibility of using replication-deficient recombinant adenoviral vectors to transfer and express genes in pancreatic acinar cells in vitro . We infected primary cultures of both isolated pancreatic acini and individual acinar cells with a recombinant adenovirus containing the coding sequence for beta-galactosidase . Our data demonstrate that recombinant adenoviruses readily infect pancreatic acinar cells in vitro . Close to 100% infection and maximal beta-galactosidase expression were obtained, when acini or acinar cells were infected with 5x10(6) or 10(6) plaque-forming units (pfu) of virus per millitre of acini or acinar cell suspension, respectively . Examination of the time-course of beta-galactosidase expression showed that there was a lag of approximately 6 h before beta-galactosidase levels increased . Thereafter beta-galactosidase expression increased rapidly . By 20 h post-infection beta-galactosidase activity had increased from undetectable levels to 2.5-3.0 units/mg of cellular protein . Acini/acinar cells maintained a robust secretory response after adenoviral infection . The cholecystokinin-octapeptide (CCK8) dose/response curves for amylase secretion for acini and acinar cells infected with 5x10(5) and 1x10(5) pfu/ml of virus, respectively, were biphasic, with maximal amylase secretion being stimulated by 1 nM CCK8 . In addition, the dose/response curves were identical to those obtained from control, sham-infected, acini/acinar cells . Our findings indicate that replication-deficient recombinant adenoviral vectors will be excellent tools to transfer and express genes in isolated pancreatic acini or acinar cells.

Bone Marrow Transplant, 1998 Jul, 22 Suppl 1, S58 - 60
Comparison of repopulating ability of hematopoietic progenitor cells isolated from human umbilical cord blood or bone marrow cells in NOD/SCID mice; Noort WA et al.; The repopulating ability and homing potential of CD34+ cells isolated from either bone marrow (BM) or umbilical cord blood (UCB) was compared in NOD/SCID mice . Mice were sublethally irradiated (3.5 Gy) and within 24 h transplanted i.v . with 10(5)-10(6) CD34+ cells . Four weeks post-transplantation blood was collected from the tail vein for detection of human cells and after 6-7 weeks the mice were sacrificed and blood, BM, thymus, lymph nodes, spleen, liver, lung and kidney were harvested and single cells suspensions were made . Human cells were detected by flow cytometry, and staining was performed for CD34, CD45, and markers of the myeloid, and lymphoid lineages . CD34+ cells from UCB successfully engrafted into the NOD/SCID mice . Eighty-five percent of cells in BM of the mice were of human origin, depending on the dose of cells injected . In all other organs these percentages were always lower, and maximally 63, 13, 3 and 2% in spleen, liver, lung and kidney, respectively . Transplantation of CD34+ cells isolated from human BM, on the other hand, resulted in a very low percentage of human cells after 6-7 weeks of transplantation, not exceeding 3% of the cell suspension . Whether this difference in repopulating ability can be explained by an intrinsic qualitative difference or by differences in quantity of stem cells within the CD34+ compartment from UCB or BM remains to be determined.

Gene, 1998 Aug 17, 216(1), 47 - 53
Characterization of the Mt4 gene from Medicago truncatula; Burleigh SM et al.; Previously we identified Mt4, a phosphate starvation inducible cDNA from Medicago truncatula which is down-regulated in roots in response to phosphate fertilization as well as colonization by arbuscular mycorrhizal (AM) fungi (AM) . Here we present further studies of Mt4 . Expression was highly sensitive to exogenous applications of phosphate fertilizer; transcripts were abundant in roots fertilized with nutrient solution lacking phosphate, reduced when fertilized with 0.02 or 0.1 mM phosphate and undetectable when fertilized with 1 or 5 mM phosphate . A time course experiment, to study the expression of Mt4 following colonization by AM fungi, revealed that Mt4 transcripts increased in uncolonized roots during the first three weeks of growth and then plateaued, while transcript levels in roots colonized with the AM fungus, Glomas versiforme, increased transiently and then decreased . Although the Mt4 gene is expressed exclusively in roots, transcripts were also detected in M . truncatula cell suspension cultures following phosphate starvation . A genomic clone containing the Mt4 gene and 1133 bp of the 5' flanking sequence was identified from a M . truncatula genomic library . The promoter region contains a conserved cis-element found in the promoters of phosphate starvation inducible genes of yeast and tomato . As Mt4 is the first cDNA reported to show independent regulation by both phosphate and mycorrhizal fungi, the genomic clone may provide a starting point from which to analyze the signal transduction pathways involved in these two processes.

J Burn Care Rehabil, 1998 Jul-Aug, 19(4), 337 - 45
Autotransplantation of epithelial cells in the pig via an aerosol vehicle; Fraulin FO et al.; A new method of delivery of epithelial suspensions with use of an aerosolization apparatus was examined in the pig . Full-thickness pig skin was harvested, and an epithelial suspension was created using standard techniques of dispase and trypsin . Twenty-four hours after skin harvest, four full-thickness wounds were created on the flanks of the pig . The control wound was sprayed with a solution without epithelial cells . The three experimental wounds were sprayed with epithelial cell suspensions (integral 10(6) cells/suspension) . Weekly evaluation with photographs, biopsies, and tracings were done for 4 weeks . At 10 weeks, the entire process was repeated with new wounds on the pig's back . Thirty-five wounds in five pigs were evaluated: 10 control (5 flank, 5 back) and 25 experimental (15 flank, 10 back) . Control wounds healed by contraction alone, with epithelium at the edges only . After 4 weeks, an open area remained . Central epithelial islands developed in experimental wounds at 2 weeks . These islands coalesced to close the wounds by 4 weeks . Histology at 1 week showed groups of epithelial cells deeply embedded in granulation tissue . These groups became immature epithelial layers on the surface by 2 weeks, and all layers of epithelium were present by 4 weeks . Overall, flank experimental wounds epithelialized sooner, but contracted at the same rate as control wounds . In conclusion, epithelial cells can be delivered by an aerosolization apparatus and remain viable and proliferative in a pig model.

Acta Vet Hung, 1998, 46(2), 191 - 7
Comparison of 3H-thymidine incorporation and CellTiter 96 aqueous colorimetric assays in cell proliferation of bovine mononuclear cells; Zolnai A et al.; A rapid colorimetric non-radioactive assay for the determination of bovine mitogen-induced lymphocyte proliferation in cell culture was evaluated using a novel tetrazolium compound (MTS) and an electron coupling reagent (PMS) provided in the CellTiter 96 kit (Promega) . The results of the new method were compared with those of the 3H-thymidine incorporation assay using parallels obtained from the same lymphocyte population . The concentrations used in the cell suspension of primary cultured lymphocytes resulted in a significant signal/background ratio when cells were prepared from peripheral blood, spleen or mesenteric lymph nodes . The same concentrations of thymocytes resulted in a weak signal even for the highest concentrations of mitogen . A good correlation was demonstrated between the results of the two methods . The non-radioactive method performed well in assays in bovine mononuclear cells derived from prolactin-treated or -untreated calves, showing a 50% lower responsiveness to mitogenic stimulation in prolactin-deprived animals.

Anticancer Res, 1998 Jul-Aug, 18(4A), 2583 - 90
Guanidinobenzoatase and UPA in high-grade human astrocytomas and after xenografting cell suspensions into the rat cerebral cortex: proteases for metastasis and disease progression; Bernstein LJ et al.; BACKGROUND: Invasion and metastasis is aided by the secretion of guanidinobenzoatase, that cleaves the link peptide to fibronectin, and urokinase plasminogen activator (uPA), which initiates a molecular cascade to activate plasmin and collagenases . This process permits malignant cell migration through the extracellular matrix . MATERIALS: Original human astrocytomas were examined for guanidinobenzoatase and uPA . Suspensions of high-grade human astrocytomas were xenografted into pockets in host cerebral cortex for 1-7 days . RESULTS: A class of guanidinobenzoatase positive cells was observed in the original human astrocytomas and in tumor masses formed in the implantation pocket and around blood vessels . Secondary foci containing guanidinobenzoatase positive cells formed around blood vessels and individual positive astrocytoma cells migrated on the glia limitans along parallel and intersecting nerve fiber fascicles and the corpus callosum . uPA and GFAP were colocalized with guanidinobenzoatase . CONCLUSION: The high-grade astrocytomas reestablish themselves and maintain their characteristics as a tissue although grafted as individual cells.

Anticancer Res, 1998 Jul-Aug, 18(4A), 2435 - 47
Glioma cell adhesion and migration on human brain sections; Giese A et al.; Within the brain, dissemination of glioma cells follows myelinated fiber tracts and extracellular matrix containing structures such as the basement membranes of blood vessels . These patterns represent the two major routes of invasion frequently observed in clinical disease . Previously, we have characterized the substrates for preferential glioma adhesion and migration on purified ECM protein . In this study sections of human brain from different anatomical regions were used as adhesive substrates and also characterized for the presence and distribution of matrix proteins . Adhesion of marker gene transfected glioma cell suspensions to different regions and anatomical structures of human brain was quantified using a computer assisted image analysis system . Monoclonal antibodies against different adhesion molecules were used to inhibit glioma cell attachment ot specific anatomical structures . In addition, glioma cell aggregates were allowed to adhere to brain sections and single cells were observed to migrate out of these aggregates . Scanning electron microscopy was used to morphologically study the preferred routes of glioma dissemination on brain sections . In brain sections different kinetics of cell adhesion to distinct structures were observed . Within 15 minutes cells adhered and spread on blood vessels and arachnoid tissue containing sections . Choroid plexus and the ventricular wall were also adhesive structures . Adhesion to cortex required 1 hour, while adhesion and spreading on myelinated fiber tracts was retarded and required several hours of incubation . The predominant matrix proteins in small vessels were found to be laminin, collagen type IV, and fibronectin . Choroid plexus and the ependyma showed a similar composition of matrix proteins . Arachnoid fibers contained different types of collagens, predominately type I and III, whereas the only matrix protein identified in the subependyma was fibronectin . Antibodies to the alpha 2, alpha 3, and beta 1 integrin subunits completely blocked adhesion to arachnoid tissue, anti-NCAM inhibited attachment to cortex . Adhesion to blood vessels in brain sections could only be inhibited to 50% by anti-integrin beta 1 . Antibodies to the av containing integrin av beta 3 also blocked 50% of adhesion to vessels . Our findings indicate that adhesion of glioma cells to brain sections most rapidly takes place on ECM protein containing regions, especially blood vessels which may serve as guiding structures for glioma dissemination.

Mol Hum Reprod, 1998 Jul, 4(7), 667 - 72
Antioxidant capacity of prostasomes in human semen; Saez F et al.; Prostasomes are human-specific lipid vesicles originating from the prostate and present in seminal plasma . They are involved in a number of biological functions such as sperm motility and immunomodulation by seminal plasma . The aim of our study was to investigate whether prostasomes play a role in the antioxidant status of seminal plasma . In cell suspensions obtained after elimination of seminal plasma by centrifugation, reactive oxygen species (ROS) as measured by luminol-enhanced chemiluminescence were mainly produced by polymorphonuclear neutrophils (PMN) . The addition of prostasomes to these cell suspensions lowered the overall ROS production in the basal state and after stimulation with phorbol ester . This action could not be explained by a ROS-scavenging capacity of the prostasomes, as demonstrated by their inability to scavenge ROS produced by 2,2'-azobis-2-amidinopropane dihydrochloride . Using electron spin resonance, we could assess the influence of prostasomes on the plasma membranes of blood PMN and show that it was characterized by an increase in the correlation-relaxation time of the probe 16-doxyl-stearic acid inserted in the membranes . Thus, prostasomes caused a rigidification of blood PMN membranes . These results strongly suggest that the effect of prostasomes in semen could result from their interaction with PMN.

Int Immunol, 1998 Jul, 10(7), 999 - 1008
Positive selection of low responsive, potentially autoreactive T cells induced by high avidity, non-deleting interactions; Chidgey AP et al.; Using a novel cell suspension model we investigated the relative abilities of nominal peptide and variants thereof to modulate de novo positive selection of lymphocytic choriomeningitis virus (LCMV)-specific TCR transgenic T cells . Confirming our earlier findings intermediate concentrations (10(-7) to 10(-5) M) of the nominal agonist peptide, p33, induced CD8 co-receptor down-modulation at the level of the entire receptor and the CD8beta chain as a consequence of high but non-deleting signal interactions . Agonist peptide variants caused down-modulation of the CD8beta chain but to a lesser degree . An antagonist peptide capable of inducing positive selection did not cause such modifications of the co-receptor . The positively selected TCRhiCD8alpha alpha and TCRhiCD8- cells were functional but not as efficient as TCRhiCD8alphabeta cells, presumably due to lower avidity interactions in the absence of the CD8beta chain or entire co-receptor . CD8beta mRNA was absent in these cells and was not up-regulated when further stimulated with fresh antigen-presenting cells pulsed with 10(-5) M p33 . Effectively our data suggest that it is not the agonist or antagonist nature of a peptide per se but the overall strength of signalling that determines whether a cell will be positively or negatively selected, or die by neglect . Furthermore the agonist/antagonist properties of peptides defined at the level of mature T cell function do not unequivocally predict their effect on positive/negative selection . The ability of the T cell to down-modulate its CD8 co-receptor in response to high but non-deleting peptide interactions during positive selection allows the survival of T cells with a broader range of affinities and represents a possible mechanism by which low responsive but potentially autoreactive cells may escape into the periphery.

Pathol Int, 1998 Jul, 48(7), 512 - 7
Activation of peripheral blood monocytes and macrophages in Kawasaki disease: ultrastructural and immunocytochemical investigation; Koga M et al.; Monocytes/macrophages are considered to play an important role in the pathogenesis of Kawasaki disease (KD) . However, the morphological and immunocytochemical features of the cells in acute KD have not been investigated . The ultrastructural and immunocytochemical characteristics of peripheral blood CD14+ monocytes/macrophages sorted by a magnetic cell sorter (MACS) during the course of KD were, therefore, studied to evaluate their role in the disease pathogenesis . Electron microscopy showed that CD14+ monocytes/macrophages from patients with acute KD had nuclei with complex shapes, apparent nucleoli and abundant intracytoplasmic granules, some of which were positive for acid phosphatase . The quantity of intracytoplasmic granules was correlated with disease severity, in terms of the duration of fever, maximum level of C-reactive protein and the presence of coronary artery lesions (CAL), suggesting that the monocytes/macrophages were activated and showed increased phagocytosis . Immunocytochemical staining of smears made from cell suspensions of sorted CD14+ monocytes/macrophages was carried out using a monoclonal antibody against tumor necrosis factor (TNF)-alpha . The cytoplasm of monocytes/macrophages from patients with acute KD was strongly positive in comparison to that of cells from control subjects, suggesting that intracytoplasmic granules secrete TNF-alpha.

J Neurosci, 1998 Aug 15, 18(16), 6176 - 85
Transplanted olfactory ensheathing cells remyelinate and enhance axonal conduction in the demyelinated dorsal columns of the rat spinal cord; Imaizumi T et al.; Olfactory ensheathing cells (OECs), which have properties of both astrocytes and Schwann cells, can remyelinate axons with a Schwann cell-like pattern of myelin . In this study the pattern and extent of remyelination and the electrophysiological properties of dorsal column axons were characterized after transplantation of OECs into a demyelinated rat spinal cord lesion . Dorsal columns of adult rat spinal cords were demyelinated by x-ray irradiation and focal injections of ethidium bromide . Cell suspensions of acutely dissociated OECs from neonatal rats were injected into the lesion 6 d after x-ray irradiation . At 21-25 d after transplantation of OECs, the spinal cords were maintained in an in vitro recording chamber to study the conduction properties of the axons . The remyelinated axons displayed improved conduction velocity and frequency-response properties, and action potentials were conducted a greater distance into the lesion, suggesting that conduction block was overcome . Quantitative histological analysis revealed remyelinated axons near and remote from the cell injection site, indicating extensive migration of OECs within the lesion . These data support the conclusion that transplantation of neonatal OECs results in quantitatively extensive and functional remyelination of demyelinated dorsal column axons.

Blood, 1998 Aug 15, 92(4), 1390 - 6
The kinetics and extent of engraftment of chronic myelogenous leukemia cells in non-obese diabetic/severe combined immunodeficiency mice reflect the phase of the donor's disease: an in vivo model of chronic myelogenous leukemia biology; Dazzi F et al.; In vitro studies have provided little consensus on the kinetic abnormality underlying the myeloid expansion of chronic myelogenous leukemia (CML) . Transplantation of human CML cells into non-obese diabetic mice with severe immunodeficiency disease (NOD/SCID mice) may therefore be a useful model . A CML cell line (BV173) and peripheral blood cells collected from CML patients in chronic phase (CP), accelerated phase (AP), or blastic phase (BP) were injected into preirradiated NOD/SCID mice . Animals were killed at serial intervals; cell suspensions and/or tissue sections from different organs were studied by immunohistochemistry and/or flow cytometry using antihuman CD45 monoclonal antibodies (MoAbs), and by fluorescence in situ hybridization (FISH) for the BCR-ABL fusion gene . One hour after injection, cells were sequestered in the lungs and liver, but 2 weeks later they were no longer detectable in either site . Similar short-term kinetics were observed using 51Cr-labeled cells . The first signs of engraftment for BV173, AP, and BP cells were detected in the bone marrow (BM) at 4 weeks . At 8 weeks the median percentages of human cells in murine marrow were 4% (range, 1 to 9) for CP, 11% (range, 5 to 36) for AP, 38.5% (range, 18 to 79) for BP, and 54% (range, 31 to 69) for BV173 . CP cells progressively infiltrated BM (21%) and spleen (6%) by 18 to 20 weeks; no animals injected with the cell line or BP cells survived beyond 12 weeks . The rate of increase in human cell numbers was higher for BP (7.3%/week) as compared with CP (0.9%/week) and AP (0 . 5%/week) . FISH analysis with BCR and ABL probes showed that some of the human cells engrafting after injection of CP cells lacked a BCR-ABL gene and were presumably normal . We conclude that CML cells proliferate in NOD/SCID mice with kinetics that recapitulate the phase of the donor's disease, thus providing an in vivo model of CML biology .

Biotechnol Prog, 1998 Jul-Aug, 14(4), 573 - 9
Low-cost serum-free medium for the BTI-Tn5B1-4 insect cell line; Donaldson MS et al.; The BTI-Tn5B1-4 insect cell line, commercially available as the High Five cell line (Invitrogen), supports higher levels of recombinant protein production compared to existing insect cell lines . Proprietary serum-free media such as ExCell 405 (JRH Biosciences), Express Five (Life Technologies), IS BAC (Irvine Scientific), and CCM3 (HyClone) are available which were developed specifically for a suspension culture of High Five cells . While these media are highly optimized, a lower cost alternative is desirable for large-scale protein production which is also serum-free and supports good cell growth (>5 x 10(6) cells/mL) and recombinant protein production (>50 mg/L of secreted protein) . The amino acid and carbohydrate metabolism of the Tn5B1-4 cells was first examined . It was found that asparagine was nearly depleted during batch growth in Ex-Cell 405, without limitations in glutamine, other amino acids, or glucose . Alanine also accumulated to about 35 mM during growth . We then extended the formulation techniques for medium development used for Spodoptera cell lines to the Tn5B1-4 cell line . A medium based on IPL-41 basal medium, Hy-Soy protein hydrolysate (Quest, International), yeastolate ultrafiltrate, a lipid-sterol emulsion, and Pluronic F-68 was developed . Dextran sulfate (100 microg/mL) was used to induce a single cell suspension culture . This medium is denoted as ISYL and performs best when supplemented with a 2.5% lipid-Pluronic F-68 mixture . Supplementation with additional aspargine in a 1.5% lipid-Pluronic F-68 mixture did not improve growth, suggesting that a lipid was growth-limiting and not an amino acid . Ex-Cell 405 and ISYL with 2.5% lipid-Pluronic F-68 supplement supported virtually identical growth rates, extent of growth (ca . 6.0 x 10(6) cells/mL) in an 80% oxygen atmosphere, and supported production of SEAP (secreted human alkaline phosphatase) at a volumetric level of about 65-70 mg/L . Thus, the less expensive ISYL medium can deliver acceptable performance and may be suitable for large-scale insect cell cultures.

J Biolumin Chemilumin, 1998 May-Jun, 13(3), 147 - 56
Differentiation of marine luminous bacteria using commercial identification plates; Makemson JC et al.; Differentiation of marine luminous bacteria using Biology GN plate combined with API 20e or the BBL Crystal ID plate inoculated with cell suspensions in artificial seawater was accomplished by comparison to type species using cluster analysis . Inoculum density affected the results from Biolog GN plates, but had less of an effect on the reactions obtained from API 20e strip or BBL Crystal ID plate . In a few cases, combination of the Biolog GN traits along with either the API 20e or Crystal ID traits was necessary to differentiate some marine luminous bacteria.

Biosci Biotechnol Biochem, 1998 Jun, 62(6), 1134 - 7
Preparation and preservation of freeze-dried cells of acetic acid bacteria with aldehyde oxidase activity; Nomura Y et al.; Freeze-dried cells of acetic acid bacteria were prepared to use as an additive for manufacturing and processing foods . When the freeze-dried cells were stored for 1 week at 5 degrees C, however, more than 50% of the original activity of aldehyde oxidase (AOX) was lost . It was found that this decrease in AOX was caused by damage to both the membrane-bound aldehyde dehydrogenase and terminal oxidase activities involved in the aldehyde oxidase electron transport system of acetic acid bacteria . The addition of 30% sucrose to the cell suspension prepared in a McIlvaine buffer (pH 6) before lyophilization was found to be effective for preventing the decrease of AOX activity . Cells freeze-dried in this way lost no AOX activity at all during first 3 weeks of storage at 5 degrees C and, even after 9 weeks, 80% of the original activity remained.

J Membr Biol, 1998 Aug 1, 164(3), 229 - 37
Ca2+ uptake through voltage-gated L-type Ca2+ channels by polarized enterocytes from Atlantic cod Gadus morhua; Larsson D et al.; The presence and localization of voltage-gated Ca2+ channels of L-type were investigated in intestinal cells of the Atlantic cod . Enterocytes were loaded with the fluorescent Ca2+ probe, fure-2/AM and changes in intracellular Ca2+ concentrations ({Ca2+}i) were measured, in cell suspensions, in the presence of high potassium levels (100 mm), BAY K-8644 (5 microM), nifedipine (5 microM) or omega-conotoxin (1 microM) . L-type Ca2+ channels were visualized on intestinal sections using the fluorescent dihydropyridine (-)-STBodipy . Depolarization of the plasma membrane produced a rapid (within 5 sec) and transient (at basal levels after 21 sec) increase in {Ca2+}i . BAY K-8644 increased the {Ca2+}i by 7.2% . Cells in a Ca2+-free buffer increased {Ca2+}i after addition of 10 mm Ca2+, and this increase was abolished by nifedipine in both depolarizing and normal medium but not by omega-conotoxin . Single cell experiments using video microscopy revealed that enterocytes remained polarized several hours after preparation and that the Ca2+ entry and extrusion occurred at specific and different regions of the enterocyte outer membrane . Fluorescent staining of L-type Ca2+ channels in the intestinal mucosa showed the most intense staining at the brushborder membrane . These results demonstrate the presence of voltage gated L-type Ca2+ channels in enterocytes from the Atlantic cod . The channels are mainly located at the apical side of the cells, and there is a polarized uptake of Ca2+ into the enterocytes . This suggests that the L-type Ca2+ channels are involved in the transcellular Ca2+ entry into the enterocytes.

Cell Biol Toxicol, 1998 Jun, 14(3), 225 - 36
Modulation of adrenal cell functions by cadmium salts . 4 . Ca(2+)-dependent sites affected by CdCl2 during basal and ACTH-stimulated steroid synthesis; Mathias SA et al.; In previous studies, nonlethal CdCl2 concentrations apparently inhibited basal Y-1 mouse adrenal tumor cell endogenous mitochondrial cholesterol conversion to pregnenolone . In addition, CdCl2 inhibited all agents stimulating both plasma membrane-dependent cAMP synthesis and 20 alpha-hydroxy-4-pregnen-3-one (20DHP) secretion . Bypassing the plasma membrane using dibutyryl-cAMP (dbcAMP) stimulated cytoplasmic cholesterol metabolism and 20DHP secretion in the presence of CdCl2 . Since CdCl2 competed at metabolic steps requiring Ca2+ in other tissues, experiments were designed to examine Cd2+ competition with Ca2+ during steroidogenesis . Sets of cells incubated with either medium or adrenocorticotropin (ACTH) with or without CdCl2 were also treated with 0, 1.0, 5.0 or 10.0 mmol/L CaCl2 in the presence or absence of EGTA, a relatively specific Ca2+, but not Cd2+, chelating agent . Another experimental cell set incubated with either medium or ACTH, with or without CdCl2, was treated with or without 1 mmol/L A23187, an ionophore specifically facilitating extracellular Ca2+ transfer across plasma membranes . Besides determining Ca2+ involvement in steroidogenesis using steroid secretion as an endpoint, we directly measured Ca2+ concentrations using intracellular fura-2 fluorescence . Following loading with 2 mumol/L fura-2, cells remained untreated or medium was infused with CdCl2, ACTH, ACTH/CdCl2 or ACTH followed after 50 s by CdCl2 . Using Ca(2+)-supplemented media, we observed that Cd2+ inhibition of ACTH-stimulated 20DHP secretion was completely reversed . Standard Ca(2+)-containing medium supplemented with Ca2+ also enhanced maximally stimulated 20DHP secretion by ACTH . 20DHP secretion by ACTH-treated and ACTH/Cd(2+)-treated cells was only reduced by EGTA, when Ca2+ was not supplemented . The ionophore A23187 increased basal and ACTH-stimulated 20DHP secretion by Cd(2+)-treated cells, suggesting that extracellular Ca2+ resources may compete against Cd2+ effects on plasma membrane cAMP synthesis and on basal cholesterol metabolism by mitochondria . No time-dependent change in Ca2+ concentrations occurred within untreated cell suspensions . ACTH stimulation caused a 25 s burst in Ca2+ concentrations before returning to basal, steady-state levels . Cd2+ also stimulated intracellular fura-2 fluorescence . Untreated cell suspensions infused with Cd2+ exhibited a continuous rise in intracellular fluorescence . ACTH/CdCl2-treated cells exhibited a hyperbolic rise in intracellular fluorescence over the 300 s study period . Cells treated with Cd2+ 50 s after ACTH treatment initially exhibited the 25 s fluorescence burst followed by a Cd(2+)-induced hyperbolic rise in intracellular Cd2+ . These fluorescence measurements suggested that cytoplasmic Ca2+ changes do not appear to be necessary for basal 20DHP synthesis and secretion; only a 25 s burst in intracellular Ca2+ is necessary to a slightly higher plateau level for stimulated 20DHP synthesis and secretion . Cd2+ freely enters the cell under basal conditions and Cd2+ entry is accelerated by ACTH stimulation . Data were consistent with Ca2+ being required for optimal stimulated steroid production and Cd2+ probably competing with Ca2+ during basal mitochondrial cholesterol metabolism and plasma membrane ACTH-stimulated cAMP generation.

Hum Reprod, 1998 Jun, 13(6), 1584 - 9
Luteal leukocytes are modulators of the steroidogenic process of human mid-luteal cells; Castro A et al.; Flow cytometry analysis of luteal cells revealed that an important proportion of these cells are leukocytes . The percentage of leukocytes was higher in the early (42 +/- 4) and late (35 +/- 3) luteal phases than in the mid-luteal (24 +/- 2) phase . However, the proportion of macrophages did not differ between the luteal stages . The flow cytometric properties correlated with cellular size and granularity were not reliable as discriminators of luteal cell subpopulations . Therefore, to assess the contribution of luteal leukocytes, these cells were completely removed from luteal cell suspensions (total cells), by a negative selection procedure (immunomagnetic separation) . The functional role of leukocytes in mid-luteal steroidogenesis was assessed, in total as well as leukocyte-depleted cells . Progesterone production was found to have increased 2.2-fold in leukocyte-depleted cell cultures, in comparison with total cells under basal conditions . However, the response to human chorionic gonadotrophin (HCG) was 36% lower under the latter conditions . Oestradiol production was not significantly modified under basal or HCG-treated conditions . In leukocyte-depleted cells, the concentration of interleukin (IL)-1beta decreased 5-fold in comparison with total cell cultures, suggesting that leukocytes are the principal source of IL-1beta . In summary, the results of the present investigation suggest functional interactions between the immune system and steroidogenic cells of the human corpus luteum.

Hum Reprod, 1998 Jun, 13(6), 1570 - 7
Simple and reliable identification of the human round spermatid by inverted phase-contrast microscopy; Verheyen G et al.; Based on the results of animal studies, round spermatid injection (ROSI) has been introduced into the clinical practice of several in-vitro fertilization (IVF) centres . The efficiency of this procedure in terms of fertilization rates and pregnancy rates, however, remains very poor . An essential aspect which does not receive enough attention is the correct identification of this type of round cell within a heterogeneous population of testicular cells . A Nikon inverted microscope equipped with phase-contrast optics (DLL) provided a clear image which allowed reliable recognition of round spermatids in cell suspensions smeared at the glass bottom of the dish . Fluorescent in-situ hybridization confirmed the haploid status of the selected cells . However, exploration of several biopsies from patients with non-obstructive azoospermia showing no spermatozoa after extensive search did not reveal any round spermatids . This observation questions whether enough effort is spent on searching for mature spermatozoa or late spermatids . Experimental investigations should precede the introduction of ROSI into the clinical practice of any IVF centre.

J Neurosurg, 1998 Aug, 89(2), 267 - 74
Effects of severity of host striatal damage on the morphological development of intrastriatal transplants in a rodent model of Huntington's disease: implications for timing of surgical intervention; Watts C et al.; OBJECT: The goal of this study was to investigate the effect of the severity of host neural damage on the morphological development of intrastriatal transplants in a rodent model of Huntington's disease . METHODS: Sprague-Dawley rats were subjected to unilateral striatal lesioning induced by administration of quinolinic acid (20 nM, 40 nM, or 90 nM) . Seven days postlesioning, intrastriatal cell suspension grafts were placed in the right striatum in some of these animals . Grafts were also placed in the right striatum of additional animals that had not been subjected to lesioning . The rats were killed and processed for morphological analysis 8 weeks after grafting . The results indicate that striatal grafts survive and grow much better when implanted into a lesioned striatum rather than into an intact striatum, as measured both by the volume and the numbers of medium-sized spiny neurons within the graft . Only a small or modest lesion is necessary to produce this effect . By some measures (such as graft volume) grafts survive less well when the lesion is more extensive . The presence of a graft reduced the extent of striatal atrophy induced by the lesions, but this effect was not caused by differences in the numbers of surviving neurons per se . CONCLUSIONS: These results have significant implications for the timing of surgical intervention and patient selection with respect to current and future clinical trials of striatal transplantation in the treatment of Huntington's disease.

