Clin Endocrinol (Oxf), 1983 Sep, 19(3), 325 - 36 Gonadotrophin and alpha subunit secretion by human 'functionless' pituitary adenomas in cell culture: long term effects of luteinizing hormone releasing hormone and thyrotrophin releasing hormone; Surmont DW et al.; The long-term effects of LHRH and TRH on gonadotrophin alpha subunit, FSH and LH secretion by cell cultures of four human chromophobic pituitary tumours have been examined . The tumours derived from one male and three female patients who presented because of visual disturbance but had no evident endocrine symptoms . Subsequent serum hormone analysis showed the FSH to be high in the male but low or normal in the post-menopausal females whereas LH levels were low in all patients . In culture, basal hormone secretion could be maintained for periods up to 63 d . All tumours secreted alpha subunit and FSH, but much lower amounts of LH . Addition of LHRH or TRH for a period of 12 to 41 d showed that alpha subunit, FSH and LH secretion were stimulated by LHRH from one tumour, by LHRH and TRH from two tumours . There was always a rapid decline in the LH secretion . The tumour which secreted FSH predominantly was stimulated by TRH . We conclude that human pituitary 'functionless' adenomas can secrete gonadotrophin alpha subunit and FSH in vitro and that secretion can be stimulated during long term releasing hormone experiments . LH secretion, however, cannot be maintained. J Neurosci, 1983 Sep, 3(9), 1818 - 23 Effect of depolarizing agents on choline acetyltransferase and acetylcholinesterase activities in primary cell cultures of spinal cord; Ishida I et al.; In cultured neurons dissociated from the spinal cord of fetal mouse, high concentrations of KCl (47 mM) increased choline acetyltransferase (CAT) activity up to 5.5-fold but suppressed acetylcholinesterase (AChE) activity to less than half the level of control cells . Veratridine (3 microM) also increased CAT activity 1.6-fold and suppressed AChE activity to the same level as that induced by high KCl . The increase of CAT activity by the depolarizing agents was blocked by Ca2+ antagonists (verapamil and high Mg2+) and in a low Ca2+ medium, whereas the suppression of AChE activity by high KCl was restored by the same procedures . The synthesis of radiolabeled acetylcholine from {14C}choline was also enhanced 4-fold by incubating cells in high KCl medium . Although the uptake of L-{3H}leucine and {14C}choline into the cells was slightly enhanced by high KCl medium, neither the total amount of protein nor the incorporation of L-{3H}leucine into protein was increased by high KCl medium . These observations indicate that depolarization increased CAT activity in a specific manner, that the activities of CAT and AChE changed inversely under several conditions, and that the effect of depolarization presumably was mediated by the entry of Ca2+ into neuronal cells . The findings raise the possibility that trans-synaptic input could play a crucial role in the development of the activity of cholinergic neurons in spinal cord. Eur J Biochem, 1983 Aug 15, 134(3), 547 - 54 Induction and characterization of a microsomal flavonoid 3'-hydroxylase from parsley cell cultures; Hagmann ML et al.; A microsomal preparation from irradiated parsley cell cultures catalyses the NADPH and dioxygen-dependent hydroxylation of (S)-naringenin {(S)-5, 7, 4'-trihydroxyflavanone} to eriodictyol (5, 7, 3', 4'-tetrahydroxyflavanone) . Dihydrokaempferol, kaempferol, and apigenin were also substrates for the 3'-hydroxylase reaction . In contrast prunin (naringenin 7-O-beta-glucoside) was not converted by the enzyme . The microsomal preparation, which also contains cinnamate 4-hydroxylase, did not catalyse hydroxylation of 4-coumaric acid to caffeic acid . 3'-Hydroxylase activity is partially inhibited by carbon monoxide in the presence of oxygen as well as by cytochrome c and NADP+ . These properties suggest that the enzyme is a cytochrome P-450-dependent flavonoid 3'-monooxygenase . Pronounced differences in the inhibition of flavonoid 3'-hydroxylase and cinnamate 4-hydroxylase were found with EDTA, potassium cyanide and N-ethylmaleimide . Irradiation of the cell cultures led to increase of flavonoid 3'-hydroxylase activity with a maximum at about 23 h after onset of irradiation and subsequent decrease . This is similar to light-induction of phenylalanine ammonialyase and cinnamate 4-hydroxylase . In contrast, treatment of the cell cultures with a glucan elicitor from Phytophthora megasperma f . sp . glycinea did not induce flavonoid 3'-hydroxylase nor chalcone isomerase but caused a strong increase in the activities of phenylalanine ammonia-lyase, cinnamate 4-hydroxylase, and NADPH--cytochrome reductase . The results prove that flavonoid 3'-hydroxylase and cinnamate 4-hydroxylase are two different microsomal monooxygenases. J Neurosci, 1983 Aug, 3(8), 1634 - 47 Morphological classification of rat cortical neurons in cell culture; Kriegstein AR et al.; Neurons in "mature" (4- to 6-week-old) dissociated cell cultures of 15-day gestational age rat fetal cortex were injected with Lucifer Yellow in order to compare their detailed morphological features with those of cortical neurons in situ, and in order to determine which features of cellular morphology were dependent on local environmental conditions . Neurons were characterized by their cell form (pyramidal, multipolar, fusiform, etc.), dendritic branching pattern, spine density, and axonal projections . The neurons in culture appeared to display all the morphological features seen in cortical neurons in situ . These characteristics appeared to be independent of whether an individual neuron grew in a dense or sparse region of the culture . In addition, examination of neurons during early differentiation indicated that many of their morphological features developed as soon as the neurons could be recognized and before extensive synapse formation occurred. J Immunol, 1983 Aug, 131(2), 869 - 76 Localized elaboration of IgA in normal and neoplastic murine mammary cell cultures: relationship to fibronectin matrix; Myrdal SE et al.; IgA secretion by the lactating mammary gland culminates a complex sequence of biologic events both within the gland and at distant sites . Because of the many extraglandular influences, it is difficult to investigate IgA secretion at the tissue and cellular levels in the intact animal . In this study, with the use of immunohistofluorescence, we have observed elaboration of IgA by primary monolayer cultures of mammary cells from the glands of mid-pregnant mice and from mammary tumors . In cultures of normal cells, IgA appeared first in vesicular structures on the upper surfaces of the monolayers . Vesicular IgA was maximal at day 5 in culture, and at that time, was observed only over mounds (domelike structures) that were covered with fibrillar fibronectin (FN), and eventually developed a subpopulation of fusiform cells . It appears that the IgA observed was secreted in vitro, that normal mammary epithelial cells must form multicellular FN-bearing structures to secrete IgA in culture, and that by mid-pregnancy the murine mammary gland contains all of the lymphoid cells required for IgA secretion . In contrast, primary cultures of mammary tumors displayed minimal amounts of IgA and FN . The small amount of cell-associated IgA that was detected on tumor cultures was not localized to any multicellular structures nor was it associated with FN, but instead appeared as minute, punctate accumulations on individual cells scattered across the epithelioid areas . This finding is consistent with the loss of specialized functions and the increased autonomy typical of malignant cells . The study in cultured cells of a function as specialized as IgA secretion should permit greater understanding of both the process itself and the despecializations that accompany malignancies of secretory epithelia. J Cell Physiol, 1983 Aug, 116(2), 221 - 6 Isozymes of creatine kinase in mammalian cell cultures; Van Brussel E et al.; Previous studies on the energy metabolism of rat myocardial cells in culture supported the hypothesis that the creatine-phosphocreatine-creatine kinase system plays an important role in the intracellular transport of energy from the mitochondria to the myofibrils and in the regulation of energy production coupled to energy utilization in this model system . Effective functional compartmentation of ATP could result from the binding of creatine kinase to cellular organelles (e.g., myofibrils and mitochondria) such that high energy charge at the myofibrils is maintained by the reverse creatine kinase reaction, while phosphocreatine is synthesized mainly at the mitochondria in the forward creatine kinase reaction . It was, therefore, essential to demonstrate the presence of mitochondrial creatine kinase in the cultured myocardial cells to support this hypothesis, particularly since the mitochondrial creatine kinase was reportedly absent in fetal hearts . Using electrophoresis on cellulose acetate strips, the mitochondrial creatine kinase isozyme, as well as MM, MB, and BB isozymes, have now been demonstrated in myocardial cultures derived from neonatal rats . The mitochondrial creatine kinase increased with age in culture and with age of animal from which the culture is derived . Furthermore, the addition of creatine to culture media stimulates its synthesis . The mitochondrial creatine kinase isozyme was not detected in nonmuscle cells in culture derived from the neonatal rat hearts, nor in L6 muscle cell line . Phosphocreatine was present in all cells, but the regulation of energy metabolism and energy shuttle by creatine-phosphocreatine-creatine kinase could be operative only in the cells where the mitochondrial creatine kinase is present . This regulatory mechanism provides for an efficient system concomitant with the continuous energy demand of the myocardium; it is not ubiquitous and its development in myocardial cells seems to be triggered postnatally. J Cell Biol, 1983 Aug, 97(2), 489 - 98 Cytoskeletal organization of the presynaptic nerve terminal and the acetylcholine receptor cluster in cell cultures; Peng HB; Whole-mount stereo electron microscopy has been used to examine the cytoskeletal organization of the presynaptic nerve terminal and the acetylcholine receptor (AChR) clusters in cultures of Xenopus nerve and muscle cells . The cells were grown on Formvar-coated gold electron microscope (EM) finder grids . AChR clusters were identified in live cultures by fluorescence microscopy after labeling with tetramethylrhodamine-conjugated alpha-bungarotoxin . After chemical fixation and critical-point drying, the cytoplasmic specializations of identified cells were examined in whole mount under an electron microscope . In the presynaptic nerve terminal opposite to the AChR cluster, synaptic vesicles were clearly suspended in a lattice of 5-12-nm filaments . Stereo microscopy showed that these filaments directly contacted the vesicles . This lattice was also contiguous with the filament bundle that formed the core of the axon . At the AChR cluster, an increased cytoplasmic density differentiated this area from the rest of the cytoplasm . This density was composed of a meshwork of filaments with a mean diameter of 6 nm and irregularly shaped membrane cisternae 0.1-0.5 micron in width, which resembled the smooth endoplasmic reticulum . These membrane structures were interconnected via the filaments . Organelles that were characteristic of the bulk of the sarcoplasm such as the rough endoplasmic reticulum and the polysomes, were absent from the cytoplasm associated with the AChR cluster . These results indicate that the cytoskeleton may play an important role in the development and/or the maintenance of the neuromuscular synapse, including the release of transmitter in the nerve terminal and the clustering of AChRs in the postsynaptic membrane. Teratology, 1983 Aug, 28(1), 109 - 22 Detection of teratogens in the Drosophila embryonic cell culture test: assay of 100 chemicals; Bournias-Vardiabasis N et al.; An in vitro assay of teratogenesis has been developed that utilizes Drosophila embryonic cell cultures . The endpoint selected in assessing the teratogenic potential of any substance