J Biol Chem, 1987 Oct 15, 262(29), 14056 - 62 Accumulation of regulatory oxysterols, 32-oxolanosterol and 32-hydroxylanosterol in mevalonate-treated cell cultures; Saucier SE et al.; In a previous publication (Saucier, S.E., A.A., Taylor, F.R., Spencer, T.A., Phirwa, S., and Gayen, A.K., J . Biol . Chem . (1985) 260, 14571-14579), we demonstrated that cultured Chinese hamster lung (Dede) cells contain 24(S),25-epoxycholesterol and 25-hydroxycholesterol in cellular concentrations within the range required to repress 3-hydroxy-3-methylglutaryl-CoA (HMG-CoA) reductase . In this paper, we show that the addition to the culture medium of a concentration of mevalonate high enough to repress the reductase by 90% resulted in the appearance of two new regulatory oxysterols . The two new sterols are believed to be 32-oxolanosterol and 32-hydroxylanosterol on the basis of high performance liquid chromatography (HPLC) retention times and mass spectrometric and nuclear magnetic resonance spectroscopic data and by NaBH4 reduction of the putative aldehyde to material which had the HPLC retention time of the putative alcohol . The cellular concentrations of 24(S),25-epoxycholesterol and 25-hydroxycholesterol were not significantly changed by the presence of mevalonate . However, there was approximately a 30% increase in total HMG-CoA reductase repressor units which can be ascribed to the 32-oxolanosterol and 32-hydroxylanosterol, where 1 unit equals the repressor activity of 1 ng of 25-hydroxycholesterol . These results support the idea that the level of HMG-CoA reductase activity in growing cell cultures is determined by intracellular oxysterol metabolites and that relatively small changes in their numbers or concentrations can alter the level of HMG-CoA reductase activity. J Cell Physiol, 1987 Oct, 133(1), 72 - 8 Erythropoiesis in murine long-term bone-marrow cell cultures: dependence on erythropoietin and endogenous production of an erythropoietic stimulating activity; Oddos T et al.; Erythropoiesis was obtained in murine long-term bone-marrow cell cultures (LTBMCs) in the presence of erythropoietin (Epo) when the medium was frequently renewed . The level of the erythropoietic differentiation was shown to be a function of the erythropoietin concentration . In response to Epo addition, an activity which stimulates CFU-E proliferation in semisolid cultures of fresh bone marrow cells was detected in the LTBMC supernatants . These results suggest that another factor, whose synthesis may be under Epo control, participates in the stimulation of erythropoiesis in vitro. In Vitro Cell Dev Biol, 1987 Oct, 23(10), 663 - 76 Elastin in a neonatal rat smooth muscle cell culture has greatly decreased susceptibility to proteolysis by human neutrophil elastase . An in vitro model of elastolytic injury; Stone PJ et al.; A neonatal rat aorta smooth muscle cell culture system with a unique elastin-rich extracellular matrix was used as a model substrate for elastases . To study the susceptibility to solubilization of insoluble elastin, cultures were incubated in the presence of human neutrophil elastase (HNE) or porcine pancreatic elastase (PPE) and in the absence of serum for periods up to 45 min . Both the incubation media and cell layers were then assessed for elastin and collagen markers, total protein, and lactate dehydrogenase (LDH) . Although HNE and PPE exhibited comparable activity against elastin purified from the cell layer, HNE exhibited a 6.7- to 25-fold reduction in its elastin solubilizing activity using intact cell layers as compared with the purified elastin, whereas PPE exhibited only a 1.5- to 2.5-fold reduction . This effect could not satisfactorily be explained as preferential inhibition of HNE activity in the culture system, because the amount of protein solubilized by HNE was 59% that of PPE . The mean elastin content of PPE-solubilized protein was 110% that of the elastin content of the corresponding cell layer; the value for HNE-solubilized protein was only 16% . Thus, the amount of elastin per microgram of solubilized protein for HNE was 15% that for PPE . Possible explanations for the greatly diminished elastolytic activity of HNE in the culture system include the preference of HNE for other substrates in the cell layer, the inability of HNE to penetrate sufficiently into the cell layer, and the presence of sulfated glycosaminoglycans in the vicinity of the elastin that act in an inhibitory fashion . Although there was extensive proteolytic damage to the extracellular matrix, LDH and DNA measurements indicated that little loss of cells or cell viability occurred . The observed differences in elastolytic activity of HNE and PPE in the culture system parallel the relative emphysema-inducing potency of the elastases in the hamster model of elastase-induced emphysema. Cell Tissue Res, 1987 Oct, 250(1), 21 - 7 Localization of acetylcholinesterase in dissociated cell cultures of the carotid body of the rat; Nurse CA; The localization of acetylcholinesterase (AChE) was investigated at the cellular and subcellular levels in dissociated cell cultures of the carotid body of the neonatal rat, prepared by the methods of Fishman and Schaffner (1984) . In the presence of iso-OMPA, which blocks nonspecific cholinesterase, staining was confined almost exclusively to glomus-cell clusters and occasional isolated cells . These clusters grow as discrete islands scattered throughout the culture and display typical catecholamine (CA) fluorescence as in vivo . AChE staining was abolished or reduced by the cholinesterase inhibitors eserine (30-100 microM), or (the poorly lipid soluble) echothiophate (8 microM) . Processing of the same culture sequentially for the demonstration of both AChE and CA revealed that glomus-cell clusters and individual glomus cells were consistently positive for both . In electron micrographs AChE reaction product was associated intracellularly with the nuclear envelope and cytoplasm of glomus cells (identified by their characteristic dense cored granules), as well as extracellularly with the boundaries of contiguous glomus cells . Significantly, reaction product occurred in some glomus cell profiles that had both dense-cored and clear (cholinergic-like) vesicles . These findings are discussed in the context of a possible dual (adrenergic/cholinergic) function status of glomus cells in the rat's carotid body. Diagn Microbiol Infect Dis, 1987 Oct, 8(2), 101 - 5 Comparison of nasopharyngeal washings and swab specimens for diagnosis of respiratory syncytial virus by EIA, FAT, and cell culture; Masters HB et al.; Respiratory secretions for viral diagnosis are often collected with nasopharyngeal (NP) swabs, although many laboratories recommend NP aspirates or washings . We compared results using NP washings and NP swabs in three diagnostic RSV tests, a rapid RSV EIA antigen test (Abbott Laboratories), an indirect fluorescent antibody test (FAT) with rabbit antiserum, and virus culture (HEp-2 cells) . Paired samples were collected from 121 children with suspected RSV bronchiolitis or pneumonia . A minitip swap was passed into the nasopharynx for 10 sec, rotated and withdrawn . The opposite nares was irrigated with approximately 1 ml of saline and aspirated using a syringe and plastic feeding tube . Fifty-one children (42%) grew RSV in culture, 49 from NP washings versus 27 from NP swabs (p less than 0.001) . Fifty-three (44%) were positive by FAT, 52 from NP washings versus 12 from NP swabs (p less than 0.001) . Fifty-eight children (48%) had positive RSV EIA tests, 57 from NP washings versus 35 from NP swabs (p less than 0.001) . Detection by EIA was more sensitive than culture regardless of the method of specimen collection . We conclude that NP washings are superior to NP swabs for RSV culture and rapid diagnosis by EIA or FAT. Anal Biochem, 1987 Oct, 166(1), 1 - 13 Use of extracellular matrix components for cell culture; Kleinman HK et al.; Extracellular matrix components when used as a substratum in vitro can greatly influence cell behavior . The response observed is dependent on the type of cell and matrix used . Cells in vitro usually respond best to the matrix components with which they are normally in contact in vivo . More differentiated phenotypes are observed and cells generally survive longer on such matrices . In some cases, the presence of such matrices allows cells to be cultured in the absence of serum and growth factors . As more investigators try the matrices and matrix components described, as well as new components and combinations of them, it is anticipated that improvement in the culture of many cells can be expected. J Clin Microbiol, 1987 Oct, 25(10), 1864 - 7 Detection of chlamydial inclusions in cell culture or biopsy tissue by alkaline phosphatase-anti-alkaline phosphatase staining; Mahony JB et al.; An immunological technique for detecting Chlamydia trachomatis and Chlamydia psittaci inclusions in infected McCoy cell cultures was developed by using a genus-specific monoclonal antibody to Chlamydia spp., rabbit anti-mouse immunoglobulin G bridging antibody, alkaline phosphatase-anti-alkaline phosphatase (APAAP) monoclonal antibody conjugate, and naphthol AS-phosphate/fast red substrate . Chlamydial inclusions stained red and were easily detected against a background of blue hematoxylin-stained nuclei . After 18 h, inclusions of C . trachomatis serovar L2 LGV434/Bu and C . psittaci strain 6BC were stained by APAAP but not by iodine or Giemsa . At 48 h inclusion counts were significantly higher in the APAAP cultures . Both the APAAP procedure and conventional staining detected 35 of 239 (15%) cultures 48 h after inoculation with urethral or endocervical specimens . However, at 24 h after inoculation 22 of 35 (63%) were positive by APAAP staining while negative by iodine . This immunostain also allowed identification of chlamydial inclusions in endometrial biopsies from patients with tubal factor infertility or pelvic inflammatory disease. Blood, 1987 Oct, 70(4), 1151 - 60 HL-T, a new cell line derived from HL-60 promyelocytic leukemia cell cultures expressing terminal transferase and secreting suppressor activity; Paietta E et al.; A cell line with immature blast cell morphology was isolated from HL-60 promyelocytic leukemia cell cultures and designated HL-T . This new cell type is biphenotypic, expressing terminal transferase (TdT) together with myelomonocytoid immunologic features . TdT enzymatic activity, undetectable in HL-60, was determined to be 140 to 180 units/10(8) HL-T cells by the dGTP-assay, approximately 20% of the activity found in lymphoblastoid cell lines . HL-T predominantly synthesize the known 58-kDa TdT-protein plus a minor 54/56-kDa doublet . The 58-kDa steady state form is nonglycosylated and is phosphorylated . Precursor antigens S3.13 and MY-10, absent on HL-60, are expressed by HL-T; however, the cells are negative for HLA-Dr . Southern blot analysis by hybridization with immunoglobulin heavy chain (JH) and T cell-receptor chain gene (T beta) probes shows JH to be in the germ-line configuration in both cell lines and the T beta gene to be in germ-line in HL-60 but to be rearranged in HL-T . Truncation of the gene encoding the granulocyte-macrophage-colony-stimulating factor (GM-CSF), as found in HL-60, is not observed in HL-T . HL-T are resistant to differentiation-induction by retinoic acid and 1,25-dihydroxyvitamin D3 . Cytogenetically HL-T share with HL-60 a deletion of the short arm of chromosome 9 at breakpoint p13, an aberration frequently found in patients with T cell leukemia . In addition, HL-T display t(8;9)(p11;p24) and trisomy 20 . Tetraploidy is observed in 80% of HL-T metaphases with aberrations identical to those in the diploid karyotype . Like HL-60, the new line shows some surface-antigenic-T cell characteristics . Despite an antigenic pattern most consistent with that of helper-inducer T cells (T4+, D44+/-, 4B4+, 2H4-, TQ1+/-), HL-T cells and their conditioned culture medium suppress antigen, mitogen, and mixed-leukocyte-culture-mediated lymphocyte proliferation. Eur J Cell Biol, 1987 Oct, 44(2), 187 - 94 Codistribution of galactosyl- and sialyltransferase: reorganization of trans Golgi apparatus elements in hepatocytes in intact liver and cell culture; Taatjes DJ et al.; The intracellular distribution of galactosyl- and sialyltransferase was investigated in rat hepatocytes of intact liver, primary monolayer cultures of freshly isolated hepatocytes, in a nontumorigenic hepatocyte cell line and in a hepatoma cell line . The two glycosyltransferases were detected by immunofluorescence using affinity-purified rabbit antibodies . Indirect double immunofluorescence showed that both terminal glycosyltransferases were identically codistributed in the same cell . This codistribution was always observed regardless of the cell type investigated, and in both stationary and migrating cells . The immunofluorescence pattern for both galactosyl- and sialyltransferase was found to be different in hepatocytes in vivo compared to hepatocytes grown in vitro . In hepatocytes of intact liver a spot-like cytoplasmic fluorescence was observed, whereas in cultured normal hepatocytes a perinuclear fluorescence from which an extensive tubular network radiated far into the cytoplasm existed . Cultured hepatoma cells also exhibited an extensive cytoplasmic fluorescence, which in contrast to the normal hepatocytes was rather diffuse . We conclude that (a) galactosyl- and sialyltransferase are codistributed in rat hepatocytes, and (b) a reorganization of (trans) Golgi apparatus elements containing both terminal glycosyltransferases occurs under conditions of in vitro growth and malignant transformation. Neuroscience, 1987 Oct, 23(1), 121 - 30 The expression and distribution of the microtubule-associated proteins tau and microtubule-associated protein 2 in hippocampal neurons in the rat in situ and in cell culture; Dotti CG et al.; Using a monoclonal antibody