Biochim Biophys Acta, 1998 Jul 23, 1381(2), 147 - 60
Expression and characterisation of single-chain antibody fragments produced in transgenic plants against the organic herbicides atrazine and paraquat; Longstaff M et al.; Single-chain antibody fragments (scAbs), which have a human C-kappa constant domain and a hexa-histidine tail attached to the carboxy terminus of the single-chain Fv (ScFv) fragments to facilitate purification, have been raised against the herbicides paraquat and atrazine and expressed in transgenic Nicotiana tabacum cv . Samsun NN . Prior to purification, the anti-atrazine scAb is expressed as up to 0.014% of soluble leaf protein and has a binding profile in ELISA, against an atrazine-bovine serum albumin (BSA) conjugate, similar to that of the scAb produced in Escherichia coli . Competition ELISA has shown that the plant-derived scAb also recognises free atrazine . Following antibody affinity purification to isolate dimers, the affinity for immobilised antigen approaches that of the parental monoclonal antibody . This was confirmed by surface plasmon resonance analysis . The purified scAb also recognises related triazine herbicides . When isolated from cell-suspension cultures, the anti-paraquat scAb binds to a paraquat conjugate in a concentration-dependent manner, with a profile similar to the parental monoclonal antibody . This is the first demonstration that functional scAbs against organic pollutants can be produced in transgenic plants and that the scAbs may be appropriate for the development of immunoassay-based detection systems .

Mol Genet Metab, 1998 May, 64(1), 44 - 51
A novel method of cell-specific mRNA transfection; Sawai K et al.; In this study, we developed a cell-specific mRNA transfection system using streptavidin-protein A (ST-PA) fusion protein and monoclonal antibodies (mAbs) . We previously reported that ST-PA fusion protein and mAb complexes can transfer certain biotinylated proteins into specific cell types . At this time, we combined an in vitro transcribed biotinylated and self-replicating Sindbis virus genomic RNA with ST-PA fusion protein and mAbs . In the presence of cationic liposomes, to prevent RNA degradation, this complex is able to transfect a reporter gene to specific cancer cells in a mAb does-dependent manner . Even in the absence of cationic liposomes, biotinylated mRNA, ST-PA fusion, and mAb complexes can transfer some types of cancer cell suspension cultures . This cell-specific transfection system is a novel method of introducing various mRNAs into cells that results in high levels of transient protein expression.

Cell Calcium, 1998 May, 23(5), 281 - 9
Heterotrimeric Gi protein is associated with the inositol 1,4,5-trisphosphate receptor complex and modulates calcium flux; Neylon CB et al.; In vascular smooth muscle, pertussis toxin (PT) inhibits thrombin-induced Ca2+ release by a mechanism independent of its effect on IP3 formation . Thus, the possibility of a direct role of G alpha i proteins in regulating IP3-sensitive Ca2+ release was investigated by examining whether G alpha i proteins are associated with the IP3 receptor complex . Purified microsomal membranes were prepared and separated by sucrose density gradient centrifugation . The relative density of {3H}-IP3 binding sites between the microsomal fractions was inversely related to the distribution of the plasma membrane marker . The relative distribution of G alpha i3 determined by immunoblotting was closely correlated with the density of {3H}-IP3 binding . Levels of G alpha i2 were more evenly distributed with highest levels present in plasma membrane-enriched fractions . IP3 receptor immunoprecipitated from triton-solubilized microsomal membranes contained G alpha i3 immunoreactivity . To determine whether G alpha i proteins influence IP3-induced Ca2+ release, the effect of PT on Ca2+ release from digitonin-permeabilized cell suspensions using Fluo-3 was examined . Exposure to PT (0.1 microgram/ml, 5 min) attenuated the initial rate of IP3 (1 microM)-induced Ca2+ release . Together, these findings are consistent with the hypothesis that a heterotrimeric G alpha i protein directly regulates IP3-dependent Ca2+ release.

J Gen Virol, 1998 Jul, 79 ( Pt 7), 1745 - 9
Bovine viral diarrhoea virus induces apoptosis in blood mononuclear cells by a mechanism largely dependent on monocytes; Lambot M et al.; The induction of apoptosis by bovine viral diarrhoea virus (BVDV) was examined in bovine peripheral blood mononuclear cells (PBMC) incubated with an antigenically homologous pair of non-cytopathic and cytopathic BVDV . Our results show that the cytopathic biotype, in contrast to the non-cytopathic counterpart, induces apoptosis in PBMC . Flow cytometry analysis of cells undergoing apoptosis revealed that: (1) monocytes constitute the major cell population undergoing apoptosis; (2) cytopathic virus also induces apoptosis in BoCD4+, BoCD8+ and BoWC1+ T cells in whole PBMC cultures but not in purified T cell suspensions; (3) the degree of apoptosis of BoCD4+ and BoCD8+ T cells incubated with the cytopathic virus was significantly enhanced by the presence of monocytes . Taken together, these results suggest that bovine monocytes play an important role in apoptosis induced by cytopathic BVDV.

Gen Comp Endocrinol, 1998 Aug, 111(2), 207 - 15
Follicle stimulating hormone increases somatic and germ cell number in the ovary during chick embryo development; Mendez-Herrera MC et al.; The aim of the present study was to evaluate the effect of human follicle stimulating hormone (hFSH) on cellular proliferation in the chick embryo ovary . Chick embryos (Babcock B300) were injected on chorioallantoic membrane with a single dose of hFSH (2.0 IU/ embryo) at Days 7, 9, or 13 of incubation or with hCG (2.0 IU/embryo) at Day 13 of incubation . At 17 days of incubation and within 24 h after hatching, left ovaries were dissected and completely dissociated . Cells from the whole ovary were classified into germ cells (primary oocytes), typical steroidogenic cells, and poorly differentiated somatic cells and counted with the aid of a hemocytometer . Aliquots of the cell suspension from the whole left ovary were analyzed by flow cytometry, in order to determine the percentage of cells at each phase of the cell cycle . In addition, samples of the suspension (1.0 x 10(6 )cells) were incubated for 2 h in basal and stimulated conditions measuring 17beta-estradiol secretion in the medium . The ovarian cell number at 17 days of incubation showed that hFSH treatment at Day 7 did not modify the cell number in any of the subpopulations evaluated; treatment at Day 9 resulted in an increase in poorly differentiated somatic cell number, without changes in steroidogenic and germ cells, whereas hFSH treatment at Day 13 augmented the number of poorly differentiated, steroidogenic, and germ cells . The percentage of cells in S-phase was increased 12 and 15 h after hFSH treatment (Day 13) . Secretion of 17beta-estradiol was increased in the hFSH-treated group (Day 13) measured at 17 days of incubation . The increase in cell number of the three subpopulations was still observed in the left ovary of the newly hatched chicken . Treatment with hCG at Day 13 of incubation did not change the number of poorly differentiated, steroidogenic, and germ cells in the left ovary, neither in the 17-day-old chick embryo nor in the newly hatched chicken . The 17beta-estradiol secretion in hCG-treated embryos was similar to controls . The present study is the first evidence of an effect of FSH on somatic and germ cell number, together with an increase in 17beta-estradiol production during chick embryo ovary development .

Biol Reprod, 1998 Jul, 59(1), 84 - 92
Developmental schedule of the postnatal rat testis determined by flow cytometry; Malkov M et al.; Analysis of the biochemical events and the genes expressed at various postnatal developmental stages in the testis of mammals is of great importance for understanding spermatogenesis in general and meiosis in particular . A prerequisite for such an analysis is the characterization of a detailed developmental schedule of the postnatal testis . In this study we used four-parameter flow cytometry analysis to determine a detailed testicular developmental schedule in rats as compared to mice . A dot plot of forward-scatter/side-scatter of testicular cell suspensions from mature animals revealed 7 distinct subpopulations within the testis . These, when analyzed by fluorescence parameters, were divided into 4 levels of fluorescence: cells containing 4d DNA, 2d DNA, and 2 levels of haploid cells . Observing the acquisition pattern of these subpopulations during postnatal development, we were able to suggest the following developmental schedule for the rat . At postnatal Days 6-7, the testis contains somatic cells and spermatogonia cells only . By Days 13-14, leptotene spermatocytes appear; by Days 17-18, zygotene spermatocytes are present; by Days 19-20 and Days 22-23, early and late pachytene spermatocytes, respectively, are seen . Haploid round spermatids first appear at Days 24-25 and elongating spermatids by Days 30-31; by Day 36, elongated spermatozoa can be found.

Br J Haematol, 1998 Jun, 101(4), 728 - 36
Novel enzymatic abnormalities in AML and CML in blast crisis: elevated leucocyte leukotriene C4 synthase activity paralleled by deficient leukotriene biosynthesis from endogenous substrate; Stenke L et al.; Leukotrienes (LT) are inflammatory mediators which can also exert regulatory effects on human myelopoiesis . We have studied the LT-producing capacity of freshly isolated leucocyte suspensions (containing blast cells in variable proportions) from 41 patients with acute myeloid leukaemia (AML) or chronic myeloid leukaemia (CML) in blast crisis (CMLbc) at diagnosis or relapse/resistant disease . Leucocyte suspensions from 19/29 AML patients (66%), and 2/12 CMLbc patients (17%; P = 0.012) demonstrated deficient capacity to synthesize LT from endogenous substrate after ionophore A23187 stimulation . Thus, these cells produced < 8 pmol LTB4+LTC4/10(6) cells (< 20% of mean LT formation in leucocyte suspensions from 18 healthy subjects) . Addition of exogenous arachidonic acid did not normalize the LT synthesis in poor-producing cell suspensions . Purified, morphologically mature granulocytes from two AML patients also failed to produce normal amounts of LT . In leucocyte suspensions from the remaining 20 AML/CMLbc patients A23187 provoked LT biosynthesis, with markedly increased production of LTC4, but decreased LTB4 formation . Furthermore, elevated conversion of exogenous LTA4 to LTC4 was noted in the patient samples, independent of their capacity to produce LT after A23187 stimulation . The percentage of blast cells in patient white blood cell differential counts correlated inversely with ionophore-induced LT synthesis, but positively with the conversion of exogenous LTA4 to LTC4 . The results suggest elevated LTC4 synthase activity and suppressed 5-lipoxygenase activity as novel enzymatic features of myeloid leukaemia patients with immature phenotype.

J Immunol Methods, 1998 Mar 1, 212(1), 113 - 23
Detection and analysis of cytokine mRNA in tissues and cell lines; Walker KB; Many normal and pathological processes have been shown to be influenced by the quantitative and qualitative nature of cytokine production . Cytokines have also been shown to be central in modulating host responses to invasion by pathogens . Therefore detection and analysis of cytokine production are critical in the understanding of host responses to a variety of immunological insults . The detection of cytokines by bioassay or immunoassay provides data from in vitro experiments and can be used to determine the circulating plasma levels of particular cytokines . However, many investigations require the detection of cytokines in small numbers of cells or directly from tissue samples . It is in this particular area that detection and analysis of cytokine gene expression by use of the sensitive and specific reverse transcription-polymerase chain reaction (RT-PCR) has application . The purpose of these protocols is to provide a practical guide to the detection of cytokine gene expression using RT-PCR techniques . Procedures for analysing mRNA expression in cell culture, cell suspensions and tissue samples will be described and pitfalls and caveats concerning the techniques will be discussed . These protocols should provide a starting point for the development of RT-PCR technology in laboratories with only limited molecular experience.

Mol Gen Genet, 1998 Jun, 258(6), 655 - 62
Cloning and expression analyses of AtMRP4, a novel MRP-like gene from Arabidopsis thaliana; Sanchez-Fernandez R et al.; In all organisms glutathione-conjugate transporters (GS-X pumps) mediate the detoxification of a number of xenobiotics by removing them from the cytosol . In addition, GS-X pumps appear to play a role in the processing of endogenous compounds . We have isolated a novel genomic clone from Arabidopsis thaliana that encodes a putative GS-X pump, AtMRP4, which is part of a recently defined gene family . The derived amino acid sequence shares high levels of similarity (55-63%) with human, yeast, and other Arabidopsis homologues . The expression of the different members of the AtMRP gene family in Arabidopsis cell suspensions after treatment with chemicals that modify glutathione metabolism (compounds that induce different types of stress and that act as herbicide antidotes- safeners- in monocotyledonous species) revealed that the members of this gene family are differentially regulated.

Br J Cancer, 1998 Jun, 77(11), 1832 - 8
Proliferation and apoptosis in malignant and normal cells in B-cell non-Hodgkin's lymphomas; Stokke T et al.; We have examined apoptosis and proliferation in lymph node cell suspensions from patients with B-cell non-Hodgkin's lymphoma using flow cytometry . A method was developed which allowed estimation of the fractions of apoptotic cells and cells in the S-phase of the cell cycle simultaneously with tumour-characteristic light chain expression . Analysis of the tumour S-phase fraction and the tumour apoptotic fraction in lymph node cell suspensions from 95 B-cell non-Hodgkin's lymphoma (NHL) patients revealed a non-normal distribution for both parameters . The median fraction of apoptotic tumour cells was 1.1% (25 percentiles 0.5%, 2.7%) . In the same samples, the median fraction of apoptotic normal cells was higher than for the tumour cells (1.9%; 25 percentiles 0.7%, 4.0%; P = 0.03) . The median fraction of tumour cells in S-phase was 1.4% (25 percentiles 0.8%, 4.8%), the median fraction of normal cells in S-phase was significantly lower than for the tumour cells (1.0%; 25 percentiles 0.6%, 1.9%; P = 0.004) . When the number of cases was plotted against the logarithm of the S-phase fraction of the tumour cells, a distribution with two Gaussian peaks was needed to fit the data . One peak was centred around an S-phase fraction of 0.9%; the other was centred around 7% . These peaks were separated by a valley at approximately 3%, indicating that the S-phase fraction in NHL can be classified as 'low' (< 3%) or 'high' (> 3%), independent of the median S-phase fraction . The apoptotic fractions were log-normally distributed . The median apoptotic fraction was higher (1.5%) in the 'high' S-phase group than in the 'low' S-phase group (0.8%; P = 0.02) . However, there was no significant correlation between the two parameters (P > 0.05).

Clin Exp Dermatol, 1998 Jan, 23(1), 14 - 8
The epidermis of chronic idiopathic lichen planus during topical treatment with the vitamin D3 analogue KH 1060; Glade CP et al.; A parallel-group, double-blind, randomised study was performed to establish the effect of the vitamin D3 analogue KH 1060, in an ointment versus vehicle only, on the epidermal cell characteristics of chronic idiopathic lichen planus; KH 1060 also has marked immunosuppressive activity . A group of 10 patients were treated for 8 weeks with either KH 1060 ointment or vehicle only . In addition to the assessment of clinical scores, keratotome biopsies were taken before and after 8 weeks' treatment . Epidermal cell suspensions were prepared with trypsin and the suspensions incubated with TO-PRO-3 (DNA marker), RKSE 60 (marker for keratin 10-positive cells) and antivimentin (marker for all non-keratinocytes) . In nine of the 10 patients, keratotome biopsies were obtained both before and after 8 weeks treatment . The vehicle alone had no significant effect on the clinical severity scores or epidermal cell characteristics . In contrast, the KH 1060 ointment resulted in a statistically significant reduction in the percentage of cells in S- and G2M phase and the percentage of vimentin-positive cells, but it did not affect the percentage of keratin 10-positive cells.

Magn Reson Imaging, 1998 May, 16(4), 423 - 34
Characterisation of erythrocyte shapes and sizes by NMR diffusion-diffraction of water: correlations with electron micrographs; Torres AM et al.; The utility of 1H nuclear magnetic resonance (NMR) diffusion-diffraction of water as a tool for characterising red cell shape was investigated . Experiments were conducted on various cell suspensions which contained different shapes/forms of erythrocytes prepared by manipulating the conditions of the suspension medium, such as osmolality, and altering metabolism to affect the adenosine triphosphate concentration . Abnormal red cells from patients with hereditary stomatocytosis and megaloblastic anemia were also studied in order to assess the practical application of this "new" technique . The results clearly show that NMR diffusion-diffraction is sensitive to very small changes in mean cell dimensions and that a "characteristic" q-space plot/profile can be ascribed to each erythrocyte form . It was also found that the homogeneity of the cell shape and/or size is an important factor that affects the intensity of the diffusion-diffraction peaks . This study demonstrates the potential of the NMR diffusion-diffraction technique as a diagnostic tool in hematology.

Int J Dev Neurosci, 1998 Feb, 16(1), 9 - 17
Adenoviral-mediated transfection of the Lac-Z gene into rat dissociated embryonic central nervous system cells before and after seeding; Cot C et al.; The adenovirus carrying a reporter gene--the Lac Z gene--is known to infect central nervous system (CNS) cells in primary cell cultures . The percentage of infected neurons with respect to the total number of neurons was studied in primary dissociated cultures as a function of the day of inoculation and the age of three rat CNS cultures: spinal cord, mesencephalon and cortex . Two methods of viral inoculation were compared: the first inoculation was performed on the cultured cell at 2, 3 or 6 days in vitro (DIV) whereas the second inoculation was performed on the cell suspensions before seeding . All the infected CNS cells has the same aspect as the control cultures . In the spinal cord and the mesencephalic cultures, the glial cells were preferentially infected, especially when the cells were inoculated at 6 DIV . In the cortical cultures, there were more infected neurons than infected glial cells . The number of CNS cells was lower when inoculation was performed at 6 DIV as compared with 3 DIV . Very few infected GABA cells were found in the cultures . A high percentage of infected neuronal cells relative to the total number of neuronal cells was found when infection of the three types of cultures was performed on the dissociated embryonic cell suspension before seeding.

Cell Vis, 1998 Jan-Feb, 5(1), 56 - 61
Flow cytometry: an overview; Villas BH; Flow cytometry is an innovative technology that measures certain cell parameters as the cells flow in a fluid stream and in single file past an analytical laser light source . Clinical applications of flow cytometry currently utilized in the laboratory include cell surface antigen determinations or immunophenotyping of hematologic cells, DNA analysis of hematopoietic malignancies and solid tumors, and measurement of CD4 (T helper/inducer cell) absolute counts and T helper/T suppressor (CD4/CD8) ratios in the evaluation of immune deficiency . Flow cytometry often offers a more rapid, sensitive, accurate, and quantitative means of analyzing a particular cell population in a heterogeneous cell suspension as compared to more traditional microscopic methods . This article is intended to provide a general understanding of the technological basis of how a flow cytometer functions as well as an overview of both current and future flow cytometric clinical laboratory applications.

Cell Vis, 1998 Jan-Feb, 5(1), 8 - 12
Morphological evidence for the location of calcitonin gene-related peptide (CGRP) immunoreactivity in rat lymphocytes; Xing L et al.; Calcitonin gene-related peptide (CGRP) is widely distributed in sensory neurons and nerve fibers . It was shown recently in our laboratory that there was CGRP-immunoreactivity (CGRP-ir) in extract of rat lymphocyte of thymus and mesenteric lymph node by radioimmunoassay and reversed-phase HPLC . The aim of this study was to detect the CGRP-ir location in the rat lymphocyte by immunocytochemical method . Single cells isolated from thymus and mesenteric lymph node of male Wistar rat (200-250 g) were suspended in RPMI-1640 medium . After adhesion to plate wall and through nylon wool fiber columns, T cell-riched suspension was obtained . Immunocytochemical ABC method was performed in cell suspension, the specific antiserum (1:200 diluted) was rabbit anti-human CGRP . The cells were examined under light microscope after smeared on glass slide by Shandon Cytospin . The results showed that some lymphocytes were CGRP-ir positive . The positive granules were seen as ring, plaque, cap or spot distributing on the surface or inside the cell . The results indicate that CGRP-ir may be located in some rat T lymphocytes . This work provides new information about the interaction between the nervous and the immune systems . The functional significance of CGRP located in lymphocytes needs to be further studied.

Gut, 1998 May, 42(5), 643 - 9
Spontaneous secretion of interferon gamma and interleukin 4 by human intraepithelial and lamina propria gut lymphocytes; Carol M et al.; BACKGROUND: Cytokines secreted by intestinal T lymphocytes probably play a critical role in regulation of the gut associated immune responses . AIMS: To quantify interferon gamma (IFN-gamma) and interleukin 4 (IL-4) secreting cells (SC) among human intraepithelial (IEL) and lamina propria (LPL) lymphocytes from the duodenum and right colon in non-pathological situations and in the absence of in vitro stimulation . PATIENTS: Duodenal and right colonic biopsy specimens were obtained from patients with no inflammation of the intestinal mucosa . METHODS: Intraepithelial and lamina propria cell suspensions were assayed for numbers of cells spontaneously secreting IFN-gamma and IL-4 by a two site reverse enzyme linked immunospot technique (ELISPOT) . RESULTS: The relatively high proportion of duodenal lymphocytes spontaneously secreting IFN-gamma (IEL 3.6%; LPL 1.9%) and IL-4 (IEL 1.3%; LPL 0.7%) contrasted with the very low numbers of spontaneously IFN-gamma SC and the absence of spontaneously IL-4 SC among peripheral blood mononuclear cells . In the basal state, both IFN-gamma and IL-4 were mainly produced by CD4+ cells . Within the colon, only 0.2% of IEL and LPL secreted IFN-gamma in the basal state, and 0.1% secreted IL-4 . CONCLUSIONS: Compared with peripheral lymphocytes substantial proportions of intestinal epithelial and lamina propria lymphocytes spontaneously secrete IFN-gamma and/or IL-4 . These cytokines are probably involved in the normal homoeostasis of the human intestinal mucosa . Disturbances in their secretion could play a role in the pathogenesis of gastrointestinal diseases.

Microvasc Res, 1998 May, 55(3), 241 - 8
Can low density lipoprotein influence microvascular caliber?
Skinner S, Locher R, Niederer E, Vetter W.
The intracellular free calcium concentration ({Ca2+}i) plays an important role in the regulation of vascular tone . In vascular smooth muscle cells (VSMCs) LDL causes changes in vascular tone by increasing {Ca2+}i . Pericytes are regarded as the microvascular counterpart of VSMCs and implicated in the regulation of microvascular cell biology under normal and pathological conditions (e.g., diabetes mellitus, arterial hypertension, arteriosclerosis) . For this reason pericytes and VSMCs were compared in their ability to increase {Ca2+}i after stimulation with LDL . Single VSMCs and pericytes were loaded with 2 microM of the Ca2+-sensitive dye Indo-1/AM . Fluorescence was recorded at 405 nm (Ca2+-bound) and 485 nm (Ca2+-free) . Cells in suspension were loaded with 2 microM of the calcium ionophore FURA-2 AM (excitation wavelengths: 340 and 380 nm, emission 505 nm) . Basal {Ca2+}i levels were significantly higher in single pericytes (165 +/- 38 nmol/L, n = 41) than in VSMCs (150 +/- 39 nmol/L, n = 40, P = 0.0038) . In cell suspensions the following values were obtained: Pericytes (113 +/- 27 nmol/L, n = 36) and VSMCs (109 +/- 26 nmol/L, n = 28), which are statistically not significant . For all concentrations of LDL used (except at 1 microg/ml n-LDL), the increase above basal values was significant and both cell types showed a clear dose-dependent reaction pattern . This study shows for the first time that pericytes and VSMCs increase their {Ca2+}i in a similar way after LDL stimulation . In analogy to aortic smooth muscle cells, our results indicate that LDL mediated {Ca2+}i changes in pericytes in the microvascular bed may cause vasoconstriction leading to impairment of blood flow in the microvasculature .

Am J Physiol, 1998 Jul, 275(1 Pt 1), G138 - 50
Origin of molecular species of diacylglycerol induced by bombesin in smooth muscle cells from rabbit rectosigmoid; Sbrissa D et al.; The source of early production of sn-1,2-diacylglycerol (DAG) has for a long time been exclusively linked to hydrolysis of phosphatidylinositol 4,5-diphosphate, which on receptor activation is hydrolyzed into DAG and inositol 1,4,5-trisphosphate . We have investigated the origin of lipid sources of DAG production in smooth muscle cells, in response to contraction induced by peptide agonists . We have performed a quantitative analysis of the molecular species of DAG formed in relation to the known molecular composition of parent phospholipids . The molecular species of phospholipids are sufficiently unique that the phospholipid origin of DAGs and its quantitative contribution to their formation can be measured by HPLC . Cell suspensions (10-15 x 10(6) cells/ml) from the circular muscle of rabbit rectosigmoid were incubated in the presence of the contractile peptide agonist bombesin (BB) at 10(-6) M . Reactions were stopped at different time intervals from 30 s to 4 min . DAGs were extracted, purified by TLC, and benzoylated with benzoic anhydride . The benzoylated DAGs were first purified by TLC and then by normal phase HPLC before they were injected onto a reverse-phase column and eluted isocratically . Furthermore, phospholipids in the lipid extract {phosphatidylinositol (PI), phosphatidylcholine (PC), phosphatidylserine (PS), and phosphatidylethanolamine (PE)} were purified by TLC and similarly analyzed after hydrolysis to DAGs with phospholipase C (PLC) . The DAG molecular species profiles for PI, PC, PS, and PE were all unique . Contraction of cells with BB gave noticeable increases (17-55%) in newly formed DAGs . The major phospholipid source of the newly formed DAGs at 30 s was only approximately 30% from PI, and the remainder was from PC . In contrast, after 4 min of BB stimulation, a decrease was seen in newly formed DAGs in the peak specific for PI hydrolysis . The data suggest that BB-induced contraction by activation of PLCs results in hydrolysis of different phospholipids . The DAGs formed as a result are qualitatively and quantitatively distinct . This could be the basis for the kinetically different pattern of sustained contraction observed with BB.

Neurosci Lett, 1998 May 15, 247(2-3), 111 - 4
Preparation of cell suspensions for co-transplantation: methodological considerations; Othberg AI et al.; The purpose of the current study was to determine the optimal strategy for preparing cell suspensions for co-transplantation . In the first experiment, the number of Sertoli cell (SC) aggregates and the number of tyrosine hydroxylase positive neurons were compared over time when cell suspensions of Sertoli or ventral mesencephalic cells were kept as a co-suspension mixed at 0 h . Cells from each suspension were dispensed onto glass slides in a manner similar to transplantation . When dispensed in this manner, the number of SC aggregates and TH-positive neurons decreased over 4 h . In experiment 2, the cell suspensions were mixed just prior to injection at each of four timepoints, the number of aggregates and TH neurons was consistent over time . Clearly this latter strategy resulted consistent recovery of both cell types for transplants up to 3 h after suspension preparation.

Methods, 1998 Jun, 15(2), 151 - 9
Cell replication rates and processes concerning antibody production in vitro are not influenced by 2.45-GHz microwaves at physiologically normal temperatures; van Dorp R et al.; Several contradictory papers concerning the effects of microwaves on living organisms and on in vitro cell suspensions have been published through the years . These papers are difficult to interpret, because temperature measurement data are often lacking . Reliable temperature measurements are important, because they enable one to determine whether the observed microwave effects are thermal or nonthermal . Therefore, a method was developed to investigate microwave effects on cellular processes, in which the temperature was precisely monitored during microwave treatment using a fiberoptic thermometer . This method involved the processes required for in vitro production of monoclonal antibodies . Monoclonal antibodies are vital ingredients in (microwave-stimulated) immunostaining techniques and ELISAs, which have become important techniques in neuroscience . The effects of 2.45-GHz microwaves on mouse myeloma and (neural) hybridoma cell replication rates and on antibody production were investigated . In addition, the effects on the cell fusion abilities of spleen lymphocytes and myeloma cells and on in vitro immunization were studied . The results of this study show no effects of microwaves on either of the processes mentioned using exposure times up to 5 h a day at a physiologically normal temperature of 37 degrees C . It was concluded that the effects of 2.45-GHz microwaves detected at higher temperatures are thermal effects and that no indications for nonthermal 2.45-GHz microwave effects exist under the exposure conditions used in the present study .

Proc Natl Acad Sci U S A, 1998 Jul 7, 95(14), 8398 - 403
Local mechanical stimulation induces components of the pathogen defense response in parsley; Gus-Mayer S et al.; Cell suspension cultures of parsley (Petroselinum crispum) have previously been used as a suitable system for studies of the nonhost resistance response to Phytophthora sojae . In this study, we replaced the penetrating fungus by local mechanical stimulation by using a needle of the same diameter as a fungal hypha, by local application of a structurally defined fungus-derived elicitor, or by a combination of the two stimuli . Similar to the fungal infection hypha, the local mechanical stimulus alone induced the translocation of cytoplasm and nucleus to the site of stimulation, the generation of intracellular reactive oxygen intermediates (ROI), and the expression of some, but not all, elicitor-responsive genes . When the elicitor was applied locally to the cell surface without mechanical stimulation, intracellular ROI also accumulated rapidly, but morphological changes were not detected . A combination of the mechanical stimulus with simultaneous application of low doses of elicitor closely simulated early reactions to fungal infection, including cytoplasmic aggregation, nuclear migration, and ROI accumulation . By contrast, cytoplasmic rearrangements were impaired at high elicitor concentrations . Neither papilla formation nor hypersensitive cell death occurred under the conditions tested . These results suggest that mechanical stimulation by the invading fungus is responsible for the observed intracellular rearrangements and may trigger some of the previously demonstrated changes in the activity of elicitor-responsive genes, whereas chemical stimulation is required for additional biochemical processes . As yet unidentified signals may be involved in papilla formation and hypersensitive cell death.

Proc Natl Acad Sci U S A, 1998 Jul 7, 95(14), 7921 - 6
Arabinogalactan-proteins from Nicotiana alata and Pyrus communis contain glycosylphosphatidylinositol membrane anchors; Youl JJ et al.; Arabinogalactan-proteins (AGPs) are a class of proteoglycans found in cell secretions and plasma membranes of plants . Attention is currently focused on their structure and their potential role in growth and development . We present evidence that two members of a major class of AGPs, the classical AGPs, AGPNa1 from styles of Nicotiana alata and AGPPc1 from cell suspension cultures of Pyrus communis, undergo C-terminal processing involving glycosylphosphatidylinositol membrane anchors . The evidence is that (i) the transmembrane helix at the C terminus predicted from the cDNA encoding these proteins is not present-the C-terminal amino acid is Asn87 and Ser97 for AGPNa1 and AGPPc1, respectively; (ii) both AGP protein backbones are substituted with ethanolamine at the C-terminal amino acid; and (iii) inositol, glucosamine, and mannose are present in the native AGPs . An examination of the deduced amino acid sequences of other classical AGP protein backbones shows that glycosylphosphatidylinositol-anchors may be a common feature of this class of AGPs.

