Cell cultures

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 be the inductive influence of afferent nervous fibers which reach hippocampal neurons in situ in the pre-cultivation period.

Endocrinology, 1987 Jun, 120(6), 2569 - 75
Regulation of differentiated thyroid function by iodide: preferential inhibitory effect of excess iodide on thyroid hormone secretion in sheep thyroid cell cultures; Becks GP et al.; Primary cultures of sheep thyroid cells have been used to study the inhibitory effects of iodide on thyroid function . Under the influence of TSH, iodide was concentrated with a cell to medium ratio of 20 . When thyroid hormone secretion was measured from cells cultured without addition of exogenous iodide, preferential T3 secretion was evident . The optimum iodide concentration for T4 and T3 synthesis and secretion was 10(-6) M . Prior exposure to 10(-5) M or more iodide decreased subsequent iodide transport in a concentration-dependent manner compared to that in cells acutely exposed to iodide . Although cell to medium ratios were decreased, intracellular iodide concentrations continued to rise with increasing external iodide concentrations, and iodide available for thyroid hormone synthesis was not in limited supply . Iodide concentrations of 10(-4) M or greater inhibited iodothyronine synthesis and thyroid hormone secretion, assessed by both assay of trichloroacetic acid-insoluble Na125I activity in cells and RIA of T4 and T3 in the medium and cell layer . An intermediate concentration of 10(-5) M iodide had a marked inhibitory effect on T4 and T3 secretion, but iodothyronine formation on thyroglobulin was only slightly affected . Our results suggest a preferential inhibitory effect of elevated iodide concentrations on thyroid hormone secretion . The adaptive advantages of this selective inhibition would allow storage of iodothyronines in times of iodide sufficiency while maintaining euthyroidism.

Cancer Res, 1987 Jun 1, 47(11), 2831 - 8
Induction of common patterns of polypeptide synthesis and phosphorylation by calcium and 12-O-tetradecanoylphorbol-13-acetate in mouse epidermal cell culture; Wirth PJ et al.; Terminal differentiation can be induced in cultured basal cells by either increasing the Ca2+ level in the medium from 0.05 to 1.4 mM or by exposure to the tumor promoter 12-O-tetradecanoylphorbol-13-acetate (TPA) . If Ca2+ and TPA act by a common mechanism, then a common pattern of protein synthesis and/or phosphorylation would be expected . Computer-assisted analysis of radioactively labeled polypeptides separated by two-dimensional-polyacrylamide gel electrophoresis was utilized to study protein synthesis and phosphorylation . Within 1 h of increasing the Ca2+ level in the medium, the synthesis of 57 polypeptides was altered by 2-fold or more . Similarly, exposure to TPA for 1 h affected the synthesis of 106 polypeptides . Sixteen polypeptides were affected by both Ca2+ and TPA; the synthesis of nine was increased and seven was decreased, with changes in the same direction for both effectors . By 4 h, the synthesis of 32 polypeptides was similarly modulated by both Ca2+ and TPA . Only one polypeptide which was increased at 1 h was still elevated at 4 h . These results suggest that a common dynamic program of protein synthesis, likely to be related to terminal keratinocyte differentiation, is induced by both Ca2+ and TPA . Overall phosphorylation of epidermal proteins was increased after 30 min of TPA treatment, but was not increased by Ca2+ at this time . Keratin polypeptides were heavily phosphorylated in low Ca2+ medium, but the level or pattern of phosphorylation of these proteins was not altered by either Ca2+ or TPA . Although phosphorylation of a minor polypeptide (pI 5.1/Mr 45,000) was increased 2-3-fold by both Ca2+ and TPA, most of the specific protein phosphorylation changes induced in keratinocytes by Ca2+ and TPA appear to be unique . Thus, if protein phosphorylation is an early signal for epidermal differentiation by each effector, only a single apparent common substrate is involved and multiple kinases are activated . Alternatively, substrate specificity of a single kinase may be differentially altered by each effector.

Isr J Med Sci, 1987 Jun, 23(6), 603 - 7
Attachment of Mycoplasma hominis to human cell cultures; Izumikawa K et al.; Clinical isolates, cell-culture contaminants, and the type strain PG21 of Mycoplasma hominis were examined for attachment to erythrocytes and human cell cultures . Strain 13428 (from blood, postpartum fever) and strain 1184 (cell culture) attached to human and guinea pig erythrocytes, but there were no differences in attachment activities between these strains . However, five M . hominis strains isolated from different tissue sites showed quantitative differences in attachment to human WiDr (intestinal carcinoma cell cultures), MRC-5 (human embryonic lung fibroblasts) and HeLa (carcinoma of cervix) cell cultures . The relative attachment activities were, in descending order: strain 1184 (cell culture), strain 11932 (cervix), strain 13428 (blood, postpartum fever), 13408 (nongonococcal urethritis), and type strain PG21 (multiple passage, originally from human rectum) . Trypsin and pronase treatment of M . hominis strain 1184 markedly reduced attachment, suggesting that surface proteins play a role in M . hominis attachment to mammalian cells . In subsequent studies, strain 1620 (septic arthritis) showed the highest attachment activity, whereas strain 1652 (surgical skin flap) and L01888 (cell culture) showed attachment activity similar to cell culture strain 1184 . The differing attachment activities of these M . hominis strains isolated from different infected sites of patients with a variety of diseases may be relevant to the virulence of these strains.

Klin Wochenschr, 1987 Jun 1, 65(11), 507 - 12
Establishment of primary cell cultures: experiences with 155 cell strains; Dietel M et al.; Cell culture systems allow the examination of cell populations in a functional state . To simulate in vivo conditions as closely as possible freshly established cell strains are superior to permanent cell lines . Different aspects for the establishment of primary cell cultures obtained from various tissues are compared: Disintegration, culture media supplemented with basal additions, special supplements (growth factors, hormones), and attachment factors . The proliferation rates of the attained cell strains were evaluated by determination of cell doubling times . Procedures for how to obtain a relatively high plating efficiency (approx . 70% in our series of 219 attempts) of primary growth in vitro are described: (1) Mechanical disintegration is superior to enzymatic digestion . If mechanical treatment alone did not produce a sufficient number of viable cells, additional digestion with collagenase/dispase revealed a higher number of proliferating primary cultures than with trypsin . (2) Proliferation of cell cultures from normal and tumorous tissues of epithelial origin was superior in Leibovitz L 15 medium (58 of 87 (67%) cases) . Cultures from mesenchymal tissues and tumors were found to have shortest cell doubling times in MEM and RPMI 1640 (16 of 23 (70%) cases) . The media were supplemented with the basal additions indicated . (3) In approx . 30% of the cases special supplements like growth factors or hormones increased cell replication, although they were almost always not essential for cell growth . (4) Attachment factors only rarely contributed to the initiation of primary monolayer cultures . The application of various culture conditions does not lead to a protocol optimal for all tissues, for all probes of the same type of tumor, or for all tumor specimens of unique differentiation.

Clin Exp Immunol, 1987 Jun, 68(3), 500 - 9
Expression of major histocompatibility complex class I and class II antigens in human Schwann cell cultures and effects of infection with Mycobacterium leprae; Samuel NM et al.; Recent experiments on rats have raised the possibility that Schwann cells can present antigens to T lymphocytes . We have investigated whether this mechanism might be relevant in leprosy by determining under what conditions human Schwann cells express class I and class II antigens, and whether infection with Mycobacterium leprae affects this expression . The distribution of these antigens was examined on human Schwann cells in dissociated cell cultures derived from human fetal peripheral nerves . We find that both Schwann cells and fibroblastic cells in these cultures normally express class I antigens but not class II antigens . When Schwann cells are infected with live Mycobacterium leprae for 48 h, 73% of Schwann cells phagocytose the bacteria . Mycobacterium leprae prevents 3H-thymidine incorporation into cultured human Schwann cells, but does not affect class I expression in these cells . Treatment of normal and Mycobacterium leprae infected cultures with gamma-interferon for 72 h induces class II expression on most Schwann cells but not on the majority of fibroblastic cells . The fact that human Schwann cells infected with Mycobacterium leprae can be induced by gamma-interferon to express class II antigens suggests that they may be able to present Mycobacterium leprae antigens to T lymphocytes and thus initiate immune responses against the bacteria . We suggest that a failure of this response, such as that seen within nerve trunks in lepromatous leprosy, is caused by deficient class II expression on Schwann cells . This deficiency in class II expression, in turn, may be caused by the reduced gamma-interferon production characteristic of lepromatous leprosy.

Gastroenterol Jpn, 1987 Jun, 22(3), 285 - 91
Arachidonic acid does not protect against sodium taurocholate damage to rat gastric epithelial cell cultures; Ota S et al.; Arachidonic acid is cytoprotective against ethanol damage in vivo . Stimulation of production of endogenous prostaglandin has been postulated as a mechanism of this protection . The current study assessed the ability of arachidonic acid to protect gastric mucosal cells directly and the effect of arachidonic acid on prostaglandin production . To determine cytoprotection, sodium taurocholate was used as a damaging agent and cell viability was assayed by 51Cr release in vitro . By the dose response curve, 5 mM sodium taurocholate was chosen for the protective study . Cells were preincubated with arachidonic acid (0.1-10 micrograms/ml) and then incubated with 5 mM sodium taurocholate . The synthetic activity of prostaglandins by cultured cells was assessed using 14C arachidonic acid prelabeled cells . Media content of prostaglandin E2 was assayed by radioimmunoassay . Cell viability was assayed by 51Cr release . Our results showed that arachidonic acid did not significantly protect cultured gastric cells against sodium taurocholate damage, while larger doses of arachidonic acid produced cell damage . Furthermore, cultured gastric cells produced mainly prostaglandin E2 and prostaglandin I2 and arachidonic acid stimulated prostaglandin E2 production dose-dependently (p less than 0.01) . In conclusion, arachidonic acid does not directly protect gastric mucosal cells in vitro . In vivo protection by arachidonic acid must be based on indirect factors such as preservation of microvasculature.

Proc Natl Acad Sci U S A, 1987 Jun, 84(12), 4303 - 7
Identification of a herpes simplex virus 1 glycoprotein gene within a gene cluster dispensable for growth in cell culture; Longnecker R et al.; The genome of herpes simplex virus 1 consists of two components, L and S, each containing unique sequences flanked by inverted repeats . Current and earlier studies have shown that 11 of the 12 open reading frames contained in the unique sequences of the S component can be deleted and are dispensable for growth in cell culture . Analyses of one recombinant virus containing a deletion in the open reading frame US7 permitted the identification of a monoclonal antibody specific for the product of this gene . The protein encoded by this gene has a predicted translated molecular weight of 41,366 and an apparent molecular weight of approximately 65,000 in denaturing polyacrylamide gels . The electrophoretic mobility of the protein synthesized by cells in the presence of inhibitory concentrations of tunicamycin is faster than that of the protein accumulating in lysates of untreated infected cells . We conclude that the product of US7 is glycoprotein subject to N-linked glycosylation, and we have designated it glycoprotein I . These studies indicate that the unique sequences of the S component encode four glycoproteins (G, D, I, and E) of which at least three (G, I, and E) are dispensable for growth in continuous lines of primate cells.

Virus Res, 1987 Jun, 7(4), 335 - 49
Effects of serial passage of Autographa californica nuclear polyhedrosis virus in cell culture; Kumar S et al.; A study of the major genomic alterations occurring during serial passage of Autographa californica nuclear polyhedrosis virus (AcNPV) in a Trichoplusia ni cell line was conducted . Progeny viruses from 24 independent passages were randomly selected and analyzed with restriction endonucleases . Specific deletion mutations were generated repeatedly in the PstI-G (7.6 to 13.1%) and the PstI-I (14.4-17.9%) regions; these mutations became predominant in the serially passaged stocks in which they arose . The deletions in the PstI-G region and two different insertions in this region were mapped to a 1 Kb PvuII-Bg/II fragment (9.85-10.70%) reflecting a high degree of sequence specificity in the initiation or selection of genomic alterations in this region . Insertional mutations were observed frequently and repeatedly within the PstI-E/HindIII-I region (33.6-37.2%) of the AcNPV genome . Individual examples of insertional mutations were observed in several other regions of the genome.

Brain Res, 1987 Jun, 430(2), 305 - 9
Quantification of synapse turnover in cell culture; Puro DG; The on and off rates of synaptogenesis can be quantitated using a cell culture system in which embryonic retinal cells form functional synapses with striated muscle cells . Quantification showed that synapse formation and termination can occur simultaneously in culture . Quantification also revealed that some retina-muscle synapses are transient, terminating within 8 h, while other synapses are much more stable . Relatively stable and transient synaptic pairs could be identified prospectively based on the strength of evoked transmission across the retina-muscle synapse.

J Virol, 1987 Jun, 61(6), 1913 - 8
Identification and mapping of human papillomavirus type 1 RNA transcripts recovered from plantar warts and infected epithelial cell cultures; Chow LT et al.; Multiple spliced transcripts of human papillomavirus type 1 were detected by electron microscopic analysis of R-loops formed with total RNA extracted from plantar warts and with poly(A)+ RNA isolated from cultured keratinocytes infected with human papillomavirus type 1 . The 5' ends of the RNAs were mapped to sites in the E7 open reading frame (ORF), just upstream of the E6 ORF and in the upstream regulatory region . Species with 5' ends in E7 accounted for over 95% of all transcripts seen . Two polyadenylation sites were used, one at the end of the early (E) region of the viral DNA, the other at the end of the late (L) region . The most abundant species had a short 5' exon of approximately 100 nucleotides spanning the junction of the E7 and E1 ORFs spliced to a 3' exon of 800 nucleotides in the region with overlapping E2 and E4 ORFs; it was polyadenylated at the end of the E region . This species probably encodes the abundant E4 protein found in plantar warts (F . Breitburd, O . Croissant, and G . Orth, Cancer Cells, vol . 5, in press; J . Doorbar, D . Campbell, R . J . A . Grand, and P . H . Gallimore, EMBO J . 5:355-362, 1986) . Other transcripts had exons spanning the E6-E7 ORFs, the E4-E5-L2-L1 ORFs, or the L1 ORF . The infrequent L1 transcript, probably the mRNA coding for the major capsid protein, had the same 5' exon in E7 as the abundant mRNA spliced from E1 and E4 ORFs, suggesting genetic regulation via the choice of the alternative polyadenylation sites or mRNA processing.

Pharm Res, 1987 Jun, 4(3), 200 - 6
Efficacy of 5-vinyl-1-beta-D-arabinofuranosyluracil (VaraU) against herpes simplex virus type 2 strains in cell cultures and against experimental herpes encephalitis in mice: comparison with acyclovir and foscarnet; Reefschlager J et al.; The sensitivity of different herpes simplex virus type 2 (HSV-2) strains to inhibition by 5-vinyl-1-beta-D-arabinofuranosyluracil (VaraU) was evaluated in comparison to 9-(2-hydroxyethoxymethyl)guanine (ACV; acyclovir) and trisodiumphosphonoformate (Na3PFA; foscarnet), using a plaque inhibition assay in primary rabbit testes (PRT) cells as well as in human embryonic lung fibroblast (HELF) cell cultures . The order of decreasing activity found was ACV much greater than VaraU greater than Na3PFA in PRT cells and ACV greater than VaraU much greater than Na3PFA in HELF cells, with 50% inhibition doses (ID50) of 1.8, 8.8, and greater than 110 microM for the three drugs in HELF cells, respectively . After 72hr of drug treatment, inhibition of HELF cell proliferation by VaraU (ID50, greater than 1000 microM) was less than that by ACV and Na3PFA, resulting in high selectivity indexes of greater than 100 against HSV-2 for VaraU and ACV . Their in vivo efficacy was assessed in a mouse encephalitis model . Using a treatment schedule of three daily intraperitoneal (ip) doses over a period of 5 days, only the survival times of mice were considerably prolonged by VaraU (150 or 300 mg/kg per day; P less than 0.05 or P less than 0.001, respectively) . In contrast, ACV treatment (150 mg/kg per day) led to a nearly complete prevention of encephalitis and death (P less than 0.001) . Similar therapy results with VaraU application through the drinking water were obtained using only one-sixth of the high ip dose (approximately 50 mg/kg per day) but over a prolonged period of treatment . Under similar conditions no therapeutic effect of oral Na3PFA was observed.

Pharmazie, 1987 Jun, 42(6), 407 - 11
{(E)-5-(2-bromovinyl)-2'-desoxyuridine--a new nucleoside analog with selective inhibitory action against herpesviruses . Studies in cell culture and animal experiments}; Reefschlager J et al.; (E)-5-(2-Bromovinyl)-2'-deoxyuridine (1; BrVUdR) inhibits the replication of herpes simplex virus type 1 (HSV-1) and of varicella-zoster virus (VZV) in vitro at concentrations of 0.01 to 0.23 mumol/l, whereas herpes simplex virus type 2 (HSV-2) is influenced only at 5.5 to 27 mumol/l . In comparison to some classical and newly developed antiherpetics, i . e . 5-iodo-2'-desoxyuridine (2; idoxuridine, IDU), 9-beta-D-arabinofuranosyladenine (4; vidarabine Ara-A), 9-(2-hydroxyethoxymethyl) guanine (5; acyclovir, ACV) and 2'-fluoro-5-iodo-1-beta-D-aracytosine (6;FIAC) the following order of decreasing activity was found:1 greater than 6 greater than 5 greater than 2 greater than 4 (against HSV-1) and 6 greater than 2 greater than 5 greater than 1 greater than 4 (against HSV-2) . The high selectivity of the antiviral effect of BrVUdR towards HSV-1 and TZV is based on the fact, that proliferation of different mammalian cell lines is inhibited by 50% only at concentrations as high as 90 to 170 mumol/l, resulting in a therapeutical index of 1000 to 10,000 . Successful treatment of an HSV-1 encephalitis in mice as well as an HSV-1 keratitis of rabbits confirmed the efficiency of 1 in experimental animal infections . No toxic side effects in both local and systemic applications were observed . Promising data from cell culture and animal experiments recommend 1 as a potential candidate for the local and systemic treatment of HSV-1 and VZV infections in man.

Cancer Res, 1987 May 15, 47(10), 2704 - 13
Cell culture of human colon adenomas and carcinomas; Willson JK et al.; Cell lines were established from colon adenomas, including tubular and villous polyps, primary adenocarcinomas, and metastases arising in patients with colon adenocarcinomas . The protocol for cultivating these diverse tissues includes primary cultivation of tissue explants on a type I collagen gel followed by nonenzymatic subculture of the epithelial outgrowth . All early passages were accomplished using low subculture ratios . Cultured cells elaborate morphological structures which are similar to features present in the tissues from which they were cultivated . Specifically, all structural features of colon epithelial cells were identified, including junction formation, prominent microvilli, and mucin secretion, in several cell lines . Five cell lines cultured from colonic neoplasms at different stages of cancer progression were selected for detailed characterization . Cells grown from two tubular polyps had normal human karyotypes . Cells from a villous polyp and all adenocarcinomas were aneuploid with stable marker chromosomes . The established cell lines exhibit distinct phenotypes based on growth characteristics in vitro and in athymic mice; and it is suggested that these cell lines represent useful models for studying the evolution of colon cancer from a benign to an aggressive cell type.

Biochim Biophys Acta, 1987 May 13, 919(1), 1 - 12
Enhanced release and synthesis of lipoprotein lipase in rat heart cell cultures exposed to high concentrations of Hepes; Friedman G et al.; While attempting to optimize conditions for synthesis of lipoprotein lipase by cultured heart cells, we encountered an unexpected rise in enzyme activity when media were supplemented inadvertently with 100 mM Hepes buffer (4-(2-hydroxyethyl)-1-piperazineethanesulphonic acid) . This finding was further investigated and optimal results were obtained at pH 7.0-7.2 . The increase in lipoprotein lipase activity was time dependent; after 3-6 h there was a rise in medium activity but cellular activity increased only after 24 h . The increased enzyme activity was defined as lipoprotein lipase by inhibition with antiserum to rat adipose tissue lipoprotein lipase . A 72-h exposure to Hepes resulted in a 30% increase in the incorporation of {35S}methionine into cellular proteins and a 2-fold increase into heparin-releasable proteins . Using heparin Sepharose chromatography and stepwise elution, a lipoprotein lipase enriched fraction was recovered with 2 M NaCl . The amount of {35S}methionine and {3H}galactose incorporated into protein of this fraction derived from Hepes-treated cells was 2-6-fold that of controls . A 4-fold increase in cellular lipoprotein lipase mass in Hepes-treated cells was shown by immunoblotting . Results obtained with Hepes-conditioned medium suggest the presence of cell-derived compounds that enhance release and subsequent synthesis of lipoprotein lipase . The effect of Hepes-conditioned medium on lipoprotein lipase resembled to some extent that of the addition of heparin . Therefore, it appears that when Hepes is first added to the culture medium, it might promote a release of heparan sulfate or related compounds, possibly by virtue of its negatively charged sulfonic acid residue . The accumulated heparan sulfate could then promote a sustained release of lipoprotein lipase into the culture medium which in turn leads to increased enzyme synthesis.

Vet Parasitol, 1987 May, 24(3-4), 301 - 4
Development of Isospora suis from pigs in primary porcine and bovine cell cultures; Lindsay DS et al.; Sporozoites of Isospora suis penetrated and developed by endodyogeny in primary porcine kidney (PPK) and primary fetal bovine kidney (PFBK) cell cultures . Motile merozoites and binucleate Type I meronts were observed in both types of cultured cells . Multinucleate Type II meronts developed in PPK cell cultures only . These multinucleate meronts were always found singly, were nonmotile and did not form merozoites.

J Androl, 1987 May-Jun, 8(3), 155 - 61
Alkaline phosphatase histochemistry discriminates peritubular cells in primary rat testicular cell culture; Chapin RE et al.; Histochemical demonstration of alkaline phosphatase activity appears to be useful in identifying rat peritubular cells in primary testicular cell culture . In both frozen sections of rat testis and Mirsky's fixed, methacrylate-embedded rat testis, the reaction product localized primarily in peritubular cells, vascular endothelium and occasionally in interstitial cells, with much smaller amounts of reaction product associated with elongating spermatids in the germinal epithelium . Occasional late-stage tubules (X-XIV) showed weak reactivity in the epithelium, associated with spermatocytes or Sertoli cells . Ultrastructurally, Gomori-method reaction product was localized to peritubular cells, lymphatics, and spermatogonia in stage VII; no staining was found consistently in Sertoli cells . In isolated cell preparations enriched for Sertoli and germ cells, 1 to 8% of the cells demonstrated alkaline phosphatase activity, while greater than 50% of the cells stained positive for alkaline phosphatase activity in peritubular-enriched fractions . The histochemical demonstration of alkaline phosphatase activity can be useful for identifying peritubular cells in primary cultures of testicular cells.

Am J Vet Res, 1987 May, 48(5), 822 - 7
Tropism of border disease virus for oligodendrocytes in ovine fetal brain cell cultures; Anderson CA et al.; Primary dissociated ovine brain cell cultures prepared from 50- or 140-day-old fetuses were inoculated with border disease virus (BDV) . The cells present in the cultures were identified, using immunofluorescence procedures and sera against various CNS cell-specific markers . These markers were glial fibrillary acidic protein, myelin basic protein, myelin-associated glycoprotein, neuron-specific enolase, neurofilament protein, and fibronectin . Double-labeling immunofluorescence techniques for visualization of BDV antigen and of the CNS cell markers were used to evaluate the pattern of individual cell susceptibility 48 hours after infection . In cultures from fetuses of both ages, about half of the infected cells were glial fibrillary acidic protein-positive astrocytes . Scattered myelin-associated glycoprotein-positive oligodendrocytes were positive for BDV antigen, but only in the infected cultures from the older fetuses . Fibronectin-positive cells were not infected with BDV . In infected and noninfected cultures, cells positive for neuron-specific enolase, myelin basic protein, or neurofilament protein were not seen . Therefore, the remaining infected cells in all the cultures were not identified by the cell-specific markers used . Results of these in vitro experiments indicate that BDV does not selectively infect oligodendrocytes, and that such a selective pattern of infection may not be the basis for the in vivo congenital hypomyelination in sheep with border disease.

Int J Radiat Oncol Biol Phys, 1987 May, 13(5), 753 - 7
Radiosensitization of primary human tumor cell cultures by N-methylformamide; Arundel C et al.; The effects of the differentiation-inducing agent N-methylformamide (NMF) on the radiation response of ten primary human tumor cell cultures were investigated . Cell survival was determined using an adhesive tumor cell culture system . NMF (1%) was added to cultures on Day 1 and was left for 6 days; cultures were irradiated with graded doses of X rays (1.0-6.0 Gy) on Day 4 . Using survival at 2.0 Gy as a comparative endpoint, eight of ten cultures tested exhibited enhanced radiosensitivity upon exposure to NMF . In sensitized cultures, the dose-enhancement factors ranged from 1.3 to 2.5 . The NMF-mediated radiosensitization did not appear to be dependent on the histologic cell type . The results presented support previous data obtained from established cell lines and suggest that NMF may offer clinical benefits against a variety of tumor types when used in combination with radiotherapy.