against the microtubule-associated protein tau we compared the distribution and the biochemical maturation of this protein in hippocampal pyramidal neurons in the rat in tau and in culture . In tissue sections from mature animals tau was localized heterogeneously within neurons . It was concentrated in axons; dendrites and somata showed little or no staining . In hippocampal cultures ranging from 12 h to 4 weeks in vitro tau was present in neurons but not in glial cells, as it is in situ . Within cultured neurons, however, tau was not compartmentalized but was present throughout the dendrites, axons and somata . Immunoblotting experiments showed that the biochemical maturation of tau that occurs in situ also failed to occur in culture . The young form of tau persisted, and the adult forms did not develop . In contrast the biochemical maturation and the compartmentalization of microtubule-associated protein 2 occurred normally in hippocampal cultures . These results show that the biochemical maturation and the intraneuronal compartmentalization of these two microtubule-associated proteins are independently controlled . Despite the non-restricted distribution of tau in hippocampal neurons in culture, and despite the presence of only the immature isoform which has a lessened stimulatory effect on microtubule polymerization, axons and dendrites appear to grow normally and to exhibit appropriate functional properties. Blood, 1987 Oct, 70(4), 932 - 42 Delineation of a novel pre-B cell component in plasma cell myeloma: immunochemical, immunophenotypic, genotypic, cytologic, cell culture, and kinetic features; Grogan TM et al.; A novel pre-B cell component in direct and cultured myeloma bone marrow material has been delineated by using immunochemistry and flow cytometry techniques . Our phenotypic studies suggest a novel hybrid expression of pre-B and plasma cell antigens with coexpression of cytoplasmic mu, common acute lymphoblastic leukemia antigen, terminal deoxynucleotidyl transferase, and plasma cell antigens (PCA-1 and PC-1) . This suggests that myeloma pre-B-like cells are aberrant malignant cells and not normal pre-B lymphocytic counterparts . With the advantage of a pure and stable source of these cells from M3 culture to allow molecular characterization, we performed one- and two-dimensional gel electrophoresis and Western blotting . We found that the cytoplasmic mu in myeloma pre-B-like cells has a molecular weight of 74,000 daltons and an isoelectric point of 6.3 and that it is strikingly homogeneous and discrete in size and charge compared with standard secretory mu, which suggests an aberrant, mutant, or monoclonal form of mu . Monoclonality was further evidenced by heavy- and light-chain immunoglobulin gene rearrangements demonstrated with JH and C kappa probes . We also established that this novel myeloma pre-B component is a major proliferative element as determined by double-labeling experiments with phenotype coupled to labeling/proliferative indexes . Our stimulatory studies indicate some capacity of these cells to mature on exposure to phorbol esters . These myeloma pre-B cells may represent the stem cell or self-renewal component in myeloma . Our establishment of these cells in long-term culture offers a considerable asset in studying the immature cells, which may be critical to the immortalization of myeloma. J Virol, 1987 Oct, 61(10), 3035 - 9 Hepatitis A virus cDNA and its RNA transcripts are infectious in cell culture; Cohen JI et al.; A full-length cDNA copy of an attenuated, cell culture-adapted hepatitis A virus (HAV HM-175/7 MK-5) genome was constructed in the PstI site of plasmid vector pBR322 . Transfection of monkey kidney cells with this plasmid failed to induce the production of hepatitis A virus (HAV) . The HAV cDNA was excised from pBR322 and inserted, without the oligo(dG) X oligo(dC) tails, into an RNA transcription vector to yield plasmid pHAV/7 . Transfection of monkey kidney cells with pHAV/7 DNA induced HAV infection . Transfection with RNA transcripts produced in vitro from pHAV/7 yielded about 10-fold more HAV than did transfection with pHAV/7 DNA . Marmosets inoculated with transfection-derived virus developed anti-HAV antibodies and had liver enzyme patterns that closely resembled the liver enzyme patterns seen in animals inoculated with virus from a comparable level of cell culture passage . Infectious RNA transcripts from HAV cDNA should be useful for studying the molecular basis of cell culture adaptation and attenuation as well as for studying specific viral functions. J Invest Dermatol, 1987 Oct, 89(4), 358 - 61 Induction of an immature T-cell phenotype in malignant helper T cells by cocultivation with epidermal cell cultures; Chu T et al.; The possible inductive effect of epidermal cells on T-cell maturation has been examined employing an in vitro cocultivation technique . Mononuclear cells from 6 patients with cutaneous T-cell lymphoma (CTCL) and from 12 healthy volunteers were studied . In the 6 CTCL patients, all showed an expansion of the helper T-cell subpopulation and in one patient with leukemic CTCL, there was almost complete replacement of peripheral blood mononuclear cells by malignant cells with a helper T-cell phenotype . Epidermal cells derived from normal human skin were cultured to confluent monolayers, and were cocultivated with the mononuclear cells from CTCL patients or normal controls for 48 h at a density of 10(6)/ml . Following cocultivation, the surface phenotype of the cells from the 12 healthy volunteers and 5 of the patients with CTCL showed no significant phenotypic change . In the patient with leukemic CTCL, however, the surface phenotype of the malignant T cells had changed, with the acquisition of the T6 antigen by the majority of the cells . Cells cocultivated in medium alone and with human fibroblast monolayers showed no change in surface phenotype . The malignant T cells from the leukemic CTCL patient failed to react in a mixed lymphocyte culture to lymphocytes from 2 different healthy donors, and showed no phenotypic change following culture with these lymphocytes, indicating that the phenotypic change seen was not due to allogeneic stimulation. Eur J Cancer Clin Oncol, 1987 Oct, 23(10), 1469 - 76 Comparison of antitumor activity of standard and investigational drugs at equivalent granulocyte-macrophage colony-forming cell inhibitory concentrations in the adhesive tumor cell culture system: an in vitro method of screening new drugs; Fan D et al.; We compared the in vitro growth inhibition of primary human tumor cells in the adhesive tumor cell culture system (ATCCS), exposed to the investigational agents caracemide, spirogermanium and taxol and to standard chemotherapy agents at equitoxic concentrations for granulocyte-macrophage colony-forming cells (GM-CFC) in vitro . Clinically active standard agents tested at up to GM-CFC 90% inhibitory concentrations (IC90) resulted in in vitro activity (greater than or equal to 50% tumor growth inhibition) in at least 30% of tumors tested . In vitro responses for taxol, caracemide and spirogermanium were 78%, 9% and 7%, respectively . This paper proposes a model that incorporates two hypotheses: (1) myelotoxic drugs which inhibit tumor growth at concentrations equal to or less than equitoxic GM-CFC ICs will demonstrate clinical activity; and (2) both myelotoxic and particular nonmyelotoxic drugs inactive in vitro at these doses will not be active clinically . If this drug screening concept is valid, taxol may be clinically more active than caracemide and spirogermanium. Am J Med Genet, 1987 Oct, 28(2), 521 - 6 Rapid cell culture procedure for tissue samples; Gray BA et al.; Cell culture is an integral part of genetic studies, including cytogenetic, metabolic, and DNA analyses . Standard culture procedures used today involve plating minced tissue explants in dishes and waiting 3 to 4 weeks for adequate growth . We describe a method that utilizes collagenase digestion of tissue biopsies, enabling cell harvest as soon as 3 days after culture initiation; this has been used successfully in our laboratory for cytogenetic and metabolic studies . Culture duration can be controlled by the cell plating density . Although collagenase digestion of tissues is commonly used in cytogenetics laboratories for tumor or chorionic villous dissociation, we have found that few labs have considered using this rapid and efficient procedure for routine tissue samples. Endocrinology, 1987 Oct, 121(4), 1390 - 9 Steroid synthesis-dependent, oxygen-mediated damage of mitochondrial and microsomal cytochrome P-450 enzymes in rat Leydig cell cultures; Georgiou M et al.; Treatment of primary cultures of rat Leydig cells with 1 mM 8-bromo-cAMP for 2 days at ambient oxygen tension (19%) caused a 59% decrease in mitochondrial cholesterol side-chain cleavage (P-450scc) activity . This decrease was completely prevented when the oxygen tension was reduced to 1% O2 or when steroid synthesis was inhibited by aminoglutethimide . When the endogenous concentration of pregnenolone was increased by inhibiting its further metabolism, P-450scc activity was reduced by 80% in unstimulated cultures and was completely eliminated in cAMP-treated cultures . These losses were prevented when cells were maintained at 1% O2 . The amount of immunoreactive P-450scc was also decreased by treatments that reduced P-450scc activity . Stimulation with cAMP also lowered microsomal C17-20 lyase activity by an oxygen-mediated, steroid synthesis-dependent mechanism . Treatment of cultures with testosterone caused a similar oxygen tension-sensitive decrease in C17-20 lyase activity . These results suggest that the enhanced loss of mitochondrial and microsomal cytochrome P-450 activities in cAMP-treated cultures is caused by the increased production of pregnenolone and testosterone, respectively, which generate reactive damaging species derived from reduced dioxygen . The increased catalytic turnover of these P-450 enzymes may also contribute to their damage . Although P-450 activities were preserved at 1% O2, the ability of cAMP-treated cells to synthesize testosterone in response to subsequent cAMP stimulation was still reduced . If, however, 25-hydroxycholesterol was supplied to these cells the decrease in testosterone-producing capacity was prevented, which demonstrates that the reduced steroidogenic capacity of cAMP-treated Leydig cells is caused, primarily, by the reduced availability of endogenous cholesterol. Rev Argent Microbiol, 1987 Oct-Dec, 19(4), 165 - 72 {Presence of mycoplasmas in cell cultures in Argentina}; Coronato S et al.; The presence of mycoplasmas in cell cultures used for virus propagation in Argentine laboratories, was studied . Samples were obtained from laboratories located in the city of Buenos Aires and provinces of Cordoba and Buenos Aires . The fluorescent stain Hoechst 33258, which specifically binds to DNA, was used for mycoplasma testing . Thirty three (65%) of 51 analyzed cell cultures (48 established lines and 3 primary cultures), were found contaminated despite that all laboratories were well equipped to work under sterile conditions . The high rate of infection observed let us conclude that the risk to work with mycoplasma contaminated cultures was not properly appreciated. J Immunol, 1987 Oct 1, 139(7), 2414 - 8 Biologic-response-modifier-induced indoleamine 2,3-dioxygenase activity in human peripheral blood mononuclear cell cultures; Carlin JM et al.; Degradation of tryptophan to kynurenine, catalyzed by indoleamine 2,3-dioxygenase (IDO), has been augmented in human epithelial cell lines treated with human interferon-gamma (HuIFN-gamma) . Several human biologic response modifiers, including HuIFN-gamma, HuIFN-beta, HuIFN-alpha, interleukin 2 (HuIL-2), and tumor necrosis factor alpha, have now been assessed for their ability to enhance tryptophan degradation in human peripheral blood mononuclear cell (PMC) cultures . PMC were isolated from normal donors, cultivated in RPMI 1640 medium containing {3H}tryptophan, and treated with individual biologic response modifiers . At various intervals, culture supernatants were removed, fractionated by reversed-phase high performance liquid chromatography, and radioactivity in resultant fractions was determined . Significantly increased amounts of tryptophan catabolites were observed after treatment with HuIFN-gamma, HuIFN-beta, HuIFN-alpha, and HuIL-2, but not human tumor necrosis factor alpha . Often, greater than 30% of available tryptophan was degraded by treated PMC cultures . Although antibodies to HuIFN-alpha, HuIFN-beta, and HuIFN-gamma specifically neutralized the induction of IDO activity in PMC by their respective HuIFN, only anti-HuIFN-gamma antibody also neutralized HuIL-2-induced IDO activity . Furthermore, T24 bladder carcinoma cells, in which IDO was induced by HuIFN-gamma but not by the other biologic response modifiers, were induced to degrade tryptophan by supernatants of HuIL-2-stimulated PMC cultures, but not by HuIFN-beta-stimulated PMC culture supernatants . Thus, whereas HuIL-2 indirectly induced IDO in PMC cultures by stimulating production of HuIFN-gamma, all cases of interferons appeared to induce IDO directly in PMC cultures. Brain Res, 1987 Sep 15, 420(2), 313 - 23 Morphological and biochemical differences expressed in separate dissociated cell cultures of dorsal and ventral halves of the mouse spinal cord; Guthrie PB et al.; The neuronal properties of separate dissociated cell cultures of dorsal and ventral halves of the embryonic mouse spinal cord (E 13.5) were investigated . Ventral-half cultures grew on a variety of substrates and in a variety of media; dorsal-half cultures required a non-neuronal feeder layer and supplemented medium for survival . The two types of cultures differed in their morphological and biochemical properties . Ventral-half neurons remained well separated on the culture plate, whereas dorsal-half neurons