Photochem Photobiol, 1998 Jun, 67(6), 714 - 9
Susceptibility to effects of UVB irradiation on induction of contact sensitivity, relevance of number and function of Langerhans cells and epidermal macrophages; Skov L et al.; Sensitization on skin exposed to acute low-dose UVB irradiation separates normal humans into two phenotypically distinct groups: One group, following sensitization on UVB-irradiated skin, develops contact sensitivity, designated UVB resistant (UVB-R) and the second group, following sensitization on UVB-irradiated skin, fails to develop contact sensitivity, designated UVB susceptible (UVB-S) . To investigate whether UVB susceptibility in humans in related to antigen-presenting activity in the skin we studied the effect of UVB irradiation on the number and function of the epidermal antigen-presenting cells in volunteers identified as UVB-R and UVB-S . Single cell suspensions of epidermal cells from control skin and skin exposed to 3 minimal erythema doses (MED) of UVB 3 days previously were stained for Langerhans cells (CD1a+HLA-DR+) and epidermal macrophages (CD1a-HLA-DR+) . The UVB exposure of the skin significantly decreased the percentage of Langerhans cells (UVB-R: n = 7, P < 0.02, UVB-S: n = 6, P < 0.03) and increased the percentage of epidermal macrophages (UVB-R: n = 7, P < 0.03, UVB-S: n = 6, P < 0.03) however to the same degree in both the UVB-R and the UVB-S group . To study the effect on Langerhans cell alloreactivity, epidermal cells were harvested immediately after UVB irradiation . However, in both UVB-R and UVB-S subjects the Langerhans cell alloreactivity was blocked to the same degree immediately after UVB irradiation compared to nonirradiated epidermal cells . To determine the effect of UVB irradiation on epidermal macrophages, epidermal cells were harvested 3 days after UVB irradiation . Irradiated epidermal cells from both UVB-R and UVB-S subjects demonstrated a strong antigen-presenting capacity compared to epidermal cells from control skin leading to activation of T cells that mainly secrete interferon (IFN)-gamma and not interleukin (IL)-4 . In conclusion we found that UVB susceptibility was not correlated with the number of Langerhans cells or epidermal macrophages in the skin at the same time of sensitization . Neither was it correlated with the capacity of Langerhans cells nor UVB-induced epidermal macrophages to activate T cells in vitro.

Cell Transplant, 1998 May-Jun, 7(3), 267 - 73
In vivo identification, survival, and functional efficacy of transplanted hepatocytes in acute liver failure mice model by FISH using Y-chromosome probe; Krishna Vanaja D et al.; Hepatocyte transplantation has excited much interest in lending temporary metabolic support to a failing liver following acute liver injury . The exact site from which they act and the clinical, biochemical, and histological changes in the recipient body following hepatocyte transplantation is yet to be worked out . The present study is an attempt to delineate location and function of transplanted hepatocytes and also the overall survival of these cells with a fluorescent in situ hybridization (FISH) technique using a Y-chromosome-specific probe in a carbon tetrachloride (CCl4)-induced mice model of fulminant hepatic failure . Fifty-five syngenic adult Swiss female mice of approximately the same age and body weight were divided into three groups . Group-1 (n = 15), which received mineral oil, served as a negative control . Group-II (n = 15) received CCl4 (3 mL/kg) 40% vol/vol in mineral oil, by gavage served as positive control for hepatic failure . Group-III (n = 25) received intrasplenic transplantation of syngenic single cell suspension of hepatocytes in Hanks medium, after 30 h of CCl4 administration . Male Swiss adult mice (n = 15) served as donors of hepatocytes . The overall survival of animals in groups I to III was 100, 0, and 70%, respectively, by 2 wk of the study period . Transplanted hepatocytes were identified by Periodic Acid Schiff (PAS) staining and confirmed with a FISH technique using the Y-chromosome probe . The majority of exogenously transplanted hepatocytes were found in the liver and spleen sections even after 1 wk of hepatocyte transplantation . Transplanted cells were mostly found to be translocated into the sinusoids of the liver . Transplanted hepatocytes were found to be beneficial as a temporary liver support in a failing liver, significantly improving the survival of the animals . In the present study, the FISH technique was used to unequivocally distinguish the transplanted cells from the host, and thus describes a model for studying the distribution and survival of the transplanted cells.

Pathobiology, 1998, 66(2), 84 - 9
Epinephrine augments specific T-cell responses to antigen in C57BL/6 (H-2b) weak-responder mice by a CD8+ lymphocyte-dependent mechanism; Rodberg GM et al.; Stress has been implicated as a factor in the pathogenesis of autoimmune disorders . In order to determine the effect of adrenergic stress on immune responses in vivo, C57BL/6 (B6; H-2b) mice, which respond weakly to hen-egg lysozyme (HEL), were immunized on day 0 with HEL (50-200 microg s.q.) and subsequently injected with epinephrine (EPI; 0.1-0.5 mg/kg s.q.) daily for up to 10 days . Controls included A/J mice (H-2k) which respond strongly to HEL . In some experiments, B6 mice were depleted of CD4+ or CD8+ lymphocytes by monoclonal antibody treatment in vivo, prior to immunization with HEL, and injection with EPI . On day 10, single cell suspensions of draining lymph nodes (LN) and spleen were examined for immune phenotype, proliferative responses to HEL, and lymphokine production . Minimal specific proliferative responses were detected in B6 mice compared to A/J mice . However, lymphocyte proliferation increased in HEL-immunized EPI-treated B6 mice but not in the A/J mice . IL-2-mediated proliferation and IL-2 secretion were both increased in the HEL-immunized EPI-treated B6 mice . The depletion of CD8+ but not CD4+ lymphocytes in vivo abrogated the effects of EPI, whereas adoptive transfer of naive CD8+ splenocytes to the CD8-depleted mice restored specific responses in the HEL-immunized EPI-treated animals . We conclude that EPI augments antigen-specific T-cell responses to HEL in B6 mice by a CD8+ T-cell-dependent mechanism.

Mol Gen Genet, 1998 May, 258(3), 306 - 14
Analysis of the Ac promoter: structure and regulation; Fridlender M et al.; The Ac-encoded transposase, a factor that is essential for the mobility of the Ac element, is expressed under the control of a promoter that lacks a conventional TATA box . The regulation of this promoter is poorly understood . We have analyzed Ac promoter structure and activity, both in vitro and in vivo, using transgenic tobacco plants and cell suspensions . A deletion analysis of the Ac 5' region showed that the minimal promoter is located within 70 bp of the major transcription initiation site (at position 334) . The minimal promoter includes the sequence TAAGAAATA at position 294 303, i.e., about 30 nucleotides upstream from the transcription start site . This sequence binds specifically to the TATA-binding protein (TBP), suggesting that it is functional as a TATA box . The regulation of the Ac promoter was studied throughout plant development . Levels of Ac mRNA were low in all tissues studied, with higher expression being observed in dividing cells . In order to test whether Ac promoter is regulated during the cell cycle, a tobacco cell suspension transformed with Ac, was grown synchronously . No differences were found in Ac mRNA levels between cells in S, G2, M, or G1 phases; however, expression was lower in the stationary phase . We conclude that Ac promoter is not cell-cycle regulated but is expressed at a higher level in dividing cells . The possible relationship between promoter features and the regulation of Ac element transposition is discussed.

J Gastroenterol Hepatol, 1998 May, 13(5), 521 - 7
Tamoxifen inhibits colorectal cancer metastases in the liver: a study in a murine model; Kuruppu D et al.; Liver metastases account for over 70% of deaths resulting from colorectal carcinoma, with survival rates varying between 6-18 months . At present, surgical resection offers the only hope for a cure, while chemotherapy, focal destructive techniques and selective internal radiation offer palliative care . Tamoxifen, a non-steroidal anti-oestrogen is primarily known for treating oestrogen receptor (ER)-positive breast cancer . Some studies suggest that tamoxifen may have beneficial effects in malignancies other than breast cancer . These inhibitory effects, which have been shown to be independent of the ER, highlight new mechanisms of therapeutic action . Using an intrasplenic animal model we report the efficacy of tamoxifen on experimental liver metastases . In this model, a dimethyl hydrazine-induced colon carcinoma cell suspension is introduced into the portal circulation via the spleen, which results in secondary tumour deposits in the liver in virtually all animals . Tamoxifen was administered at a dose of 1 mg/kg suspended in 1.0% methyl cellulose . The control group received an equal volume of the vehicle . The reagents were administered s.c . on the day of metastases induction and were continued daily over a 4 week period . The effect of tamoxifen on tumour growth was assessed by stereology and bromodeoxyuridine immunohistochemistry at selected time points . Data were assessed by a multiple analysis of variance where P < 0.05 was considered significant . In the control group the volume of metastases increased from 44 +/- 41 mm3 at day 10 to 517 +/- 380 mm3, 1394 +/- 598 mm3 and 2082 +/- 675 mm3 by days 16, 22 and 28, respectively . Daily administration of tamoxifen exerted an inhibitory effect on tumour growth during the first 3 weeks, recording a volume of 421 +/- 299 mm3 by day 22 compared with the control group at that time point (P = 0.00004) . The inhibitory effect diminished by the fourth week recording a tumour volume of 1344 +/- 674 mm3 by day 28 . Inhibition of tumour growth at day 22 coincides with a reduction of cells in the S phase of the cell cycle . The percentage of brdU-positive nuclear profiles in metastases of tamoxifen-treated mice at 3 weeks was 35.87 +/- 5.60% compared with 48.01 +/- 3.96% in the control group (P = 0.001) . These data suggest that tamoxifen has a potent inhibitory action on colorectal liver metastases by exerting an effect on cell proliferation.

Br J Dermatol, 1998 Apr, 138(4), 644 - 8
Leucoderma treated by transplantation of a basal cell layer enriched suspension; Olsson MJ et al.; The aim of this study was to test the usefulness of a melanocyte-enriched cell suspension for the treatment of leucoderma . After removal of a superficial (4-30 cm2) skin sample, the cells were mechanically separated in a trypsin-EDTA solution, centrifuged and washed in a melanocyte medium . The melanocyte-enriched epidermal cell suspension devoid of stratum corneum and stratum granulosum was then applied to the dermabraded depigmented skin . The 26 patients treated had piebaldism (three), vitiligo vulgaris (17), segmental vitiligo (three), halo naevi (one), naevus depigmentosus (one) and chemical leucoderma (one) . In patients with widespread piebaldism we found that by diluting the cell suspension the recipient area could be increased to up to 10 times the size of the donor area with the same good results as without or with less dilution . In patients with vitiligo areas of between 50 and 90 cm2, the recipient areas were increased three- to fivefold in the donor area . Patients with piebaldism, segmental vitiligo and halo naevi healed completely, as did most patients with vitiligo . In naevus depigmentosus no effect was seen . Our new method for treatment of leucoderma has the advantage that cell culture is not needed and that it is more suitable than epidermal sheet grafts when several small areas are to be treated.

Clin Sci (Lond), 1998 Apr, 94(4), 437 - 45
Neutrophil cathepsin G modulates platelet P-selectin expression and inhibits P-selectin-mediated platelet-neutrophil adhesion; Ilton MK et al.; 1 . Close contact between platelets and neutrophils modulates their cellular interactions in thrombotic and inflammatory states, with stimulation of P-selectin expression on platelets by agonists such as thrombin and neutrophil-derived cathepsin G being critical in mediating platelet-neutrophil adhesion . This study compared the effects of thrombin and cathepsin G on platelet P-selectin expression and on P-selectin-mediated platelet-neutrophil adhesion . 2 . Washed platelets and platelet-neutrophil mixed cell suspensions (platelet/neutrophil ratio, 10:1) were incubated with either the supernatant of activated neutrophils, purified cathepsin G or thrombin . Platelet P-selectin expression and platelet adhesion to neutrophils was quantified by flow fluorocytometric analysis . 3 . The supernatant from activated neutrophils stimulated platelet P-selectin expression comparable to that produced by purified cathepsin G or thrombin . P-selectin expression induced by both activated neutrophil supernatant and purified cathepsin G was completely inhibited by alpha 1-antichymotrypsin, a specific inhibitor of cathepsin G . Unlike thrombin, which induced maximum platelet P-selectin expression by 10 min, sustained to 120 min, cathepsin G induced an initial large increase in platelet P-selectin expression, followed by a progressive reduction over 30-60 min to baseline levels . 4 . Co-incubation of neutrophils with thrombin-stimulated platelets resulted in a significant increase in P-selectin-mediated platelet-neutrophil adhesion, which was completely inhibited by preincubation of neutrophils with anti-sialyl Lewis(x) monoclonal antibody . Thrombin produced maximum platelet-neutrophil adhesion by 10 min which remained stable over 120 min . In contrast, cathepsin G-stimulated platelets did not adhere to neutrophils over 120 min of co-incubation . Addition of cathepsin G to thrombin-stimulated platelets caused a progressive reduction over 30-60 min to baseline levels of platelet-neutrophil adhesion . 5 . Neutrophil-derived cathepsin G is a potent platelet activator, but unlike thrombin it causes a time-dependent loss of platelet P-selectin expression and inhibits P-selectin-mediated platelet-neutrophil adhesion . Therefore, cathepsin G may modulate thrombin-mediated platelet-neutrophil adhesive interactions in inflammation and thrombosis.

Leukemia, 1998 Jun, 12(6), 882 - 6
CD34+/CD36- cells from myelodysplasia patients have a limited capacity to proliferate but can differentiate in response to Epo and MGF stimulation; Brada SJ et al.; Myelodysplasia (MDS) is mostly characterized by a normal or increased number of normoblasts in the bone marrow and an impaired in vitro colony formation . In the present study we analyzed whether this might be due to a disconnection between proliferation and differentiation . CD34+/CD36- sorted bone marrow cells of 18 MDS patients were cultured in a clonogenic and suspension culture assay in the presence of erythropoietin (Epo) and mast cell growth factor (MGF) . Burst-forming units erythroid (BFU-E, 75 +/- 88/10(4) CD34+ cells, X +/- s.d.) and colony-forming units E (CFU-E) were observed in eight of the 13 cases (62%) with refractory anemia with or without ring sideroblasts (RA and RARS) and one of the five cases with RA with excess of blasts or in transformation (RAEB and RAEB-T) . Suspension cultures with CD34+/CD36- sorted cells with Epo plus MGF demonstrated an 8.9 +/- 6.5-fold expansion after 7 days in cases with >10 BFU-E/10(4) CD34+/CD36- cells while cases with <10 BFU-E/10(4) CD34+/CD36- cells demonstrated 1.0 +/- 0.8-fold expansion especially in cases with RAEB/RAEB-T . FACS and morphology analysis after 7 days of suspension culture demonstrated partial differentiation along the erythroid lineage in cases with RA/RARS (75%) and RAEB/RAEB-T (66%) reflected by the presence of erythroblasts and normoblasts with variable expression of CD34, CD36 and Glycophorin A . In cases with erythroid colony formation 69 +/- 24% of the cells were CD34-/CD36+ and in cases with <10 BFU-E/10(4) CD34+ cells 18 +/- 16% of cells were CD34-/CD36+ . Iron staining showed the presence of ring sideroblasts in two cases with RARS indicating that the cells originate from the abnormal erythroid clone . Finally, it was shown that cases with an impaired proliferative response demonstrate an enhanced binding of Annexin-V on CD34+ cells during the first days of the cell suspension culture phase . These results suggest that a defect in the proliferative response is most pronouncedly expressed in MDS whereas a subpopulation of cells retain the capacity to differentiate between transition to a terminated stage.

J Clin Invest, 1998 Jun 15, 101(12), 2650 - 7
Regulation of proliferation of human colonic subepithelial myofibroblasts by mediators important in intestinal inflammation; Jobson TM et al.; An increase in myofibroblast number may be necessary for wound healing but may also lead to postinflammatory scarring . We have, therefore, studied the role of mediators important in inflammatory bowel disease in regulating proliferation of human colonic myofibroblasts . Using primary cultures of these cells, we have shown increases in {3H}thymidine incorporation in response to platelet-derived growth factor (EC50 = 14 ng/ml), basic fibroblast growth factor (EC50 = 2.2 ng/ml), and epidermal growth factor (EC50 = 1.1 ng/ml) . Coulter counting of cell suspensions demonstrated increases in cell number with these growth factors along with insulin-like growth factor-I and -II . In addition the proinflammatory cytokines IL-1beta and TNF-alpha produced increases in {3H}thymidine incorporation . IL-1beta and platelet-derived growth factor together produced an increase in {3H}thymidine greater than either agonist alone; this effect was not, however, seen when we examined changes in cell numbers . Finally, we demonstrate a mechanism whereby these responses may be downregulated: vasoactive intestinal peptide (1 microM) elevates cyclic AwMP in these cells 4 . 2-fold over control and produces a dose-related inhibition of platelet-derived growth factor-driven proliferation with a maximum inhibition of 33% at 1 microM.

Neuroreport, 1998 May 11, 9(7), 1313 - 6
MK-801 does not enhance dopaminergic cell survival in embryonic nigral grafts; Schierle GS et al.; Two groups of hemiparkinsonian rats received grafts of embryonic ventral mesencephalon with or without the addition of the NMDA receptor antagonist (+)dizocilpine hydrogen maleate (MK-801) . When added to the cell suspension, a 10 microM concentration of MK-801 did not enhance the survival of tyrosine hydroxylase positive neurones in the grafts . These findings suggest that cell death occurring during nigral transplantation is not primarily due to excitotoxicity.

Nat Biotechnol, 1996 Sep, 14(9), 1129 - 32
Methyl jasmonate-induced overproduction of paclitaxel and baccatin III in Taxus cell suspension cultures; Yukimune Y et al.; Taxus cell culture may be an alternative source of paclitaxel and related taxane production . Significantly increased amounts of paclitaxel and baccatin III were observed in cultured cells of Taxus species after exposure to methyl jasmonate . Among the three species of Taxus tested, Taxus media showed the highest paclitaxel content while Taxus baccata showed the highest baccatin III content when 100 microM of methyl jasmonate was added to the culture media . Furthermore, the activities of methyl jasmonate and related substances for inducing paclitaxel production were compared in cell suspension cultures of T . media . Methyl jasmonate and its free acid showed the strongest promoting activity . Reduction of the keto group at the C-3 position greatly reduced this activity . cis-Jasmone, which does not have a carboxyl group at the C-1 position, had almost no activity . These results suggest that these two regions of methyl jasmonate are important for promoting the production of paclitaxel and related taxanes in Taxus cell cultures.

J Investig Dermatol Symp Proc, 1996 Apr, 1(1), 78 - 81
Reduction of epidermal abnormalities and inflammatory changes in psoriatic plaques during treatment with vitamin D3 analogs; van de Kerkhof PC; Vitamin D3 analogs interfere with various aspects of epidermal growth, inflammation, and cellular differentiation . Most data are derived from in vitro studies . In the present review, the in vivo effects of vitamin D3 analogues on the psoriatic plaque are discussed . Calcipotriol, tacalcitol, and calcitriol in ointment modulate aspects of epidermal growth, differentiation, and inflammation . Immunohistochemical studies suggest that the inflammatory changes might be more expressed after treatment with calcitriol and tacalcitol . Flow cytometric quantification of the percentage of cells in SG2M phase and of keratin 10-positive cells revealed that calcipotriol reduced both indices significantly during treatment of psoriatic plaques . Flow cytometric analysis of epidermal cell suspensions using triple labeling for epidermal proliferation, expression of keratin 10, and vimentin permits a quantitative assessment of DNA synthesis selectively in the basal cells of the epidermis, an estimation of the distribution of the basal and suprabasal compartments, and a quantification of the distribution of mesenchymal and nonmesenchymal cells . Using this approach, the interference of tacalcitol with growth control of basal cells was demonstrated . Remarkably, recompartmentalization of basal and suprabasal cells and mesenchymal and nonmesenchymal cells proved to be inconspicuous during this treatment.

J Investig Dermatol Symp Proc, 1996 Apr, 1(1), 72 - 7
The vitamin D3 analog calcipotriol suppresses the number and antigen-presenting function of Langerhans cells in normal human skin; Dam TN et al.; Local activation of T lymphocytes appears to play an important role in psoriasis and autoimmune skin disease . 1 alpha,25-dihydroxyvitamin D3 and the vitamin D3 analog calcipotriol have been shown to inhibit immune induction in vitro . The purpose of the present study was to investigate the in vivo effect of calcipotriol on Langerhans cells in normal human skin and to determine the effect of 1,25-dihydroxyvitamin D3 and calcipotriol on isolated Langerhans cells to induce autologous T-cell proliferation . Using confocal laser scanning microscopy of epidermal suction blister roofs, it was found that application of calcipotriol cream to normal human skin for 4 d resulted in a dose-dependent decrease in the number of CD1a+ cells with a dendritic morphology and in the number of dendrites per cell . The suppressive effect of calcipotriol on Langerhans cells was as strong as that of the potent corticosteroid mometasonfuroate . In Langerhans cell-enriched cell suspensions (60-97% pure) isolated from normal human skin, 1,25-dihydroxyvitamin D3 and calcipotriol (10(-8)-10(-7) M) significantly suppressed their ability to stimulate antigen-dependent T-cell proliferation . Furthermore, the vitamin D receptor was detected by Western blot analysis in the isolated Langerhans cells . Neither immunohistochemical studies nor flow cytometry of Langerhans cells showed any change in the human leukocyte antigen-DR expression after 48 h culture with antigen with or without calcipotriol . It is proposed that the inhibitory effects of the vitamin D3 on Langerhans cells may induce immunosuppression in the skin.

Rinsho Byori, 1998 May, 46(5), 479 - 85
{Determination of pyrazinamide susceptibility for Mycobacterium tuberculosis by use of Middlebrook culture media and comparison with results of pyrazinamidase test}; Yamane N et al.; We developed a new test method to determine pyrazinamide (PZA) susceptibility for Mycobacterium tuberculosis in an acidified Middlebrook 7H9 broth (pH6.0), and evaluated in comparison with the agar proportion method of the National Committee for Clinical Laboratory Standards (NCCLS) M24-T and with pyrazinamidase assay . The test method is based on a culture in 4 ml of the modified Middlebrook 7H9 broth containing 100, 200 and 400 micrograms PZA/ml, respectively . First, the cell suspension was adjusted to a McFarland #1 turbidity, and then diluted 1:10 . After mixing, 0.1 ml of the diluted cell suspension was inoculated and incubated at 36 +/- 1 degrees C in an ambient air . After 7 day-incubation, the test broth was read in comparison with the growth control . When a significant growth at 100 micrograms PZA/ml or an attenuated growth at 100 micrograms PZA/ml but a significant growth at 400 micrograms PZA/ml were observed, the test isolate was interpreted as being PZA-resistant . When PZA-susceptible and PZA-resistant ATCC reference strains were repeatedly tested, the results obtained were highly precise and accurate . A total of 65 clinical isolates were tested, the results indicating 95.4% of agreements with the agar proportion method and 90.8% with pyrazinamidase assay . There found six discrepant results of 13 resistant isolates; three were susceptible by the agar proportion and all the six were positive by pyrazinamidase assay . Accordingly, we can conclude that, in place of radiometric Bactec System, our newly developed test method is an accurate, practical, rapid and nonradiometric alternative to determine PZA susceptibility for M . tuberculosis in clinical mycobacteriology laboratories.

J Virol Methods, 1998 Apr, 71(2), 211 - 8
In situ amplification of the Epstein-Barr virus genome in cell suspensions; Fares F et al.; Epstein-Barr virus (EBV) is distributed widely throughout the world . Apart from a association with two geographically-restricted malignancies (Burkitt's lymphoma and nasopharyngeal carcinoma), EBV is thought to be implicated in the etiology of B-cell lymphoma in immunocompromised individuals . In these patients, monitoring the viral load in serum can provide useful information on the timing of the instigation of antiviral therapy, i.e . as soon as a rise is detected . PCR technology, owing to its high sensitivity, is used frequently in such situations . In order to gain further insight into the nature of the peripheral blood cells carrying the viral genome on a cell-by-cell basis, an in situ amplification technique was developed as a model using two cell lines growing in suspension, with the aim of distinguishing between EBV-positive and EBV-negative cells . Preliminary experiments were undertaken subsequently on clinical samples from patients with infectious mononucleosis and patients with lymphoma indicating that this technique might be useful clinically.

Cell Immunol, 1998 Feb 25, 184(1), 74 - 82
Differential regulation of rejection of small intestinal and skin allografts in rats by injection of antibodies to ICAM-1 or the integrins alpha 4, alpha L, or beta 2; Gorczynski RM et al.; Female Lewis (LEW) rats received orthotopic small intestinal transplantation (SIT), or tail skin grafts from female (Lewis x Brown Norway)F1 (LBNF1) rats, along with peritransplant portal venous (pv) infusion of LBNF1 bone marrow-derived dendritic cells derived from male donors . All animals received im injection with cyclosporin A (5 mg/kg) for 3 consecutive days following transplantation . In some cases rats received intravenous injections, at 2-day intervals, with 1 mg of monoclonal antibodies to ICAM-1 or the integrins alpha 4, alpha L, or beta 2, or combinations of these reagents . Cells were harvested from the recipient rats at different times posttransplantation, and single cell suspensions were analyzed by FACS for expression of CD3+, CD4+, CD8+, alpha beta TcR+, and gamma delta TcR+ cells . Other tissue samples were used for histopathological assessment of rejection . We also investigated donor-specific and third-party (Wistar-Furth, Wi) restimulation of host lymphocytes from MLN, PLN, and PP for production of different cytokines in vitro . Of the various antibodies tested, only anti-alpha 4, but not anti-alpha L, -beta 2, nor -ICAM-1 led to further increased graft survival of LBNF1 SIT beyond that seen with pv-infused cells alone (30 days vs 19 days), while the combination of anti-alpha L (or beta 2) and ICAM-1 produced further significantly increased survival of skin grafts (30 days vs 21 days) . For both SIT and skin-grafted animals increased graft survival was associated with decreased production of IL-2 and IFN-gamma and increased production of IL-4 and IL-10 from tissues local to the graft (PP and draining LN, respectively), with less significant alterations in tissues distant to the graft (PLN for SIT, and MLN for skin grafts) . While, as reported previously, pv-immunized SIT rats showed increased gamma delta TCR+ cells within the SIT in association with increased graft survival, treatment with anti-alpha 4 diminished this increase in gamma delta TCR+ cells, while simultaneously increasing SIT survival . Nevertheless, the bias toward increased IL-10 production, and decreased IFN-gamma production, from cells of animals showing increased survival was maintained . These data suggest that local graft infiltration with gamma delta TCR+ cells following pv immunization is not necessary for prolongation of survival in this model system, although functional changes in the local cytokines milieu may be important.

Biotechnol Prog, 1998 May-Jun, 14(3), 425 - 33
Kinetics of ferrous iron oxidation by Leptospirillum bacteria in continuous cultures; van Scherpenzeel DA et al.; The oxidation of ferrous iron by Leptospirillum bacteria was studied in a continuous culture in the dilution rate range 0.009-0.077 h-1 and could be described with a rate equation for competitive ferric iron inhibition kinetics in terms of the ferric/ferrous iron ratio in the solution . The ferrous iron oxidation in the continuous culture was followed by means of oxygen and carbon dioxide concentration analyses in reference air and off-gas . From these measurements the oxygen consumption rate, rO2, the carbon dioxide consumption rate, rCO2, the biomass concentration, Cx, and the biomass specific oxygen consumption rate, qO2, in the culture were determined . The ferrous iron concentration in the culture was below accurate levels to determine with the usual titrimetric method and was therefore derived from measuring the solution redox potential . The degree of reduction balance was used to check the theoretically expected relation between the rates of ferrous iron, -rFe2+, oxygen, -rO2, and biomass, rx . The maximum biomass yield and maintenance coefficient on oxygen are Yoxmax = 0.047 mol of C/mol of O2 and mo = 0.057 mol of O2/(mol of C.h) . The maximum specific oxygen consumption rate, qO2,max = 1.7 mol of O2/(mol of C.h), the affinity coefficient, Ks/Ki = 0.0005 mol of Fe2+/mol of Fe3+, and the maximum specific growth rate, micromax = 0.069 h-1, Ks/Ki = 0.0004, were fitted from the measured data . For several dilution rates, off-line respiratory measurements with cell suspension from the continuous culture were carried out in dynamic BOM-Eh measurements . The dissolved oxygen and redox potential were measured simultaneously and monitored . The measured value of qO2,max varied between 2.3 and 1.7 mol/(mol of C.h) . The value of Ks/Ki = 0.0007 was equal in all experiments . The measured values of qO2 in the continuous culture were well described with the kinetics determined in dynamic BOM-Eh measurements . It was concluded that dynamic BOM-Eh measurements are a convenient method to determine the kinetics of continuous culture grown Leptospirillum bacteria.

Arch Pathol Lab Med, 1998 Jun, 122(6), 539 - 44
CD10 expression in follicular lymphoma and large cell lymphoma is different from that of reactive lymph node follicles; Almasri NM et al.; BACKGROUND AND OBJECTIVES: CD10 is a proteolytic enzyme expressed on the surface of germinal center cells and lymphomas derived from these cells . There is a well-known association between CD10 expression and lymphomas of follicular center cell origin . However, the reported frequency of CD10 positivity in follicular lymphomas is widely variable, and no studies have addressed the importance of assessing the intensity of CD10 expression in the diagnosis of these tumors . In this study, we utilized flow cytometry to determine differences in CD10 expression in lymphomas and follicular hyperplasias . METHODS: Cell suspensions from 61 follicular lymphomas, 43 diffuse large B-cell lymphomas, and 44 lymph nodes with follicular hyperplasia were analyzed simultaneously with phycoerythrin-labeled anti-CD20 and fluorescein isothiocyanate-labeled anti-CD10 . RESULTS: CD10 expression was mainly observed on B cells and was detected in 89% of follicular lymphomas, 56% of diffuse large B-cell lymphomas, and 55% of lymph nodes showing follicular hyperplasia . In follicular hyperplasia, two subpopulations of B cells displaying dim and bright CD20 expression were recognized . CD10 expression was restricted to the bright CD20-positive cells, which accounted for an average of 16% of B cells . In CD10-positive follicular and diffuse large B-cell lymphoma cases, a significantly higher proportion of B cells (73%) coexpressed CD10 . Furthermore, the intensity of CD10 expression in follicular lymphoma and diffuse large B-cell lymphoma was much higher than that of follicular hyperplasia . CONCLUSIONS: Quantitative flow cytometry can detect significant differences in CD10 expression between normal follicular cells and follicular or diffuse large B-cell lymphoma . The use of CD10 intensity of expression as well as the fraction of CD10-expressing B cells should help distinguish reactive from neoplastic B-cell processes.

