Cesk Farm, 1990 May, 39(3), 134 - 5 {Possibilities in the use of cell cultures in carcinogenicity tests}; Plaisner V; The cell transformational test, consisting in morphological transformation of the human embryonal lung cells, is a rapid and sensitive method of identification of chemical carcinogens . The test is capable of revealing carcinogens undetectable by other methods and it makes it possible to avoid mistakes due to inter-species differences in the biotransformation of the drugs tested. Mutagenesis, 1990 May, 5(3), 241 - 9 Expression of xenobiotic-metabolizing enzymes in propagatable cell cultures and induction of micronuclei by 13 compounds; Glatt H et al.; Activities of various xenobiotic-metabolizing enzymes were determined in 18 cell lines . Activities of cytochrome P450 reductase, microsomal epoxide hydrolase and glutathione transferase were detectable in all lines . The highest values were similar to the activities found in freshly isolated rat hepatocytes . Catalase activity was also present in all 12 investigated cell lines . Activity of UDP-glucuronosyl transferase was high in some lines, but low or undetectable in others . Activity of cytosolic epoxide hydrolase was not measurable in most lines, and was low in the others . Metabolism of benzo{a}pyrene was observed in eight out of nine examined lines, no activity being found in V79 cells . V79 and three epithelial cell lines were then used as target cells in a genotoxicity assay in which the frequency of micronucleated cells was determined . In V79 cells, 7,12-dimethyl- benz{a}anthracene, benzo{a}pyrene, benzo{a}pyrene-trans-7,8-dihydrodiol, aflatoxin B1, N-nitrosomorpholine and 2-acetylaminofluorene showed negative responses, whereas N-methyl-N'-nitro-N-nitrosoguanidine, 9-hydroxybenzo{a}pyrene, 2-nitrofluorene, dibenz{a,h}anthracene 1,2-catechol, dibenz{a,h}anthracene, 1,2-quinone hydroquinone and p-benzoquinone proved positive in the test . All 13 compounds, however, induced micronuclei in rat intestinal cells (IEC-17 and IEC-18) and in embryonal human liver cells (HuFoe-15) . Thus, these epithelial cell lines are capable of activating and detecting a broad spectrum of chemically diverse genotoxic compounds . They may also be useful for the detection of hazardous compounds whose active metabolites are not able to penetrate from the extracellular space into the indicator cell. Aust Vet J, 1990 May, 67(5), 182 - 6 Vaccination of sheep with cell culture grown orf virus; Pye D; Orf virus, derived from contagious pustular dermatitis (scabby mouth) lesions in sheep, was adapted to cell culture and subsequently evaluated as a potential vaccine for sheep . The traditional vaccine virus, prepared from the infected scabs of orf virus lesions in sheep, was used to vaccinate sheep by scratching with an applicator (mounted pins) dipped in virus . Less than 10 TCID50 (50% tissue culture infectious doses) of virus was required to produce large lesions (greater than 5 mm diameter) which developed during a period of 10 to 14 d prior to onset of healing which was complete by 28 to 30 d . A serum neutralising antibody response was also detected and protection against challenge by application of virulent virus to abraded skin was demonstrated in that challenge lesions developed and healed more quickly (14 d against 30 d) . However, cell culture-adapted virus required more than 10(5) TCID50 to induce even small lesions (less than 2 mm diameter) . An antibody response could not be detected and no evidence of protection against challenge with virulent virus was demonstrated . In contrast, a recent field isolate has yielded a cell culture-adapted virus preparation that readily infects sheep, produces large lesions, detectable antibody and protects against challenge . This isolate is distinct from the traditional vaccine strain on the basis of restriction enzyme analysis but provides cross-protection in sheep inmmunisation and challenge studies . These results demonstrate that a cell culture produced scabby mouth vaccine is feasible. Neurochem Res, 1990 May, 15(5), 501 - 5 Effect of ferric nitrilotriacetate on predominantly cortical glial cell cultures; Swaiman KF et al.; Cultured glial cells were exposed to ferric nitrilotriacetate (Fe-NTA) at varying concentrations . Studies of the exposed glial cells were performed at days 29 and 36 post-conceptional age (culture days 8 and 15) . In addition to morphologic studies, biochemical assays including {3H}-flunitrazepam (FLU) specific binding, Ro5-4864-displaceable 3H-FLU binding, and protein determinations were performed . At day 29 post-conceptional age, significant decreases in 3H-FLU specific binding, Ro5-4864-displaceable 3H-FLU binding, and protein determinations were discernible only in the presence of 100 microM Fe-NTA . At day 36 post-conceptional age 3H-FLU specific binding was significantly decreased at 20, 60, and 100 microM Fe-NTA concentrations, while Ro5-4864-displaceable 3H-FLU binding and protein determinations were significantly reduced at 60 and 100 microM Fe-NTA concentrations . The effects of Fe-NTA exposure appear to be both concentration and duration-of-exposure related . When compared to previously reported neuronal cell culture studies utilizing 3H-FLU specific binding, Ro5-4864-displaceable 3H-FLU binding, and protein determinations, glial cells appear to be significantly more resistant to chelated iron exposure. J Pharmacol Exp Ther, 1990 May, 253(2), 892 - 8 Neurochemical and toxic effects of 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine and 1-methyl-4-phenylpyridine to rat serotonin neurons in dissociated cell cultures; Friedman LK et al.; Dissociated cell cultures from the pontine area of embryonic rat brain were used to study the sensitivity of serotonin (5-hydroxy-tryptamine (5-HT)) neurons to the neurotoxins 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) and 1-methyl-4-phenylpyridine (MPP+) . Treatment with MPTP (up to 100 microM) for 7 days did not cause degeneration of 5-HT neurons . A 50% inhibition of {3H}5-HT uptake caused by 100 microM MPTP was a direct effect on the 5-HT uptake carrier, reversed by washing for 7 days . Incubation of cultures with MPTP increased the intraneuronal levels of 5-HT and reduced the levels of 5-hydroxyindoleacetic acid, suggesting a reduction in 5-HT metabolism . MPTP reduced monoamine oxidase activity in the cultures, which probably led to the reduction in 5-HT metabolism . Exposure to MPP+ (0.5-10 microM) for 4 to 7 days decreased {3H}5-HT uptake and induced loss of neurons stained with antibodies against 5-HT . Comparison between 5-HT and dopamine (DA) neurons indicated a differential sensitivity to MPP+ toxicity with DA neurons being more susceptible . Analysis of the competition of MPP+ with the natural substrates for uptake sites of 5-HT and DA neurons demonstrated higher affinity of MPP+ for DA compared to 5-HT neurons . The lower affinity of MPP+ for 5-HT neurons could be responsible for the accumulation of lower MPP+ levels observed in pontine cultures and explain the resistance of 5-HT neurons to this toxin. Am J Physiol, 1990 May, 258(5 Pt 1), G774 - 87 Characterization of mucous cell synthetic functions in a new primary canine gastric mucous cell culture system; Boland CR et al.; Mucin is a critical component of the protective layer secreted by gastrointestinal mucous cells . A detailed understanding of the molecular processing of gastric mucin and the physiology of its secretion has been limited by the lack of an adequate model for their study . We have developed a primary culture system of canine gastric mucous cells that has permitted us to study their synthetic and secretory functions . It was found that {3H}glucosamine used for metabolic labeling studies was incorporated into both mucin and lipid components of gastric mucus . To measure mucin with this model, a new immunoassay was developed to quantitate canine gastric mucin . Mucin was purified from the canine stomach, a polyclonal antibody was generated, and an enzyme-linked immunosorbent assay for gastric mucin was established . Mast cells were frequent contaminants of the gastric mucous cell preparation, and two methods were developed to limit their contamination . A new culture system has been developed for the study of gastric mucous cells . These cells synthesize and secrete both mucin and phospholipids . This system will permit us to study the molecular processing of mucin and the physiology of its production and release. Cancer Genet Cytogenet, 1990 May, 46(1), 107 - 13 Use of conditioned media in cell culture can mask cytogenetic abnormalities in acute leukemia; Sun GX et al.; Conditioned media (CM) from a human lung adenocarcinoma cell line expressing interleukins 1 and 6 (IL-1, IL-6), granulocyte (G), macrophage (M), and GM colony-stimulating factors (G, M, GM-CSF) and transforming growth factor beta (TGF beta) were used to stimulate growth of bone marrow (BM) cells from 18 persons with leukemia, myelodysplastic syndrome, or lymphoma . The objective was to increase numbers of analyzable metaphases and to enhance the likelihood of detecting cytogenetic abnormalities . Although more mitotic cells were observed with CM, the detection rate of cytogenetic abnormalities decreased in 12 of 18 cases . These data indicate that use of CM for cytogenetic analyses may favor growth of normal versus leukemia cells and mask cytogenetic abnormalities. J Med Virol, 1990 May, 31(1), 5 - 12 History, precedent, and progress in the development of mammalian cell culture systems for preparing vaccines: safety considerations revisited; Hilleman MR; The use of cell substrates to propagate viruses or recombinant plasmids for vaccine productions has been the subject of long evolutionary conflict, primarily from the standpoint of product safety, and especially from the viewpoint of cancer induction . The present concern is for safety of vaccines made using transformed or neoplastic mammalian cells that may contain endogenous contaminating viruses or integrated gene sequences from oncogenic viruses . There is also concern for use of plasmid vectors employing promoter elements from oncogenic viruses . The principal concern for safety lies with retention of residual DNA in the vaccine, especially since induction of cancer is a single-cell phenomenon, and a single functional unit of foreign DNA integrated into the host cell genome might serve to induce cell transformation as a single event or part of a series of multifactorial events . Current proposed standards for vaccines would permit contamination with up to 100 pg of heterologous DNA per dose . This is equivalent to about 10(8) "functional lengths" of DNA . Total safety would seem to require complete absence of DNA from the product . While preparation of biologicals used to treat serious disease might demand the use of mammalian cells, this is not the situation with vaccines that are given prophylactically to persons who might be given equally efficacious vaccines produced in bacterial cells or in yeast that have attributes for greater safety . Careful assessment of safety and risk vs . benefit of continuous mammalian cell-produced vaccines should be made by technically expert scientists in the relevant disciplines and a consensus needs to be evolved in the scientific community at large. Cancer, 1990 May 1, 65(9), 2006 - 13 A cell culture, chromosomal and quantitative DNA analysis of a metastatic epithelioid sarcoma . Deletion 1p, a possible primary chromosomal abnormality in epithelioid sarcoma; Stenman G et al.; The chromosomal banding pattern and the in vitro growth characteristics of a metastatic epithelioid sarcoma are described . The cultured tumor cells retained growth characteristics as well as ultrastructural and immunohistochemical properties similar to the cells of the primary tumor . Cytogenetic analysis revealed a modal range in the diploid-hypodiploid region, a finding which was corroborated by quantitative DNA determinations of both the primary tumor and a lymph node metastasis . Fourteen different marker chromosomes were identified . The most frequent clonal rearrangement was a 1p-marker resulting from a short arm terminal deletion, i.e., del (1) (p21-22) . A similar 1p- marker has previously been observed in an established epithelioid sarcoma cell line . The finding of an apparently identical 1p-marker in two of two analyzed epithelioid sarcomas suggests that this rearrangement may be a primary cytogenetic abnormality in epithelioid sarcoma . An elevated ras p21 expression was demonstrated using immunohistochemical methods . The possible involvement of the N-ras gene and/or a tumor suppressor in the 1p deletion is considered. Carcinogenesis, 1990 May, 11(5), 817 - 22 A reassessment of methylcholanthrene transformation in the C3H10T1/2 cell culture system; Chen AC et al.; We previously demonstrated that four tumorigenic methylcholanthrene (MCA) transformed cell lines derived from C3H10T1/2 cells each contain a common G34----T nucleotide alteration in the c-Ki-ras gene . In contrast, a non-tumorigenic MCA transformant does not contain this mutation . We have now examined 75 newly isolated MCA transformants of C3H10T1/2 cells for their degree of morphological transformation, the presence of the c-Ki-ras G34----T mutation, colony formation in soft agar, and tumorigenicity in nude mice . Although many of these new MCA transformants exhibit morphological characteristics indistinguishable from previously isolated tumorigenic MCA transformants, none contain the G34----T mutation in the c-Ki-ras gene . Only one newly isolated MCA transformant can grow in soft agar . Of 14 tested, none of the new MCA C3H10T1/2 transformants are tumorigenic in nude mice. J Immunol, 1990 May 1, 144(9), 3569 - 73 Decreased steady state c-myc mRNA in activated T cell cultures from old humans is caused by a smaller proportion of T cells that transcribe the c-myc gene; Gamble DA et al.; The proliferative