involves detection of interference with normal muscle and/or neuron differentiation . In the validation phase of this project, 100 chemicals were tested . With drugs for which extensive reliable mammalian data are available, the results in the Drosophila assay equate rather favorably with those observed in animals and humans (i.e., a low percentage of false positives and false negatives has been obtained) . In an effort to determine if strain differences exist and also to establish that the system shows a dose response, cultures from three wild-type Drosophila strains (Canton S, Canton S109, and Oregon R) were tested . Dose-response differences were observed when diethylstilbestrol, diphenylhydantoin, imipramine, testosterone, and tolbutamide were added to the cultures . These results suggest that the Drosophila assay, with further testing and refinements, might be capable of identifying agents of high teratogenic potential by their effect on neurons and muscle differentiation . Furthermore, sensitive strains might be used to study mechanisms of abnormal development and gene involvement in teratogenic resistance. Biophys J, 1983 Aug, 43(2), 183 - 90 Transepithelial transport in cell culture . A theoretical and experimental analysis of the biophysical properties of domes; Tanner C et al.; Dissociated cells of transporting epithelia, when cultured on an impermeant substratum, form polarized monolayers frequently characterized by the presence of domes . If the assumption is made that the monolayer exhibits a uniform stretch modulus of elasticity and tension of cell-dish adhesion, Ta, then biophysical properties of the epithelium can be predicted . We have shown that for such epithelia, domes should (a) have circular bases, (b) be sections of spheres with a constant height to radius, h/r, ratio, (c) have a dome-wall tension, Tw, that is constant, and (d) have a dome volume that is a function of radius alone . Additionally, a Laplace equation derived for this geometry predicted the hydrostatic pressure from within to outside domes as a decreasing function of radius alone . By microscopy, domes had predominantly circular bases and were found to be sections of spheres with a constant height, h, to radius, r, ratio of 0.684 . Using the Laplace equation derived for this geometry and measurements of delta P and r, the tension of cell-dish adhesion, Ta, and dome-wall tension, Tw, were found to be constants of 6.60 and 7.08 torr, respectively . Combining the constants for Ta and h/r ratio, and the fact that domes are sections of spheres, delta P and dome volume were shown to be known functions of radius alone . In addition, the modulus of elasticity of the epithelium was calculated to be 4.82 X 10(3) dyn/cm2. Br J Exp Pathol, 1983 Aug, 64(4), 354 - 60 Fractionation of human tumour-associated cells by centrifugal elutriation to establish lymphocyte, macrophage and tumor cell cultures from a single tumour biopsy; Moore K et al.; The application of the Beckman centrifugal elutriator system for the simultaneous isolation of lymphocytes, macrophages and tumour cells from enzyme disaggregated tumour-associated cells or from peritoneal aspirates is described . Using this technique we have been able to prepare cultures of these cells from single human tumour biopsies . Overall cellular recovery was always in excess of 70% and the purity of cell fractions varied between 89 and 99% . Lymphocyte fractions were found to be free of tumour cell contamination and this allowed lymphocyte cultures in T-cell growth factor containing medium to be established . Using this approach it should prove possible to investigate the clonal effector functions of tumour infiltrating lymphocytes against autologous and allogeneic tumour cells. Exp Hematol, 1983 Aug, 11(7), 601 - 10 Variation amongst K562 cell cultures; Dimery IW et al.; K562 cell cultures were obtained from three laboratories (A, B and C) outside our institution, and were designated according to source as K562A, K562B or K562C . The cultures obtained were constitutive or "wild type" K562 cell cultures, not cloned sublines . These cell cultures were compared with respect to growth kinetics, cell surface protein markers, surface antigens, cytogenetics and hemoglobin production . Morphology, growth kinetics in liquid suspension culture, cloning efficiency in soft agar culture, binding of anti-K562 monoclonal antibodies, and the majority of cell surface proteins were generally similar . In contrast, several important differences were observed: (1) hemoglobin synthesis induced by hemin was significantly different among K562A, B and C, K562A being most sensitive (P less than 0.05); (2) whereas more than 90% of K562A or C cells appeared to be Philadelphia chromosome (Ph1)-positive, less than 15% of K562B cells contained a Ph1; (3) membrane proteins (93 and 85 kilodalton) were identified in K562A, whereas only the 93 kilodalton protein was detected in K562B and neither of the proteins were detected in K562C . Our results indicate that K562 cells maintained in different laboratories can undergo tangible changes which may influence experimental results obtained in studies using these cells. Am J Vet Res, 1983 Aug, 44(8), 1456 - 9 Cell culture-derived Babesia bovis vaccine: sequential challenge exposure of protective immunity during a 6-month postvaccination period; Kuttler KL et al.; Cell culture-derived soluble Babesia bovis vaccine admixed with a saponin adjuvant was administered in 2 doses, 3 weeks between doses, to 18 yearling heifers . An additional 9 heifers served as nonvaccinated controls . Comparable groups of these animals were challenge exposed at 131 and 178 days after vaccination, using 1 X 10(8) B bovis organisms contained in freshly collected blood from a splenectomized calf with ascending parasitemia . Challenge exposure led to no deaths of vaccinated animals, whereas 4 of the control (nonvaccinated) animals died of disease . All clinical and hematologic variables examined indicated that the vaccinated animals had gained immunologic protection in comparison with the control group . An immune recognition as manifested by an anamnestic humoral response to challenge exposure was in evidence in the vaccinated animals. Neuroendocrinology, 1983 Aug, 37(2), 111 - 6 Neonatal rat hypothalamus cell culture: neuron subpopulations secrete immunoreactive beta-endorphin but not immunoreactive ACTH; Lolait SJ et al.; Primary cultures of dissociated hypothalamic cells were prepared from 1-day-old rat neonates . Studies with cell-specific antisera revealed the presence of neurons, glial cells, oligodendrocytes and fibroblast-like cells . By immunohistochemistry, two morphologically distinct cells in culture were positive for immunoreactive beta-endorphin (IR-beta-EP) . The medium derived from these cultures contained radioimmunoassayable IR-beta-EP, but not IR-ACTH . These data suggest that, in rat neonatal hypothalamic cultures, two subpopulations of cells exist which store and secrete IR-beta-EP, but not IR-ACTH. Am J Vet Res, 1983 Aug, 44(8), 1588 - 92 Virus-induced interferon production in canine lymphoid cell cultures; Tsai SC et al.; Virus-induced interferon (IFN) production in canine lymphoid cells was studied, using Newcastle disease virus as principal inducer . It was found that spleen cells at a concentration of 5 X 10(6) cells/ml with Newcastle disease virus at a multiplicity of infection of 1, incubated at 37 C in 5% CO2 for 24 hours, produced highest titers of IFN . Among the lymphoid cells from different tissues, IFN production was in the order of spleen = bone marrow greater than thymus greater than mesenteric lymph node greater than or equal to peripheral blood lymphocytes . Macrophages did not produce IFN, and virus-induced IFN production in spleen cells did not depend on the presence of phagocytic mononuclear cells . These optimal conditions and macrophage independence are different from those of mitogen-induced IFN production in canine lymphoid cells. J Rheumatol, 1983 Aug, 10(4), 595 - 601 Scleredema adultorum of Buschke: a clinical, pathologic, and cell culture study of two patients; Christy WC et al.; Two patients with scleredema of Buschke are presented . The second patient developed scleredema after a febrile drug reaction . Biopsies of both involved and uninvolved skin were obtained for histologic examination and cell culture studies . Immunofluorescent studies of the biopsy specimens revealed staining with IgG, IgM and C3 at the dermal-epidermal junction in the involved skin of the first patient . This has not been previously reported . Cell culture studies revealed that fibroblasts from the involved skin produced more glycosaminoglycans (GAG) than uninvolved skin and most of the GAG produced was hyaluronic acid . Possible pathogenic mechanisms for this unusual condition are discussed. In Vitro, 1983 Aug, 19(8), 642 - 50 Human retinal pigment cell culture; Aronson JF; Human retinal pigment epithelium (RPE)-derived cell lines were established from RPE-covered choroid tissue fragments, which had been generated by culture on nontissue culture plastic . Two phenotypes were apparent in a given line: (a) a compact cell which formed domes and ultimately melanosomes before being sloughed; and (b) a squamous cell which was often elongated and which bound antibody to human keratins . This latter cell did not become black or form domes . The average number of cell doublings for the 13 lines tested was between 15 and 40 when cultured in a modified Eagle's minimum essential medium containing 10% fetal bovine serum . Cell lines newly established from material that had been in culture for more than 6 months had normal mitotic chromosomes and still developed areas with strongly pigmented cells when refed . Normal human epithelial cell lines of this kind may be useful in studies of cell aging and defining change associated with the development of neural cells from ectoderm. Clin Endocrinol (Oxf), 1983 Aug, 19(2), 193 - 206 Bioassay of thyroid-stimulating immunoglobulins using human thyroid cell cultures: optimization and clinical assessment; Bidey SP et al.; Primary monolayer cultures of human thyroid cells have been used to investigate the intracellular cyclic AMP response to thyroid-stimulating immunoglobulins (TSIg) prepared from the sera of patients with Graves' disease . In particular, attention was directed towards dose-response characteristics obtained under differing incubation conditions, and an optimized incubation procedure based upon conditions consistent with the maximal precision of TSIg measurement was developed . The magnitude of the cyclic AMP response to a tested TSIg preparation was shown to be greatest after the longest incubation period investigated, 16 h, for all doses of TSIg investigated . The greatest precision of TSIg measurement, as determined by the lowest relative error, was obtained at an immunoglobulin (Ig) dose of 1 mg/ml, and when incubated at this dose level for 16 h, all Ig-enriched fractions prepared from the sera of 28 newly-diagnosed patients with Graves' disease significantly (P less than 0.05) raised the intracellular cyclic AMP level of thyroid cell monolayers . After thionamide drug therapy, the incidence of TSIg detection declined to 42% (5 of 12 patients) . TSIg bioactivity was also found in 9 of 14 (64%) patients with euthyroid exophthalmos and in 4 of 11 (36%) cases of non-toxic goitre . In contrast, Ig's prepared from the sera of normal euthyroid volunteers were devoid of TSIg bioactivity. Virology, 1983 Jul 30, 128(2), 310 - 8 Multiple genetic variants arise in the course of replication of foot-and-mouth disease virus in cell culture; Sobrino F et al.; The genetic heterogeneity generated upon passage of foot-and-mouth disease virus (FMDV) in cell culture has been evaluated by T1-oligonucleotide fingerprinting of genomic RNA . Plaque-purified FMDV O-S7 and C-S8 were propagated by serial low multiplicity infections of BHK-21 (c-13) or IBRS-2 (c-26) cells . In independent parallel passage of the same virus, different oligonucleotide variations were fixed in the RNAs . T1-oligonucleotide fingerprinting of RNA from 34 individual viral clones derived from two passaged populations shows an extensive heterogeneity, with some mutations present in only one of the cloned