Clin Immunol Immunopathol, 1987 May, 43(2), 163 - 73
Functional effects of epidermal cell culture supernatants (ECCS) on human B-cell activation; Penso D et al.; Epidermal cells (EC) were cultured without stimulation and the effect of these EC culture supernatants (ECCS) on human in vitro B-cell response was determined . Supernatants obtained between Days 5 and 7 were able to replace monocytes in the antibody response to the particulate antigen trinitrophenyl-polyacrylamide (TNP-PAA) . These results were obtained when highly monocyte-depleted cultures (less than 0.5% peroxidase-positive cells) were used and were reproduced with supernatants from several different EC cultures . ECCS could not substitute for T cells in the T-dependent response to TNP-PAA . They contained an interleukin 1 (IL-1) activity but no interleukin 2 or B-cell growth factor (BCGF) activities . We tested the effect of ECCS on the proliferative response of highly monocyte-depleted B cells cultured at low cell density costimulated with anti-u antibody and BCGF . ECCS had no BCGF-like activity of its own but did potentiate the effect of BCGF . Thus EC cultures produce IL-1-like factor(s) which act directly on the early stages of B-cell activation.

J Clin Microbiol, 1987 May, 25(5), 763 - 7
Comparison of nasopharyngeal aspirate and nasopharyngeal swab specimens for respiratory syncytial virus diagnosis by cell culture, indirect immunofluorescence assay, and enzyme-linked immunosorbent assay; Ahluwalia G et al.; Paired nasopharyngeal aspirate (NPA) and nasopharyngeal swab (NPS) specimens obtained from each of 32 hospitalized infants with X-ray-confirmed pneumonia (91%) or bronchiolitis were tested for respiratory syncytial virus (RSV) infection by virus culture, the indirect immunofluorescent-antibody (IFA) technique, enzyme-linked immunosorbent assay (ELISA; Ortho Diagnostic Systems, Inc.), and spot hybridization with a human genomic probe to quantitate cellular DNA . RSV was isolated in cell cultures from 72% (23 of 32) of patients by using NPA specimens compared with 47% (15 of 32) by using NPS specimens . With tissue culture positivity as the reference test, the sensitivities of the ELISA on NPA and NPS specimens were found to be 69% (16 of 23) and 61% (14 of 23), respectively, with a specificity and a positive predictive value from both sites of 100% . The sensitivities of the IFA technique compared with the cell culture on NPA and NPS specimens were 61% (14 of 23) and 52% (12 of 23) with specificities of 89 and 78% and positive predictive values of 96 and 92%, respectively . Despite the recovery of significantly more cells (as shown by detection of more cellular DNA by using NPA specimens), virus was detected by the IFA technique or ELISA at similar frequencies in paired specimens . However, virus was recovered more often from NPA than NPS specimens by cell culture, and ELISA optical density readings and the number of RSV-positive fluorescing cells were greater for NPA specimens . NPA specimen collection was less traumatic for the patient, was an easier procedure for the physician to perform, and provided a superior laboratory specimen for RSV diagnosis than the NPS technique.

Epilepsia, 1987 May-Jun, 28(3), 214 - 21
Effects of anticonvulsants on cell growth and enzymatic and receptor binding activity in a neuroblastoma x glioma hybrid cell culture; Slesinger PA et al.; The effects of anticonvulsants on markers of growth, intracellular enzymes, and synaptic functions were evaluated using a rapidly dividing cholinergic neuroblastoma x glioma hybrid cell-line (NG108-15) . Cell cultures were exposed for 4 days to phenobarbital, phenytoin, carbamazepine, or valproic acid . Anticonvulsant concentrations added to the media were selected to produce free levels in the cell media that were equivalent to free levels in humans ranging from therapeutic to very toxic . Free levels of anticonvulsants in the toxic range affected cell number, protein content, and neurochemical markers . However, only valproic acid and phenytoin reduced cell growth at therapeutic free drug concentrations . Valproic acid was the only medication to act as a differentiating agent, significantly increasing the activity of choline acetyltransferase, beta-galactosidase, and muscarinic cholinergic receptor binding . These results emphasize the importance of performing drug studies at appropriate free drug concentrations and suggest that valproic acid differs from other commonly prescribed anticonvulsants by having both a growth-suppressing and a differentiating effect.

Mol Gen Mikrobiol Virusol, 1987 May, (5), 40 - 4
{Adaptation of hepatitis A virus strain (JaM-55) originally pathogenic for monkeys to a cell culture}; Andzhaparidze AG et al.; The results of adaptation of hepatitis A viral strain JaM-55 to the culture of embryo kidney cells FRhk-4 from macaque Rhesus are presented . The viral strain was isolated from a M . fascicularis suffering from spontaneous hepatitis . Before inoculating the cell culture the virus was passaged twice in the M . arctoides capable of reproducing hepatitis . FRhk-4 cell line inoculation by the monkey liver extract, containing the strain HAV-YaM-55, resulted in isolation of single viral particles of hepatitis A in the preparations obtained at the first 3 passages by the 28-31 day of cultivation . Beginning from the fourth passage the abrupt increase in the number of viral particles and hepatitis A antigen was registered . There were no traces of cytopathogenic effect at any level of viral passages in the inoculated cell culture . The adapted virus contains hepatitis A viral RNA identified by spot hybridization with the cloned cDNA of hepatitis A virus.

J Virol Methods, 1987 May, 16(1-2), 139 - 46
BK virus in cell culture: infectivity quantitation and sequential expression of antigens detected by immunoperoxidase staining; Flaegstad T et al.; We applied an indirect immunoperoxidase staining method for the detection of BK virus (BKV) antigens in Vero cells . By immunoperoxidase staining of sequential samples, expression of BKV T- and structural antigens was first detectable 72 h postinfection (pi) . The initial low number of antigen positive cells was slowly increasing until 6 d pi . Thereafter, a steady progression was observed until total cytopathogenic effect 30 d pi . Viral hemagglutinins were detected 11 d pi in both the culture medium and in the cell lysate . Passage of the preparations into fresh Vero cells and immunoperoxidase detection demonstrated the presence of infectious virions in both the cell lysates and the medium from 2 d pi . The number of infectious particles in both media and cell lysates correlated to the number of IP stained cells in the sequential staining . We suggest that this type of assay might be used to examine the effects, in vitro, of interferons and other virostatic drugs of BKV and other viruses.

Proc Natl Acad Sci U S A, 1987 May, 84(9), 2678 - 82
Hepatitis B virus (HBV) particles are produced in a cell culture system by transient expression of transfected HBV DNA; Yaginuma K et al.; An in vitro system for the production of hepatitis B virus (HBV) particles was established by the transient expression of transfected HBV DNA using a human hepatocellular carcinoma cell, HuH-7, as a recipient . The 3.6- and 2.2-kilobase transcripts observed were similar to those in virus-infected liver cells . Both transcripts revealed the microheterogeneity of their 5' ends . The formation of virus-related particles subsequent to the RNA transcription was demonstrated . The core particles observed in the cytoplasm and the virus particles secreted in the culture medium contained the replicative intermediates of HBV DNA and banded at densities of 1.35-1.36 g/cm3 and 1.22-1.24 g/cm3, respectively . Furthermore, the in vitro mutagenesis of the template HBV DNA demonstrated that the P gene as well as the C gene products were essential for the production of HBV particles.

J Neurosci Methods, 1987 May, 20(1), 83 - 90
Quantitative determination of glutamate mediated cortical neuronal injury in cell culture by lactate dehydrogenase efflux assay; Koh JY et al.; Measurement of lactate dehydrogenase (LDH) activity released to the extracellular bathing media has been found to be a simple yet quantitative method for assessing glutamate mediated central neuronal cell injury in cortical cell culture . Extracellular LDH is both chemically and biologically stable; the magnitude of LDH efflux in the cultures correlates in a linear fashion with the number of neurons damaged by glutamate exposure.

Vopr Virusol, 1987 May-Jun, 32(3), 360 - 2
{Isolation of the human rotavirus in a cell culture of green monkey kidneys}; Vasil'eva VI et al.; Properties of a virus isolated from a patient with rotavirus gastroenteritis (the diagnosis verified by direct electron microscopy) were studied . The virus was identified as a rotavirus in tissue culture neutralization and complement fixation tests . It was shown to replicate through a significant number of passages in continuous cell cultures (Vero, RH) as well as in primary cell cultures of human and animal origin (human embryo kidney, green monkey kidney); some features associated with rotavirus propagation are described.

Vopr Virusol, 1987 May-Jun, 32(3), 307 - 12
{Accumulation of the infectious virus and the viral antigen during the multiplication of the hepatitis A virus in an embryonic kidney cell culture of the rhesus macaque (FRhK-4)}; Kusov IuIu et al.; Growth characteristics of the HAS-15 strain of human hepatitis A virus (HAV) in rhesus monkey foetal kidney cell line (FRhK-4) are described . The conditions optimal for the accumulation of infectious HAV and viral antigen (HAAg) in the infected cells and tissue culture fluids were studied . The production of infectious HAV occurred in the first stage while in the second stage predominantly HAAg was accumulating intracellularly . Serological properties of the cultivated HAV proved to be very similar to those of the HAV occurring in hepatitis A patients.

Scand J Clin Lab Invest, 1987 May, 47(3), 207 - 13
Application of an enzyme-linked immunoassay for the measurement of pregnancy zone protein (PZP) in cell culture supernatants and sera; Povlsen JV et al.; A simple and sensitive enzyme-linked immunosorbent assay (ELISA) measuring specifically the pregnancy zone protein (PZP) was constructed . The assay range was 2.0-500 micrograms/l . The intra-assay coefficient of variation (CV%) was 5.9% at the level of 100 micrograms/l and 3.5% at 10 micrograms/l . The imprecision between runs was 4.5% at 100 micrograms/l and 7.6% at 10 micrograms/l . Recovery of the native PZP standard added to serum-free cell culture medium was 98.1 +/- 3.7% (mean +/- SD), and recovery from serum of women in late pregnancy was 96.0 +/- 9.3% . Recovery from PZP-chymotrypsin (PZP-CT) complexes added to serum-free medium was 141 +/- 4.3% . There was no detectable cross-reactivity between the anti-human PZP antibody and human alpha 2-macroglobulin (alpha 2-M) . The dose-response of two PZP standards and the PZP serum concentrations of 100 blood donors were determined . Furthermore, the serum level of PZP from 11 patients suffering from IgA myeloma was quantitated and found within the normal range when compared to serum levels of healthy blood donors of the same age and sex . Finally, supernatants from serum-free cultures of different human peripheral blood mononuclear cell (PBM) subpopulations were assayed . Neither of them were found to exhibit any detectable increase in PZP concentration during culture, but cultures of monocytes were found to produce alpha 2-M.

Biochim Biophys Acta, 1987 Apr 2, 928(1), 56 - 62
Adaptations of neurotransmitter synthesis to chronic hypoxia in cell culture; Feinsilver SH et al.; Tyrosine hydroxylase and tryptophan hydroxylase are widely held to be rate-limiting for the synthesis of the catecholamines and serotonin, respectively . Both enzymes are oxygen-requiring and kinetic properties suggest that oxygen availability may limit synthesis of these neurotransmitters in the brain . Using pheochromocytoma cells as a cell culture model for catecholamine synthesis, and neuroblastoma cells as a model for serotonin synthesis, enzyme activity was measured under control and hypoxic conditions . Both tyrosine hydroxylase and tryptophan hydroxylase activity increased substantially with chronic exposure but not with acute exposure . In the case of tyrosine hydroxylase, increased enzyme content with hypoxia accounts for increased activity . This suggests a mechanism for the maintenance of neurotransmitter synthesis with chronic hypoxia . Measurement of intracellular metabolites revealed no change in dopamine or norepinephrine in hypoxic pheochromocytoma cells, consistent with a simple adaptive mechanism . However, in neuroblastoma cells, hypoxia was associated with an increase in serotonin concentration . The reasons for this are still unclear.

J Clin Endocrinol Metab, 1987 Apr, 64(4), 713 - 7
Angiotensin II promotes prolactin release from normal human anterior pituitary cell cultures in a calcium-dependent manner; Malarkey WB et al.; Renin and angiotensin II (AII) have been demonstrated in the mammalian central nervous system, and AII has been found to promote PRL release in the rat and monkey . We added AII to monolayer cultures of human anterior pituitary cells and found significant PRL release by 30 min with concentrations of AII as low as 10(-10) M . This AII-induced PRL release was inhibited by the specific AII antagonist saralasin . AII-induced PRL release was a calcium-dependent process, since the calcium channel blockers verapamil and nifedipine as well as the calcium-calmodulin antagonist R2471 significantly inhibited AII-induced PRL release . Prostaglandins E2, A2, and F2 alpha also inhibited AII-induced PRL release . The significance of this latter observation is not clear, however, as indomethacin, an inhibitor of the cyclo-oxygenase prostaglandin metabolic pathway, had no effect on AII-induced PRL release . In light of recent immunohistochemical evidence of the presence of renin, angiotensinogen, and converting enzyme in human lactotrophs, our data support the concept that AII may be an important autocrine regulator of PRL secretion.

Cancer Res, 1987 Apr 1, 47(7), 1751 - 5
Transient requirement for prolactin as a growth initiator following treatment of autonomous Nb2 node rat lymphoma cell cultures with butyrate; Gout PW; A previous study described the development of cultures of Nb2 node rat lymphoma cells which specifically require a lactogenic hormone, e.g., prolactin (PRL), as a growth factor . In the present study, sublines of these cultures have been established, including clones, which do not depend on exogenous lactogens for growth; the cells do not appear to produce autocrine, PRL-like substances . Although these autonomous cells grew readily in the complete absence of lactogens, their growth rate was stimulated (up to approximately 30%) by PRL concentrations greater than or equal to 0.1 ng/ml . When cultures of the lactogen-dependent and of the cloned, autonomous lymphoma cells were incubated for 3 days with sodium n-butyrate (NaBT; 2 mM), cells were arrested in the G1 phase of the cell cycle, as shown by flow cytometric analysis . A substantial proportion of the G1-arrested cells was viable and readily resumed growth, within 1 day, when transferred to NaBT-free medium containing PRL (1-100 ng/ml) . The resumption of growth of the cells from the lactogen-dependent cultures was critically dependent on PRL; in its absence cells lysed . Surprisingly, the rapid recovery of the cells from the lactogen-independent cultures also depended on the presence of PRL in the medium; in its absence growth did not resume for at least 2.3 days . The acquired need of the NaBT-treated, autonomous cells for PRL was only transient, since such cells reverted fully to the PRL-independent state within about 3 days of culturing in PRL-containing, NaBT-free medium . It is proposed, as a working hypothesis, that the autonomous lymphoma cells can be mitogenically activated by two different pathways, one requiring exogenous lactogens and another which is independent of lactogens; the latter pathway recovers much more slowly from the treatment with NaBT than the lactogen-dependent pathway . This model could explain the sensitivity of the autonomous lymphoma cells to PRL and their transient dependency on exogenous lactogens during their recovery from NaBT treatment . NaBT would appear to be a useful agent for studying the mechanism(s) by which the autonomous lymphoma cells circumvent the need for mitogenic lactogens.

Pharmazie, 1987 Apr, 42(4), 245 - 8
{Prediction of LD50 values by cell culture}; Halle W et al.; Alternative approaches to toxicity tests with animals-more precisely defined as complementary methods-serve for reducing the number of animal experiments . The determination of cytotoxicity with cell cultures could be an important complementary method to get more information about a general basal toxicity of a substance in an organism during the course of an acute toxicity experiment . We employed the endothelial cell line BKEz-7 in a general (i.e . nonspecific) cytotoxicity test . As a quantitative marker of cytotoxic effects we determined the inhibition concentration for a fifty per cent reduction of the cell number per culture (IC50) . the IC50 values of 28 compounds were compared with the corresponding LD50 values in the acute toxicity experiments . In these comparisons we included the determination of the lethality index (LI = IC50/LD50), the correlation analyses and the application of a single linear regression model . The following results were obtained . Between the IC50 and LD50 values a positive correlation exists at the significance level 1 - alpha = 0.95 . After plotting the regression line in a coordinate system, a novel graphic prediction of LD50 estimations is possible with a relatively high accuracy . On the basis of tenfold steps of concentrations a new scale of cytotoxicity was attributed to the internationally employed classification of LD50 . In the cytotoxicity test with the cell line BKEz-7 a broad spectrum of substance classes can be tested . The cytotoxicity test and the comparison of IC50 and LD50 p.o . as described here contribute to reduce the number of animals in the acute toxicity experiments.

J Clin Lab Immunol, 1987 Apr, 22(4), 201 - 3
A simple method for detecting anti-tubular membrane antibodies in rat lymphoid cell cultures; Fierro C et al.; The application of enzyme-linked immunosorbent assay (ELISA) to short cell cultures has proved to be useful in detecting immunoglobulins secreted to supernatants . This paper describes a modified ELISA to detect and quantify the production of specific anti-tubular basal membrane IgG released in vitro by lymphoid cells from Brown Norway rats with tubulointerstitial nephritis . This method uses 4-methyl-umbelliferyl-phosphate as substrate and allows to detect approximately concentrations 100 times less than the detectable concentration of visibly colored substrates . This method eliminates the need for mitogens, is precise and reproducible and the use of 4-methyl-umbelliferyl-phosphate allows a substantial increase in sensitivity.

Cell Struct Funct, 1987 Apr, 12(2), 165 - 71
Ganglioside composition of growth cone-deficient nerve cell cultures; Asou H et al.; The ganglioside concentration and composition in growth cone-deficient nerve cells, induced by inclusion of cytochalasin B (CB) are compared with those of 2-day-old control cells from primary cultures of embryonic rat cerebral cortex . Ganglioside GM1 and GD1a are the major gangliosides in the growth cone . Ganglioside GM1 may be one of the membrane components of growth cones that function in neural recognition during development.

Hybridoma, 1987 Apr, 6(2), 219 - 28
Simple and rapid purification of monoclonal antibodies from cell culture supernatants and ascites fluids by hydroxylapatite chromatography on analytical and preparative scales; Bukovsky J et al.; A simple and rapid method for the purification of murine and human monoclonal antibodies from ascites fluids and cell culture supernatants is described . The method, based on the use of hydroxylapatite (HAP) column chromatography, is applicable on both analytical and preparative scales . In our work on purification of monoclonal antibodies, we have found that the combination of a single step elution of impurities followed by linear gradient elution of antibody provides an excellent purification of the antibody from cell culture and ascites fluids . The procedure provides very good resolution at high flow rates . The cell culture supernatant can be pumped on the preparative column at the rate of 2-3 ml/min without any measureable back pressure . The binding is independent of the flow rate . This method has been successfully used to purify several monoclonal antibodies of different subtypes from cell culture supernatants.

J Neurosurg, 1987 Apr, 66(4), 588 - 94
DNA in meningioma tissues and explant cell cultures . A flow cytometric study with clinicopathological correlates; Ironside JW et al.; Flow cytometry was performed on stored frozen tissues and explant cell cultures from 39 meningiomas using ethidium bromide and mithramycin in a selective staining technique for deoxyribonucleic acid (DNA) . The ploidy index and percentage of cells in the G0/G1, S, and G2/M phases were calculated for each specimen . The results were compared with the age and sex of the patients; the site, the histological subtype, and mitotic rate of the neoplasms; and the estrogen- and progesterone-receptor levels assayed in cytosol-enriched supernatants from cryostat-cut sections . Sixteen neoplasms (41%) were aneuploid . These included two recurrent neoplasms, seven of the eight neoplasms from patients with multiple meningiomas, and three clinically aggressive neoplasms (one hemangiopericytic and two anaplastic meningiomas) . Significant correlations were found between values for the ploidy index (r = 0.75, p less than 0.01), the percentage of S-phase cells (r = 0.82, p less than 0.01), and the percentage of G2/M-phase cells (r = 0.69, p less than 0.05) in vivo and in vitro . The results support the suggestion that flow cytometry for DNA in meningiomas may be of value in predicting the behavior of these neoplasms, and indicate that under controlled conditions explant cell cultures may provide a useful model for the proliferative characteristics of meningiomas in vivo.

Mol Toxicol, 1987 Apr-Sep, 1(2-3), 209 - 16
Comparative cytotoxicity of aflatoxin B1 and saxitoxin in cell cultures; Gabliks J et al.; Human and animal cell cultures were evaluated for their susceptibility to two environmental toxins found as contaminants in human food supplies: aflatoxin B1, a hepatotoxin produced by the mold Aspergillus flavus, and saxitoxin, a paralytic neurotoxin produced by the marine dinoflagellate Gonyaulax catenella . Both toxins cause food poisoning in humans and other animals . The acute cytotoxicity of both toxins was measured and compared by inhibition of cell growth and by progressive cytopathogenicity resulting in cell destruction . Aflatoxin B1 was cytotoxic to all of the 11 primary kidney cultures derived from susceptible animals . The cell growth inhibition 10% values (TD10) ranged from 0.02 to 6.0 micrograms/ml: mouse (TD10 = 0.02 micrograms), guinea pig (0.03 micrograms), rat (0.07 micrograms), hamster (0.16 micrograms), monkey (0.1 microgram), human (0.7-1.5 micrograms), chick (0.05 micrograms), and duck (6.0 micrograms) . The corresponding TD50 levels were about 10 times higher concentrations and caused cell destruction within 2 d . Saxitoxin did not induce cytotoxicity manifestations in cultures derived from susceptible species--mouse kidney, human carcinoma HeLa line, chick embryo, and goldfish fin (CAR) cell line--at high concentration levels up to 5 micrograms/ml . When the same toxin preparation at only 1 microgram was injected into mice, the animals died immediately . The results indicate that animal cell cultures are useful for studies of general cytotoxins that affect common essential metabolism but cannot be used to detect environmental toxins that cause toxic manifestations by an interference with specific physiological functions of organ systems.

J Endocrinol, 1987 Apr, 113(1), 103 - 10
Inhibin-like activity in Sertoli cell culture media and testicular homogenates from rats of various ages; Ultee-van Gessel AM et al.; The influence of age on testicular inhibin in untreated, neonatally hemicastrated and prenatally irradiated rats was studied using in-vivo and in-vitro experiments . In testicular cytosols prepared from 1-, 7-, 14-, 21-, 42- and 63-day-old rats concentrations of testicular inhibin could be measured with an in-vitro bioassay method using dispersed pituitary cells . Preparations of testicular cytosols caused a dose-dependent suppression of pituitary FSH secretion, whereas no effects were found on LH secretion . Testicular content of inhibin increased gradually with age, while after 14 days of age a relatively large increase of peripheral FSH concentrations occurred in all experimental groups . Neonatal hemicastration or prenatal irradiation resulted in decreased inhibin content of the testis and increased plasma FSH levels . The production of inhibin activity by Sertoli cells obtained from 7-, 14-, 21-, 42- and 63-day-old normal rats was measured during a 24-h incubation period on the third day of culture . The inhibin production per 10(6) plated Sertoli cells decreased rapidly after 14 days of age and the lowest production of inhibin was found in Sertoli cells from rats of 63 days of age . After pre-incubation with ovine FSH significantly larger amounts of inhibin activity were detected in spent media from 21-day-old rat testes . In contrast, suppression of inhibin production was found after pre-culture in the presence of testosterone at most of the ages studied . These data from in-vivo and in-vitro experiments indicate that a reciprocal relationship exists between pituitary FSH secretion and inhibin production before the age of 21 days.(ABSTRACT TRUNCATED AT 250 WORDS)

J Clin Endocrinol Metab, 1987 Apr, 64(4), 783 - 8
Identification of inhibin secreted by cynomolgus monkey Sertoli cell cultures; Noguchi K et al.; An inhibin was identified in the media of primary Sertoli cell-enriched cultures from the cynomolgus monkey, Macaca fascicularis, and some of its biochemical properties were studied . Conditioned monkey Sertoli cell culture medium (m-SCCM), when added to pituitary cells from 6-week-old male rats, inhibited the basal secretion of FSH but not that of LH . This specificity was lost after the addition of GnRH; mSCCM inhibited not only FSH but also LH release, determined by both RIA and mouse interstitial cell bioassay, from pituitary cells exposed for 6 h to 10 nM GnRH . FSH-inhibiting activity persisted when m-SCCM was boiled for 30 min, but activity was lost after incubation for 1 h at 37 C with 0.1% trypsin . m-SCCM inhibin activity was completely retained by Concanavalin A-Sepharose and could be eluted with 0.2 M alpha-methyl-D-glucoside . Gel filtration high pressure liquid chromatography with a Superose-12 column revealed inhibin activity between 20-60K, with the greatest activity at 40K . Our results indicate that primate Sertoli cells produce an inhibin-like factor which could play a role in controlling gonadotropin secretion in males.