response of T cells is known to decrease with age of the T cell donor . We now report that this proliferative defect affects both major subsets (CD4+, CD8- and CD4-, CD8+) of peripheral T cells from old humans . Furthermore, this proliferative defect can be detected within the first hours after addition of mitogen by a reduction in the steady state levels of c-myc mRNA in T cell cultures from old donors . Lymphocytes from old humans cultured with PHA have less than 50% of the level of c-myc message than do such cultures from young donors . Nuclear run-on assays suggest that the decreased steady state level of c-myc mRNA in cultures from old donors is caused by reduced transcription of the c-myc gene in T cells from old donors . The age-associated defect in transcription of the c-myc gene affects the second exon to a greater extent than the first, noncoding exon . Individual T lymphocytes from old donors that do express c-myc message, detected by in situ hybridization, have the same intracellular level of c-myc message as T lymphocytes from young donors . These data add additional support for the hypothesis that the proliferative defect of T lymphocytes from old humans is caused by the smaller fraction of T cells from old as compared with young humans that can be activated by mitogens to enter the G1 phase of the cell cycle. Diagn Microbiol Infect Dis, 1990 May-Jun, 13(3), 241 - 4 Comparison of enzyme immunoassay, shell vial culture, and conventional cell culture for the rapid detection of herpes simplex virus; Johnston SL et al.; Specimens submitted for the detection of herpes simplex virus (HSV) were inoculated into conventional cell-culture tubes and fresh MRC-5 shell vials . The shell vial centrifugation cultures (SVCs) were examined at 16 hr postinoculation for HSV by using type-specific monoclonal antibodies (SVC-FA); they were also analyzed for HSV antigen by using an enzyme-linked immunoassay (SVC-ELISA) . Mink Lung (ML) and rhabdomyosarcoma (RD) cells were used in the cell-culture tubes . Of 182 specimens, 35 (19%) were positive in cell-culture tubes, 16 (9%) were positive by SVC-ELISA, and 22 (12%) were positive SVC-FA . All specimens that were positive by SVC-ELISA, SVC-FA, and in culture tubes displayed cytopathic effect (CPE) at 16 hr . Those specimens that had a negative ELISA and/or FA result and were positive in culture were evaluated for the time in which it took to detect CPE . At 16 hr, 48% of the positive tubes were detected; at 40 hr (second day), 83% of the positive tubes were detected; and by the third day, 94% were detected . The RD cell line displayed CPE at the same time or earlier than ML cells did in 92% of the positive cases . The sensitivity of the SVC-ELISA at 16 hr was 46% with 100% specificity . The sensitivity of the SVC-FA at 16 hr was 63% with 99% specificity . Given the increased sensitivity, rapid display of CPE, and reduced cost and handling time of cell cultures, our laboratory found that rapid SVC-ELISA and SVC-FA procedures for HSV detection have no clinical or laboratory advantage. J Clin Microbiol, 1990 May, 28(5), 864 - 8 Sensitive solid-phase immune electron microscopy double-antibody technique with gold-immunoglobulin G complexes for detecting rotavirus in cell culture and feces; Wu B et al.; A new solid-phase immune electron microscopy double-antibody colloidal-gold technique (SPIEMDAGT) was developed and compared with direct electron microscopy, direct immune electron microscopy, and enzyme immunoassay for detecting rotavirus . Guinea pig and rabbit antirotavirus antisera were used as capture and detector antibodies, respectively, and goat anti-rabbit immunoglobulin G-gold complexes were employed as a label . Animal rotavirus in cell culture media and human virus in stool specimens were detected by this method . On average, SPIEMDAGT detected 800 times more virus particles than direct electron microscopy and 45 times more particles than direct immune electron microscopy and yielded 20% more positives than enzyme immunoassay . SPIEMDAGT could detect not only viral antigen associated with morphologically recognizable particles but also antigen present when whole virus particles were not visible. Am J Vet Res, 1990 May, 51(5), 723 - 5 Evaluation of age-related effects on the antiviral activity of interferon and induction of 2-5A synthetase in testicular cell cultures derived from swine of various ages; Bosworth BT et al.; The antiviral activity of recombinant DNA-derived bovine alpha 1(-1) interferon on an established swine testicular cell line and primary testicular cell cultures derived from swine of various ages (2 days, 3 weeks, and 5 weeks) was determined . Bovine interferon induced a dose-dependent increase in 2-5A synthetase in testicular cells, regardless of the source of the cells . Furthermore, interferon inhibited replication of vesicular stomatitis virus to an equivalent extent in all testicular cell cultures . The results indicate that 2-5A synthetase is a reliable marker of interferon activity in swine testicular cell cultures and that the induction of 2-5A synthetase and antiviral effects of recombinant bovine interferon in primary testicular cell cultures are not dependent on the age of the donor animal. Mol Endocrinol, 1990 May, 4(5), 721 - 6 Rapid stimulatory effect of activin-A on messenger RNA encoding the follicle-stimulating hormone beta-subunit in rat pituitary cell cultures; Attardi B et al.; Activin, a dimer of the beta-subunits of inhibin, stimulates FSH secretion by cultured rat pituitary cells . Both the cell content of FSH and total FSH (secreted plus intracellular) are increased by activin, suggesting an effect on FSH biosynthesis . To test this idea directly, we examined the effect of purified human activin-A of recombinant DNA origin (rhactivin-A) on steady state levels of mRNAs for the gonadotropin subunits in pituitary cell cultures prepared from adult male rats . A preliminary study of the time course of rhactivin-A action indicated that the first significant effect on FSH secretion was observed at 6 h, with maximal stimulation occurring at 24-72 h . A small (20-30%), but significant, increase in LH secretion was also observed by 24 h . For RNA analysis, pituitary cell-cultures were treated for 2-72 h with a maximally effective concentration (50 ng/ml) of rhactivin-A . FSH secretion in rhactivin-A-treated cultures was elevated by 2- to 2.5-fold . Intracellular FSH increased gradually from 24-72 h . Recombinant human activin-A stimulated FSH beta mRNA levels at all times examined; FSH beta mRNA levels in activin-treated cultures were already twice those in control cultures at 2 h, and the magnitude of this effect remained constant up to 72 h . Recombinant human activin-A brought about small increases in secretion and cell content of LH and free glycoprotein alpha-subunit and in LH beta and alpha mRNAs at various times . Thus, the increases in gonadotropin release and cell content stimulated by rhactivin-A can be accounted for by increases in the gonadotropin subunit mRNAs.(ABSTRACT TRUNCATED AT 250 WORDS) Dtsch Z Mund Kiefer Gesichtschir, 1990 May-Jun, 14(3), 224 - 8 {Bone regeneration following the implantation of osteoblasts from cell cultures}; Lang H et al.; An animal model utilizing 26 inbred rats was aimed at the question if the regeneration of bone tissue is enhanced by implantation of osteoblasts previously cultured in vitro . Bone tissue was harvested from inbred rats (Lewis) and a cell line of rat osteoblasts was established . The osteoblasts were cultured in vitro and multiplied in three passages . Characterization of the cells was by various methods . Monocortical defects were created in the distal portion of the rat femurs by drilling; and the cells, embedded in 25% bone gelatine, were reimplanted into these defects . After observation periods of varying lengths, the femurs were examined radiographically and bone growth in the defect area was evaluated histologically . The results of this study indicated that there was an initial effect of the reimplanted cultured osteoblasts on the bone growth pattern . At a later point in time this effect could no longer be demonstrated in the young, healthy rats. Int J STD AIDS, 1990 May, 1(3), 199 - 204 Comparison of Pharmacia Chlamydia EIA, IDEIA and cell culture in the detection of urogenital chlamydial infection; Bygdeman SM et al.; Two commercial test kits, Pharmacia Chlamydia EIA (PhEIA) and IDEIA Chlamydia Test, for the identification of Chlamydia trachomasis and McCoy cell culture were compared in urogenital specimens . The sediments of the transportation buffers of specimens with discordant results were investigated for elementary bodies (EB) with fluorescein-labelled antichlamydial antibodies . The prevalence of chlamydial infection among the men was 16% (48 of 293), 47 culture positive and one EB positive, and among the women 10% (10 of 97), 10 culture positive . In men, the sensitivity of PhEIA, IDEIA and culture was 71%, 40% and 98%, respectively . In women, irrespective of site, corresponding figures were 100%, 80% and 100% . The specificity and positive predictive values were 100% for both enzyme immunoassays in men and women . The low sensitivity of IDEIA could not be explained by the degree of infection as measured by the number of inclusion bodies in cell culture, the presence of antigen as measured by the number of EBs or the sampling order. Int J STD AIDS, 1990 May, 1(3), 187 - 90 Comparison of an enzyme immunoassay (Ortho) with cell culture and immunofluorescence for the detection of genital chlamydial infection; Mumtaz G et al.; An enzyme immunoassay (EIA) test (Ortho Diagnostic Systems Ltd) was evaluated against cell culture for the detection of chlamydial genital infection . Specimens were obtained from 409 patients (204 men and 205 women) . Sensitivity, specificity, predictive value of a positive result (PVP) and predictive value of a negative result (PVN) for the new test compared to cell culture were respectively 73.1%, 93.8%, 63.3% and 96% for men and 80%, 95.6%, 71.4% and 97.2% for women . Discrepancies were further evaluated by repeating the EIA, and by direct immunofluorescence (IF) on the EIA transport buffer . The sensitivity, specificity, PVP and PVN of the EIA against the combination of cell culture and direct IF were respectively 76.7%, 96%, 76.7% and 96% for men, and unchanged for women . Overall agreement between the EIA and the combination of cell culture and direct IF was 93.4% . The EIA is rapid and simple to perform and does not require elaborate equipment. J Clin Lab Immunol, 1990 May, 32(1), 41 - 7 A high density cell culture system for generation of human lymphokine-activated killer (LAK) cells for clinical use in adoptive immunotherapy; Shimizu K et al.; A high density cell culture system has been developed for large-scale production of lymphokine-activated killer (LAK) cells from peripheral blood lymphocytes (PBLs) of malignant tumor patients . The system consists of a culture bag, which has two compartments separated by a semipermeable membrane, and an external rotator . The system allows for a long-term, at least 4 weeks, culture of LAK cells at high cell density in the inner compartment . The collected PBLs were first divided between the two culture bags and cultured without harvesting for 7-10 days to obtain LAK cells . Half of the LAK cells from each bag was administered to patients twice a week for clinical trials . Culture of the remaining half was continued following addition of a fresh culture medium . LAK cells were transferred to patients alternatively from each bag for the following 2-3 weeks . The total number of LAK cells administered amounted to 3.9-9.8 (mean 5.8) times more than the PBLs collected by leukapheresis (n = 10) . The 5 x 10(6)/ml of PBLS of the initial concentration reached a maximum of 2 x 10(7)/ml . Our system does not need for a CO2 incubator . Cytotoxicity of the LAK cells was evaluated in 4 hr 51Cr release assays . Mean cytotoxicity at maximum cell density was 95.4 +/- 3.2% against ONS-12 (a human glioma cell) and 84.8 +/- 3.0% against Daudi cells (n = 10), but gradually decreased to about 50% at the end of fourth week of the culture period . Cell viability of the LAK cells was normally over 80% through the entire culture period.