Eur J Endocrinol, 1998 May, 138(5), 567 - 73
Intra-adrenal factors are not involved in the differential control of cortisol and adrenal androgens in human adrenals; Fearon U et al.; The differential control of adrenal androgens and cortisol may be due to intra-adrenal factors, which may be age- or sex-related, or due to extra-adrenal factors, such as circulating hormones . The purpose of this study was to identify any intrinsic differences that may exist in steroidogenic production occurring within adrenals obtained from males and females, and any maturational differences that may evolve with age . Using human adrenals from 48 transplant donors (32 males, 16 females; ages 5-60 years), the influences of age and sex on basal production of and ACTH-stimulated cortisol, androstenedione and dehydroepiandrosterone (DHEA) were examined in freshly prepared adrenal cell suspensions . Basal and ACTH-stimulated cortisol, androstenedione and DHEA production were similar in adrenals from males and females and did not correlate significantly with age when the whole group was examined . When steroidogenesis in male and female adrenals was examined separately against age, a significant correlation was observed only for basal and ACTH-stimulated androstenedione in adrenals from males in the younger age group, 5-30 years (basal: r=0.84, P=0.0001; ACTH-stimulated: r=0.52, P=0.007) . Examination of the relationships between the steroids disclosed that the basal and ACTH-stimulated cortisol/androgen ratios did not correlate significantly with age, but the androstenedione/DHEA ratio showed a significant direct relationship with age in males only (basal: r=0.53, P=0.006; ACTH-stimulated: r=0.5, P=0.01) . These data suggest that the influences of sex and age are minor in the modulation of adrenal steroidogenesis and support the concept that extra-adrenal factors dominate in the differential modulation of adrenal androgens and cortisol . The relationship between the androstenedione/ DHEA ratio and increasing age in men is consistent with the recently reported stimulatory effect of testosterone on adrenal steroidogenesis by induction of the conversion of DHEA to androstenedione.

Neurosci Lett, 1998 Apr 17, 246(1), 1 - 4
A lateralised grip strength test to evaluate unilateral nigrostriatal lesions in rats; Dunnett SB et al.; A modified grip strength meter was designed to enable the separate measurement of lateralised grip strength in the two forelimbs of rats, in order to allow assessment of deficits in animals with unilateral lesions and grafts within the basal ganglia . Unilateral 6-hydroxydopamine lesions of the dopaminergic nigrostriatal bundle induced a significant asymmetry, marked by an increased grip strength on the side contralateral to the lesion . Lesioned animals with additional implants of embryonic nigral cell suspensions into the dopamine-denervated neostriatum showed a reduced (but not significant) deficit and did not differ from control performance . The lateralised nature of the deficit excludes explanation based on global activational changes; rather the unilateral deficit may provide a simple test of unilateral 'rigidity' in a widely used rodent model of Parkinson's disease.

Phytochemistry, 1998 May, 48(1), 113 - 7
Induction of furanocoumarin biosynthesis in Glehnia littoralis cell suspension cultures by elicitor treatment; Kitamura Y et al.; Cell suspension cultures were established from Glehnia littoralis plants belonging to two different geographic strains . When the cells were treated with yeast extract, they started to produce and excrete furanocoumarins into the culture medium; a major component, bergapten, and a minor one, xanthotoxin, were detected and identified by HPLC and GC/MS . Changes in phenylalanine ammonia-lyase (PAL) activity and furanocoumarin production after elicitor treatment were traced, showing that PAL activity increased rapidly, reached a maximum after 24 h, and then declined to the normal level after 96 h which preceded the induced bergapten production . The induced-PAL activity of the cultured cells established from an S-type plant which accumulated trace amounts of furanocoumarins was about 50% of that in the cultured cells from an N-type plant that accumulated more than 0.1% furanocoumarins in the underground parts . However, the elicited production of bergapten was about six times higher in the cell cultures from the S-type plant . Addition of the PAL inhibitor 2-aminoindan-2-phosphoric acid (AIP) at 10 microM suppressed the induction of PAL activity and furanocoumarin production.

Biochem J, 1998 Apr 15, 331 ( Pt 2), 623 - 30
Histones and basic polypeptides activate Ca2+/cation influx in various cell types; Gamberucci A et al.; Histone H2A (1-10 microg/ml) added to Ehrlich ascite cell suspensions promoted: (i) Ca2+ influx, but no apparent intracellular Ca2+ mobilization; (ii) plasma-membrane depolarization and Na+ influx in Ca2+-free medium, which were recovered by Ca2+ readmission; (iii) influx of other cations such as Ba2+, Mn2+, choline+ and N-methyl-d-glucamine+, but not of propidium+, ethidium bromide and Trypan Blue . H2A-induced Ca2+ influx and cell depolarization were: (i) blocked by La3+ and Gd3+, but not by various inhibitors of receptor-activated Ca2+-influx pathways/channels; (ii) mimicked by various basic polypeptides, with Mr>4000; (iii) prevented or reversed by polyanions such as polyglutamate or heparin; (iv) present in other cell types, such as Jurkat, PC12 and Friend erythroleukaemia cells, but virtually absent from rat hepatocytes and thymocytes . We conclude that cationic proteins/polypeptides, by interacting in a cell-specific manner with the cell surface, can activate in those cells putative non-selective Ca2+ channels and membrane depolarization.

Biochem J, 1998 Apr 15, 331 ( Pt 2), 513 - 9
Purification and partial characterization of a peroxidase from plant cell cultures of Cassia didymobotrya and biotransformation studies; Vitali A et al.; An acidic peroxidase (EC 1.11.1.7) produced by cell suspension cultures of Cassia didymobotrya (wild senna) was purified from culture medium collected on the 29th day . The enzyme was shown to be a glycoprotein with a pI of 3.5, a molecular mass of approx . 43 kDa by SDS/PAGE and 50 kDa by gel filtration . The N-terminal sequence was very similar to those of other plant peroxidases . The peroxidase was characterized by a high specificity towards coniferyl alcohol and other natural phenolics such as guaiacol and ferulic and caffeic acids . These findings suggest that the enzyme is involved in lignification processes of the cell wall . Moreover, the enzyme was able to catalyse the oxidation of 4,3',4'-trihydroxychalcone and 4, 3',4'-trihydroxy-3-methoxychalcone to the corresponding 3, 3'-biflavanones, as mixtures of racemic and meso forms.

Plant Mol Biol, 1998 May, 37(1), 15 - 24
A diverse family of phenylalanine ammonia-lyase genes expressed in pine trees and cell cultures; Butland SL et al.; Using degenerate PCR primers that target evolutionarily conserved sequences in pal genes, we show that in the gymnosperm, Pinus banksiana, phenylalanine ammonia-lyase (PAL) is encoded by a multigene family of at least eight to ten loci . Five classes of pal sequence were easily distinguished among 28 clones sequenced from the products of PCR amplification of haploid genomic DNA . The dominant sequence from each class was named, yielding pal1 to pal5 loci . These genes shared 68.8% to 94.0% nucleotide identity over the 366 bp region compared . All of pal1 to pal5 were expressed in cell suspension cultures treated with a fungal elicitor and all but pal3 were expressed in differentiating xylem tissue of a mature tree . Only pall was expressed in unelicited cell cultures . While these P . banksiana genes are quite divergent, they are still more similar to each other than to any angiosperm pal gene cloned to date . For its roles in development and defense, PAL production in P . banksiana is coordinated from a large, diverse multigene family . We discuss evidence suggesting that other pines have similar pal gene family structures.

Surg Endosc, 1998 Jun, 12(6), 828 - 34
Traumatic handling of the tumor independent of pneumoperitoneum increases port site implantation rate of colon cancer in a murine model; Lee SW et al.; BACKGROUND: Reports of port site tumor recurrences after laparoscopic-assisted resection of colon tumors have raised concerns about the safety of laparoscopic cancer surgery . Tumor cell suspension studies in animals have implicated the CO2 pneumoperitoneum (pneumo) in the etiology of port tumors . Unfortunately, in several ways, the cell suspension model is unrealistic and does not permit assessment of how tumor cells become liberated from the primary tumor . The purpose of this study was to establish a more realistic splenic tumor model and to determine the relative importance of the CO2 pneumo and excessive surgical manipulation in the development of port site and incisional tumor recurrences . METHODS: Splenic tumors were established in female Balb/C mice (n = 134) via a subcapsular injection of 10(5) C-26 colon adenocarcinoma cells (0.1 ml volume) via a left-flank incision at the initial procedure . Ten days later, the animals were reexplored via a 1-cm left subcostal incision . Those with isolated splenic tumors (95%) were randomized into one of four groups: (a) control, (b) CO2 pneumo, (c) crushed tumor, or (d) crushed tumor with pneumo . Ports were placed in the left lower, right lower, and right upper quadrants of each mouse . In groups 1 and 2, the mice underwent a meticulously performed splenectomy; in groups 3 and 4, the tumor capsule was crushed intraabdominally prior to splenectomy . In groups 1 and 3, the subcostal incision was closed and the ports were removed after 15 min of anesthesia . Following splenectomy, group 2 and group 4 mice underwent closure of the subcostal incision and a 15-min CO2 pneumo (4-6 mm Hg) after which the ports were removed . Twelve days later, the mice were killed and examined for abdominal wall tumor implants . RESULTS: Significantly more animals in group 3 (crushed tumor) developed port site and incisional tumors than those in group 1 (control) (p < 0.002 for both comparisons) . The same results were found when group 4 (crush plus pneumo) was compared to group 2 (pneumo) (p < 0.002 for both comparisons) . Regarding the port wounds, when the ports are considered individually (number of ports with tumors/total number of ports for each group), there were significantly more port tumors in the two crush groups than in the noncrush groups . No significant differences were noted when the port site and incisional tumor rates for group 1 (control) and group 2 (pneumo) were compared or when the results for group 2 (crush) and group 4 (crush pneumo) were compared . CONCLUSIONS: A splenic tumor model was successfully established . When compared to meticulous technique, purposefully traumatic handling of the splenic tumor before resection resulted in significantly more port wound and incisional tumors . In contrast, the addition of a pneumo after splenectomy did not significantly influence the incidence of port tumors in either the "good" or the "poor" technique groups . These results suggest that surgical technique plays a larger role in the development of port site tumors than the CO2 pneumoperitoneum.

Obstet Gynecol, 1998 Jun, 91(6), 987 - 92
Leukocytes in the cervix: a quantitative evaluation of cervicitis; Stern JE et al.; OBJECTIVE: To quantify the numbers of leukocytes in the normal cervix and relate these numbers to the diagnosis of cervicitis . METHODS: Isolated cell suspensions were prepared from cervical tissue recovered at hysterectomy from 37 women who had no obvious cervical disease . The percentages of CD45+ cells (leukocytes) in these preparations were determined using immunofluorescence-based flow cytometric analysis . These percentages were compared with the pathologist's assessment of cervicitis . RESULTS: Leukocytes were present in all cervical samples tested . For endocervical samples, the mean (+/- standard error of the mean {SEM}) percentage of CD45+ cells was 12.4 +/- 1.9% of cells in patients with a diagnosis of cervicitis (n = 16) and 9.1 +/- 1.1% in patients without cervicitis (n = 17) . For ectocervical samples, the mean (+/- SEM) percentage was 14.8 +/- 3.0% in those with cervicitis (n = 16) and 9.5 +/- 1.6% in those without cervicitis (n = 19) . The differences between samples from patients with cervicitis and those without cervicitis were not statistically significant at the .05 level . Intra- and interassay variabilities were 5.7 +/- 1.2% and 7.3 +/- 1.6%, respectively . CONCLUSION: Our study demonstrates there is a resident population of leukocytes in the cervix . Leukocyte number did not relate clearly and consistently to the diagnosis of cervicitis made by the pathologist . We suggest that the resident population of leukocytes, in the absence of other indicators of infection, may confuse determinations of cervicitis.

J Dermatol, 1998 Apr, 25(4), 211 - 21
The proliferation and differentiation of neonatal epidermal melanocytes in F1 hairless mice of HR-1 x HR/De in serum-free culture; Furuya R et al.; To investigate the characteristics of the proliferation and differentiation of epidermal melanocytes in F1 hairless mice of HR-1 x HR/De parents in vitro, cell suspensions of the neonatal epidermis were cultured in a serum-free medium supplemented with dibutyryl adenosine 3',5'-cyclic monophosphate (DBcAMP) and/or basic fibroblast growth factor (bFGF) . The differentiation of melanocytes was induced by treatment with DBcAMP . In contrast, the sustained proliferation of melanoblasts was induced by combined treatment with DBcAMP and bFGF . The melanoblasts could be subcultured in serum-free medium supplemented with the two factors in the presence of keratinocytes, but not in their absence . This is the first report of successful culture of melanoblasts and melanocytes from hairless mice; it is expected to be useful in understanding the mechanism of the development of pigmented spots in the epidermis of (HR-1 x HR/De)F1 mice, which are reported to be induced by repeated exposure to ultraviolet light B.

Kidney Int, 1998 Jun, 53(6), 1647 - 53
Clusterin protects against oxidative stress in vitro through aggregative and nonaggregative properties; Schwochau GB et al.; Perturbations of cell interactions, an early event in acute renal injury, have important pathophysiologic consequences . We hypothesized that promotion of cell interactions protects cells from injury . To test this hypothesis, a single cell suspension of LLC-PK1 cells (porcine proximal tubular cell line) treated with albumin (control) was compared to cells aggregated with fibrinogen or purified human clusterin (aggregation graded 0 to 4) . Following aggregation, the cells were injured with 1.5 mM hydrogen peroxide (H2O2) for three hours . Cell aggregation induced by clusterin but not fibrinogen protected against oxidant injury by H2O2 . Complete abrogation of cytotoxicity occurred at a clusterin concentration of 2.5 micrograms/ml, which resulted in an aggregation score of 1 . In the absence of aggregation, clusterin at concentrations of 20 and 50 micrograms/ml, but not lower doses, partially protected against injury induced by H2O2 . Cell aggregation induced by both clusterin and fibrinogen partially protected against endogenously generated oxidant stress induced by incubating LLC-PK1 cells with aminotriazole and 1-chloro-2,4-dinitrobenzene (CDNB) . In conclusion, clusterin protects against models of oxidant stress in vitro, whether generated by exogenously administered hydrogen peroxide, or from endogenously produced peroxide, and such protective effects can accrue from aggregative and nonaggregative properties of clusterin.

J Biochem (Tokyo), 1998 Jun, 123(6), 1017 - 23
Efficient induction of hepatocyte spheroids in a suspension culture using a water-soluble synthetic polymer as an artificial matrix; Yamada K et al.; The preparation of hepatocyte spheroids by adding a water-soluble synthetic polymer as an artificial matrix was performed in a cell suspension system . Cell-aggregation was promoted without cytotoxicity by adding Eudragit (a copolymer of methacrylic acid and methylmethacrylate) to the culture medium . Spheroid-like cell aggregates, whose liver functions were enhanced, were effectively formed in the presence of 0.1% Eudragit, independent of the cultural substratum . Moreover, the mass preparation of spheroids could be achieved with a high production yield by means of a suspension culture in a spinner flask . In this case, the polymer protected the cells from damage due to agitation . The spheroids induced with Eudragit expressed high liver functions, such as albumin secretion, ammonia removal, and urea synthesis . On histological observation, the spheroids showed a well-developed cell adhesion apparatus and bile canaliculi . In addition, a higher calcium ion concentration in the cells of spheroids was observed compared with in monolayer cells.

Blood, 1998 Jun 1, 91(11), 4282 - 91
A directly spliced exon 10-containing CD44 variant promotes the metastasis and homotypic aggregation of aggressive non-Hodgkin's lymphoma; Yakushijin Y et al.; Variants of the CD44 cell-surface adhesion molecule include additional sequences encoded by combinations of exons from the membrane proximal domain (exons 6-14) . Preliminary studies suggest that these additional variable membrane proximal sequences may alter the ligand specificity, glycosylation, and biologic function of CD44 . In earlier studies, we found that primary extranodal and widely disseminated aggressive non-Hodgkin's lymphomas (NHLs) and normal activated B cells expressed a directly spliced exon 10-containing variant (CD44ex10), whereas normal resting B cells expressed larger exon 10-containing variants (CD44ex10-14 and CD44ex7-14) . To obtain additional information regarding the function of exon 10-containing CD44 variants in aggressive NHL, we generated aggressive NHL transfectants that expressed CD44ex10, CD44ex10-14, CD44ex7-14, the standard CD44 isoform (CD44H), or vector alone, and evaluated the local tumorogenicity, aggregation, and metastatic potential of these transfectants . CD44ex10 aggressive NHL transfectants were more likely to cause local tumor formation in nude mice than transfectants expressing the larger exon 10-containing variants, CD44H, or vector alone . In addition, cell suspensions derived from CD44ex10 local tumors exhibited far greater homotypic aggregation than those obtained from other CD44 or vector-only local tumors . In nude mice that received CD44ex10 transfectants, distant metastases were also significantly more likely to develop than in animals that were given either the CD44ex10-14, CD44ex7-14, CD44H, or vector-only transfectants . These data provide the first evidence that the directly spliced exon 10-containing CD44 variant (CD44ex10) has a unique biologic function in aggressive NHL.

Planta, 1998 May, 205(1), 92 - 9
Evidence for secretion of vacuolar alpha-mannosidase, class I chitinase, and class I beta-1,3-glucanase in suspension cultures of tobacco cells; Kunze I et al.; We have investigated the possibility that vacuolar proteins can be secreted into the medium of cultured cells of Nicotiana tabacum L . Time-course and balance-sheet experiments showed that a large fraction, up to ca . 19%, of vacuolar alpha-mannosidase (EC 3.2.1.24) and vacuolar class I chitinase (EC 3.2.1.14) in suspension cultures accumulated in the medium within one week after subculturing . This effect was most pronounced in media containing 2,4-dichlorophenoxyacetic acid (2,4-D) . Under comparable conditions only a small fraction, 1.8-5.1% of the total protein and ca . 1% of malate dehydrogenase (EC 1.1.1.37), which is localized primarily in the mitochondria and cytoplasm, accumulated in the medium . Pulse-chase experiments showed that newly synthesized vacuolar class I isoforms of chitinase and beta-1,3-glucanase (EC 3.2.1.39) were released into the medium . Post-translational processing, but not the release of these proteins, was delayed by the secretion inhibitor brefeldin A . Only forms of the proteins present in the vacuole, i.e . mature chitinase and pro-beta-1,3-glucanase and mature beta-1,3-glucanase, were chased into the medium of tobacco cell-suspension cultures . Our results provide strong evidence that vacuolar alpha-mannosidase, chitinase and beta-1,3-glucanase can be secreted into the medium . They also suggest that secretion of chitinase and beta-1,3-glucanase might be via a novel pathway in which the proteins pass through the vacuolar compartment.

Biochim Biophys Acta, 1998 Mar 30, 1391(2), 247 - 55
Ionophore A23187-induced leukotriene biosynthesis in equine granulocytes-neutrophils, but not eosinophils require exogenous arachidonic acid; Lindberg A et al.; Equine granulocyte suspensions, mainly consisting of neutrophils, failed to produce detectable amounts of leukotrienes when stimulated with calcium ionophore A23187 alone, whereas leukotrienes were dose-dependently formed in control incubations with human granulocytes . In contrast, ionophore A23187 initiated synthesis of leukotrienes B4 and C4 in equine granulocytes when added in combination with low concentrations of exogenous arachidonic acid . Similarly, ionophore A23187 provoked leukotriene biosynthesis when added alone to human whole blood, whereas addition of exogenous arachidonic acid was a prerequisite for ionophore A23187-induced leukotriene formation in equine whole blood . Leukotriene biosynthesis was provoked by A23187 alone after addition of homologous platelets to equine granulocyte suspensions . After separation of equine neutrophils and eosinophils, purified eosinophil suspensions produced LTC4 after stimulation with ionophore A23187 alone, whereas exogenous arachidonic acid was required for ionophore-induced LTB4 formation in purified neutrophil suspensions . Leukotriene synthesis in both eosinophils and neutrophils was suppressed by the 5-lipoxygenase activating protein (FLAP) inhibitor, MK-886 . Exogenous arachidonic acid was needed for ionophore-induced leukotriene synthesis also in bovine granulocytes, but was not a prerequisite for the production of leukotrienes in porcine granulocytes or in rat and rabbit white blood cell suspensions . The results indicate differences in the mechanisms regulating leukotriene synthesis in equine neutrophils, as compared to human granulocytes or equine eosinophils, and suggest that elevation of intracellular calcium is an insufficient stimulus to provoke utilisation of endogenous arachidonic acid for leukotriene synthesis in equine neutrophils .

Allergy, 1998 Mar, 53(3), 302 - 6
Factors determining spontaneous histamine release from human basophils purified with Percoll gradients and Dynabeads; Nielsen HV et al.; Identification of factors influencing histamine release from purified and cultured basophil leukocytes is important for proper interpretation of results obtained on histamine release . This paper describes factors that influence spontaneous histamine secretion from human basophil leukocytes purified on Percoll gradients, followed by negative selection with Dynabeads . Anti-IgE and recombinant human interleukin-3 were used as model stimulants, and the purified basophil leukocytes were stimulated for 10 min and 6 h . The effect of the following conditions was examined: Percoll temperature, cell-suspension density, and serum in the media . The results showed that low Percoll temperature, high cell-suspension density, and the presence of serum in the media decreased spontaneous histamine release and increased maximal net histamine release upon stimulation.

Cryobiology, 1998 Mar, 36(2), 124 - 55
Measurement of water transport during freezing in cell suspensions using a differential scanning calorimeter; Devireddy RV et al.; A new technique using a differential scanning calorimeter (DSC) was developed to obtain dynamic and quantitative water transport data in cell suspensions during freezing . The model system investigated was a nonattached spherical lymphocyte (Epstein-Barr virus transformed, EBVT) human cell line . Data from the technique show that the initial heat release of a prenucleated sample containing osmotically active cells in media is greater than the final heat release of an identical sample of osmotically inactive or lysed cells in media . The total integrated magnitude of this difference, Deltaqdsc, was found to be proportional to the cytocrit and hence also to the supercooled water volume in the sample . Further, the normalized fractional integrated heat release difference as a function of temperature, Deltaq(T)dsc/Deltaqdsc, was shown to correlate with the amount of supercooled cellular water which had exosmosed from the cell as a function of subzero temperature at constant cooling rates of 5, 10, and 20 degrees C/min . Several important limitations of the technique are (1) that it requires a priori knowledge of geometric parameters such as the surface area, initial volume, and osmotically inactive cell volume and (2) that the technique alone cannot determine whether the heat released from supercooled cellular water is due to dehydration or intracellular ice formation . Cryomicroscopy was used to address these limitations . The initial cell volume and surface area were obtained directly whereas a Boyle-van't Hoff (BVH) plot was constructed to obtain the osmotically inactive cell volume Vb . Curve fitting the BVH data assuming linear osmometric behavior yielded Vb = 0.258V0; however, nonlinearity in the data suggests that the EBVT lymphocyte cells are not "ideal osmometers" at low subzero temperatures and created some uncertainty in the actual value of Vb . Cryomicroscopy further confirmed that dehydration was the predominant biophysical response of the cells over the range of cooling rates investigated . One notable exception occurred at a rate of 20 degrees C/min where evidence for intracellular ice formation due to a DSC measured heat release between -30 and -34 degrees C correlated with a higher end volume but no darkening of the cells during cryomicroscopy . For the cooling rate tested (5 degrees C/min) the cryomicroscopy data correlated statistically very well with the DSC water transport data . A model of water transport was fit to the DSC water transport data and the average (5, 10, and 20 degrees C/min) biophysical parameters for the EBVT lymphocytes were found to be Lpg = 0.10 micro m/min-atm, ELp = 15.5 kcal/mol . Finally, the decrease in heat release from osmotically active cells measured by the DSC during repetitive freezing and thawing was found to correlate strongly with the viability of the cells measured during identical freeze/thaw protocols with cryomicroscopy . This shows the additional ability of the technique to assess freeze/thaw injury . In summary, this DSC technique is a promising new approach for measuring water transport in cellular systems during freezing .

Eur Arch Otorhinolaryngol, 1998, 255(4), 211 - 5
Magnetic cell separation for purification of human oral keratinocytes: an effective method for functional studies without prior cell subcultivation; Formanek M et al.; In studying human oral keratinocytes, it would be very helpful to obtain a pure population of cells without prior in vitro expansion . An immunomagnetic separation technique, or magnetic cell separation (MACS), was modified for efficient purification of human oral keratinocytes . Subsequent to two-step enzymatic digestion, the cell suspension was labelled with a mouse anti-CD45 (pan-leukocyte) monoclonal antibody (MoAb) to stain mononuclear cells . In a second step a rat anti-mouse antibody conjugated with colloidal superparamagnetic particles was used . Labelled cells were retained in the magnetic field of a permanent magnet on columns containing a ferromagnetic matrix . The unlabelled, unretained cells were further examined by flow cytometry analysis, enzyme-linked immunosorbent assay and polymerase chain reaction . After the MACS procedure, unretained cells showed a strong positivity for the lu-5 MoAb (as a marker for pan-cytokeratin) and were negative for anti-vimentin (to mark mesenchymal cells), for anti-CD45 MoAb and for melanocyte-detecting antibodies, thus representing pure keratinocytes (> 98%) . Purified keratinocytes maintained full viability (> 91%) and functional capacities . {3H}thymidine uptake and epidermal growth factor (EGF) receptor expression were unaltered when compared with the non-separated cell population . Furthermore, interleukin-1 alpha was detected at the protein and RNA levels in keratinocytes immediately after MACS enrichment . Our findings show that MACS appears to be a useful tool for purification of oral keratinocytes and allows for further functional studies without prior subcultivation of cells.

Eur J Anaesthesiol, 1998 Mar, 15(2), 158 - 60
Antioxidant activity of propofol in blood from anaesthetized patients; Stratford N et al.; The antioxidant capacity of plasma taken from 10 patients before and during propofol anaesthesia was measured . Mean total plasma antioxidant activity fell from 1.73 to 1.64 mM L-1 trolox equivalents (P = 0.047) . This was caused by haemodilution since mean haemoglobin concentration fell from 13.0 to 12.5 g dL-1 (P = 0.016) . The time to 50% haemolysis (H50) of 10% red blood cell suspensions induced by 2,2'azo-bis(2-amidinopropane) dihydrochloride (ABAP) was measured in blood from six patients . There was no change when red cells alone were studied, but when the patients' plasma was added to red cell suspensions (producing 10% plasma and 10% red cell suspensions), mean H50 increased from 291 to 308 min (P = 0.049) . Despite there being no overall increase in plasma antioxidant activity, the lipid soluble component of blood antioxidant activity appears to be increased by propofol.

Cancer Lett, 1998 Apr 24, 126(2), 193 - 7
Augmentation of anti-tumor effects of methotrexate by distilled water on Dunn osteosarcoma in mouse air pouch; Yoshida K et al.; The anti-tumor effects of hypoosmotic solution of MTX in distilled water (DW) on Dunn osteosarcoma were evaluated in mouse air pouches . Dunn osteosarcoma cell suspension (1 x 10{5} cells in 0.1 ml of medium) was inoculated into the mouse subcutaneous air pouch that had formed 7 days after the initial injection of air . Two hours after the inoculation of tumor cells, 5 ml of various concentrations of MTX (from 0 to 1 x 10{-3} M) dissolved in DW or PBS were injected into the air pouch . Five minutes later, the entire solution in the air pouch was aspirated . The mice were sacrificed 3 weeks after the inoculation of tumor cells and the air-pouch tissue was transected in the coronal plane with the largest area of tumor mass . The sections were stained with H&E and the area was measured with the NIH Image program . The largest area of tumor mass in the air pouch treated with 1 x 10(-3) M of MTX in DW was 11.8+/-3.4 mm2 (N = 5), which was significantly (P < 0.005) smaller than that in PBS (51.7+/-8.3 mm2) . These findings suggested that hypoosmotic solution in DW might augment the anti-tumor effect of MTX on sarcoma cells.

J Biol Regul Homeost Agents, 1997 Oct-Dec, 11(4), 133 - 6
IL-1 alpha, IL-1 beta and psoriasis: conflicting results in the literature . Opposite behaviour of the two cytokines in lesional or non-lesional extracts of whole skin; Bonifati C et al.; A still debated question in the field of the cytokine network in psoriasis is represented by contrasting data reported on the local amount of IL-1 beta amounts, of IL-1 in this dermatosis . In fact previous studies have suggested that there were decreased Il-1 alpha amounts at the lesional level but increased nonfunctional IL-1 beta concentrations as compared to the non-lesional and normal epidermis . However, recent data suggest that IL-1 alpha and, to a lesser extent, IL-1 beta amounts, are both increased and biologically active in the epidermal cell suspension of lesional psoriatic skin as compared to those of normal skin . The data reported in the present paper show that IL-1 alpha levels are decreased in psoriatic lesional extracts of whole skin (mean 2.9 +/- 2 pg/mg) as compared to non-lesional (mean 6.7 +/- 6.2 mg/mg; p = 0.02) or normal skin (mean 13.8 +/- 9.4 pg/mg; p = 0.0002) . IL-1 alpha concentrations were also significantly lower in the non-lesional skin than in normal skin (p = 0.02) . In contrast, the IL-1 beta levels (mean 1.2 +/- 0.74 pg/mg were higher in the lesional samples than in the non-lesional ones (mean 0.5 +/- 0.4 pg/mg; p = 0.0004) or in normal skin (mean 0.4 +/- 0.2 pg/mg; p = 0.004) . No differences in IL-1 beta levels were observed between non-lesional and normal skin (p = 0.3) . In addition both IL-1 alpha and IL-1 beta are directly correlated with the disease severity and each other . Our data, extending the Il-1 determination to the whole skin, seem to confirm the previously reported findings at the epidermis level and provide new light on possible interpretation of literature discrepancies.

J Magn Reson, 1998 Mar, 131(1), 92 - 6
Quantification of the contribution of extracellular sodium to 23Na multiple-quantum-filtered NMR spectra of suspensions of human red blood cells; Knubovets T et al.; 23Na double-quantum-filtered (DQF) NMR enables the detection of anisotropic motion of sodium ions due to their interaction with ordered structures in biological tissues . Using the technique, anisotropic motion was found for sodium ions in mammalian red blood cell suspensions (RBC) and the effect was shown to correlate with the integrity of membrane cytoskeleton . In the present study relative contributions to the DQF and triple-quantum-filtered (TQF) spectra of sodium bound to anisotropic and isotropic binding sites in the intra- and extracellular sodium pools (Na content being 15 and 150 mM, respectively) of human RBC were quantified for different hematocrits . DQF spectra were measured by a modified Jeener-Broekaert pulse sequence which enabled exclusive detection of anisotropically moving sodium ions . The relative contributions of the extracellular sodium to the TQF and DQF spectra decreased as the hematocrit increased, but their efficiency relative to the sodium content increased . The contribution of the extracellular sodium to the TQF signal was found to dominate the spectrum of the RBC suspension at all hematocrits studied . The contribution of the extracellular sodium to the DQF was significantly smaller than that to the TQF and was only 22% at a high hematocrit of about 90% .