J Med Virol, 1987 Apr, 21(4), 329 - 37
Use of monoclonal antibodies for the diagnosis of cytomegalovirus infection by the detection of early antigen fluorescent foci (DEAFF) in cell culture; Stirk PR et al.; A pool of seven monoclonal antibodies, each reactive with cytomegalovirus (CMV) early antigens, was used in an indirect immunofluorescence method for the rapid detection of CMV-infected fibroblasts following inoculation with clinical specimens . A total of 1,639 specimens were examined, and the results were compared with those of conventional isolation procedures . The detection of CMV by early antigen fluorescent foci (DEAFF) was found to be comparable, both in terms of specificity and sensitivity, to that of conventional cell culture . Its great advantage, however, is the rapidity with which results are achieved . Thus, results were available from the DEAFF test within 24 hours of receipt of the specimens as compared to a mean of 16 days for cell culture . This single rapid assay for the detection of CMV in clinical samples may be performed by any laboratory familiar with cell culture techniques and in our hands is the preferred diagnostic method for CMV.

In Vitro Cell Dev Biol, 1987 Apr, 23(4), 317 - 22
N-terminal amino acid sequence of leukemia derived growth factor (LGF) from human erythroleukemia cell culture; Mihara A et al.; A human erythroleukemia cell line, K-562 T1, was adapted to a protein-free chemically defined medium; that is, the medium does not contain any proteins such as exogenous hormones, growth factors, serum and serum albumin . The K-562 T1 cells which can proliferate in a protein-free medium are one of the model systems suitably supporting the autocrine hypothesis, which claims that cancer cells produce and respond to their own growth factors . The K-562 T1 cells were cultured in a protein-free medium at large scale and the growth factors were purified from the conditioned medium . It was found that K-562 T1 cells produce at least two growth factors; one is LGF-I (leukemia-derived growth factor-I) which can stimulate the proliferation of a wide range of human leukemia cell lines and the other is LGF-II (leukemia-derived growth factor-II), which can contribute to the growth of fibroblasts . LGF-I was purified using QAE-Sephadex, Bio Gel P-60 and Mono S FPLC . The purified protein was found to be homogenous by SDS-polyacrylamide gel electrophoresis and NH2-terminal sequence analysis . The molecular weight of LGF-I was 20,000 by SDS-polyacrylamide gel electrophoresis . The 30 NH2-terminal residues of LGF-I are the same as that of ubiquitin . Ubiquitin is a protein found in eukaryotic cells with molecular weight of 8,600 . In the nucleus ubiquitin is conjugated to histone 2A to form the nuclear protein A24 which may play a role in regulation of chromatin structure, and in the cytoplasm is part of an ATP-dependent non-lysosomal proteolytic pathway . However, its physiological significance has not yet been fully resolved . Ubiquitin purified from bovine thymus did not show cell proliferating activity for any cells tested . The results suggest that LGF-I is a new autocrine growth factor with a molecular weight of 20,000 daltons, containing ubiquitin at the NH2-terminal end.

J Gen Virol, 1987 Apr, 68 ( Pt 4), 1183 - 6
Enhanced production of a human spumavirus (Retroviridae) in semi-permissive cell cultures after treatment with 5-azacytidine; Hotta J et al.; Infection by a human spumavirus of human foetal diploid lung (HFDL) cells was found to be productive with virus titres ranging from 10(3) to 10(5) p.f.u./ml . In contrast, infection of recovered amnion (RA) aneuploid cells resulted in a persistent infection with less than 100 p.f.u./ml infectious virus produced . The decreased sensitivity of RA cells to the spumavirus was not due to the failure of virus to penetrate into the cell since infectious virus was not produced even after transfection of infectious proviral DNA . The effect of 5-azacytidine, an inhibitor of DNA methylation, on virus replication was examined . Whereas virus production in HFDL cells was not affected, there was a 100-fold increase in virus yield in RA cells treated with the drug for at least 48 h and maximum virus yields were obtained 4 days post-infection.

In Vitro Cell Dev Biol, 1987 Apr, 23(4), 279 - 87
Characteristics of long-term human epithelial cell cultures derived from normal human fetal kidney; Gibson-D'Ambrosio RE et al.; Epithelial cell cultures were prepared from normal human fetal kidney and established in long-term culture . The growth characteristics and production of keratin, and alkaline phosphatase (AP) and gamma-glutamyl transpeptidase (GGT) activities were compared in a modified minimal essential medium (mMEM), D-valine-containing modified alpha-MEM (mALPHA) and L-valine mALPHA . The mean number of cumulative population doublings (CPDL) was significantly (P less than 0.001) enhanced with the L-valine mALPHA (40.8 CPDL) over that achievable in mMEM (14.2 CPDL) or D-valine mALPHA (18.3 CPDL) media . In all three media, greater than 95% of the cells in culture produced keratin throughout the life span of these cultures . Surface-associated fibronectin was absent in these cell cultures . AP and GGT activities increased as a function of subpassage and time in culture, with the greatest activity in the L-valine mALPHA . The expression of these renal cell-associated functions suggests that these cells in culture are proximal tubule epithelial cells . The conditions and procedures described in this paper can provide a human kidney epithelial cell culture system for studying human renal function, metabolism, cytotoxicity, genotoxicity, and transformation.

Biochem Biophys Res Commun, 1987 Mar 30, 143(3), 863 - 71
Vitamins E and K induce aryl hydrocarbon hydroxylase activity in human cell cultures; Chen YT et al.; Two fat soluble vitamins, Vitamins E and K, when added into culture medium, were found to increase aryl hydrocarbon hydroxylase activity in human cultured cells . The extent of induction in a hepatoma-derived cell line (Hep G2) by these vitamins is of similar magnitude to those cells receiving benz{a}anthracene; whereas in a mammary tumor-derived cell line (MCF-7), benz{a}anthracene is the best inducer for the hydroxylase activity . The increase of the hydroxylase activity is associated with increased levels of a specific mRNA coding for polynuclear aromatic hydrocarbons-induced form of cytochrome P-450 with Vitamins E and K treatment . The size of the induced mRNA is 3.3 kilobase which is the same as that of benz{a}anthracene treatment.

J Biol Chem, 1987 Mar 25, 262(9), 4024 - 33
Alpha-melanocyte-stimulating hormone regulation of tyrosinase in Cloudman S-91 mouse melanoma cell cultures; Fuller BB et al.; alpha-MSH (melanocyte-stimulating hormone) causes an increase in tyrosinase activity (O-diphenol-O2 oxidoreductase; EC 1.14.18.1) in Cloudman S-91 mouse melanoma cell cultures following a lag period of approximately 9 h . Treatment of cells with 2 X 10(-7)M alpha-MSH for 6 days results in a 90-fold increase in the specific activity of the enzyme . The hormone-mediated increase in tyrosinase activity is dependent upon continued transcription since the enzyme induction is suppressed by either cordycepin (1 microgram/ml) or alpha-amanitin (10 micrograms/ml) . Immunoprecipitation analysis of pulse-labeled tyrosinase from control and MSH-treated cultures (48-h exposure) has demonstrated that MSH stimulates tyrosinase synthesis by approximately 4-fold, a level of induction which does not correspond to the observed 14-fold increase in enzyme activity . When immunotitration curves were developed from cell extracts of control and MSH-treated cultures using immunoprecipitation and competitive enzyme-linked immunosorbent assay protocols, evidence for the presence of immunologically active but catalytically less active enzyme in untreated melanoma cell cultures was demonstrated . Degradation rates of tyrosinase were found to be similar in control cultures or in cells treated with MSH for up to 48 h . Taken together, these results suggest that in addition to stimulating tyrosinase synthesis, MSH may also promote an increase in the catalytic efficiency of the enzyme.

J Neuropathol Exp Neurol, 1987 Mar, 46(2), 130 - 40
Hyperosmotic urea reversibly opens the tight junctions between brain capillary endothelial cells in cell culture; Dorovini-Zis K et al.; The effect of hyperosmotic solutions of urea on primary cultures of bovine brain microvessel endothelial cells was examined . Confluent monolayers of cells positive for Factor VIII-related antigen were obtained by seven days in culture . The cells were: incubated in media containing 1 M, 2 M or 3 M urea and horseradish peroxidase (HRP) for various periods of time and then examined by transmission electron microscopy . Exposed to hypertonic urea solutions and 14C-3-O-methyl-D-glucose for determination of the intracellular water space . In control cultures endothelial cells were bound together by tight junctional complexes over 91% of which excluded HRP . In experimental cultures 82% of the interendothelial clefts became permeable to HRP after one minute of incubation with 3 M urea . The degree of cell shrinkage corresponded well with the extent of junctional opening . In monolayers examined 24 hours following removal of urea from the media more than 63% of the intercellular clefts were impermeable to HRP . These observations indicate that hyperosmotic solutions of urea reversibly open the tight junctions between brain microvessel endothelial cells in tissue culture . Decrease in cell volume appears to be linked to the increased junctional permeability.

Farmakol Toksikol, 1987 Mar-Apr, 50(2), 121 - 4
{Use of cell cultures in the pharmacological experiment}; Strelkov RB et al.; A statistical method is proposed which makes it possible to evaluate quantitatively the significance and reliability of the effects of pharmacological agents during their study on cell cultures using a little scope of material samples . The method substantially reduces the expenditures of the researcher's time and labour.

An Esp Pediatr, 1987 Mar, 26(3), 215 - 7
{Cell cultures of the epithelium of the bile ducts: a possible model for the study of atresia of the bile ducts}; Schier F et al.; The pathogenic mechanism of biliary atresia is still unknown . Assuming that primary lesion is localized at the epithelial layer of the biliary ducts, authors describe a technique of culturing biliary duct cells in monolayer fashion . Light and electron microscopy demonstrate the epithelial origin of the cultured cells . These cells can be used to test some of the pathogenic theories in vitro hitherto proposed.

J Med Virol, 1987 Mar, 21(3), 201 - 5
Isolation of Inoue-Melnick virus from human meningioma-derived cell cultures and detection of antibody in patients with meningioma; Inoue YK et al.; Inoue-Melnick virus (IMV) was isolated from six of seven human meningioma-derived cell cultures, while the virus was not isolated from six other brain tumor cell cultures . Sera of 145 consecutive neurosurgical inpatients were tested for IMV-neutralizing antibody . Of 26 patients with meningioma, 22 were positive for IMV antibody (84.6%) . Of the remaining 119 patients, 16 were positive.

Exp Hematol, 1987 Mar, 15(3), 226 - 33
Plasma erythropoietin assay by a fetal mouse liver cell culture method with special reference to effective elimination of erythroid colony inhibitor(s) in plasma; Sakata S et al.; Methods for the elimination of an inhibitor(s) of erythroid colony formation from plasma were examined in an attempt to measure genuine plasma erythropoietin (Epo) activities with an erythroid colony-forming assay using fetal mouse liver cells . Acid-boiling-chloroform (ABC) treatment was concluded to be the best method because the plasma thus treated stimulated colony formation most and contained the least protein . The dose-response curve for the plasma was parallel to that for the standard Epo preparation . The "erythroid colony-stimulating activity" in the plasma was completely additive to that in the standard Epo, and appeared to be a relatively heat-stable and acid protein with an isoelectric point lower than 5.0 . These results suggest that the activity in the plasma is identical to that in the standard Epo . Stability of the plasma Epo activity was dependent on storage temperature and enhanced by adding 1% bovine serum albumin (BSA) . Average Epo titers for normal adult, full-term cord, and murine plasmas, all ABC-treated and with 1% BSA added, were 192.4, 184.5, and 150.6 mU/ml, respectively . These values were much higher than those measured by the in vivo standard polycythemic mouse assay.

J Virol Methods, 1987 Mar, 15(4), 313 - 22
Purification of infectious canine parvovirus from cell culture by affinity chromatography with monoclonal antibodies; Rimmelzwaan GF et al.; Immuno affinity chromatography with virus neutralizing monoclonal antibodies, directed to the haemagglutinating protein of canine parvovirus (CPV) was used to purify and concentrate CPV from infected cell culture . The procedure was monitored by testing the respective fractions in an infectivity titration system, in an ELISA, in a haemagglutination assay and by negative contrast electron microscopy to quantify CPV or CPV antigen . The degree of purification was further estimated by testing the fractions for total protein content in a colorimetric method, for bovine serum albumin content in an ELISA and by SDS-PAGE . Over 99% of the contaminating proteins proved to be removed, and 20% or 70-90% of infectious CPV or CPV antigen, respectively, was recovered.

Ann Inst Pasteur Immunol, 1987 Mar-Apr, 138(2), 181 - 99
T-cell-dependent modulation of the polyclonal B-lymphocyte responses in normal spleen cell cultures stimulated by lipopolysaccharide; Bjorklund M et al.; The in vitro polyclonal B-cell proliferative and plaque-forming cell (PFC) responses to the T-independent (TI) mitogen lipopolysaccharide (LPS) are increased by the addition of normal syngeneic splenic T cells . Normal irradiated Lyt-2- T cells also alter the IgG subclass distribution from the typical predominance of IgG3 and IgG2b PFC to the appearance of IgG1, IgG2a and IgA PFC in T-cell-depleted spleen cell (SC) cultures . Furthermore, secondary LPS blast cultures yield increased PFC responses when co-cultured which syngeneic fresh normal T cells which, even in the absence of mitogen, induce PFC responses in such activated B cells . As LPS blasts induce normal syngeneic T cells to proliferate and significant numbers of L3T4+ blast cells are found in LPS-stimulated normal spleen cell cultures, we conclude that T cells actively participate in the regulation of these responses . The significance of these findings for the regulation of TI responses in vivo by "autoreactive" T cells is considered.

Mutat Res, 1987 Mar, 177(1), 95 - 100
Studies on mutagen-sensitive strains of Drosophila melanogaster . X . Repair of radiation-induced DNA damage in primary cell cultures after irradiation with X-rays; Vegt GB et al.; The repair of X-ray-induced DNA lesions in repair-deficient mutant strains was studied as a way of investigating the mechanism of the induction of genetic damage . Genetic effects on the recovery of X-ray-induced damage by the repair-deficient strains ebony (photoreactivation repair-deficient) and mus(1)101D1 (post-replication repair-deficient) were interpreted as impaired repair of single- and double-strand DNA breaks . We investigated the repair of X-ray-induced DNA breaks and alkaline-labile sites in primary cell cultures of ebony and mus(1)101D1 and in cultures of their control strains . No significant differences were found between the repair rates in the mutants and control strains . This indicates that the genetic effects of these mutants are not due to an impaired rate of repair of DNA breaks.

J Virol Methods, 1987 Mar, 15(4), 329 - 30
Rapid diagnosis of herpes simplex virus infections in conventional and shell vial cell cultures using monoclonal antibodies; Winter GF et al.; One hundred specimens for herpes simplex virus (HSV) isolation were tested in parallel by conventional and by centrifugation-enhanced cell culture, followed by identification using monoclonal antibodies to HSV-1 and HSV-2 . Sensitivity was comparable by the two methods; conventional culture was only marginally slower and was easier to fit into the routine of a busy laboratory . It is, therefore, advocated for HSV detection in clinical specimens.

J Virol Methods, 1987 Mar, 15(4), 323 - 8
Demonstration of hepatitis A virus in cell culture by electron microscopy with immunoperoxidase staining; Asher LV et al.; Virus-like particles were demonstrated by electron microscopy in BS-C-1 cells infected with hepatitis A virus (HAV) . Particles were usually enclosed within vesicles and accompanied by myelin-like membranous structures . Less often they were seen free in the cytoplasm . They were never observed in the nucleus . By immunoperoxidase staining particles were found to contain HAV antigens . These antigens were also found in the membrane of the vesicles surrounding the masses of particles and adjacent parts of the mitochondrial membranes . Our results demonstrate the usefulness of an electron microscopic immunocytochemical technique to study replication of HAV.

Gastroenterology, 1987 Mar, 92(3), 669 - 77
Prostaglandin protects against taurocholate-induced damage to rat gastric mucosal cell culture; Terano A et al.; Prostaglandins protect gastric mucosa against noxious agents, but it is unknown whether this protection includes a direct action on the cells themselves, this action is limited to damaging agents that inhibit prostaglandin synthesis, or cellular cyclic adenosine monophosphate is the mediator . The present study tested these questions in cultured gastric mucous epithelial cells . The effect of 16,16-dimethyl prostaglandin E2 on cellular cyclic adenosine monophosphate level and the effect of 16,16-dimethyl prostaglandin E2, dibutyryl cyclic adenosine monophosphate, and isobutyl methyl xanthine on taurocholate-induced damage to cultured rat gastric mucosal cells was determined . As parameters of cell damage, the trypan blue dye exclusion test and 51Cr-release were employed . Taurocholate significantly increased 51Cr-release in a dose-dependent manner and decreased the number of viable cells . 16,16-Dimethyl prostaglandin E2 (1.0 microM) diminished the cell damage caused by 10 mM taurocholate (p less than 0.01) and increased cyclic adenosine monophosphate levels . Prostaglandin F2 alpha but not prostaglandin I2 was also cytoprotective . Addition of dibutyryl cyclic adenosine monophosphate (1.0 mM) and isobutyl methyl xanthine while significantly increasing cyclic adenosine monophosphate levels did not significantly reduce taurocholate-induced cell damage . Thus, in vitro 16,16-dimethyl prostaglandin E2 directly protects gastric mucous cells against taurocholate-induced injury, direct prostaglandin cytoprotection is not limited to damaging agents that inhibit prostaglandin synthesis, and cyclic adenosine monophosphate levels do not correlate with gastric mucosal cell damage and may not be involved in the direct protective effect of prostaglandins.

J Virol, 1987 Mar, 61(3), 829 - 39
Deletion mutants in the gene encoding the herpes simplex virus type 1 immediate-early protein ICP0 exhibit impaired growth in cell culture; Sacks WR et al.; We report the construction and characterization of deletion mutants in the herpes simplex virus type 1 gene encoding the immediate-early protein ICP0 . In the event that ICP0 proved to play an essential role in virus replication, ICP0-transformed Vero cells were generated to serve as permissive hosts for such mutants . Two mutants, dlX0.7 and dlX3.1, were isolated in these cells by a marker rescue-transfer procedure involving the rescue of an ICP4 deletion mutant and the simultaneous insertion of a linked deletion in the ICP0 gene . Mutant dlX0.7 contained a 700-base-pair deletion in both copies of ICP0 . The deletion lay entirely within the transcript specified by the gene . dlX0.7 induced the synthesis of an ICP0-specific mRNA that was approximately 0.7 kilobases smaller than the corresponding mRNA specified by wild-type virus . The 3.1-kilobase deletion in both copies of the ICP0 gene in mutant dlX3.1 removed the majority of the transcriptional-regulatory signals and coding sequences, retaining only sequences at the 3' end of the gene . As expected, no ICP0-specific mRNA was detected in dlX3.1-infected Nero cells (G418-resistant Vero cells) . Both mutants grew in all cells tested, although their burst sizes were 10- to 100-fold lower than that of wild-type virus . Although the plaque sizes of dlX0.7 and dlX3.1 were equally small on Nero and ICP0-transformed cells, the plating efficiency of the mutants was 15- to 50-fold greater on ICP0-transformed cells than on Nero cells . The mutants exhibited modest interference with the growth of wild-type virus in mixed infections, an effect that was abolished by UV irradiation of the mutants, implying that interference required viral gene expression . Polypeptide profiles generated by the mutants in Nero cells were qualitatively similar to that of wild-type virus . Quantitatively, only slight reductions in the levels of certain late viral polypeptides were observed, a phenomenon also borne out by analysis of viral glycoproteins . Both mutants induced the synthesis of significant, although reduced, levels of viral DNA relative to wild-type virus . Taken together, the results demonstrate that ICP0 is not essential for productive infection in cell culture but that this protein plays a significant role in viral growth, as indicated by the impaired abilities of the mutants to replicate.

Dev Biol, 1987 Mar, 120(1), 101 - 11
Cell lineages in peripheral nervous system ontogeny: medium-induced modulation of neuronal phenotypic expression in neural crest cell cultures; Ziller C et al.; Neural crest, taken from cephalic and trunk levels of quail embryos, was grown in vitro in conventional tissue culture medium (Dulbecco's modified Eagle's medium containing 15% fetal calf serum and either 2 or 15% chick embryo extract (CEE} or in a chemically defined serum- and CEE-free medium . Depending on the conditions employed, different types of neuronal or neuronlike cells developed in the cultures . Thus, in medium containing 15% CEE, adrenergic cells (identified by tyrosine hydroxylase immunoreactivity and catecholamine histofluorescence) emerged after 5-6 days . These cells lacked tetanus toxin binding sites and did not react with an antibody directed against 70-kDa neurofilament protein . In the fully defined medium, a neuronal cell type exhibiting neurofilament and substance P (SP) immunoreactivity differentiated from noncycling precursors within 1 or 2 days of culture . If serum was added to the medium, the neurites disintegrated and the neuronal cells ultimately died . By sequentially culturing neural crest, first in the wholly synthetic medium for 1-3 days and then in the conventional medium supplemented with serum and 15% CEE, the disappearance of the SP-positive neurons was followed, several days later, by the emergence of adrenergic cells . The majority of these cells and/or their precursors were found to undergo cell division in culture . We conclude that the cells expressing the adrenergic phenotype (characteristic of the sympathetic nervous system) and those displaying SP immunoreactivity, comparable to a category of neurons in dorsal root and cranial sensory ganglia, derive from distinct sets of precursors . Our results reinforce the contention, deduced from in ovo transplantation experiments (see N . M . Le Douarin, (1984) In Cellular and Molecular Biology of Neuronal Development (I . Black, Ed.), pp . 3-28 . Plenum, New York), that at least two lineages, from which sensory and autonomic cell types are derived respectively, are segregated early during neural crest ontogeny and have extremely different survival and trophic requirements.

J Immunol Methods, 1987 Feb 26, 97(1), 11 - 7
Use of a liquid cell culture assay to quantify the elimination of Burkitt lymphoma cells from the bone marrow; Philip I et al.; A new liquid cell culture assay for propagating Burkitt lymphoma (BL) cells in the presence of excess allogeneic irradiated bone marrow (BM) has been standardized . The Burkitt cell lines used in this assay were newly established from the tumours of our patients and were either EBV(+) or EBV(-) . The phenotypic characteristics of lines were similar to those of the original malignant tumours . With the help of this liquid assay it was possible to evaluate the efficacy of in vitro procedures designed to completely eliminate malignant cells from harvested BM prior to autologous transplantation (i.e., immunomagnetic depletion or antibody-complement-mediated cytolysis) . With six out of the seven cell lines tested, the assay permitted the detection of as few as one BL cell in 4 X 10(6) normal BM cells and the measurement of up to 5 log BL cell elimination from 1% contaminated samples.

J Biol Chem, 1987 Feb 25, 262(6), 2869 - 74
Transforming growth factor beta is a bifunctional regulator of replication and collagen synthesis in osteoblast-enriched cell cultures from fetal rat bone; Centrella M et al.; Transforming growth factor beta (TGF beta) stimulates cell replication in fetal rat calvariae, and studies with isolated bone cells suggest that the primary mitogenically responsive cell is of the osteoblast lineage . The effect of TGF beta on bone cell replication is biphasic and depends on both the TGF beta concentration and cell density in monolayer culture . After 23 h of treatment, DNA synthesis in confluent cells is progressively enhanced by 0.15-15 ng/ml TGF beta; but in subconfluent cells, 15 ng/ml is less than maximal; and in sparse cell cultures, it is inhibitory . At all cell densities, however, 15 ng/ml TGF beta stimulates collagen synthesis, an effect which is more pronounced when DNA synthesis rates are declining . Furthermore, 1 mM hydroxyurea, which blocks the mitogenic effect of TGF beta by 85%, only minimally influences the increase in collagen synthesis . Cytoplasmic slot blot analysis reveals alterations in the amount of type I collagen mRNA in TGF beta-treated cells, suggesting that control is exerted, at least in part, at the transcriptional level . Since TGF beta is found in culture medium conditioned by bone explants and in bone tissue extracts, these results support that TGF beta is an important and multifunctional autocrine regulator of bone formation.

Anal Biochem, 1987 Feb 15, 161(1), 26 - 31
Comparison of sample preparation methods for the high-performance liquid chromatographic analysis of cell culture extracts for triphosphate ribonucleosides and deoxyribonucleosides; Harmenberg J et al.; We compared four different procedures for the purification and concentration of nucleoside triphosphates in cell extracts prior to HPLC analysis . Two methods involved precipitation, with either acetonitrile or calcium fluoride . The acetonitrile procedure yielded reasonable recovery and sufficient purity for the subsequent HPLC analysis . The calcium fluoride coprecipitation procedure gave both good recovery and purity; but the recovery was shown to be dependent on the concentration of the nucleoside triphosphates . The other two methods involved small Sep-Pak cartridges . The silica cartridge procedure yielded unfavorable recoveries in periodate-treated cell extracts, apparently due to poor solubility of nucleoside triphosphates in the requisite solvents . The strong anion exchange cartridge procedure yielded both good recovery and purity . This procedure was found to be fast, efficient, and reliable for purifying and concentrating nucleotides in cell extracts.