(ABSTRACT TRUNCATED AT 250 WORDS) Matrix, 1990 May, 10(2), 98 - 111 Purification of hexabrachion (tenascin) from cell culture conditioned medium, and separation from a cell adhesion factor; Aukhil I et al.; We describe a protocol for purifying hexabrachion from conditioned medium of cell cultures, using gel filtration chromatography on Sephacryl 500, followed by anion-exchange chromatography on a Mono Q column, followed optionally by a second gel filtration or zone sedimentation on glycerol gradients . The protocol has several advantages over previous procedures based on affinity chromatography on monoclonal antibodies . Perhaps foremost, the protein is never exposed to the denaturing solvents that are required for elution from the antibody column . The Mono Q column also separated hexabrachion from a prominent cell adhesion activity that eluted with the hexabrachion on the first gel filtration, and co-sedimented with hexabrachions on glycerol gradients . The cell adhesion fractions showed several bands between 190 and 400 kDa . A single band at 220 kDa stained prominently with a polyclonal antibody against mouse EHS laminin, and a band at 190 kDa stained with a monoclonal antibody against s-laminin . The purification protocol gave hexabrachion at high concentration and with no detectable contamination by fibronectin or laminin . The highest yield of hexabrachion (1-4 mg from 400 ml of conditioned medium) was from human glioblastoma cell cultures, but the same procedure allowed us to purify and characterize the rat hexabrachion . Protein purified from primary cultures of rat embryo fibroblasts showed approximately equal amounts of three subunit sizes: 280, 230, and 220 kDa . These different subunits, presumably derived from alternative RNA splicing, appeared to be segregated into large and small hexabrachions, which could be separated on glycerol gradients. J Virol Methods, 1990 May, 28(2), 147 - 54 An in-vivo infectivity assay for cloned retroviruses lacking a susceptible cell culture; Gak E et al.; The lymphoproliferative disease virus (LPDV) of turkeys is the retroviral agent of etiology of a rapidly developing, naturally occurring, lymphoproliferative process . Recently we have molecularly cloned the viral genome . The lack of a susceptible cell culture which can sustain LPDV replication hampered the analysis of the infectious capability of the cloned genome . Based on the efficient in-vivo replication of LPDV we have developed a sensitive in-vivo approach aimed at establishing the infectious capability of the cloned provirus . According to this approach, peripheral leukocytes withdrawn from 3-week-old turkeys were transfected with the cloned DNA and the transfected leukocytes were re-injected into the turkey from which they had been obtained . The injected leukocytes enabled the efficient expression of the viral genome and the release into the blood stream of LPDV virions, which thereafter could travel to their appropriate in-vivo target lymphoid cells and start multiple replication cycles, resulting in the development of a detectable viremia . The applicability of this in-vivo assay for other cloned viral genomes is discussed. Blood, 1990 May 1, 75(9), 1862 - 9 Fetal calf serum contains activities that induce fetal hemoglobin in adult erythroid cell cultures; Constantoulakis P et al.; Cultures of peripheral blood or bone marrow erythroid progenitors display stimulated production of fetal hemoglobin . We investigated whether this stimulation is due to factors contained in the sera of the culture medium . Comparisons of gamma/gamma + beta biosynthetic ratios in erythroid colonies grown in fetal calf serum (FCS) or in charcoal treated FCS (C-FCS) showed that FCS-grown cells had significantly higher gamma/gamma + beta ratios . This increase in globin chain biosynthesis was reflected by an increase in relative amounts of steady-state gamma-globin mRNA . In contrast to its effect on adult cells, FCS failed to influence gamma-chain synthesis in fetal burst forming units-erythroid (BFU-E) colonies . There was a high correlation of gamma-globin expression in paired cultures done with C-FCS or fetal sheep serum . Dose-response experiments showed that the induction of gamma-globin expression is dependent on the concentration of FCS . These results indicate that FCS contains an activity that induces gamma-globin expression in adult erythroid progenitor cell cultures. Trends Biotechnol, 1990 May, 8(5), 131 - 6 Biomaterials aspects of porous microcarriers for animal cell culture; Cahn F; Porous microcarriers are new support materials with important advantages in both industrial cell-culture processes and the culture of cells of medical importance . Porous microcarriers are now commercially available with internal architecture and surface chemistry suitable for culture of both anchorage-dependent and anchorage-independent animal cells. Brain Res, 1990 Apr 9, 513(1), 74 - 80 Autoradiographic visualization of muscarinic receptors on rat paratracheal neurons in dissociated cell culture; James S et al.; An autoradiographic method was used to determine the distribution of muscarinic receptors on cells cultured from the trachealis muscle of 12-13-day-old rats . Cells identified in these culture preparations included neurones, fibroblasts, smooth muscle, and glial and epithelial cells . The cultured cells were incubated with the specific, irreversible ligand {3H}propylbenzylylcholine mustard, and the autoradiographs generated showed that most, if not all, of the paratracheal neurones observed in these cultures were specifically labelled . Both the neuronal cell body and associated neurites were evenly labelled over their entire surface . Neither the pattern nor the density of neuronal labelling appeared to be influenced by close association with other cultured cell types . Autoradiographic grains for muscarinic receptors also appeared to be uniformly distributed over smooth muscle cells and epithelial cell groups in culture . In contrast, no specific labelling was associated with cultured fibroblasts, glial cells and other non-neuronal supporting cells . The precise localization of muscarinic receptors on different cell types in culture may prove to be useful knowledge in the design of an effective and specific antimuscarinic bronchodilator. J Chromatogr, 1990 Apr 6, 526(2), 447 - 59 Determination of cocaine and norcocaine in plasma and cell cultures using high-performance liquid chromatography; Bouis P et al.; A new simple high-performance liquid chromatographic (HPLC) method was developed for the determination of cocaine and norcocaine . Cocaine and norcocaine in biological samples were buffered to pH 9.0, extracted with diethyl ether and reextracted in a 0.1% aqueous solution of tetramethylammonium hydrogen sulfate (TMAHS) with a theoretical yield of extraction of 100% . The HPLC elution of cocaine and norcocaine was performed using a Spherisorb RP-18, 100 mm x 4.6 mm I.D., 5 microns particle size column with a mobile phase containing acetonitrile-0.1% TMAHS aqueous solution (60:40) . The compounds were entirely separated, and a reliable limit of quantitation was set at 20 ng/ml when extracted from 0.5 ml of plasma . No interference with 26 other drugs was found . Cocaine and norcocaine stability studies showed that their half-lives in human plasma incubated at 37 degrees C were 50.8 and 43.2 min, respectively . In contrast, plasma from dogs or rats exhibited only weak or no enzymatic esterase activity towards cocaine and norcocaine resulting in less rapid degradation . Hydrolysis could be efficiently inhibited with sodium fluoride and prevented by storage of the sample at -20 degrees C . The highly sensitive assay also allowed the assessment of the oxidative metabolism pathway of cocaine to norcocaine in primary rat hepatocyte cultures. Environ Health Perspect, 1990 Apr, 85, 71 - 80 Type II pneumocytes in mixed cell culture of human lung: a light and electron microscopic study; Bingle L et al.; Alveolar Type II epithelial cells dedifferentiate rapidly in vitro . Studies with animal tissue suggest that cell-cell and extracellular matrix-cell interactions are important in the retention of Type II cell morphology in vitro . Thus, in this study with human tissue, alveolar Type II cells, alveolar macrophages, and spindle cells were prepared from the same sample of lung (obtained following lobectomy for cancer, n = 3), cocultured on glass cover slips or tissue culture plastic, and studied by light microscopy with scanning (SEM) and transmission (TEM) electron microscopy for 8 days . The primary cell isolates contained approximately 45% Type II cells; the remainder were macrophages or unidentifiable cells . Clusters, made up of a single layer of cuboidal Type II cells around a central core of connective tissue (largely collagen and some elastic tissue), formed above a monolayer of spindle cells . The Type II cells were morphologically similar to those seen in vivo . The cells were still cuboidal at 8 days but had lost their lamellar bodies, which were released into the medium via the apical surface . The clusters increased in size with time (area, microns 2: day 1, 29(5-143) x 10(2); day 8, 63(10-311) x 10(2); mean(range); p less than 0.02) without changing in number per culture, suggesting Type II cell proliferation . This may have been due to factors produced by the other cells and adherence to the extracellular matrix (ECM); (free collagen fibers, present in the original preparation, spindle cells, and/or Type II cells could be responsible for presence of ECM) . We propose this as a useful model for the study of human Type II epithelial cells in vitro. Environ Health Perspect, 1990 Apr, 85, 119 - 27 Clara cell cultures from the mouse and their reaction to bronchiolar toxins; Richards RJ et al.; The major aim of this study was to determine if small numbers of freshly isolated mouse Clara cells could be used to rapidly screen the toxic effects of a number of diverse pulmonary toxins . A short-term (20 hr) culture of functionally competent (nitotetrazolium reductase positive) Clara cells was developed . In this culture the Clara cells were allowed to attach to an extracellular matrix in 96-well multiwell plates containing a culture medium of DCCM 1 and Ultroser G (0.4%) . Pulmonary toxins (a total of 26 agents with concentrations ranging from 10(-7) M to 10(-3) M) were examined for their ability to reduce the attachment efficiency of functionally competent Clara cells and TD50 values (the amount of toxin required to reduce normal attachment efficiency by 50%) were calculated . With the possible exception of some halogenated hydrocarbons, the simple toxicity test in vitro correlated well with the known effects of the bronchiolar necrotic agents in vivo . For 13 compounds studied there was a direct correlation between TD50 values in vitro and LD50 values (mostly oral) in rodents in vivo, the correlation coefficient of the regression line being 0.783. Sb Lek, 1990 Apr, 92(4), 118 - 22 {Testing bronchogenic carcinoma cells for sensitivity to cytostatics in vitro and in cell cultures}; Zatloukal P et al.; The authors submit the first experience assembled in Czechoslovakia with testing of the sensitivity of bronchogenic carcinoma cells to cytostatics by the HTCA method (Human Tumor Clonogenic Assay) . Based on work by A . W . Hamburger, S . L . Salmon and others the authors elaborated their own modification of the method of cell cultivation in a double layer of soft agar, which can be used in small laboratories attached to clinical departments . The sensitivity tests were made in eight bronchogenic carcinomas--four adenocarcinomas and four epidermoid carcinomas . For the tests five cytostatics were used: 5-fluorouracil, methotrexate, cis-platinum, vincristine and vinblastine . Because of contamination the results in three tested adenocarcinomas could not be evaluated . The evaluated specimen of adenocarcinoma was sensitive to 5-fluorouracil and methotrexate, but was resistant to the remaining tested cytostatics . In the group of epidermoid carcinomas one specimen was completely resistant and one completely sensitive to all tested cytostatics . In one specimen the test with 5-fluorouracil could not be evaluated, and the specimen was resistant to the remaining cytostatics . The last specimen in this group was resistant to cis-platinum and sensitive to the remaining cytostatics. Atherosclerosis, 1990 Apr, 81(3), 183 - 9 Correlation between cholesterol content in circulating immune complexes and atherogenic properties of CHD patients' serum manifested in cell culture; Tertov VV et al.; Blood serum of most patients with coronary heart disease (CHD) caused a 2-5-fold increase in the total cholesterol content of smooth muscle cells cultured from unaffected human aortic intima, i.e . possessed an atherogenic potential manifested in culture . Removal of immunoglobulins G and M from an atherogenic serum brought about a fall in its atherogenic potential . The serum deficient in immunoglobulins A retained its ability to induce the cholesterol accumulation in cells . Treatment of the CHD patients' serum with 2.5% polyethylene glycol 6000 removed the circulating immune complexes . The serum subjected to this treatment lost its atherogenicity, i.e . failed to increase the cholesterol content in cultured cells . Incubation of smooth muscle cells derived from human aortic intima with circulating immune complexes isolated from an atherogenic patients' serum caused a 1.5-3-fold rise in the intracellular cholesterol . Blood sera of most (89%) CHD patients was characterized by a high cholesterol content in circulating immune complexes . More than 75% of healthy subjects and patients without stenosis of coronary arteries had low level of cholesterol in immune complexes . Blood sera atherogenicity manifested in culture directly correlated with the cholesterol level of circulating immune complexes (r = 0.90) . These findings suggest that the atherogenicity of CHD patients blood serum is due to cholesterol-containing immune complexes. J Bone Miner Res, 1990 Apr, 5(4), 337 - 43 Influence of experimental conditions on osteoblast activity in human primary bone cell cultures; Chavassieux PM et al.; Primary bone cell cultures were derived from human bone explants . Cellular activity was characterized by the alkaline phosphatase (AP) activity, osteocalcin, and type I and III collagen secretions in the supernatant . The determination of bone cell activity was performed in three different wells . No significant difference was noted between wells: the coefficient of variation was 8.0 +/- 2.9% for AP activity, 18.3 +/- 1.9% for osteocalcin secretion, and 22.5 +/- 14.3% for collagen . The AP activity and osteocalcin secretion significantly decreased with the number of passages: they were the highest after the first passage . Between each subject, the coefficient of variation was 85% for AP activity and osteocalcin secretion and 63 and 57% for type I and III collagen secretion, respectively . The AP activity did not differ with the age or sex of the donor . In contrast, osteocalcin secretion was significantly lower in females than in males . In males, osteocalcin significantly decreased with the age of the donor (r = -0.61; p less than 0.05) . Cellular activity did not depend on the site of the biopsy . When bone explants from one donor were cultured in two different petri dishes, the activity of cells was similar in both dishes, except in one case . Primary cell cultures derived from human bone explants are the only model providing untransformed osteoblastlike cells of human origin . Because of the experimental conditions, some factors may have influenced the cellular activity and they must be taken into account to validate further in vitro studies. Invest Ophthalmol Vis Sci, 1990 Apr, 31(4), 689 - 95 Neutral glycolipids of migrating and nonmigrating rabbit corneal epithelium in organ and cell culture; Panjwani N et al.; It is generally believed that plasma membrane glycoconjugates influence corneal epithelial cell migration after wounding . Previous studies have focused on the role of glycoproteins in this event . The present study was designed to determine whether migration-specific glycolipids are synthesized by epithelium of healing rabbit corneas . Migrating and nonmigrating rabbit corneal epithelia were incubated with {3H}-galactose in an organ culture system for 48 hr . At the end of the labeling period, a neutral glycosphingolipid (NGSL) fraction was isolated from each radiolabeled epithelium and was analyzed by thin-layer chromatography . Three radiolabeled NGSL components, M1, M2 and M3 (M1-M3), were present in significantly higher amounts in the extracts of migrating as compared to nonmigrating epithelium . Chromatographic mobility of M3 was similar to that of a standard glucosylceramide; M1 and M2 migrated more slowly than M3 . For characterization of the migration-related NGSL, a large amount of the starting material is required . Experiments, therefore, were conducted using cell cultures of rabbit corneal epithelium . Confluent (nonmigrating) cell cultures of rabbit corneal epithelium were found to synthesize either minimal or undetectable amounts of NGSL M1-M3 . In contrast, we found that the NGSL M1-M3 are synthesized as major components by sparse (migrating) corneal epithelial cell cultures . Components M1-M3 were synthesized as major components by sparse cultures even in the absence of cell mitosis . This suggests that the increased synthesis of components M1-M3 by sparse cell cultures may be related to cell migration rather than cell mitosis.