Prostate, 1998 May 15, 35(3), 185 - 92
Role of alphaII(b)beta3 integrin in prostate cancer metastasis; Trikha M et al.; BACKGROUND: Integrins participate in cell-cell and cell-matrix interactions . In this study we determined whether alphaII(b)beta3 integrin is involved in metastasis of human prostate adenocarcinoma cells . METHODS: Prostate adenocarcinoma PC-3 and DU-145 cell lines express alphaII(b)beta3 . Northern blotting, 5'-RACE, and immunofluorescent localization confirmed expression of alphaIIb integrin in prostate adenocarcinoma cells . We used orthotopic/ectopic site of implantation and lung colonization assays in SCID mice to determine whether alphaII(b)beta3 participates in metastatasis of tumor cells . RESULTS: Immunofluorescent localization of alphaIIb integrin in fibronectin-adherent DU-145 and PC-3 cells is remarkably different . In DU-145 cells the integrin localizes to focal contact sites, whereas it is predominantly intracellular in PC-3 cells . Both tumor cell lines are tumorigenic when implanted subcutaneously or intraprostatically in SCID mice, but only DU-145 cells injected intraprostatically metastasize . Flow cytometry with a mAb directed to alphaII(b)beta3 revealed higher expression of alphaII(b)beta3 in DU-145 tumor cell suspensions isolated from the prostate when compared to DU-145 tumor cells from the subcutis . Function-blocking mAbs to alphaII(b)beta3 inhibit lung colonization of tail vein-injected DU-145 cells . CONCLUSIONS: Altogether, the data suggest that alphaII(b)beta3 integrin participates in the metastatic progression of prostatic adenocarcinoma.

Cytometry, 1998 May 1, 32(1), 18 - 27
Flow cytometric evaluation of bone marrow differentials in rats with pharmacologically induced hematologic abnormalities; Criswell KA et al.; Previously, flow cytometric determination of peroxidase activity, cell size, and reactivity to lymphocyte antibodies were used to produce bone marrow differentials in untreated rats . In the present study, abnormal hematologic profiles were induced with erythropoietin (EPO), recombinant murine stem cell factor (rm-SCF), granulocyte-macrophage stimulating factor (GM-CSF), and cyclophosphamide (CP) . Manual and flow cytometric data showed comparable levels of erythroid and myeloid hyperplasia in EPO- and rm-SCF/GM-CSF-treated animals, respectively . In CP-treated animals, flow cytometric data revealed significant decreases in cellularity at concentrations of CP > or = 5 mg/kg . In contrast, 20 mg/kg CP were necessary to induce microscopically apparent hypoplasia in histologic bone sections, showing that the automated methodology was a more sensitive indicator of bone marrow hypocellularity than was the more conventional manual method . Megakaryocyte counts were consistently higher by flow cytometer than by manual counts performed on cytocentrifuge preparations made from the same cell suspensions but were similar to megakaryocyte counts performed on histologic sections of femur, indicating that the automated methodology produced a more accurate reflection of true megakaryocyte numbers . Induction of hematologic abnormalities in the present study showed that manual bone marrow differentials can be replaced with the more efficient and reliable flow cytometric method in most preclinical toxicology studies.

AIDS Res Hum Retroviruses, 1998 Apr, 14 Suppl 1, S57 - 62
The mucosal immune system in the human female reproductive tract: potential insights into the heterosexual transmission of HIV; Yeaman GR et al.; Using isolated cell suspensions and in situ techniques, we have partially characterized the organization, functional capacity, and sex hormone regulation of the mucosal immune system in the human female reproductive tract . Isolated cells suspensions have been used to demonstrate that the uterus contains antigen-presenting cells that are functionally able to present antigen to autologous tetanus toxoid-specific T cells . Immunophenotypic analyses of the female reproductive tract by three-color immunofluorescent staining has been used to show that lymphoid aggregates, which are absent in postmenopausal women, develop in the uterine endometrium during the menstrual cycle in premenopausal women . Lymphoid aggregates are composed of a B lymphocyte core surrounded by numerous CD8+CD4- T lymphocytes and an outer halo of macrophages . Macrophages, CD4+ and CD8+ T cells, and CD56+ NK cells are distributed throughout the uterine endometrium . In contrast, the Fallopian tube, cervix, and vagina, which lack lymphoid aggregates, contain CD8+ and CD4+ T cells as well as macrophages . The female reproductive tract has also been analyzed for the presence of antigen-independent CD3+ T lymphocyte cytolytic function by an anti-CD3 MAb-mediated redirected lysis assay . High levels of CD3+ T lymphocyte cytolytic activity were demonstrated in cervix and vagina and independent of stage of the menstrual cycle . In the uterus, cytolytic activity changed with endocrine state . In postmenopausal women the uterine endometrium had CD3+ T lymphocytes with high cytolytic activity, whereas premenopausal women had CD3+ T lymphocytes with moderate cytolytic potential during the proliferative phase to low/no cytolytic activity during the secretory phase of the menstrual cycle . In studies to determine whether the upper reproductive tract could be infected with HIV-1, we found on the basis of nef expression and p24 release that epithelial cells from the Fallopian tube, and from the uterus and cervix, are infectable . These studies demonstrate that the human female reproductive tract is an inductive site for immune responses and the cell-mediated immunity is present throughout the female reproductive tract . These studies further indicate that the Fallopian tube and uterus are potential entry sites for HIV-1 infection and that uterine immune cell architecture as well as cytolytic activity are under hormonal control.

Planta Med, 1998 Apr, 64(3), 270 - 2
Improved taxol yield in cell suspension culture of Taxus wallichiana (Himalayan yew)
Jha S, Sanyal D, Ghosh B, Jha TB.
Cell culture of Taxus wallichiana Zucc (= T . baccata ssp wallichiana Zucc . Pilg.) (Himalayan yew) has been established and taxol-producing cell lines selected by cell line cloning . Cell line NC110 derived from needle leaf of a 40-year-old tree growing in Darjeeling Himalayas produced 0.018% taxol in B5 basal medium supplemented with 2,4-D (2 mg/l), kinetin (0.5 mg/l), and casein hydrolysate (0.5%) . This cell line has been maintained for over 2 years . Significant enhancement in the level of taxol (0.05%) was obtained in this cell line by supplementation of the basal medium with 5 mg/l of IAA-phenylalanine instead of 2,4-D without adversely affecting cell growth . IAA-glycine also enhanced taxol level (0.03%) while IAA alone (1-10 mg/l) was ineffective in inducing taxol accumulation . Using three different cell lines with different taxol-producing capacities, it has been demonstrated that 2,4-D and IAA-phenylalanine when present alone favour growth and taxol production but when combined enhance biomass to a maximum (six-fold in NC110) without enhancing taxol accumulation, suggesting that a two-stage culture may be beneficial for optimising taxol accumulation in cell culture of T . wallichiana.

Clin Endocrinol (Oxf), 1998 Feb, 48(2), 129 - 34
Human ACTH hypersensitivity syndrome associated with abnormalities of the ACTH receptor gene; Hiroi N et al.; OBJECTIVE: Activating mutations of the ACTH receptor have not been previously described . We investigated a 69-year-old woman with normal blood cortisol but undetectable blood ACTH concentrations . The aim of this study was to evaluate her hypothalamo-pituitary-adrenal axis by measuring circadian variation in blood ACTH and cortisol, and by performing CRH and ACTH stimulation and dexamethasone suppression tests . We also examined biological activity of her circulating blood ACTH using bovine adrenocortical cell suspensions and ACTH receptor gene structure by Northern blotting analysis . RESULTS: Random plasma cortisol concentrations ranged from 182 to 328 nmol/l, while ACTH concentrations were always undetectable . After an intravenous bolus injection of human CRH 100 micrograms, plasma ACTH rose slightly, while plasma cortisol increased appropriately . ACTH stimulation tests revealed that a small amount of ACTH (5 ng/kg b.w.) had the maximal cortisol stimulatory activity, and even smaller amounts of ACTH (0.5 and 0.05 ng/kg b.w.) produced significant increases in cortisol levels . ACTH bioassay of the patient's plasma demonstrated weak biological activity in the HPLC fractions which corresponded to the band of synthetic human ACTH 1-39 . The ACTH receptor coding region was amplified by polymerase chain reaction using the leucocyte genomic DNA . There were two base mutations; cysteine 21-->arginine and serine 247-->glycine in the sequences coding for the first extramembranous N-terminal domain and the third extramembranous loop of the ACTH receptor . CONCLUSIONS: This patient with normal blood cortisol but undetectable ACTH levels showed increased adrenocortical sensitivity to ACTH and two point mutations in the ACTH receptor gene . This study, therefore, reports a previously undescribed syndrome--ACTH hypersensitivity syndrome--and provides insights into the molecular mechanism of ACTH receptor action.

Clin Cancer Res, 1998 Apr, 4(4), 887 - 94
An orthotopic model of human pancreatic cancer in severe combined immunodeficient mice: potential application for preclinical studies; Mohammad RM et al.; Pancreatic adenocarcinoma is one of the most incurable and least understood of all human cancers . It is the fourth leading cause of cancer-related mortality in males (after lung, prostate, and colon) and in females (after lung, breast, and colon) in the United States with <2-3% of patients surviving >5 years . In an attempt to search for more effective therapies for this disease, we report here, for the first time, an effective treatment, the combination of gemcitabine and auristatin-phenethylamine (PE), against an orthotopic implantation of a human pancreatic adenocarcinoma cell line (HPAC) in severe combined immunodeficient (SCID) mice . Tumor implantation was performed by injecting 100 microl of the HPAC cell suspension (1 x 10(6) cells) directly into the pancreas of 5-week-old SCID mice . After implantation, tumor formation was checked twice a week . All palpable tumors were detected within 21 days (100% take rate), and tumors were confirmed histologically to be pancreatic adenocarcinoma . For the subsequent efficacy trial, tumor-bearing SCID mice were randomized into four groups with five mice in each group . One served as a control, the second received gemcitabine alone (2.5 mg/kg/injection i.p.), the third received auristatin-PE alone (2.0 mg/kg/injection i.v.), and the fourth group received the combination of gemcitabine (i.p.) and auristatin-PE (1.5 mg/kg/injection i.v.) . All animals were euthanized 7 days after the completion of their treatments, and the pancreases were resected . Histological examination revealed the tumors to be adenocarcinoma . The tumors were composed of diffuse sheets of cells interrupted by glandular spaces containing secretory material . Cytologically, the tumor cells were large, pleomorphic, and hyperchromatic . Many cells contained intracellular lumina containing mucin . Immunohistochemical studies showed strong p21WAF1 (p21) expression but no immunoreactivity with p53 and Her-2/neu antibodies . The mean pancreatic weight in the gemcitabine/auristatin-PE combination group was significantly (P = 0.014) lower (0.84 +/- 0.639 g) when compared with those of the control (2.91 +/- 1.19 g) and gemcitabine alone (1.84 +/- 0.796 g; P = 0.064) groups . In addition, the mean weight in the combination group approached statistical significance when compared with the auristatin-PE group alone (1.16 +/- 0.635 g; P = 0.028) . We conclude that the combination of gemcitabine and auristatin-PE is an effective treatment against HPAC tumors in this xenograft model and more effective than treatment with either gemcitabine or auristatin-PE alone and could be considered for future animal studies with pancreas cancer and/or for human clinical trials.

Sangre (Barc), 1998 Feb, 43(1), 25 - 9
{Correlation of flow and static cytometry; their application to the study of anaplastic lymphomas}; Marcos B et al.; PURPOSE: DNA study by cytometric methods is one of the prognosis factors considered in malignant tumours . Flow cytometry (FCM) was the most frequently used techniques in cell suspensions . Image cytometry (ICM) was also applied in cellular smears and it is possible to measure the results with an Image Analyzer, which supposes a substancial advantage over DNA studies . To confirm the results and correlation of the two techniques a controversial subtype of lymphoid tumour was selected: Anaplastic large cell lymphoma (ALCL) . MATERIAL AND METHODS: Fifty four cases of ALCL (23 classical type and 31 ACL-Hodgkin related) were studied . Cytometry was performed in paraffin-embedded tissues previously dewaxed, rehydrated and minced . FCM was done in suspensions incubated with ribonuclease A and stained with propidium iodide in an EPICS-C flow cytometer . ICM study was performed in Feulgen-stained smears and measured by an Image Analyzer CAS-200 . RESULTS: All cases were aneuploid . ALCL were 30.5% hypodiploid (HpD) and 69.5% hyperdiploid (HrD) by FCM; 43.5% HpD and 56.5% HrD by ICM . ALCL-HR were 58% HpD and 42% HrD by FCM; 68% HpD and 32% HrD by ICM . There was a lack of correlation of 22% between both methods but it was not statistically significant . CONCLUSIONS: We can conclude the obtained results by FCM and ICM are almost similar.

Am J Physiol, 1998 Apr, 274(4 Pt 2), R1031 - 8
Functional role of ecto-ATPase activity in goldfish hepatocytes; Schwarzbaum PJ et al.; Extracellular {gamma-32P}ATP added to a suspension of goldfish hepatocytes can be hydrolyzed to ADP plus gamma-32Pi due to the presence of an ecto-ATPase located in the plasma membrane . Ecto-ATPase activity was a hyperbolic function of ATP concentration ({ATP}), with apparent maximal activity of 8.3 +/- 0.4 nmol P(i).(10(6) cells)-1.min-1 and substrate concentration at which a half-maximal hydrolysis rate is obtained of 667 +/- 123 microM . Ecto-ATPase activity was inhibited 70% by suramin but was insensitive to inhibitors of transport ATPases . Addition of 5 microM {alpha-32P}ATP to the hepatocyte suspension induced the extracellular release of alpha-32P(i) {8.2 pmol.(10(6) cells)-1.min-1} and adenosine, suggesting the presence of other ectonucleotidase(s) . Exposure of cell suspensions to 5 microM {2,8-3H}ATP resulted in uptake of {2,8-3H}adenosine at 7.9 pmol.(10(6) cells)-1.min-1 . Addition of low micromolar {ATP} strongly increased cytosolic free Ca2+ (Ca2+i) . This effect could be partially mimicked by adenosine 5'-O-(3-thiotriphosphate), a nonhydrolyzable analog of ATP . The blockage of both glycolysis and oxidative phosphorylation led to a sixfold increase of Ca2+i and an 80% decrease of intracellular ATP, but ecto-ATPase activity was insensitive to these metabolic changes . Ecto-ATPase activity represents the first step leading to the complete hydrolysis of extracellular ATP, which allows 1) termination of the action of ATP on specific purinoceptors and 2) the resulting adenosine to be taken up by the cells.

Oral Microbiol Immunol, 1997 Dec, 12(6), 323 - 8
Acid tolerance and acid-neutralizing activity of Porphyromonas gingivalis, Prevotella intermedia and Fusobacterium nucleatum; Takahashi N et al.; The tolerance to acid and the acid-neutralizing activity of three important periodontopathic bacteria, Porphyromonas gingivalis, Prevotella intermedia and Fusobacterium nucleatum were studied . P . gingivalis strains grew only at neutral pH and did not utilize glucose, whereas strains of P . intermedia and F . nucleatum could grow under acidic conditions and increased their growth by utilizing glucose . P . gingivalis tended to raise the culture pH during growth . P . intermedia and F . nucleatum raised the culture pH during growth in the absence of glucose, while in the presence of glucose they decreased the pH . Resting cell suspensions of all the bacteria raised the pH in the presence of tryptone and casamino acids . Acid-neutralizing activity was confirmed by measuring base production at a fixed pH with a pH-stat . During neutralization, the cells produced cytotoxic substrates, ammonia and organic acids (butyric, isobutyric and isovaleric acids by P . gingivalis; isovaleric and succinic acids by P . intermedia; propionic and butyric acids by F . nucleatum) . These findings suggest that deamination of amino acids into ammonia and organic acids occurs simultaneously with base production, resulting in acid neutralization . These results could partially explain the survival of P . intermedia and F . nucleatum in both supragingival and subgingival plaque and the apparent restriction of P . gingivalis to subgingival plaque . The former bacteria may aid in creation of an environment fostering colonization of subgingival plaque by P . gingivalis.

Oral Microbiol Immunol, 1998 Apr, 13(2), 97 - 105
Identification of hemolytic activity in Prevotella intermedia; Beem JE et al.; Hemolysin production was measured in strains of Prevotella intermedia . Zones of beta-hemolysis were detected on agar plates supplemented with either sheep, rabbit or human erythrocytes . A standard tube assay was performed on cell suspensions of the organism to measure hemolytic activity, which was found to be dose dependent, eliminated by heat treatment, and saturable with increasing concentrations of blood . Growth-phase experiments suggested that hemolysin production was increased during logarithmic growth and was reduced during stationary phase . Cell fractionation, performed on several strains of P . intermedia, localized the activity in the outer membrane and in cell vesicles . The biological implication of this study is that P . intermedia, by virtue of its hemolytic activity, is capable of liberating the hemoglobin from erythrocytes, thereby acquiring an essential nutrient, iron, for its metabolism.

Appl Environ Microbiol, 1998 May 1, 64(5), 1884 - 9
Oxidation of Methyl-Substituted Naphthalenes: Pathways in a Versatile Sphingomonas paucimobilis Strain
Dutta TK, Selifonov SA, Gunsalus IC.
Aromatic compounds with alkyl substituents are abundant in fossil fuels . These compounds become important environmental sources of soluble toxic products, developmental inhibitors, etc . principally through biological activities . To assess the effect of methyl substitution on the completeness of mineralization and accumulation of pathway products, an isolate from a phenanthrene enrichment culture, Sphingomonas paucimobilis 2322, was used . Washed cell suspensions containing cells grown on 2,6-dimethylnaphthalene in mineral medium were incubated with various mono-, di-, and trimethylnaphthalene isomers, and the products were identified and quantified by gas chromatography and mass spectrometry . The data revealed enzymes with relaxed substrate specificity that initiate metabolism either by methyl group monoxygenation or by ring dioxygenation . Congeners with a methyl group on each ring initially hydroxylate a methyl, and this is followed by conversion to a carboxyl; when there are two methyl groups on a single ring, the first reaction is aryl dioxygenation of the unsubstituted ring . Intermediates are channeled to primary ring fission via dihydrodiols to form methyl-substituted salicylates . Further evidence that there are multiple pathways comes from the fact that both phthalate and (methyl)salicylate are formed from 2-methylnaphthalene.

Brain Res Brain Res Protoc, 1998 Mar, 2(3), 215 - 22
Acetylcholine determination of microdialysates of fetal neocortex grafts that induce recovery of learning; Miranda MI et al.; The microdialysis technique for acetylcholine (ACh) first became possible when sensitive and specific assays for ACh (pmol/sample range) were developed {G . Damsma, B.H.C . Westerink, P . de Boer, J.B . de Vries, A.S . Horn, Determination of basal acetylcholine release in freely moving rats by transstriatal dialysis coupled to on-line HPLC analysis: pharmacological aspects, Life Sci . 43 (1988) 1161-1168; G . Damsma, B.H.C . Westerink, A . Imperato, H . Rollema, J.B . de Vries, A . S . Horn, Automated brain dialysis of acetylcholine in freely moving rats: detection of basal acetylcholine, Life Sci . 41 (1987) 873-876; P.E . Potter, J.L . Meek, N.H . Neff, Acetylcholine and choline in neural tissue measured by HPLC with electrochemical detection, J . Neurochem . 41 (1983) 188-194; B.H.C . Westerink, G . Damsma, Determination of acetylcholine in microdialysates by HPLC and electrochemical detection, Neurosci . Protocols 20 (1993) 1-9.} . In the present protocol, the microdialysis technique was used to correlate ACh release with the recovery of the ability to acquire a conditioning taste aversion (CTA), by fetal brain grafts in insular cortex (IC) lesioned rats {M.I . Miranda, A.M . Lopez-Colome, F . Bermudez Rattoni, Recovery of conditional taste aversion induced by fetal neocortex grafts . In vivo correlation of acetylcholine levels, Brain Res . 759 (1997) 141-148} . Three groups of IC lesioned rats showing disrupted CTA received cell suspension grafts of fetal tissue dissected from either the IC or occipital cortex (OC) of 16-day-old rat fetuses . One of the groups of IC-grafted animals was tested after 15 days post-graft; the other groups, IC- and OC-grafted animals, were tested after a recovery time of 45 days, as well as the groups of lesioned and unoperated animals used as control . After the CTA test, guide cannulas were stereotaxically implanted into the IC of all groups . Two days later, microdialysis was performed to determine the extracellular levels of ACh inside the graft . The dialysates were analyzed by high-performance liquid chromatography and electrochemical detection . The ACh was converted by the enzyme acetylcholinesterase to choline, and subsequently by choline oxidase to hydrogen peroxide {J.L . Meek, C . Eva, Enzymes adsorbed on an ion exchanger as a post-column reactor: application to acetylcholine measurement, J . Chromatogr . 317 (1984) 343-347.} . The reactor with these enzymes was placed between the analytical column and the electrochemical detector . The hydrogen peroxide produced was detected with a platinum electrode, and choline was determined concurrently . We believe that the application of free-moving microdialysis as a method to measure the cholinergic levels inside the transplant at two post-graft periods, is a good, direct technique to correlate the effects of ACh levels from the fetal grafts in lesioned rats .

Hum Reprod, 1998 Mar, 13(3), 634 - 8
Short-term in-vitro culture and cryopreservation of spermatogenic cells used for human in-vitro conception; Aslam I et al.; Testicular cell suspensions were prepared from obstructive and non-obstructive azoospermic men and were cultured in vitro for 96 h as (i) mixed cell populations and (ii) isolated homogeneous populations of primary spermatocytes, round spermatids and elongating spermatids . The cells lost their viability gradually during the first 24 h period . By 72 h almost 90% of the cells were non-viable . Isolated pure fractions showed better viability at each time interval (P < 0.0005) . Throughout the culture period primary spermatocytes, elongating spermatids and other non-spermatogenic cells showed no change in their morphology, but almost 22% of round spermatids showed growth of flagella . Most of the round spermatids developed their flagella during the first 4-8 h period of culture . Isolated pure round spermatids showed better flagellar growth compared with mixed cell suspensions (P < 0.0005) . The spermatogenic cells were successfully cryopreserved . However, when mixed spermatogenic cell suspensions were cryopreserved, more cells lost their viability compared with when isolated pure fractions were cryopreserved (P < 0.0005).

Leuk Lymphoma, 1997 Dec, 26 Suppl 1, 1 - 11
Selection and transplantation of autologous hematopoietic CD34+ cells for patients with multiple myeloma; Lemoli RM et al.; Here we review our recent experience addressing the issue of positive selection and transplantation of hematopoietic CD34+ cells to reduce neoplastic contamination in peripheral blood (PB) autografts from patients with multiple myeloma (MM) . We evaluated PB samples from 30 pretreated MM patients following the administration of high dose cyclophosphamide (Cy; 7g/m2 or 4g/m2) and granulocyte-colony stimulating factor (G-CSF), for collection of circulating stem cells (PBSC) to support hematopoietic reconstitution following myeloablative radio-chemotherapy . Twenty six patients showed adequate mobilization of CD34+ progenitor cells and were submitted to PBSC collection . Circulating hematopoietic CD34+ cells were highly enriched by avidin-biotin immunoabsorption, cryopreserved, and used to reconstitute BM function after myeloablative therapy in 13 patients . The median purity of the enriched CD34+ cell population was 89.5% (range 51-94%) with a 75-fold increase compared to the pretreatment samples . The median overall recovery of CD34+ cells and CFU-GM was 58% (range 33-95%) and 45% (range 7-100%), respectively . Positive selection of CD34+ cells resulted in 2.5-3 log of plasma cells and CD19+ B-lineage cells depletion as determined by immunofluorescence studies, although DNA analysis of CDR III region of IgH gene demonstrated the persistence of minimal residual disease (MRD) in 5 out of 6 patient samples studied . Myeloma patients were reinfused with enriched CD34+ cells after myeloablative therapy consisting of total body irradiation (TBI, 1000 cGy) and high dose Melphalan (140 mg/m2) or Melphalan (200 mg/m2) alone . They received a median of 5 x 10(6) CD34+ cells/kg and showed a rapid reconstitution of hematopoiesis: the median time to 0.5 x 10(9) neutrophils, 20 and 50 x 10(9) platelets/L of PB was 10, 11 and 12 days, respectively . When we analyzed the immunological reconstitution of this group of patients, we observed a rapid and full recovery of total lymphocyte and NK cell count, although the absolute CD4+ cell count was lower than pretreatment level . These results, as well as other clinically significant parameters, did not significantly differ from those of patients (=13) receiving unmanipulated PBSC following the same pretransplant conditioning regimen . The results of this trial demonstrate that positive selection of CD34+ cells reduces the contamination of myeloma cells from the apheresis products up to 3 log and provides a cell suspension capable of restoring a normal hematopoiesis after a TBI-containing conditioning regimen . Based on this pilot trial, we have recently started a clinical study involving a double autotransplant, conditioned with melphalan (200 mg/m2) followed by melphalan (140 mg/m2) and busulphan (14 mg/kg), supported by the reinfusion of highly purified CD34+ cells.

J Comp Physiol {B}, 1998 Mar, 168(2), 81 - 6
Effects of vertebrate growth factors on digestive gland cells from the mollusc Pecten maximus L.: an in vitro study; Giard W et al.; In relation with the digestive cycle, the digestive gland cells of bivalve molluscs undergo a sequence of cytological changes which is controlled by external and internal effectors such as putative gastrointestinal hormones and growth differentiation factors . A tissue dissociation method was developed to investigate the in vitro effect of the vertebrate growth and differentiation factors: insulin, insulin growth factor I (IGF-I), basic fibroblast growth factor (bFGF) and epidermal growth factor (EGF) on the digestive gland cells of the scallop Pecten maximus . All these vertebrate peptides induced a dose-dependent increased incorporation of 3H-leucine and 14C-uridine in whole digestive gland cell suspensions . However, after Percoll density gradient purification of the digestive cells, only stem and undifferentiated enriched cell fractions were responsive to the different peptides . In addition, insulin and IGF-I, but not EGF and bFGF, stimulated 3H-leucine incorporation in control dispersed mantle edge cells . These results suggest that insulin-related peptides could work as general growth promoting factors in molluscs . On the other hand, EGF and bFGF, or at least their molluscan counterparts, may be efficient growth differentiation factors in the regenerative processes occurring in the digestive gland of molluscs.

Biosci Rep, 1997 Dec, 17(6), 547 - 56
Apoptosis dependent decrease of the intramembrane ion traffic in cultured mouse fibroblasts shown by conductivity dispersion; Bonincontro A et al.; We have investigated the intramembranal ion traffic in apoptotic 3T6 cells in culture . Apoptosis was induced by various treatments, such as serum deprivation, high density growth and hydrogen peroxide at subnecrotic doses . Cell death was assessed by nucleosomal DNA fragmentation, single cell electrophoresis, immunofluorescence and histological staining . To study the modifications of membrane structure and function, we adopted a well established biophysical strategy based on the measurement of the electrical conductivity of cell suspensions, as a function of the frequency of the electrical field applied to the sample . A comparison between the conductivity of normal and apoptotic cell suspensions shows that programmed cell death causes a decrease of membrane conductivity which indicates a diminished intramembranal ion traffic . Our results strongly suggest that one of the early events in the triggering of apoptosis is represented by an overall reduction of plasma membrane function . Finally, our results are in agreement with the idea that the nucleus is not the sole target of the apoptotic process.

Plant Cell Physiol, 1998 Feb, 39(2), 235 - 40
Characterization of a cDNA encoding CuZn-superoxide dismutase from the liverwort Marchantia paleacea var . diptera; Tanaka K et al.; Suspension-cultured cells of the liverwort Marchantia paleacea var . diptera contain a cytosolic CuZn-superoxide dismutase (SOD) whose N-terminal amino acid sequence is similar to those of the isozymes found in chloroplasts of higher plants {Tanaka et al . (1996) Plant Cell Physiol . 37: 523} . A cDNA (MSODCc) encoding the cytosolic CuZn-SOD was isolated from cDNA library constructed from a liverwort cell suspension culture . The deduced amino acid sequence showed a higher degree of homology with the sequences of CuZn-SODs in chloroplasts than those in the cytosol of higher plants and an unique additional peptide in the C-terminal region, but no plastid transit sequence . Northern blotting using MSODCc as a probe and immunoblot analysis with antiserum against the enzyme revealed that the steady state level of transcript was not affected by copper, but both CuZn-SOD protein and its activity increased.

J Immunol, 1998 Apr 15, 160(8), 3776 - 82
The actin-bundling protein fascin is involved in the formation of dendritic processes in maturing epidermal Langerhans cells; Ross R et al.; Dendritic cells (DC) are characterized by their unique potential to prime naive T cells . Epidermal Langerhans cells (LC), the DC resident in the epidermis, gain this immunostimulatory capacity following Ag contact in vivo or during in vitro culture of epidermal cell suspensions . To analyze differential gene expression in maturing LC, we constructed a highly representative cDNA library of cultivated LC (cLC) in lambda ZAP II containing 18 x 10(6) independent clones . This library was screened with freshly isolated Langerhans cell (fLC)- and cLC-derived probes for cLC-specific cDNAs . The cDNAs identified were sequenced and analyzed by database searches . Two cDNA fragments were identified as fragments of fascin, indicating that fascin is differentially expressed in LC . By competitive RT-PCR, we confirmed that fascin is highly expressed in cLC cultivated for 1, 2, and 3 days, while no signals were obtained with fLC . Western blot and immunofluorescence analysis revealed cLC-specific expression of fascin on the protein level as well . Fascin is known to be involved in the organization of the actin cytoskeleton in cytoplasmatic extensions of nerve growth cones . Its differential expression in maturing LC coincides with the formation of numerous dendritic projections in LC . Their formation was inhibited by incubation of LC with fascin antisense oligonucleotides during cultivation . Therefore, we conclude that fascin is necessary for the formation of the dendritic processes of maturing Langerhans cells and may thus influence T cell-LC interaction.