genomes analyzed . Some FMDV variants are phenotypically distinct in that they yield increased progeny in infections of cell monolayers . From the number of variant sequences it can be estimated that each infectious RNA in the population differs in two to eight mutations from the average parental sequence . Thus, passaged FMDV populations consist of a pool of variants, an observation previously made with phage Q beta (E . Domingo, D . Sabo, T . Taniguchi, and C . Weissmann, Cell 13, 735-744, 1978) . The FMDV genome must be described as a fluctuating distribution of sequences due to its high mutability . This may be the basis of the extensive genetic and antigenic diversity of this virus in nature. Neurosci Lett, 1983 Jul 29, 38(2), 181 - 6 Neonatal retinal ganglion cell cultures of high purity: effect of superior colliculus on their survival; Armson PF et al.; A method is described for obtaining retinal ganglion cell (RGC) cultures of high purity . RGC were retrogradely labelled in vivo with either the fluorescent dye True Blue and horseradish peroxidase (HRP), or with FITC-conjugated HRP . Following dissociation, RGC were separated from the intrinsic cells of the retina using fluorescent activated cell sorters, on the basis of their greater size and fluorescent intensity . When the sorted cells were cultured, RGC could be subsequently identified by their HRP labelling . Using such criteria, cultures in which 75% of the cell population consisted of RGC could be regularly obtained . This represents a 150-fold increase in RGC concentration over whole retinal cultures . The survival of the sorted RGC could be greatly enhanced when they were cocultured with their target tissue, the superior colliculus. In Vitro, 1983 Jul, 19(7), 576 - 88 Repeated establishment of diploid epithelial cell cultures from normal and partially hepatectomized rats; Herrinig AS et al.; A number of liver epithelial cell cultures were established from 10 to 12-d-old sucklings, 6 to 8-wk-old young adults, or from 2 to 18-month-old partially hepatectomized rats . Improvements in the methods for cell isolation and culture yielded replicating cells from every experiment and they were maintained for different periods with regular passages . The proliferative potential in vitro of adult rat liver cells could be increased if the rats were subjected to partial hepatectomy before cell isolation . In the early passages, the majority of the cells were found to have a true diploid karyotype as studied by the Giemsa-banding technique . Under the culture conditions described, a very high percentage of cells remained in the diploid range for, in most cases, at least 4 months and in some cases for up to 6 months . Afterward, the karyotype was unstable, but no "crisis" period was seen before the cells became aneuploid . Until this time, the growth characteristics of the cells also followed a normal pattern showing density dependent inhibition of division and a lack of markers associated with malignancy . The cultured liver cells exhibited a number of liver specific properties when they were maintained as a confluent monolayer . The early passages of diploid epithelial cell cultures derived from normal and regenerating rat liver are good models for studies of the regulation of cell division and the changes that are related to carcinogenesis. Exp Lung Res, 1983 Jul, 5(1), 37 - 48 Differentiation-arrested rat fetal lung in primary monolayer cell culture . I . Development of a differentiation-arrested and growth-supporting culture system using carbon-stripped bovine fetal calf serum; Tanswell AK et al.; In vitro observations of fetal lung maturation are complicated by continued epithelial cell maturation in culture . Removal of trace quantities of cortisol from the culture system has been reported elsewhere by Torday to prevent epithelial cell differentiation, but also prevent cell growth . In this report we have developed monolayer cell cultures from 18-, 19-, 20-, and 22-day-gestation fetal rat lung in a culture system stripped of steroid and thyroid hormones . These cultures demonstrate lamellar body development comparable to that seen in vivo at the gestational age at which cultures were developed; the gestation-dependent increase in precursor incorporation into saturated lecithin observed in these cultures is similar to that reported in in vivo studies, yet cell growth in culture has been preserved. J Neurochem, 1983 Jul, 41(1), 291 - 4 Induction of a stress protein in developing cell cultures of the rat cerebellum; Pearce BR et al.; We examined the ability of developing cerebellar cell cultures to synthesize a 71,000 MW stress protein (SP71) in response to heat shock and Cd2+ treatment . The induction of SP71 synthesis appeared to be dependent on both the age of the culture and the stressor used . Heat shock induced SP71 synthesis in freshly prepared cells and in cell cultures at each age examined, whereas Cd2+ was effective only in cultures at 7 days of age and older . These findings are discussed with reference to the development of various cell types in these cultures. Neurochem Res, 1983 Jul, 8(7), 847 - 52 Growth of neural cell cultures in a chemically defined, serum-free culture medium; Kumar S; The composition of a serum-free, completely chemically defined culture medium which supports active growth of dissociated neural-cells in culture is described . This serum-free medium can also be used to grow many types of human cell lines without modification . It is the first report which describes the development of a wholly chemically defined, synthetic culture medium for growth of neural cells. Q J Exp Physiol, 1983 Jul, 68(3), 329 - 35 Serotonin, and mouse spinal neurones in cell culture; Green KA et al.; Two different responses to serotonin have been observed . One response was a depolarization accompanied by a decrease in membrane conductance . This response was enhanced at depolarized potentials and reduced at hyperpolarized potentials; the apparent conductance change was also reduced at hyperpolarized potentials indicating some voltage sensitivity of the response . The other response was a depolarization accompanied by an increased membrane conductance . The response was enhanced at hyperpolarized potentials and reversed to a hyperpolarization at -35 to -60 mV . The total number of responsive neurones was small (5%) . This might be explained by a deficiency of serotonergic input to the recorded cells, since it was shown autoradiographically that very few neurones in the cultures used exhibited a specific high-affinity uptake for the transmitter, and hence probably contained it. Microbiologica, 1983 Jul, 6(3), 199 - 205 Enhancement of the spontaneous development of autoreactive B cells by PPD in mouse peritoneal cell cultures; Campa M et al.; The effect of tuberculin purified protein derivative (PPD) on the development of plaque-forming cells (PFC) against bromelain-treated syngeneic mouse red blood cells (Br-MRBC) was studied in peritoneal cell cultures . The finding that PPD enhances the development of PFC to Br-MRBC, even under conditions where cell division is blocked by mitomycin C treatment, suggests that cell proliferation does not represent a necessary prerequisite for differentiation of precursor cells into autoantibody-forming cells. J Steroid Biochem, 1983 Jul, 19(1A), 235 - 9 5 alpha-dihydrotestosterone formation and its functional significance in rat anterior pituitary, subpopulations of gonadotrophs and cell cultures; Denef C; The present paper reviews the work in the author's laboratory on the conversion of testosterone to 5 alpha-reduced metabolities and its regulatory control in intact pituitary, in enzymatically dispersed pituitary cells and in enriched populations of gonadotrophs isolated by unit gravity sedimentation . The data show that 5 alpha-reductase is located mainly in gonadotrophs and the possible physiological significance is discussed . A new culture method, suitable to study the regulation of 5 alpha-reductase and its biological significance in vitro, is presented. Biull Eksp Biol Med, 1983 Jul, 96(7), 83 - 6 {Induction by mycotoxins of somatic mosaicism in Drosophila and DNA repair in mammalian liver cell cultures}; Belitskii GA et al.; The genotoxic activity of four mycotoxins has been studied . High level of somatic mutagenesis in imaginal discs of Drosophila melanogaster larvae and DNA repair synthesis in human embryo and adult rat liver cell cultures were inducible only by highly carcinogenic aflatoxin B1 . Patulin, a weak direct-action carcinogenic substance, slightly elevated the mutagenesis in somatic cells of Drosophila but did not induce DNA repair synthesis in liver cell cultures . Citrinin that did not exhibit any carcinogenic properties when used alone and stachybotrotoxin with non-reported carcinogenic activity appeared inactive in the test-systems applied . The possibilities of rapid recognition of carcinogenic mycotoxins by detecting their genotoxic properties are discussed. Cancer Res, 1983 Jul, 43(7), 3212 - 8 Involvement of both syn- and anti-dihydrodiol-epoxides in the binding of 7, 12-dimethylbenz(a)anthracene to DNA in mouse embryo cell cultures; Sawicki JT et al.; 7,12-Dimethylbenz(a)anthracene (DMBA):deoxyribonucleoside adducts, from enzymic hydrolyses of DNA from mouse embryo cells exposed in culture to {3H}DMBA, can be separated into two fractions on the basis of whether or not they bind to the phenyl boronic acid residues of Servacel DHB . This suggests that these two fractions of adducts are derived from anti and syn bay-region dihydrodiol-epoxides, respectively . The fluorescence spectra and interactions of the major components of these two fractions with borate ions substantiate this interpretation . These findings indicate that both syn- and anti-dihydrodiol-epoxides make a substantial contribution to DMBA binding to DNA in mouse embryo cells . For a given mouse embryo cell preparation, the relative contributions of each of these dihydrodiol-epoxides to DNA binding did not vary substantially with DMBA dose, with incubation time with DMBA, or in growing versus confluent cultures, although there were differences between one cell preparation and another. Can J Comp Med, 1983 Jul, 47(3), 352 - 7 Effects of cortisone, cytochalasin B and cycloheximide on strains of Chlamydia psittaci in cell cultures; Tessler J; Vero and McCoy cell cultures were tested for their susceptibility to Chlamydia psittaci in the presence of several antimetabolites, such as cortisone acetate, cytochalasin B and cycloheximide . Vero cells were more susceptible than the McCoy cell cultures as demonstrated by cytopathic changes, fluorescent antibody activity, and titer of C . psittaci . Although all of the antimetabolites increased these parameters, a mixture of cortisone and cytochalasin B was the most effective. Exp Lung Res, 1983 Jul, 5(1), 49 - 60 Differentiation-arrested rat fetal lung in primary monolayer cell culture . II . Dexamethasone, triiodothyronine, and insulin effects on different gestational age cultures; Tanswell AK et al.; Differentiation-arrested and hormone-depleted monolayer cultures were developed from rat fetal lungs of 18-, 19-, 20-, and 22-days gestation . Incorporation of {3H}-choline into saturated phosphatidylcholine increased, whereas the rate of cell division decreased, with advancing gestational age . Both functions were modified by physiological concentrations of glucocorticoids, thyroid hormones, and insulin . Dexamethasone (0.055-5.5 nM) increased {3H}-choline incorporation into total saturated phosphatidylcholine in immature cultures only, but caused secretion in mature (day-22) cultures . Triiodothyronine (0.055-5.5 nM) increased {3H}-choline incorporation into total and secreted saturated phosphatidylcholine at all gestational ages . Insulin (5-50 microU/ml) inhibition of {3H}-choline incorporation into saturated phosphatidylcholine was evident only in mature cultures . Dexamethasone (0.55 nM), triiodothyronine (5.5 nM), and insulin (50 microU/ml) also had gestation-dependent effects on cell division. Proc Natl Acad Sci U S A, 1983 Jul, 80(13), 4008 - 11 Renal mesangial cell cultures as a model for study of erythropoietin production; Kurtz A et al.; Mesangial cells derived from isolated glomeruli