(ABSTRACT TRUNCATED AT 250 WORDS) J Cell Physiol, 1990 Apr, 143(1), 26 - 38 Contraction of vascular smooth muscle in cell culture; Murray TR et al.; The use of cultured vascular smooth muscle cells for the study of events related to excitation and contraction of smooth muscle has been limited by the inability to reliably induce contractile responses after subculturing of the cells . This limitation has been overcome by the cell culture preparation described herein . We demonstrate that appropriate responses to both smooth muscle agonists and vasodilators were preserved in cells that were serially subcultured . Fetal bovine pulmonary artery and aortic cell cultures were established following enzymatic dispersion of the medial portion of freshly harvested vessels . At various times after isolation, cells were transferred to microscope coverslips coated with a polymerized silicone preparation (polydimethyl siloxane) . Tension forces generated by the cells were manifested as wrinkles and distortions of this flexible growth surface . Visual evidence of cell contraction in the form of increased wrinkling was documented for cells exposed to angiotensin II, carbachol, and KCl . Decreases in cell tension occurred following treatment with isoproterenol, and those relaxing effects were overcome by subsequent treatment with the agonist carbachol . The contractile responses did not diminish with prolonged maintenance in culture or repeated subculturing . Phosphorylation of the light chains on the contractile protein myosin was also measured as a biochemical index of agonist-induced contraction . Cells depolarized with KCl or exposed to carbachol showed increased myosin phosphorylation when analyzed by 2-dimensional gel electrophoresis . The responses remained intact through 7 passages and 9 weeks in culture . These results show that cultured vascular smooth muscle cells do not necessarily undergo a phenotypic modulation with loss of contractility under prolonged maintenance in culture. Biochem Cell Biol, 1990 Apr, 68(4), 804 - 7 Acidic extracellular environment induces only a subset of heat-shock proteins in primary mouse kidney cell cultures; Khandjian EW; Exposure of primary mouse kidney cell cultures to acidic medium (pH 5.5) induced the expression of a 70 kilodalton (kDa) protein . This protein was identified as the major inducible heat-shock protein 70 (hsp70) by immunoprecipitation with anti-hsp70 serum and Northern blot analysis with a hsp70 cDNA probe . Maximum induction of the 70-kDa protein at pH 5.5 after 240 min was about 30% of that observed after 60 min of thermal treatment at 43 degrees C . In addition, there was an apparent induction of the glucose-regulated proteins (GRPs) of 76-78 and 98-100 kDa, but not of the other hsps . This subset induction of the heat-shock response by acidic medium suggests that different mechanisms are responsible for the induction of the various families of hsps. Eur J Drug Metab Pharmacokinet, 1990 Apr-Jun, 15(2), 159 - 63 Cell culture techniques for the study of drug transport; Wilson G; The growth of differentiated cell monolayers on microporous filters is providing powerful new techniques for investigating the transport of drug and delivery systems across defined cellular barriers, and for discriminating between different routes and mechanisms . The growth, characterization and potential use of these systems is illustrated by studies on the human Caco-2 cell system which provides an in vitro model of the intestinal epithelial barrier . This system, still in the early stages of characterization and development, displays a number of carrier-mediated and vesicular transport systems found in the intestine in vivo, and is thus providing a useful system for studying the intestinal transport of drugs including peptides and proteins. Endocrinology, 1990 Apr, 126(4), 1895 - 903 Islet cell culture in defined serum-free medium; Clark SA et al.; A serum-free, hormone- and factor-supplemented, defined medium was developed which maintains functional activity of primary cultures of adult islet cells and continuous islet cell lines . Medium supplements examined included proteose peptone (PP), transferrin (TrFe), insulin-like growth factor-I (IGF-I), an insulinotropic fragment of human GH {hGH-(6-13)}, ethanolamine (EA), phosphoethanolamine (PEA), and human serum albumin (HSA) . Glucose-stimulated insulin secretion from islet monolayers was determined after culture in serum-free supplemented medium by either 2- to 3-h static incubations or in a superfusion system with either low (2.8-8.3 mM) or stimulatory (16.7-19.4 mM) glucose concentrations . Glucose-induced secretion was not sustained after 3-4 days of culture in medium supplemented with PP (0.5 mg/ml) and TrFe (10 micrograms/ml) alone . Addition of T3 did not restore glucose-induced secretion, although a combination of T3 and IGF-I or of T3, IGF-I, and PRL (10(-10)-10(-9) M) maintained glucose-induced insulin secretion for 1 month . No beneficial effects were noted with hGH-(6-13) . The beta-cell lines HIT-T15 and RINr 1046-38 were used to screen for a potential replacement for PP, the undefined component of the serum-free medium . A combination of HSA (1 mg/ml), EA (50 microM), and PEA (50 microM) provided a replacement for PP . In fact, insulin secretion from HIT-T15 cells was significantly better after culture in medium supplemented with HSA, EA, PEA, TrFe, T3, IGF-I, and PRL than in medium with PP, TrFe, T3, IGF-I, and PRL . HSA (1 mg/ml), EA (50 microM), and PEA (50 microM) in combination with TrFe (10 micrograms/ml), T3 (0.1 nM), IGF-I (0.65 nM), and PRL (1 nM) were used in studies with primary islet monolayers . After 3 weeks of culture islet monolayers were superfused, and the biphasic glucose-induced insulin secretion of cells maintained in defined medium was indistinguishable from the insulin secretion of cells maintained in medium with 5% fetal bovine serum . These studies indicate that adult rat beta-cells retain biphasic glucose-induced insulin secretion after extended culture in defined serum-free medium . The defined medium was also useful for cultures of RINr 1046-38 and HIT-T15 cells and should provide a basis for formulating media for islet cells from higher mammals, including man. Gastroenterology, 1990 Apr, 98(4), 936 - 54 Gastrinoma in vitro: morphological and physiological studies of primary cell cultures; Gower WR Jr et al.; Functional gastrin-containing tumor cells were maintained for up to 8 wk without fibroblastoid cell overgrowth . Short-term cultures consisted mainly of colonies composed of small polygonal cells, 70%-90% of which stained positive for immunoreactive gastrin . Cultures exhibited limited growth but viability remained high for 2-3 wk . Culture medium contained component I, and gastrin 34, 17, and 14 . With time the major C-terminal gastrin species in medium changed from gastrin 17 at 3 days to gastrin 34 at 5 wk . Extracts of cultured cells contained gastrin 34, 17, and 14; gastrin 17 was the major form detected at all times . Ultrastructurally, cultured tumor cells retained morphological integrity for several weeks; however, with time changes in the appearance of the secretory granules accompanied by evidence of cellular retrodifferentiation were gradually observed . Secretin, gastrin-releasing peptide, 8-bromoadenosine 3':5'-cyclic monophosphate, and phorbol, 12-myristate, 13-acetate stimulated the release of gastrin from cultured cells in a time-dependent fashion . Secretin, bombesin, gastrin-releasing peptide, L-tryptophan, and ethylamine stimulated gastrin release in a dose-dependent fashion . Somatostatin 14 inhibited secretin, bombesin, and gastrin-releasing peptide stimulated gastrin release but did not alter basal release . Cultured cells demonstrated de novo gastrin synthesis, evidenced by their ability to incorporate radiolabeled amino acids into immunoadsorbable gastrinlike material . Primary cultures of gastrin-containing tumor cells free from stromal contamination offer unique advantages for studies of factors that regulate the synthesis and secretion of gastrin and may prove of potential value for studies on cell differentiation and growth. J Clin Microbiol, 1990 Apr, 28(4), 643 - 5 Comparison of a serum replacement (Omni Serum) and fetal bovine serum in cell cultures used to isolate herpes simplex virus from clinical specimens; Johnston S et al.; Traditionally, fetal bovine serum (FBS) has been the principal component in media used in the growth and maintenance of cell cultures . Recent shortages have affected the cost and availability of FBS to clinical laboratories . Furthermore, lot-to-lot variability can affect cell culture performance and growth . We evaluated a commercially available serum replacement (Omni Serum; Advanced Biotechnologies Inc., Columbia, Md.) for use in the growth of cell cultures and for use in maintenance media used for the isolation of herpes simplex virus from clinical specimens . Cells (rhabdomyosarcoma and mink lung) raised on 5% Omni Serum grew as well as those grown on 10% FBS . The sensitivity of the Omni-raised cells to herpes simplex virus that had been isolated from 111 clinical specimens was equal to that of the cells raised and maintained with FBS . Cells grown with 10% FBS and maintained with 2% Omni Serum displayed the same sensitivity and integrity in tubes (rhabdomyosarcoma and mink lung) and vials (MRC-5 cells) as cells grown with 10% FBS and maintained with 5% FBS . This study indicates that Omni Serum is an acceptable substitute for FBS in maintenance media for cell culture tubes and vials used for viral isolation from clinical specimens. Cell Biol Toxicol, 1990 Apr, 6(2), 205 - 17 Histopathology and cell culture characteristics of liver cells from grc- and grc+ rats given diethylnitrosamine; Smith GJ et al.; The histopathological response and cell culture characteristics of liver cells from the R16 (grc-) strain of rats, which carries an MHC-linked deletion, were examined one week after a single intraperitoneal injection of 200 mg/kg body weight diethylnitrosamine (DEN) and were compared with the response of liver cells from wild type (grc+) rats . The DEN exposure induced hydropic/vacuolar changes in the parenchymal cells and a limited proliferation of oval cells in the periportal areas of the livers of both grc+ and grc- rats . Primary culture of collagenase-digested livers consisted of parenchymal, bile ductular and oval-related cells as determined by cell-specific immunohistochemistry . Subpassaged cells from grc+ rats exhibited oval cell ultrastructural morphology, inducible histochemical staining for gamma-glutamyl transpeptidase (GGT), and DEN-associated onset of anchorage-independent growth . Primary cultures of liver cells from R16 rats consistently failed to form cell strains upon subpassage. Herz, 1990 Apr, 15(2), 139 - 45 Antiatherosclerotic effects of calcium antagonists . Study in human aortic cell culture; Orekhov AN et al.; To investigate the effects of calcium antagonists on atherosclerotic cellular indices, {3H}thymidine incorporation and intracellular cholesterol content, primary culture of cells isolated from subendothelial intima of human atherosclerotic aorta was used . Among tested drugs were: verapamil, nifedipine, diltiazem, papaverin, nicardipine, D-600, cinnarizine, PN 200 110 and PY 108 068 . Verapamil proved to be the most effective . It significantly reduced the total intracellular cholesterol and sharply decreased the incorporation of {3H}thymidine . With respective efficacy verapamil was followed by nifedipine and PY 108 068 . Neither beta-blocker (metoprolol) nor nitrate (nitroglycerin) modified antiatherosclerotic effects of calcium antagonist (nifedipine) . Calcium agonist Bay K 8644 which facilitates the penetration of calcium into cells caused the accumulation of intracellular cholesterol and stimulated cell proliferation . Simultaneous addition of nifedipine