Dis Colon Rectum, 1998 Feb, 41(2), 141 - 6
Effects of pneumoperitoneum on tumor implantation with decreasing tumor inoculum; Wu JS et al.; INTRODUCTION: The aim of this study was to determine the effect of pneumoperitoneum on the rate of trocar-site implantation with decreasing inoculum of cancer cells . METHODS: A total of 0.5 ml of GW-39 human colon cancer cell suspensions at 1 percent (approximately 3.2 x 10(5) cells) and at 0.5 percent (approximately 1.6 x 10(5) cells; v/v) were injected into the abdomen of hamsters through a midline incision . Animals in each group were randomized to receive either pneumoperitoneum (1 percent = 33; 0.5 percent = 43) or not (1 percent = 32; 0.5 percent = 39) . Gross and microscopic tumor implants were documented seven weeks later at four trocar sites . RESULTS: In the 1 percent group, pneumoperitoneum significantly increased trocar-site tumor implants from 50 to 71 percent (P < 0.001) . Pneumoperitoneum also resulted in the following: 1) more frequent involvement of all four concurrent sites (38 vs . 10 percent; P < 0.02); 2) more frequent palpable tumors (13 vs . 5 percent; P < 0.01); 3) larger tumor mass (2.1 +/- 0.6 g vs . 0.2 +/- 0.1 g; P < 0.02) . In the 0.5 percent group, pneumoperitoneum did not significantly increase trocar-site tumor implants, and it did not result in a larger tumor mass . The percent increase in trocar-site implants owing to pneumoperitoneum was influenced by the amount of tumor inoculum (21 percent in the 1 percent group; 10 percent in the 0.5 percent group) . The mass of palpable tumor implants after pneumoperitoneum decreased with decreased inoculum: 1 percent = 2.1 +/- 0.6 g; 0.5 percent = 0.3 +/- 0.1 g (P < 0.0001) . CONCLUSIONS: Pneumoperitoneum significantly increased both tumor implantation rate and mass when approximately 3.2 x 10(5) colon cancer cells were injected into the peritoneal cavity . These effects of pneumoperitoneum diminished with one-half as many tumor cells injected in the peritoneal cavity.

Exp Brain Res, 1998 Apr, 119(3), 345 - 55
Comparison of mesencephalic free-floating tissue culture grafts and cell suspension grafts in the 6-hydroxydopamine-lesioned rat; Meyer M et al.; Ventral mesencephalon (VM) of fetal rat and human origin grown as free-floating roller-tube (FFRT) cultures can survive subsequent grafting to the adult rat striatum . To further explore the functional efficacy of such grafts, embryonic day 13 ventral mesencephalic tissue was grafted either after 7 days in culture or directly as dissociated cell suspensions, and compared with regard to neuronal survival and ability to normalize rotational behavior in adult rats with unilateral 6-hydroxydopamine (6-OHDA) lesions . Other lesioned rats received injections of cell-free medium and served as controls . The amphetamine-induced rotational behavior of all 6-OHDA-lesioned animals was monitored at various time points from 18 days before transplantation and up to 80 days after transplantation . Tyrosine hydroxylase (TH) immunostaining of the histologically processed brains served to assess the long-term survival of grafted dopaminergic neurons and to correlate that with the behavioral effects . Additional cultures and acutely prepared explants were also fixed and stored for histological investigation in order to estimate the loss of dopaminergic neurons in culture and after transplantation . Similar behavioral improvements in terms of significant reductions in amphetamine-induced rotations were observed in rats grafted with FFRT cultures (127%) and rats grafted with cell suspensions (122%), while control animals showed no normalization of rotational behavior . At 84 days after transplantation, there were similar numbers of TH-immunoreactive (TH-ir) neurons in grafts of cultured tissue (775 +/- 98, mean +/- SEM) and grafts of fresh, dissociated cell suspension (806 +/- 105, mean +/- SEM) . Cell counts in fresh explants, 7-day-old cultures, and grafted cultures revealed a 68.2% loss of TH-ir cells 7 days after explantation, with an additional 23.1% loss after grafting, leaving 8.7% of the original number of TH-ir cells in the intracerebral grafts . This is to be compared with a survival rate of 9.1% for the TH-ir cells in the cell-suspension grafts . Immunostaining for the calcium-binding proteins calretinin, calbindin, and parvalbumin showed no differences in the neuronal expression of these proteins between the two graft types . In conclusion, we found comparable dopaminergic cell survival and functional effects of tissue-culture grafts and cell-suspension grafts, which currently is the type of graft most commonly used for experimental and clinical grafting . In this sense the result is promising for the development of an effective in vitro storage of fetal nigral tissue, which at the same time would allow neuroprotective and neurotrophic treatment prior to intracerebral transplantation.

J Clin Endocrinol Metab, 1998 Apr, 83(4), 1299 - 305
Localization, characterization, and second messenger coupling of pituitary adenylate cyclase-activating polypeptide receptors in the fetal human adrenal gland during the second trimester of gestation; Yon L et al.; The distribution and pharmacological properties of pituitary adenylate cyclase-activating polypeptide (PACAP) receptors were studied in the fetal human adrenal gland during the second trimester of gestation . Autoradiographic studies, using {125I}PACAP27 as a radioligand, revealed that PACAP-binding sites are exclusively located on chromaffin cells of adrenals from fetuses 14-20 weeks old . Biochemical characterization of binding revealed the occurrence of a single class of PACAP-binding sites with a dissociation constant value of 0.32-0.74 nmol/L and a binding capacity of 0.30-0.81 pmol/mg wet tissue . PACAP27 and PACAP38 were equipotent in competing for {125I}PACAP27 binding (IC50 = 0.28-0.64 nmol/L and 0.15-0.81 nmol/L, respectively), and the Hill coefficients were close to 1 . In contrast, vasoactive intestinal polypeptide was much less efficient in displacing the tracer (IC50 = 4-362 nmol/L), and the Hill coefficients were less than 0.6 . PACAP38 induced a dose-dependent increase in cAMP production in fetal human adrenal cell suspension (ED50 = 0.07 +/- 0.02 nmol/L), as well as in cells maintained in culture for 5 days (5.4 +/- 1.8 nmol/L) . In contrast, PACAP38 induced a modest increase in inositol phosphate formation . These data indicate that type I PACAP receptors are present in the early stages of the human medulla organization during the process of migration of chromaffin cells from the periphery to the central part of the gland . The present results suggest that PACAP could be involved in the regulation of the human adrenochromaffin cells during ontogenesis.

Semin Surg Oncol, 1998 Apr-May, 14(3), 189 - 201
In vitro concentration response studies and in vitro phase II tests as the experimental basis for regional chemotherapeutic protocols; Link KH et al.; The theoretical pharmacologic benefit of regional vs . systemic chemotherapy is defined and the concentration response behavior of cytostatic drugs and their optimal exposure times are described with human cancer cell lines (HT29, NMG64/84) and fresh human tumor cell suspensions in the human tumor colony assay (HTCA) . The theoretical pharmacological advantages are 5.8 to 6 for adriamycin (ADM), 8 for cisplatinum (CDDP), 6.3 for epidoxorubicin (EPI), 22 to 58 for 5-fluorouracil (5FU), 4.6 for mitomycin C (MMC), and 6.3 for mitoxantrone (NOV) . The drugs differed in their cytotoxic potency in vitro and thus also potential efficacy for regional chemotherapy; however, all but 5-fluorodeoxyuridine (5FUDR) exerted cytotoxicity dependent on exposure time and concentration . On average, elevation of the test concentrations by 1 lg doubled responses in fresh human tumor cell suspensions . From these results and clinical considerations, optimal times were defined for the regional chemotherapy strategies of hepatic artery infusion, intraperitoneal instillation, and chemoembolisation as performed at our institution.

J Immunol, 1997 Dec 1, 159(11), 5560 - 7
Functional properties of human intestinal mast cells cultured in a new culture system: enhancement of IgE receptor-dependent mediator release and response to stem cell factor; Bischoff SC et al.; Culture of human mast cells (MC) in vitro has only been possible to date in the presence of 3T3 fibroblasts . The aim of the present study was to maintain freshly isolated human MC in culture without addition of feeder cells and to study their functional properties . We isolated cell suspensions containing 1 to 11% MC from human intestinal tissue and cultured them in standard medium . MC survived in culture for about 2 wk without cytokine supplementation and for several months with supplementation of medium with stem cell factor (SCF) . SCF selectively supported MC survival, whereas the number of contaminating cells declined rapidly during culture . Most interestingly, we found that histamine and leukotriene release induced by IgE receptor cross-linking was substantially enhanced in cultured MC compared with that in MC stimulated directly after cell isolation . Cultured MC, but not freshly isolated MC, released mediators in response to SCF in a concentration-dependent fashion provided that the cells were cultured in SCF-free medium . These findings demonstrate that human MC isolated from intestinal tissue can be maintained in culture in vitro for several weeks . After culture they have different functional properties, which might resemble more closely the functional status of human intestinal MC in vivo than that of freshly isolated cells.

J Immunol, 1997 Dec 1, 159(11), 5483 - 91
Augmentation of dendritic cells in murine organ donors by Flt3 ligand alters the balance between transplant tolerance and immunity; Steptoe RJ et al.; Treatment of mice with the recently cloned hemopoietic growth factor Flt3 ligand (FL; 10 microg/day for 10 days) resulted in a large increase in myeloid lineage cells within the liver . While the number of nonparenchymal cells (NPC) harvested from liver increased about 9-fold, a 90-fold increase was observed in the proportion of CD11c+ dendritic cells (DC) recovered from NPC following overnight (18-h) culture in granulocyte-macrophage CSF . In contrast, only a 50% increase was seen in CD11c+ cells within heart single cell suspensions and in the number of DC obtained from hearts after 18-h culture . Liver NPC and heart cell suspensions freshly isolated from 10-day FL-treated animals exhibited increased T cell allostimulatory capacity compared with controls . Overnight cultured DC from livers of FL-treated animals expressed both higher levels of costimulatory molecules (CD80 and CD86) and allostimulatory activity than those from controls . Heart-derived DC also displayed enhanced stimulatory capacity . Pretreatment of organ donors with FL for either 5 or 10 days before transplant of organs to normal recipients abrogated the spontaneous liver allograft acceptance normally observed and resulted in delayed or acute graft rejection (median survival times, 40 and 12 days, respectively) . Heart rejection was significantly accelerated by pretreatment of donors with FL for 5 or 10 days (median survival times, 8 and 7 days, respectively, vs 12 days in controls) . These novel findings reveal the potent immunologic adjuvant properties of FL in vivo . They also show that substantial augmentation of the number of potential allostimulatory cells in donor organs before transplantation favors rejection rather than tolerance induction.

Eur J Cell Biol, 1998 Feb, 75(2), 184 - 91
Effects of genic substitution at the agouti, brown, albino, dilute, and pink-eyed dilution loci on the proliferation and differentiation of mouse epidermal melanocytes in serum-free culture; Hirobe T et al.; To examine the effects of coat-color genes on the proliferation and differentiation of mouse epidermal melanocytes, we cultured epidermal, cell suspensions derived from neonatal skins of C57BL/10JHir (black) and its congenic mice carrying agouti, brown, albino, dilute, and pink-eyed dilution genes in a serum-free medium supplemented with dibutyryl adenosine 3',5'-cyclic monophosphate . The proliferative rates of agouti, brown and dilute black melanocytes were similar to that of black melanocytes, while those of albino and pink-eyed black melanocytes were about one-half of that of black melanocytes . The morphology of albino and pink-eyed black melanocytes, though nonpigmented, was similar to black melanocytes; namely, dendritic, polygonal or epithelioid . Dilute black melanocytes also possessed the similar morphology, whereas their melanosomes were accumulated in the perinuclear region . Dopa-melanin depositions after dopa reaction in brown and dilute black melanocytes were greater than in black and agouti melanocytes . Although dopa-melanin depositions were not observed in albino melanocytes, about 8% of pink-eyed black melanocytes were positive to dopa reaction . Silver depositions after combined dopa-premelanin reaction in agouti, brown and dilute black melanocytes were similar to that in black melanocytes . Although albino melanocytes were devoid of silver depositions, about 25% of pink-eyed black melanocytes were positive to the reaction . Pyrrole-2,3,5-tricarboxylic acid (PTCA, degradation product of eumelanin) contents in agouti and dilute black melanocytes were slightly lower than in black melanocytes, while that in brown melanocytes was reduced to one-third . In contrast, PTCA contents in albino and pink-eyed black melanocytes were reduced to less than 0.5% . Aminohydroxyphenylalanine (AHP, degradation product of pheomelanin) contents did not differ among these melanocytes . These results suggest that the coat-color genes exert their influences on the proliferation and differentiation of mouse epidermal melanocytes by affecting tyrosinase activity, melanosome maturation and transport, and eumelanin synthesis.

Mol Cell Biochem, 1998 Mar, 180(1-2), 193 - 9
Effects of nucleoside transport inhibitors and adenine/ribose supply on ATP concentration and adenosine production in cardiac myocytes; Kalsi KK et al.; Adenosine plays an important role in protection of the heart before, during and after ischemia . Nucleoside transport inhibitors (NTI) increase adenosine concentration without inducing ischemia by preventing its uptake and metabolism in cardiac cells . However, prolonged effects of nucleoside transport inhibitors on adenosine and nucleotide metabolism and its combined effect with nucleotide precursors has not been established in cardiomyocytes . The aim of this study was to investigate the effect of two nucleoside transport inhibitors, dipyridamole (DIPY) and nitrobenzylthioinosine (NBTI) alone or combined with adenine and ribose on adenosine production and ATP content in cardiomyocytes . Rat cardiomyocytes were isolated using collagenase perfusion technique . Isolated cell suspensions were incubated for up to 480 min with different substrates and inhibitors as follows: (1) control; (2) 100 microM adenine and 2.5 mM ribose; (3) 10 microM DIPY; (4) 1 microM NBTI; (5) DIPY, adenine and ribose and (6) NBTI, adenine and ribose . Five microM EHNA (erythro-9(2-hydroxy-3-nonyl)adenine, an inhibitor of adenosine deaminase) was added to all incubations . After incubation, extracts of myocyte suspension were analysed by HPLC for adenine nucleotides and metabolite concentrations . ATP content decreased in cardiomyocytes after 8 h of incubation with DIPY, while no change was observed with NBTI or without inhibitors . Adenosine concentration increased with both DIPY and NBTI . In the presence of adenine and ribose an elevation in ATP concentration was observed, but no significant change in adenosine content . In the presence of DIPY or NBTI together with adenine and ribose, an enhancement in cardiomyocyte ATP concentration was observed together with an increase in adenosine content . This increase in adenosine production was especially prominent with DIPY . In conclusion, dipyridamole causes a decrease in ATP concentration in isolated cardiomyocytes by mechanisms other than nucleoside transport inhibition . Addition of adenine/ribose with dipyridamole prevents the depletion of ATP . Combination of adenine/ribose with nucleoside transport inhibitors may also further enhance adenosine concentration and thus, could be more effective as pharmacological agents for treatment.

Cancer Genet Cytogenet, 1998 Apr 15, 102(2), 114 - 24
Fluorescence in situ hybridization to assess aneuploidy for chromosomes 7 and 8 in hematologic disorders; Wyandt HE et al.; Stored, fixed cell suspensions of bone marrows from 70 patients karyotyped over a three-year period for myelodysplastic syndrome (MDS) or related hematologic conditions were retrospectively studied in two series using centromeric probes for chromosomes 7 and 8 . Series I consisted of patient samples with numerical and/or structural abnormalities of chromosomes 7 or 8, matched with chromosomally normal samples from about the same time period . Series II consisted of consecutive MDS patient samples as well as patient samples in which one or more cells had numerical or structural abnormalities of 7 and 8 . In both series, probes for chromosomes 7 and 8 were applied in each case and at least 100 nuclei were scored for each probe for the distribution of one, two, or three signals . Twenty-seven cases had clonal abnormalities by routine cytogenetics (RC): 12 with monosomy 7; one with monosomy 8; five with trisomy 8; nine with clonal abnormalities other than 7 or 8 aneuploidy . Eleven cytogenetically normal cases gave abnormal interphase FISH (IF) results; one was subsequently confirmed by metaphase FISH analysis to have a clonal structural abnormality of chromosome 7; one case with a trisomy 8 clone, in remission by RC, showed 35% of cells by IF with three signals for chromosome 8; one case had heteromorphic chromosomes by FISH . Of eight remaining cases, five (four with -7 and one with +8 by IF) were among 22 cases of cytogenetically normal MDS . Three remaining cases (two with +8 and one with both +7 and +8 by IF) had AML or MPD . The high rate of possible undetected monosomy 7, among MDS cases in particular, suggests all MDS cases should be screened by IF.

Dev Genes Evol, 1998 Jan, 207(7), 417 - 26
Induction of blood cells in Xenopus embryo explants; Miyanaga Y et al.; A Xenopus-specific anti-leukocyte monoclonal antibody designated XL-2 was isolated and used to identify leukocytes in tailbud embryos and activin A-treated explants of blastula animal cap . XL-2 bound to a 135-kDa polypeptide in western blots of protein extracts from adult thymocytes, tailbud embryos, tadpoles, and explants . In cell suspensions, it immunostained the cell surface of all types of adult leukocytes including lymphocytes, monocyte/macrophages, thrombocytes, and granulocytes . At embryonic stage 24, immunocytochemistry revealed XL-2-positive leukocytes, the earliest time at which such cells have been recognized . Whole-mount staining of tailbud embryos and tadpoles showed a widely dispersed population of XL-2-reactive leukocytes, many of which had elongated shapes and ameboid pseudopodia . In activin A-treated animal caps, XL-2 recognized a subpopulation of cells within the lumen of the central fluid-filled cavity as well as cells in the interstitium of mesenchymal and mesothelial components of the explant . Together, activin A and human interleukin-11 induced 100% of explants to form lumenal blood cells . Compared to activin A alone, murine stem cell factor plus activin A significantly increased the numbers of XL-2-reactive leukocytes and erythrocytes . These results support the view that activin A induces leukocyte and erythrocyte progenitors during Xenopus embryogenesis.

J Leukoc Biol, 1998 Apr, 63(4), 451 - 5
Collagenase expression by normal human eosinophils; Shlopov BV et al.; Collagenolytic activity was detected in extracts from human blood eosinophilic granulocytes . To characterize this collagenase, we compared extracts from isolated populations of eosinophils and neutrophils . Significant collagenase activity against type I and II collagens was present in extracts from both cell populations . Although collagenase activity was present in eosinophils, the cells did not stain with antibodies specific for fibroblast, neutrophil collagenase, or collagenase-3 . In contrast, neutrophils immunostained positively with antibody to neutrophil collagenase . Western blot analysis confirmed the presence of immunoreactive protein in neutrophil extracts but not in the eosinophil extracts . Reverse transcription-polymerase chain reaction using primers specific for all three known collagenases of an eosinophil cell suspension from peripheral blood that had 3% contamination with immature neutrophils showed a polymerase chain reaction product only with neutrophil collagenase oligonucleotide primers, but not with fibroblast collagenase or collagenase-3 primers . Eosinophil collagenase would appear to have a unique antigenic structure and may represent a new enzyme.

Curr Eye Res, 1998 Mar, 17(3), 231 - 7
Environmental factors influence P . aeruginosa binding to the wounded mouse cornea; Chen L et al.; PURPOSE: To determine whether environmental factors or bacterial viability affect the binding of two strains of P . aeruginosa to mouse cornea . METHODS: Scarified corneas were placed in organ culture and inoculated with P . aeruginosa cell suspensions containing either ATCC 19660 or PAO1 bacterial strains classed as cytotoxic or invasive, respectively . Eyes were incubated in vitro for 1 h after bacterial application at different pH or temperature conditions or in PBS containing various divalent cations . The adhesion of heat-killed or formalin-fixed bacteria was tested similarly . Scanning electron microscopy (scanning EM) was used to quantitate adherent bacteria . RESULTS: P . aeruginosa ATCC 19660 showed an increase in binding at pH 8.0, favored higher temperatures and required both calcium and magnesium for optimum binding . Adherence of PAO1 was enhanced at pH 6.5 and decreased at pH 8.0 . This strain favored binding at lower temperatures and did not require either divalent cation for optimum binding . In addition, the presence of magnesium ions resulted in reduced binding for this strain . Both strains exhibited less binding ability after formalin fixation or heat killing . CONCLUSION: Environmental factors and bacterial viability are important factors which influence the ability of both cytotoxic and invasive strains of P . aeruginosa to bind to the scarified cornea.

J Hypertens, 1998 Jan, 16(1), 51 - 4
Single pericytes and pericytes in suspension are stimulated in a similar way by low-density lipoprotein; Skinner S et al.; BACKGROUND: Pericytes are regarded as the microvascular counterpart of smooth muscle cells and implicated in the regulation of blood pressure at the microvascular level . Ca2+ plays an important role in biochemical processes involved in blood pressure regulation and can be activated by low-density lipoprotein (LDL) cholesterol . OBJECTIVE: To determine whether stimulation either of single cells or of cells in suspension by LDL would produce any difference in the increase in cytosolic free calcium levels ({Ca2+}i) . DESIGN AND METHODS: Single pericytes were loaded with 2 micromol/l of the Ca2+-sensitive dye Indo-1/AM . The Indo-1 fluorescence was recorded at 405 nm (Ca2+-bound) and 485 nm (Ca2+-free) after stimulation with LDL . Pericytes in suspension were loaded with 2 micromol/l of the Ca2+-sensitive dye FURA-2/AM . The FURA-2 fluorescence kinetics were recorded at 340-380 nm . Ratios of fluorescence at the two wavelengths were transformed to {Ca2+}i . RESULTS: Basal {Ca2+} levels appeared to be higher in single cells (148+/-13 nmol/l, n = 20) than they were in cells in suspension (128+/-8 nmol/l, n = 25; P= 0.0078) . After stimulation with LDL the increase in {Ca2+}i in both systems was about 220% above baseline . A clear dose dependency was seen for both systems . CONCLUSIONS: Single pericytes and pericytes in suspension increase their {Ca2+}i after stimulation with LDL dose-dependently . Even though single-cell measurements revealed some technical limitations, their responses were comparable to those obtained in a cell suspension . In analogy to aortic smooth muscle cells, our results indicate that LDL might also play a blood-pressure-regulatory role in the microvasculature.

Zentralbl Veterinarmed B, 1998 Feb, 45(1), 25 - 9
Selective loss of BoLA class I determinants from the lymphocyte surface after acid treatment; Horin P et al.; Acid treatment of bovine lymphocytes by a buffered solution of 0.263 m citric acid and 0.123 M Na2HPO4 at pH = 3.0, originally described for human and murine lymphocytes, selectively eliminated the antigenicity of MHC (BoLA) class I determinants also from bovine lymphocytes . The viability of acid-treated cell suspension was higher than 90% . The reactivity of acid-treated lymphocytes with BoLA class I typing alloantisera was lost in microcytotoxicity test, while their reactions with cross-reactive anti-HLA class II, anti-BoCD2 and BoCD5 monoclonal antibodies, and with antisera detecting two non-MHC lymphocyte alloantigenic specificities (BoLY w1 and R') remained unchanged in the microcytotoxicity and/or indirect immunofluorescence tests . The results thus show that this approach of modulating cell surface expression of MHC class I determinants may be used in cattle.

Endocrinology, 1998 Apr, 139(4), 2085 - 91
Glucagon-like-peptide-1 secretion from canine L-cells is increased by glucose-dependent-insulinotropic peptide but unaffected by glucose; Damholt AB et al.; Glucagon-like peptide-1(7-36)amide (GLP-1) is a potent insulinotropic peptide released from the small intestine . To investigate the regulation of GLP-1 secretion, we established a GLP-1 release assay based on primary canine intestinal L-cells . The ileal mucosa was digested with collagenase/EDTA to a single cell suspension and enriched for L-cells by counterstream centrifugal elutriation . We performed release assays on the cultured cells after 36 h, and GLP-1 in the supernatant was determined by enzyme-linked immunoabsorbent assay (ELISA) . Glucose-dependent insulinotropic peptide (GIP) dose dependently stimulated the release of GLP-1 and resulted in a 2-fold increase at 100 nM GIP . This effect was fully inhibited by 10 nM somatostatin . However, neither basal or GIP stimulated GLP-1 secretion were affected by ambient glucose concentrations from 5-25 mM . The receptor-independent secretagogues beta phorbol myristate acetate and forskolin dose dependently increased the secretion of GLP-1; effects inhibited by staurosporine and H8 respectively . Costimulation with GIP and phorbol ester, but not forskolin, resulted in an additive response . Furthermore, the effect of GIP could be inhibited by H8 but not by staurosporine . These results indicate that glucose does not directly stimulate canine L-cells . It is more probable that glucose releases GIP from the upper intestine that in turn stimulates GLP-1 secretion . The ability of GIP to stimulate GLP-1 secretion is probably mediated through activation of protein kinase A.

Anat Rec, 1998 Mar, 250(3), 351 - 65
Class III beta-tubulin isotype (beta III) in the adrenal medulla: III . Differential expression of neuronal and glial antigens identifies two distinct populations of neuronal and glial-like (sustentacular) cells in the PC12 rat pheochromocytoma cell line maintained in a Gelfoam matrix system; Katsetos CD et al.; BACKGROUND: The rat PC12 pheochromocytoma cell line provides an established system for the study of neuronal differentiation . To our knowledge, glial differentiation has not been reported in this cell line . METHODS: We have studied, by immunohistochemistry and immunoblotting, the presence of neuronal cytoskeletal antigens {class III beta-tubulin isotype (beta III), microtubule associated proteins MAP2, MAP1B and tau, and different neurofilament (NF) protein components}, and synaptophysin in comparison with the glial fibrillary acidic protein (GFAP) and S-100 protein in the PC12 cell line . In three different experiments, PC12 cells were maintained in a three-dimensional gelatin foam (Gelfoam) matrix system for up to 34 days with and without treatment with 1 mM dibutyryl cyclic (dc)AMP . Immunohistochemistry was performed on explants ranging from 2 to 32 days-in vitro, which were fixed in either Bouin's solution, 70% ethanol, or 10% neutral-buffered formalin and embedded in paraffin . Immunoblotting was performed on Gelfoam explants with a panel of antibodies against all aforementioned neuronal and glial markers . Additional immunoblot experiments using anti-GFAP and anti-beta III monoclonal antibodies in cell suspensions and homogenates from PC12 monolayer cultures were carried out to compare growth conditions in relation to the expression of these proteins . RESULTS: Beta III and MAP2 were demonstrated by immunohistochemistry and immunoblotting of PC12 explants maintained for up to 32 days in Gelfoam matrices with and without treatment with dcAMP . Intense filamentous and granular beta III staining of PC12 cells was observed in dcAMP-treated cultures concomitant with neuronal morphologic alterations (neuritogenesis and ganglionic phenotype) . In untreated cultures, beta III staining was present in less differentiated cells, as well in cells undergoing neuritic development . The neuronal phenotype of PC12 cells was confirmed by staining for MAP2, tau, and NF proteins, as well as for synaptophysin . The presence of beta III, MAP2, MAP1B, tau, and NF proteins was confirmed by immunoblotting . Clusters of GFAP-positive and S-100 protein-positive spindle cells, phenotypically distinct from the chromaffin-like or neuronal cells, were demonstrated in Gelfoam explants at 5-30 days in vitro . In 30-day-old cultures treated with dcAMP, there was strong filamentous GFAP and diffuse S-100 protein staining in an increased number of sustentacular-like PC12 cells . GFAP staining was corroborated by immunoblotting of explants maintained under identical conditions in vitro . In contrast, immunoblots performed on homogenates from PC12 suspension and monolayer cultures were GFAP-negative . CONCLUSIONS: Neuronal and glial-like, presumed sustentacular, phenotypes were demonstrated in PC12 cells grown in Gelfoam matrices with and without treatment with dcAMP for up to 34 days . To our knowledge, the occurrence of glial differentiation in the PC12 line is a hitherto unreported finding . Adult rat medullary sustentacular cells are known to express S-100 and GFA proteins (Suzuki and Kachi, Kaibogaku Zasshi-Anat 70(2): 130-139, 1995), and the organ culture system employed in our study may well have favored this direction of differentiation.

Ann Clin Lab Sci, 1998 Jan-Feb, 28(1), 34 - 42
Applicability of direct in situ reverse transcription-polymerase chain reaction on bone marrow smears; Chang CY et al.; In situ reverse transcription (RT)-polymerase chain reaction (PCR) is a promising laboratory tool for biomedical investigation at the molecular level in tissues . Direct in-cell amplification of the breakpoint cluster region (BCR)-Abelson (ABL) fusion transcript of chronic myeloid leukemia (CML) has recently been accomplished in Italy using bone marrow mononuclear cell suspensions . The goals of this study are to determine if in situ RT-PCR amplification is possible on bone marrow spirate smears and to demonstrate any unique factors in this procedure . A commercially available method was used because of the existence of published protocols for adaptation . Bone marrow (BM) aspirate smears (n = 17) from patients with CML in blast crisis (positive case material) or other hematological malignancies (negative case material) were evaluated . Satisfactory amplification of the BCR-ABL fusion transcript occurred, and distinct blue cytoplasmic granules that varied in intensity were found in most CML blasts . The negative case materials lacked the specifically amplified granular signals . Overall signal strength and backgrounds were readily affected by the quality of the specimen as well as by changes in assay parameters . In conclusion, the direct in situ RT-PCR technique is applicable for bone marrow aspirate smear evaluation . However, it remains an investigative tool until optimization for sensitivity, specificity, and accuracy can be achieved.

Crit Care Med, 1998 Mar, 26(3), 470 - 6
Segmental bioelectrical impedance analysis improves the prediction for extracellular water volume changes during abdominal surgery; Tatara T et al.; OBJECTIVE: To determine whether the segmental multifrequency bioelectrical impedance analysis may improve the prediction for intraoperative changes in extracellular water volume (deltaECW) compared with whole body multifrequency bioelectrical impedance analysis in abdominal surgical patients . DESIGN: Prospective, consecutive sample . SETTING: Surgical operative patients in a university-affiliated city hospital . PATIENTS: Thirty patients who underwent elective gastrointestinal surgery . INTERVENTIONS: Multifrequency bioelectrical impedance analysis was conducted preoperatively (before the induction of anesthesia) and postoperatively (after recovery from anesthesia) . Resistance values fitted at zero frequency (R0) in the whole body and in each body segment (arm, trunk, and leg) were determined by performing nonlinear curve-fitting and subsequent extrapolation . DeltaECW values were estimated from the whole body resistance between wrist and ankle using two different prediction formulas . In segmental multifrequency bioelectrical impedance analysis, however, ECW was obtained as the sum of each body segment (arms, trunk, and legs) using the equation newly derived from the cell suspension theory . DeltaECW estimated from both measurements were compared with net fluid balances during surgery . MEASUREMENTS AND MAIN RESULTS: R0 in whole body and all body segments significantly decreased after surgery (p < .0001) . The most striking decrease in post/preoperative ratios was found in the R0 in the trunk . The post/preoperative ratio of the R0 value in the trunk was significantly lower than the post/preoperative ratio of the R0 value in the leg (p = .0007) . DeltaECW from segmental multifrequency bioelectrical impedance analysis was similar to net fluid balance (r2 = .80, bias = -0.03 L), whereas whole body multifrequency bioelectrical impedance analysis resulted in considerable underestimations of deltaECW (r2 = .50, .51, bias = 0.95, 0.53 L) . CONCLUSIONS: The difference in the prediction of deltaECW between whole body and segmental multifrequency bioelectrical impedance analysis may be explained by the significant decrease in the resistance of the trunk, which contributed only minimally to the whole body resistance . Segmental multifrequency bioelectrical impedance analysis provides a better approach to predict ECW changes in critically ill patients with nonuniform fluid distribution.