tended to aggregate . Lucifer yellow fills showed that ventral-half neurons were substantially larger and had more processes than dorsal-half neurons . Because of the large size and good separation of the neurons, ventral-half cultures provide an especially attractive system for electrophysiologic and morphologic studies . Ventral-half cultures were highly enriched for choline acetyltransferase (ChAT) activity and had more neurons that stained for intracellular acetylcholinesterase (AChE); dorsal-half cultures were enriched for glutamic acid decarboxylase (GAD) activity, and high-affinity gamma-aminobutyric acid (GABA) uptake . The clear differences between the two cultures indicate that many morphological and biochemical properties are already specified on embryonic day 13.5. Cancer Res, 1987 Sep 15, 47(18), 4924 - 31 Use of lymph in cell culture to model hormonal and nutritional constraints on tumor growth in vivo; Rubin H et al.; Transformed BALB/3T3 cells, which proliferate without restraint in culture, consistently produce rapidly growing sarcomas when 10(5) or more cells are inoculated into nude mice but produce sarcomas, of widely varying latent periods and growth rates, or negatives when 10(4) or fewer cells are inoculated . In an attempt to simulate the in vivo constraints on tumor development, these cells were cultured on plastic surfaces in concentrations of lymph up to 100% . Calf lymph was less effective in supporting multiplication than calf serum at all concentrations up to about 50% . The rate of cell multiplication progressively decreased with increasing concentrations of both fluids above 50% . Nonetheless, rapid multiplication could be achieved even in 100% serum or lymph by supplementing them with the high concentrations of nutrients used in the synthetic medium MCDB 402 . Supplementation of cystine and glutamine was essential for the growth-enhancing effects of the other nutrients . When the cells were suspended in agar, lymph was much less effective than serum in promoting colony formation even when both were supplemented with cystine and glutamine, or with all the constituents of the synthetic medium . We conclude that part of the low efficiency of tumor production and reduced growth rate of the transformed cells in mice resulted from a combination of (a) the paucity of growth factors in interstitial fluid, (b) the marked reduction in concentration of essential amino acids encountered by the cells in passing from culture into mice, and (c) the fact that cells multiplying in s.c . space do so without benefit of attachment to a solid substratum . Other factors, such as the growth inhibiting effects of direct contact with quiescent muscle and connective tissue cells, remain to be evaluated. Brain Res, 1987 Sep 8, 420(1), 1 - 10 Quinolinate is a weak excitant of cortical neurons in cell culture; Peters S et al.; Defined concentrations of quinolinate (QUIN) were administered to murine cortical neurons in culture impaled for intracellular recording . In physiological recording medium containing 1 mM Mg, concentrations of QUIN up to 2 mM had minimal effect on membrane potential and input resistance; only at higher concentrations did QUIN produce consistent depolarizations, which were accompanied by apparent increases in membrane resistance . In the absence of Mg, responses to QUIN were larger and were accompanied by decreases in membrane resistance, but QUIN was still a weak neuroexcitant, exhibiting an ED50 of greater than 1 mM . Phthalic, dipicolinic and nicotinic acids, structural analogues of QUIN, were even less potent neuroexcitants . The relationship between QUIN depolarization amplitude and membrane potential was linear in the absence of Mg, but in the presence of 1 mM Mg showed a non-linearity consistent with the voltage-dependent Mg block of N-methyl-D-aspartate (NMDA) receptor-mediated responses described by others . QUIN responses had a marked dependence on the presence of extracellular Na, and an extrapolated reversal potential of +12 mV, consistent with the large involvement of an Na influx . The responses were attenuated by the selective NMDA receptor antagonists, DL-2-amino-5-phosphonovalerate and ketamine, as well as by the broad spectrum antagonist kynurenate, but not by L-glutamate diethylester or gamma-D-glutamylaminomethyl sulfonate, compounds reported to block quisqualate or kainate receptors . The present study is consistent with the suggestion of other workers that QUIN neuroexcitation is mediated in large part by an Na influx through cation permeable NMDA-activated channels, but provides new quantitative data suggesting that the potency of QUIN as a cortical neuroexcitant is low . This low potency may argue against a role for QUIN as a traditional fast excitatory neurotransmitter. J Assoc Off Anal Chem, 1987 Sep-Oct, 70(5), 844 - 9 Determination of cytotoxic trichothecenes in corn by cell culture toxicity assay; Porcher JM et al.; The great sensitivity of some cell species to toxins has been adapted to a direct biological determination of trichothecene contamination of food and feeds . The murine spleen lymphocyte stimulated by PHA (Phaseolus vulgaris phytohaemagglutinin) appeared to be the most convenient cells because of their particular sensitivity to cytotoxic trichothecenes and the opportunity to translate this cytotoxicity to immunosuppressive hazard, one of the most important concerns for trichothecenes . In this paper, the use of cell cultures was adapted for a survey of corn . The toxins were extracted by aqueous methanol, and the extract was defatted with hexane and purified on a silica gel/Florisil column . This extract was then used for a gas chromatographic (GC) determination and the biological test . The growth of cells was measured by the incorporation of tritiated thymidine (3H Tdr), and the inhibition was expressed by the IC50: concentration of corn extract inhibiting by half the 3H Tdr incorporation . We have tested pure toxins, control corn, corn spiked with T-2 toxin, corn experimentally inoculated with toxigenic Fusarium strains, and naturally contaminated corn . A good correlation exists between IC50 and the T-2 toxin concentration as determined by GC analysis . The response is not affected by the presence of zearalenone or by small amounts of deoxynivalenol . A quantitative evaluation of cytotoxic trichothecenes in corn is valuable in the range of 100 ppb to 10 ppm, expressed as T-2 toxin equivalents . The result is obtained in 48 h. Vaccine, 1987 Sep, 5(3), 208 - 10 Pre-exposure studies with purified chick embryo cell culture rabies vaccine and human diploid cell vaccine: serological and clinical responses in man; Nicholson KG et al.; Clinical reactions and neutralizing antibody responses to six pre-exposure regimens of purified chick embryo cell culture rabies vaccine (PCECV) and human diploid cell strain rabies vaccine (HDCSV) were studied in 177 volunteers . Antibody kinetics, height of the response and persistence of antibody over two years were virtually identical after PCECV and HDCSV . An antibody response was detected in all subjects on day 14 when the highest titres were found after two intramuscular (i.m.) 1.0 ml doses of a schedule of immunization on days 0, 7 and 21 . In comparison, a schedule of immunization on days 0, 28 and 56 ultimately evoked the highest titres 21 days after the final injection, but antibody persisted equally well over two years with either schedule . Neutralizing antibody titres were lower after intradermal (i.d.) vaccination with 0.1 ml compared to 1.0 ml i.m . on days 0, 7 and 21, but when given on days 0, 28 and 56 the responses were comparable . Three subjects with a personal or family history of atopy developed urticarial lesions after PCECV . Both vaccines were otherwise well tolerated. J Dairy Sci, 1987 Sep, 70(9), 1967 - 80 A novel system for mammary epithelial cell culture; McGrath MF; A method is described for the isolation and density gradient enrichment of mammary epithelial fragments from pregnant, nonlactating bovine tissue . Immunocytochemical analysis prior to and following culture revealed specific staining with antibodies to keratin, indicating that these cells are epithelial in nature . Fragments enriched for epithelium could be stored in liquid nitrogen for extended periods prior to culture . When cast within a three-dimensional matrix of collagen gel, the mammary fragments grew as branching, duct-like structures and displayed a 4-fold increase in cell number during 10 to 12 d of culture. J Cell Physiol, 1987 Sep, 132(3), 453 - 62 Cytogenetic evaluation of human endothelial cell cultures; Nichols WW et al.; Cytogenetic evaluation of serially subcultivated human endothelial cells revealed significant differences between cultures derived from fetal umbilical cords and cultures derived from various vessel sites in adults . A rapid increase in the prevalence of polyploid cells, to levels of 100% in many cases, was detected in human umbilical vein endothelial cell cultures but not in endothelial cell cultures from adult vessels . Because the development of polyploidy has been viewed as one signpost of in vitro senescence, it may be that these in vitro observations of high levels of polyploidy are a reflection of the fact that umbilical tissue is at the end of its in vivo developmental lifespan when studied . Consistent karyotypic alterations also were observed in two clones from adult human abdominal aorta, even though these cultures exhibited low percentages of polyploid cells . Cultures of one clone exhibited a trisomy of chromosome 11, on which there are at least three onc gene loci, and a deletion of chromosome 13 through band q14 . A loss of band 13q14 is a prezygotic chromosomal lesion known to predispose to retinoblastoma . In the other clone, two cell populations were observed, and each displayed a chromosomal abnormality . A trisomy of the long arm of chromosome 2 was noted in one cell population via a marker chromosome involving 2 and 14 . The other cell population exhibited an abnormality of chromosome 2 . Neither of these karyotypic alterations was detected in the parent culture from which the clones were derived . The results reported in this study have both practical and theoretical implications . The high incidence of polyploidy in serially cultivated umbilical cultures as well as the occurrence of chromosomal changes in umbilical and aortic cultures testify to the need for cytogenetic monitoring of cell cultures even though they are derived from presumably normal tissue . Cytogenetic changes in the endothelium may be important in atherogenesis and other pathologic states . The conversion of diploid endothelial cells into polyploid endothelial cells may provide a convenient model cell system for studying mechanisms of the development of polyploidy in cells and their relationship to in vitro senescence. Cancer Res, 1987 Sep 1, 47(17), 4548 - 51 Effects of the aromatase inhibitor 7 alpha-(4'-amino)phenylthio-4-androstene-3,17-dione in MCF-7 human mammary carcinoma cell culture; Brueggemeier RW et al.; 7 alpha-Substituted 4-androstene-3,17-diones are effective inhibitors of aromatase in vitro . The microsomal P-450 enzyme complex has a greater affinity for several of these inhibitors than for the substrate androstenedione, with 7 alpha-(4'-amino)phenylthio-4-androstene-3,17-dione (7 alpha-APTA) being the most potent competitive inhibitor of the series . The effects of 7 alpha-APTA were examined in MCF-7 human mammary carcinoma cell culture . 7 alpha-APTA inhibited aromatase activity in the MCF-7 human mammary carcinoma cell line with an ED50 value of 25.07 nM (+/- 7.71) . The inhibitor did not bind to the estrogen receptor of the MCF-7 cells in vitro . In addition, 7 alpha-APTA did not stimulate growth of the MCF-7 cells when compared with controls . No induction of progesterone receptors was observed in MCF-7 cultures grown in the presence of inhibitor at concentrations ranging from 10 pM to 1 microM . Thus, the steroid 7 alpha-APTA is an effective inhibitor of aromatase in intact MCF-7 cells . Furthermore, it does not demonstrate any significant hormonal effects on the cells nor does this agent produce any general toxicological effects. J Virol, 1987 Sep, 61(9), 2885 - 90 Recombinant vaccinia viruses carrying the N gene of human respiratory syncytial virus: studies of gene expression in cell culture and immune response in mice; King AM et al.; The construction and characterization of vaccinia virus recombinants carrying the nucleocapsid (N) protein gene of human respiratory syncytial (RS) virus are described . Recombinant viruses were constructed that contained the N gene oriented either positively or negatively with respect to the 7.5-kilodalton vaccinia virus promoter . In addition, a positively oriented recombinant was constructed that lacked an out-of-frame AUG codon in the 5'-terminal noncoding region . In HEp-2 cells, both positive-orientation recombinants induced the synthesis of a protein which comigrated with N protein and was precipitated by antisera to RS virus . Sera from mice immunized with these recombinants specifically precipitated the RS virus N protein . Analysis of mRNA and protein expressed from the recombinant N genes showed that deletion of the upstream AUG codon markedly improved the efficiency of protein synthesis . Mice were vaccinated with the high-expressing recombinant and subsequently challenged with live RS virus . The results of these experiments demonstrated that the immune response to N protein afforded a significant degree of protection against RS virus disease. J Lab Clin Med, 1987 Sep, 110(3), 355 - 61 Modification of iron uptake and lipid peroxidation by hypoxia, ascorbic acid, and alpha-tocopherol in iron-loaded rat myocardial cell cultures; Hershko C et al.; The ability of ascorbic acid, alpha-tocopherol, and hypoxia to modify iron uptake, chelation, and toxicity as manifested by the generation of malonyldialdehyde (MDA) was studied in myocardial cell cultures obtained from newborn rats . Exposure to 20 micrograms/ml iron provided as 59Fe-ferric ammonium citrate in serum-free Ham F-10 culture medium resulted in the accumulation of 39% of the iron within 24 hours and a 10- to 12-fold increase in cellular MDA . Hypoxia (1% oxygen) resulted in a more than twofold increase in iron uptake but only minor changes in cellular MDA concentrations . Ascorbic acid and alpha-tocopherol (1 mg/ml) had opposing effects on iron uptake and MDA production . Ascorbic acid reduced 24-hour iron uptake by 73% (P less than 0.001) whereas alpha-tocopherol increased iron uptake by 19% (P less than 0.025) . In contrast, cellular MDA after iron loading increased by 86% with the addition of ascorbate, and was reduced by 75% with alpha-tocopherol (P less than 0.001) . The ratio of increase in cellular MDA relative to percent iron uptake (lipid peroxidation ratio) was 7.29 with iron loading plus ascorbate vs . 