J Biol Chem, 1987 Feb 5, 262(4), 1748 - 55
Cyclic AMP decreases the phosphorylation state of myelin basic proteins in rat brain cell cultures; Ulmer JB et al.; Previous work has suggested that myelin basic proteins are phosphorylated prior to their appearance in the myelin sheath (Ulmer, J . B . and Braun, P . E . (1984) Dev . Neurosci . 6, 345-355) . In order to corroborate this finding we have examined the phosphorylation of myelin basic proteins in rat brain cell cultures containing 14-17% oligodendrocytes . Incorporation of 32P into the 14-, 17-, 18.5-, and 21.5-kDa myelin basic proteins was observed in cells incubated with 32P at 7, 14, and 21 days in culture . Myelin basic proteins in 14-day cells incorporated 32P linearly until at least 120 min after the addition of isotope . The apparent half-life of myelin basic protein phosphate groups was determined to be approximately 80 min in pulse-chase experiments . However, this value may be an overestimation due to the presence of significant levels of acid-soluble radioactivity in the cells throughout the chase period . The presence of dibutyryl cAMP or 8-bromo-cAMP in the incubation medium substantially inhibited the incorporation of 32P into the myelin basic proteins at all time points studied . The presence of dibutyryl cAMP in the chase medium in pulse-chase experiments resulted in an increase in the turnover rate of {32P} phosphate in the myelin basic proteins . These results indicate that cAMP decreases the phosphorylation state of myelin basic proteins in oligodendrocytes by inhibiting the phosphorylation and/or stimulating the dephosphorylation of myelin basic proteins.

Neurology, 1987 Feb, 37(2), 319 - 22
Effect of anticonvulsant drugs on glutamate neurotoxicity in cortical cell culture; Koh JY et al.; Pretreatment with anticonvulsants partially protects animals against the brain damage induced by intraparenchymal injection of kainate, an analogue of the neurotransmitter glutamate . In murine cortical cell culture, high concentrations of phenobarbital, diazepam, phenytoin, or GABA itself did not prevent glutamate-induced neuronal loss . Addition of a glutamate receptor antagonist (gamma-D-glutamyl glycine) did reduce glutamate neurotoxicity . The in vivo protective effect of anticonvulsant drugs against the toxicity of excitatory amino acids must be indirect.

J Cell Biol, 1987 Feb, 104(2), 363 - 70
Changes in the number of chick ciliary ganglion neuron processes with time in cell culture; Role LW et al.; The purpose of this study was to describe the shape of chick ciliary ganglion neurons dissociated from embryonic day 8 or 9 ganglia and maintained in vitro . Most of the neurons were multipolar during the first three days after plating, with an average of 6.0 processes extending directly from the cell body . The neurons became unipolar with time . The remaining primary process accounted for greater than 90% of the total neuritic arbor . This striking change in morphology was not due to the selective loss of multipolar cells, or to an obvious decline in the health of apparently intact cells . The retraction of processes was neither prevented nor promoted by the presence of embryonic muscle cells . Process pruning occurred to the same extent and over the same time course whether the cells were plated on a monolayer of embryonic myotubes or on a layer of lysed fibroblasts . Process retraction is not an inevitable consequence of our culture conditions . Motoneurons dissociated from embryonic spinal cords remained multipolar over the same period of time . We conclude that ciliary ganglion neurons breed true in dissociated cell culture in that the multipolar-unipolar transition reflects their normal, in vivo, developmental program.

Fed Proc, 1987 Feb, 46(2), 290 - 4
Muscle cell culture as a tool in animal growth research; Allen RE; Muscle cell culture techniques have been used for several years in research on muscle growth and development . Several types of culture systems have been devised, including primary cultures from embryonic or postnatal muscle and myogenic cell lines . In addition, serum-free and serum-containing media have been developed to address specific muscle development questions . Many of these questions center around muscle cell differentiation and muscle cell physiology; and, more recently, muscle cell cultures have been used as bioassay tools for examining growth physiology in domestic animals . In our laboratory, skeletal muscle satellite cells have been studied in vitro to evaluate the effect of several protein hormones and growth factors on satellite cell proliferation and differentiation . Of the hormones examined, only the insulin-like growth factors/somatomedins and fibroblast growth factor have been shown to have a stimulatory effect on proliferation that could be physiologically significant . None of the major anterior pituitary hormones interacted directly with satellite cells to stimulate proliferation . With advances in serum-free medium formulations and cell separation techniques, more information can be obtained from experiments with muscle cell cultures . With appropriate design and interpretation, our knowledge of muscle growth in domestic animals will be expanded.

Cancer Genet Cytogenet, 1987 Feb, 24(2), 191 - 204
Interphase cell flow cytometry as a means of monitoring genomic size in normal and neoplastoid cell cultures; Hoehn H et al.; The DNA specific fluorescence of mass cultures and clones derived from human skin and bladder tumor tissue was assayed by flow cytometry . In order to detect and quantitate small fluorescence intensity changes, cytogenetically defined triploid or diploid human fibroblast strains were cocultivated, harvested, and stained with the cell strain of unknown karyotype . The triploid standard (derived from human abortus tissue) proved chromosomally unstable at high passage level . Fifteen male, female, and 45,X strains displayed target-to-standard cell fluorescence ratios commensurate with their respective chromosome constitutions . Interstrain variation was highest among the 45,X strains, although mosaicism could not be detected by conventional cytogenetics . Interclonal fluorescence variation was two- to ten-fold higher among the tumor-derived clones tested . Chromosome counts and subcloning experiments indicate that this increased fluorescence variation is due to genome size variation . The clonal evolution of genome size differences was observed in subclones of chromosomally divergent parental clones . These observations suggest that well controlled flow cytometry can adequately resolve subtle degrees of genome size variation in cultivated human cells . The technique is especially suited for monitoring genome size changes in cultivated tumor cells.

Tsitologiia, 1987 Feb, 29(2), 221 - 6
{Viability and proliferative activity of a Chinese hamster cell culture exposed to nitrosoureas in exponential and stationary growth phases}; Makarova GF et al.; The lethal effect of antitumor nitrosourea chloroethyl derivatives on proliferating (exponential phase of growth) and non-proliferating (stationary phase of growth) cells is observed at a concentration 5-fold less than that of methyl derivatives revealed by the colony-formation technique . 1,3-bis(2-chlororoethyl)-1-nitrosourea is equally effective towards proliferating and non-proliferating cells, but chlorozotocin exerts a primary cytotoxic effect on proliferating cells . 1-methyl-1-nitrosourea at low concentration causes death more readily of proliferating cells than non-proliferating ones . However, studies on proliferative activity during the first hours after treatment with 1-methyl-1-nitrosourea revealed drug sensitivity in cells being at the early stationary phase of growth.

J Anim Sci, 1987 Feb, 64(2), 615 - 22
A statistically standardized muscle cell culture bioassay measuring the effect of swine serum on muscle cell proliferation; Kotts CE et al.; We have developed a statistically standardized bioassay for quantifying the effect of swine serum on the proliferation rate of cultured L6 myogenic cells . The intra-assay coefficient of variation for this assay is 2.5% . Over 29 experiments, the relationship between the assays response to 2.5% control swine serum and its response to 2.5% serum plus 10(-7) M insulin is linear (r2 = .9) . This relationship can be used to adjust experimental data obtained in different experiment to a common control value so that valid inter-assay comparisons can be made . The inter-assay coefficient of variation is reduced four- to five fold by this adjustment procedure . We believe this standardized assay provides a useful system for identifying and isolating unknown growth factors that affect muscle growth rate in an economically important species.

Mol Cell Endocrinol, 1987 Feb, 49(2-3), 173 - 9
The structure of tissue on cell culture-extracted thyroglobulin is independent of its iodine content; Delain E et al.; The major protein synthesized in vitro by the ovine thyroid cell line OVNIS 6H is the prothyroid hormone thyroglobulin . Purified from serum-free cell culture media using sucrose gradient centrifugation, the thyroglobulin dimer was analysed for iodine content and observed by electron microscopy . In their usual medium, the OVNIS 6H cells produce a very poorly iodinated thyroglobulin containing 0.05 I atom per molecule . When cultured with methimazole or propylthiouracil, two inhibitors of iodide organification, less than 0.007 I atom/molecules was found . These molecules purified from cell cultures were compared to those purified from ovine thyroid tissue containing 26 I atoms/mol . Despite large differences in iodine content, the three preparations all consist of 19 S thyroglobulin dimers with the classical ovoidal shape . The variability in size measurements remains in a 2% range for all thyroglobulin types . Consequently, no real significant variation can be found between the highly iodinated thyroglobulin isolated from tissue, and the poorly or non-iodinated thyroglobulins isolated from cells cultured with or without methimazole or propylthiouracil.

J Clin Microbiol, 1987 Feb, 25(2), 434 - 6
Comparison of HEp-2 cell culture and Abbott respiratory syncytial virus enzyme immunoassay; Bromberg K et al.; Respiratory syncytial virus (RSV) is the leading cause of lower respiratory tract infection in children . Rapid identification of RSV infections would allow for specific chemotherapy . We evaluated a means of rapid diagnosis, the Abbott enzyme immunoassay (EIA), by using 314 stored nasopharyngeal aspirates . RSV antigens were identified in 62 of 66 RSV culture-positive specimens . An additional 37 specimens from which RSV was not isolated were positive in the EIA . Of these, 29 were confirmed as truly positive by a blocking assay, for a total of 95 (66 + 29) positive specimens . The sensitivity of the EIA for total positive samples was 96% (91/95) versus 69% (66/95) for cell culture . The specificity of the EIA was 96% (211/219) . In these stored specimens, Abbott EIA was superior to cell culture for the detection of RSV.

Proc Natl Acad Sci U S A, 1987 Feb, 84(3), 871 - 5
Immortality of cell cultures derived from brains of mice and hamsters infected with Creutzfeldt-Jakob disease agent; Manuelidis EE et al.; Isolates from six patients with Creutzfeldt-Jakob disease (CJD) were injected into various strains of hamsters and mice, and the infective agent was propagated . Serially passaged cultures were established from these CJD agent-infected brains and from uninfected control brains . All healthy cultures (21 out of 21) from CJD agent-infected brains became immortal and/or transformed . In contrast only 3 out of 13 normal brain cultures became immortal, and the rest died out with serial propagation in vitro . The fact that permanent cell lines were readily derived from multiple rodent strains and all CJD isolates tested suggests that a transforming capability is an intrinsic property of CJD agents . This conclusion is supported by demonstrations of in vitro cell transformation by CJD infectious brain fractions . Although the molecular mechanism of transformation events associated with the CJD agent is not presently known, a provocative possibility is that the CJD agent has a direct effect on the host genome by mechanisms analogous to those known for slowly oncogenic retroviruses.

Eur J Cancer Clin Oncol, 1987 Feb, 23(2), 187 - 93
Representativity of human mammary tumor cell cultures: DNA-cytophotometry as a method for checking tumour cell characteristics; Stolwijk JA et al.; Short term cultures (3-6 days) of 40 primary human mammary carcinomas were prepared and compared with the original tumours from which they were derived . As a criterion the nuclear DNA Frequency Distribution Pattern (FDP), cytophotometrically measured, was used . Comparisons were made between the FDPs of smears of freshly-cut tumour surfaces and their cultures . Twenty-nine (73%) cultures showed FDPs identical with the smears . Eleven cultures (27%) showed gross shifts in ratios between different peaks or showed a complete loss of one or more peaks in the FDP and were classified as not representative . Our results show that it is necessary to check primary mammary tumour cultures to determine whether or not they are representative of the original tumour . This is especially so if conclusions are to be drawn from the cultures about the original tumour . Analysis of FDPs in the cell islands of the cultures (migration of cells from attached clumps) resulted in a better understanding of the FDPs found in the smears . We showed that cultures of human mammary tumours either can be composed of cell islands with identical FDPs (diploid or aneuploid) or may show heterogeneity between different cell islands within one tumour.

Exp Neurol, 1987 Feb, 95(2), 323 - 35
Apolipoprotein E synthesis in neurofibrosarcoma and schwannoma cell cultures from two individuals with neurofibromatosis; Gebicke-Haerter PJ et al.; Apolipoprotein E is expressed in neurofibrosarcoma and Schwannoma cell cultures derived from two patients with different types of neurofibromatosis, but not in six cell cultures derived from the benign neurofibromatosis neurofibromas . In addition, a cell culture derived from a nonneurofibromatosis human malignant astrocytoma showed apolipoprotein E expression . Although all cells in either the neurofibrosarcoma or Schwannoma cultures appeared morphologically similar (suggesting homogeneity), apolipoprotein E was immunochemically detected in the perinuclear region of only half of the cells . Thus, production of apolipoprotein E in neurofibromatosis-associated neurofibroma tumors may be a marker for a specific subclass of transformed cells . The expression of apolipoprotein E in glial cell neoplasms is possibly related to an alteration in their lipid metabolism.

Vet Microbiol, 1987 Feb, 13(2), 153 - 7
Detection of bovine virus diarrhea virus in cell culture using an immunoperoxidase technique; Katz JB et al.; An indirect immunoperoxidase staining technique was developed for identifying cell cultures infected with bovine virus diarrhea virus . Infected cell monolayers stained intensely while uninfected monolayers remained colorless . Immunoperoxidase staining was as sensitive as direct immunofluorescence in detecting endpoint dilutions of virus suspensions . Using the immunoperoxidase technique, infected monolayers were detectable by macroscopic, as well as microscopic, observation.

J Clin Microbiol, 1987 Feb, 25(2), 421 - 2
Early testing of cell cultures for detection of hemadsorbing viruses; Minnich LL et al.; Hemadsorption of primary monkey kidney cell cultures was commenced at 1 day after inoculation to evaluate how rapidly influenza A virus, influenza B virus, and parainfluenza virus types 1, 2, and 3 could be detected by this method from respiratory specimens . Overall, 38% of all isolates could be detected within 24 h of inoculation, and 69% could be detected within 48 h . All influenza A viruses and all but one influenza B virus were detected by day 3.

J Clin Microbiol, 1987 Feb, 25(2), 268 - 72
Physicochemical characterization of porcine pararotavirus and detection of virus and viral antibodies using cell culture immunofluorescence; Terrett LA et al.; A cell culture immunofluorescence (CCIF) assay was optimized for detection of porcine pararotavirus (group C rotavirus) in intestinal contents . The greatest viral infectivity was observed when MA104 cells (5 days after subculturing) were rinsed and refed in serum-free medium before inoculation, pancreatin was added to the inocula, and the inocula were centrifuged onto the cells . Gentamicin treatment of pararotavirus samples to reduce bacterial contamination also reduced the viral infectivity of these samples for MA104 cells . An indirect CCIF assay was used to determine the prevalence of pararotavirus and rotavirus antibodies in pig sera . In pigs from four herds, pararotavirus antibodies were detected in 100% (68 of 68) of adults and 59% (24 of 41) of weanling pigs, while 86% (24 of 28) of nursing pigs from 12 herds had pararotavirus antibodies . The physicochemical properties of pararotavirus were examined and compared with those of group A rotaviruses by using the CCIF assay to quantitate in vitro changes in viral infectivity . Pararotavirus was inactivated (greater than or equal to 99% reduction in titer) by heating to 56 degrees C for 30 min, was slightly labile at pH 3 (16 to 34% reduction in titer), and was stable at pH 5 (0 to 17% reduction in titer) and in either (3 to 19% reduction in titer) . One group A rotavirus (Gottfried strain) was stable at 56 degrees C (0% reduction in titer), whereas the OSU strain of group A rotavirus was inactivated at this temperature (99% reduction in titer).

J Virol, 1987 Feb, 61(2), 509 - 15
Differences in biological activity and structural protein VP1 phosphorylation of polyomavirus progeny resulting from infection of primary mouse kidney and primary mouse embryo cell cultures; Ludlow JW et al.; Both primary mouse kidney and primary mouse embryo cells in culture were used for polyomavirus progeny production . Examination of polyomavirus virion structural integrity revealed that mouse embryo cell progeny contained a threefold greater population of unstable particles when compared with mouse kidney cell progeny . Differences in biological activity between these two progeny virion types were also shown . Mouse kidney cell progeny compared with mouse embryo cell progeny exhibited a 10-fold greater ability to agglutinate guinea pig erythrocytes, a 3-fold lower ability to become internalized into monopinocytotic vesicles, and a 2-fold lower ability to initiate a productive infection based on positive nuclear immunofluorescence when mouse embryo host cell cultures were used . The mouse kidney progeny were also found to bind to host cells less specifically than the mouse embryo cell progeny . When these two progeny virion types were labeled in vivo with 32P and subjected to isoelectric focusing followed by sodium dodecyl sulfate-polyacrylamide gel electrophroesis in the second dimension, differences in the phosphorylation pattern of the major virus-encoded structural protein VP1 species were observed . It was revealed that species D and E of mouse kidney cell progeny were phosphorylated to the same degree, while mouse embryo cell progeny species E and F were phosphorylated equally . These data suggest that the host cells play a role in modulating the biological activity of the virus by affecting the degree and site-specific phosphorylation of the major capsid protein VP1 which may influence the recognition of virus attachment proteins for specific cellular receptors.

J Neurosci, 1987 Feb, 7(2), 357 - 68
Glutamate neurotoxicity in cortical cell culture; Choi DW et al.; The central neurotoxicity of the excitatory amino acid neurotransmitter glutamate has been postulated to participate in the pathogenesis of the neuronal cell loss associated with several neurological disease states, but the complexity of the intact nervous system has impeded detailed analysis of the phenomenon . In the present study, glutamate neurotoxicity was studied with novel precision in dissociated cell cultures prepared from the fetal mouse neocortex . Brief exposure to glutamate was found to produce morphological changes in mature cortical neurons beginning as quickly as 90 sec after exposure, followed by widespread neuronal degeneration over the next hours . Quantitative dose-toxicity study suggested an ED50 of 50-100 microM for a 5 min exposure to glutamate . Immature cortical neurons and glia were not injured by such exposures to glutamate . Uptake processes probably do not limit GNT in culture, as the uptake inhibitor dihydrokainate did not potentiate GNT . Possibly reflecting the lack of uptake limitation, glutamate was found to be actually more potent than kainate as a neurotoxin in these cultures, a dramatic reversal of the in vivo potency rank order . Some neurons regularly survived brief glutamate exposure; these possibly glutamate-resistant neurons had electrophysiologic properties, including chemosensitivity to glutamate, that were grossly similar to those of the original population.

Biosci Rep, 1987 Feb, 7(2), 151 - 7
Investigations on myelinogenesis in vitro: I . The occurrence of endogenous protein kinase and its role in the phosphorylation of myelin basic proteins in "myelin-like membranes" isolated from cerebral cell cultures; Shanker G et al.; The presence of a protein kinase capable of phosphorylating endogenous as well as exogenously added myelin basic proteins has been demonstrated in a myelin-like membrane fraction isolated from reaggregating and surface adhering, primary cultures of cells dissociated from embryonic mouse brain . Only the large and small components of myelin basic proteins were found to be phosphorylated when myelin-like membrane fraction was incubated with {gamma-32P}ATP . The protein kinase endogenous to the myelin-like membrane fraction was mainly of the cyclic AMP independent type . There was very little cyclic AMP dependent or cyclic GMP dependent protein kinase activities in this myelin-like fraction . Although the myelin basic proteins were the only endogenous proteins phosphorylated, protein kinase of the myelin-like membrane was capable of catalyzing the phosphorylation of exogenous substrates, such as histones.

Immunology, 1987 Feb, 60(2), 167 - 72
Effect of interferons on the inhibition of human natural killers by primary monolayer cell cultures; Heiskala M; Natural killer (NK) activity is inhibited by the contact of peripheral blood lymphocytes with primary monolayer cell cultures of both benign and malignant origin . In this study the effect of interferons (IFNs) on the inhibition has been analysed . Both alpha IFN- and gamma IFN-pretreated peripheral blood lymphocytes are effectively inhibited by monolayer target cells . IFN treatment of lymphocytes does not change cytotoxicity against the inhibitory target cells, although it enhances reactivity against NK-sensitive target cells . Treatment of monolayer cells with interferons significantly reduces their inhibitory capacity . However, diminished inhibition of NK activity by the IFN-treated target cells is not associated with increased lysis, probably due to their decreased sensitivity to natural killer cytotoxic factors (NKCF) . In 18.5% of the cases studied, monolayer target cells induced endogenous IFN production in lymphocytes . In these cases no inhibition of the NK activity of the effector cells was seen . According to the results of this paper, IFNs have a dual effect on the NK regulatory system: they enhance the NK activity of the effector cells against NK-sensitive target cells, and they change the NK resistant target cells in a way that makes them less inhibitory to NK activity but simultaneously more resistant to the toxic factors secreted by NK cells.

J Neurochem, 1987 Feb, 48(2), 522 - 8
Atrial natriuretic factors stimulate accumulation and efflux of cyclic GMP in C6-2B rat glioma and PC12 rat pheochromocytoma cell cultures; Fiscus RR et al.; Atrial natriuretic factors (ANFs) were tested for their effects on cyclic GMP production in two neurally derived cell lines, the C6-2B rat glioma cells and the PC12 rat pheochromocytoma cells . These cell lines were selected because both are known to possess high amounts of the particulate form of guanylate cyclase, a proposed target of ANF in peripheral organs . Previous studies from our laboratory have shown that ANF selectively activates particulate, but not soluble, guanylate cyclase in homogenates of a variety of rat tissues and that one class of ANF receptor appears to be the same glycoprotein as particulate guanylate cyclase . In the present study we found that four analogs of ANF stimulate cyclic GMP accumulation in both C6-2B and PC12 cells with the rank order of potency being atriopeptin III = atriopeptin II greater than human atrial natriuretic polypeptide greater than atriopeptin I . Atriopeptin II (100 nM) for 20 min elevated cyclic GMP content in C6-2B cells fourfold and in PC12 cells 12-fold . Atriopeptin II (100 nM) for 20 min also stimulated the efflux of cyclic GMP from both C6-2B cells (47-fold) and PC12 cells (12-fold) . Accumulation of cyclic GMP in both cells and media was enhanced by preincubation with the phosphodiesterase inhibitor 3-isobutyl-1-methylxanthine (250 microM) . After 20 min of exposure to atriopeptin II, cyclic GMP amounts in the media were equal to or greater than the amounts in the cells.(ABSTRACT TRUNCATED AT 250 WORDS)

Biochem Biophys Res Commun, 1987 Jan 30, 142(2), 496 - 500
The effects of ethanol on cellular calcium content in primary myocardial cell cultures from offspring of sedentary and swim-trained pregnant rats; Butler AA et al.; Primary myocardial cell cultures obtained from offspring of swim-trained (T) and sedentary (S) Sprague-Dawley rat mothers (dams) were used to evaluate the influence of exercise training and ethanol on cellular calcium (45Ca) content . The pregnant rats swam in water maintained at 37 degrees C 6 days/week during gestation . The dams swam continuously for 30 min on the first day of gestation . The swimming time was increased by 5 min until the rats swam continuously for 1.5 hr . After the cultures had been in incubation for 4 days, the cells were treated with 600, 800, and 1000 mg% ethanol for 30 min and 1 hr . 45Ca content (nmol/mg protein) of the untreated controls and all but one of the ethanol treated groups from the T cultures (1000 mg%) were significantly elevated over the comparable groups from the S cultures for both 30 min and 1 hr of incubation (p less than or equal to 0.05) . The data suggest that exercise during pregnancy induces adaptations in myocardial cell cultures from the offspring such that 45Ca content levels are elevated which may provide protection against ethanol toxicity.

Med J Aust, 1987 Jan 5, 146(1), 27 - 9
Prenatal cytogenetic diagnosis . Amniotic cell culture versus chorionic villus sampling; Bell JA et al.; Chorionic villus sampling is under evaluation throughout the world . Amniocentesis with amniotic cell culture is reliable for the diagnosis of certain types of genetic disease in the second trimester, and has been the subject of extensive clinical and laboratory audit . Chorionic villus sampling has the advantage of the early diagnosis (for example, at 10 weeks) of chromosomal abnormalities, a shorter delay with results after the diagnostic procedure, and is, for some couples, a more socially and morally acceptable method of antenatal diagnosis . In current experience, the disadvantages of chorionic villus sampling include an increased fetal loss and an increased diagnostic error rate . Another factor is the early diagnosis of fetuses with chromosomal abnormalities--a proportion of whom would have miscarried spontaneously before being detected at an amniocentesis at 16 weeks . This has implications for an increased rate of therapeutic terminations of pregnancy . Chorionic villus sampling and amniotic cell culture are discussed and comparisons are drawn that concern the clinical advantages and diagnostic issues which are inherent in each method of prenatal diagnosis.

Undersea Biomed Res, 1987 Jan, 14(1), 11 - 9
High hydrostatic pressure potentiation of the toxic effects of chromate in cell culture; Syversen T et al.; Development of hyperbaric welding has created the need for new occupational health standards . We have used cell cultures to investigate effects of high pressure chromate on the toxicity of the welding-fume component, chromate . The results indicate that pressure caused rounding-up of cells and changes in F-actin filaments, and that these effects were related to the extent of high pressure and independent of exposure duration . When exposing the Flow 5000 cells to chromate (1-5 microM) the amount of disrupted F-actin fibers was found to depend on the concentration applied and the duration of exposure . The amount of cell-associated chromate was the same for 1 h exposure at 1 and 150 bar . The fraction of rounded cells after exposure to 1 microM chromate and 100 bar for 18 h was higher than would be expected if the effects were purely additive.