of rat kidney were grown as homogeneous cell lines in culture . They released, into the culture medium, erythropoietin that had free terminal galactosyl residues and was therefore not active in vivo . The production of erythropoietin by these cells was significantly enhanced by either lowering the PO2 in the incubation atmosphere or by adding cobalt chloride to the culture medium . Therefore, mesangial cells in culture may be considered as an in vitro system in which the regulation of erythropoietin production can be studied under well-defined conditions. Avian Dis, 1983 Jul-Sep, 27(3), 594 - 601 Comparative sensitivities of oviduct and tracheal organ cultures and chicken embryo kidney cell cultures to infectious bronchitis virus; Pradhan HK et al.; In vitro studies with organ (oviduct and trachea) and chicken embryo kidney cell cultures were attempted to assess the pathogenicity of locally isolated infectious bronchitis virus (IBV-P:120) initially isolated from the oviduct of young chicks . In oviduct cultures infected with IBV, ciliary movements decreased as early as 24 hours postinoculation (PI), and on the 6th day ciliary movements ceased completely . Cytopathic changes were also noticed . Immunofluorescent antigen was detected from 1 to 6 days PI, the maximum being on the 3rd day . The characteristic microscopic changes in the oviduct explants were reduced by 24 hours PI and had completely ceased on the 5th day . Cytopathic effect and immunofluorescent antigen were present from 1 to 8 days PI, being maximum on the 5th day . Histological changes marked by loss of cilia, rounding of the epithelial cells, degeneration, and sloughing were detected from 2 to 8 days PI . Low-embryo-passaged (EP-7) IBV did not produce cytopathic effect on the chicken embryo kidney cell cultures . On the contrary, high-embryo-passaged (EP-14) virus produced cytopathic effect at the third tissue-culture-passage level. J Natl Cancer Inst, 1983 Jul, 71(1), 39 - 43 Cytomegalovirus isolations from cell cultures derived from Epstein-Barr virus-associated nasopharyngeal carcinoma; Desgranges C et al.; Cytomegalovirus (CMV) was isolated in cell cultures derived from 2 of 11 nasopharyngeal carcinoma (NPC) biopsy specimens from North African patients . All these cases were Epstein-Barr virus (EBV)-associated NPC . Morphologic cytopathic changes and viral replication not associated with EBV were observed after 2 months in culture . Virus identification was achieved by immunofluorescence studies, and cell culture antigens were tested by the use of complement fixation and indirect hemagglutination . All these NPC patients had been infected by herpes simplex virus, varicella-zoster virus, and CMV, but the antibody titers determined by complement fixation and immunofluorescence were normal . CMV, which is not associated with this cancer, could nevertheless favor carcinogenesis in facilitating fusion between epithelial cells and EBV-positive lymphocytes. Virologie, 1983 Jul-Sep, 34(3), 191 - 6 Investigation of the effect of cellular and viral nucleic acids on certain virus infections . Note 2 . Effect of nucleic acids on virus multiplication in cell cultures; Iliescu R et al.; Treatment of cell cultures with different natural nucleic acids prior to inoculation of herpes simplex virus type 1 led in certain cases to an obvious reduction in infectant titer . The reduction was maximum at a dose of 50 micrograms nucleic acid/culture tube and it was not dependent on the nature of the nucleic acid preparation . The antiviral effect of nucleic acids was enhanced by complexation with intercalation agents such as ethidium bromide or violamycin BI . No detectable amounts of interferon could be made evident in cell cultures treated with chromosomal DNA under conditions leading to a reduction by 1.75 log in infectant titer. Ren Physiol, 1983 Jul-Aug, 6(4), 163 - 70 Localization by immunofluorescent microscopy of several collagen types and of a basement membrane proteoglycan in rat glomerular epithelial and mesangial cell cultures; Foidart JB et al.; Confluent cultures of rat glomerular epithelial and mesangial cells were studied by immunofluorescent microscopy, using affinity-purified antibodies directed against collagen of type I-V or an antiserum directed against a basement membrane (BM) proteoglycan . The epithelial cells were stained by antibodies directed against type I, IV and V collagen, whereas the mesangial cells were stained by all the antibodies directed against the different collagenous antigens tested . Therefore, only mesangial cells contained antigenic determinants of type III collagen . On the contrary, both cell types possessed BM proteoglycan antigens . The data suggest that rat glomerular epithelial and mesangial cells may be implicated in the biosynthesis of different components of normal (and pathological) glomerular BM. J Clin Lab Immunol, 1983 Jul, 11(3), 149 - 54 Immunoregulatory mechanisms in pregnancy . II . Further characterization of suppressor lymphocytes induced by alpha-fetoprotein in lymphoid cell cultures; Toder V et al.; Nonspecific suppressor cell (SPC) activity has been induced in vitro by preculturing splenocytes from normal mice in the presence of mouse amniotic fluid (MAF) and alpha-fetoprotein for 5 days or more . In adoptive transfer experiments in vivo, these AFP-precultured SPC were shown to reduce the humoral response of mice to sheep red blood cells and the cell-mediated cytotoxic response to allogeneic tumor cells . In mixing experiments in vitro, using freshly explanted splenocytes, the AFP-precultured splenocytes abrogated the generation of specific cytotoxic T-lymphocytes in primary mixed lymphocyte-tumor cell cultures . Supernatants of such precultured cells had at best only a marginal effect . These suppressor cells were found to be nylon-wool nonadherent and their effect could be almost completely abolished by treatment with anti-Thy-1, 2 serum plus complement . SPC precursors were found to be sensitive to cyclophosphamide (in vivo) and to hydrocortisone (in vivo and in vitro) . At the same time, they are resistant to different doses of radiation. J Cell Biol, 1983 Jul, 97(1), 244 - 51 The 50- and 58-kdalton keratin classes as molecular markers for stratified squamous epithelia: cell culture studies; Nelson WG et al.; The keratins are a highly heterogeneous group of proteins that form intermediate filaments in a wide variety of epithelial cells . These proteins can be divided into at least seven major classes according to their molecular weight and their immunological reactivity with monoclonal antibodies . Tissue-distribution studies have revealed a correlation between the expression of specific keratin classes and different morphological features of in vivo epithelial differentiation (simple vs . stratified; keratinized vs . nonkeratinized) . Specifically, a 50,000- and a 58,000-dalton keratin class were found in all stratified epithelia but not in simple epithelia, and a 56,500- and a 65-67,000-dalton keratin class were found only in keratinized epidermis . To determine whether these keratin classes can serve as markers for identifying epithelial cells in culture, we analyzed cytoskeletal proteins from various cultured human cells by the immunoblot technique using AE1 and AE3 monoclonal antikeratin antibodies . The 56,500- and 65-67,000-dalton keratins were not expressed in any cultured epithelial cells examined so far, reflecting the fact that none of them underwent morphological keratinization . The 50,000- and 58,000-dalton keratin classes were detected in all cultured cells that originated from stratified squamous epithelia, but not in cells that originated from simple epithelia . Furthermore, human epidermal cells growing as a monolayer in low calcium medium continued to express the 50,000- and 58,000-dalton keratin classes . These findings suggest that the 50,000- and 58,000-dalton keratin classes may be regarded as "permanent" markers for stratified squamous epithelial cells (keratinocytes), and that the expression of these keratin markers does not depend on the process of cellular stratification . The selective expression of the 50,000- and 58,000-dalton keratin classes, which are synthesized in large quantities on a per cell basis, may explain the high keratin content of cultured keratinocytes. J Cell Biol, 1983 Jul, 97(1), 153 - 65 Capillary endothelial cell cultures: phenotypic modulation by matrix components; Madri JA et al.; Capillary endothelial cells of rat epididymal fat pad were isolated and cultured in media conditioned by bovine aortic endothelial cells and substrata consisting of interstitial or basement membrane collagens . When these cells were grown on interstitial collagens they underwent proliferation, formed a continuous cell layer and, if cultured for long periods of time, formed occasional tubelike structures . In contrast, when these cells were grown on basement membrane collagens, they did not proliferate but did aggregate and form tubelike structures at early culture times . In addition, cells grown on basement membrane substrata expressed more basement membrane constituents as compared with cells grown on interstitial matrices when assayed by immunoperoxidase methods and quantitated by enzyme-linked immunosorbent inhibition assays . Furthermore, when cells were grown on either side of washed, acellular amnionic membranes their phenotypes were markedly different . On the basement membrane surface they adhered, spread, and formed tubelike structures but did not migrate through the basement membrane . In contrast, when seeded on the stromal surface, these cells were observed to proliferate and migrate into the stromal aspect of the amnion and ultimately formed tubelike structures at high cell densities at longer culture periods (21 d) . Thus, connective tissue components play important roles in regulating the phenotypic expression of capillary endothelial cells in vitro, and similar roles of the collagenous components of the extracellular matrix may exist in vivo following injury and during angiogenesis . Furthermore, the culture systems outlined here may be of use in the further study of differentiated, organized capillary endothelial cells in culture. J Biol Chem, 1983 Jun 25, 258(12), 7644 - 7 Dexamethasone decreases the amounts of type I procollagen mRNAs in vivo and in fibroblast cell cultures; Sterling KM Jr et al.; Dexamethasone treatment of neonatal chicks resulted in a time- and dose-dependent selective decrease of skin collagen synthesis . Total RNA of chick skin was isolated and hybridized to the cloned cDNAs pCg54 for pro-alpha 1 (I) mRNA and pCg45 for pro-alpha 2(I) mRNA . RNA isolated from the total skin of chicks receiving various doses of dexamethasone had dose-related decreases of pro-alpha 1 (I) and pro-alpha 2(I) mRNAs . The decrease of type I procollagen mRNAs for various doses of dexamethasone were similar to the decreases observed for collagen synthesis in vivo . Dexamethasone treatment of chick skin and chick lung fibroblasts resulted in a selective decrease of procollagen synthesis . A dose-related decrease of procollagen synthesis was observed with chick skin fibroblasts . Dexamethasone-treated chick skin and chick lung fibroblasts had decreased levels of pro-alpha 1 (I) and pro-alpha 2(I) mRNAs as determined by solid support hybridization with pCg54 and pCg45 . The dexamethasone-mediated decreases of type I procollagen mRNAs in skin fibroblasts and lung fibroblasts were similar to the decreases observed in procollagen synthesis. J Neurophysiol, 1983 Jun, 49(6), 1442 - 58 Synaptic interactions between mammalian central neurons in cell culture . II . Quantal Analysis of EPSPs; Nelson PG et al.; The presynaptic release mechanism involved in excitatory synaptic connections between neurons in cell cultures of fetal mouse spinal cord were studied by statistical analysis of intracellularly recorded postsynaptic responses . Quantal parameters were determined for the EPSPs evoked in spinal cord (SC) neurons by stimulation of either other SC or dorsal root ganglion (DRG) neurons . Transmitter release was manipulated by varying the Ca2+ and Mg2+ content of the culture medium . The release process was represented better by binomial than by Poisson statistics . A method was