0.13 with iron loading plus alpha-tocopherol, a 56-fold difference between the two extremes . In vitro deferoxamine treatment for 3 hours resulted in a 53% reduction in the radioactive iron content of iron-loaded heart cells and a 40% reduction in MDA . Simultaneous deferoxamine and ascorbate or alpha-tocopherol treatment did not affect iron mobilization, but had a profound effect on MDA concentrations . Ascorbic acid prevented entirely the beneficial effect of deferoxamine on MDA concentrations in iron-loaded cells, whereas alpha-tocopherol potentiated the effect of deferoxamine.(ABSTRACT TRUNCATED AT 250 WORDS) Basic Res Cardiol, 1987 Sep-Oct, 82(5), 454 - 64 The development of beat-rate synchronization of rat myocyte pairs in cell culture; Jongsma HJ et al.; When two spontaneously beating neonatal rat heart cells in tissue culture were allowed to grow together they synchronized their originally independent beats to a common rhythm, as measured with an opto-electronic technique . Both single isolated cells and cell pairs exhibited a highly irregular beating pattern . Beating irregularity was strongly and positively correlated with mean interbeat interval . Synchronization of beating occurred in 50% of the pairs studied within one beating interval . In the remaining cell pairs, the first synchronized beat was followed by a 4-65 s period of partial synchronization . The time difference between contraction moments of two cells in a pair respective to each other (latency) changed upon synchronization from a random value to a fixed value . In a few cases the latency decreased during 20 to 30 s after the first synchronized beat before a steady-state value was reached . The mean interbeat interval (IBI) of the synchronized cell pairs was governed by the mean IBI of the originally faster beating cells . In 83% of the cases the mean IBI of the cell pairs was between that of the originally isolated beating cells . We conclude from the experiments described that physical coupling (i.e . gap junction formation) is virtually complete before beating synchronization occurs. J Virol, 1987 Sep, 61(9), 2896 - 901 Herpes simplex virus 1 protein kinase is encoded by open reading frame US3 which is not essential for virus growth in cell culture; Purves FC et al.; Earlier reports have described a novel protein kinase in cells infected with herpes simplex or pseudorabies viruses . These novel enzymes were characterized by their acceptance of protamine as a substrate and by their differential chromatographic behavior in anion-exchange chromatography . We report that this activity was not present in extracts of uninfected cells or of cells infected with a mutant constructed so as to contain a deletion in the US3 open reading frame mapping in the small component of herpes simplex virus 1 DNA . The activity was present in extracts of cells infected with wild-type virus and with a recombinant in which the US3 open reading frame had been rescued . Our results are consistent with the observation reported earlier that the coding sequences predict an amino acid motif common to protein kinases and lead to the conclusion that the US3 open reading frame encodes a virus-specific protein kinase that is not required for virus growth in cells in culture. In Vitro Cell Dev Biol, 1987 Sep, 23(9), 633 - 40 Activation of tyrosinase in mouse melanoma cell cultures; Fuller BB; Tyrosinase activity increased in Cloudman S-91 mouse melanoma cell homogenates incubated at 37 degrees C for a minimum of 8 h . Enzyme activity continued to increase for 48 h at which time the maximal level of activation was observed . Activation did not occur at 4 degrees C and did not occur in the cytosol fraction of the cell, suggesting that the response was localized to melanosomes . The activated enzyme was resistant to solubilization with the nonionic detergent, Triton X-100, and preparation of homogenates in this detergent did not inhibit the temperature-dependent activation of the melanosomal fraction of the cell . The activation process increased the Vmax of tyrosinase 10-fold and lowered the Km by a factor of 2 as determined by the tyrosine hydroxylase assay . The increase in tyrosinase activity was detectable by three assay methods: tyrosine hydroxylation, melanin synthesis, and by tyrosine decarboxylation . The formation of melanin, however, was found to be 1/20 that of either tyrosine hydroxylation or decarboxylation, a finding which suggests that the melanin pathway may be blocked at 5,6-dihydroxyindole . The "self-activation" response could not be mimicked by incubating cell homogenates with cyclic AMP-dependent protein kinase . Activated tyrosinase could be inhibited by the addition of fresh cell extracts, a finding which suggests that tyrosinase inhibitors may be present in these cells. Mol Endocrinol, 1987 Sep, 1(9), 595 - 603 Noncoordinate regulation of de novo synthesis of cytochrome P-450 cholesterol side-chain cleavage and cytochrome P-450 17 alpha-hydroxylase/C17-20 lyase in mouse Leydig cell cultures: relation to steroid production; Anakwe OO et al.; The role of cAMP in the regulation of the amount and synthesis of cytochrome P-450 cholesterol side-chain cleavage (P-450scc) and cytochrome P-450 17 alpha-hydroxylase/C17-20 lyase (P-450(17 alpha) was investigated in mouse Leydig cell cultures . In the absence of cAMP, the amount of immunoreactive P-450(17 alpha) decreased to less than 5% by day 4 and was undetectable between days 7 and 11 . In contrast, the amount of immunoreactive P-450scc remained relatively constant throughout the same period . Treatment of Leydig cell cultures for 4 days with 0.05 mM 8-bromo-cAMP initiated on day 7 increased the amount of P-450(17 alpha) with relatively little effect on the amount of P-450scc . The rate of de novo synthesis of each of the P-450 enzymes was studied by determining {35S}methionine incorporation into newly synthesized protein . In the absence of cAMP, de novo synthesis of P-450(17 alpha) ceased while the rate of de novo synthesis of P-450scc increased with time in culture between days 2 and 11 . Treatment with cAMP initiated on day 7 of culture caused a time-dependent increase in the rate of de novo synthesis of P-450(17 alpha) on days 9 and 11 equivalent to 40% and 60%, respectively, of that observed in freshly isolated Leydig cells . The rate of de novo synthesis of P-450scc was increased 2-fold relative to untreated cultures on days 9 and 11 . De novo synthesis of P-450(17 alpha) ceased when cAMP was removed on day 11 and restored when cAMP was added again on day 13 of culture.(ABSTRACT TRUNCATED AT 250 WORDS) J Virol Methods, 1987 Sep, 17(3-4), 311 - 8 Comparison of in situ DNA hybridization and immunological staining with conventional virus isolation for the detection of human cytomegalovirus infection in cell cultures; Janssen HP et al.; The sensitivity of immunochemical staining and in situ DNA hybridization for the detection of human cytomegalovirus (HCMV) was compared with that of virus isolation . Human diploid fibroblasts were infected with serial, 10-fold dilutions of HCMV strain AD169 and examined at various intervals between 1 and 42 days after inoculation, using the three methods being compared . HCMV-DNA was detected by in situ hybridization using a biotin-labeled HCMV probe and CMV early antigen (EA) by immunochemistry using a specific monoclonal antibody . During the first 2 days after inoculation detection of EA appeared to be the most sensitive method . After the fifth day the sensitivity of the immunochemical and in situ hybridization methods was similar and equalled that of conventional virus isolation . However, 2-5 times more HCMV-DNA than HCMV-EA positive cells were detected . Our results indicate that both detection of HCMV-EA by immunological staining and HCMV-DNA by in situ hybridization are suitable methods for rapid and sensitive detection of HCMV infections. J Steroid Biochem, 1987 Sep, 28(3), 267 - 72 Effects of 2-hydroxyoestradiol, oestradiol and testosterone on FSH-induction of catecholamine- and gonadotrophin-responsive progesterone biosynthesis in rat granulosa cell cultures; Hudson KE et al.; Catecholamine- and gonadotrophin-responsive progesterone biosynthesis increases during FSH-induced granulosa cell maturation . In this study, we compared in vitro effects of interrelated classes of follicular steroid on both endpoints: an androgen (testosterone), an oestrogen (oestradiol) and an oestradiol metabolite (2-hydroxyoestradiol) . Granulosa cells from diethylstilboestrol-pretreated, immature rat ovaries were cultured for 48 h (pretreatment) in serum-free medium containing human FSH with or without steroid . The cell monolayers were then washed and reincubated for a further 24 h (test-treatment) in fresh medium with and without a catecholamine (isoproterenol or norepinephrine) or a gonadotrophin (FSH or hCG); test-treatment culture medium was collected and assayed for progesterone content . At concentrations up to 10(-6) M, each steroid enhanced dose-dependently basal and catecholamine-responsive progesterone production with a ranked potency-order of testosterone greater than 2-hydroxyoestradiol much greater than oestradiol . All three compounds also enhanced FSH and hCG responsiveness but this endpoint was affected most markedly by oestradiol . Pretreatment in the presence of specific antiandrogen (hydroxyflutamide; SCH16423) blocked the stimulatory effects of testosterone and 2-hydroxyoestradiol but did not inhibit stimulation by oestradiol, highlighting similarities between the actions of testosterone and the catechol oestrogen distinct from that of oestradiol . Test-treatment in the presence of beta-adrenergic antagonist (propranolol) blocked the stimulatory action of isoproterenol and reduced the response to FSH, suggesting the involvement of beta-adrenergic receptors in FSH as well as catecholamine action on steroidogenesis . We conclude that testosterone and catechol oestradiol have major effects on FSH-induced development of catecholamine responsive steroidogenesis whereas oestradiol mainly affects gonadotrophin responsiveness in this granulosa cell culture system . All three steroid types could interact with gonadotrophins and locally produced catecholamines to influence granulosa cell function in vivo. J Gen Virol, 1987 Sep, 68 ( Pt 9), 2339 - 46 The ultrastructure of cell cultures infected with border disease and bovine virus diarrhoea viruses; Gray EW et al.; The morphology of border disease virus and bovine virus diarrhoea virus in infected bovine embryonic testis cells was examined by electron microscopy . Particles which appeared to be mature virions of both viruses were similar, being roughly circular and approximately 46 nm in diameter with a 20 to 25 nm core . Virus replication took place totally within the cytoplasm in association with structures formed from modified endoplasmic reticulum. Endocrinology, 1987 Sep, 121(3), 965 - 71 Calcium-dependent control of corticotropin release in rat anterior pituitary cell cultures; Abou-Samra AB et al.; The role of calcium in the stimulation of ACTH secretion by CRF and other regulators was studied in rat anterior pituitary cells . Incubation of cultured pituitary cells in normal calcium with CRF, vasopressin, angiotensin II, or norepinephrine increased the rate of ACTH release for up to 45 min and then became constant for up to 3 h . In the absence of extracellular calcium, the initial rate of stimulated secretion was unaffected, but after 45 min the secretion rate decreased by 40% for CRF and to a greater extent for the other stimuli . Addition of calcium after 90 min in calcium-free medium restored the CRF-stimulated ACTH release rate to the control value . The absence of extracellular calcium had no effect on CRF-stimulated cAMP accumulation, but intracellular calcium depletion by preincubation of the cells with EGTA completely inhibited CRF-stimulated cAMP production and ACTH release . The voltage-dependent calcium channel antagonist nitrendipine and the calcium channel agonist BK 8644 had little effect on the CRF-stimulated ACTH release rate, while they, respectively, inhibited and enhanced the stimulation by vasopressin and high potassium . In calcium-depleted cells incubated with the calcium ionophore A23187, CRF stimulation of cAMP production and ACTH release were dependent upon extracellular calcium concentrations from 0.1-100 microM . These findings have defined two phases in the stimulation of ACTH release by CRF and cAMP-independent stimuli in cultured pituitary cells: an early phase with a rapid increase in the ACTH release rate which is independent of extracellular calcium, and a late phase of constant secretion rate, with partial extracellular calcium dependence for the stimulation by CRF and complete calcium dependence for the stimulation by non-cAMP-mediated stimuli. Cancer Res, 1987 Aug 15, 47(16), 4329 - 34 Growth-stimulating effect of pharmacological doses of glucocorticoid on androgen-responsive Shionogi carcinoma 