In Vitro Cell Dev Biol, 1987 Jan, 23(1), 57 - 62
Placental-derived mitogenic factor for human fetal adrenocortical cell cultures; Simonian MH et al.; The human fetal adrenal cortex is one of the largest fetal organs and synthesizes precursors for placental estrogen production as part of the feto-placental unit . The factors controlling the rapid growth of the human fetal adrenal cortex during the second and third trimesters are not known . Placental regulation of the growth of human fetal adrenocortical cell cultures from second trimester fetuses was studied . A placental-derived mitogenic factor (PDMF) was detected in tissue homogenates of 14 to 22 week human placentas and stimulated adrenocortical cell number and {3H}thymidine incorporation into DNA 5-8 fold . PDMF has been partially purified by ammonium sulfate precipitation and anion exchange chromatography . PDMF is a heat sensitive protein with disulfide bonds required for activity . The growth stimulation by PDMF was significantly greater than that for basic or acidic fibroblast growth factor by 25-50% and epidermal growth factor by 3-4 fold . The placental hormones, progesterone, estriol, estradiol, placental lactogen and chorionic gonadotropin, either alone or in combination did not stimulate fetal adrenocortical cell growth, except for a 41% cell number increase by progesterone . Platelet-derived growth factor and insulin-like growth factors I and II were not mitogenic for these cells . These results show that the placenta contains a potent growth factor for human fetal adrenocortical cell cultures . This implies a direct role for the placenta in control of this fetal organ's growth, which would make the human feto-placental unit a bi-directional relationship.

Carcinogenesis, 1987 Jan, 8(1), 59 - 66
Separation by ion-pair high-performance liquid chromatography of the glucuronide, sulfate and glutathione conjugates formed from benzo{a}pyrene in cell cultures from rodents, fish and humans; Plakunov I et al.; A large proportion of the metabolites formed from benzo{a}pyrene (BP) in cell cultures from rodents, fish and humans result from conjugation of an oxidized metabolite of BP with sulfate, glucuronic acid or glutathione (GSH) . To improve the analysis of these metabolites, a reversed-phase ion-pair h.p.l.c . system using a step gradient of methanol:tetrabutyl-ammonium bromide in ammonium formate buffer has been developed for the separation of these three classes of conjugates . This system separated 3-hydroxy-BP glucuronide and sulfate conjugates and resolved them from GSH conjugates of BP 4,5-oxide, 7,8-oxide and 7,8-diol-9,10-epoxide . Cultures of early passage Syrian hamster, Wistar rat and Sencar mouse embryo cells, a bluegill fry (BF-2) cell line and a human hepatoma cell line (HepG2) were exposed to {3H}BP for 24 h . Medium samples from each were extracted with chloroform: methanol:water, and the water-soluble metabolites were analyzed by ion-pair h.p.l.c . The largest peak of metabolites in the media from cell cultures from rodents and the bluegill fry cell line co-eluted with the glucuronic acid conjugate of 3-hydroxy-BP . These phenol-glucuronides represented 48-62% of the total water-soluble metabolites in the fish and rodent cell cultures . Treatment of this material with beta-glucuronidase released 3-hydroxy-BP and 9-hydroxy-BP in ratios from 3:4 to 13.3:1 in various cultures . Media from the bluegill fry cell line and the mouse embryo cell cultures also contained a peak of BP-diol glucuronides; treatment of these peaks with beta-glucuronidase released mainly BP-7,8-diol . In HepG2 cells, 40% of the water-soluble metabolites were identified as sulfate conjugates of 3-hydroxy-BP and 9-hydroxy-BP . No glucuronic acid conjugates of BP metabolites were detected in HepG2 cells . Only small amounts of the water-soluble metabolites from these cell cultures eluted in the same volumes as the synthetic GSH conjugate of BP-4,5-oxide, BP-7,8-oxide and BP-7,8-diol-9,10-oxide . These studies indicate that conjugation with glucuronic acid represents a major pathway of formation of water-soluble metabolites from BP in cells derived from a number of species and demonstrate the value of this ion-pair h.p.l.c . system for the analysis of conjugates formed from BP.

J Neuropathol Exp Neurol, 1987 Jan, 46(1), 57 - 71
Analysis of hemangiopericytic meningiomas by immunohistochemistry, electron microscopy and cell culture; Nakamura M et al.; Intracranial hemangiopericytic meningiomas (HM) from seven patients were examined by immunostaining, transmission electron microscopy (TEM), scanning electron microscopy (SEM) and cell culture . Positive staining for Factor VIII-related antigen was restricted to capillary endothelial cells . There was no reaction with anti-glial fibrillary acidic protein (GFAP) serum or anti-S-100 protein serum . In these neoplasms TEM displayed extracellular basement membrane-like material (BMLM), cytoplasmic intermediate filaments (IF) associated with a dense body, dilated rough-surfaced endoplasmic reticulum containing BMLM, a small area of interdigitation of cell membranes, and a unique intercellular punctate or linear density . By SEM these tumors had intercellular shell-like or reticular structures and irregularly branched capillaries which were compressed by ellipsoidal- or carrot-shaped tumor cells . Short-term monolayer culture showed rapid and vigorous growth of tumor cells, and the formation of pseudolumens but not of whorls . The TEM of cultured cells also showed cytoplasmic IF associated with the dense body . By SEM the cultured cells were flat and had a discoid nucleus with conspicuous nucleolar hillocks . Our results show that HM are mainly poorly specialized mesenchyme-related tumors of the meninges; some possess a potential for aggressive growth and some for differentiation into smooth muscle cells . Further study is needed to determine their histogenesis.

Cancer Res, 1987 Jan 1, 47(1), 263 - 8
Growth-stimulating effect of pharmacological doses of estrogen on androgen-dependent Shionogi carcinoma 115 in vivo but not in cell culture; Noguchi S et al.; Shionogi carcinoma 115 (SC115) had been accepted for 20 years as an androgen-dependent mouse mammary tumor, the growth of which is stimulated only by androgen . However, we very recently found that the growth of SC115 tumors in vivo is stimulated not only by physiological doses of androgen but also by pharmacological doses of estrogen through the estrogen receptor system . In the present study, the growth-stimulative effect of estrogen on an androgen-dependent cloned cell line (SC-3) derived from SC115 cells, which showed androgen- and estrogen-dependent growth in vivo, was examined in vitro . In serum-supplemented medium (Eagle's minimum essential medium containing 2% steroid-free fetal calf serum), testosterone or 5 alpha-dihydrotestosterone (10(-9)-10(-6) M) significantly stimulated the growth of SC-3 cells (3.2-fold increase in cell number at day 10 in culture containing 10(-8) M androgens) and changed the shape of SC-3 cells from epithelial to spindle (fibroblast-like), whereas 17 beta-estradiol (10(-12)-10(-6) M) even in high concentrations had no such effects on SC-3 cells . Contrary to the effect of 17 beta-estradiol in vivo, 17 beta-estradiol as well as cyproterone acetate (10(-8)-10(-6) M) inhibited the growth-stimulative effect of testosterone (10(-8) M) on SC-3 cells in a dose-dependent manner in the serum-supplemented medium . The anti-androgen and 17 beta-estradiol also showed comparable competitive effects on {3H}testosterone binding to androgen receptor in SC-3 cells . In serum-free medium {Ham's F-12:Eagle's minimum essential medium (1:1, v/v) containing 0.2% bovine serum albumin}, testosterone {10(-8) M} also markedly stimulated the growth of spindle-shaped SC-3 cells, and epidermal growth factor (1 ng/ml) enhanced the growth-stimulative effect of testosterone, whereas 17 beta-estradiol (10(-8)-10(-6) M) in the absence or presence of epidermal growth factor had no growth-stimulative effect on SC-3 cells . We conclude that the growth of SC115 cells is stimulated by either physiological doses of androgen or pharmacological doses of estrogen in vivo but only by androgen in cell culture.

Tissue Cell, 1987, 19(2), 197 - 206
Characterization of microvascular cell cultures from normotensive and hypertensive rat brains: pericyte-endothelial cell interactions in vitro; Herman IM et al.; We used specific markers and fluorescence microscopy to identify and characterize cerebrovascular cells . Cultures were derived from brain microvessels isolated from normotensive (Wistar Kyoto, WKY) and spontaneously hypertensive (SHR) rat brains prior to, coincident with and following the onset of chronic hypertension . Endothelial cells were characterized using di-acyl LDL and non-muscle isoactin-specific antibodies . Cerebrovascular pericytes were identified with the anti-muscle and non-muscle actin antibody staining . Using this combination of cell culture and fluorescence localization, we have been able to demonstrate that brain pericytes are tightly associated with the endothelial cells of the hypertensive-prone and hypertensive cell cultures, but not with the normotensive endothelial cultures . While the endothelial-pericyte ratio in the hypertensive-prone microvascular cultures was between 5:1 and 10:1, the number of pericytes associated with the hypertensive rat brain cultures increased two to five times (2:1-1:1) . Muscle and non-muscle actin antibody staining localized the spindle-shaped pericytes of the hypertensive microvascular colonies . Pericytes were found overlaying and encircling the endothelial cells . Normotensive pericytes were not endothelial-associated . Whereas the hypertensive pericyte is devoid of stress fibers, the normotensive pericyte is a larger, spread-out cell possessing numerous stress fibers rich in muscle and non-muscle actin . These results provide the first evidence that the etiology and inception of cerebrovascular disease may be pericyte-related and suggest that pericyte contraction could play a pivotal role in regulating the flow of blood within the brain microcirculation.

Dev Biol Stand, 1987, 66, 391 - 401
Purification of proteins from cell culture supernatants; Menge U et al.; Desired proteins excreted by animal cells usually reach rather low concentrations in the culture supernatant and have to be purified from an excess of serum proteins which are added to the animal cell culture to maintain its viability and/or productivity . Defined media offer among others the advantage of an easier purification procedure . In addition lysis of cells may contribute also to the complexity of the protein mixture encountered . If fetal calf serum or new born calf serum is used in cultivation, bovine serum albumin and globulins will be the most abundant protein in the supernatant . The interaction of albumin especially with hydrophobic proteins represents significant problems for the effective purification of minor constituent . Isolation of a desired protein follows the general scheme: concentration: by precipitation, ultrafiltration, batch adsorption or partition in aqueous phase system; enrichment: by chromatography or partition; high resolution purification: by chromatography and/or immuno adsorption; final concentration and finishing: pyrogen removal, sterilization, formulation . Biochemical engineering aspects of proteins purification are discussed using human interferon-beta produced in a recombinant mouse cell line as an example . The process developed encloses extraction of interferon-beta from the production medium in aqueous two-phase systems and subsequent recovery of interferon-beta from the top phase by high pressure liquid chromatography.

Dev Biol Stand, 1987, 66, 227 - 40
Bubble free cell culture aeration with porous moving membranes; Lehmann J et al.; For the cultivation of mammalian cells a new aeration system was developed . Its construction with hydrophobic porous Accurel hollow fiber membranes and the use in reactors with 1 and 20 l reaction volume will be presented and introduced by means of the cultivation of a genetically manipulated mouse cell line . The mass transfer capacity of this system is demonstrated as a function of gas flow rates through the membrane and of the membrane movement.

Dev Biol Stand, 1987, 66, 155 - 60
Serum and growth factors in cell culture media--an introductory review; Griffiths JB; Amongst this bewildering array of growth factors, serum replacements and serum-free media is there a system which works well for most cell types used in biotechnology? If so is there a short-cut for novices in the field to be able quickly to select the right formulation . I hope the following discussion will enable those with experience in this field to share that experience so that we can get an indication of which compound works for which cell under which conditions . Also the cost in both yield and monetary terms needs to be kept in perspective as one option is to produce these very expensive factors from genetically engineered bacteria . Discussion points should include the question of why so many medium formulations still include serum at 0.1 or 0.5% i.e . what does this small quantity of serum provide and does it mean that in fact we cannot have a completely protein-free media to achieve optimal growth? In which case, what is the minimal acceptable level? An adaptation procedure to low-serum media is accepted as essential but in fact the time-scale for this varies enormously from laboratory to laboratory and the question must be asked "is this based on quantitative or empirical investigation"? I hope the following panel discussion will answer these questions as the correct use of growth factors is of vital importance to the future development of cell product technology . It is probably true to say that with the correct blend of these compounds all cell types can be grown in culture as successfully as the ubiquitous fibroblast.

Dev Biol Stand, 1987, 66, 13 - 22
Safety issues related to the use of recombinant DNA-derived cell culture products . I . Cellular components; Palladino MA et al.; Anti-thymocyte serum (ATS) treated newborn rats were used to assess the tumorigenic potential of mammalian cell and mammalian cell culture derived substances . Injection of as much as 100 micrograms of Chinese hamster ovary (CHO) DNA, an amount in excess of 10(8) fold more than might be present in one dose of a typical final product derived from mammalian cell cultures, failed to initiate a tumor in immunosuppressed animals . In addition, injection of 10 micrograms of activated oncogene cloned from a human bladder carcinoma was also insufficient to initiate a tumor in immunosuppressed animals . Injection of some but not all CHO cell lines did result in tumor growth which upon isoenzyme analysis was verified to be of hamster origin . Of importance was the finding that recombinant tissue plasminogen activator (rt-PA) and hepatitis B surface antigen (HBsAg) vaccine failed to induce tumors in either normal or immunosuppressed rats . The results suggest that the presence of minute quantities of CHO derived nucleic acid fragments in these final products have no discernable tumorigenic potential.

Eur J Pediatr, 1987 Jan, 146(1), 27 - 30
Cell cultures of bile duct epithelium and the pathogenesis of biliary atresia; Schier F et al.; The pathogenesis of biliary atresia is unknown . The authors describe a technique for culturing extrahepatic bile duct epithelial cells of human and bovine origin in monolayer cell cultures . Light-, electron microscopy and immunohistological studies prove the epithelial nature of the cultured cells . Inoculation of the cells with reovirus 3 showed no destruction; adenovirus 6, herpes simplex and polio virus 1 and 2 destroyed the cells within 24 h . The cells produce a growth factor maintaining the integrity of the cells, even in the absence of serum.

Thymus, 1987, 9(1), 45 - 60
In vitro bone marrow cell migration to supernatants prepared from thymic epithelial cell cultures; Taubenberger JK et al.; Thymic epithelial cell cultures were established from neonatal CBA/J mice by inhibition of fibroblast overgrowth . Epithelial cells were identified by their cobblestone appearance in culture, by the presence of keratin, and by ultrastructural analysis demonstrating desmosomes . Supernatants were prepared by incubation of confluent cultures of these cells in serum-free media . Using blind well chambers, these supernatants were chemoattractive to murine bone marrow cells, enriched for immature lymphoid cells, and decreased the myeloid to lymphoid ratio in the migrating cell population . Interleukin 2 had no effect as a modulator of this chemoattraction, nor did it possess chemoattractive properties . Supernatants prepared from epidermal growth factor stimulated thymic epithelial cells possessed significantly enhanced chemoattractive properties to bone marrow cells, and was found to be a thymic epithelial cell mitogen . Supernatants from serum-free cultures of thymocytes also induced a significant migration of bone marrow cells but were found to enrich for mature myeloid cells.

Tsitologiia, 1987 Jan, 29(1), 66 - 72
{Effect of amines on the binding, internalization and degradation of epidermal growth factor and the induction of DNA synthesis in a 3T3 cell culture}; Nikol'skii NN et al.; The role of intracellular processing of epidermal growth factor (EGF) in the induction of proliferation of quiescent Swiss 3T3 cells was studied using various inhibitors . The number of amines (dansylcadaverine, chloroquine, cystamine, 5-methoxytryptamine) dimethylurea and monensin were shown to block the mitogenic effect of EGF . The majority of these substances while used in concentrations sufficient to inhibit the proliferation do not significantly influence 125I-EGF binding and internalization . The level of EGF degradation was reduced only by chloroquine . The inhibitory effect of amines and monensin on the generation of proliferative signal was supposed to take place at the stages of EGF processing in "specialized" endosomes and in Golgi apparatus.

Med Microbiol Immunol (Berl), 1987, 176(1), 27 - 35
Subtype-specific identification of influenza virus in cell cultures with FITC labelled egg yolk antibodies; Doller PC et al.; We report on results obtained with a direct immunofluorescence test for subtype-specific identification of influenza virus in detached cells of MDCK cultures after inoculation of 281 clinical specimens from patients with influenza-like disease . Influenza virus antibodies were produced in eggs from immunized hens and labelled with FITC . In 157 cases CPE was found in MDCK cells . A total of 57 cases of influenza A (H3N2), 86 cases of influenza A (H1N1), and 14 cases of influenza B were identified . In 33 cases of influenza A (H1N1) infection with massive CPE guinea pig but not chicken erythrocytes were agglutinated by the cell culture supernatants . The single step immunofluorescence test described proved easy to perform and results were obtained within 1 h after CPE was observed in contrast to the conventional HIT which is very time-consuming.

Biorheology, 1987, 24(6), 585 - 8
Studies of tracheal secretion using serous cell cultures and monoclonal antibodies; Basbaum CB et al.; The glycoconjugate composition of tracheal secretions varies with physiological and pathophysiological parameters . Believing that these differences might be explained by metabolic or regulatory modifications of particular cell types, we have developed strategies for biochemical analysis at the cellular level . We have produced monoclonal antibodies whose determinants are restricted to a single secretory cell type (serous, mucous, or goblet cell granules, or ciliated cell glycocalyx) . By enzyme immunoassay (ELISA), we have characterized four of the antibodies biochemically, and have also used the antibodies as quantitative molecular probes to detect release of antigen from mixed cell explants . Four of the antigens are carried by carbohydrate moieties of high molecular weight glycoproteins . Western blot analysis shows their molecular weight in reducing gels (SDS-PAGE) to exceed 200 kD . When used in parallel with pulse-chase labeling studies, the antibodies are both more sensitive and specific (than bound radioactivity) in detecting gland or goblet cell secretion in response to autonomic drugs or proteases . We have also isolated and cultured serous gland cells for physiological and biochemical studies . These cells express serous cell phenotype as reflected by ultrastructure, histochemistry, and lysozyme activity . Biochemical analysis of their secretory products reveals glycoconjugate components which are heterogeneous with respect to both molecular weight and charge . Radiolabeled secretory products eluting in the void volume of Sepharose C1 4B were completely degraded by chondroitinase ABC . This indicates that the major glycoconjugate produced by serous cell is a proteoglycan resembling chondroitin sulfate.

Eur J Respir Dis Suppl, 1987, 153, 59 - 67
Cell culture approaches to the investigation of human airway ion transport; Boucher RC et al.; Several techniques for the study of human airway epithelial ion transport employing cultured cells were explored . Human nasal epithelial cells were cultured in serum-free, hormone supplemented media . For bioelectric characterization of ion transport functions of cultured cells, cells were inoculated into heterologous grafts, and implanted into immunocompromised mice, or onto collagen membranes maintained in vitro . Cells populating either preparation exhibited a pattern of Na+ and Cl- transport similar to that of freshly excised nasal specimens . Differences between preparations were observed for absolute transport rates, tissue resistance, and morphology . We conclude that (1) cell culture techniques will be useful in investigating ion transport activities of human pulmonary epithelia from normal and abnormal lungs; and (2) the selection of specific culture techniques should be guided by the nature of the epithelial functions tested.

Blood Cells, 1987, 12(3), 647 - 55
Characterization of "fetal-type" acetylcholinesterase in hemin-treated K562 cell culture; Bartha E et al.; The alteration of acetylcholinesterase (ACHE) activity, a marker enzyme of erythroid differentiation, was studied during the hemin-induced erythroid differentiation of K562 human leukemia cells in suspension culture . The kinetics of postinduction differentiation was followed by determining the hemoglobin (Hb) content and the ACHE activity of cells . Embryonic hemoglobins as well as small quantities of fetal Hb (HbF) were synthetized by stimulated cells . The peaks of ACHE activity preceded the highest level of Hb content and, following induction, reached their pinnacles at 72 and 120 hours, respectively . These data indicate that ACHE activity is an earlier and more sensitive marker for hemin-induced erythroid differentiation of K562 cells than is elevated Hb content . Electrophoretic mobility of ACHE from hemin-treated cells proved to be the fetal type, but after incubation with neuraminidase, the rate of migration decreased to the level of the adult type enzyme.

Acta Endocrinol Suppl (Copenh), 1987, 281, 21 - 6
Modulation of class-II antigen expression in human thyroid epithelial cell cultures; Wenzel BE et al.; The modulation of HLA-D expression of thyroid epithelial cells (TEC) was studied in vitro by means of immunofluorescence . Under serum-free culture conditions, TSH and TSH-receptor antibodies induce HLA-D on TECs derived from GD-patients . Serum-free culture conditions provide a higher availability of TSH-receptors by a 'right side right' polarity of the cellular morphology . There was no evidence for IFN-gamma producing cell contaminations on GD-TECs . TSH in contrast to IFN-gamma does not induce HLA-DQ on TECs . HLA-DQ is not displayed by spontaneously class-II antigen expressing GD-TECs . Methimazole as well as perchlorate do not suppress HLA-D expression of TECs.

Differentiation, 1987, 36(1), 47 - 56
The mesangial cell culture: a tool for the study of the electrophysiological and pharmacological properties of the glomerular mesangial cell; Nobiling R et al.; Cultured rat glomerular mesangial cells (MC) were evaluated as a tool for reliable electrophysiological measurements as well as for fluorimetric determinations of intracellular Ca++ . They had a resting potential similar to that observed in cultured vascular smooth muscle cells (VSMCs), in VSMCs of mouse kidney arterioles, or in glomerular--presumably mesangial--cells of kidney slices . The comparison with the other cell types was carried out in order to look for features distinguishing them from these cells, e.g., active and passive electrical membrane properties or electrical membrane responses to vasoactive pharmacological agents . In MCs, as well as in the other cell types, the average membrane potential was approx . -50 mV . The vasoconstrictor peptides angiotensin II (ANG II) and arginine-vasopressin (AVP) caused depolarizations that could be blocked by the respective specific inhibitors of these compounds . The agonist-induced depolarizations have to be attributed, at least in part, to a Ca++ inward current . Norepinephrine, if any, had only a weak action upon MCs, whereas isoproterenol either did not influence the membrane potential or hyperpolarized the cells . Other substances tested, which had no influences upon the membrane potential, were neuropeptide Y and atriopeptin 3 . As to their resting electrical properties and their responses to pharmacological agents, cultured mesangial cells did not differ from glomerular, i.e., most probably mesangial, cells in the kidney slice . The difference between mesangial cells and VSMCs consists in their reaction to noradrenaline . Whereas VSMCs respond with a marked depolarization, the noradrenaline effect upon MCs in culture and in the kidney slice is either absent or very weak . Repeated passage of the cells (more than six passages) led to a gradual loss of their responsiveness to the agonists, indicating reduced receptor expression which may be interpreted as dedifferentiation . This held for both cultured MCs and VSMCs . Fluorimetric measurements using the Ca++-specific indicators quin-2 and fura-2 were performed with a purpose-developed, ultrasensitive photon-counting microspectrofluorimeter . Individual MCs as well as isolated glomeruli responded to the vasoconstrictors ANG II and AVP with an increase in Ca++-dependent fluorescence indicating that these agents indeed depolarize the cells partly via a Ca++ influx and increase cytosolic free Ca++.(ABSTRACT TRUNCATED AT 400 WORDS)

Vet Med Nauki, 1987, 24(9), 9 - 16
{Replication of a local strain of swine parvovirus in swine kidney cell cultures}; Peev Ia et al.; The kinetics of replication of strain of parvovirus in swine was investigated in the primary cell cultures of swine kidney . The morphologic changes were traced in inoculated cultures by microscopic observation and the replication of the virus in the cells by immune-fluorescent examinations . The quality of the virus in the cell monolayer and in the nutritive medium, in the different periods after the infection, was determined by hemagglutination test . By the immune-fluorescent examinations, the virus was proved still in the first hours, while the first morphologic changes of the monolayer were determined 72 hours after the infection . The nuclear fluorescent appears at the 12-th hour and increases its intensity 24 hours after the infection . At the 72-nd hour and later predominates the cytoplasmatic fluorescent . The cell monolayer produces virus up to the 96-th hour, and the virus titer reaches its maximum 120 hours after the inoculation, which is the most appropriate moment for the yield of virus.

Jpn J Ophthalmol, 1987, 31(3), 501 - 9
Dome formation in cell cultures of chick embryo retinal pigment epithelium and effects of ouabain and 2,4-dinitrophenol thereon; Kishida K et al.; Cells from chick embryo retinal pigment epithelium were cultured on glass slips . The primary culture cells formed confluent cell layers which were studded with a number of domes . The domes had usually appeared by the fourth day of culture and were susceptible to 3 X 10(-6) M ouabain and 10(-4) M 2,4-dinitrophenol, indicating that fluid was transepithelially transported from the apical to the basal side by means of an energy-requiring and ouabain-sensitive mechanism . Other than domes, small blisters appeared after 4-10 days of culture . They were also susceptible to the metabolic inhibitors.