Matrix Biol, 1998 Jan, 16(6), 295 - 306
Flow cytometric analysis of recombinant human osteogenic protein-1 (BMP-7) responsive subpopulations from fetal rat calvaria based on intracellular osteopontin content; Zohar R et al.; The bone morphogenetic proteins (BMPs) are characterized by their ability to induce both chondrogenic and osteogenic differentiation of mesenchymal cells in vivo and in vitro . Primary cultures of fetal rat calvarial cells contain a broad spectrum of osteogenic cells at various stages of differentiation, but the responsive subpopulations are incompletely characterized . To identify responsive cells in osteogenic cell differentiation, we have treated fetal rat calvarial cells with recombinant osteogenic protein-1 and used flow cytometric analyses of intracellular osteopontin, and of cartilage and bone nodule formation, to evaluate the effects . When administered as a single dose at confluence, osteogenic protein-1 stimulated bone nodule formation in fetal rat calvarial cultures in dose-dependently way . To determine the response of osteogenic subpopulations at two discrete stages of differentiation, fetal rat calvaria cells were cultured for 2 days (proliferative stage) or 12 days (early mineralization stage) and treated with 100 ng/ml recombinant osteogenic protein-1 for 12 h before analysis by flow cytometry . Flow cytometry analyses of cell suspensions revealed that osteogenic protein-1 increased the total protein content of cells, and selectively increased the mean expression of osteopontin and the size and granularity of osteopontin expressing cells, particularly at day 12, consistent with a stimulation of osteogenic differentiation and matrix formation . Pulse administration of 100 ng/ml osteogenic protein-1 to sorted, osteopontin-negative subpopulations enriched for stem cells reduced by more than four-fold the number and size of bone nodules while promoting chondrogenesis and adipogenesis . In contrast, a pulse administration of osteogenic protein-1 to more differentiated, large osteopontin-positive cells increased bone nodule formation two-fold . Continuous administration of 100 ng/ml osteogenic protein-1 to the large osteopontin-positive and small osteopontin-negative cell populations obliterated bone nodule formation and promoted chondrogenesis . We conclude that pulse administration of osteogenic protein-1 promotes osteogenic differentiation of cells committed to the osteogenic lineage, whereas undifferentiated periosteal cells are induced to differentiate along the chondrogenic pathway . In contrast, continuous exposure to osteogenic protein-1 promotes chondrogenesis in populations of committed osteogenic cells and in undifferentiated periosteal cells.

Arch Biochem Biophys, 1998 Feb 15, 350(2), 169 - 82
Loss of glutathione, ascorbate recycling, and free radical scavenging in human erythrocytes exposed to filtered cigarette smoke; Maranzana A et al.; Exposure of human erythrocytes to filtered cigarette smoke in vitro inhibited their capacity to reduce dehydroascorbic acid (ascorbate recycling activity) . Glucose uptake was not affected, implying that dehydroascorbic acid transport was not inhibited by the smoke treatment . The intracellular reduction of cationic nitroxide free radicals, which provides a measure of ascorbate recycling, was also inhibited by cigarette smoke . A major factor in the inhibition of free radical reduction was glutathione depletion . However, glutathione depletion alone could not account for the inhibition of free radical reduction because a restoration of the glutathione pool in hemolyzed cells only partially restored free radical reduction activity . Another factor inhibiting free radical reduction was a lowering of pH, which was attributed mainly to the uptake of CO2 and was reversible by restoring the physiological pH . Exogenous glutathione spared both intracellular glutathione and free radical reduction activity . The rate of depletion of intracellular glutathione was similar to that of extracellular glutathione, indicating that the erythrocyte membrane did not significantly attenuate thiol-reactive species in smoke . Protein thiols were also depleted by cigarette smoke, but to a much lesser extent than was glutathione . Ascorbate was relatively unaffected by cigarette smoke; significant intracellular ascorbate levels remained after glutathione was barely detectable . Autooxidizable reducing agents, capable of reducing both reduced piperidinyl (Tempo) and pyrrolidinyl (Proxyl) nitroxides partitioned from filtered cigarette smoke into aqueous solutions . Attempts to detect cigarette smoke-derived oxidants in buffer solutions or in cell suspensions with a prereduced Tempo nitroxide, whose oxidation properties resemble those of ascorbate, were unsuccessful . The results of this study suggest that chemical modification of glutathione is a major damage mechanism of filtered cigarette smoke, whereas free radical oxidations are relatively insignificant .

Int J Exp Pathol, 1997 Aug, 78(4), 245 - 57
Abnormal development and differentiation of macrophages and dendritic cells in viable motheaten mutant mice deficient in haematopoietic cell phosphatase; Nakayama K et al.; In mice homozygous for the 'viable motheaten' (mev) mutation, numbers of macrophage progenitor cells, particularly monocytes, were markedly increased in the bone marrow and spleen . Increased mobilization of these precursor cells to peripheral tissues and their differentiation to macrophages were evidenced by striking increases in macrophage numbers . Immunohistochemical double staining of tissue sections and flow cytometry analyses of single cell suspensions from these mice demonstrated CD5 (Ly-1)-positive macrophages in the peritoneal cavity, spleen and other tissues . Ly-1-positive macrophage precursor cells were demonstrated in the peritoneal cavity of the mev mice and developed in the omental milky spots . The development of marginal metallophilic and marginal zone macrophages was poor in the splenic white pulp and related macrophage populations were absent in the other lymphoid tissues . The numbers of epidermal Langerhans cells in the skin and T cell-associated dendritic cells in the thymic medulla, lymph nodes, and the other peripheral lymphoid tissues were decreased . However, increased numbers of dendritic cells accumulated in the lungs, liver, and kidneys . These abnormalities in development and differentiation of macrophages and dendritic cells may be ascribed to the deficiency in haematopoietic cell SHP-1 tyrosine phosphatase or may be a secondary consequence of abnormal microenvironments, (either constitutive or in response to inflammatory stimuli) in the haematopoietic and lymphopoietic organs and tissues of these mice.

Diabetes Metab, 1997 Dec, 23(6), 511 - 8
Decrease in insulin and insulin-like growth factor I (IGF-I) binding to erythrocytes from patients with cystic fibrosis; Dooghe C et al.; Cystic fibrosis, an autosomal recessive disease affecting exocrine glands, is associated in many cases with a severe undernutritional state, growth retardation and glucose intolerance . To obtain a better definition of the possible defects of insulin and insulin-like growth factor I (IGF-I) receptors, we investigated 125I-insulin and 125I-IGF-I binding to erythrocytes from patients with cystic fibrosis (n = 23) and controls (n = 13) . Erythrocytes were isolated by Ficoll-Hypaque gradient centrifugation, and hormone binding was performed in cell suspensions of 3 x 10(9) cells/ml . Cystic fibrosis patients displayed a statistically significant 33% and 40% (p < 0.05) decrease of insulin and IGF-I binding, respectively, compared to controls . These alterations were due to an almost 50% reduction in the binding capacity of the high-affinity receptor compartment . Affinity constants were modified to a lesser extent, except for a two-fold decrease in K1 of the high-affinity compartment of insulin receptors . Interestingly, the decrease in insulin binding was proportional to the degree of growth failure . The statistical significance of hormone binding alterations was assessed in terms of the graphic distribution of individual affinity constants and binding capacity values . Although variable, 50 to 60% of cystic fibrosis patients displayed alterations in stoichiometric binding parameters located outside the area described by the 95% tolerance interval of controls . A major reduction in insulin and IGF-I binding in conditions of low and normal insulin and IGF-I plasma levels, respectively, as well as the correlation with the degree of growth failure in patients with cystic fibrosis, may contribute to an understanding of the pathogenesis of insulin resistance and glucose abnormalities in undernutritional states.

Prostate, 1998 Feb 15, 34(3), 169 - 74
Surgical orthotopic implantation allows high lung and lymph node metastatic expression of human prostate carcinoma cell line PC-3 in nude mice; An Z et al.; BACKGROUND: Prostate cancer is the second leading cause of male death in the United States . When diagnosed, nearly half the cases have metastatic lesions . An animal model of human prostate cancer demonstrating spontaneous metastasis from the orthotopic site after tumor implantation should be of great help for us to understand the disease and to formulate treatment strategy . We report here a high metastatic model of human prostate cancer PC-3 . METHODS: We developed microsurgical techniques, termed surgical orthotopic implantation (SOI), to implant histologically intact tumor tissues orthotopically in immunodeficient mice . In this study intact tissue of the human prostate cancer cell line PC-3, harvested from a subcutaneous tumor in a nude mouse, was implanted to the ventral lateral lobes of the prostate gland in a series of nude mice . Mice were sacrificed when found moribund, and autopsy and histology were performed subsequently . RESULTS: A high frequency of lymph node and lung metastasis was noted upon histological examination . The extensive and widespread lung metastasis following orthotopic implantation of PC-3 is, to the best of our knowledge, the first report in the literature . CONCLUSIONS: In contrast to orthotopic injection of cell suspensions, no multiple metastatic cell selection was necessary after SOI for significant expression of the metastatic potential of PC-3 . We conclude that the stromal tissue architecture maintained in the implanted tumor played a critical role in tumor growth and progression.

Obstet Gynecol, 1998 Mar, 91(3), 329 - 35
Identification of placental cytokine-producing cells in term and preterm labor; Steinborn A et al.; OBJECTIVE: To determine if the production of proinflammatory cytokines by placentally derived macrophages changes with term and preterm labor and to examine if changes in antigen expression of these cytokines can be detected by immunohistologic methods . METHODS: Enzymatically dispersed placental cell suspensions of the trophoblastic villi, obtained from 16 women with spontaneous term delivery, 16 women with elective cesarean delivery without any labor, and 22 preterm delivering women with labor unresponsive to tocolysis, were fractionated by magnetic-associated-cell-sorting, on the basis of CD11b-antigen expression . Positively and negatively sorted cell fractions were cultured and concentrations of interleukin-6, interleukin-1beta, and tumor-necrosis-factor-alpha were measured in the culture supernatants . Immunohistologic staining was used for identification of cytokine-producing cells within placental tissues . RESULTS: Positively sorted cells obtained from term (median 2027 pg/mL, P = .037) and preterm (median 3628 pg/mL, P = .001) laboring women produced significantly elevated amounts of tumor-necrosis-factor-alpha compared with nonlaboring (median 1088 pg/mL) women at term . Negatively sorted cell fractions obtained from term (median interleukin-1beta 162 pg/mL, P = .031, median interleukin-6 3134 pg/mL, P = .004) and preterm (median interleukin-1beta 934 pg/mL, P = .003, median interleukin-6 5695 pg/mL, P = .001) laboring women produced significantly elevated amounts of interleukin-1beta and interleukin-6 compared with nonlaboring (median interleukin-1beta 29 pg/mL, median interleukin-6 135 pg/mL) women at term . Immunohistologic staining revealed that tumor-necrosis-factor-alpha activity was localized in isolated stromal cells, whereas interleukin-1beta and interleukin-6 were predominantly found in endothelial cells within placental villi . CONCLUSION: The source of labor-associated release of tumor-necrosis-factor-alpha from placental tissues are macrophages, whereas interleukin-1beta and interleukin-6 are released from placental endothelial cells.

Plant Physiol, 1998 Feb 1, 116(2), 659 - 69
Oxidative Burst and Hypoosmotic Stress in Tobacco Cell Suspensions
Cazale AC, Rouet-Mayer MA, Barbier-Brygoo H, Mathieu Y, Lauriere C.
Oxidative burst constitutes an early response in plant defense reactions toward pathogens, but active oxygen production may also be induced by other stimuli . The oxidative response of suspension-cultured tobacco (Nicotiana tabacum cv Xanthi) cells to hypoosmotic and mechanical stresses was characterized . The oxidase involved in the hypoosmotic stress response showed similarities by its NADPH dependence and its inhibition by iodonium diphenyl with the neutrophil NADPH oxidase . Activation of the oxidative response by hypoosmotic stress needed protein phosphorylation and anion effluxes, as well as opening of Ca2+ channels . Inhibition of the oxidative response impaired Cl- efflux, K+ efflux, and extracellular alkalinization, suggesting that the oxidative burst may play a role in ionic flux regulation . Active oxygen species also induced the cross-linking of a cell wall protein, homologous to a soybean (Glycine max L.) extensin, that may act as part of cell volume and turgor regulation through modification of the physical properties of the cell wall.

Anat Rec, 1998 Feb, 250(2), 182 - 9
Appearance and development of lymphoid cells in the chicken (Gallus gallus) caecal tonsil; Gomez Del Moral M et al.; BACKGROUND: We have analyzed by electron microscopy, immunohistochemistry, and flow cytometry the development of chicken caecal tonsil, the largest lymphoid organ of avian gut-associated lymphoid tissue (GALT) . METHODS: White Leghorn chickens of different ages obtained from a local supplier were routinely processed by transmission electron microscopy . For both immunohistochemistry and flow cytometry, we tested a battery of specific monoclonal antibodies (mAbs) to chicken cell markers on caecal cryosections or cell suspensions, respectively . RESULTS: A rudimentary caecal tonsil occurs at the end of incubation . The organ grows just after birth, reaching the adult condition 4 days later . Firstly (4 days to 2 weeks), it contains predominantly T lymphocytes, principally TcR alphabeta+ and CD4+ cells, which occupy largely the named caecal diffuse lymphoid tissue . In adult tonsils (6-week-old chickens) however, B lymphocytes, mainly expressing either IgM or IgA, predominate . They occur in both the subepithelial zone and the germinal centers, in which there are also a few T cells . After 2 weeks the CD8+ lymphocytes gradually become more numerous than CD4+ cells . In the tonsillar epithelium CD8+TcRgammadelta+ T cells, CD8+TcRgammadelta-alphabeta-, presumably NK cells, and a few B lymphocytes are the main cell subpopulations . CONCLUSIONS: Chicken caecum grows fast after hatching . The diffuse lymphoid tissue largely contains TcR alphabeta CD4+ or CD8+ cells . CD8+ cells of caecal epithelium represent gammadelta T cells or NK cells . B lymphocytes which occur in the subepithelial zone, germinal centers, and, in few numbers, the caecal epithelium predominantly express either IgM or IgA.

Tsitologiia, 1997, 39(7), 560 - 5
{The structural organization of suspension grafts of human embryonic midbrain in the rat brain}; Bystron' IP et al.; Dynamics of survival, differentiation and migration of human fetal cells after their transplantation into the rat brain without immunosuppression was studied using routine histology, electron microscopy and immunocytochemistry . A cell suspension prepared from the ventral mesencephalon of human embryos 7-8 weeks of gestation was injected into the striatum of rats-recipients . The graft development depends on the intensity of immune reaction . Under a weak reaction, the viability and differentiation of human embryo cells in the rat brain were observed within the three months of the experiment . The grafted cells conserve their mediator specificity, some of them being seen to migrate into the host (rat) tissue beyond the graft . In the transplant neuropil various types of cell contacts were observed, including synapses . The described method makes its possible to study the human nervous tissue histogenesis in an abnormal environment.

Invest Ophthalmol Vis Sci, 1998 Feb, 39(2), 336 - 43
Isolation of human conjunctival mast cells and epithelial cells: tumor necrosis factor-alpha from mast cells affects intercellular adhesion molecule 1 expression on epithelial cells; Cook EB et al.; PURPOSE: To isolate and purify mast cells and epithelial cells from human cadaveric donor conjunctival tissue and to characterize interactions between these cell types in vitro . METHODS: Monodispersed cell suspensions obtained by enzymatic digestion of conjunctival tissue were applied to a single-density Percoll gradient . Epithelial cells obtained from the top layer of the gradient were cultured to confluence . Mast cells obtained from the pellet were equilibrated in culture medium and further purified using a two-step Percoll gradient . Using reverse transcription-polymerase chain reaction (RT-PCR), RNA from the purified mast cell preparation was probed for tumor necrosis factor-alpha (TNF alpha) message . Fluorescence activated cell sorting (FACS) analysis of intracellular immunostained mast cells was used to detect the TNF alpha protein . An examination for intercellular adhesion molecule 1 (ICAM-1) on epithelial cells was performed after 24-hour incubations with either recombinant TNF alpha supernatants from calcium ionophore A23187 (CaI)-stimulated mast cells or appropriate controls using FACS analysis . RESULTS: Highly purified human conjunctival mast cells and epithelial cells (each > 95%) were obtained from human cadaveric donor tissue . RT-PCR analysis of purified mast cell RNA revealed the expression of TNF alpha mRNA . An evaluation of mast cells for intracellular protein demonstrated positive staining for tryptase and TNF alpha . ICAM-1 was found on purified epithelial cells, and incubation of epithelial cell monolayers with supernatants from Cal-stimulated mast cells resulted in upregulation of this receptor . This upregulation was blocked by incubation with TNF alpha-neutralizing antibody . CONCLUSIONS: This work provides the methods for isolating and purifying mast cells and epithelial cells from human donor tissue and the opportunity for studying mechanisms of conjunctival inflammation by evaluating the interactions between these cells.

Planta, 1998 Feb, 204(2), 234 - 41
Regulatory role of microsomal 3-hydroxy-3-methylglutaryl-coenzyme A reductase for shikonin biosynthesis in Lithospermum erythrorhizon cell suspension cultures; Lange BM et al.; The carbon skeleton of the naphthoquinone pigment shikonin, which is produced in Lithospermum erythrorhizon Sieb . et Zucc . cell-suspension cultures, is partly derived from the isoprenoid biosynthetic pathway . The requirement of 3-hydroxy-3-methylglutaryl-coenzyme A reductase (HMGR, EC 1.1.1.34), a key enzyme of the mevalonate route to isoprenoids, for shikonin synthesis was investigated . Conserved regions of sequences from plant HMGR genes were used to design polymerase chain reaction (PCR) primers for the cloning of a cDNA fragment from L . erythrorhizon . The resulting 443-bp clone was used as a probe for Northern analyses and hybridized to an mRNA of approx . 2.5 kb . Under shikonin-producing conditions, microsomal HMGR enzyme activity as well as mRNA level closely correlated with the accumulation of shikonin derivatives . White light, which inhibits shikonin formation, was shown to strongly suppress HMGR gene expression . The results presented here indicate that HMGR plays a significant role in the regulation of shikonin biosynthesis and that the control appears to act at the transcriptional level.

Clin Exp Immunol, 1998 Feb, 111(2), 450 - 6
Indirect demonstration of the lifetime function of human thymus; Marusic M et al.; The aim of this study was to test the hypothesis that human thymus maintains its function as the site of early T cell development throughout life, but to a progressively diminishing extent . Mononuclear cell suspensions prepared from the samples of 39 human thymuses were analysed for the total number of cells per gram of thymus tissue, percentage of single marker-positive CD2, CD4 and CD8 cells, percentages of double-positive CD4CD8 and CD2CD8 cells, double-negative CD4CD8 cells, absolute numbers of these cells per gram of tissue, and extent of the in vitro proliferation upon stimulation with concanavalin A (Con A), phytohaemagglutinin (PHA) and pokeweed mitogen (PWM) mitogens . The main outcome measures were flow cytometric data on thymus lymphoid cell composition (according to CD classification), expressed as percentages and numbers of cells per gram of thymus tissue . The total number of mononuclear cells expressed per gram of thymus tissue exponentially decreased with age . The slope of none of the analysed cell subpopulations differed from the slope of the line constructed for age-related decline of the total number of mononuclear cells (-0.024 on a semilogarithmic scale) . The thymuses of all ages contained all analysed cell subpopulations in approximately the same proportions: percentages of these cell subpopulations did not change with age, except for all CD4+ (P=0.017) and double-positive CD4+CD8+ (P=0.016) cells, which tended to decrease with age . The extent of proliferation of thymus cells upon stimulation with T and B cell mitogens was unrelated to age . We conclude that the thymus retains its function as the site of differentiation of T lymphocytes throughout life . With respect to the number of involved lymphoid cells, the function exponentially decreases with age.

Mol Microbiol, 1998 Jan, 27(2), 437 - 53
PrhA controls a novel regulatory pathway required for the specific induction of Ralstonia solanacearum hrp genes in the presence of plant cells; Marenda M et al.; The Ralstonia solanacearum hrp gene cluster is organized in five transcriptional units . Expression of transcriptional units 2, 3 and 4 is induced in minimal medium and depends on the hrp regulatory gene hrpB, which belongs to unit 1 . This regulatory gene also controls the expression of genes, such as popA, located to the left of the hrp cluster . Here, we show that, upon co-culture with Arabidopsis thaliana and tomato cell suspensions, the expression of the hrp transcriptional units 1, 2, 3 and 4 is induced 10- to 20-fold more than in minimal medium . This induction is not triggered by diffusible signals but requires the presence of plant cells . Moreover, we show that this specific plant cell induction of hrp genes is controlled by a gene, called prhA (plant regulator of hrp genes), located next to popA . This gene codes for a putative protein of 770 amino acids, which shows similarities with TonB-dependent outer membrane siderophore receptors . Expression of prhA and hrp genes is not regulated by iron status, and we postulate that iron is not the signal sensed by PrhA . In prhA mutants, the induction of hrpB and other hrp genes is abolished in co-culture with Arabidopsis cells, partially reduced in co-culture with tomato cells and not modified in minimal medium . prhA mutants are hypo-aggressive on Arabidopsis (accessions Col-0 and Col-5) but remain fully pathogenic on tomato plants, suggesting that the co-culture assays mimic the in planta conditions . A model suggesting that PrhA is a receptor for plant specific signals at the top of a novel hrp regulatory pathway is discussed.

Br J Cancer, 1998 Feb, 77(4), 595 - 604
Development of a new metastatic human breast carcinoma xenograft line; Mehta RR et al.; Xenografts originated from human tumours offer the most appropriate research material for in vivo experimental research . However, primary human breast carcinomas are difficult to grow when transplanted in athymic mice: tumour take is less than 15% . Recently, we have achieved 60% tumour take by injecting tumour cell suspensions mixed with Matrigel . Human breast xenografts originated from primary breast carcinoma also frequently show the potential to metastasize spontaneously . In the present study, we generated a human breast carcinoma xenograft line (UISO-BCA-NMT-18) that shows 100% tumorigenicity and 80-100% lung metastasis when transplanted s.c . in athymic mice . We have studied in detail the characteristics of the xenograft and the patient's tumour from which the xenograft line originated . Both the xenograft and the patient's tumour showed intense staining for mutant p53 nuclear protein, and high expression of U-PA, PAI and u-PAR . In vivo growth of the xenograft is stimulated by exogenous supplementation of oestrogen . This xenograft is continuously growing in mice and has shown 80-100% metastasis for the last three successive in vivo passages . This well-characterized, oestrogen-responsive, metastatic breast carcinoma xenograft line will provide excellent research material for metastasis-related research.

Plant Mol Biol, 1998 Feb, 36(3), 343 - 51
Expression of extensin genes is dependent on the stage of the cell cycle and cell proliferation in suspension-cultured Catharanthus roseus cells; Ito M et al.; To isolate cDNAs expressed at a specific phase of the cell cycle in a higher plant, we performed differential screening of a cDNA library prepared from the S-phase cells of synchronized cultures of Catharanthus roseus . Sequence analysis shows that two of the identified cDNAs, cyc15 and cyc17, encode extensins that represent a family of cell wall hydroxyproline-rich glycoproteins . Protein sequences deduced from the two cDNAs contain the characteristic pentapeptide repeat sequence, Ser-Pro-Pro-Pro-Pro, which is commonly observed in extensins . The protein sequences also share several other extensin characteristics such as the presence of a N-terminal signal peptide and a high content of Tyr and Lys residues . When C . roseus cell suspension cultures were synchronized by phosphate starvation, the mRNAs of both cyc15 and cyc17 were transiently expressed during the S and G2 phases of the cell cycle . However, significant amounts of the mRNAs also accumulated in phosphate-starved cells arrested in the G1 phase . In asynchronous cultures, both genes were expressed during the stationary phase, when cell proliferation ceased . The observed patterns of expression suggest that the extensin genes, cyc15 and cyc17, are under two types of regulation: one that depends on the stage of the cell cycle and another that is induced during the growth arrest . Thus, the products of these genes may function both during the progression through the cell cycle and in the strengthening of the cell wall after cell division.

J Pediatr Gastroenterol Nutr, 1998 Feb, 26(2), 194 - 9
Increased levels of prostaglandins and nitric oxide in esophageal mucosa of children with reflux esophagitis; Zicari A et al.; BACKGROUND: Prostaglandin E2 (PGE2) is said to be both protective and detrimental for esophageal mucosal integrity . Nitric oxide (NO) controls several esophageal neuromuscular functions, including relaxation of the lower esophageal sphincter . The purpose of this study was to verify PGE2 and NO levels in esophageal mucosa of children with reflux esophagitis . METHODS: The patients were 10 children, age range 7 to 12 years, affected by reflux esophagitis . The control subjects were 10 children, age range 6 to 11 years, with recurrent abdominal pain . Tissue fragments obtained by esophageal biopsies were placed in a culture medium and processed to obtain a cell suspension . Cells were incubated for 24 hours at 37 degrees C . Thereafter, supernatants were collected and divided into aliquots to determine the amounts of PGE2 and NO metabolites . RESULTS: Esophageal cells obtained from reflux esophagitis patients synthesize and release a significantly higher (p < 0.01) amount of PGE2 and NO (PGE2 1.9 +/- 0.56 ng/10(6) cells per 24 hours; NO 124.94 +/- 18.36 microM/10(6) cells per 24 hours) than did the control group (PGE2 0.66 +/- 0.14 ng/10(6) cells per 24 hours; NO 68.03 +/- 12.3 microM/10(6) cells per 24 hours) . CONCLUSIONS: These results suggest that in esophageal mucosa, PGE2 and NO, in low concentrations, are protective, whereas, at high doses, they can be harmful . Higher amounts of PGE2 and NO in the esophageal mucosa of reflux esophagitis patients suggest that similar noxious stimuli trigger the inducible forms of the respective enzyme.

J Clin Gastroenterol, 1997, 25 Suppl 1, S164 - 8
Studies on gastric mucosal cell injury induced by Helicobacter pylori; Mitani-Ehara S et al.; The cause of gastric cell injury induced by Helicobacter pylori was investigated in vitro using gastric mucosal cells derived from male Japanese white rabbits . To evaluate the contribution of the potent urease activity of H . pylori to gastric mucosal cell injury, the supernatant of the H . pylori bacterial pellet, solubilized with N-octyl-glucoside, was added to the gastric mucosal cell suspension . Cell injury was assessed by lactate dehydrogenase (LDH) release into the extracellular fluid . Treatment of cells with H . pylori extracts together with urea resulted in high levels of LDH release, suggesting definite gastric mucosal cell injury, and elevation of ammonia concentration was also observed . In contrast, incubation with H . pylori extracts alone or urea solution alone did not result in increased LDH release or elevated ammonia concentrations . The degree of LDH release from gastric mucosal cells due to H . pylori extracts in the presence of urea was similar to that induced by administration of the same amount of exogenous ammonia . The addition of acetohydroxamic acid, a potent specific urease inhibitor, remarkably inhibited ammonia production, the elevation of pH of extracellular fluid, and LDH release in a dose-dependent manner . These results suggest that ammonia produced by potent urease activity of H . pylori in the presence of urea plays an important role in the pathogenesis of gastric mucosal cell injury.

J Mol Cell Cardiol, 1997 Nov, 29(11), 2865 - 71
Phosphatidic acid: a potential signal transducer for cardiac hypertrophy; Dhalla NS et al.; Phosphatidic acid (PA) is mainly formed by the hydrolysis of phosphatidylcholine due to the activation of phospholipase D (PLD) . PA is also generated by phosphorylation of diacylglycerol (DAG) due to the action of DAG kinase and is converted to DAG under the action of PA phosphohydrolase . Most of the positive inotropic agents which are known to stimulate cardiac hypertrophy, have been shown to increase the level of PA in cardiac sarcolemma . Although the growth factor-like effect of PA has been recognized in a wide variety of tissues, there is a lack of similar information in adult cardiomyocytes . By using single cardiomyocytes, we have now shown that PA increased the basal {Ca2+}i level without significant effect on the amplitude of Ca2+ transients . PA (10-50 mu M) also increased the {Ca2+}i in cardiac cell suspension . PA has also been shown to stimulate protein synthesis in cardiomyocytes, which is inhibited by a PKC inhibitor as well as a Ca2+ chelator . PA at the concentration of 1-50 mu M was observed to stimulate the activity of PLC in cardiac sarcolemma; this effect was attenuated by a PLC inhibitor . Since DAG, formed due to the activation of PLC, is considered to play a crucial role in regulating the activity of protein kinase C (PKC), the positive feedback effect of PA on this pathway may be essential for maintaining the sustained elevation in the activity of PKC during the development of cardiac hypertrophy . In view of these observations and other facts available in the literature, it is suggested that PA may be a potential signal transducer for the development of cardiac hypertrophy .

Verh Dtsch Ges Pathol, 1997, 81, 306 - 11
Cytogenetics on released DNA fibers; Vaandrager JW et al.; DNA fibers can be released from cell suspensions and frozen tissue and can be used as a template for hybridization with multiple differently labeled probes . Simultaneous hybridization with 5-10 adjacent or partly overlapping probes generates a highly specific "color barcode" for individual DNA segments . Rearrangements in this barcode can be easily detected and mapped . The resolution of DNA fiber FISH is between 2 and 500kb . In mixing experiments of cell lines with different structural abnormalities, we found a sensitivity of approximately 10% . We applied DNA fiber FISH for detection of t(11;14) in mantle cell lymphoma (MCL) and immunoglobulin (Ig) class switching in hairy cell leukemia (HCL) . Using a barcode for the Ig and BCL-1 loci at 14q32 and 11q13, we detected and mapped a t(11;14) breakpoint in 35 of 36 MCL . In 5 cases complex mono-allelic rearrangements at both sides of the cyclin D1 gene were identified . In 13 HCL with phenotypic evidence of Ig class switching, including 2 cases with solely IgD, fiber FISH revealed concordant Ig class switch deletions . In most cases both alleles were affected . These results indicate that DNA fiber FISH is a very powerful method to detect and map structural DNA alterations.