115 in vivo in mice and in cell culture; Omukai Y et al.; It has been generally accepted for 20 years that the growth of Shionogi carcinoma 115 (SC115) is stimulated only by androgen . However, we recently found that the growth of SC115 cells is also stimulated by pharmacological doses of estrogen in vivo but not in cell culture . In the present study, the growth-stimulatory effect of glucocorticoid on SC115 cells was examined . In castrated mice, daily injections of high doses of dexamethasone (100 micrograms/mouse) markedly stimulated the tumor growth, and the growth approached that found in normal males . However, daily injections of physiological doses of dexamethasone (4 micrograms/mouse) or high doses of epitestosterone, progesterone, or cholesterol (200-5000 micrograms/mouse) did not enhance the tumor growth in castrated mice . The androgen dependency, growth speed, steroid receptors, and histological type of the tumors grown by pharmacological doses of glucocorticoid were not significantly different from those of the original SC115 tumors grown by androgen . In a serum-free medium {Ham's F-12:Eagle's minimum essential medium (1:1, v/v) containing 0.1% bovine serum albumin}, the proliferation of SC-3 cells (a cloned cell line from SC115 cells) was markedly (by up to 25-fold) stimulated by 10(-10)-10(-8) M testosterone, whereas the proliferation was only slightly but significantly (by up to 3.3-fold) stimulated by 10(-8)-10(-5) M dexamethasone . The present findings demonstrate that the growth of SC115 cells in vivo and in cell culture is significantly stimulated by physiological doses of androgen or pharmacological doses of glucocorticoid. Anal Biochem, 1987 Aug 15, 165(1), 56 - 8 Automatic nitrous oxide synchronization of mitotic human cell cultures; Downes CS et al.; Large numbers of human cells can be reversibly arrested in mitosis by high-pressure nitrous oxide . The optimum schedule for this arrest requires that the high-pressure block be started around midnight to provide a mitotic population that can be released the next morning to progress in synchrony through the next cell cycle . We describe a simple and safe device which can be set up at the end of the day and automatically exposes the cells to high-pressure nitrous oxide overnight. Cancer Res, 1987 Aug 15, 47(16), 4402 - 6 Interspecies differences in the major DNA adducts formed from benzo(a)pyrene but not 7,12-dimethylbenz(a)anthracene in rat and human mammary cell cultures; Moore CJ et al.; Mammary epithelial cells from rats and humans show both quantitative and qualitative species- and carcinogen-specific differences in their abilities to activate benzo(a)pyrene (B(a)P) and 7,12-dimethylbenz(a)anthracene (DMBA) . Previous studies of the DNA binding of these compounds in mammary epithelial cells demonstrated that rat cells bound relatively more DMBA than B(a)P to DNA under identical treatment conditions, while the opposite pattern was exhibited by human mammary epithelial cells . The specific DNA adducts formed in these cells after 24-h incubations with {3H}DMBA and {3H}B(a)P were analyzed to determine if there were qualitative as well as quantitative differences in the amounts of individual adducts . Similar proportions of specific DMBA-DNA adducts were found in both rat and human cells, although the total amount of adducts formed was significantly higher in the rat cells . In contrast, an essentially qualitative species-specific difference was observed in the major B(a)P-DNA adduct present in the rat and human cells . The major B(a)P adduct formed in the human mammary epithelial cells was identified as the (+)-anti-B(a)P-7,8-dihydrodiol-9, 10-epoxide(BPDE)-deoxyguanosine adduct . However, this adduct was formed at very low levels in the rat mammary epithelial cells . The rat cells contained a large proportion of syn-BPDE adducts, and other unidentified B(a)P-DNA adducts . The high level of the (+)-anti-BPDE-deoxyguanosine adduct in the human but not the rat mammary cells is consistent with the potential role of (+)-anti-BPDE in the high mutagenic activity of B(a)P in the cell-mediated mutagenesis assays using the human mammary cells as activators, and the low mutagenic activity of B(a)P when rat cells were used as activators . The quantitative differences in the activation of DMBA by cells from these two species are also consistent with the cell-mediated mutagenic activities of DMBA using these cells as activators . These results suggest that the higher carcinogenic activity of DMBA compared to B(a)P in the rat mammary gland may not be indicative of the relative carcinogenic potencies of these compounds for human mammary cells. J Biol Chem, 1987 Aug 5, 262(22), 10574 - 81 Characterization of the core protein of the large chondroitin sulfate proteoglycan synthesized by chondrocytes in chick limb bud cell cultures; Haynesworth SE et al.; The core protein of high buoyant density proteoglycans synthesized by chondrocytes in stage 24 chick limb bud mesenchymal cell cultures was cleaved with cyanogen bromide to produce 17 resolvable peptides on sodium dodecyl sulfate-polyacrylamide slab gels . Of these peptides, 10 appear to originate from the chondroitin sulfate-rich region, 2 appear to be derived from the keratan sulfate-rich region, and 5 seem to be derived from the hyaluronic acid-binding region . The peptides from the chondroitin sulfate-rich region are almost all large (200 to 64 kDa) . In contrast, the peptides from the keratan sulfate-rich and hyaluronic acid-binding regions are relatively small (47 to 12 kDa) . One peptide from the hyaluronic acid-binding region appears to contain mannose-rich N-linked oligosaccharides as inferred from its observed binding by concanavalin A . A different hyaluronic acid-binding region peptide and one of the keratan sulfate-rich peptides were shown to contain disulfide bonds and therefore may be involved in contributing to the tertiary structure of the hyaluronic acid-binding region . Based on these observations, a map of the chick chondrocyte proteoglycan core protein has been constructed. Antimicrob Agents Chemother, 1987 Aug, 31(8), 1238 - 42 Antiherpesvirus activity of 9-(4-hydroxy-3-hydroxy-methylbut-1-yl)guanine (BRL 39123) in cell culture; Boyd MR et al.; The activity of 9-(4-hydroxy-3-hydroxymethylbut-1-yl)guanine (BRL 39123) against several herpesviruses was compared with that of acyclovir (ACV) . In plaque reduction tests with clinical isolates of herpes simplex virus type 1 (HSV-1), herpes simplex virus type 2 (HSV-2), and varicella-zoster virus, mean 50% inhibitory concentrations (IC50S) (n = number tested) for BRL 39123 were 0.4 (n = 17), 1.5 (n = 13), and 3.1 (n = 5) micrograms/ml, respectively . Corresponding IC50S for ACV were 0.2, 0.6, and 3.8 micrograms/ml . Cytomegalovirus was relatively resistant to BRL 39123 (IC50, 51 micrograms/ml), but equid herpesvirus 1, bovid herpesvirus 2, and felid herpesvirus 1 were susceptible (IC50S, 1.6, 1.2, and 0.9 micrograms/ml, respectively) . BRL 39123 was inactive against an HSV-1 strain which does not express thymidine kinase activity, but a DNA polymerase mutant selected for resistance to ACV was sensitive to BRL 39123 (IC50, 1.5 micrograms/ml) . In contrast to the results from plaque reduction tests, BRL 39123 was more active than ACV against HSV-1 and of equal activity against HSV-2 in virus yield reduction assays in MRC-5 cells . After treatment of HSV-infected cultures for short periods, BRL 39123 was considerably more effective than ACV at reducing virus replication, and furthermore, after removal of extracellular BRL 39123, virus replication remained depressed for long periods, whereas such persistent activity was not observed with ACV . Neither compound significantly affected MRC-5 cell replication at 100 micrograms/ml, but at 300 micrograms/ml BRL 39123 was more inhibitory than ACV. Cancer Res, 1987 Aug 1, 47(15), 4032 - 7 Stereochemical specificity in the metabolic activation of benzo(c)phenanthrene to metabolites that covalently bind to DNA in rodent embryo cell cultures; Pruess-Schwartz D et al.; Benzo(c)phenanthrene (BcPh) has only weak carcinogenic activity in rodent bioassays . However, bay-region diol-epoxides of BcPh have the highest tumor-initiating activities of all hydrocarbon diol-epoxides tested to date . To determine whether BcPh is metabolically activated to bay-region diol-epoxides that bind to DNA in cells, Sencar mouse, Syrian hamster, and Wistar rat embryo cell cultures were exposed to {5-3H}-BcPh, and the BcPh-deoxyribonucleoside adducts formed were analyzed by immobilized boronate chromatography and reverse-phase high-performance liquid chromatography . Greater than 74% of the BcPh-deoxyribonucleoside adducts formed in all 3 species resulted from reaction of (4R,3S)-dihydroxy-(2S,1R)-epoxy-1,2,3,4-tetrahydro-BcPh {(-)-BcPhDE-2} with DNA to yield deoxyadenosine and deoxyguanosine adducts in a ratio of 3:1 . A much smaller proportion of BcPh-deoxyribonucleoside adducts were formed by reaction of (4S,3R)-dihydroxy-(2S,1R)-epoxy-1,2,3,4-tetrahydro-BcPh {(+)-BcPhDE-1} with deoxyadenosine . No BcPh-deoxyribonucleoside adducts arising from either (+)-BcPhDE-2 or (-)-BcPhDE-1 were detected . The absence of adducts from these isomers of BcPhDE was not due to failure of these isomers to react with DNA in cells, for reaction of (+/-)-BcPhDE-1 or (+/-)-BcPhDE-2 with DNA in solution or in hamster embryo cell cultures resulted in the formation of DNA adducts from both the (+)- and (-)-enantiomers of each BcPhDE . These results indicate that both the (+)- and (-)-3,4-dihydrodiols of BcPh are formed and that their metabolic activation to diol-epoxides occurs with high stereospecificity in cells from all 3 species of rodents . The finding that the major DNA-binding metabolite is (-)-BcPhDE-2, the diol-epoxide with the (R,S)-diol-(S,R)-epoxide absolute configuration that is associated with high carcinogenic activity of diol-epoxides of other hydrocarbons, demonstrates that these cells are able to activate BcPh to an ultimate carcinogenic metabolite . The fact that a high proportion of the BcPh-DNA adducts are deoxyadenosine adducts suggests that BcPh has DNA-binding properties similar to those of the potent carcinogen 7,12-dimethylbenz(a)anthracene . The stereospecificity observed in the metabolic activation of BcPh to DNA-binding metabolites and the reaction of these metabolites with both deoxyguanosine and deoxyadenosine suggest that studies of the interactions of BcPh with DNA in vivo may be a valuable approach for establishing the role of specific activation pathways and DNA adducts in tumor induction. J Virol, 1987 Aug, 61(8), 2627 - 30 Characterization of capsid and noncapsid proteins of B19 parvovirus propagated in human erythroid bone marrow cell cultures; Ozawa K et al.; The major capsid and noncapsid proteins of the pathogenic parvovirus B19, propagated in vitro, were detected by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, immunoprecipitation, and immunoblot of the erythroid fraction of infected human bone marrow cell cultures . There were two capsid proteins of 58 kilodaltons (kDa; the major species) and 84 kDa (the minor species) . Newly synthesized capsid viral proteins were present in the supernatants of infected cultures . The major noncapsid protein of 77 kDa was localized to the nucleus. Tsitologiia, 1987 Aug, 29(8), 911 - 6 {Comparison of the effect of the epidermal growth factor (EGF) and its cyanogen bromide derivative on EGF receptor phosphorylation and uridine uptake in a cell culture}; Nesterov AM et al.; In contrast to the intact EGF, a cyanogen bromide derivative of EGF (EGF-CNBr) does not induce an increase in uridine phosphorylation rate in 3T3 cells, the ability of the EGF-CNBr to stimulate autophosphorylation of the EGF-receptor in A-431 cells being reserved . EGF and EGF-CNBr were used in concentrations promoting their equivalent binding with EGF receptor in both the series of experiments, which was necessary because of a decreased affinity to binding EGF-CNBr . Thus, the EGF-induced receptor autophosphorylation was not enough for uridine kinase activation . The differences between EGF and EGF-CNBr cellular processing made it possible to discuss potential ways of uridine-kinase activity regulation during the early period of stimulation of quiescent cell cultures. Diagn Microbiol Infect Dis, 1987 Aug, 7(4), 265 - 8 Rapid detection of influenza virus in cell culture by indirect immunoperoxidase staining with type-specific monoclonal antibodies; Swenson PD et al.; Seventy respiratory specimens culture-positive for influenza virus were stored at -70 degrees C and retested in duplicate by virus isolation and by indirect immunoperoxidase (IPA) staining of cell monolayers 24 hr postinoculation using influenza type-specific monoclonal antibodies . The IPA stain was positive for 24 of 28 specimens from which influenza virus was reisolated and for eight specimens from which influenza virus was not reisolated. Invest Radiol, 1987 Aug, 22(8), 678 - 84 Cytostatic effects of radiographic contrast media in synchronized cell cultures; Nordby A et al.; The cytostatic effects of conventional high osmolal ionic contrast media (meglumine-calcium metrizoate and Na-metrizoate) and new low osmolal nonionic contrast media (iohexol and iopamidol) in synchronized cell cultures were tested . The cell-cycle prolongation was most pronounced when the contrast media were added in the G1 phase, but there was also a marked effect when the contrast media were added in the S phase or late in the G2 phase . The cytostatic effect even persisted into the first cell cycle following the termination of the exposure . All four contrast media exerted effects stronger than that of equiosmolal saline . Iohexol and iopamidol produced a more severe effect than meglumine-calcium metrizoate and Nametrizoate at equal osmolality . Thus, the cytostatic effect of contrast media cannot be explained only by hypertonicity; the