derived for obtaining the probability of release and the number of release elements . The quantal content and the number of release elements were substantially higher for the SC-SC connection than for the DRG-SC connection . This was partially compensated for by a larger quantal amplitude for the DRG-SC connection . There was some indication that the probability of release was higher for the SC-SC connection . The relationship between transmitter output and effective external Ca2+ ion concentration was approximately linear. J Neurophysiol, 1983 Jun, 49(6), 1428 - 41 Synaptic interactions between mammalian central neurons in cell culture . I . Reversal potential for excitatory postsynaptic potentials; Macdonald RL et al.; Intracellular recording and stimulation techniques were used to study the electrical properties of neurons in cell cultures from fetal mouse spinal cord (SC) . The morphology of SC neurons and the distribution on SC neurons of boutons formed by synaptically connected SC or dorsal root ganglion (DRG) neurons were demonstrated with horseradish peroxidase (HRP) injection . Postsynaptic polarization in conjunction with synaptic activation of SC neurons was used to determine the reversal potential for excitatory postsynaptic potentials (EPSPs) . Tetraethylammonium ions were injected postsynaptically in order to obtain reversal of the EPSPs . Both SC-SC and DRG-SC excitatory connections could be reversed by postsynaptic depolarization . The average reversal potential for the SC-SC EPSP was -4 +/- 12.2 (SD) mV and that for the DRG-SC EPSP was +8 +/- 7.9 (SD) mV, a statistically significant difference (Wilcoxon two-sample rank; P less than 0.05) . Scatter was quite large, particularly for the SC-SC connection . While some neurons gave clear electrophysiological evidence of significant dendritic effects, the average total electrotonic length was small (0.58 +/- 0.65 (SD) of a length constant) . The morphological extent of the dendrites of SC neurons was substantially less than that of mature motoneurons in vivo . We concluded that both SC-SC and DRG-SC EPSPs were mediated by a conventional conductance increase and that most synaptic input was not far removed electrically from the recording site in the neuron cell body. Biochem Genet, 1983 Jun, 21(5-6), 443 - 52 Isolation of parafluorophenylalanine-resistant mutants from HeLa cell cultures; Yim LK et al.; This report describes a method to isolate temperature-conditional phenylalanine transport mutants from the transformed human cell line HeLa . Using ultraviolet light as a mutagenic agent and DL-parafluorophenylalanine (PFPA), a poisonous analogue of L-phenylalanine, as a selective agent, mutagenized cells were selected for survival in the presence of PFPA at a temperature of 39 degrees C . Survivors of the mutagenesis and selection procedures were removed from the culture dishes by trypsin and cloned at a temperature of 35 degrees C . Seven of these lines isolated demonstrated continued resistance to PFPA at 39 degrees C . These lines were tested for uptake of L-phenylalanine at an external concentration of 100 microM and for continued resistance to PFPA at two concentrations . Cells were tested at 35 and at 39 degrees C . The data were compared to those obtained for the parental HeLa cell line under identical conditions . The seven mutant cell lines demonstrated varying resistances to PFPA and varying levels of accumulation of L-phenylalanine when tested at 35 and 39 degrees C . Three mutant lines were additionally tested for L-phenylalanine tRNA charging levels and for transport of L-arginine . The lines had parental cell levels of tRNA charging and L-arginine transport which suggest that the induced genetic defect affects a specific L-phenylalanine transport system. Mol Cell Endocrinol, 1983 Jun, 30(3), 313 - 28 Primary monolayer cell culture of bovine parathyroids: effects of calcium, isoproterenol and growth factors; MacGregor RR et al.; Cell aggregates of bovine parathyroid tissue were prepared by limited collagenase digestion and placed in culture in Weymouth's MB752/1 (calcium = 3.3 mg/100 ml) containing 5% fetal bovine serum and supplemented with insulin alone, or insulin, hydrocortisone, transferrin and epidermal growth factor . Only insulin was required for the maintenance of PTH secretion over a 9-day period . The cell aggregates spread to form monolayer in 3-5 days . The majority of the cells in monolayer were polygonal with well-defined borders . Nuclei were round and the cytoplasm was free of vacuoles . Cell cultures responded to secretory stimulation by low calcium or by isoproterenol with increases in the secretion of PTH and SP-1 . At low calcium, about 18% of both the cellular PTH and SP-1 was secreted per hour, and up to 50% of the cell content of these proteins was released per hour upon stimulation by isoproterenol and low calcium combined . The responses to calcium and isoproterenol decreased as a function of time in culture, and calcium responses often disappeared completely by 10 days of culture . When cells were cultured in medium containing a higher (5 mg%) than standard concentration of calcium between days 3-6 of culture, the degree of secretory inhibition attainable with high calcium was greater than that of cells cultured in the standard medium . When secreted hormonal peptides were separated by SDS-gel electrophoresis prior to RIA, it was found that the secretion of intact hormone was sensitive to calcium . For every molecule of PTH secreted into the medium, 1.5-2 mole-equivalents of carboxyl fragments were also released . Calcium control of fragment release was not as stringent as that of PTH release. Lab Invest, 1983 Jun, 48(6), 749 - 54 Cell proliferation in normal and atherosclerotic human aorta . II . Autoradiographic observation on deoxyribonucleic acid synthesis in primary cell culture; Orekhov AN et al.; Proliferative potentiality of cells from atherosclerotic and nonatherosclerotic human aorta was evaluated in culture by the incorporation of radioactive thymidine . Thymidine index was determined autoradiographically in primary cultures of intimal and medial cells isolated from zones of fatty infiltration, fatty streaks, atherosclerotic plaques, and uninvolved areas . The thymidine index in medial cultures was higher than in the intimal ones and was unrelated to the type of lesion . In intimal cultures obtained from zones of fatty infiltration and fatty streaks the thymidine index exceeded the normal value by 4.5- and 3-fold, respectively . In cell cultures of the plaque and unaffected intima the thymidine index was the same. Infect Immun, 1983 Jun, 40(3), 1214 - 7 Cytopathogenicity of Naegleria fowleri for rat neuroblastoma cell cultures: scanning electron microscopy study; Marciano-Cabral F et al.; Neuroblastoma cells were inoculated with Naegleria fowleri Lee and examined for cytopathology at various periods post-inoculation by scanning electron microscopy . By 18 h post-inoculation, approximately 50% of neuroblastoma cells were nonviable, as evidenced by trypan blue exclusion and light microscopic examination . This cytopathology resulted from piecemeal consumption of target cells mediated by a sucker apparatus extending from the surface of N . fowleri. In Vitro, 1983 Jun, 19(6), 471 - 8 A serum-free, chemically-defined medium for function and growth of primary neonatal rat heart cell cultures; Mohamed SN et al.; A serum-free, chemically defined medium for supporting rhythmic contraction, maximum survival, and moderate growth of cardiac cells was achieved by using a combination of hormones and growth supplements in a mixture of equal volumes of Ham's F12 and Dulbecco's modified Eagle's medium . The hormones and growth supplements included insulin, transferrin, selenium, fetuin, bovine serum albumin, hydrocortisone (HC), L-thyroxine (T4), and epidermal growth factor (EGF) . Cardiac cells were grown on fibronectin-precoated plates using the above serum-free medium . Cells grown in this medium exhibited a higher beating rate and were maintained for a longer time compared to those cells grown in serum . The effects of T4, EGF, and HC on beating rate and survival time of both cultures of mixed cell population and enriched myoblast cell population were studied . In the enriched myoblast cell cultures grown in serum-supplemented medium, the beating rate ranged from 40 to 200 beats/min, and these cultures survived for 30 d . When these enriched cell cultures were grown in serum-free hormone-supplemented medium, the beating rate ranged from 190 to 240 beats/min, and these cultures survived for more than 90 d . These results show that some hormones affect growth, whereas others affect function. Antimicrob Agents Chemother, 1983 Jun, 23(6), 808 - 13 Activity of 1-(2'-fluoro-2'-deoxy-beta-D-arabinofuranosyl)thymine against herpes simplex virus in cell cultures and rabbit eyes; Trousdale MD et al.; A new antiviral compound 1-(2'-fluoro-2'-deoxy-beta-D-arabinofuranosyl)thymine (2'-fluoro-5-methyl-ara-uracil {FMAU}), was compared with acyclovir and idoxuridine in vitro against two strains of both herpes simplex virus type 1 (HSV-1) and HSV-2 . Determinations of the 50% effective dose varied slightly with each strain and with the host cells employed . The 50% effective dose for FMAU and acyclovir against HSV-1 ranged from 0.1 microM to 0.5 to 0.6 microM in rabbit kidney cells and from 0.5 microM to 0.6 to 0.78 microM in Vero cells . Beginning 4 days post-inoculation, topical FMAU therapy given five times per day to rabbits with acute herpetic keratitis either suppressed or delayed the severity of corneal epithelial involvement, conjunctivitis, iritis, and corneal clouding . Responses to treatment with FMAU were similar to those obtained with acyclovir and significantly better than those attained with idoxuridine and vidarabine . At 30 to 40 days after the end of treatment, rabbit eyes were subjected to iontophoresis with epinephrine in an attempt to induce reactivation and enhance detection of previously latent HSV-1 . Latent HSV-1 was detected in 67 to 92% of trigeminal ganglia in FMAU-treated animals and in 90% of placebo-treated animals. J Neurophysiol, 1983 Jun, 49(6), 1459 - 68 Synaptic interactions between mammalian central neurons in cell culture . III . Morphophysiological correlates of quantal synaptic transmission; Neale EA et al.; The statistical properties of excitatory synaptic transmission between neurons in cell cultures of fetal mouse spinal cord were studied and the anatomical extent of these connections demonstrated by horseradish peroxidase (HRP) injection of the presynaptic cell . In conjunction with previous experiments (7), we have correlated the number of boutons involved in a given synaptic connection with the physiologically determined number of release elements . The number of boutons is somewhat greater than the number of release elements, and our results are supportive of the conclusion of others (1, 2) that the physiologically defined release element corresponds to the bouton . We interpret this to suggest that a rate-limiting mechanism may operate at the level of the bouton to allow release of no more than one quantal unit of transmitter with each presynaptic action potential. In Vitro, 1983 Jun, 19(6), 443 - 52 Ultrastructural and biological characterization of human choroid cell cultures transformed by Simian Virus 40; Carruba G et al.; A human diploid cell line of choroid origin was isolated from the retrouveal portion of an enucleated eye and designated HC . After 10 passages, when the proliferative capacity of HC cells decreased, they were infected and transformed by Simian Virus 40 (SV40) . A proliferating long-term cultured cell line designated HC/SV40 was established and it has been maintained as monolayer for more than 100 passages so far . The two cell lines, HC and HC/SV40, were compared for growth characteristics, capacity to form colonies in soft agar, presence of nuclear T-antigen, and ultrastructure . Cytogenetic analysis was also performed to determine the presence of chromosomal aberrations due to the permanent viral transformation of the cell line . The results indicate that HC/SV40 should be considered the transformed counterparts of HC cells because they are morphologically similar to the