and Bay 8644 led to the inhibition of the agonist's atherogenic effect . Thus, facilitation of calcium influx into cells causes atherosclerotic alterations in the arterial cells; atherogenic calcium effects are inhibited by calcium channel blockers . Furthermore, based on the results of application of blood plasma from patients treated with calcium antagonists or beta-blocker to primary cultures of atherosclerotic cells, it can be assumed that calcium antagonists affect an anti-atherosclerotic and beta-blockers an atherogenic action. J Anim Sci, 1990 Apr, 68(4), 1158 - 69 Protein metabolism in chicken muscle cell cultures treated with cimaterol; Young RB et al.; Primary muscle cell cultures were prepared from the leg muscle of 12-d broiler chicken embryos . The partitioning agent cimaterol (10(-6) to 10(-10) M) was added on d 1 and each day thereafter, and cells were studied after 7 d in culture . Cimaterol had no effect at any level either on the percentage of nuclei within multinucleated myotubes or on the total number of nuclei within myotubes . At 10(-7) M cimaterol, the quantity of the myofibrillar protein fraction was increased by 25.1 +/- 8.0% (P less than .05) and the quantity of myosin heavy chain was increased by 30.9 +/- 4.5% (P less than .05) . To understand the basis for the increase in myofibrillar protein, the incorporation rate of {3H}Leu was measured in pulse labeling experiments . The apparent synthesis rate of the soluble protein fraction and the crude myofibrillar fraction was not significantly increased by cimaterol; however, cimaterol levels greater than 10(-8) M caused a 10 to 12% increase (P less than .05) in the incorporation rate of {3H}Leu into myosin heavy chain . The effect of cimaterol on release of {3H}Leu from prelabeled protein also was assessed in pulse-chase experiments; the apparent rate of protein degradation was inhibited by 10 to 15% (P less than .05) at the higher levels of cimaterol . Dot blot analysis indicated that the quantity of myosin heavy chain mRNA was elevated in cimaterol-treated cultures . Thus, the increased quantity of myofibrillar proteins in embryonic broiler muscle cell cultures is the combined result of a stimulation in the rate of protein synthesis and an inhibition in the rate of protein degradation. Clin Invest Med, 1990 Apr, 13(2), 71 - 6 The effect of Trichomonas vaginalis and the role of pH on cell culture monolayer viability; Garber GE et al.; In vitro, Trichomonas vaginalis produces contact-dependent cytotoxicity in which the monolayers are disrupted and monolayer cells die . In contrast, when cell-free filtrates of T . vaginalis culture supernatants are applied to a monolayer, detachment occurs but cell viability is maintained . Growth of T . vaginalis in culture results in a dramatic fall in pH . Both in systems where T . vaginalis grew in physical contact with McCoy cells and where they were physically separate, monolayers still disrupted; but the rate of death of McCoy cells significantly decreased when pH was rigidly controlled . This would indicate that cell death attributed to contact-dependent cytotoxicity is in part due to the drop in pH when T . vaginalis is grown in vitro. Neurosci Lett, 1990 Mar 26, 111(1-2), 58 - 63 Rabbit retinal Müller cells in cell culture show gap and tight junctions which they do not express in situ; Wolburg H et al.; Retinae of early postnatal rabbits were enzymatically dissociated and explanted in a culture system . The prospective myelinated region was discarded in order to avoid the presence of astrocytic or mesenchymal cells . After about 14 days in vitro (DIV), outgrowing glial (Muller) cells formed what light optically appeared to be confluent monolayers but by electron microscopy was shown to consist of flat epithelioid cells which overlapped considerably by extension of cytoplasmic tongues . Applying the freeze-fracture technique, apposed membranes of these cells were demonstrated to express infrequently but consistently both gap and tight junctions . This kind of junctions has never been observed on the membrane of rabbit Muller cells in situ . In comparison with Muller cell membranes in situ, the density of intramembrane particles was considerably reduced . Orthogonal arrays of particles which are characteristic elements of Muller cells in situ were not detected . Our results suggest that in homogeneous cell culture, Muller cells form some kind of epithelium-like specialized intercellular junctions . This situation resembles that of closely related glial cell types which form homogeneous layers in situ as e.g . retinal pigment epithelium cells expressing tight junctions, and marginal astrocytes being coupled by extensive gap junctions. Neurosci Lett, 1990 Mar 26, 111(1-2), 222 - 7 Depolarization regulates selectively the expression of different opioid receptors: a decreased number of kappa receptors in chronically activated guinea pig brain cell cultures; Barg J et al.; Previous studies about the differential effect of chronic membrane depolarization on the expression of mu and delta opioid receptors prompted us to investigate whether the same treatment regulates also the expression of kappa opioid receptors . Embryonic guinea pig brain cells that exhibit in culture a high density of kappa receptors were treated for different time periods with potassium chloride, and the number of these receptors was determined with the universal opioid ligand {3H}diprenorphine . In a second set of experiments the cultures were treated with the sodium channel activator veratridine and opioid receptors were determined with the selective kappa ligand {3H}U69,593 . The results indicate that chronic membrane depolarization (for 3 or 6 days) decreases significantly the number of kappa receptors, with no effect on the affinity of the ligands . Taken together with our previous reports, it is suggested that neuronal activation has a selective and possibly opposite regulatory role on the expression of the 3 opioid receptors. J Immunol, 1990 Mar 1, 144(5), 1563 - 70 Analysis of thymic stromal cell subpopulations grown in vitro on extracellular matrix in defined medium . II . Cytokine activities in murine thymic epithelial and mesenchymal cell culture supernatants; Eshel I et al.; Two morphologically distinct primary cultures of murine thymic stroma were established and found to be of epithelial (MTEC) and mesenchymal (MTMC) origin . These cultures were generated by selective conditions of tissue disruption and were maintained on extracellular matrix in defined medium . Culture supernatants (CS) from these cultures (EC-CS and MC-CS respectively), were tested for cytokine production and for effects on thymocyte maturation . Both supernatants displayed the activities of IL-3 and of granulocyte/macrophage-CSF and not of IL-1, -2, -4, or IFN . In addition they were found to be mitogenic to murine thymocytes in a "spontaneous" {3H}TdR incorporation assay . The two supernatants differed, however, in their effect on Con A stimulation . EC-CS had a strong enhancing effect, both when used for preincubation (18 h) before Con A stimulation or when present simultaneously with it . MC-CS had a small inconsistent effect under these conditions . Also EC-CS enhanced IL-2 and IL-3 production by thymocytes . The responsive thymocyte subpopulation was the one that does not bind peanut agglutinin . CS of an established thymic epithelial cell line displayed only part of these activities at a considerably lower level . CS from primary kidney cell culture was completely devoid of activity . The results suggest that primary thymic stromal cell cultures, cultivated under the defined conditions described here, may better preserve physiologic secretory activities, and probably also other cell functions, compared with established cell lines . Furthermore, the results are compatible with the hypothesis that the soluble factors, secreted by thymic stromal cells, are active on either very early or late stages of thymic differentiation, whereas the main intrathymic stages of differentiation are conceivable dependent primarily on direct contact with stromal cells. J Virol Methods, 1990 Mar, 27(3), 287 - 94 Comparison of fortified calf serum, serum substitutes and fetal calf serum with or without extenders for propagation of cell cultures for virus plaque assays; Dahling DR et al.; Two studies comparing sewage-isolated and laboratory stock viruses were conducted to determine if alternative forms of serum or serum extenders could be used in place of fetal bovine serum without a significant loss of viral titer . In the first study, BGM cells were grown in standard MEM-L15 medium which was supplemented with Nuserum, Sigma serum replacement (CPSR-1), HyClone defined iron supplemented calf bovine serum, fetal bovine serum (FBS) or FBS supplemented with either SerXtend or Mito serum extenders . Comparison of virus titers showed that CPSR-1 gave the best overall results and was comparable to FBS . Of the serum extenders, only SerXtend improved virus recovery from sewage samples . In the second study, all sera were tested with and without SerXtend . In these experiments, SerXtend enhanced virus sensitivity of the BGM cell line grown in the HyClone serum but reduced the sensitivity of those cultured in Sigma serum . In both series, the growth of BGM cells was monitored for 12 weeks and all test products were shown to support long-term cell growth. Am J Physiol, 1990 Mar, 258(3 Pt 1), C489 - 94 Vector-free gravity disrupts synapse formation in cell culture; Gruener R et al.; Terrestrial organisms evolved under and are subjected to the constancy of gravity . The organisms having adapted to this environmental factor, it is possible that embryonic development may be modified by exposure to altered gravity . To test the effects of gravity on embryonic development, we monitored the formation of nerve-associated acetylcholine receptor patches (NARPs) as an index of synaptogenesis . Embryonic spinal neuron and myotomal myocyte cocultures were placed in a horizontally rotating clinostat . From the cell's perspective, this results in the cancellation of the gravitational vector because of continuous averaging, thus mimicking the reduced gravitational force encountered in space . NARPs from cultures in which nerve-muscle contact was established before the onset of rotation were unaffected . In contrast, cultures in which nerve contact took place during rotation showed a marked inhibition of NARPs . Moreover, in the myocytes which did exhibit NARPs, the area of the patch was significantly reduced compared with control sister cultures . Several paradigms were used to ascertain that these findings did not result simply from loss of contact between neurites and myocytes, accelerated diffusion of a putative aggregating factor secreted by neurites, or from turbulence in the medium . Our data suggest that the process of synapse formation is sensitive to the gravitational vector . Embryonic development of the nervous system, in space, may therefore be markedly different from that normally occurring on earth. J Cell Physiol, 1990 Mar, 142(3), 505 - 13 Tumor promoters retard the loss of a transient subpopulation of cells in low passage Syrian hamster cell cultures; Ueo H et al.; Early passage normal diploid Syrian hamster (SH) fetal cell cultures contain a transient subpopulation of contact-insensitive (CS-) cells which lack density-dependent inhibition of cell division . The size of this CS- subpopulation decreases during in vitro passage by conversion of the CS- cells to contact-sensitive (CS+) cells . Approximately 10-15 population doublings after the frequency of the CS- cells has declined to below 0.001%, mass cultures cease proliferating and exhibit cellular senescence . Cultures with higher initial numbers of CS- cells exhibit longer in vitro proliferative life spans than cultures with smaller initial numbers of CS- cells . Active tumor promoting phorbol esters (12-O-tetra-decanoyl-phorbol-13-acetate {TPA} and phorbol-12,13-didecanoate {PDD}) retard the decline in the proportion of CS- cells during in vitro passage, while the inactive tumor promoting phorbol ester, 4 alpha-phorbol-12,13-didecanoate (4 alpha PDD) has no effect on the rate of loss of the CS- cells . In addition, continuous treatment from secondary culture with TPA or PDD extends by approximately twofold the in vitro proliferative life span of SH fetal cell cultures . Treatment must, however, begin at passage 1 or 2 when the CS- cells are still present . After the proportion of the CS- cells has decreased to less than 0.001% as in passage 6 cultures, promoters have no effect on the life span of the culture . This finding that exposure to promoters results in both a prolonged maintenance of the CS- cellular subpopulation, as well as an extension of in vitro proliferative life span, suggests that the conversion of CS- cells to CS+ cells is involved in the mechanism of in vitro senescence. FEMS Microbiol Lett, 1990 Mar 1, 56(1-2), 217 - 21 NMR studies of a bacterial cell culture medium (LB broth): cyclic nucleotides in yeast extracts; Rayner MH et al.; The composition of LB broth (tryptone, yeast extract and NaCl) was investigated by 1H,31P-NMR spectroscopy, FPLC and gel electrophoresis . An unexpected finding was the high level of 2'3'-cyclic nucleotides, detected by characteristic 31P-NMR resonances in the region 20-21 ppm, originating from the yeast component . 