contrast media must have an additional specific cytostatic effect . When the cytostatic effect was related to iodine concentration, the new low osmolal nonionic contrast media influenced the cell cycle less than the conventional high osmolal ionic contrast media. Am J Obstet Gynecol, 1987 Aug, 157(2), 394 - 9 Detection of Chlamydia trachomatis cervical infection: a comparison of Papanicolaou and immunofluorescent staining with cell culture; Quinn TC et al.; We compared tissue culture with Papanicolaou-stained cervical smears, endocervical cytologic smears stained with immunofluorescent monoclonal antibody, and a direct immunofluorescent stain of cervical specimens (MicroTrak) for detection of Chlamydia trachomatis in cervical specimens . Fifty-one (21%) of 245 women had positive cultures for C . trachomatis, 14 (27%) of whom had clinical evidence of cervicitis . With the criteria of intracytoplasmic coccoid inclusion bodies within metaplastic cells, 45 (34%) of 130 Papanicolaou smears were read as suggestive for C . trachomatis . Seventeen of the 45 positive Papanicolaou smears were positive on culture and 28 were negative (sensitivity 54%, specificity 71%) . In contrast, 48 of 51 women with positive cultures and one woman with a negative culture had positive immunofluorescent-stained cytologic smears (sensitivity 94%, specificity 99%, with positive predictive value of 98%) . Similarly, 47 of 51 women with positive cultures also had positive results with MicroTrak direct immunofluorescent stain, with only one positive specimen in 196 women with negative cultures (sensitivity 92%, specificity 99%, with positive predictive value of 98%) . This study demonstrates that immunofluorescent staining of cervical specimens or of cytologic smears is a more sensitive and specific method than routine Papanicolaou smear for detection of chlamydia infection in a high-prevalence population. Endocrinol Jpn, 1987 Aug, 34(4), 615 - 20 Increases in phenolic and nonphenolic ring deiodinase activity due to serum in monolayer liver cell cultures; Sorimachi K et al.; When monkey hepatocarcinoma cells (NCLP-6E) were treated with 10% of various serum preparations for 24 h at 37 degrees C, nonphenolic ring deiodinase activity increased 2.0- to 2.3-fold . Phenolic ring deiodinase activity also increased 0.9- to 2.1-fold . Dialysis of the sera enhanced the effect on deiodinase activities in some preparations, but reduced activity in other serum preparations . Similarly, a 1.3- to 3.1-fold increase in phenolic ring deiodinase activity was observed in rat hepatoma cells (R-Y121B) . In this case, dialysis usually reduced the effect of the sera . It is concluded that both large molecule(s) (undialyzable) and small molecule(s) (dialyzable) in serum contribute to the regulation of phenolic and nonphenolic ring deiodinase activity in NCLP-6E and R-Y121B cells. Scand J Immunol, 1987 Aug, 26(2), 119 - 27 Autologous red blood cells potentiate antibody synthesis by unfractionated human mononuclear cell cultures; Rugeles MT et al.; We have tried to determine the most favourable conditions for the in vitro induction of specific antibody (Ab) responses to tetanus toxoid (TT) and keyhole limpet haemocyanin (KLH) . Human peripheral blood mononuclear cells (PBMNC) were obtained from normal volunteers and stimulated with PWM, TT, KLH, and mixtures of PWM and antigens in the presence or absence of autologous red blood cells (RBC) (1:50 ratio of PBMNC/RBC) . The cultures were harvested on day 11; immunoglobulins were determined immunonephelometrically and Ab levels by ELISA with human antibodies used for calibration . While anti-TT responses were easy to induce with PBMNC from recently boosted individuals, the production of anti-TT from PBMNC obtained from non-recently boosted individuals was only possible when PBMNC were stimulated with TT and PWM in the presence of autologous RBC . Similarly, anti-KLH responses were easier to induce with PBMNC from an immune donor; maximal response was observed after stimulation with PWM + KLH in the presence of autologous RBC . Stimulation of primary anti-KLH responses with PBMNC from non-immune donors was only successful when the cells were stimulated with KLH + PWM in the presence of autologous RBC . The potentiation of human B-cell responses with autologous RBC can be abrogated by pretreatment of PBMNC with anti-CD2 antibodies and is associated with increased expression of IL-2 receptors and increased production of gamma interferon (IFN-gamma) . However, addition of IFN-gamma in different doses and at different times to PWM-stimulated PBMNC cultures was not as effective as addition of RBC in enhancing the production of immunoglobulin and antibody. J Clin Microbiol, 1987 Aug, 25(8), 1401 - 5 Impact of cell culture sensitivity and virus concentration on rapid detection of herpes simplex virus by cytopathic effects and immunoperoxidase staining; Zhao LS et al.; Tremendous interest has been generated in the commercial kits now available that incorporate herpes simplex virus isolation in cell culture with immunoperoxidase staining for viral antigen detection . Most studies comparing commercial kits with conventional cell culture techniques have found the kits to be less sensitive . However, different cell cultures were used for the two methods . In this study, mink lung, rabbit kidney, MRC-5, and Vero cells were compared for reisolation of herpes simplex virus from clinical specimens in which viral infectivity titers were concurrently determined . When specimens contained high titers of infectious virus, the cell system used made little difference and all specimens were detected by immunoperoxidase staining at 48 h postinoculation . However, when specimens contained low concentrations of virus, the differences in sensitivity between cell systems became apparent in rapidity of detection and overall isolation rate . Mink lung and rabbit kidney cells were both more sensitive than MRC-5 cells; Vero cells were significantly less sensitive than the other cells tested . The application of immunoperoxidase staining shortened the time to virus detection and lessened, but did not eliminate, the differences between the cell systems . Cytopathic effects alone in the most sensitive cell system equaled or exceeded immunoperoxidase staining applied in less-sensitive cell cultures. J Virol, 1987 Aug, 61(8), 2407 - 19 In situ molecular hybridization for detection of Aleutian mink disease parvovirus DNA by using strand-specific probes: identification of target cells for viral replication in cell cultures and in mink kits with virus-induced interstitial pneumonia; Alexandersen S et al.; Strand-specific hybridization probes were utilized in in situ molecular hybridization specifically to localize replicative form DNA of Aleutian mink disease parvovirus (ADV) . Throughout in vitro infection, duplex replicative form DNA of ADV was located in the cell nuclei . Single-stranded virion DNA and capsid proteins were present in the nuclei early in infection, but were later translocated to the cytoplasm . In neonatal mink, ADV causes acute interstitial pneumonia, and replicative forms of viral DNA were found predominantly in alveolar type II cells of the lung . Viral DNA was also found in other organs, but strand-specific probes made it possible to show that most of this DNA represented virus sequestration . In addition, glomerular immune complexes containing intact virions were detected, suggesting that ADV virions may have a role in the genesis of ADV-induced glomerulonephritis. J Pharmacol Exp Ther, 1987 Aug, 242(2), 692 - 8 Taxol is converted to 7-epitaxol, a biologically active isomer, in cell culture medium; Ringel I et al.; The hydrolysis products of taxol have been isolated by high-performance liquid chromatography and identified by nuclear magnetic resonance and mass spectroscopy . In contrast to taxol, the major hydrolysis product, baccatin III, has little cytotoxic activity and does not promote in vitro microtubule assembly . In cell culture medium, the concentration of taxol decreases with time and 7-epitaxol, which exhibits properties comparable to those of taxol both on cells and on in vitro microtubuli polymerization, is formed . Baccatin III is found in small quantities in the cell medium, although it is barely detectable within the cells . It is concluded that 7-epitaxol is the major derivative of taxol found in cells and that its presence does not alter, in a major way, the overall biological activity of taxol. Microb Pathog, 1987 Aug, 3(2), 79 - 86 Coronavirus mouse hepatitis virus (MHV)-A59 causes a persistent, productive infection in primary glial cell cultures; Lavi E et al.; MHV-A59 causes a chronic demyelinating disease in mice which is accompanied by persistence of viral genome in white matter . As part of the investigation into the mechanism of viral persistence, infection of glial cells, probable targets for chronic infection, was studied by the use of mixed glial, enriched oligodendrocyte and enriched astrocyte cultures . Following MHV-A59 infection in vitro, approximately 10% of oligodendrocytes and 30% of astrocytes expressed viral antigens in the absence of overt cytopathic effect . All cultures released infectious virus for the lifetime of the cultures, for at least 45 days in the case of mixed glial cultures . Cultures derived from previously infected mice were similar to those infected in vitro with respect to percentage of cells expressing viral antigen and levels of infectious virus produced . These results show (1) that glial cells are early sites of infection in vivo as well as sites of infection in vitro cultures, and (2) that glial cells support a non-lytic but productive infection in vitro and thus may contribute to viral persistence in vivo. Arch Toxicol, 1987 Aug, 60(6), 403 - 14 Limb bud cell cultures for estimating the teratogenic potential of compounds . Validation of the test system with retinoids; Kistler A; Mesenchyme cells, derived from embryonic limb buds, cultured at high cell density, multiply and differentiate into chondrocytes . Using alcian blue, a stain specific for cartilage proteoglycans, the degree of chondrogenesis can be visualized in the micromass cultures as well as quantified by extraction of the stain and spectrophotometric determination of its absorbance . In the presence of active retinoids chondrogenesis is concentration-dependently inhibited . For comparison of the activity of the various retinoids the concentration needed to reduce alcian blue staining by 50% was estimated . In order to validate whether the activity in limb bud cells can predict the teratogenic potential in vivo, the in vitro activity of 25 retinoids was compared with their in vivo teratogenicity observed mainly in rodents . For retinoids which were already in the biologically active form like those with a free carboxylic acid endgroup, there was a good quantitative correlation between the in vitro and in vivo activity . In contrast, the ethylester analog etretinate was slightly active and the ethylamine analog motretinide inactive in vitro but both were teratogenic in vivo . This finding may indicate that these retinoids were not metabolized to the active form in vitro . In conclusion, these results suggest that the limb bud cell culture system may be useful for a preliminary testing to select non-teratogenic retinoids . For the risk assessment in humans, however, the in vitro result should be verified in animal studies. Proc Natl Acad Sci U S A, 1987 Aug, 84(15), 5469 - 73 Dynorphin A selectively reduces a large transient (N-type) calcium current of mouse dorsal root ganglion neurons in cell culture; Gross RA et al.; Opioid receptors are differentially coupled to ion channels . Mu- and delta-opioid receptors are coupled to calcium- and/or voltage-dependent potassium channels and kappa-opioid receptors are coupled to voltage-dependent calcium channels . Using the single-electrode voltage-clamp technique, we investigated the effect of the kappa-opioid receptor agonist dynorphin A on somatic calcium currents of mouse dorsal root ganglion (DRG) neurons in culture . Three different calcium currents were recorded: a small transient current activated positive to -60 mV; a large, inactivating current activated positive to -50 mV; and a moderate, slowly inactivating current activated positive to -40 mV . The first was less sensitive to cadmium block than the others . These calcium currents were similar to those described in other cells, which have been designated T, N, and L calcium currents, respectively . The opioid peptide dynorphin A reduced calcium current by selectively reducing the large inactivating (N) calcium current . Naloxone, an opioid receptor antagonist, reversed this action of dynorphin A . N calcium current is the predominant calcium current in DRG neurons . If N calcium channels are present in primary afferent terminals, and if they are coupled to kappa-opioid receptors as in the soma, these results suggest a mechanism by which dynorphin A inhibits calcium influx and neurotransmitter release. Arch Toxicol, 1987 Jul, 60(5), 388 - 93 Effects of toxic chemicals on the release of pyrimidine compounds in cell culture; Uziel M et al.; Exposure of hamster embryo cells and BF lymphoblastoid cells to 18 known toxic substances and four nominally nontoxic substances results in the release of pyrimidines (and their nucleosides) into the culture medium . The extent of release is dependent on the specific chemical and the specific cells present in the assay . BF cells are not affected by exposure to benzo(a)pyrene, while the hamster embryo cells exhibit enhanced excretion on exposure to benzo(a)pyrene . This difference in response may be due to the difference in endogenous aryl hydrocarbon hydroxylase (BaP) activity . In contrast, diethylstilbestrol, which is metabolized by a peroxidase-mediated enzyme system, causes enhanced excretion in both cell types . Direct alkylating agents and Ni(+2) salts also cause enhanced excretion in both cell types . We have used concentrations of chemicals that give a 5% enhanced excretion as the criterion of low-dose response . Within the range of concentrations tested, chromate induces enhanced excretion in BF cells but not the HEC cells, and Pb(+2) induces enhanced excretion in HEC cells but not the BF cells . Benzene, dimethylnitrosamine, and Mg(+2) did not affect either cell type . 