J Exp Zool, 1998 Feb 15, 280(3), 265 - 7
Production of germ-line chimeras by transfer of cryopreserved gonadal primordial germ cells (gPGCs) in chicken; Tajima A et al.; Gonadal primordial germ cells (gPGCs) were collected from gonadal anlage of 5-day-old White Leghorn (WL) embryos . Collected gPGCs were suspended in freezing medium containing 10% dimethyl sulphoxide (DMSO) . The cell-suspension was frozen at 1 degree C/min until the temperature reached -80 degrees C; cells were then immersed in liquid nitrogen at -196 degrees C and stored up to 3 mo . Approximately 100 frozen/thawed gPGCs were injected into the dorsal aorta of each Barred Plymouth Rock (BPR) embryo from which blood was drawn prior to germ-cell injection . The injected embryos were incubated until hatched, and hatched chicks were raised until sexually mature . Upon reaching sexual maturity, a progeny test was performed by mating recipient chicks with normal BPR of the opposite sex . Of 840 progeny produced from 4 gPGC recipients, 74 chicks (8.8%) were phenotypically white . Present results demonstrate that frozen/thawed gPGCs collected from gonadal anlage are capable of producing germ-line chimeras in chicken.

J Biochem Biophys Methods, 1997 Dec 3, 35(3), 185 - 95
A polarographic method for measuring dissolved nitric oxide; Jensen BO et al.; A polarographic method for measuring the concentration of authentic nitric oxide (NO) in aqueous solutions is described . When solutions of NO were injected into aqueous solutions containing dissolved oxygen, NO reacted with oxygen to give nitrite . The amount of nitrite formed in this reaction (analyzed by capillary electrophoresis) was compared with the amount of oxygen consumed (measured by polarography) . We observed a linear relationship between the amount of consumed oxygen and the amount of nitrite formed in the measured range (20-100 nmol NO-2) (R2 = 0.89) . These results demonstrate that polarographic measurements of the amount of oxygen consumed in the reaction with NO could be used to estimate the concentration of dissolved NO in authentic NO solutions . There are several advantages to measuring consumed oxygen by polarographic methods: The method is simple and can be performed immediately prior to utilization of the NO solutions, it does not require any NO calibration standards and it fully discriminates between NO and oxidation products of NO . An additional advantage is, therefore, that stock solutions of NO can be stored for an indefinite period of time prior to measurement . Both the preparation of authentic NO solutions and the measurement of their NO content are therefore simplified . We also demonstrate that a slow infusion of authentic NO solution to cell suspensions in vitro makes it possible to study the rapid, reversible biological effects of NO.

J Clin Pathol, 1997 Nov, 50(11), 957 - 9
Analysis of TH1 and TH2 cytokine production in low grade B cell gastric MALT-type lymphomas stimulated in vitro with Helicobacter pylori; Hauer AC et al.; Previous studies have suggested that the dependence of low grade B cell gastric lymphoma on infection of the gastric mucosa with Helicobacter pylori results from help provided by H pylori specific tumour infiltrating T cells . ELISPOT analysis was used to characterise functional subpopulations of tumour infiltrating T cells . The production of the TH1 cytokine interferon gamma and TH2 cytokines interleukin (IL)-4, IL-5, and IL-10 were measured in tumour cell suspensions from two cases of low grade B cell gastric lymphoma, one case of thyroid gland lymphoma, and one case of salivary gland lymphoma . Cells were assayed on day 0 and following 24 hours incubation either in culture medium or with a range of strains of H pylori . There was a dominant TH1-type (pro-inflammatory) response consistent with the TH1 response observed in H pylori gastritis.

Tissue Antigens, 1998 Jan, 51(1), 67 - 71
Comparison of serological and molecular typing for HLA-A and -B on cord blood lymphocytes; Poli F et al.; HLA class I typing by standard microcytotoxicity testing has been unsatisfactory for 14.5% of 1644 cord blood samples . In this study, we evaluated the capacity of PCR-SSP in solving problems in HLA-A,B typing with serological methods . With this aim we have compared serology with PCR-SSP in 100 cord blood samples with doubtful or unreliable HLA-A,B typing . PCR-SSP was successful in amplifying HLA-A,B alleles in all 100 cord blood samples . Forty-six typings gave discrepant results with the 2 methods (serology and PCR-SSP) . Typings were considered discrepant also in the case of inability to define a split . For 19 specimens, no serological conclusion was drawn due to high mortality of the cell suspension, while PCR-SSP allowed the definition of a clear typing . In 6 cases it was necessary to infer information from serology to define the current typing . Finally, in 3 other cases it was impossible to exclude or attribute the antigen/allele B67 or B4802 . PCR-SSP for HLA-A,B can improve the overall reliability of HLA-A,B typing requiring a small amount of blood although, with the set of sequence specific primers adopted, a number of alleles are still poorly defined.

Planta, 1997, 203(3), 349 - 61
Are the characteristics of betanidin glucosyltransferases from cell-suspension cultures of Dorotheanthus bellidiformis indicative of their phylogenetic relationship with flavonoid glucosyltransferases?
Vogt T, Zimmermann E, Grimm R, Meyer M, Strack D.
Uridine 5'-diphosphoglucose:betanidin 5-O- and 6-O-glucosyltransferases (5-GT and 6-GT; EC 2.4.1) catalyze the regiospecific formation of betanin (betanidin 5-O-beta-glucoside) and gomphrenin I (betanidin 6-O-beta-glucoside), respectively . Both enzymes were purified to near homogeneity from cell-suspension cultures of Dorotheanthus bellidiformis, the 5-GT by classical chromatographic techniques and the 6-GT by affinity dye-ligand chromatography using UDP-glucose as eluent . Data obtained with highly purified enzymes indicate that 5-GT and 6-GT catalyze the indiscriminate transfer of glucose from UDP-glucose to hydroxyl groups of betanidin, flavonols, anthocyanidins and flavones, but discriminate between individual hydroxyl groups of the respective acceptor compounds . The 5-GT catalyzes the transfer of glucose to the C-4' hydroxyl group of quercetin as its best substrate, and the 6-GT to the C-3 hydroxyl group of cyanidin as its best substrate . Both enzymes also catalyze the formation of the respective 7-O-glucosides, but to a minor extent . Although the enzymes were not isolated to homogeneity, chromatographic, electrophoretic and kinetic properties proved that the respective enzyme activities were based on the presence of single enzymes, i.e . 5-GT and 6-GT . The N terminus of the 6-GT revealed high sequence identity to a proposed UDP-glucose: flavonol 3-O-glucosyltransferase (UF3GT) of Manihot esculenta . In addition to the 5-GT and 6-GT, we isolated a UF3GT from D . bellidiformis cell cultures that preferentially accepted myricetin and quercetin, but was inactive with betanidin . The same result was obtained with a UF3GT from Antirrhinum majus and a flavonol 4'-O-glucosyltransferase from Allium cepa . Based on these results, the main question to be addressed reads: Are the characteristics of the 5-GT and 6-GT indicative of their phylogenetic relationship with flavonoid glucosyltransferases?

Gene Ther, 1997 Oct, 4(10), 1061 - 8
Double-copy bicistronic retroviral vector platform for gene therapy and tissue engineering: application to melanoma vaccine development; Wiznerowicz M et al.; The efficient genetic modification of solid tumors in situ to stimulate therapeutic immune responses against them is currently under active investigation, but is not yet possible using existing gene transfer technologies . Thus, ex vivo/in vivo vaccination strategies have been proposed in which the patient's tumor is surgically excised, single cell suspensions are prepared, the therapeutic genes are introduced and then the gene-modified cells, after being gamma-irradiated, are injected back into the patient . However, even with high-efficiency gene delivery systems, this is a labor-intensive process . Moreover, it is often difficult to obtain sufficient numbers of gene-modified primary tumor cells during short-term culturing . On the other hand, extended in vitro passaging of primary tumor explants may alter their immunophenotypic properties . One approach to overcome these limitations would be to design universal vaccines consisting of standardized gene-transduced neoplastic cell lines or mixtures of gene-transduced cell lines to be combined with autologous tumor samples if available . Melanoma, which is notable for being one of the most immunogenic human malignancies, represents a cancer where shared tumor-associated antigens have been identified . We developed and analyzed several different retroviral vectors for their ability to stably express exogenous genes at high levels in a panel of melanoma cell lines . All vectors contained a reporter gene (nlslacZ) encoding beta-galactosidase with a nuclear localization signal and the neomycin phosphotransferase (neo) gene as selectable marker . One vector, DCCMV, which carried a bicistronic nlslacZ-neo transcriptional unit under the control of the human cytomegalovirus immediate-early promoter in the U3 region of its 3' LTR, was found to perform consistently better than the other vectors . The DCCMV vector, which is an extreme example of the double-copy class of retroviral vectors, was subsequently used to generate melanoma cell lines constitutively secreting human interleukin-6 or a soluble form of the human interleukin-6 receptor for potential use in a phase II clinical vaccine trial for the treatment of melanoma patients . The DCCMV vector design may also be useful in gene therapy applications where the intent is to implant polymer-encapsulated cell lines genetically engineered to stably express high levels of bioactive proteins.

J Histochem Cytochem, 1997 Dec, 45(12), 1715 - 22
Immunomagnetic isolation of rat bone marrow-derived and peritoneal mast cells; Jamur MC et al.; Mast cells are difficult to purify from heterogeneous cell populations and to preserve, especially for pre-embedding immunostaining at the ultrastructural level . We have developed a technique that permits the isolation of a pure population of mast cells suitable for immunocytochemical studies . A rat mast cell-specific monoclonal antibody (MAb AA4) conjugated to tosylactivated Dynabeads 450 was used to immunomagnetically separate mast cells from rat bone marrow and peritoneal cell suspensions . Approximately 85% of the mast cells were recovered in the positive population that comprised virtually pure mast cells . After microwave fixation, morphological examination showed that the cells were intact and retained their ultrastructural detail . Mast cells in all stages of maturation were immunolabeled with a panel of antibodies after immunomagnetic separation . The combination of immunomagnetic separation followed by immunostaining should prove useful for the study of mast cell maturation and for the characterization of other specific cell types that are present in tissues in only limited numbers.

Rev Neurol, 1997 Sep, 25(145), 1383 - 6
{The obtaining of neural cell suspensions . Their application in neurobiological studies}; de la Cuetara-Bernal K et al.; INTRODUCTION: Techniques of intracranial grafting have become powerful tools used in the study of the mechanisms of regeneration and plasticity taking place in the central nervous system (CNS) . Suspensions of foetal neural cells have been successfully used as intracerebral grafts in deep regions of the brain . OBJECTIVE: The objective of this paper is to describe the methodology used by our group to obtain and prepare suspensions of cells from different parts of rat embryo brain (midbrain, striatum and septum) used for experimental transplants in animal models . MATERIAL AND METHODS: We give a detailed description of the steps involved in the preparation of tissue in the form of a suspension of cells . These steps include the obtention of tissue at the optimum stage of development, dissection of the donor tissue, enzymatic treatment and mechanical dissociation until a suspension of cells is obtained . Viability counts showed a high percentage (more than 95%) of viable cells in the suspensions thus obtained . Cellular viability is affected by different factors and is a prerequisite for good survival of the graft . CONCLUSION: The methodology described permits obtention of suspensions of foetal neural cells which are optimal for use in studies both in vitro and in experimental transplants.

Br J Dermatol, 1997 Sep, 137(3), 376 - 80
Presence of somatostatin in normal human epidermis; Gaudillere A et al.; Somatostatin (SOM) is a ubiquitous peptide which is responsible for the inhibition of numerous biological functions . SOM is described as an antiproliferative molecule and an inhibitor of exocrine or endocrine secretion from a variety of tissues, including pancreas, gastrointestinal tract, central and peripheral nervous system . Mediation of SOM effects can be indirect or direct, respectively, through other molecules or receptors on target cells . We have searched for the presence of SOM in the epidermis using immunofluorescence, confocal laser scanning microscopy, radioimmunoassay, and chromatography . Immunofluorescence and confocal laser scanning microscopy studies were performed using rabbit antiserum anti-SOM and mouse monoclonal antibody directed to CD1a Langerhans cell (LC) marker disclosed with fluorescein or tetramethylrhodamine isothiocyanate conjugates . SOM was extracted from whole skin or epidermal cell suspension or LC-enriched suspensions and analysed by radioimmunoassay . We used an antiserum which was reactive for the 6-11 portion of native SOM . Chromatographic columns were performed on extracts from whole skin . The epidermis was SOM immunoreactive . LC were immunoreactive for SOM and the staining was membranous . SOM was extracted from the whole skin at about 0.13 +/- 0.02 fmol/mg of tissue (mean +/- SEM) . The SOM concentration in epidermal cell suspensions was 1.5 +/- 0.9 fmol/10(6) cells . Data obtained with LC-enriched suspensions showed large variations between donors . Extracts from skin showed one peak with an elution profile like that of 14 amino acid SOM . This study demonstrates that 14 amino acid SOM is expressed in normal human epidermis.

Br J Dermatol, 1997 Sep, 137(3), 367 - 75
Multiparameter flow cytometry as a tool to evaluate antipsoriatic therapy; Glade CP et al.; Objective comparison of different antipsoriatic therapies requires quantitative assessment of disease severity . However, clinical assessment with the widely used Psoriasis Area and Severity Index (PASI) introduces inaccuracy . An alternative is the quantitative analysis of different epidermal cell parameters using multiparameter flow cytometry . Our aim in the present study was to compare the clinical and flow cytometric approach to monitor disease activity and to evaluate antipsoriatic efficacy . Clinical scores for erythema, induration and scaling were assessed and biopsies for flow cytometric analysis were obtained from the psoriatic plaques of 89 patients before and after treatment with different therapeutic regimens consisting of vitamin D3 analogues and corticosteroids . In total, 219 epidermal cell suspensions were analysed using triple-labelling, with the simultaneous staining of markers for epidermal proliferation (DNA dye TO-PRO-3), differentiation (antikeratin 10), and inflammation (antivimentin) . Correlation analysis was performed on 166 paired values obtained from 83 patients . A highly significant correlation was observed between erythema and the percentage of vimentin-positive cells, between scaling and the percentage of keratin 10 positive keratinocytes, and between induration and the number of basal keratinocytes in the S- and G2M phase, when all 166 biopsies were assessed . The correlation remained in the same range if the analysis was restricted to the 83 pretreatment biopsies . In contrast to the clinical scores, the flow cytometric analysis permitted a clear separation between the antiproliferative and anti-inflammatory or keratinization-enhancing effects of antipsoriatic treatment . The vitamin D3 analogues proved to exert a mainly antiproliferative effect . The combination of calcipotriol and a topical corticosteroid improved all cell biological markers substantially, and clobetasol monotherapy had a powerful effect on these markers . In conclusion, multiparameter flow cytometry has been shown to be a sensitive tool to evaluate the growth inhibiting, anti-inflammatory and keratinization-enhancing effects of antipsoriatic therapies.

Plant Mol Biol, 1997 Oct, 35(3), 261 - 9
Cloning of two plant cDNAs encoding a beta-type proteasome subunit and a transformer-2-like SR-related protein: early induction of the corresponding genes in tobacco cells treated with cryptogein; Petitot AS et al.; We report the successful combination of mRNA differential-display reverse-transcription PCR (DDRT-PCR) and 5'-rapid amplification of cDNA ends (5'-RACE) in order to isolate full-length cDNAs corresponding to genes activated in tobacco cells treated with cryptogein within 60 min . Cloning and sequencing of two cDNAs, called 'tcI 7' and 'tcI 14' (for tobacco cryptogein-induced), allowed the identification of open reading frames . Deduced amino-acid sequences of 'tcI 7' and 'tcI 14' showed significant homologies with a beta-type proteasome subunit and a transformer-2-like serine/arginine-rich (SR) ribonucleoprotein, respectively . The accumulation of mRNAs corresponding to 'tcI 7' started 30 min after the addition of cryptogein to tobacco cell suspensions and continued up to 180 min, whereas the accumulation of 'tcI 14' corresponding mRNAs was transitory between 30 and 60 min . These results indicated a transcriptional activation of the corresponding genes early after elicitation of tobacco cells by cryptogein . The biological significance of this activation remains to be elucidated.

Radiat Res, 1998 Feb, 149(2), 142 - 6
Indirect mechanisms contribute to biological effects produced by decay of DNA-incorporated iodine-125 in mammalian cells in vitro: clonogenic survival; Walicka MA et al.; We have examined whether mammalian cells in vitro can be protected against the lethal effects of irradiation by Auger electrons emitted from DNA-incorporated 125I . Chinese hamster V79 lung fibroblasts were cultivated in the presence of 5-{125I}iodo-2'-deoxyuridine (125IdU) for 18 h and resuspended in ice-cold medium in the presence or absence of 10% dimethyl sulfoxide (DMSO) . DNA-incorporated 125I activity was measured and the cells were plated for survival . A portion of the cell suspensions were also stored on ice to accumulate 125I decays for 6 to 48 h, after which the cells were plated to determine survival . Storage on ice up to 48 h without radioactivity reduced plating efficiency from 67 +/- 4% (SEM) to 20 +/- 1% . DMSO had a protective effect on colony formation, as the respective cloning efficiencies were 83 +/- 3% and 72 +/- 12% at 0 and 48 h . The survival curves for 125IdU-labeled cells are exponential with D0 = 36 +/- 2 decays per cell in the absence of DMSO and 195 +/- 20 decays per cell in the presence of DMSO . Thus the dose modification factor (DMF) at 37% survival for 10% DMSO is 5.4 +/- 0.6 for DNA-incorporated 125I . In reference experiments, a DMF of 2.5 +/- 0.8 was measured for cells irradiated with 137Cs gamma rays . These results indicate that the radiotoxicity of Auger electrons from 125I decay in mammalian cells is caused mainly by an indirect mechanism(s).

Int J Radiat Oncol Biol Phys, 1998 Jan 15, 40(2), 467 - 76
Partial volume rat lung irradiation: an evaluation of early DNA damage; Khan MA et al.; PURPOSE: 1 . To investigate early DNA damage induced in rat lung cells following single-dose, partial-volume irradiation (lung base and lung apex) . 2 . To determine the variation in DNA damage in different lung regions . 3 . To investigate the possible mechanisms associated with early DNA damage after lung irradiation . METHODS AND MATERIALS: The whole lung or the upper or lower half of the entire lung of Sprague-Dawley rats was exposed to 10 Gy 60Co gamma rays . The animals were sacrificed at various times up to 42 h after irradiation . A trypsin-digested lung cell suspension was prepared and cells that attached to slides in the initial 24-h period were then grown in the presence of culture medium with cytochalasin-B for a further 72 h . Radiation-induced DNA damage was quantified in the cells (primarily fibroblasts) from both irradiated and unirradiated lung regions by using a well-characterized micronucleus assay . RESULTS: When the lungs were removed at 16-18 h after whole-lung irradiation, about 0.85 micronuclei (MN) per binucleate cell (BNC) were observed in the lung cells of the irradiated animals, compared to 0.02 MN/BNC in the lung cells of the controls . When only the lung base was irradiated, the frequency of micronuclei was 0.85 MN/BNC compared to 0.43 MN/BNC observed in cells from the irradiated lung apex . Of particular interest was the finding that the unirradiated lung apex also showed a large frequency of micronuclei (0.43 MN/BNC) after the irradiation of the lung base, whereas the unirradiated lung base showed only a marginal (approximately 2-fold) increase relative to the spontaneous frequency following irradiation of the lung apex . The changes in the frequency of micronuclei varied with the time at which the lungs were removed from the rats for early times, but had stabilized by 18 h after irradiation . Normal (unirradiated) cells grown in filtered or unfiltered conditioned media obtained from irradiated cell cultures showed an insignificant marginal increase in the number of micronuclei relative to the spontaneous frequency . Lung cells obtained from the lung base or the lung apex of healthy controls and irradiated separately in vitro showed no regional differences in the induction of micronuclei . Cells from the lungs of rats injected with superoxide dismutase, within 1 h prior to irradiation of the lung base, and processed 16-18 h after irradiation showed a reduction in the number of MN in the shielded lung apex, indicating the possible involvement of oxygen radicals . CONCLUSIONS: These data indicate that cells in the lung base sustain more DNA damage than those in the lung apex when either region is irradiated; however, when the whole lung, is irradiated, the lung damage observed is similar in the two regions . Also, out-of-field effects are observed for the lung apex but not the lung base . Possible mechanisms include a clastogenic (chromosome damaging) factor produced in the plasma following irradiation and/or the production of oxygen radicals by activated lymphocytes/monocytes . The partial blocking of the DNA damage, observed in the unirradiated lung apex following irradiation of the lung base, by superoxide dismutase, suggests that oxygen radicals are involved in this out-of-field effect . These radicals are likely produced as a result of the induction of inflammatory cytokines, such as tumor necrosis factor (TNF) and interleukin-1 (IL-1) by the irradiation . The reason for the lack of an out-of-field effect in the lung base when the lung apex is irradiated is unknown, but may be due to the greater volume of lung irradiated in the lower lung field, because this is likely to affect the level of cytokines produced . Alternatively, it may reflect cytokines produced as a result of the partial liver irradiation that occurs with the lower lung field.

Acta Neuropathol (Berl), 1998 Jan, 95(1), 85 - 97
Time-dependent expression of donor- and host-specific major histocompatibility complex class I and II antigens in allogeneic dopamine-rich macro- and micrografts: comparison of two different grafting protocols; Brandis A et al.; Neural transplantation, as a therapeutic approach to Parkinson's disease, still requires allogeneic graft material and raises questions of immunosuppression and graft rejection . The present study investigated the time course of major histocompatibility complex (MHC) expression and astrocytic response in allogeneic dopaminergic grafts, comparing two different grafting protocols . Adult 6-hydroxydopamine-lesioned Lewis 1.W rats received intrastriatal cell suspension grafts from the ventral mesencephalon of DA rat fetuses, either as single 1-microliter macrograft via metal cannula or as four micrografts of 250 nl/deposit via a glass capillary . No immunosuppression was administered . Immunohistochemistry was performed at 1, 3, 6, and 12 weeks after grafting, using antibodies against donor- and host-specific MHC class I and II antigen, glial fibrillary acidic protein (GFAP) and tyrosine hydroxylase (TH) . Most animals showed good allograft survival up to 12 weeks after transplantation with no signs of rejection . Reinnervation of the lesioned striatum by TH-positive neurites was observed from 3-6 weeks on . Expression of donor-specific MHC class I was comparably low in both allogeneic grafting groups, while host MHC class I and II reaction as well as astrocytic response tended to be higher in the macrografted animals . Donor MHC class II was not observed at any time point . It is concluded that intraparenchymal allografts of fetal mesencephalic cell suspensions can survive well in the rat Parkinson model without immunosuppression for at least 12 weeks, and that the expression of moderate amounts of donor-specific MHC class I antigen does not suffice to initiate a rejection process . In addition, the microtransplantation approach may reduce the level of trauma and subsequent MHC and GFAP expression and may, thereby, minimize the risk of graft rejection.

Mol Cell Biochem, 1997 Dec, 177(1-2), 251 - 5
Culturing of human vascular endothelial cells strongly affects their endothelin-1 and prostacyclin production; Ranta V et al.; Cultured human umbilical vein endothelial cells (HUVECs) are a widely used model to study the regulation of endothelial production of vasoactive substances such as endothelin-1 (ET-1) and prostacyclin (PGI2) in human . As even short term culturing is known to affect the function of many cell types, we studied whether there are differences in the production of ET-1 and PGI2 between freshly isolated HUVECs and HUVECs cultured for two passages, and whether variation in cell density affects the production of ET-1 and PGI2 by these cells . At confluency, freshly isolated HUVECs produced only from one-tenth to one-fifth of ET-1, but 46-86 times more PGI2 (p < 0.001), when compared to respective productions by similar amounts of cultured HUVECs . When the cell density of freshly isolated HUVECs was lowered either by diluting the cell suspension or by plating the same amount of cells on different size wells, the production of ET-1 increased: lowering cell density to one-tenth led to 18 fold increase in ET-1 production (p < 0.001) . PGI2 production was not affected by cell density . Thus our data imply that the production of both ET-1 and PGI2 are differently regulated in freshly isolated and cultured HUVECs, and that cell density is an important determinant in the regulation of ET-1 production.

Biophys J, 1998 Jan, 74(1), 82 - 9
Thermal imaging of receptor-activated heat production in single cells; Zohar O et al.; Changes in enthalpy (i.e., heat content) occur during the diverse intracellular chemical and biophysical interactions that take place in the life cycle of biological cells . Such changes have previously been measured for cell suspensions or cell-free biochemical extracts by using microcalorimetry, thermocouples, or pyroelectric films, all of which afford minimal spatial or temporal resolution . Here we present a novel thermal imaging method that combines both diffraction-limited spatial (approximately 300 nm) and sampling-rate-limited time resolution, using the temperature-dependent phosphorescence intensity of the rare earth chelate Eu-TTA (europium (III) thenoyltrifluoro-acetonate) . With this thermosensitive dye, we imaged intracellular heat waves evoked in Chinese hamster ovary cells after activation of the metabotropic m1-muscarinic receptor . Fast application of acetylcholine onto the cells evoked a biphasic heat wave that was blocked by atropine, and after a brief delay was followed by a calcium wave . Atropine applied by itself produced a monophasic heat wave in the cells, suggesting that its interactions with the receptor activate some intracellular metabolic pathways . The thermal imaging technique introduced here should provide new insights into cellular functions by resolving the location, kinetics, and quantity of intracellular heat production.

Arch Biochem Biophys, 1998 Jan 15, 349(2), 205 - 15
Molecular characterization of tobacco squalene synthase and regulation in response to fungal elicitor; Devarenne TP et al.; The enzyme squalene synthase (SS) represents the first commitment of carbon from the general isoprenoid pathway toward sterol biosynthesis and is a potential point for regulation of sterol biosynthesis . The isolation and characterization of tobacco (Nicotiana tabacum) squalene synthase (TSS) cDNA and genomic DNA clones, as well as determination of the steady state level of TSS mRNA in response to elicitor treatment, were investigated . cDNA clones for TSS were isolated from poly (A)+ RNA using a reverse transcription/polymerase chain reaction (RT/PCR) method . A 1233-bp cDNA clone was generated that contained an open reading frame of 411 amino acids giving a predicted molecular mass of 46.9 kDa . Comparison of the TSS deduced amino acid sequence with currently described SS from different species showed the highest identify with Nicotiana benthamiana (97%), followed by Glycyrrhiza glabra (81%), Arabidopsis thaliana (74%), rat (40%), and yeast (37%) . Expression of a soluble form of the TSS enzyme with enzymatic activity in Escherichia coli was achieved by truncating 24 hydrophobic amino acids at the carboxy terminus . Characterization of genomic TSS (gTSS) revealed a gene of 7.086 kb with a complex organization of small exons and large introns not typical of plant genes . Southern blot hybridization indicated only two copies of the SS gene in the tobacco genome . Treatment of tobacco cell suspension cultures with a fungal elicitor dramatically reduced TSS enzymatic activity, lowering it to zero within 24 h . Analysis of TSS mRNA levels, by RNA blot hybridization and primer extension assays, in elicitor-treated cells indicated that the transcript level remained largely unchanged over this 24-h period . These results suggest that the suppression of TSS enzyme activity in elicitor-treated cells may result from a posttranscriptional modification of TSS.

Br J Haematol, 1997 Dec, 99(4), 777 - 83
Rheological analysis of the formation of rosettes by red blood cells parasitized by Plasmodium falciparum; Chu Y et al.; Red blood cells infected by mature malarial parasites of the species Plasmodium falciparum can adhere to non-parasitized red cells (rosetting) and also to endothelial cells (cytoadhesion) . To investigate how the circulation might influence rosetting, we studied formation of rosettes in cell suspensions sheared in a cone-and-plate viscometer, and the ability of flowing non-parasitized cells to bind to parasitized cells already adherent to a surface . After rosettes of strain R29 had been disrupted with fucoidan, they reformed slowly under stationary conditions but more rapidly in suspensions sheared at low stress (about 0.1-0.2 Pa) . Strain Malayan Camp gave a lower rosetting frequency which actually increased at low shear . Increasing shear stress was associated with reduction in rosette formation, although rosetting occurred at >1 Pa, suggesting that rosettes could form in the systemic circulation . Rosetting inhibited adhesion of flowing parasitized cells to immobilized platelets (which express the cytoadhesion receptor CD36), as evidenced by increased adhesion after disruption of rosettes . The de-rosetted adherent cells parasitized by R29 supported only a low level of rosetting when non-parasitized cells were flowed over them at a wall shear of 0.1 Pa, with little increase if the stress was decreased to 0.05 Pa . Rosettes formed in the circulation might obstruct microvessels and inhibit cytoadhesion if they reached venules . However, if cytoadhesion occurred before rosetting, then adherent cells should not efficiently form rosettes.

J Biol Chem, 1997 Dec 26, 272(52), 33319 - 26
Inhibition of the iron-induced ZmFer1 maize ferritin gene expression by antioxidants and serine/threonine phosphatase inhibitors; Savino G et al.; Two pathways have been implicated in the regulation of maize ferritin synthesis in response to iron . One of them involves the plant hormone abscisic acid (ABA) and controls the expression of ZmFer2 gene(s) . Another pathway, ABA-independent, has been characterized in a de-rooted maize plantlet system and involves an oxidative step . The ZmFer1 maize ferritin gene is not regulated by ABA, and it is shown in this paper that the corresponding mRNA accumulates in de-rooted maize plantlets and BMS (Black Mexican Sweet) maize cell suspension cultures in response to iron via the oxidative pathway described previously . To investigate ZmFer1 gene regulation further, the BMS cell system has been used to develop a transient expression assay using a ZmFer1-beta-glucuronidase fusion . Both iron induction and antioxidant inhibition of ZmFer1 gene expression were observed in this system . Using Northern blot analysis and transient expression experiments, it was shown that both okadaic acid and calyculin A, two serine/ threonine phosphatase inhibitors, specifically inhibit ZmFer1 gene expression . These data indicate that an okadaic acid-sensitive protein phosphatase activity is involved in the regulation of the ZmFer1 ferritin gene in maize cells, and this activity is required for iron-induced expression of this gene.

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