31P-NMR resonances for cyclic nucleotides were observed during the autolysis of Saccharomyces cerevisiae cells, and in model reactions of RNase with RNA. Am J Vet Res, 1990 Mar, 51(3), 344 - 8 Effect of growth in cell cultures and strain on virulence of Mycoplasma hyopneumoniae for swine; Zielinski GC et al.; In an attempt to develop better methods for consistent induction of pneumonia in naturally born swine, using cultures of Mycoplasma hyopneumoniae, fifty 6-week-old, naturally born pigs from a respiratory disease-free herd were used in 3 trials . Pigs inoculated with Mycoplasma hyopneumoniae strain 232 (passage 21) grown for 1 passage or 5 passages in Eagle minimal essential medium plus 20% porcine serum, with or without human lung fibroblasts, had a mean (+/- SD) value range between 5.4 +/- 3.6 and 9.2 +/- 2.1% of consolidated lung area . In the second trial, pigs inoculated 1, 2, or 3 days in succession with strain 232 grown in Eagle medium or Friis mycoplasmal medium with 20% porcine serum had between 5.1 +/- 7 and 8.7 +/- 4.3% of consolidated lung area . In the third trial, virulence of Mycoplasma hyopneumoniae strains 144L (p27), 11 (p26), J (p60), and 232 (p27) grown in Friis mycoplasmal medium was compared . Pigs inoculated with those strains had 5.1 +/- 4.1, 2.6 +/- 3.1, 0, and 4.3 +/- 4% of consolidated lung area, respectively . Significant differences were not found in consolidated lung area among groups in trials 1 and 2, and among groups of pigs inoculated with M hyopneumoniae strains 144L, 11, and 232 in trial 3 . Pneumonia was not detected in pigs inoculated with strain J in trial 3. In Vitro Cell Dev Biol, 1990 Mar, 26(3 Pt 1), 285 - 90 Growth factor binding and bioactivity in human kidney epithelial cell cultures; Gansler T et al.; Insulinlike growth factors (IGF) and epidermal growth factor (EGF) are produced in renal tissue, as are specific receptors for these hormones . To evaluate the significance of these observations to regulation of renal tubular cell proliferation, we have examined the interaction of IGF and EGF with cultured human proximal tubular epithelial cells (HPT) . HPT cells showed specific binding of IGF-1, insulin, and EGF . IGF-1 binding was inhibited by antibody to the type 1 IGF receptor (alpha-IR3) . Insulin receptors and type 1 IGF receptors were identified by bifunctional cross-linking . IGF-1, insulin, and EGF stimulated {3H}thymidine incorporation by 77, 73, and 87%, respectively . Half maximal stimulation by IGF-1, insulin, and EGF were produced with 4 X 10(-9) M, 2.5 X 10(-8) M, and 8 X 10(-10) M concentrations of these hormones . Alpha-IR3 inhibited stimulation of thymidine incorporation by IGF-1 and insulin but had no effect in EGF-stimulated thymidine incorporation . EGF and high concentrations of insulin both stimulated cell proliferation by 83 and 79%, respectively . These data are consistent with regulation of tubular epithelial proliferation by IGF-1, insulin, and EGF and suggest that the mitogenic activity of both insulin and IGF-1 is mediated by the type 1 IGF receptor. Anal Biochem, 1990 Mar, 185(2), 265 - 9 A radiolabel-release microwell assay for proteolytic enzymes present in cell culture media; Rucklidge GJ et al.; A modified method for the measurement of proteolytic enzyme activity in cell culture-conditioned media has been developed . Using the release of 3H-labeled peptides from 3H-labeled gelatin the method is performed in microwell plates . The substrate is insolubilized and attached to the wells by glutaraldehyde treatment, thus eliminating the need for a precipitation step at the end of the assay . The assay is sensitive, reproducible, and convenient for small sample volumes . The effect of different protease inhibitors on activity can be assessed rapidly allowing an early characterization of the enzyme . It can also be adapted to microplate spectrophotometric analysis by staining residual substrate with Coomassie blue. J Pharm Pharmacol, 1990 Mar, 42(3), 191 - 3 Characterization of cysteinyl-leukotriene formation in primary astroglial cell cultures; Seregi A et al.; The formation and composition of cysteinyl-leukotrienes (LT) in primary astroglial cell cultures prepared from newborn rat brain has been studied . Small amounts of cysteinyl-LT determined in terms of LTC4-like material in the supernatants of the cultures, became detectable after stimulation of the cells with 10(-5) M ionophore A23187 . Cysteinyl-LT formation increased with time, reaching about 600 pg (mg protein)-1 after 60 min incubation . In contrast, considerable thromboxane (TX) B2 synthesis was found at 5 min following A23187-stimulation (about 30 ng TXB2 (mg protein)-1) . The synthesis of cysteinyl-LT was abolished by 5 x 10(-5) M nordihydroguaiaretic acid (NDGA) . Irrespective of the duration of incubation, blockage of prostanoid synthesis by 10(-6) M indomethacin did not result in increased cysteinyl-LT production . Reversed phase HPLC combined with radioimmunological detection showed that, after 60 min incubation in the presence of A23187, LTC4 and LTD4 accounted for practically all the LTC4-like immunoreactive material in the supernatants of cell cultures . No significant amounts of LTE4 could be detected . The results show that astrocytes may contribute to brain LTC4 and LTD4 synthesis . However, the cellular site of cerebral LTE4 formation seems to be other than the astroglia. J Neurosci, 1990 Mar, 10(3), 1025 - 34 A vasoactive intestinal peptide-like cotransmitter at cholinergic synapses between rat myenteric neurons in cell culture; Willard AL; Intracellular recording and immunochemical techniques were used to study synaptic transmission between individual pairs of rat myenteric plexus neurons in cell culture . This report describes the synaptic connections made by "dual function" presynaptic neurons that evoked slow postsynaptic depolarizations (slow EPSPs) in the same neurons in which they also evoked fast nicotinic cholinergic EPSPs . The slow EPSPs occurred only when presynaptic neurons were stimulated at frequencies of 5 Hz or higher . During the slow EPSPs, slope input resistance increased . The slow EPSPs were not detectably voltage-dependent, and they reversed sign at the estimated K+ equilibrium potential, suggesting that they resulted from a synaptically mediated decrease in resting K+ conductance . Several lines of evidence suggested that dual-function neurons evoke slow EPSPs by releasing a vasoactive intestinal peptide (VIP)-like cotransmitter . (1) Immunocytochemical staining revealed VIP-like immunoreactivity in all physiologically identified dual-function neurons . (2) Responses to exogenously applied VIP mimicked the slow EPSPs . (3) Superfusion of cultures with anti-VIP antisera blocked the slow EPSPs reversibly, as did application of desensitizing doses of VIP . These findings suggest that during periods of increased activity, subsets of cholinergic myenteric neurons release a VIP-like cotransmitter that enhances postsynaptic excitability . The effects of the cotransmitter may help to compensate for decreases in nicotinic EPSPs that occur during increased presynaptic activity. AIDS Res Hum Retroviruses, 1990 Mar, 6(3), 411 - 6 Suppression of HIV-1 reverse transcriptase activity by mycoplasma contamination of cell cultures; Vasudevachari MB et al.; The detection of HIV-1 in human peripheral blood lymphocytes is routinely carried out by cocultivation of test cells with normal peripheral blood mononuclear cells (PBMC) . The presence of virus is evidenced by cytologic observation of syncytia or by detecting viral reverse transcriptase (RT) and/or p24 antigen in the culture supernatant fluid . Syncytia formation is almost always associated with the presence of virus as measured by RT, although many RT-positive cultures do not form syncytia . As part of a large screening program, we identified three cultures that showed syncytia but were RT negative . The basis for these discrepant observations was contamination of cultures with mycoplasma that interfered with the RT assay and thereby obscured virus detection . Treatment of cultures with BM-cycline removed mycoplasma contamination and restored RT activity . The present findings indicate the need for caution in the interpretation of negative RT results during HIV-1 isolation and especially in cultures that show evidence of syncytia formation. J Pathol, 1990 Mar, 160(3), 259 - 69 The behaviour of human oral squamous cell carcinoma in cell culture; Prime SS et al.; This study examined the initial behaviour of 48 human oral squamous cell carcinomas (SCC) in cell culture . The early outcome of these cultures (contamination, absence of cell growth, epithelial cell senescence/fibroblast overgrowth, extended keratinocyte growth) did not reflect the clinical characteristics of the tumours of origin . Four new human oral SCC cell lines were characterized more extensively . Each cell line was immortal, 3T3-independent, and expressed low degrees of anchorage independence (CFE less than 4 per cent) . Two of the four cell lines were tumorigenic in athymic mice . All of the cell lines expressed keratin intermediate filaments and two showed weak co-expression of vimentin . A wide range of keratins were expressed by the tumour xenografts; cornified keratins (K1, K10) were only expressed in the absence of K19 and vimentin, and vice versa . The nuclear:cytoplasmic ratio and the degree of serum independence correlated with each other and with the STNMP clinical grading of the tumours of origin. J Virol Methods, 1990 Mar, 27(3), 269 - 76 A solid phase reverse transcriptase micro-assay for the detection of human immunodeficiency virus and other retroviruses in cell culture supernatants; Somogyi PA et al.; A simple and rapid solid-phase reverse transcriptase assay was developed based on the use of poly(rA):oligo(dT)12-18 as template primer immobilized on DEAE cellulose paper squares to detect human immunodeficiency virus (HIV) and/or other retroviruses in cell culture supernatants . It was found that PEG (per se) -up to 4% concentrations (w/v)--did not inhibit reverse transcriptase activity . Optimal conditions of the assay were determined . This solid-phase technique is much faster and more convenient than the methods described previously. J Gen Virol, 1990 Mar, 71 ( Pt 3), 561 - 7 Neutralization of Egtved virus pathogenicity to cell cultures and fish by monoclonal antibodies to the viral G protein; Lorenzen N et al.; Egtved virus, the rhabdovirus causing viral haemorrhagic septicaemia in rainbow trout, was analysed at the antigen level with a future subunit vaccine in mind . Three monoclonal antibodies to the viral G protein were characterized with respect to neutralizing activity at the cell culture level, as well as their ability to protect rainbow trout fingerlings against virus infection following passive immunization . Two antibodies showed strong protective activity in fish . Only one of these antibodies was able to neutralize viral infectivity in vitro . Reduction of disulphide bonds in the G protein abolished reactivity of this antibody in immunoblotting, whereas antigen deglycosylation did not influence the binding ability of any of the antibodies . These data suggest that the G protein contains linear as well as non-linear, carbohydrate-free epitopes, which are involved in the protection against Egtved virus . However, an indirect influence of oligosaccharide side chains on epitope formation could not be excluded, since in situ inhibition of glycosylation prevented the binding of the protecting antibodies in immunofluorescence. Biotechnol Prog, 1990 Mar-Apr, 6(2), 121 - 8 Chemical decomposition of glutamine in cell culture media: effect of media type, pH, and serum concentration; Ozturk SS et al.; The chemical decomposition of glutamine to ammonia and pyrrolidonecarboxylic acid was studied at 37 degrees C in a pH range of 6.8-7.8 in different media preparations containing various amounts of fetal bovine serum . The media type influenced the decomposition rate, and the first-order rate constants increased with increasing pH values . The serum concentration had little or no effect on the decomposition rate . The importance of chemical decomposition of glutamine on the analysis of glutamine and ammonia metabolism was illustrated by an example of batch cultivation of a hybridoma cell line . The difference between the apparent uptake rate of glutamine and the actual uptake rate (which is corrected for the chemical decomposition) is shown to be as high as 200% . Similar discrepancy between the apparent and actual ammonia production rate is observed . Mathematical analysis was carried out to develop the relationship between the apparent and actual glutamine uptake and ammonia production rates . The analysis reveals that there are three important dimensionless parameter ratios that govern the difference between the apparent and actual glutamine uptake and ammonia production rates. J Biol Chem, 1990 Feb 25, 265(6), 3432 - 5 Expression of chimeric cDNAs in cell culture defines a region of UDP glucuronosyltransferase involved in substrate selection; Mackenzie PI; The cDNAs encoding two forms of UDP glucuronosyltransferase have been expressed in cultured cells to demonstrate that one form, UDPGTr-3, glucuronidates testosterone, whereas the second form, UDPGTr-4, is mainly active toward etiocholanolone (Mackenzie, P . I . (1986) J . Biol . Chem . 261, 14112-14117; Mackenzie, P . I . (1987) J . Biol . Chem . 