Differentiation, 1987, 36(1), 71 - 85
Intestinal tissue and cell cultures; Kedinger M et al.; The culture of animal cells and tissues is a widely used technique in the field of cellular and molecular biology; one of the most interesting aspect being linked to the study of the mechanisms of cell differentiation . In the specific case of intestinal epithelial cells, various tissue culture technologies have proved to be important tools for the study of precise facets related to intestinal function, pathology and differentiation . Concerning this latter aspect, organ culture experiments have brought about interesting data on the hormonal or nutritional control of intestinal maturation . Nevertheless, the study of the precise mechanisms underlying epithelial proliferation and/or differentiation at the cellular level needs more adequate cell culture model systems . One of them has been described for two cell lines derived from human colonic adenocarcinomas, in which the cells can be induced to achieve enterocytic-like differentiation . Up to date, none of the continuous cell lines starting from normal undifferentiated cells have allowed generation of morphological or functional enterocytic polarity . In contrast, primary cell cultures which allow maintenance of a more physiological environment for the epithelial cells like contacts with their in vivo counterparts, mesenchymal cells or extracellular matrix molecules, have proved to be promising approaches.

Exp Cell Biol, 1987, 55(5), 225 - 36
Differences in growth factor sensitivity between primary and transformed murine cell cultures revealed by BrdU/Hoechst flow cytometry; Kubbies M et al.; Spontaneous cell transformation is a common feature of all murine cell cultures grown in vitro for extended periods of time . During early passages, these cultures show either progressively reduced growth or complete cessation of growth; after such a 'crisis' they display increasing growth rates and unlimited lifespan . Here we use a novel bromodeoxyuridine/Hoechst flow-cytometric technique to examine the growth response of diploid and transformed cells of the murine species Micromys minutus under a variety of growth conditions . After spontaneous transformation, growth factor exposure results in increased G0/G1 cell recruitment and higher growth rates than shown by the nontransformed diploid cell fraction . Despite clonal heterogeneity, this difference is seen at all fetal calf serum (FCS) concentrations, although it is most pronounced with low serum . Epidermal growth factor and insulin are shown to act synergistically and promote growth equal to exposures of transformed cultures to 10% FCS . The observed differences in growth factor response between diploid and aneuploid cells could explain the reported lack of a classical growth crisis in growth factor-supplemented media during the spontaneous in vitro transformation of primary cell cultures.

Vet Med Nauki, 1987, 24(8), 13 - 6
{Experiments to identify Mycoplasma isolates from cell cultures}; Petkova K et al.; Identified were various species of Mycoplasma organisms isolated from three continuous cell lines--BHK-21, SPZF, and TT . On the base of cultural, biochemical, and serological investigations the isolates were defined as M . bovirhinis, M . arginini, and A . laidlawii . The bovine sera, used to culture the cell lines were shown to be the basic source of contamination with Mycoplasma organisms . This made it necessary to carry out a preliminary study on each batch of serum for Mycoplasma contamination prior to its use for laboratory and productional needs.

Ann Endocrinol (Paris), 1987, 48(5), 352 - 5
{Hormonal control of the differentiation of hypothalamic neurons in cell culture}; Puymirat J; Hypothalamic cell cultures represent a suitable model for studying the role of hormones during the development of the hypothalamus . In vitro studies have shown that oestradiol increases the neurite length in some hypothalamic nuclei and the number of LH-RH neurons . Triiodothyronine had no effect on the number of neurons but controlled the size and the neurite length of hypothalamic dopaminergic neurons . In contrast, mesencephalic DA neurons are regulated differently by T3 . The effects of hormones in culture are correlated with the presence of specific nuclear binding sites . At last, serum-free cultures have demonstrated the importance of the interaction between hormones (T3, corticosterone) and others diffusible factors (polyunsaturated fatty acids) on the potassium evoked release of thyroliberin and on the maturation of synapses.

Mol Toxicol, 1987-88 Fall, 1(4), 313 - 34
Search for cell culture systems with diverse xenobiotic-metabolizing activities and their use in toxicological studies; Glatt H et al.; Many toxic effects are not caused by the administered compound itself, but are due to metabolites . All cell types express some xenobiotic-metabolizing enzymes, but levels and patterns are very variable . Critical metabolic steps may occur within the target cell and/or at other sites . This complex situation is difficult to mimic in vitro . The further problem is that cells that are taken into culture tend to rapidly cease the expression of important xenobiotic-metabolizing enzymes . Part of the problem may be solved by the addition of exogenous metabolizing systems, for example, in the form of freshly isolated hepatocytes, crude subcellular preparations, or purified enzymes . In these systems, the plasma membrane of the target cell may act as a barrier for the active metabolite and thereby lead to false negative results . The alternative is the use of metabolically active target cells . We therefore screened 18 cell lines for monooxygenase, cytochrome P-450 reductase, epoxide hydrolase, glutathione transferase, and UDP-glucuronosyl transferase activities . In further studies, IEC-17, IEC-18, and HuFoe-15 cells showed their capabilities of activating a broad spectrum of structurally heterogenous promutagens, as indicated by the induction of micronuclei . These cells, however, were not suited for the study of a more relevant genetic end point, the induction of hereditary functional changes (gene mutations), implying that a compromise had to be made on the level of the toxicodynamics . In the second approach, cDNAs encoding the rat cytochromes P-450IA1 and P-450IIB1, set under the control of a constitutive promoter, were transfected into V79 Chinese hamster cells, which do not express cytochromes P-450 but are ideal target cells for gene mutation assays . The resulting substrains (XEM1, XEM2, XEM3; SD1) stably expressed cytochromes P-450IA1 and P-450IIB1, respectively, and showed the corresponding monooxygenase activities . Aflatoxin B1, cyclophosphamide, dibutylnitrosamine, and benzo{a}pyrene mutated SD1 and/or XEM1 and XEM2 cells, but were inactive in parental V79 cells . The mutagenicity of benzo{a}pyrene 7,8-trans-dihydrodiol was about 1000 times more potent in XEM1 and XEM2 cells than in SD1 and V79 cells . Other promutagens were inactive in V79 as well as in the genetically engineered daughter lines . This system therefore is not yet optimal in general screening for the detection of new mutagens, but appears ideal in the identification of critical xenobiotic-metabolizing enzymes for a given mutagen.

Brain Dev, 1987, 9(6), 588 - 92
Cell culture study on neurofibromatosis; Hayashi A et al.; Cells obtained on culture from normal appearing skin (NFns) and from neurofibromas of neurofibromatosis patients (NFnf), and from normal skin of normal donors (Normal) were studied . We measured the growth rate, maximal cell density, radiation sensitivity and resistance to 3-nitrotyrosine of each of the three groups of strains . The growth rate and maximal cell density of fibroblasts derived from neurofibromas of the neurofibromatosis patients were significantly lower than those in normal donors . The neurofibromatosis cells showed no X-ray hypersensitivity, as compared to normal controls . We also did not observe resistance to 3-nitrotyrosine of the cells derived from normal appearing skin of the neurofibromatosis patients . However, cells derived from neurofibromas of the neurofibromatosis patients showed resistance to 3-nitrotyrosine . This phenomenon may explain the slow growth rate of the latter cells.

Chem Biol Interact, 1987, 64(1-2), 71 - 82
Effects of pretreatment with benzo{a}pyrene on the stereochemical selectivity of metabolic activation of benzo{a}pyrene to DNA-binding metabolites in hamster embryo cell cultures; Smolarek TA et al.; The proportions of individual benzo{a}pyrene (BaP)-DNA adducts present in rodent embryo cell cultures change with the length of time of exposure to BaP; the major alteration is an increase in the proportion of (+)-anti-7,8-dihydroxy-9,10-epoxy-7,8,9,10-tetrahydroBaP (BaPDE)-deoxyguanosine (dG) adduct (Sebti et al., Cancer Res., 45 (1984) 1594-1600) . To determine if this change in the BaP-DNA adducts could result from the induction of enzymes involved in oxidation of BaP, hamster embryo cell cultures were exposed to acetone or BaP for 24 h and then the medium was replaced with fresh medium containing {3H}BaP . After 5 h the BaP-pretreated cells had a 30% higher level of binding of BaP to DNA and formed a greater proportion of (+)-anti-BaPE-dG adduct than the acetone-pretreated control group . Cells pretreated for 24 h with BaP and then exposed to {3H}BaP and Actinomycin D for 5 h had a lower level of binding of BaP to DNA and a lower amount of (+)-anti-BaPDE-deoxyguanosine adduct than cells pretreated with acetone and exposed to {3H}BaP for 5 h . In contrast, pretreatment for 24 h with BaP plus Actinomycin D followed by a 5-h exposure to {3H}BaP resulted in a decrease in overall binding of BaP to DNA but had no effect on the amount of (+)-anti-BaPDE-deoxyguanosine adduct . Actinomycin D treatment had no significant effect on either the total amount of BaP metabolized, the formation of primary and water-soluble BaP metabolites, or cell viability, but reduced {3H}uridine incorporation into RNA by more than 65% at all times . These results suggest that induction of specific isozymes of cytochrome P-450 may be involved in the time-dependent increase in the proportion of (+)-anti-BaPDE-DNA adducts in BaP-treated cells . The state of induction of specific isozymes of cytochrome P-450 and the ability of the BaP dose applied to induce them may be major factors in determining the proportion of BaP metabolized to (+)-anti-BaPDE, the most carcinogenic stereoisomer of BaPDE.

Dev Biol Stand, 1987, 66, 503 - 9
A rapid and sensitive method for the detection of mycoplasmas in infected cell cultures using 6-methyl purine deoxyriboside; Whitaker AM et al.; 6MPDR causes the death of cells lightly infected with mycoplasmas . This is brought about by the action of the mycoplasmal enzyme adenosine phosphorylase which converts 6MPDR to highly toxic metabolites . If the medium conditions are adjusted to favour the growth of mycoplasmas by the addition of pig serum to the medium, infections as low as 1 mycoplasma per 200,000 cells can be detected within 7 days . This finding enables mycoplasma tests to be carried out rapidly and reliably by untrained personnel.

Dev Biol Stand, 1987, 66, 367 - 75
Process-scale purification from cell culture supernatants: monoclonal antibodies; Ostlund C et al.; A method for processing monoclonal antibodies (mAb) from large volumes of cell culture supernatants using recently developed high performance and fast flow chromatography media is described . A high-antibody producing mouse hybridoma cell line was adapted to low serum containing medium (1% foetal calf serum) for the production of anti-tissue Plasminogen Activator (anti-tPA) monoclonal antibody (murine subclass IgG1) . The process consisted of three main chromatographic steps: desalting, cation exchange on S Sepharose Fast Flow and gel filtration on Superose 6 prep grade . With this process for the purification of anti-tPA monoclonal antibody, the final product was greater than 95% pure with a total recovery of 75% i.e . 1.4 g was recovered in 0.345 l from 35 l of culture supernatant originally containing 1.9 g of mAb . The adaptation of this process for purification of other monoclonal antibodies is discussed.

Horm Res, 1987, 25(2), 80 - 7
Ectopic corticotrophin-releasing-factor and growth hormone releasing factor secretion: diagnosis using human pituitary cell culture; Adams EF et al.; Cell culture of human pituitary tissue has been used to diagnose a patient with Cushing's syndrome due to ectopic secretion of corticotrophin-releasing factor (CRF; case 1) and a case of acromegaly associated with ectopic secretion of a growth-hormone releasing factor (GRF; case 2) . In both patients a pituitary tumour was not detected . Case 1 had a small cell carcinoma and symptoms of the ectopic ACTH syndrome, but in culture the carcinoma failed to secrete detectable ACTH . However, the culture medium used to maintain this carcinoma in vitro was found to contain a substance which stimulated ACTH secretion by human pituitary corticotrophs in cell culture . Radioimmunoassays and HPLC indicated that this substance had similar elution characteristics to human CRF and cross-reacted with antiserum to ovine CRF . Case 2 was found to have a lung tumour, the removal of which led to regression of her acromegalic symptoms . In culture, this tumour did not secrete GH, but did secrete a GRF . We conclude that the Cushing's syndrome and acromegaly, in cases 1 and 2, respectively, were due to ectopic secretion of CRF and GRF leading to hyperstimulation of the pituitary gland.

Microbiol Immunol, 1987, 31(2), 139 - 46
Effect of cytolytic infection on maintenance of resistance to HVJ (Sendai virus) in an altered BHK cell culture; Yokoo J et al.; Altered baby hamster kidney (BHK-R) cells were serially cultured in the continuous presence of hemagglutinating virus of Japan (HVJ) . These cells showed a distinct resistance to superinfection with the homologous HVJ . This resistance of BHK-R cells gradually disappeared after serial passages in the presence of ultraviolet-irradiated HVJ particles which lost infectivity but still preserved hemagglutinating and neuraminidase activities . When BHK-R cells were serially cultured in the presence of a temperature-sensitive mutant of HVJ at non-permissive temperature, the cells also lost the resistance . The resistance of BHK-R cells remained unchanged, even after prolonged incubation in virus-free maintenance medium under the conditions of no cell division . It was suggested that killing of virus-sensitive cells, which were generated during cell proliferation, was required for maintenance of the resistance.

Vopr Virusol, 1987 Jan-Feb, 32(1), 71 - 4
{Suppression of rotavirus SA-11 reproduction by protease inhibitors in cell culture}; Bukrinskaia AG et al.; The effect of proteases inhibitors, epsilon-amino-caproic acid and gordox, on reproduction of rotavirus SA-11 in MA-104 cells was studied by enzyme immunoassay . These inhibitors were shown to exert an inhibiting effect on rotavirus reproduction.

Antimicrob Agents Chemother, 1987 Jan, 31(1), 76 - 80
Inhibiting effect of (RS)-9-{4-hydroxy-2-(hydroxymethyl)butyl}guanine on varicella-zoster virus replication in cell culture; Abele G et al.; The activity and mode of action of the new nucleoside analog (RS)-9-{4-hydroxy-2-(hydroxymethyl)butyl}guanine (2HM-HBG) against varicella-zoster virus (VZV) were determined . In cell culture, replication of different strains of VZV was inhibited to 50% by 0.4 to 0.7 microM 2HM-HBG, while 685 microM was required to inhibit 50% of the DNA synthesis in uninfected human lung fibroblasts . A thymidine kinase-negative VZV strain was not inhibited by 100 microM 2HM-HBG . Inhibition of VZV replication was not reversible after 7 to 14 days of incubation, depending on the multiplicity of VZV . 2HM-HBG was shown to be selectively phosphorylated by purified VZV thymidine kinase, with an inhibition constant of 32.5 microM . The antiviral activity of 2HM-HBG in cell culture was decreased by the addition of deoxythymidine and deoxycytidine but not by other ribo- or deoxyribonucleosides.

Experientia Suppl, 1987, 51, 243 - 8
Lysosome response and cytoskeleton alteration in cell cultures exposed to airborne lead; Orsi EV et al.; The results indicate that the delay in acridine orange loss by lysosomes exposed to UV can serve as a sensitive probe to the lead content of the cell milieu and the induced responses . This lysosome stabilization is in agreement with the reported decreased release of lysosome enzymes by lead in rat cerebral tissue . The lead induced lysosome stabilization reported here is undoubtedly pathologic in view of the cytoskeleton alteration that usually accompanied the introduction of lead in the cell media either from laboratory sources or natural conditions.

J Neurosci Res, 1987, 17(3), 225 - 34
Dehydroepiandrosterone and its sulfated derivative reduce neuronal death and enhance astrocytic differentiation in brain cell cultures; Bologa L et al.; Human studies of dehydroepiandrosterone (DHEA) and dehydroepiandrosterone sulfate (DHEA-S) have shown age-related changes in serum levels of these two sex hormone precursors . The levels of both DHEA and DHEA-S are characterized by monotonic decreases after puberty in females and after 20-24 yr of age in males . Further studies have shown that DHEA and DHEA-S levels are significantly low or close to minimal at ages when the incidence of senile dementia of Alzheimer's type (SDAT) begins to increase . We propose that DHEA and DHEA-S play a significant role in normal function of neuronal cells and that supplementation with them may prevent neuronal loss and/or damage . In the present study, using methods of immunocytochemistry, autoradiography, and scanning electron microscopy, we show that a supplement of as little as 10(-8) M DHEA or DHEA-S greatly increases neuronal survival and differentiation and reduces astroglial proliferation rates in mouse brain cells in cultures . These results suggest that correcting the DHEA and the DHEA-S deficit may prevent and/or improve the SDAT condition in humans.

Cancer Immunol Immunother, 1987, 24(2), 180 - 3
Lysis of autologous tumor cells by blood lymphocytes activated in autologous mixed lymphocyte tumor cell culture--no correlation with the postsurgical clinical course; Vanky F et al.; Mixed lymphocyte tumor cell cultures (MLTC) were initiated with cells collected at the time of surgery from 62 patients with the following diagnoses: 12 squamous cell carcinoma, 14 adenocarcinoma of the lung, 17 osteosarcomas, and 16 soft tissue sarcomas . The lytic effect generated against autologous tumor cells was tested on the 7th day . These patients had been part of previous evaluation, which revealed that the lysis of autologous tumor cells by freshly collected lymphocytes correlated with the postsurgical clinical course . Patients with long survival exhibited autotumor lysis without previous activation . Blood lymphocytes of about half of the primarily nonreactive patients were stimulated for lytic function in MLTC . However, this parameter showed no correlation with the clinical course.

Vet Med Nauki, 1987, 24(10), 8 - 13
{Electron microscopy research on cytopathic rotaviruses in cell cultures}; Georgiev GK et al.; Studied was the virus reproduction in cell cultures of MA-104 of the referent rotavirus strain Lincoln, and of the isolated from calves rotavirus Trebich 248/82 and Dolno-sakhrane 39/82 . Observed were the following main morphologic structures: electron-dense oval fractions, fractions of with single and double membrane, fractions with supercapsular membrane and electronic-densely granulated zones--viroplasms . There were not established considerable differences between the referent and the isolated from calves rotavirus strains in our country.

Arch Immunol Ther Exp (Warsz), 1987, 35(4), 501 - 10
The influence of temperature on interferon in cell cultures of homoiothermic and heterothermic mammals; Kandefer-Szerszen M et al.; Subnormal temperature was found to depress the production of interferon by cultures of fibroblasts of homoiotherms and heterotherms after virus or poly I.poly C induction . However, in comparison to human (HEF) and mouse (MEF) fibroblasts (homoiotherms) induced with NDV-R or poly I.poly C, interferon production in fibroblasts of spotted sousliks (SL) (heterotherm) and in the aneuploid line of mouse origin (L929) exhibits a greater cold resistance . In contrast to HEF and MEF cells in SL and L929 cells interferon was produced even at 21 degrees C after induction with NDV-R and at 26 degrees C after induction with poly I.poly C . A comparison of alpha and gamma interferon production by mouse and spotted souslik leukocytes did not reveal such distinct differences . At a low temperature (26 degrees C) the production of NDV-R induced interferon was depressed, but it was higher after induction with LPS in both types of leukocytes . PHA and Con A induced interferon was produced in both types of leukocytes only at 37 degrees C . These data confirm that alpha, beta and gamma interferon induction is triggered by different mechanisms and suggest a resistance to cold of beta interferon production in heterothermic animals.

Comp Immunol Microbiol Infect Dis, 1987, 10(3-4), 163 - 6
Susceptibility of various cell culture systems to pseudorabies virus; Onyekaba C et al.; A comparative study was carried out to determine the susceptibility of five different cell lines to pseudorabies virus (PRV), a herpes virus of pigs . The cell systems tested were swine testicle (ST), mink lung (ML), equine dermal (ED), porcine kidney (PK15), and bovine turbinate (BT) cells . Virus titers obtained were 10(4.88), 10(4.38), 10(3.75), 10(2.63), and 10(0.25) for ML, ST, PK15, BT and ED cells, respectively indicating that ML, ST, and PK15 are optimal cell lines for the growth of PRV whereas BT and ED are not very sensitive.

Arch Virol, 1987, 97(3-4), 373 - 7
Persistence of encephalitogenic arboviruses in brain cell culture . Brief report; Amor S et al.; Virus recovery from brain cultures of mice infected with either Semliki Forest and/or Langat depended on the time interval between inoculation of either virus . Mixed infections may alter the course of a disease.

Ann Rech Vet, 1987, 18(3), 255 - 9
{Influence of concanavalin A on the attachment of the coronavirus of transmissible gastroenteritis in cell cultures}; Nguyen TD et al.; Effect of Concanavalin (ConA) on attachment of three strains of Transmissible Gastroenteritis coronavirus (TGE) of swine was investigated in cell culture . Whatever the virus strain, reduction of virus plaques number is observed when viral suspension is incubated with cells together or after addition of ConA . Intensity of inhibition is related with ConA concentration . ConA treatment of preinfected cell cultures has no effect on plaque formation . Addition of alpha-methyl-D-mannoside inhibits the ConA activity . Treatment of cells with metaperiodate has no effect on plaque formation and ConA activity . Our results suggest that ConA inhibits formation of plaques by TGE coronavirus.

Exp Cell Biol, 1987, 55(3), 164 - 72
Polyadenylate polymerase activity in stationary and growing cell cultures; Kazazoglou T et al.; Soluble polyadenylic acid (poly(A} polymerase content of stationary and growing cell populations from a variety of cell lines was determined . Cell populations from stationary cultures presented poly(A) polymerase values with a mean of 31 +/- 12 enzyme units/mg protein . The mean value for growing cell populations were 62 +/- 18 enzyme units per mg protein . A statistically significant difference was found between stationary and growing cell populations from the variety of cell lines examined (p less than 0.1) . The observed differences in poly(A) polymerase levels persisted after fractionation of the crude extracts and revealed two molecular forms of enzyme activity with a net charge difference in stationary and growing cell cultures.

Arch Virol, 1987, 96(3-4), 283 - 7
Replication of avian encephalomyelitis virus in chick embryo neuroglial cell cultures . Brief report; Nicholas RA et al.; Avian encephalomyelitis virus replicated to relatively high titres in chick embryo neuroglial cell cultures, as measured by the indirect fluorescent antibody test, but induced few cytopathic effects . This cell system should provide a good source of virus for serological tests and vaccines.

Arch Immunol Ther Exp (Warsz), 1987, 35(4), 541 - 6
Histamine- and serotonin-releasing activity of the supernatants from guinea pig spleen cell cultures; Sulowska Z et al.; The action of supernatants from cultivated in vitro guinea pig spleen cells on the mast cells of guinea pigs, rats and hamsters was studied . It was found that supernatants from guinea pig spleen cell cultures are potent to release histamine from mast cells of the examined populations in a dose-dependent fashion . Histamine release from heterologous mast cells (especially from rat pleural mast cells) was significantly higher than that from homologous mesenteric mast cells . It was also demonstrated (on rat mast cells) that guinea pig spleen cell supernatants possessed not only histamine but 5-hydroxytryptamine releasing activity as well . Rat pleural mast cells were more sensitive and released more serotonin after challenge with spleen cell supernatants than peritoneal cells.

Exp Cell Biol, 1987, 55(3), 152 - 63
Initiation of primary cell cultures from human intracranial tumors on extracellular matrix from bovine corneal endothelial cells; Westphal M et al.; Tissue specimens from 105 human gliomas and 57 human meningiomas were obtained at surgery, dissociated into single cells and small cell aggregates and then plated onto plain plastic tissue culture dishes and dishes which had been precoated with an extracellular matrix (ECM) derived from bovine corneal endothelium . In 80% of the glioma cases we observed a marked improvement in initial plating efficiency, colony formation and speed of attachment when cells were plated on ECM . In 5 cases cells attached only to the ECM-coated dishes but remained afloat in the untreated dishes . In addition it could be noted that over the first 2 days, those cells which had been initiated on ECM showed more signs of morphological differentiation, i.e., extension of cytoplasmic processes or formation of fiber networks between cell groups . If adaptation occurred and proliferation began in vitro, either immediately or after a several days' lag phase, both the ECM-cultured cells as well as those which slowly had adapted to culture on plastic could be passed on to untreated culture ware and perpetuated thereon . In the case of well-differentiated low-grade gliomas where no growth in culture took place, the cultures on ECM could at least be used for initial experiments in the primary cultures (P0) . Meningiomas usually attached well to both, plastic or ECM . In 50% of our cases the plating efficiency was higher on ECM but after successful initial culture, the delay until the cells on plastic reached confluence in comparison with those on ECM was 1 or 2 days . Again there were 2 cases in which the cells would not plate on plastic . Here the cells which after 1 day were still afloat plated to more than 80% within the first 2 h after transfer to ECM . In all cases the cells from plastic and ECM cultures were indistinguishable and could be passed onto untreated dishes henceforth . In later culture stages ECM offers several advantages: It is easier to shift cells to serum-free defined culture conditions, the cells will grow at a faster rate on ECM when in higher passages and the maximal number of passages possible is higher on ECM.

C R Acad Sci III, 1987, 305(8), 295 - 300
{Particles with retrovirus appearance and reverse transcriptase activity in cell cultures derived from lymph node biopsies in Hodgkin's disease}; Lesser J et al.; Long term cultures of Reed-Sternberg like cells were obtained from lymph node biopsies of two patients suffering from Hodgkin's disease . As soon as the 15th day of culture, a weak magnesium-dependent reverse transcriptase activity was observed in the cell culture supernatants . Retroviral-like particles were observed in cell cultures as well as in their supernatants.