latter but can grow in soft agar, possess T-antigen, and show a pattern of karyotypic changes similar to that induced by SV40 in human fibroblasts . The choroid origin of HC and HC/SV40 cell lines was confirmed by the presence, in their cytoplasm, of typical electron dense granules . Their neural origin will make these cell lines very useful for neuropharmacological and differentiation studies. Biol Reprod, 1983 Jun, 28(5), 1207 - 15 Male sexual development in the nonhuman primate . III . Sertoli cell culture and age-related differences; Lee BC et al.; Homogenous preparations of primary Sertoli cell cultures were obtained from the testes of the macaque of different ages . The characteristics of Sertoli cells were confirmed by electron microscopy . Sertoli cell cultures were segregated into three developmental age groups: prepubertal, pubertal, and adult . The highest response to follicle-stimulating hormone {FSH (NIH-FSH-S13) as measured by cAMP and testosterone to estradiol conversion occurred in Sertoli cells from pubertal animals, whereas the responses were diminished in cells from both younger and older animals . Specific binding of 125I-human FSH was also increased in Sertoli cells prepared from pubertal animals when compared to cells from the other two age groups . These data demonstrate: 1) the utility of primate Sertoli cells as an in vitro model, and 2) the age-related differences in monkey Sertoli cell response to FSH and to specific FSH receptor binding. Hum Immunol, 1983 Jun, 7(2), 59 - 65 Cell culture density modulates the incorporation of HLA antigens by enveloped viruses; Azocar J et al.; Uninfected, as well as feline leukemia virus (FeLV) infected human cells cultured under high cell density conditions undergo changes in the expression of major histocompatibility complex (MHC) antigens, as determined by indirect trace binding radio immunoassay (RIA) using monoclonal anti-HLA antibodies and by decreased sensitivity to complement mediated cytotoxicity by anti-HLA alloantibodies . FeLV particles produced by the viral infected cells are also sensitive to neutralization by anti-HLA antibodies, suggesting that enveloped viral particles incorporated MHC antigens in the viral envelope . The amount of HLA antigens expressed in the viral enveloped, closely reflects the expression of HLA antigens by the virus-producer lymphoid cells . FeLV-infected HsB-2 (T) and SB (B) lymphoid cells cultured under high cell concentration condition show decreased expression of some HLA antigens (A2, B12, B17), and the viral particles produced by those cells also incorporate lower amounts of such antigens . Our results, based on the findings that human lymphoid cells (uninfected, as well as FeLV infected) show decreased expression of some HLA membrane determinants when grown under high cell density conditions, indicate that no viral selective mechanism operates in the incorporation of HLA determinants by enveloped viruses . Instead, our results suggest that viruses pick up MHC antigens from the host cell membrane according to the concentration of those antigens on the surface of the cells at the time of virus budding. Am J Physiol, 1983 Jun, 244(6), F724 - 8 Cell culture of renal epithelium derived from rabbit microdissected cortical collecting tubules; Currie MG et al.; Cortical collecting tubules were dissected from rabbit kidney and cultured in a hormonally defined serum-free medium . Morphologic studies of the cultured cells derived from the collecting tubule indicated that the cells maintained their epithelial nature . These studies also revealed the presence of two distinct cell types that closely resemble the principal and intercalated cell types of the cortical collecting tubule . Several biochemical characteristics of the cultured cells were found to be similar to previously reported values for the cortical collecting tubule . The cells retain hormonal responsiveness to antidiuretic hormone (ADH), as demonstrated by a 12-fold increase in cAMP in response to ADH . Cultured cortical collecting tubule cells produce prostaglandins, with prostaglandin E2 as the predominant cyclooxygenase product . This study presents the first morphologic and biochemical characterization of cortical collecting tubule epithelial cells grown in culture. J Virol, 1983 Jun, 46(3), 860 - 70 Analysis of extracellular West Nile virus particles produced by cell cultures from genetically resistant and susceptible mice indicates enhanced amplification of defective interfering particles by resistant cultures; Brinton MA; {3H}uridine-labeled extracellular West Nile virus (WNV) particles produced by cell cultures obtained from genetically resistant C3H/RV and congenic susceptible C3H/HE mice were compared by sucrose density gradient centrifugation as well as by analysis of the particle RNA . Defective interfering (DI) WNV particles were observed among progeny produced during acute infections in both C3H/RV and C3H/HE cells . Although only a partial separation of standard and DI particles was achieved, the DI particles were found to be more dense than the standard virions . Particles containing several species of small RNAs consistently constituted a major proportion of the total population of virus progeny produced by C3H/RV cells, but a minor proportion of the population produced by C3H/HE cells . Decreasing the multiplicity of infection or extensive plaque purification of the WNV inoculum decreased the proportion of small RNAs found in the progeny virus . The ratio of DI particles to standard virus observed in progeny virus was determined by the cell type used to grow the virus . The ratio could be shifted by passaging virus from one cell type to the other . Homologous interference could be demonstrated with WNV produced by C3H/RV cells but not with virus produced by C3H/HE cells . Continued passage of WNV in C3H/HE cells resulted in a cycling of infectivity . However, passage in C3H/RV cells resulted in the complete loss of infectious virus . Four size classes of small viral RNA, with sedimentation coefficients of about 8, 15, 26, and 34S, were observed in the extracellular particles . A preliminary analysis of these RNAs by oligonucleotide fingerprinting indicated that the smaller RNAs were less complex than the 40S RNA and differed from each other . The data are consistent with the conclusion that WNV DI particles interfere more effectively with standard virus replication and are amplified more efficiently in C3H/RV cells than in congenic C3H/HE cells . The relevance of these findings to the further understanding of genetically controlled resistance to flaviviruses is discussed. Brain Res, 1983 Jun, 284(2-3), 193 - 204 An immunohistochemical study of two myelin-specific proteins in enriched oligodendroglial cell cultures combined with an autoradiographic investigation using {3H}thymidine; Roussel G et al.; The aim of the present work was to examine the possible relationship between proliferation and expression of 2 myelin specific proteins in cultured oligodendroglial cells . Mixed cultures of glial cells, from newborn rat brain, containing astroglia and oligodendroglia were grown in 2 different culture media, minimum Eagle's medium and Waymouth's medium both supplemented with 10% calf serum in presence or absence of adult rat brain soluble extract . The proliferative activity of the cells was followed over a 28-day period by autoradiography after radioactive thymidine incorporation . It was found that in cultures grown in Waymouth's medium the proportion of oligodendroglial cells was higher and that proliferation was more active than in minimum Eagle's medium . Addition of brain extract elicited a stimulation of the proliferation of the cells in the 2 basal media . Under all conditions W1 protein appeared earlier than MBP by immunofluorescent visualization . Some oligodendroglial cells synthesizing W1 protein were still able to proliferate . MBP appears to be a marker of a later stage of cell maturation since very few MBP-positive cells incorporated tritiated thymidine . More cells contained MBP in the presence of brain extract . These results suggest that oligodendroglial cell maturation proceeds by steps, the step of W1 protein expression is compatible with proliferation while that of MBP expression appears at the end of the proliferation phase. J Lipid Res, 1983 May, 24(5), 533 - 40 Effects of human brain cell culture conditions on {14C}glucosamine radioactivity incorporation into gangliosides; Liepkalns VA et al.; Human glioma cells (12-18) and fetal neural cells (CH II) in culture were exposed for 20 hr to {14C}glucosamine to determine the level and distribution of radiolabel incorporated into gangliosides . Cells of identical passage levels at two stages of growth, preconfluent and confluent, were preincubated for 0 to 60 hr in serum-free medium (SFM) . Both higher cell densities and longer incubations in SFM caused a change in the amounts and patterns of radiolabeled gangliosides . Preincubation for 60 hr in SFM caused an increase (P less than 0.05) in the percent of total recovered ganglioside radiolabel in GM1 of CH II cells, from 10.5 to 16.7% in preconfluent cells and from 14.1 to 31.9% in confluent cells . Conversely, the proportion of radiolabel in GM3 and GM2 decreased with longer preincubations in SFM . A similar preincubation of glioma cells caused an increase in the proportion of label into GD1a of both preconfluent and confluent cells (P less than 0.02) from 4 to 11% of the total ganglioside radioactivity . Higher cell densities also resulted in consistently higher percent (of total ganglioside) incorporation into GD1a of 12-18 cells (P less than 0.05) and GM1 of CH II (P less than 0.01) . These results show that there is a shift in the incorporation of precursor label into more complex gangliosides under conditions associated with the arrest of cell division . These phenomena may represent a regulatory response of the ganglioside biosynthetic apparatus to changes in extracellular environment and cell contact. In Vitro, 1983 May, 19(5), 429 - 34 Amphibian cell culture: established fibroblastic line from embryos of the discoglossid frog, Bombina orientalis; Ellinger MS et al.; A new amphibian cell line, Bor II, is described . It was initiated from Stage 20 embryos of the discoglossid frog, Bombina orientalis . In early passages the cell line had an epithelioid morphology . Beginning at or around subcultivation 16, a more fibroblastic cell type emerged and rapidly became predominant . These later passage cells were able to proliferate in low (1.0%) serum, displayed frequent overlaps, and lacked postconfluent inhibition of cell division . The cell line was unable to survive at 37 degrees C, but grew vigorously within a temperature range of 20 degrees to 30 degrees C . The presence of two distinctive marker chromosomes in an otherwise diploid karyotype should make these cells useful for nuclear transplantation studies. Hoppe Seylers Z Physiol Chem, 1983 May, 364(5), 563 - 73 Retinol does not affect cell growth in fibroblast cell cultures; Holst A et al.; Retinol-free fetal calf serum for cell cultures was obtained by UV irradiation and by removal of retinol-binding protein (RBP) by gel-filtration . RBP from bovine serum was purified in a simple two-step procedure . This made it possible to replenish the culture medium with retinol in its physiological i.e . RBP-bound form . Retinol-free and retinol-containing medium was used to assay growth of 3T3 and L929 fibroblast cells . None of these cell lines showed a significant change in growth rate or saturation density that could be attributed to free or RBP-bound retinol. Toxicology, 1983 May, 27(1), 55 - 69 DNA breaks induced by micromolar concentrations of dimethylnitrosamine in liver primary cell cultures from untreated and phenobarbital treated rats; Mendoza-Figueroa T et al.; Direct genotoxic effects of the alkylating agent dimethylnitrosamine (DMN) have been difficult to detect in several short-term tests . We simplified our method to detect DNA breaks induced by DMN in rat liver primary cell cultures, and increased its sensitivity about 150 times by changing the conditions of ultracentrifugation and exposure to DMN . Additionally we increased 4 times the sensitivity of the improved assay