7,12-Dimethylbenzo(a)anthracene, anthracene, benzo(a)anthracene, phenylazoaniline, N-methyl, N-nitroso, N'-nitroguanidine, dioxane, and pyrene cause enhanced excretion in the hamster embryo cells while benzo(e)pyrene, ZnSO4 and cholesterol do not cause enhanced excretion in the hamster embryo cells . Of those chemicals causing enhanced excretion, the concentration range bracketing 5% enhanced excretion approximated low-dose exposures reported to result in toxic responses like cancer, teratogenesis or pulmonary disease. Prenat Diagn, 1987 Jul, 7(6), 383 - 8 A simple method for preparing prometaphase chromosomes from amniotic fluid cell cultures; Cheung SW et al.; A simple method for preparing prometaphase chromosomes from amniotic fluid cell cultures is described . The technique is based upon several key steps including: (1) reduced colcemid concentration, (2) reduced exposure to trypsin-EDTA, and (3) maintaining cells in single suspension by adjusting cell concentration appropriately . Chromosomes with banding resolution up to 800 bands per haploid set can be routinely produced . The described methodology is particularly useful in defining and establishing the clinical significance of subtle structural aberrations. Microbiologica, 1987 Jul, 10(3), 301 - 9 Cultivation of a pig parvovirus in various cell cultures; Ferrari M et al.; The susceptibility of several established cell lines of pig (LLC-PK1 = pig kidney; MPK = minipig kidney; PK15 = pig kidney; ESK = embryonic swine kidney), bovine (EBTr = embryonic bovine trachea), monkey (MA-104 = fetal rhesus monkey kidney) and human (HEL-299 = embryonic human lung) origin to porcine parvovirus was studied . The primary pig kidney cell cultures (pPK) were included in the study as the reference cell system . From the results it appeared that the virus only replicated in cell lines originated from swine . In particular the MPK and ESK cell lines showed a susceptibility similar to that observed for pPK cell cultures . Intranuclear inclusions and plaques were also induced in these cell systems . It appeared therefore that MPK and ESK cell lines both possess all the requirements for use in pig parvovirus studies. Invest Radiol, 1987 Jul, 22(7), 603 - 7 Short-term effects of radiographic contrast media on monolayer cell cultures and hepatocytes; Nordby A et al.; Short-term effects of nine different contrast media, saline, sucrose, and mannitol on monolayer cell cultures and isolated rat hepatocytes were studied . Conventional high osmolal ionic contrast media (Na-metrizoate, Na-iothalamate, meglumine/Na-diatrizoate, meglumine-calcium-metrizoate) and the new, low-osmolal, nonionic (metrizamide, iopamidol, iohexol) and ionic dimer (meglumine/Na-ioxaglate) were tested . Dilutions of different contrast media at the same final osmolality produced similar effects on cultured cells and on isolated hepatocytes as assessed by the leakage of cytoplasmic lactate-dehydrogenase . This short-term toxicity seemed to be a function of the osmolality and of the exposure time . The effect of saline, sucrose, and mannitol was equal to that of contrast media at the same osmolality. Mol Cell Endocrinol, 1987 Jul, 52(1-2), 125 - 35 Vectorial secretion of transferrin and androgen binding protein in Sertoli cell cultures: effect of extracellular matrix, peritubular myoid cells and medium composition; Janecki A et al.; We examined the effect of various culture conditions on the polarized secretion of androgen binding protein (ABP) and transferrin (Trf) by Sertoli cells (Sc) in vitro . Sc from 18-day-old rats were cultured as confluent monolayers on permeable membranes in two-compartment chambers for up to 11 days . Coating of the membranes with extracellular matrix (ECM) components: collagen IV + laminin (CL) or reconstituted basement membrane (RBM) enhanced ABP and Trf secretion (200% and 150%, respectively), with RBM being more effective than CL in stimulating ABP but not Trf secretion . Neither CL nor RBM significantly influenced the relative amounts of ABP and Trf secreted into the outer (OC) and inner (IC) compartments of the culture chamber (OC/IC ratio) . All of these effects were not significantly influenced by the presence of testosterone and serum . Co-culture of Sc with peritubular myoid cells (Pc) significantly increased the secretion of both ABP and Trf, although the magnitude of stimulation and the time-response patterns were different for each protein . Co-culture with Pc also dramatically increased the OC/IC ratios for ABP and Trf . Testosterone (10(-6) M) enhanced the Pc effects . In cultures of Sc alone, presence of 2% fetal bovine serum increased the OC/IC ratios, whereas testosterone had no effect . Based on these results, we suggest a possible role of Pc in the regulation of Sc polarized secretions. J Biol Regul Homeost Agents, 1987 Jul-Sep, 1(3), 119 - 25 Lymphokine-induced cytotoxic cell generation in thymus and spleen cell cultures; Biasi G et al.; The effect of a supernatant (SN-A) obtained from PMA-stimulated IL-2 producing EL-4 cells on cytotoxic cell induction in thymocyte and splenocyte cultures was evaluated . SN-A, but not recombinant IL-2 alone, induced cytotoxic cell differentiation in thymus cell cultures, thus indicating that factors distinct from IL-2 are required for effector cell generation . INF-gamma takes part in the process, and Ia+ accessory cell presence is also strictly required in thymus cultures for lymphokine-induced cytotoxic cell generation . Cytotoxic activity in thymocyte cultures was due only to Thy 1+ Lyt 2+ cells having a broad spectrum of target cell specificity, while in spleen cell cultures an effector population with NK activity was also generated. Gene Anal Tech, 1987 Jul-Aug, 4(4), 75 - 85 Cytogenetic methodologies for gene mapping and comparative analyses in mammalian cell culture systems; Modi WS et al.; Presented here are the detailed methods employed in our laboratory for gene mapping and cytogenetic analyses in human beings, in the domestic cat, and in other mammalian species . Induced in the procedures are: 1) establishment of primary fibroblast and lymphoid cell cultures; 2) heterologous cell fusion for production of rapidly proliferating cell hybrids; 3) cellular transformation of primary fibroblasts using an oncogenic retrovirus; 4) cell synchronization for high-resolution banding of promethaphase chromosomes; 5) chromosome-banding procedures, including G-banding, alkaline G-11, and Q-banding; and 6) in situ hybridization of radiolabeled molecular clones to metaphase chromosomes for regional gene localization. Steroids, 1987 Jul-Sep, 50(1-3), 281 - 95 Aromatase activity in human skin fibroblasts grown in cell culture; Berkovitz GD et al.; Recent studies in this laboratory have described an unusual kindred in which gynecomastia resulted from abnormally elevated levels of extraglandular aromatase activity . In order to better understand the molecular mechanisms responsible for the abnormal aromatase activity in these and other patients, we explored the aromatase activity of genital skin fibroblasts . Our studies demonstrate that the kinetic parameters for aromatase in skin are similar to those of other cultured cells and suggest that skin is an important site of extraglandular aromatase activity . These cells also contain 5 alpha-reductase activity and androgen receptors and are, therefore, a model for androgen action and metabolism . For example, they provided a system for the study of the potency and specificity of the aromatase inhibitors 4-OHA and MDL 18,962 . Finally, the influence of DEX on aromatase in genital skin fibroblasts differs in some important respects from the pattern of control observed in adipose tissue stromal-vascular cells . These findings suggest that investigating the molecular mechanisms for the regulation of aromatase in skin may provide unique information about the control of the enzyme. J Clin Microbiol, 1987 Jul, 25(7), 1275 - 9 Cell culture compared with broth for detection of Trichomonas vaginalis; Garber GE et al.; Trichomonas vaginalis can be grown in cell culture . We studied the growth kinetics of T . vaginalis in McCoy cell culture compared with that in a conventional broth medium (Diamond TYI-S-33 medium supplemented with 10% heat-inactivated bovine serum {TYI}) . In the presence of McCoy cells and two parts cell culture medium to one part TYI, a peak concentration of 2 X 10(6) to 6 X 10(6) T . vaginalis per ml was consistently achieved with inocula as low as three T . vaginalis cells per ml . Without cells, this medium did not support growth of T . vaginalis . T . vaginalis in TYI in 1-ml vials with or without McCoy cells demonstrated poor growth . In tubes containing 10 ml of TYI, inocula grew to 2 X 10(6) to 6 X 10(6) T . vaginalis per ml, but at least 3 X 10(3) T . vaginalis per tube was required to initiate growth . Thus, in vitro, cell culture was more sensitive than TYI broth in detecting low numbers of T . vaginalis . In a subsequent clinical comparison of broth and cell culture for isolation of T . vaginalis from 188 vaginal specimens and 21 urethral specimens from men, the results were in agreement for 206 specimens (98.6%) . There were no situations in which culture was negative and a saline preparation showed motile trichomonads . For women, using a positive culture as the indicator of true positivity, the sensitivity of detection of T . vaginalis was 83% with the Pappenheim stain and 77% with saline preparations . These studies show that cell culture can be used for isolation of T . vaginalis from clinical specimens; it gave results comparable to those of broth culture for the group of mainly symptomatic women . Further studies should be performed to determine its utility in clinical populations such as asymptomatic women and men with and without symptoms, in which T . vaginalis is more likely to be present in low numbers. J Cell Biol, 1987 Jul, 105(1), 465 - 71 Beta-type transforming growth factor specifies organizational behavior in vascular smooth muscle cell cultures; Majack RA; In culture, vascular smooth muscle cells (SMC) grow in a "hill-and-valley" (multilayered) pattern of organization . We have studied the growth, behavioral organization, and biosynthetic phenotype of rat aortic SMC exposed to purified platelet-derived growth regulatory molecules . We show that multilayered growth is not a constitutive feature of cultured SMC, and that beta-type transforming growth factor (TGF-beta) is the primary determinant of multilayered growth and the hill-and-valley pattern of organization diagnostic for SMC in culture . TGF-beta inhibited, in a dose-dependent manner, the serum- or platelet-derived growth factor-mediated proliferation of these cells in two-dimensional culture, but only when cells were plated at subconfluent densities . The ability of TGF-beta to inhibit SMC growth was inversely correlated to plating cell density . When SMC were plated at monolayer density (5 X 10(4) cells/cm2) to allow maximal cell-to-cell contact, TGF-beta potentiated cell growth . This differential response of SMC to TGF-beta may contribute to the hill-and-valley pattern of organization . Unlike its effect on other cell types, TGF-beta did not enhance the synthesis of fibronectin or its incorporation into the extracellular matrix . However, the synthesis of a number of other secreted proteins was altered by TGF-beta treatment . SMC treated with TGF-beta for 4 or 8 h secreted markedly enhanced amounts of an Mr 38,000-D protein doublet whose synthesis is known to be increased by heparin (another inhibitor of SMC growth), suggesting metabolic similarities between heparin- and TGF-beta-mediated SMC growth inhibition . The data suggest that TGF-beta may play an important and complex regulatory role in SMC proliferation and organization during development and after vascular injury. J Submicrosc Cytol, 1987 Jul, 19(3), 445 - 54 The effect of OH(.) radicals generated by Fenton reaction on the growth and cartilage differentiation in limb bud cell culture; Zsupan I et al.; A consistent chondrogenesis takes place in micro high-density cultures of chick limb bud mesenchyme cells stage 22-24 . The effect of an increased generation of OH(.) free radicals by Fenton reaction was tested in these cultures . Components of Fenton reaction (i.e., ferrous iron in form of ADP-Fe2+ complex in 0.1 mM concentration, or 0.2 mM H2O2, or the combination of these components with each other, as well as with 0.2 mM ascorbate) were supplemented to the culture medium after the first 24 h . ADP-Fe2+ complex resulted in a drastic decrease of the frequency and confluence of the cartilage nodules seen in light microscope, accompanied electron microscopically by a strikingly increased frequency of occurrence of lipofuscin-like, residual bodies in the cells . Biochemical methods revealed a significant decrease of both the DNA and glycosaminoglycan contents (to 48.6 and 20.7% of the controls, respectively), in day 6 cultures . H2O2 alone caused similar alterations of the cultures, whereas the combination of it with ADP-Fe2+ complex proved to be lethal for the cells . Ascorbate when added to the ADP-Fe2+-treated cultures displayed a slight protective effect for the glycosaminoglycan content but not for DNA . The results are interpreted in terms of free radical theory of aging. J Clin Microbiol, 1987 Jul, 25(7), 1323 - 4 Evaluation of three types of cell culture for recovery of adenovirus from clinical specimens; Krisher KK et al.; The performance characteristics of HEK, HDF, and MK cells for