262, 9744-9749) . In order to localize areas of the polypeptide chain involved in substrate selection, the 5' regions of UDPGTr-3 and -4 cDNAs were exchanged to form two chimeric cDNAs . A 53-kDa protein was synthesized in COS cells transfected with the chimeric UDPGTr-3.4 cDNA, which encodes the amino-terminal 298 residues of UDPGTr-3 and the carboxyl-terminal 232 residues of UDPGTr-4 . This protein glucuronidated testosterone rather than etiocholanolone and had a faster electrophoretic mobility when transfected COS cells were cultured in the presence of tunicamycin, an inhibitor of N-linked glycosylation . The unglycosylated variant produced by this treatment also glucuronidated testosterone . In contrast, a 50-kDa protein that was more active toward etiocholanolone as substrate was synthesized in COS cells transfected with UDPGTr-4.3, a chimeric cDNA that encodes the amino-terminal region of UDPGTr-4 joined to the carboxyl-terminal region of UDPGTr-3 . The electrophoretic mobility of this chimeric protein was unaffected by tunicamycin treatment . These results demonstrate that amino acid sequences that specify substrate specificity are localized in the amino-terminal half of the UDP glucuronosyltransferase polypeptide chain and that the presence of N-linked oligosaccharide chains on the protein does not affect the choice of substrate. Neurosci Lett, 1990 Feb 16, 109(3), 253 - 8 Molluscan insulin-related neuropeptide promotes neurite outgrowth in dissociated neuronal cell cultures; Kits KS et al.; The formation of neurites in isolated neurones of the snail Lymnaea stagnalis in primary culture was studied . The insulin-related neuropeptide (MIP: Molluscan insulin-related peptide) produced by the neuroendocrine light green cells (LGCs) of Lymnaea stimulated neurite formation, both in isolated unidentified central neurons and in the LGCs . The effect of MIP was dose dependent . It was significant from the second day of culture and amounted up to an 8-fold increase in neurite outgrowth after 3 days . The results add a functional aspect to the evolutionary relationship of MIP with mammalian insulin and insulin-related peptides and suggest that the LGCs, which stimulate growth, are also involved in development of the nervous system. Biochem J, 1990 Feb 15, 266(1), 41 - 6 Characterization and localization of progesterone 5 alpha-reductase from cell cultures of foxglove (Digitalis lanata EHRH); Wendroth S et al.; Progesterone 5 alpha-reductase, which catalyses the reduction of progesterone to 5 alpha-pregnane-3,20-dione, was isolated and characterized from cell cultures of Digitalis lanata (foxglove) . Optimum enzyme activity was observed at pH 7.0, and the enzyme had an apparent Km value of 30 microM for its substrate progesterone . The enzyme needs NADPH as reductant, which could not be replaced by NADH . For NADPH, the apparent Km value is 130 microM . The optimum temperature was 40 degrees C; at temperatures below 45 degrees C, the product 5 alpha-pregnane-3,20-dione was reduced by a second reaction to 5 alpha-pregnan-3 beta-ol-20-one . Progesterone 5 alpha-reductase activity was not dependent on bivalent cations . In the presence of EDTA, 0.1 mM-Mn2+ had no influence on enzyme activity, whereas 0.1 mM-Ca2+, -Co2+ and -Zn2+ decreased progesterone 5 alpha-reductase activity . Only 0.1 mM-Mg2+ was slightly stimulatory . EDTA and thiol reagents such as dithiothreitol stimulate progesterone 5 alpha-reductase activity . By means of linear sucrose gradient fractionation of the cellular membranes, progesterone 5 alpha-reductase was found to be located in the endoplasmic reticulum. J Immunol Methods, 1990 Feb 9, 126(2), 213 - 22 A cell culture system that enhances mononuclear cell IgE synthesis induced by recombinant human interleukin-4; Claassen JL et al.; A new culture system is described in which recombinant human interleukin-4 (rhIL-4) consistently induces the synthesis of large quantities of IgE by human blood mononuclear cells (MNC) . Unfractionated MNC were cultured in complete Iscove's modified Dulbecco's medium (C-IMDM), composed of IMDM enriched with human transferrin, bovine insulin, bovine serum albumin, oleic acid, palmitic acid, linoleic acid, and fetal calf serum (FCS) . Under these culture conditions, MNC from four donors synthesized mean quantities of IgE of 76 ng/ml at plateau after stimulation with rhIL-4 in concentrations ranging from 0.04 to 80 ng/ml (plateau rhIL-4 concentrations were 5 ng/ml or greater) . In contrast, rhIL-4 failed to induce significant IgE synthesis at any of those doses of rhIL-4 in parallel MNC cultures performed in RPMI 1640 supplemented with FCS (RPMI 1640) . Additional optimal conditions for the induction of IgE synthesis in this system were a MNC concentration of 1-2 X 10(6)/ml and a culture time of 18 days . Variability was noted in the amount of IgE produced by different donors (CV 0.22) and by the same donor when tested on different occasions (mean CV 0.21), but no donor's MNC failed to produce significant IgE in response to rhIL-4 when cultured in C-IMDM . The geometric mean IgE production induced by optimal IL-4 concentrations for the entire group of 16 subjects was 36.8 ng/ml IgE, with the lowest day 18 mean IgE concentration for any donor being 10.6 ng/ml and the highest 372.2 ng/ml . The enhanced rhIL-4-induced IgE synthesis supported by C-IMDM was due to the combined effects of the added enrichment factors and not to differences in the viabilities of MNC cultured in C-IMDM and RPMI 1640 . This culture system will alleviate the problems of inconsistent and low quantities of IgE induced by IL-4 that confound most current culture systems used to examine rhIL-4-induced IgE synthesis . It will, thereby, facilitate further investigation of the regulation of human IgE synthesis. Arch Biochem Biophys, 1990 Feb 1, 276(2), 382 - 9 Relationships among dolichyl phosphate, glycoprotein synthesis, and cell culture growth; Kabakoff BD et al.; Following treatment of Chinese hamster ovary cells with inhibitors of mevalonate biosynthesis in the presence of exogenous cholesterol, the cellular concentration of phosphorylated dolichol and the incorporation of {3H}mannose into dolichol-linked saccharides and N-linked glycoproteins declined coincident with a decline in DNA synthesis . Addition of mevalonate to the culture medium increased rates of mannose incorporation into lipid-linked saccharides and restored mannose incorporation into N-linked glycoproteins to control levels within 4 h . After an additional 4 h, synchronized DNA synthesis began . Inhibition of the synthesis of lipid-linked oligosaccharides and N-linked glycoproteins by tunicamycin prevented the induction of DNA synthesis by mevalonate, indicating that glycoprotein synthesis was required for cell division . The results suggest that the rate of cell culture growth may be influenced by the level of dolichyl phosphate acting to limit the synthesis of N-linked glycoproteins. J Bone Miner Res, 1990 Feb, 5(2), 165 - 71 Transfection of calcitonin gene regulatory elements into a cell culture model of the C cell; Cote GJ et al.; Calcitonin gene expression in the TT cell line can be regulated by phorbol esters, cAMP, glucocorticoids, and 1,25-dihydroxyvitamin D3 . To further study the regulation of this gene we have sequenced 1460 bases 5' to the start of calcitonin gene transcription . This DNA sequence contains cis consensus elements for both phorbol ester- and cAMP-responsive elements . To study the role of these elements, calcitonin 5' flanking DNA was coupled to the human growth hormone gene as a reporter and transiently transfected into TT cells, a human thyroid C cell line . Treatment of transfected TT cells stimulated a two- to fivefold increase in reported gene product expression, confirming the existence of functional cAMP- and phorbol ester-dependent enhancers within the calcitonin 5' flanking sequence. Eur J Immunol, 1990 Feb, 20(2), 389 - 95 Interleukin 2, 4 and 5 are sequentially produced in mitogen-stimulated murine spleen cell cultures; Cardell S et al.; Lymphokine production was analyzed in murine spleen lymphocytes stimulated with different T cell mitogens . Using in situ hybridization, frequencies of cells and the kinetics of production of interleukin (IL) 2, 4 and 5 were analyzed . The different mitogens varied in their ability to induce the three interleukins . IL2 was most successfully induced with a high dose of the calcium ionophore A23187 combined with phorbol 12-myristate 13-acetate (PMA) . Significant frequencies of cells containing IL4 or IL5 mRNA were found among cells stimulated with an anti-CD3 antibody together with PMA, or pokeweed mitogen . The combination of anti-CD3 and PMA induced relatively high frequencies of all three cytokines . The production was sequential with the highest levels of IL2 mRNA present during the first 24 h, IL4 mRNA reaching a peak on day 2 and finally IL5 peaking on day 3 . When cells that had been stimulated with mitogens in vitro were restimulated, the lymphokines were produced more rapidly . The order of production was maintained with IL2 mRNA reaching a maximum already at 3 h of culture, IL4 mRNA at 8 h and IL5 mRNA at 24 h. Immunology, 1990 Feb, 69(2), 277 - 81 The effect of human placental protein 14 (PP14) on the production of interleukin-1 from mitogenically stimulated mononuclear cell cultures; Pockley AG et al.; Crude human decidual tissue extracts containing placental protein 14 (PP14) were shown to inhibit the production of interleukin-1 beta (IL-1) from mitogenically stimulated mononuclear cell cultures . The inhibition was dose-dependent over the range of PP14 concentrations investigated (0-8.0 mg/l) and was effective on both phytohaemagglutinin-(PHA) and lipopolysaccharide- (LPS)-induced IL-1 secretion . Using these culture systems, a PP14 concentration of 1.0 mg/l induced a 34% suppression of IL-1 secretion following LPS stimulation and 22% following PHA stimulation . For PHA stimulation the suppression of IL-1 secretion was effective throughout the culture period investigated (0-89 hr) . Individual crude decidual extracts inhibited the incorporation of {3H}thymidine into PHA-stimulated lymphocytes, such inhibition being partially reversed by the addition of exogenous recombinant IL-1 to the cultures . These results suggest that the previously reported immunosuppressive activity of PP14 may be mediated by the suppression of IL-1 secretion. Bone Miner, 1990 Feb, 8(2), 145 - 56 Effects of acidic fibroblast growth factor and epidermal growth factor on subconfluent fetal rat calvaria cell cultures: DNA synthesis and alkaline phosphatase activity; Nicolas V et al.; The effects of acidic fibroblast growth factor (aFGF) and epidermal growth factor (EGF) were examined in subconfluent fetal rat calvaria cell cultures, in the presence of 2% serum . Maximal effect of aFGF and EGF on DNA synthesis measured by {3H}thymidine incorporation was observed after 18 h . aFGF stimulated DNA synthesis by 3.5-fold with an ED50 of 0.75 ng/ml while a 2.3-fold EGF stimulation was recorded with an ED50 of 0.067 ng/ml . 5-Bromo-2-deoxyuridine staining showed a higher stimulation of proliferation in the scattered cells than in the cell clusters . An 18 h aFGF or EGF treatment decreased alkaline phosphatase (ALP) activity by 40 and 23%, respectively, as compared with control cultures . This inhibition was more pronounced after 48 h in the presence of the effectors but no modification of the ALP electrophoretic mobility was observed . These data suggest that aFGF is a less potent mitogen than EGF and a higher inhibitor of ALP activity in fetal rat calvaria cell culture. Arch Biochem Biophys, 1990 Feb 1, 276(2), 390 - 5 Phytoalexin synthesis in soybean: purification and characterization of NADPH:2'-hydroxydaidzein oxidoreductase from elicitor-challenged soybean cell cultures; Fischer D et al.; An NADPH:2'-hydroxydaidzein oxidoreductase (HDR) from elicitor-challenged soybean cell cultures was purified to apparent homogeneity by a five-step procedure . The purification procedure included affinity adsorption on Blue Sepharose and elution of the enzyme with NADP+ . It was shown by gel filtration and by sodium dodecyl sulfate-polyacrylamide gel electrophoresis that HDR consists of only one polypeptide, which has a Mr about 34,700 . The pH optimum of the reaction was 7.0 . Apparent Michaelis constants determined for 2'-hydroxydaidzein, 2'-hydroxyformononetin, and NADPH were, respectively, 50, 60, and 56 microM . A low conversion of 2'-hydroxygenistein to the corresponding isoflavanone was also observed but isoflavones lacking a 2'-hydroxyl group and various other flavonoids did not serve as substrates . Enzymatically derived 2'-hydroxydihydrodaidzein gave a positive CD spectrum at 328 nm, which shows its 3R stereochemistry . Antibodies against HDR were raised in rats. Exp Cell Res, 1990 Feb, 186(2), 210 - 7 The impact of maternal serum on development of enolase activity in fetal rat brain cell culture; Shambaugh GE 3rd et al.; The effect of gestational age on serum-mediated changes in enolase activity was tested in a fetal rat brain cell culture . After 6 days exposure to graded concentrations (10 and 20%) of nonpregnant female rat sera, enolase activity in brain cell cultures increased from 2.83 +/- 0.03 to 3.74 +/- 0.19 mumol/min/mg protein, P less than 0.01 . By contrast, similar concentrations of 20-day maternal serum progressively decreased enzyme activity from 1.52 +/- 0.14 to 1.19 +/- 0.08 mumol/min/mg protein . The inhibitory effect was apparent at 14 days gestation and became progressively greater during late gestation to reach a maximum at 20 days . Combining equal concentrations of 20-day pregnant with either nonpregnant or adult male serum neutralized the inhibitory activity . When serum from 20-day pregnant rats was partitioned by a dialysis membrane with a 50,000 MW pore size, inhibitory activity could be similarly neutralized by male or nonpregnant female serum . When 20-day maternal serum was passed successively through filters with a greater than 300,000, 100,000, and 50,000 MW exclusion, the inhibitory activity was apparent in all fractions excluded by a molecular weight of 50,000 . No inhibition was apparent in fractions that were not excluded by 50,000 MW pore size . Inhibition of enolase activity was greatest in the fraction with MW greater than 300,000 . Binding of IGF II could also be demonstrated in this fraction . Binding of IGF II was evident in the fraction greater than 100,000 MW but could not be demonstrated in fractions with a lower molecular weight . The presence of mRNA for IGF II in 20-day fetal rat brain cell cultures was evident when total cellular RNA was analyzed by an RNAase protection assay . It is proposed that a high-molecular-weight component of maternal serum in late gestation can bind endogenously generated IGF II . Such binding, by depleting the necessary growth factors, could inhibit in vitro