by isolating hepatocytes from rats treated with phenobarbital (PB) . Treatment for 24 h with 60 microM and 13.5 microM DMN of hepatocytes isolated from untreated and PB-treated rats, respectively, decreased the molecular weight of DNA by 50% . After 24 h exposure to 13.5 microM {14C}DMN, hepatocytes from PB-treated rats incorporated 3 times more radioactivity into trichloroacetic acid precipitable material than hepatocytes from untreated rats . Also PB-treatment increased remarkably cytotoxic effects of DMN while it did not modify the cytotoxicity nor the genotoxicity of the direct-acting alkylating agent N-methyl-N'-nitro-N-nitrosoguanidine . These results show that DMN is more genotoxic for hepatocytes from PB-treated rats, and suggest that the enhanced genotoxicity is probably due to an augmented metabolism of DMN by these cultures . Our improved assay of DNA breaks as an indicator of DMN genotoxicity is now as sensitive but faster to perform than hepatocyte-mediated mutagenesis . It could be used to explore genotoxic effects of other alkylating agents and the action of microsomal enzyme modifiers on genotoxicity. J Steroid Biochem, 1983 May, 18(5), 581 - 4 Androstenedione-mediated inhibition of 11 beta-hydroxylation in monolayer cell cultures of fetal calf adrenals; Baird A et al.; The possibility that the formation of androstenedione by fetal calf adrenal cells in culture is linked to their decreased ability to form cortisol and corticosterone was investigated . Fetal calf adrenal cells metabolise radioactive adrostenedione to two major products which coelute on thin layer chromatography with 11 beta-hydroxyandrostenedione and 11 beta-hydroxytestosterone . When the cells are incubated with 11-deoxycortisol or 11-deoxycorticosterone in the presence of androstenedione there is a dose dependant inhibition of cortisol and corticosterone formation . Further studies with progesterone showed an accumulation of 11-deoxycortisol and 11-deoxycorticosterone in cells incubated simultaneously with androstenedione . The results demonstrate that exogenous androstenedione can have dramatic effects on steroidogenesis in the fetal calf adrenal and suggest that the accumulation of androstenedione in the medium of cultured andrenocortical cells is responsible, at least in part, for the decreased formation of cortisol and corticosterone. J Neurosci Methods, 1983 May, 8(1), 51 - 60 A solid-phase beta-galactosidase ELISA for detecting and quantifying monoclonal antibody binding to dissociated cell cultures of postnatal rodent cerebellum; Gard AL et al.; A solid-phase, indirect beta-galactosidase-linked immunoassay (ELISA) is described for screening large numbers of monoclonal antibodies that recognize cell surface antigens of primary monolayer cerebellar cultures . Target cultures were prepared from perikaryal suspensions of postnatal rodent cerebellum seeded into poly-L-lysine pre-coated, flat-bottom microtiter wells and fixed with glutaraldehyde after growth in vitro . Hybridoma supernatants were then incubated on these cultures . After the addition of beta-galactosidase-linked anti-mouse IgG F(ab')2 fragments, antigen-positive supernatants were detected with the enzyme substrate o-nitrophenyl-beta-D-galactopyranoside . Using a monoclonal antibody specific for rat brain Thy-1 glycoprotein, this solid-phase ELISA was found to be useful in quantifying changes in the developmental expression of cerebellar surface antigens in these cultures. Biull Eksp Biol Med, 1983 May, 95(5), 89 - 91 {Intrasplenic xenotransplantation of human fetal pancreatic islet cell cultures in rats with experimental diabetes mellitus}; Bliumkin VN et al.; Islet cell cultures obtained from the pancreas of human embryos were transplanted to the spleen pulp of rats with alloxan diabetes mellitus . During 1-2 weeks after transplantation, 6 of the 8 recipients manifested a decrease in glycemia to normal or almost normal . The antidiabetic effect of xenotransplantation of islet cell cultures was well preserved throughout the entire observation period (up to 4 months) . Two recipient rats with stable normoglycemia were subjected to splenectomy . One week after operation the animals manifested the recurrent grave diabetic status . Histological study of the removed spleen has shown the red pulp to contain the accumulations of implanted islet cells. J Mol Cell Cardiol, 1983 May, 15(5), 301 - 17 Long-term cell culture of adult mammalian cardiac myocytes: electron microscopic and immunofluorescent analyses of myofibrillar structure; Nag AC et al.; Adult rat heart was dissociated into a single-cell suspension by a retrograde perfusion technique with collagenase and hyaluronidase in Krebs-Ringer phosphate buffer . Long-term culture of these isolated single cardiac muscle cells was established for up to 45 days . Transmission electron microscopy and immunofluorescence analysis with monoclonal antibodies to cardiac myosin were used to examine sequentially the external and internal structural organization of the cardiac myocytes . Most of the cardiac myocytes exhibited prominent alterations in their external and internal structural organization during the first two weeks of culture . As they attached to the substrate and spread out, the myocytes assumed various shapes and sizes, with the exception of a few which maintained their original cylindrical shape . Electron microscopy of 2 to 4-day cultures revealed that most of the muscle cells contained disorganized myofibrils and surface blebs with enclosed mitochondria and myofilaments, which were eventually extruded from the cytoplasm . With progressive culture, the cardiac myocytes appeared to lose myofibrillar material; fewer myofilaments or sacromere fragments with interfibrillar mitochondria were observed in the sarcoplasm . Such cells resembled cultured embryonic or neonatal cardiac myocytes . However, some muscle cells retained closely packed, well organized myofibrils characteristic of freshly dissociated or in vivo cardiac myocytes . Immunofluorescence microscopy demonstrated that the cultured cardiac myocytes were strongly myosin positive throughout their morphological changes and subsequent maintenance in culture . Two patterns of fluorescence were observed in these cells in correlation with the fine structural evidence for myofibrillar distribution . One pattern exhibited bright fluorescence near the central region of the cell with a more weakly diffuse fluorescence throughout the cytoplasm; the other pattern was characterized by bright fluorescence throughout the sarcoplasm . Most of the myocytes retained their contractility throughout the culture period excepting the initial 24 to 48 h of cell attachment and flattening . These studies demonstrate the feasibility of maintaining contractile cardiac muscle cells from adult rats for at least 1 1/2 months in monolayer culture, although some variability in myofibrillar organization has been observed. Clin Endocrinol (Oxf), 1983 May, 18(5), 473 - 83 Hyperthyroidism due to a TSH secreting pituitary adenoma: case report, treatment and evidence for adenoma TSH by morphological and cell culture studies; Mashiter K et al.; A 36-year-old woman with recurrent hyperthyroidism, inappropriately elevated serum TSH, and an 8 mm pituitary microadenoma is described . Transsphenoidal adenomectomy rapidly reduced serum TSH to normal and restored the euthyroid state with retention of other anterior pituitary functions . Tissue removed at operation was examined by light and electron microscopy and cell culture . The tissue was neoplastic, composed of irregular often elongated cells which immunostrained positively only with antisera to beta-TSH . The cells contained small granules (100-170 nm) usually along the cell membrane . In cell culture TSH alone was secreted and the rate of secretion declined with time . We conclude that the patient had a TSH secreting microadenoma as a cause of her hyperthyroidism. J Clin Microbiol, 1983 May, 17(5), 834 - 9 Radioimmunofocus assay for quantitation of hepatitis A virus in cell cultures; Lemon SM et al.; A new method is described for the quantitation of hepatitis A virus in cell cultures, based on the immune autoradiographic detection of foci of infected cells (radioimmunofoci) developing beneath an agarose overlay 14 days after the inoculation of petri dish cultures of continuous African green monkey kidney cells (BS-C-1) . The number of foci developing in each culture was linearly related to the dose of hepatitis A virus (either HM-175 or PA-21 strain) inoculated . Focus development was prevented by prior incubation of virus with specific antisera, and the specificity of the radiolabeled antibody reaction was confirmed in competitive blocking experiments . This new assay method retains many of the advantages of conventional plaque assays for virus . Compared with existing end-dilution methods for the quantitation of hepatitis A virus, the radioimmunofocus assay offers greatly improved accuracy and comparable sensitivity, yet is relatively rapid and highly conservative of reagents. J Gen Virol, 1983 May, 64(Pt 5), 1101 - 10 Rotavirus persistence in cell cultures: selection of resistant cells in the presence of foetal calf serum; Chiarini A et al.; The Lincoln strain of bovine rotavirus, cytocidal for bovine AU-BEK cells, can establish in the same cell cultures in the presence of foetal calf serum (FCS) a persistent infection that depends on selection of highly resistant cells . In fact, after the induction of the carrier state only a small fraction of the cell population was infected . The parental and the carried viruses are not demonstrably different, the cultures are resistant to superinfection by the homologous virus, the cultures can be cured by antiviral serum in the medium and uninfected resistant cell clones can be selected . The presence of FCS was essential during induction and maintenance of persistence . Its effects appear to be exerted not on the control of virus replication in fully sensitive cells but on the proliferation and selection of the resistant cells. Endocrinology, 1983 May, 112(5), 1874 - 6 Differential effect of protein synthesis inhibition on TSH desensitization at different stages of primary thyroid cell culture; Rapoport B et al.; In contrast to our previous experience with cultured thyroid cells, cycloheximide, actinomycin D and nicotinamide did not prevent TSH-induced desensitization in dog thyroid cells in primary culture for only one day . With continued duration of culture prevention of TSH desensitization by these agents did emerge, but asynchronously . Thus on the second day of primary culture, while cycloheximide and actinomycin D prevented TSH desensitization, nicotinamide remained ineffective . On the third day of primary culture all three agents blocked TSH desensitization . Examination of precursor incorporation into newly synthesized DNA, RNA and protein revealed a temporal association between the appearance of susceptibility to inhibition of TSH desensitization and an increase in DNA and protein synthesis . These data provide an explanation for the discrepant reports regarding the effect of protein synthesis inhibitors on TSH desensitization. Acta Virol, 1983 May, 27(3), 282 - 5 Reproduction of Lassa virus in different cell cultures; Lukashevich IS et al.; Sierra Leone strain of Lassa virus was growing to high titres of 10(5)-10(6) plaque forming units (PFU) per ml in Vero, L and swine kidney cell lines as well as in diploid human cells and primary human embryo kidney cells . As many as 80% of the cells became infected as demonstrated by the immunofluorescence (IF) technique . In BHK-21, CV-1, HeLa, FL, HEp-2 and dog kidney cell lines, the virus reproduced to lower titres (10(4)-10(5) PFU per ml), whereas in primary chick embryo fibroblasts it did not multiply at all . The virus formed plaques under agar overlay only in CV-1 and Vero cells. J Biol Chem, 1983 Apr 10, 258(7), 4424 - 33 Metabolism of covalent receptor-insulin complexes by 3T3-L1 adipocytes . Synthesis and use of photosensitive insulin analogs to study insulin receptor metabolism in cell culture; Reed BC; To facilitate labeling cell surface insulin receptors and analyzing their metabolism by 3T3-L1 adipocytes, a characterization of both the interaction of photosensitive insulin analogs with 3T3-L1 adipocytes and the conditions for photocross-linking these derivatives to the insulin receptor are described . The synthesis and purification of two photoaffinity analogs of insulin are presented . Both B29-lysine- and A1-glycine-substituted N-(2-nitro-4-azidophenyl)glycyl insulin compete with 125I-insulin for binding to 3T3-L1 adipocytes, and the B29-derivative retains a biological activity similar to that for native insulin . An apparatus developed for these studies permits photolysis of cells in monolayer culture using the visible region of the lamp emission spectrum . Activation of the photoderivative by this apparatus occurs with a half-life of approximately 15 s and permits rapid photolabeling of a single species of receptor of 300,000 Da . The conditions for photolabeling permit a measurement of the turnover of covalent receptor-insulin complexes by 3T3-L1 adipocytes in monolayer culture . Degradation of this complex occurs as an apparent first order process with a half-life of 7 h . A comparison with previous studies (Reed, B . C., Ronnett, G . V., Clements, P . R., and Lane, M . D . (1981) J . Biol . Chem 256, 3917-3925; Ronnett, G . V., Knutson, V . P., and Lane, M . D . (1982) J . Biol . Chem . 