J Neurosci Res, 1987, 17(3), 220 - 4
Investigations on myelinogenesis in vitro: developmental expression of myelin basic protein mRNA and its regulation by thyroid hormone in primary cerebral cell cultures from embryonic mice; Shanker G et al.; The concentration of myelin basic protein (MBP) mRNA in primary cultures of cells dissociated from embryonic mouse cerebra and grown in the presence of varying amounts of thyroid hormone was measured using a 32P-labeled cDNA probe and a dot-blot procedure . The cDNA probe contained 1.85 kilobases of the gene for MBP . The concentration of mRNA specific for MBP in control cells grown on a medium containing normal (euthyroid) calf serum increased with increasing age of culture . The greatest increase occurred between 15 and 35 days in culture (5.25-fold increase); whereas between 35 and 50 days in culture, the rate of accumulation slowed to yield a net increase of MBP mRNA of only 10% . The quantity of MBP mRNA was drastically diminished at all ages studied when the cells were grown from the sixth day onward on a medium containing hypothyroid calf serum . Although the amount of MBP mRNA in hypothyroid-treated cells did increase, the change in concentration was less (3.43-fold), and it peaked earlier (at 30 days) . Unlike the euthyroid cells, after 30 days the MBP mRNA actually fell in the hypothyroid-treated cells . If hypothyroid media were supplemented with triiodothyronine (T3) on the eighth day in culture, the quantity of MBP mRNA in the cells was restored almost completely to the levels found in the control euthyroid cells at all ages . Therefore, the regulation of the synthesis of MBP by thyroid hormone is at least in part a pretranslational event; that is, thyroid hormone adjusts the concentration of the mRNA specific for MBP.

Folia Haematol Int Mag Klin Morphol Blutforsch, 1987, 114(1), 42 - 61
{The value of cell cultures in leukemias, aplastic anemias and bone marrow transplantations}; Schulze E et al.; In a survey different methods of culture are represented for the purpose of identifying and quantifying haemopoietic stem cells (CFU-GEMM, CFU-GM, CFU-E/BFU-E) in human bone-marrow or peripheral blood respectively . On the basis of findings from international medical literature their validity is explained in the diagnostics and prognosis of some haematological diseases, such as acute and chronic myeloic leukemia, aplastic anemia, preleukemia . Special attention is given to their significance within bone-marrow transplantation . Their importance in evaluating transplantations after their preceding in-vitro manipulation as to the separation of rest tumour and T-cells is particularly referred to.

Acta Histochem Suppl, 1987, 34, 57 - 75
Identification of cytoskeletal structures in hormone producing lung cancer cell cultures; Broers JL et al.; In order to investigate the intermediate filament protein content of hormone producing lung tumor cell cultures a panel of 16 different cytokeratin antisera were tested using immunocytochemical and biochemical techniques on lung carcinoma cell cultures from different origin . These included three cell cultures derived from small cell lung carcinoma, two large cell carcinoma cell cultures, and two cell cultures derived from squamous cell carcinomas . Flow cytometric analysis of the cell cultures demonstrated that all cell lines examined were aneuploid with DNA-indices ranging from 1.7 to 3.1 rimes the DNA-content of normal human lymphocytes . In both immunofluorescence and immunoperoxidase techniques six out of seven cell cultures reacted with most of the cytokeratin antisera used in a filamentous manner, while a large cell carcinoma cell culture did not react with any of the cytokeratin antisera used . None of the cell cultures examined reacted with the antibodies to neurofilament proteins, suggesting that none of these (neuro)hormone producing cell cultures were of neural origin . All cell cultures which were growing as adherent cell cultures did express vimentin . The cell culture that grew with cells floating in aggregates did not express this intermediate filament protein while a subline which did attach, expressed vimentin . This findings strongly indicates the relation between growth pattern in vitro (floating vs . adherent) and the expression of vimentin . No reaction was found with antisera to desmin and GFAP . The presence of cytokeratins and vimentin in most cell cultures could be confirmed using one- and two-dimensional gel electrophoresis . Cytokeratins 7, 8, 18 and 19 were most commonly present.

Nat Immun Cell Growth Regul, 1987, 6(1), 1 - 11
Inhibition of human natural killer activity by monolayers of primary cell cultures; Heiskala M et al.; Some primary and continuous cell cultures were tested for their capacity to regulate human natural killer (NK) activity . Primary cultures of endothelial cells, fetal fibroblasts, adult fibroblasts, amnion epithelial cells, renal parenchymal cells, and ovarian carcinoma cells inhibited NK activity when peripheral blood lymphocytes were preincubated on target cell monolayers for 18 h before testing the cytotoxicity against K-562 . The supernatants of the inhibiting cell cultures were not suppressive . Prostaglandins or suppressive lymphocytes were not involved in the phenomenon . The binding capacity of the effector cells was not changed, suggesting that the suppressive signal was targeted at the cytolytic machinery of NK cells . The down-regulating capacity of the cell cultures weakened significantly during subculturing in vitro, and continuous cell lines were not inhibitory . The inactivation of NK cells may be one of the mechanisms by which target cells are protected from NK activity.

Eur J Immunol, 1987 Jan, 17(1), 21 - 6
Carrier-specific T cells sufficient for the expression of multiple isotypes in B cell cultures; Kotloff DB et al.; A modified splenic fragment assay was used to assess the role of antigen-specific helper T cells in B cell isotype expression . Limiting numbers of carrier-specific helper T cells from lines or clones were injected along with a source of B cells into lethally irradiated unprimed recipients . The incidence of lodging of the T cell lines in recipient spleens at 18 h was determined by autoradiography to be 1.5 to 4.3% of the injected cells . These T cells were necessary and sufficient for the generation of T-dependent B cell responses within splenic fragments cultured in vitro with specific antigen . A comparison of isotypic responses from splenic and Peyer's patch B cells generated with the same T cell population revealed that a high proportion of the response from Peyer's patch B cells consisted of IgA antibody exclusively (46-57%) while the percentage of such responses from splenic B cells was much lower (7-10%) . Thus, the isotype pattern of the response reflected the B cell source . Experiments in which cloned hemocyanin-specific T cells provided help to T-depleted spleen cells within splenic fragments from athymic recipients indicated that a single specificity of helper T cell is both necessary and sufficient to support the generation of antibody responses consisting of multiple isotypes . Isotype-specific T cells do not appear to be required in this system.

J Invest Dermatol, 1987 Jan, 88(1), 33 - 6
Epidermal cell culture using Sephadex beads coated with denatured collagen (cytodex 3); Katayama H et al.; Epidermal cell culture using microcarriers of Sephadex beads coated with denatured collagen (cytodex 3) was performed . Epidermal basal cells (above 95%) obtained from human skin by trypsinization were cultivated statically on the beads in 96-well culture plates . Proliferation was rapid and great in synchronous waves during 2-7 days after inoculation . The growth rate depended on the inoculation cell population densities . When cells were inoculated at 7.75 X 10(4)/well, the maximum increase was 3.6-fold and at 1.69 X 10(4)/well and 0.32 X 10(4)/well, 2.5-fold and 2.1-fold, respectively . Differentiation was assessed visually on a hemocytometer . The percentage of basal cells of the total cells present in each well was reduced from 98% (on inoculation) to approximately 25% in a week and thereafter . In 1 month after inoculation, cells with keratohyaline-like granules were observed at 12% . The attachment of cells to the beads was rather loose . Cells were supposed to be attached to denatured collagen on the beads via fibronectin contained in the serum of the medium, because denatured collagen had the property to bind strongly to fibronectin . Loose attachment made it possible to harvest intact cells without the use of trypsin . This cell culture system with such new characteristics will be a useful tool for studying epidermal cell biology and biochemistry.

Strahlenther Onkol, 1986 Dec, 162(12), 785 - 92
{Effect of cisplatin on the recovery from sublethal and potentially lethal radiation damage in cell culture}; Ziegler W; The effect of Cisplatin upon the recovery from sublethal damage during fractionated irradiation and upon the recovery from potentially lethal damage after a single dose has been investigated . In three mammalian cell lines, Cisplatin did not influence the recovery from sublethal and potentially lethal damage . There were differences among the three cell lines in their ability to recover from radiation damage . However, a significant quantitative difference between the recovery from sublethal and potentially lethal damage in any one cell line could not be found.

Cancer Res, 1986 Dec, 46(12 Pt 1), 6156 - 9
cis-Diamminedichloroplatinum(II)-induced sister chromatid exchange: an indicator of sensitivity and heterogeneity in primary human tumor cell cultures; Tofilon PJ et al.; The effect of cis-diamminedichloroplatinum(II) (cPt) on sister chromatid exchange (SCE) induction was determined in 13 human primary tumor cell cultures . Primary cultures were derived from surgical specimens of solid tumors composed of a variety of histologies . Three to 16 days after biopsy, depending on the growth rate, cultures were treated with graded concentrations of cPt for 1 h and the SCE assay was performed . SCE dose-response curves (SCEs induced per chromosome versus cPt concentration) showed a wide range in cPt sensitivities that was not dependent on histology . SCE frequency histograms showed that several of the primary cultures contained both cPt-sensitive and -resistant cells . For six of the cultures, the SCEs induced per chromosome at 15 microM cPt were plotted versus the IC90 determined from a survival assay . A line fit to those points yielded a correlation coefficient of -0.74 . These results show a relationship between the activity of cPt in the SCE assay and in the survival assay, which suggests that SCE analysis may be useful for predicting cPt sensitivity . In addition, characterization of cellular heterogeneity in cPt sensitivity using the SCE assay may provide additional information useful in the prediction of tumor response to treatment.

Infect Immun, 1986 Dec, 54(3), 913 - 6
Selective infection of astrocytes by Chlamydia trachomatis in primary mixed neuron-glial cell cultures; Levitt D et al.; Both human biovars of Chlamydia trachomatis were able to productively infect primary cultures of fetal rat brain cells . Infected brain cells released bacteria that reinfected McCoy cells well as other cultured brain cells . The chlamydiae infected cultured astrocytes but were never observed to grow inside neurons, suggesting a selective susceptibility of specific brain cells to chlamydial infections.

Endocrinology, 1986 Dec, 119(6), 2695 - 9
Somatomedin-C substitutes for insulin for the growth of mammary epithelial cells from normal virgin mice in serum-free collagen gel cell culture; Imagawa W et al.; We investigated the effect of somatomedin C (SM-C) on the growth of mouse mammary ductal epithelial cells in collagen gel culture . Epithelial cells, isolated by collagenase digestion of whole glands, were placed into primary serum-free collagen gel cell culture for 10-12 days, during which SM-C was added alone or in combination with other growth-promoting factors . Previous work has shown that these cells require a superphysiological concentration of insulin (10 micrograms/ml) for optimum growth in serum-free medium (a 1:1 mixture of Ham's F-12 and Dulbecco's Modified Eagle's medium) containing epidermal growth factor (EGF) . When SM-C (1-250 ng/ml) alone was added to serum-free basal medium containing EGF, it stimulated growth (at concentrations greater than 25 ng/ml) to at least the same extent as insulin at 10 micrograms/ml . There was no additive stimulation of growth when optimal concentrations of insulin and SM-C were added together . The nonadditive stimulation at optimal concentrations of these hormones may indicate that the previous requirement for a superphysiological concentration of insulin for maximum growth was due to low affinity binding of insulin to the SM-C receptor . Rat insulin-like growth factor II (Collaborative Research) at 50-200 ng/ml did not stimulate growth in the presence or absence of insulin . SM-C could not stimulate growth alone . The presence of EGF or mammogenic hormones (progesterone and PRL) was required.

J Bone Miner Res, 1986 Dec, 1(6), 489 - 95
Effect of vitamin D metabolites on the expression of alkaline phosphatase activity by epiphyseal hypertrophic chondrocytes in primary cell culture; Hale LV et al.; The effects of three vitamin D3 metabolites, 25-hydroxyvitamin D3 (25-(OH)D3), 1 alpha,25-dihydroxyvitamin D3 (1 alpha, 25-(OH)2D3), and 24R,25-dihydroxyvitamin D3 (24R,25-(OH)2D3) on the activity of alkaline phosphatase (AP), a key enzyme involved in biomineralization, have been studied in primary cultures of chicken epiphyseal growth plate chondrocytes . Dosages of 1 alpha, 25-(OH)2D3 (10(-12) to 10(-7) M) caused a progressive, dosage- and time-dependent decrease in cellular AP levels, IC50 occurring at approximately 10(-12) M . In contrast, 24R,25-(OH)2D3 at 10(-13) to 10(-10) M stimulated cellular AP activity, half-maximal stimulation occurring at about 10(-13) M . At higher levels (10(-10) to 10(-7) M), 24R,25-(OH)2D3 caused progressive reduction in AP activity . Maximal effects of 24R,25-(OH)2D3 were evident 48 h after administration of the metabolite . 25-(OH)D3 initially (24 h) caused a weak, dosage-dependent decrease in cellular AP activity, but after 48-72 h, low levels (10(-13) to 10(-11) M) caused a dosage-dependent increase in AP activity . Higher levels of 25-(OH)D (greater than 10(-10) M) were clearly inhibitory to AP . These findings reveal that the AP activity of growth plate chondrocytes is exquisitely sensitive to both 1 alpha,25- and 24R,25-(OH)2D3 but the response to each is in opposite directions . The paradoxical response of the cells to 25-(OH)D3 can be explained if the metabolite is slowly metabolized by a 24-hydroxylase to 24R,25-(OH)2D3 leading to stimulation of cellular AP.(ABSTRACT TRUNCATED AT 250 WORDS)

Hum Genet, 1986 Dec, 74(4), 453 - 5
Cell culture studies on neurofibromatosis (von Recklinghausen) . V . Monosomy 22 and other chromosomal anomalies in cultures from peripheral neurofibromas; Krone W et al.; Cell cultures grown from peripheral neurofibromas of three patients suffering from sporadic peripheral neurofibromatosis (NF) were analysed cytogenetically at early in vitro passages . The NF-cultures exhibited a 6.7-fold higher frequency of aneuploid mitoses, including pseudodiploids, than the control cultures derived from the skin of three healthy donors . The predominant numerical anomaly was monosomy 22 . Several, as yet unidentified marker chromosomes occurred in the NF-cultures, which also showed a much higher level of unstable chromosomal anomalies . The role of monosomy 22 in tumorigenesis of meningiomas and neurofibromas is discussed.

Proc Natl Acad Sci U S A, 1986 Dec, 83(23), 9269 - 73
Modulation of gamma-aminobutyric acid-mediated inhibitory synaptic currents in dissociated cortical cell cultures; Vicini S et al.; Inhibitory gamma-aminobutyric acid-mediated synaptic currents were studied in dissociated primary cultures of neonatal rat cortex with the whole-cell patch-clamp technique . Immunocytochemical staining of the cultures showed the presence of a large number of glutamic acid decarboxylase-containing neurons, and electrical stimulation of randomly selected neurons produced in many cases chloride-mediated and bicuculline-sensitive inhibitory synaptic currents in postsynaptic cells . The amplitude and decay time of the inhibitory synaptic currents were increased by flunitrazepam and decreased by the beta-carboline derivative methyl 6,7-dimethoxy-4-ethyl-beta-carboline-3-carboxylate, two high-affinity ligands for the allosteric regulatory sites of gamma-aminobutyric acid receptors . The imidazobenzodiazepine Ro 15-1788, another high-affinity ligand of the gamma-aminobutyric acid receptor regulatory sites that has negligible intrinsic activity, blocked the action of flunitrazepam and beta-carboline . However, Ro 15-1788 also increased the decay rate of the inhibitory synaptic currents . This might suggest that an endogenous ligand for the benzodiazepine-beta-carboline binding site is operative in gamma-aminobutyric acid-mediated synaptic transmission.

Biochem Biophys Res Commun, 1986 Nov 26, 141(1), 46 - 52
Differential heat shock response of primary human cell cultures and established cell lines; Richter WW et al.; The influence of hyperthermia on the cellular growth and protein synthesis pattern from primary human brain tumour cells and skin fibroblasts was compared with established and experimentally transformed tumour cell lines . Primary cell cultures did not show any visible morphological changes after 42 degrees C treatment, whereas in immortalized cell lines usually 90% of the cells were found in suspension . Enhanced expression of the major heat shock protein (hsp 70) was found in all heat-treated cells . In contrast to the primary cell cultures, established and transformed cell lines synthesized a protein with an apparent molecular mass of 70 kDa and an isoelectric pH of 7.0 as early as 3 h after the initial hyperthermal treatment.

J Immunol Methods, 1986 Nov 20, 94(1-2), 169 - 79
Measurement of immunoglobulin concentration in cell culture supernates by computer-assisted ELISA; Slade HB et al.; The enzyme-linked immunosorbent assay (ELISA) is used extensively in immunologic research for obtaining quantitative estimates of immunoglobulin concentration in cell culture supernates . Through incorporation of a microcomputer for data acquisition, storage and rapid calculation of results, a substantial reduction in total assay time may be realized . Described here are a set of menu-driven programs written in Basic for the IBM-PC which provide advantages over existing software in simplicity, versatility and accuracy . Hardware requirements are minimal . These programs should encourage greater flexibility in terms of the size and complexity of experimental designs.

Immunol Lett, 1986 Nov 3, 13(6), 289 - 94
Histamine-releasing activity of lymphocyte supernatants of guinea pig spleen cell cultures; Brzezinska-Blaszczyk E et al.; We demonstrated the production of a histamine releasing factor (HRF) by 24-h cultures of guinea pig spleen cells which were stimulated or not with specific antigen (ovalbumin, OA) or mitogen (phytohemagglutinins or concanavalin A) . HRF induced the release of histamine from homologous mesenteric mast cells in a dose-dependent fashion . The HRF-induced histamine release was not high compared to the release induced by calcium ionophore A23187, but higher than that induced by compound 48/80, polymyxin B and con canavalin A . The mast cells from sensitized guinea pigs released histamine when challenged with OA . We found that HRF-induced histamine release was additive to that induced by antigen, when both agents were added simultaneously to sensitized mast cells . The phenomenon was most significant when a suboptimal dose of antigen was used . Moreover, we did not observe any differences in the magnitude of HRF-induced histamine release between the mast cells from nonsensitized and sensitized guinea pigs . The time course of histamine release induced by HRF was significantly slower than that with specific antigen (10 min and 45 sec, respectively) . Our results may suggest that HRF acts on mast cells through a different not immunological mechanism.

Vopr Virusol, 1986 Nov-Dec, 31(6), 712 - 7
{Nucleolus-forming regions in the chromosomes in continuous cell cultures}; Tsareva AA et al.; Localization of nucleoli-forming (NF) areas in chromosomes of continuous cell cultures of African green monkey kidney Vero, CV-1, GMK, BGM, BS-C-1 . Macaca rhesus kidney LLC-MK2, dog kidney MDCK, pig kidney SPEV, PK-15 was studied . The NF-areas in chromosomes of African green monkey, M . rhesus, and pig cell cultures were shown to be localized in sites of secondary chromosomal stangulation, and there was also a definite correlation between the intensity of staining of NF-areas and sizes of the zone of secondary strangulation . In chromosomes of MDCK cell line, NF-areas are located in telomere ends of six-nine chromosomes, and association of two chromosomes by NF-areas was observed . The possibility of using the method of identification of nucleoi-forming areas for the evaluation of productivity of continuous cell cultures is discussed.

Anticancer Res, 1986 Nov-Dec, 6(6), 1329 - 36
Influence of inoculation sites and tumor cell culture techniques on the phenotype of a mixed cartilage- and bone-producing mouse mammary tumor; Lespagnard L et al.; We have previously reported the characterization of an intraperitoneally (IP) transplantable bone-forming MXT tumor . However, the question was unresolved as whether the bone-forming cells originated from either the host animal or from the neoplasm itself . The present work attempts to answer this question by studying the influences of inoculation sites (subcutaneously, SC; intraperitoneally, IP; in the brain, IB; intracranially, ICR) on both the cartilage- and bone-forming tumor phenotypes . Furthermore the influence of cell culture procedures (two- and three- dimensional cultures) on these phenotypes was investigated . SC administered MXT cancer cells never produce bone-forming tumors, suggesting the existence in the dermis of substance(s) inhibitory to the formation of cartilage or bone . On the contrary, our data clearly demonstrate that bone-forming tumors can be obtained by either IP route, in a way which mimics endochondral ossification, or in the brain (IB), a region usually devoid of connective tissue . This observation substantiates the hypothesis according to which the tumor itself is able to produce osseous tissue . Another main finding is the increasing occurrence of skeletal tissues produced by cells proceeding from three-dimensional culture . Finally, ICR and IB tumors exerted a bone-lytic action against the host skull suggesting that tumor cells either produce osteolytic substances (prostaglandins, enzymes) and/or that they contain various cell types exhibiting different properties toward osteogenesis . This model offers new perspectives for studying the mechanisms of both normal and pathologic osteogenesis.

Tsitol Genet, 1986 Nov-Dec, 20(6), 403 - 9
{Study of the chondriome in SPEV cell culture after treatment with actinomycin D}; Shornikova MV et al.; It is shown that the 12-hour treatment of cell with actinomycin D (AMD) in the concentration of 0.05 microgram/ml disturbs a correlation between the morphological cycle of mitochondria and phases of the mitotic cell cycle which is characteristic of intact cells . An increase in the total number of mitochondria independent of phase is observed in all the cells in comparison with intact cells . At the same time a decrease in the amount of branched organellae and appearance of giant mitochondria are discovered . All mitochondria are in the condense form . These changes, perhaps, are a result of the inhibition of the rRNA synthesis in the nucleus and of the protein synthesis in the cell found with it by AMD . The possibility of the immediate interaction of AMD with membrane components of the cell, which induces changes in the ion concentrations and peroxidation of the membrane lipids is not excluded.

Res Vet Sci, 1986 Nov, 41(3), 391 - 6
Porcine parvovirus: propagation in microcarrier cell culture and immunogenic evaluation in pregnant gilts; Rivera E et al.; Porcine parvovirus was propagated in PK-15 cells cultured in roller bottles or on microcarrier beads . After inactivation, the virus was used as antigen in the preparation of vaccines . The immunogenic potency and safety of the vaccines were evaluated in specific pathogen free pregnant gilts and guinea pigs . Experimental challenge tests determined the efficacy of the vaccine in preventing porcine parvovirus transplacental infection . Neither viral antigens nor specific antibodies were detected in fetuses from vaccinated gilts . In contrast, fetal death and, or, mummification occurred when unvaccinated gilts were infected . Both virus and, or, antibodies were also detected in fetuses from these unvaccinated gilts . Serum conversion after vaccination was assayed by microserum neutralisation using guinea pig erythrocytes as cell indicators and by haemagglutination inhibition tests . Viral antigens in fetal tissues were detected using ELISA, the immunobeads technique, the haemagglutination test and by virus isolation.

Radiobiologiia, 1986 Nov-Dec, 26(6), 800 - 2
{DNA synthesis in a stationary HeLa cell culture following gamma irradiation}; Synzynbys BI et al.; Residual incorporation of 3H-thymidine into acid insoluble fraction was inhibited a few hours and stimulated 24 hours following gamma-irradiation (60Co) of a stationary culture of HeLa cells with doses of 5 to 50 Gy . The dose-response curve for the stimulated incorporation reached a maximum at a dose of about 10 Gy . Hydroxyurea (10 mM) was shown to suppress the incorporation . The authors suggest that ionizing radiation induces a transfer of resting cells to the S phase-like state.

J Androl, 1986 Nov-Dec, 7(6), 355 - 66
Transferrin-mediated transcellular transport of 59Fe across confluent epithelial sheets of Sertoli cells grown in bicameral cell culture chambers; Djakiew D et al.; The transferrin-mediated transcellular transport of 59Fe across confluent epithelial sheets of Sertoli cells grown on Millipore filters was investigated . These filters had been impregnated with reconstituted basement membrane and suspended in bicameral (two houses) culture chambers . After five days of culture, Sertoli cells from 10-day-old rats formed basally-located tight junctional complexes . Concomitantly, there was an increase in electrical resistance and the epithelial sheet became impermeable to lanthanum nitrate . The rate of passage of {3H}inulin across the epithelial sheet was considerably less than passage across a filter alone, a filter impregnated with reconstituted basement membrane or an epithelial sheet pretreated with 2 mM EGTA . We conclude from these permeability studies that the tight junctional complexes between Sertoli cells formed an effective transepithelial permeability barrier . Following addition of human serum {59Fe}transferrin to media bathing the basal cytoplasm of the cells, rat testicular {59Fe}transferrin was immunoprecipitated from apical media overlying the Sertoli cells . Cross-reactivity of the rabbit anti-rat transferrin antibody with human serum transferrin was less than 0.001% . Substitution of the primary antibody with normal rabbit serum reduced the amount of immunoprecipitable rat testicular {59Fe}transferrin to 20% of normal levels . Prior fixation of the Sertoli cell epithelial sheet in 2.5% glutaraldehyde, addition of a 100-fold excess of holotransferrin to the basal media, and incubation of the Sertoli cell epithelial sheet at 4 C all reduced the immunoprecipitable rat testicular {59Fe}transferrin in apical media to levels below that for the non-specific binding of the primary antibody . From these studies we conclude that 59Fe is shuttled across Sertoli cells by two different forms of transferrin . Serum transferrin delivers the 59Fe to the basal cytoplasm of the Sertoli cells . The 59Fe dissociates from the serum transferrin, is delivered to testicular transferrin, and is subsequently secreted from the apical surface of the epithelial sheet of Sertoli cells as testicular {59Fe}transferrin.