adenovirus isolation were examined for eye and respiratory tract specimens . HEK cells were superior to HDF and MK cells in terms of both speed of virus detection and sensitivity. J Clin Microbiol, 1987 Jul, 25(7), 1316 - 9 Serial propagation of porcine group C rotavirus (pararotavirus) in primary porcine kidney cell cultures; Terrett LA et al.; A porcine group C rotavirus was adapted to serial propagation in roller tube cultures of primary porcine kidney cells with high concentrations of pancreatin . Infected cells were identified by immunofluorescence staining of cell monolayers . Only group C rotavirus particles were observed in culture supernatants by immune electron microscopy. Anticancer Res, 1987 Jul-Aug, 7(4B), 717 - 9 Cell cultures derived from Wilms' tumour animal model; Murphy GP et al.; Several cell cultures were established from a transplantable Wistar/Furth rat Wilms' tumour . Following cloning by the limiting dilutions method, three morphologically distinct types of cells were identified and preserved for further study of growth regulation in the nephroblastoma model. Virologie, 1987 Jul-Sep, 38(3), 185 - 93 {Study of the multiplication in cell cultures of two strains of para-influenza virus type 3 . III . Multiplication in BHK 21 cells}; Isaia G et al.; Study was conducted on the multiplication of two strains (C243 and D) of parainfluenza virus type 3 in BHK 21 cells . Multiplication curve of the virus was established and immunohistochemical aspects of the process were investigated . Chronological study of successive steps of the formation and development of viral components allowed to see that the virus multiplication rate is low in this cell system . The parainfluenza antigen became detectable by immunofluorescence in the infected cell perinuclear region after a relatively long eclipse period (18 h) and synthetized virus has few RNA and induced no inclusion information in the cytoplasm or the nucleus . However, an important nuclear participation was noted: 72 h after inoculation, nuclear fluorescence was observed, as well as a nuclear DNA rising and frequent aberrant mitoses . Comparison between the two investigated strains led to the observation that the autochthonous D strain induced more frequent aberrant mitoses and more important cell destruction than the C243 one . Differences were also noted as regards the infecting and hemagglutinating titers. Brain Res, 1987 Jul, 431(1), 99 - 109 Developmental capacities of avian embryonic dorsal root ganglion cells: neuropeptides and tyrosine hydroxylase in dissociated cell cultures; Xue ZG et al.; Dorsal root ganglia (DRG) from quail embryos of 10-15 days of incubation (E10-15) contain a subpopulation of cells, distinct from postmitotic neurons, that can, under suitable conditions of culture in vitro, differentiate into neuron-like cells that display a variety of adrenergic properties, including tyrosine hydroxylase (TH) immunoreactivity (Xue et al., Proc . Natl . Acad . Sci . U.S.A., 82 (1985) 8800-8804) . The present study was undertaken to determine whether other markers typical of autonomic sympathetic nerve cells are also expressed in the same system . Cells immunoreactive for vasoactive intestinal polypeptide (VIP) and neuropeptide Y (NPY) were found to differentiate continually from non-dividing precursors in all cultures of dissociated E10 quail DRG grown in the presence of chick embryo extract . Whereas VIP was already present (in a minute number of cells) in DRG in situ, NPY could not be detected before 3 days of culture, when it appeared simultaneously with TH . Double immunostaining experiments showed that most VIP-positive cells and about half the NPY-positive cells also displayed TH-immunoreactivity . On the other hand, there was no overlap between the substance P-containing neuronal population and any of the cells containing TH, NPY or VIP . These observations are pertinent to the problem of the segregation of autonomic and sensory cell lines during peripheral nervous system ontogeny. Brain Res, 1987 Jun 23, 414(1), 177 - 81 A vasoactive intestinal polypeptide system in retinal cell cultures: immunocytochemistry and physiology; Fukuda M et al.; The purpose of this study was to search for vasoactive intestinal polypeptide (VIP)-like immunoreactivity and VIP-mediated effects in cultures containing cells from the mammalian retina . VIP-like immunoreactivity was detected by indirect immunocytochemistry within 6 days after plating dissociated retinal cells from embryonic day-19 rats . In electrophysiological experiments, VIP was found to facilitate evoked transmission at cholinergic synapses formed by retinal neurons in culture. Rev Esp Fisiol, 1987 Jun, 43(2), 215 - 21 {Proliferation kinetics of MCF-7 cell cultures . II . Synchronization and cell recruitment induced by hormones}; Olea N et al.; The culture of MCF-7 cells in well known experimental conditions and the analysis of the cellular uptake of 3HTdR under the influence of estrogens (17-beta-E2) and antiestrogens (OH-TAM) at precise concentration levels (10(-6)/10(-9) M) have made possible the study of the growing kinetic process of the cellular cultured population and the determination of the action of such hormonal preparations on the same process . The main findings are as follows: the growing rate of the cultured cells appears to slow down in presence of OH-TAM; the reason for this slowing growth seems to be the "blockade" of the cultured cells in the last part of G1 phase, a phenomenon that has proved to be dose-dependent; by the stimulating effect of the 17-beta-E2, the MCF-7 cells, synchronized in G1, progress simultaneously in their vital cycle during, at least, three divisory cycles; and this "recruitment" effect seems to be characteristic of the estrogen since the synchronization process fades or disappears when the 17-beta-E2 is absent from the culture medium. Rev Esp Fisiol, 1987 Jun, 43(2), 209 - 14 {Proliferation kinetics of MCF-7 cell cultures . I . Relative influence of estrogens and antiestrogens on the growth of the cell population}; Villalobos M et al.; The cellular hormone dependent cell line MCF-7 is a tumoral model of mammary cancer the growth kinetics of which operating under the influence of varied and opposed hormonal factors (estrogens and antiestrogens at precise concentration levels) has provided the means of knowing the action mechanisms of such agents . In this study, carried out with cultured MCF-7 cells under well defined experimental conditions, it has been shown that: 1) antiestrogens (OH-TAM) seem to be opposed to the growing process of the cellular population the elements of which, under the influence of OH-TAM, double the value of the parameter TD (Doubling Time); 2) estrogens (17-beta-E2) cancel out this effect and promote the growth of MCF-7 cells whether OH-TAM is previously or simultaneously added to the culture medium; 3) the observation of this estrogenic action needs accurate experimental conditions without which the effect may not be seen. Agents Actions, 1987 Jun, 21(1-2), 203 - 8 Investigations on the mode of action of the fungus toxin orellanine on renal cell cultures; Heufler C et al.; The effects of the fungal nephrotoxin orellanine, of 2,2'-bipyridine and of 4,4'-bipyridine on monolayers of LLPCK1-cells were tested . It is shown by the E.C.50 on growing cells that orellanine is the most toxic of the tested bipyridyls . Orellanine causes disruption of confluent monolayers and decreases the activities of membrane bound alkaline phosphatase and of cytosolic lactate dehydrogenase . Also 3H-leucine and 3H-thymidine incorporation are reduced . In contrast to this, ATP- and NADPH-levels remain constant . The cell membrane is not affected . This indicates an intracellular mechanism of action. Cell Differ, 1987 Jun, 21(1), 63 - 8 Stimulation in cell culture of mesenchymal cells of newt limb blastemas by EDGF I or II (basic or acidic FGF); Albert P et al.; After amputation of a newt limb, a blastema forms on the amputation plane and later differentiates to regenerate all the missing parts of the limb . Proliferation of blastema cells is under the control of severed nerves which deliver a 'neurotrophic factor' (NTF) of unknown nature . In order to characterize this factor we use a primary culture of blastema mesenchymal cells; changes in mitotic index after 48-h colchicine treatment indicate mitogenic activity of potential growth substances . These cells, which are stimulated by nerve extracts (mitotic index X 6), were tested with two purified growth factors extracted from bovine retina or brain (EDGF I = basic FGF and EDGF II = acidic FGF) . We show that these two growth factors stimulate proliferation of blastema cell cultures in a dose-dependent manner . Maximal stimulation was obtained at 3 pM for EDGF I (mitotic index X 5.7) or 300 pM for EDGF II (mitotic index X 4.9) . So it appears that these two growth factors have a mitogenic activity on blastema mesenchymal cells similar to that obtained with nerve extracts . The fact that two different growth factors can stimulate these cells raises the question of whether both are present in NTF and/or whether there are receptors to both EDGF I and EDGF II on mesenchymal cell membranes. J Neurosci, 1987 Jun, 7(6), 1809 - 15 Neuronal acetylcholine receptors: fate of surface and internal pools in cell culture; Stollberg J et al.; Chick ciliary ganglion neurons have nicotinic ACh receptors that mediate synaptic input to the cells . Ultrastructural studies with a monoclonal antibody that recognizes the neuronal ACh receptor have previously shown that, in addition to a predominantly synaptic location for the receptors on the neuron surface in vivo, substantial amounts of intracellular receptor are present as well . Here we report that intracellular receptor and smaller receptor-related components make up at least two-thirds of the total antibody binding sites associated with the ciliary ganglion neurons in cell culture . The intracellular sites for the most part represent integral membrane components that bind to concanavalin A when solubilized, indicating that the components are glycosylated . Sucrose gradient analysis shows that the intracellular material includes a 10 S component, likely to represent assembled receptor, along with species sedimenting in the 5-9 S range . Blocking the surface sites with unlabeled antibody and measuring the appearance of the new receptor on the cell surface with radiolabeled antibody indicates that the receptors are inserted into the plasma membrane at a rate equivalent to about 4% of the total surface receptor per hour . The transit time for newly synthesized receptor to reach the cell surface appears to be 2-3 hr . These observations suggest that about 5% of the intracellular receptors are transported to the cell surface in culture . The half-life of ACh receptors in the plasma membrane was estimated by 2 different approaches to be about 22 hr . Surface and internal sites respond in a qualitatively similar way to external agents that specifically modulate cholinergic receptors.(ABSTRACT TRUNCATED AT 250 WORDS) J Neurosci, 1987 Jun, 7(6), 1639 - 47 Phorbol esters: voltage-dependent effects on calcium-dependent action potentials of mouse central and peripheral neurons in cell culture; Werz MA et al.; The beta-phorbol esters 12-O-tetradecanoylphorbol-13-acetate (TPA) and phorbol 12,13-dibutyrate (PDBu), which activate protein kinase C, were applied to mouse dorsal root ganglion (DRG) and cerebral hemisphere neurons grown in primary dissociated cell culture . Phorbol esters did not modify the membrane potential or input resistance of either type of neuron . To assess the effects of beta-phorbol esters on voltage-dependent conductances, the effects of PDBu and TPA on action potentials evoked from these neurons were determined . The neurons were bathed in a solution containing 5 mM tetraethylammonium and action potentials that contained sodium and calcium components were evoked . When applied at resting membrane potential and at more negative potentials, PDBu and TPA reversibly increased action potential duration . The alpha-phorbol ester 4-alpha-phorbol, which does not activate protein kinase C, did not modify action potential duration . The effects of the beta-phorbol esters, however, were voltage-dependent . When the neurons were depolarized to membrane potentials less negative than -50 mV, PDBu and TPA reduced action potential duration . The effects of both PDBu (10 nM-1 microM) and TPA (100 pM-100 nM) on action potential duration were dose-dependent . The prolongation of action potentials produced at large negative potentials may be due to a reduction in voltage-and/or calcium-dependent potassium conductance, since the prolongation was associated with a reduction in the potassium-dependent afterhyperpolarization; following membrane depolarization in control solution, action potential duration was increased for several minutes, while the afterhyperpolarization was reduced and, following this prolongation, phorbol esters no longer prolonged the action potentials.(ABSTRACT TRUNCATED AT 250 WORDS) Biull Eksp Biol Med, 1987 Jun, 103(6), 738 - 41 {Morphological characteristics of hippocampal neurons developing in cell cultures}; Khaspekov LG et al.; Using observation of living cells and neurohistological methods, we have investigated growth dynamics, fasciculation of fibers and morphogenesis of neurons in hippocampal cell cultures of 18-19-day-old mouse embryos . The development of the system of neuronal outgrowths with predominant growth of apical dendrite started on the 4th-6th day of cultivation and resulted in the formation of pyramidal neurons possessing the main morphological signs of pyramidal hippocampal neurons developing in situ . Thus, the morphogenetic programme of dendrite development can ensure the in vitro formation of neurons of a certain morphological phenotype . One of the possible prerequisites of the programme realization appears to