growth and development of enolase activity. J Immunol, 1990 Feb 1, 144(3), 952 - 9 Mechanism for transforming growth factor beta and IL-2 enhancement of IgA expression in lipopolysaccharide-stimulated B cell cultures; Lebman DA et al.; Transforming growth factor beta (TGF-beta), but not IL-2, causes LPS-stimulated surface (s)IgA- cells to express sIgA . Although there is a progression of sIgA- cells to sIgA+ cells and then to IgA-secreting cells, there is not a parallel change in ratio of membrane to secreted form of alpha-mRNA . In fact, the secreted form of alpha-mRNA is always the predominant form even before the expression of sIgA . However, at least some of the secreted alpha-mRNA transcripts are sterile . The increase in sIgA expression and the induction of sterile transcripts indicate that TGF-beta enhances H chain class switching to IgA as opposed to allowing the growth and maturation of cells precommitted to IgA secretion . The addition of IL-2 to cultures with TGF-beta results in a 5- to 10-fold increase in IgA secretion compared to cultures to which only TGF-beta was added . In these cultures IL-2 increases neither the proportion nor the total number of sIgA+ cells suggesting that IL-2 acts to increase IgA secretion . However, IL-2 does not cause a change in the ratio of secreted to membrane form of alpha-mRNA nor does it lead to an increase in the steady state level of alpha-mRNA comparable to the increase in secreted IgA . Thus, it appears that regulation of transcription of IgA as sIgA- cells proliferate and undergo H class switching and maturation does not follow the same sequence as is seen when sIgM+ cells proliferate and mature to Ig-secreting cells . Furthermore, the data suggest that maturation to high level secretion is controlled posttranscriptionally. Immun Infekt, 1990 Feb, 18(1), 9 - 10 {Antibodies to cell culture cells as an interference factor in chlamydia serology}; Oehme A et al.; Antibodies against Chlamydia trachomatis were detected with the immunoperoxidase assay (IPA) in 32 patients suffering from rheumatic diseases . In this test the cells infected with Chlamydia trachomatis serotype L2 were the antigen . In addition all sera were tested for anticellular antibodies, using non-infected cells as antigen . 13 out of these 32 IPA-positive patients (41%) were also positive with respect to anticellular antibodies. Dtsch Zahnarztl Z, 1990 Feb, 45(2), 82 - 6 {Cell cultures of human osteoclasts for testing biomaterials}; Lambrecht JT; A method for isolating and culturing osteoclast-like cells from cancellous bone material collected from external iliac crest bone of patients is described . Aseptic techniques were used for comminution of the bone material, treatment with collagenase and separation of the bone cells from the bulk bone through a nylon filter . The bone cells were cultured on various surfaces for ten days . Cell motility, mobility and fusion was be observed along with tartrate-resistant acidic phosphatase activity in a majority of the cells soon after they had been cultured . These large cells attached to human cortical bone fragments, where they produced resorption lacunae in vitro . These morphologic and functional characteristics indicate that the cells we had isolated were, in fact, human osteoclasts . SEM studies of these cells on various biomaterials (titanium, hydroxyl apatite, tricalcium phosphate) revealed different morphologic characteristics varying with the substrate used and allowing conclusions as to substrate acceptance . Large areas of cell contact and cell proliferation suggest a favorable response to the materials applied. Neuropathol Appl Neurobiol, 1990 Feb, 16(1), 57 - 68 Pathogenicity of Semliki Forest virus for the rat central nervous system and primary rat neural cell cultures: possible implications for the pathogenesis of multiple sclerosis; Atkins GJ et al.; The neurovirulent L10 strain of Semliki Forest virus (SFV) causes extensive neuronal damage in the central nervous system (CNS) of infected rats, and is probably the cause of death . The avirulent A7 and M9 strains do not cause extensive neuronal damage, but do induce immune-mediated CNS demyelination . In primary CNS cell cultures derived from rats, L10 multiplies more rapidly in neurons than avirulent strains, but infection with both virulent and avirulent strains causes depletion of oligodendrocytes from mixed glial cell cultures . It is proposed that the immune-mediated demyelination, which follows infection with avirulent strains, is induced by phagocytosis of myelin debris from infected oligodendrocytes, and the presentation of antigens derived from such debris to T-helper lymphocytes . Based on these and previous results, a scheme for the pathogenicity of defined strains of SFV is proposed . The applicability of this scheme to the understanding of human demyelinating disease such as multiple sclerosis is discussed. Acta Endocrinol (Copenh), 1990 Feb, 122(2), 255 - 62 Internalization of calcitonin receptors in primary rat kidney cell cultures; Schneider HG et al.; Cultured rat kidney cells possess specific calcitonin receptors and a calcitonin-responsive adenylate cyclase . At 37 degrees C bound 125I-salmon calcitonin becomes increasingly resistant to acid washing . If 125I-salmon calcitonin is removed from the medium after binding, bound hormone decreases over the next 5 h . Monensin, which blocks lysosomal processing, inhibits the decrease of bound hormone . These data indicate that calcitonin receptors are internalized after binding of hormone in kidney cells . Cycloheximide prevents internalization, when it is administered 4 h before 125I-salmon calcitonin binding is studied . Pre-incubation of the cells with 10(-7) mol/l unlabelled salmon calcitonin decreases specific binding; recovery of binding needs 48 h to occur . The long time interval for recovery makes it unlikely that calcitonin receptors recycle . This is the first demonstration that normal rat kidney cells internalize calcitonin after binding . It might contribute to the loss of calcitonin bioactivity which is seen after continuous administration. J Med Virol, 1990 Feb, 30(2), 97 - 102 Detection of human cytomegalovirus early and late antigen and DNA production in cell culture and the effects of dimethyl sulfoxide, dexamethasone, and DNA inhibitors on early antigen induction; Li SB et al.; Recently a conventional method for the laboratory diagnosis of human cytomegalovirus (HCMV) infection was improved by using centrifugation culture to enhance viral adsorption and by detecting HCMV early antigen and DNA . Comparison of the sensitivity of three rapid methods using commercial diagnostic reagents for the detection of HCMV early antigen (EA), late antigen (LA), and DNA was quantitatively evaluated in centrifugation cultures of human fibroblast cells (MRC-5) infected with HCMV . HCMV-EA was first detected 4 hours after infection, and the number of antigen-positive cells increased rapidly thereafter . Using biotinylated DNA probe, viral DNA was first detected 12 hours postinfection; the number of DNA-positive cells increased slowly . HCMV-LA was first seen 48 hours postinfection, and the number of LA-positive cells also increased thereafter . Thus detection of HCMV-EA was the most rapid and sensitive method for HCMV diagnosis . Several chemical compounds have been used to enhance HCMV replication . The effect of dimethyl sulfoxide (DMSO), dexamethasone (DEX), 5-bromo-2-deoxyuridine (BrdU), 5-fluoro-deoxyuridine (FdU), and cytosine arabinoside (Ara-C) on HCMV-EA induction was evaluated in centrifugation cultures of MRC-5 cells infected with HCMV . Infected cells treated with 1% DMSO alone or with DMSO plus DEX (10(-5) M) have been shown to increase the number of HCMV-EA-positive cells three- to fivefold over the untreated control cultures . The enhancing effects of Ara-C, BrdU, and BrdU plus FdU were demonstrated only occasionally. J Endocrinol, 1990 Feb, 124(2), 255 - 60 Effect of high-density lipoprotein on bovine granulosa cells: progesterone production in newly isolated cells and during cell culture; O'Shaughnessy PJ et al.; It has been proposed that changes in steroidogenesis which occur during early development of the corpus luteum may be due to increased availability of lipoproteins . Bovine follicular fluid, however, contains significant amounts of high-density lipoprotein (HDL), and granulosa cells are exposed to this lipoprotein before ovulation . To determine whether bovine granulosa cells can utilize HDL the effects of this lipoprotein on freshly isolated, non-luteinized granulosa cells and on granulosa cells undergoing luteinization in serum-free culture were examined . Cells were isolated from non-atretic, antral follicles and cultured for 12 h in 10% (v/v) lipoprotein-deficient serum to allow cell attachment . After this time cells were cultured in serum-free medium . During culture the cells underwent functional luteinization as assessed by an increase in basal progesterone output (9.6-fold in 7 days) which was associated with a marked increase in activity of cholesterol side-chain cleavage and loss of aromatase activity . Dibutyryl cyclic AMP (dbcAMP) increased basal production of progesterone about twofold but HDL alone had no effect . Addition of HDL plus dbcAMP, in contrast, caused a very marked stimulation (up to ten times) of basal steroidogenesis . This trophic effect of HDL and dbcAMP lasted at least 2 weeks . Activity of cholesterol side-chain cleavage was stimulated (threefold over basal) by dbcAMP during culture but HDL was without effect, alone or with dbcAMP . Addition of HDL (in the presence or absence of dbcAMP) to freshly isolated granulosa cells had no significant stimulatory effect on progesterone production over 12 h in six experiments, and in two of these experiments a significant inhibitory effect was seen.(ABSTRACT TRUNCATED AT 250 WORDS) Mol Pharmacol, 1990 Feb, 37(2), 137 - 43 Benzo(a)pyrene inhibits epidermal growth factor binding and receptor autophosphorylation in human placental cell cultures; Guyda HJ et al.; Studies investigated the effects of benzo(a)pyrene (BP) treatment on epidermal growth factor (EGF) receptor binding and kinase activity in human placental cell cultures . Specific binding of 125I-EGF to cells from early gestation placentae was significantly decreased by 37 and 60% following exposure to 1 and 10 microM BP, respectively, for 24 hr . In contrast, cells cultured from term placentae showed no inhibitory effect of either concentration of BP . Specific binding of 125I-labeled insulin and insulin-like growth factors-I and -II to early gestation cells was decreased only 15-18% at 10 microM BP, which indicates that loss of membrane receptors appears to be selective for EGF . Scatchard analysis of early gestation cells revealed that BP was associated with a dose-dependent loss in the number of high affinity EGF binding sites . Evidence from cross-linking and autophosphorylation experiments confirmed that the Mr 170,000 binding protein was decreased in a dose-dependent manner following BP treatment . In comparison, term placental cells exhibit a 26% loss of EGF receptor autophosphorylation without alteration in binding following exposure to 10 microM BP . Thus, early gestation cells exhibit a BP-related down-regulation of EGF receptors, whereas term placental cells show receptor desensitization . No adverse effect of BP treatment was observed on the incorporation of {35S} methionine into proteins secreted by early gestation cells . Further experiments compared the effects of BP with the related poly-cyclic compounds beta-naphthoflavone, alpha-naphthoflavone, and 3-methylcholanthrene . In early gestation cells, EGF binding and receptor autophosphorylation were measurably decreased at 10 microM concentrations of these polycyclic compounds, but to a lesser extent than observed with BP . In term placental cells, however, EGF binding was unchanged or increased, whereas receptor autophosphorylation was decreased 10-26% . Thus, exposure of term placental cells to these polycyclic compounds leads to a dissociation between EGF binding and receptor protein kinase activity . Finally, aryl hydrocarbon hydroxylase activity was induced 20- to 200-fold in early placental cells exposed to BP, beta-naphthoflavone, and 3-methylcholanthrene . In summary, the direct effects of BP and related compounds observed on placental EGF receptors may indicate altered function of EGF in the regulation of cell growth and differentiation in the human placenta. Mol Endocrinol, 1990 Feb, 4(2), 349 - 55 Follicle-stimulating hormone regulation of androgen-binding protein messenger RNA in sertoli cell cultures; Hall SH et al.; FSH plays an important role in testicular Sertoli cell differentiation and function in spermatogenesis . Previous studies using rat androgen-binding protein (ABP) as a marker of FSH action on Sertoli cells have demonstrated in vivo and in vitro regulation of ABP . We now have extended these studies to examine FSH regulation of ABP mRNA using Northern blot hybridization . In the immature rat testicular ABP mRNA {1.7- and 2.3-kilobase (kb) species} increased with age and reached a maximum 20 days postpartum, coincident with an increased plasma FSH concentration . To determine the direct effect of FSH on Sertoli cells, we examined ABP mRNA in vitro . In Sertoli cell-enriched cultures FSH was found to maintain the major 1.7-kb ABP RNA transcript level over 5 days of treatment in a dose-dependent manner, whereas in the absence of FSH, ABP mRNA declined with time in culture . The ABP mRNA maintenance by FSH was accompanied by higher concentrations of secreted immunoreactive ABP, which declined in the absence of FSH . This FSH effect on ABP mRNA and secreted ABP was mimicked by the cAMP analog (Bu)2cAMP . After the decline of ABP mRNA during culture, administration of FSH did not result in a detectable increase in the 1.7-kb ABP mRNA within 3 days, wherea