Anat Rec, 1986 Nov, 216(3), 416 - 22
Adipocyte development in primary rat cell cultures: a scanning electron microscopy study; Richardson RL et al.; This study utilized scanning electron microscopy (SEM) techniques to observe primary cultures of stromal-vascular (SV) cells derived from postnatal rat inguinal adipose tissue . Cells were grown on collagen-coated, fibronectin-coated, or uncoated glass coverslips . Coverslips were normally fixed in glutaraldehyde, osmium tetroxide, dehydrated, and critical-point-dried . Other coverslips were frozen in isopentane (cooled in LN2) and dried or fixed in Baker's formalin for demonstration of inosine diphosphatase (IDPase) by X-ray microprobe analysis (XRMA) . Adipocyte morphologies were similar on all substrates . At 2 days of culture, actin cables were detected extending from developing adipocytes . No difference in actin cable structure, cellular shape, or lipid accumulation was observed among the different substrates . Some stromal cells did not accumulate lipid but proliferated into a multilayer by 9 days in culture . Inosine diphosphatase was detected in the Golgi apparatus of developing adipocytes utilizing the technique of XRMA . This study demonstrates the potential for using SEM and XRMA techniques to define morphological features and cytochemical markers of adipocytes in vitro and the response of primary cultured rat SV cells to other attachment substrates.

Brain Res, 1986 Nov, 395(1), 110 - 3
Effects of AF64A on {3H}acetylcholine synthesis in neuron-enriched primary brain cell cultures; Koppenaal DW et al.; {3H}Acetylcholine (ACh) synthesis was measured in primary neuronal cultures from neonatal rat brains . Neuronal {3H}ACh synthesis was blocked by hemicholinium-3 and depended on the age of the cultures, increasing for ca . 10 days, and eventually declining . The irreversible inhibitor AF64A (10 or 30 microM) inhibited {3H}ACh synthesis from {3H}choline at concentrations (10 or 30 microM) with affecting choline acetyltransferase activity . Nine-day-old cultures recovered 90% of their {3H}ACh synthesis within 7 days after AF64A, while 13-day-old cultures never recovered . These results suggest that the turnover of neuronal choline transporters is age-related.

Virology, 1986 Nov, 155(1), 267 - 70
Characterization of a variant virus isolated from neural cell culture after infection of mouse coronavirus JHMV; Taguchi F et al.; Our previous experiments showed that a variant virus with a larger envelope glycoprotein encoded by a larger mRNA3 (cl-2) multiplied predominantly in the brain of rats after wild type (wt) JHMV infection (F . Taguchi, S . Siddell, H . Wege, and V . ter Meulen, 1985, J . Virol . 54, 429-435) . We could isolate similar but not identical variant virus after infection of cultured neural cells from rat brain with wt JHMV (designated CNS virus), which also had a larger mRNA3 and produced larger envelope E2 glycoprotein in infected cells . CNS virus multiplied to a higher degree in cultured astrocytes from rat than wt JHMV and cl-2 . During infection with these variant viruses in neural cells, virus populations generated did not change, in contrast to consistent selection of viruses with larger mRNA3 after wt JHMV infections . CNS virus produced abundant mRNA2a as well as 65K glycoprotein while the productions of both were trace in cl-2 infected cells . The present experiments, together with our previous observation, suggest that the larger E2 glycoprotein may be of importance for the replication in rat brain cells.

J Surg Res, 1986 Nov, 41(5), 463 - 72
The cytotoxic effect of surgical glove powder particles on adult human vascular endothelial cell cultures: implications for clinical uses of tissue culture techniques; Sharefkin JB et al.; Clinical use of autogenous endothelial cell (EC) seeding of vascular prostheses (VP) would require reliable methods for EC harvest for immediate seeding or primary culture in a hospital or operating room setting . Observation of glove powder particles (GPP) in failed primary adult human saphenous vein EC (AHSVEC) cultures led us to study the effect of surgical GPP on cultured AHSVEC . Addition of GPP to the culture medium of growing ASHVEC cultures reduced the cell counts in a dose-dependent fashion; the mean concentration of GPP required to produce a greater than 50% decrease in cell number was 1.5 +/- 0.8 (SD) X 10(4) GPP/ml (N = 10 experiments), equivalent to a mean dose of 36 micrograms glove powder per milliliter . The effect was seen within 24 hr of addition of GPP and was not due to interference with EC attachment and spreading or to changes in medium osmolality, pH, glucose, electrolyte, Ca2+, or Mg2+ content . Instead, the effect appeared to be due to a filterable toxin added during the final rubber-vulcanizing stage of glove manufacture, since pure cornstarch particles and epichlorhydrin-treated pure cornstarch did not prevent culture growth, whereas 0.2 micron filtrates of medium incubated with GPP taken directly from gloves were lethal . We conclude that filterable cytotoxic substances from GPP may be an avoidable cause of failure in EC seeding of VP, and may affect surgical wound healing as well.

Cytometry, 1986 Nov, 7(6), 551 - 7
The fate of the primary diploid population during spontaneous transformation of growth factor-supplemented murine cell cultures; Kubbies M et al.; Primary cultures derived from lung and renal tissue of the newborn harvest mouse (Micromys minutus) were serially passaged in media supplemented with epidermal growth factor, hydrocortisone, transferrin, insulin, and triiodothyronine . Although these growth factor supplements eliminated the growth crisis commonly encountered during the initial stages of murine primary cultures, the original diploid cell fraction clearly underwent such a "crisis"; the truly diploid cells invariably disappeared as these cultures reached 20 to 40 population doublings . They were replaced, either gradually or precipitously, by various heteroploid cell fractions . In three of four independent cultures, these "established" cells were hypotetraploid and appeared to be derived from a small number of progenitors already present during the very early (precrisis) culture stages . In contrast to rather frequent DNA changes displayed by clones and subclones derived from the various heteroploid cell lineages, the predominant components of the established mass cultures displayed a highly constant DNA fluorescence pattern . Our results suggest that primary murine cell cultures develop heteroploid cell lineages even if the initial growth crisis is mitigated by growth factor supplements . These heteroploid cells appear to respond more efficiently to stimulation by various growth factors than the primary diploid cell population.

Endocrinology, 1986 Nov, 119(5), 1933 - 8
Progesterone antagonizes the ability of porcine ovarian inhibin to sensitize ovine pituitary cell culture to luteinizing hormone-releasing hormone: dependence on ovaries in vivo; Batra SK et al.; Progesterone (P4) and a porcine follicular preparation of inhibin (MGRA-IV) have opposite actions on regulation of the ability of LHRH to release LH in ovine pituitary cell culture . Both P4 and inhibin change the response to LHRH . The ability of inhibin to sensitize cultures to LHRH (126-273%) was greatly inhibited (up to 100%) in the presence of P4 (10(-7) M) . The inhibitory action of P4 on LHRH-stimulated and inhibin-sensitized LHRH-stimulated LH secretion in ovine pituitary cell culture was dependent on the presence of ovaries in vivo . P4 inhibited 68% of LHRH-stimulated LH secretion in pituitary cultures from intact ewes . However, when cultures were prepared from pituitaries collected on days 9, 21, and 42 after ovariectomy, P4 inhibited LHRH-stimulated LH secretion by only 36%, 13%, and 0%, respectively . Ovariectomy had no effect (P greater than 0.05) on the sensitizing action of inhibin on LHRH-stimulated LH secretion, but ovariectomy did cause a time-dependent decline in the inhibitory action of P4 on inhibin-sensitized LHRH-stimulated LH secretion . Furthermore, when cultures were prepared from pituitaries collected from ewes ovariectomized for 35 days but treated with estradiol implants, both LHRH-stimulated and inhibin-sensitized LHRH-stimulated LH secretion were inhibited as well by P4 as in pituitary cultures from intact ewes . These results suggest that although P4 can completely inhibit the sensitizing action of inhibin on LHRH-stimulated LH secretion, its inhibitory action is dependent on the presence of ovaries or estradiol in vivo.

J Natl Cancer Inst, 1986 Nov, 77(5), 1125 - 35
Tumor progression in nude mice and its representation in cell culture; Rubin H et al.; Varying dilutions containing from 10(6) to 10(3) spontaneously transformed Balb/3T3 cells were inoculated into nude mice {N:NIH(S)II} . Less than half the mice inoculated with 10(3) cells developed tumors . The higher concentrations of cells produced visible tumors in all mice within 2-3 weeks, and these tumors grew rapidly to large sizes . Some tumors initiated by the lower concentrations of cells arose quickly, but others were greatly delayed in onset, then grew slowly, if at all, for several weeks before a rapid acceleration . The delayed acceleration can be considered a form of tumor progression . When first explanted into culture, cells from the early tumors multiplied somewhat more slowly than the parental cells that initiated the tumors, but narrowed the gap in a few weekly passages . By contrast, only a small fraction (less than or equal to 0.001) of cells from the longest delayed tumors could sustain multiplication in culture, although flow cytometry revealed them to have been a rapidly multiplying population when explanted . A relatively large fraction of these explanted tumor cells incorporated a 1-hour pulse of {3H}thymidine into DNA, although at a low rate . The shift to culture apparently slowed progress through the S-period of the cell cycle . The multiplication rate of cell populations from the delayed tumors increased in successive passages in culture . There was great heterogeneity in growth capacity among clones of the tumor cells . The growth rates of some clones declined to the point of extinction, those of others remained constant for several weeks, while those of still others steadily increased in growth rate . The low initial cloning efficiency of cells from the delayed tumors and the heterogeneity of growth rates among the clonable cells indicate that selection plays a major role in the increase of the growth capacity of the cell population . The steady increase in growth rates within clones suggests that physiological adaptation also contributes to the progressive growth of the tumor populations in culture . The results constitute a rationale for using the progressive growth of cells in culture as a model system for discriminating the types of cellular changes that underlie tumor progression.

Endocrinology, 1986 Nov, 119(5), 1929 - 32
A direct pituitary action of progesterone on basal secretion of follicle-stimulating hormone in ovine cell culture: dependence on ovaries in vivo; Batra SK et al.; Progesterone (P4) inhibits FSH production by 60-70% in cell cultures of pituitaries from normal ewes . This communication reports that P4 is much less effective in inhibiting FSH production in cultures prepared from pituitaries of ovariectomized ewes . P4 (10(-7) M) inhibited 61 +/- 5% of basal FSH secretion in cultures from intact anestrous ewes . When cultures were prepared from pituitaries collected on day 9, 21, or 42 after ovariectomy, P4 inhibited FSH secretion by only 46 +/- 3%, 16 +/- 2%, and 10 +/- 2%, respectively . One group of ewes received implants of 17beta-estradiol (E2) at the time of ovariectomy . When cultures were prepared from these ewes 35 days after ovariectomy, P4 inhibited FSH secretion 56 +/- 5%, not significantly different (P greater than 0.05) from inhibition in normal cultures . Furthermore, under similar culture conditions, E2 and a porcine ovarian inhibin preparation inhibited FSH secretion regardless of the length of time after ovariectomy . These in vitro results suggest that ovariectomy causes a time-dependent decrease in pituitary responsiveness to P4 in vivo . Since E2 can maintain P4 sensitivity in pituitaries from ovariectomized ewes, E2 may be the only ovarian factor required to maintain pituitary responsiveness to P4 in vivo.

Vopr Virusol, 1986 Nov-Dec, 31(6), 723 - 9
{Enhancement of alphavirus reproduction in cell cultures at an alkaline pH}; Zhirnov OP; Replication of different alphaviruses, among them Semliki Forest, Sindbis, and Venezuelan equine encephalomyelitis viruses, was studied in chick embryo fibroblasts and hamster BHK cells at different pH in a range of 6.8 to 7.9 . Incubation of the infected cell cultures in a mild alkaline medium (pH 7.5-7.9) resulted in a marked activation of both one-cycle and multi-cycle reproduction of the alphaviruses under study as compared with that in the neutral medium (pH 6.8-7.2) . At an alkaline pH, the rate of amplification and level of synthesis of viral proteins in the infected cells increased markedly, while virus adsorption on cell receptors decreased by 30-50% . It is recommended to use nutrient media with mild alkaline pH for stabilization of alphaviruses reproduction in cell cultures.

J Neurosci, 1986 Nov, 6(11), 3385 - 92
High concentrations of N-acetylaspartylglutamate (NAAG) selectively activate NMDA receptors on mouse spinal cord neurons in cell culture; Westbrook GL et al.; We examined the membrane action of the endogenous dipeptide and putative neurotransmitter N-acetylaspartylglutamate (NAAG) on the excitatory amino acid receptors of cultured mouse spinal cord neurons using electrophysiological methods . Responses to NAAG (1 microM-5 mM) were compared to those elicited by N-methyl-D-aspartate (1 microM-1 mM) and L-glutamate (0.5-500 microM) . Under voltage clamp, concentration-response curves of agonist-evoked currents demonstrated that NAAG was much less potent than either L-glutamate or N-methyl-D-aspartate (NMDA), so that inward currents could be evoked only at NAAG concentrations above 300 microM . Analysis of the dipeptide by high-pressure liquid chromatography showed no evidence of contamination by excitatory amino acids, suggesting that NAAG has an intrinsic, although weak, neuroexcitatory action on spinal neurons . Previous studies have shown that activation of NMDA receptors produces a voltage-dependent response . The current-voltage relationship of responses evoked by NAAG was also voltage-dependent . The peptide-activated conductance decreased with hyperpolarization in the presence of extracellular Mg2+, such that little inward current could be evoked at a membrane potential of -80 mV . In addition, responses to NAAG were completely antagonized by 250 microM DL-2-amino-5-phosphonovaleric acid, a specific NMDA-receptor antagonist . Application of NAAG in Mg2+-free medium resulted in an inward current with a large increase in membrane current noise . The spectral density function of this current noise could be fitted with a single Lorentzian with a decay time constant near 5 msec and a calculated single-channel conductance of 50-60 pS.(ABSTRACT TRUNCATED AT 250 WORDS)

J Neurosci, 1986 Nov, 6(11), 3284 - 9
Excitatory synaptic transmission between interneurons and motoneurons in chick spinal cord cell cultures; O'Brien RJ et al.; We have examined the development of synaptic transmission between interneurons and motoneurons in spinal cord cell cultures . Unitary excitatory synaptic currents and complex bursts of excitatory currents develop rapidly: EPSCs (excitatory postsynaptic currents) were detected in 100% of the motoneurons by the 4th day after plating . Inhibitory synaptic currents develop more slowly: IPSCs (inhibitory postsynaptic currents) were detected in only 10% of the motoneurons on day 5 and 40% on day 8 . During the 1st and 2nd days in vitro, 24% of the motoneurons tested were dye (Lucifer Yellow) coupled to nearby interneurons . The incidence of dye coupling declined during the first week in culture . No coupling was observed between motoneurons . Our data imply that both G1 and G2 receptors are activated at each synapse . The amplitude of spontaneous excitatory synaptic currents did not change when the motoneuron was hyperpolarized from -50 to -80 mV . This behavior is similar to that of currents induced by glutamate, an agonist that activates 2 types of receptors (G1 and G2) on motoneurons . In addition, a concentration of 2-amino-5-phosphonovaleric acid sufficient to inhibit all G1 receptors only partially inhibited the excitatory synaptic currents . Given the conductance of G1 and G2 channels and the ratio of channels activated during unitary EPSCs, we estimate that as few as 25 G1 channels and 5 G2 channels may mediate excitatory interaction between interneurons and motoneurons during the first week in culture.

J Neurophysiol, 1986 Nov, 56(5), 1242 - 56
Active and inactive central synapses in cell culture; Pun RY et al.; Synaptic interactions between pairs of spinal cord (SC) neurons and between dorsal root ganglion neurons and SC neurons were studied in dissociated cell cultures prepared from fetal mouse . Combined injection of horseradish peroxidase into presynaptic neurons and Lucifer yellow into postsynaptic neurons allowed detailed correlation of morphological-physiological analyses of synaptically linked cells . Statistical analysis of trains of evoked EPSPs under conditions of high and of low transmitter output was used to determine the number of physiological release elements, n, involved in a given synaptic connection . When n was compared with the number of boutons subserving a synaptic connection, it was found that in 80% of cases the number of boutons was equal to or greater than the number of release elements . In some cases, the bouton count was more than fivefold greater than n . The simplest explanation is that, in general, one bouton can release no more than one quantum of transmitter and, in a significant proportion of synaptic connections, a large fraction of boutons do not participate in the release process . Theoretical consideration and analysis of the electrotonic structure of some of the neurons studied indicate that the dendritic location of synaptic inputs does not affect our results . Variations in the probability of release, p, may contribute to the apparent disparity between n and bouton number . If so, this variation must be large with many boutons having a very low p, difficult to distinguish experimentally from zero.

J Cell Biol, 1986 Nov, 103(5), 2017 - 24
The tyrosine phosphorylation substrate p36 is developmentally regulated in embryonic avian limb and is induced in cell culture; Carter C et al.; The 36-kD protein-tyrosine kinase substrate p36 has been variously postulated to be involved in membrane-cytoskeletal interactions, membrane traffic, and the regulation of phospholipase A2, and its phosphorylation may play some role in malignant transformation by avian sarcoma viruses . Because embryonic tissues are resistant to transformation by avian sarcoma viruses, we have examined the expression of p36 in the developing avian embryonic limb . The level of p36 increased progressively from day 5 to day 14 of development . It was largely absent from day-5 mesenchyme, and was induced during the differentiation of mesenchymal cells into connective tissue and cartilage, but was not induced in differentiating muscle . In contrast, p36 was detected in ectodermal cells at all developmental stages examined . When day-5 limbs were dissociated and cultured, p36 was induced in all adherent cells, beginning at 2-4 h after plating, and reaching levels comparable to those observed with intact day-14 limb tissue within 48 h . The accumulation of p36 in culture was dependent on substratum adherence, suggesting that its stability is regulated by cell attachment or spreading . These findings are consistent with a structural or mechanical role for p36.

J Neurosci, 1986 Nov, 6(11), 3229 - 41
Noradrenergic neurons from the locus ceruleus in dissociated cell culture: culture methods, morphology, and electrophysiology; Masuko S et al.; We have developed a dissociated primary cell culture of noradrenergic neurons from the locus ceruleus of postnatal (1- to 5-d-old) mice or rats . Slices of the brain stem were made on a Vibratome . Then the region of locus ceruleus, which was identified by observing the slices under a dissecting microscope, was dissected out from the slices . The removed fragments of brain slices were dissociated and cultured up to 3 weeks on a non-neuronal feeder layer, which consisted predominantly of astroglial cells, or on a fibronectin-treated collagen substratum . After 2 weeks of culture, about 70% of total neuronlike cells revealed positive catecholamine histofluorescence, indicating that they were probably noradrenergic neurons . About 98% of large- and medium-sized cultured neurons (soma diameter greater than or equal to 20 microns) was histofluorescence positive . The fluorescence-positive cells had long processes rich in varicosities, and the shape of their soma was either multipolar or fusiform . Electron microscopy using permanganate fixation revealed that the varicosities along their processes had small granular vesicles, which may contain norepinephrine . Physiological properties of these noradrenergic neurons were investigated with intracellular microelectrodes or with the whole-cell version of the patch clamp . We observed that many cells were producing spontaneous firing . Many of these spontaneously firing cells had no obvious contact with neighboring cells . The neurons were depolarized when glutamate was applied by pressure ejection . They also responded to GABA and glycine with either hyperpolarization or depolarization, and these responses were antagonized by picrotoxin and strychnine . Application of substance P generally produced depolarization with an increase in input resistance . The neurons responded with hyperpolarization to somatostatin, beta-endorphin, and enkephalin . This culture system will become a useful tool for elucidating the cellular and molecular properties of the central noradrenergic neurons.

Eur J Biochem, 1986 Oct 15, 160(2), 297 - 304
Control of ribosome biosynthesis in plant cell cultures under heat-shock conditions . Ribosomal RNA; Nover L et al.; The immediate block of ribosome biosynthesis in heat-shocked tomato cell cultures is primarily caused by the complete inhibition of pre-rRNP processing . Depending on the heat-shock conditions synthesis of pre-rRNP goes on, though at a reduced level . Synthesis and/or preservation of pre-rRNP during heat shock as well as its efficient processing in the recovery period are thoroughly improved by preconditioning of cells to the hyperthermic treatment . Such preinduced cultures are characterized by their content of preformed heat-shock proteins, whose dominant representative (hsp 70) becomes highly enriched in the characteristic granular rRNP material observed in nucleoli of heat-shocked cells . This is shown by immune fluorescence staining and microautoradiography.

J Immunol, 1986 Oct 15, 137(8), 2726 - 32
Coexistence of a chemotactic factor and a retroviral P15E-related chemotaxis inhibitor in human tumor cell culture supernatants; Wang JM et al.; Two sets of seemingly contradictory evidence have been reported concerning the effects of tumor cell products on the regulation of monocyte migration in vitro and presumably the extravasation of macrophages into tumors in vivo . The present study was designed to explore the relationship between chemotactic and anti-chemotactic products related to tumor cells: a tumor-derived chemotactic factor (TDCF) and retroviral P15E-related inhibitor(s) of chemotaxis . Culture supernatants of the human 8387 sarcoma and SW626 ovarian carcinoma were depleted of P15E-related antigens with immobilized anti-P15E monoclonal antibodies . This treatment produced a significant and consistent increase of the polarizing and chemotactic activity in the tumor cell supernatants . The material eluted from Sepharose-bound anti-P15E antibodies was devoid of chemotactic and polarizing activity and suppressed the polarization and migration of monocytes in response to chemoattractants . These results demonstrate the coexistence in culture supernatants of two human tumor cell lines of factors with opposite influences on monocyte chemotaxis . The data suggest that the entry of monocytes into neoplastic tissue may be regulated by the interplay of chemotactic and anti-chemotactic principals produced by tumor cells.

Z Hautkr, 1986 Oct 15, 61(20), 1433 - 42
{Epidermal cell cultures--significance for wound coverage in the human}; Bonnekoh B et al.; Epithelial sheets can be cultivated from isolated epidermal cells; in this way, it is possible to increase the cell number considerably . H . Green and co-workers were the first to make use of such epithelia for the autologous covering of burn wounds . We modified this method and report on our experiences with this technique in a patient with small skin defects.

Biol Reprod, 1986 Oct, 35(3), 761 - 72
Isolation of cyclic protein-2 from rat seminiferous tubule fluid and Sertoli cell culture medium; Wright WW et al.; Cyclic Protein-2 (CP-2), a stage-specific secretory product of the rat seminiferous epithelium, has been isolated from seminiferous tubule fluid (STF) and Sertoli cell culture medium . Isolation from STF was accomplished by mixing STF with radiolabeled proteins secreted by Stage VI-VII seminiferous tubules and sequential fractionation of these proteins by hydroxylapatite, DEAE-agarose, and quaternary amine ion-exchange chromatography . Radiolabeled proteins were used to identify the chromatographic fractions that contained CP-2 . Through use of these procedures, a highly purified preparation of radioinert CP-2 was obtained from seminiferous tubule fluid . Cyclic Protein-2 was also isolated from Sertoli cell culture medium, indicating that the Sertoli cell is its most likely source . Preliminary characterization of CP-2 was conducted . First, CP-2 appeared to be highly enriched in methionine . Second, the molecular weight of CP-2 was found to be 20,000 . Third, analysis by reverse-phase hydrophobic chromatography indicated that CP-2 was relatively hydrophobic . We conclude that CP-2 is a small hydrophobic glycoprotein secreted in vivo and in vitro in a stage-specific manner by Sertoli cells.

Neurosurgery, 1986 Oct, 19(4), 495 - 501
Hematoporphyrin derivative photocytotoxicity of human glioblastoma in cell culture; Wharen RE Jr et al.; The parameters of hematoporphyrin-derivative (HpD) photocytotoxicity of human glioma cells in cell culture were studied to determine the optimum wavelength and power density of light, to investigate the influence of tissue oxygenation, and to evaluate the role of singlet oxygen and free radicals in producing cell death . Cell survival curves demonstrated a relative killing efficiency of 12:1 for violet compared to red light . Eighty joules of red light were required to produce 100% cell kill at an HpD concentration of 10 micrograms/ml, a level of HpD that has been quantitated in biopsies from patients receiving HpD photoradiation therapy . No difference in cellular killing efficiency was observed for power densities of red light varying from 10 to 100 mW/cm2 . Cytotoxicity was directly related to O2 tension from 12 to 490 torr with a slight but consistent increase in cell kill at O2 tensions from 7 to 12 torr . Cytotoxicity was effectively quenched by beta-carotene, whereas mannitol had no effect, indicating that cytotoxicity is probably mediated via a mechanism involving singlet oxygen . This information may serve as a basis for more effective application of HpD photoradiation therapy and for designing protocols to study the efficacy of such therapy.

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