257, 4285-4291) indicates that in a "down-regulated" state, 3T3-L1 adipocytes degrade covalent receptor-hormone complexes with kinetics similar to those for the degradation of dissociable receptor-hormone complexes. In Vitro, 1983 Apr, 19(4), 355 - 60 Partial reversal by sodium ascorbate of hyperoxia-induced damage to HEp-2 cell cultures; Bornside GH et al.; Hyperoxia induced cellular damage was used as an experimental model system for examining the ameliorative role of antioxidants . Multiplication of HEp-2 cells in monolayer culture was inhibited after exposure to 100% O2 either hyperbarically at 3 atm absolute (atma) or normobarically at 1 atma for periods from 15 s to 4 h . The inhibition was characterized by a slower rate of replication for a period from 1 to 3 d after exposure than in unexposed cultures, and then massive cellular death . Less killing followed exposure to normobaric O2 than to hyperbaric O2, and the shorter the period of exposure to hyperoxia the less killing . Addition of 100 micrograms/ml of sodium L-ascorbate to unexposed cultures enhanced growth (cell number at 6 d) almost twofold . When added ascorbate was present only during hyperoxic exposure (but not afterward), subsequent growth in air was enhanced 1.6-fold . However, when cells were exposed without added ascorbate, there was from 2 to 12-fold greater growth in air in the presence of the added ascorbate (as compared to exposed controls) . This greater growth was always only a partial reversal of the lethal effect resulting from hyperoxia . Addition of 25 micrograms/ml catalase did not affect control or exposed cultures . Addition of ascorbate plus catalase was not as effective as ascorbate alone in promoting growth; the catalase moiety antagonized some of the growth enhancing influence of ascorbate . This suggests that extracellular H2O2 was not a factor in the lethal effect resulting from hyperoxia. Mutat Res, 1983 Apr, 120(1), 61 - 7 The effects of cell-culture density on cell inactivation by benzo{a}pyrene-4,5-oxide; Newman CN; Initial survival levels of CHO-K1 monolayers following exposure to benzo{a}pyrene-4,5-oxide were found to depend upon the culture density of exponential-phase cells during the exposure portion of the experiment . A continuous increase in the surviving fraction (measured by cloning ability) was observed when culture treatment densities were above 2 x 10(4) cells/cm2 (approximately 15% of maximum density) . This effect was not observed with ultraviolet-irradiated cells nor due to increased repair times, frequently observed in plateau (density)-inhibited cultures . Furthermore, treated cells unable to grow at cloning densities (8-15 cells/cm2) continued to grow and divide for at least 9 population doublings following exposure provided post-exposure culture densities were maintained at 6.6 x 10(3) cells/cm2 or greater. Appl Biochem Biotechnol, 1983 Apr, 8(2), 115 - 26 Cell culture on polymers prepared by radiation-induced polymerization of various glass-forming monomers; Yoshii F et al.; The growth of cells on polymers prepared by the radiation polymerization of monomethacrylate and dimethacrylate was investigated . Cell growth was affected greatly by such properties of the polymers as water content, wettability, and porosity . Growth was promoted remarkably by rinsing the polymers with warm water at 60-70 degrees C and by irradiation of polymers with an electron beam . Cell growth decreased with increasing oxyethylene length (n) in the polymerized dimethacrylate of same series, CH2C(CH3)CO(OCH2CH2)nOCOC(CH3)CH2 . A decrease in the hydrophilicity of the polymer increased cell growth rate . Formation of pore structures in the polymer films also increased the cell growth. Antonie Van Leeuwenhoek, 1983 Apr, 49(1), 31 - 40 Comparison of various atmospheric conditions for isolation and subcultivation of Mycoplasma hyorhinis from cell cultures; Polak-Vogelzang AA et al.; The efficiency of aerobic incubation was compared with incubation under various oxygen and carbon dioxide conditions for the isolation and subcultivation of three strains of Mycoplasma hyorhinis from VERO-cell cultures and subcultivation of three laboratory strains . Under anaerobic conditions with a low oxidation-reduction potential (at or below -115 mV) as obtained in jars, with catalysts, containing mixtures of 5%-10% CO2 in H2, very poor or no growth of any of the six M . hyorhinis strains was observed . When traces of oxygen were present (that is, under conditions with higher oxidation-reduction potentials, e.g . when omitting the catalyst in the above gas mixtures or in 5% CO2 + 95% N2) isolation from cell cultures was successful in most tests, but subcultivation of these primary isolates was seldom possible under these semi-anaerobic conditions . However, in most cases these primary isolates could be subcultivated aerobically, although aerobic conditions were unsatisfactory for isolation in about half of the experiments . Isolation of M . hyorhinis was optimal in 5% O2 + 95% N2, under which condition the isolates could also always be subcultivated . Isolation failed occasionally when 5% O2 + 5% CO2 + 90% N2 was used, thus indicating that 5% CO2 was slightly inhibitory . 5% CO2 in air and 10% CO2 either in air, H2 or N2 were also inadequate for isolation from cell cultures . In contrast to the findings with these cell culture-adapted M . hyorhinis strains, the laboratory strains could be subcultivated easily under all conditions tested except those with an oxidation-reduction potential at or below -115 mV; 100% CO2 was inhibitory for all 6 strains . Our findings may partly explain why M . hyorhinis is often considered "non-cultivable" on artificial media once adapted to cell cultures . The findings emphasize the need to employ also a micro-aerophilic condition (5% O2 in 95% N2) in the examination of cell cultures for mycoplasma. J Pathol, 1983 Apr, 139(4), 431 - 40 The behaviour of human ameloblastoma tissue in cell culture; Prime SS et al.; Human ameloblastoma tissue was investigated using cell culture techniques, transmission electron microscopy and fluorescent microscopy . Cultured cellular morphology was dependent on the type of substratum, with polygonal cells predominant on collagen substrata in contrast to an elongated cellular morphology on glass substrata . The presence of tonofilaments and desmosomes confirmed the epithelial origin of these cells . The distribution of Con A surface receptors and cytoplasmic actin in the same cell was studied using a double fluorochrome technique . Incubation with fluorescein isothiocyanate-labelled Con A at 37 degrees C for increasing time periods resulted in the Con A receptors showing progressive changes in staining patterns from clusters, to caps to perinuclear globules . Sequential changes in cytoplasmic actin, labelled by a specific anti-actin auto-antibody traced with rhodamine-labelled goat anti-human globulin, corresponded to the Con A staining patterns. Jpn J Med Sci Biol, 1983 Apr, 36(2), 85 - 95 A serum-deprived human embryo pancreatic islet cell culture system: a culture method suitable for double-label antibody technique to detect virus infection of beta cells; Sato S et al.; By the use of serum-free Eagle's MEM instead of serum-supplemented culture medium in human embryo islet cell culture, it was possible to increase the proportion of beta cells identified by the indirect fluorescent antibody (IFA) technique . With the combination of the serum-deprived culture system and the double-label antibody technique, it was possible to show that the human embryo beta cells in culture were susceptible to infection with human papovavirus BK (BKV) . Furthermore, this combination enabled us to assay quantitatively the BKV-infected beta cells, and it was shown that the proportion of BKV-infected beta cells in the islet cultures from three subjects were not significantly different either in BKV tumor antigen (T-Ag)-positive ratio or in BKV virion antigen (V-Ag)-positive ratio . The quantitative assay also showed that the percentages of BKV T-Ag- or V-Ag-positive cells in insulin-positive cells and in insulin-negative cells were almost the same. J Biol Stand, 1983 Apr, 11(2), 91 - 7 Poliovirus and polio antibody assay in HEp-2 and Vero cell cultures; Albrecht P et al.; HEp-2 cell cultures were about three to 30 times more sensitive for poliovirus titration than Vero cells . Attenuated strains induced a complete cytopathic effect in HEp-2 but not in Vero cells . For polio antibody titration, HEp-2 and Vero cells were equally suitable . A high degree of sensitivity and reproducibility of virus neutralization was achieved in tests utilizing a low virus dose and serum-virus incubation overnight at 36 degrees C . Staining of infected trays with crystal violet obviated reading of viral CPE under the microscope and expedited the evaluation of larger-scale tests. J Clin Microbiol, 1983 Apr, 17(4), 666 - 8 Detection of Chlamydia trachomatis inclusions in Mccoy cell cultures with fluorescein-conjugated monoclonal antibodies; Stamm WE et al.; We compared two methods for identification of Chlamydia trachomatis inclusions in McCoy cell monolayers: conventional iodine staining and immunofluorescence staining with monoclonal antibodies against the species-specific major outer membrane protein antigen of C . trachomatis . Among 878 urethral and cervical specimens tested in parallel, the immunofluorescence method detected eightfold more inclusions per monolayer, identified a higher proportion of positive specimens on first passage (98 versus 62% by iodine staining; P less than 0.01), and improved overall sensitivity (98% of total positive specimens detected versus 84% by iodine staining; P less than 0.01) . Improved sensitivity was most evident in specimens with low numbers of inclusions . Compared with conventional iodine staining, immunofluorescence staining with monoclonal antibodies improves sensitivity and offers more rapid detection of chlamydial inclusions in cell culture. Brain Res, 1983 Apr, 283(2-3), 205 - 13 Insulin-like growth factor I (IGF I) stimulates DNA synthesis in fetal rat brain cell cultures; Lenoir D et al.; Addition of insulin, IGF I or IGF II to serum-free cultures of fetal rat brain cells (gestation day 15/16) significantly stimulates DNA synthesis . The dose-response curves show that IGF I is more potent than insulin; half maximal stimulation of {3H}thymidine incorporation is obtained at about 0.4 nM IGF I and 14 nM insulin, respectively . Cultures initiated 2 days later (gestation day 17/18) showed a decreased responsiveness to both peptides . No additive effect was observed after combined addition of both peptides at near-maximal doses . Both peptides show a latency of action of about 12-18 h . In the presence of either IGF or insulin, neuronal as well as glial enzymes are increased, suggesting that neuronal and glial precursor cell division is influenced . IGF I and IGF II interact with a specific binding site for which insulin competes very weakly; however IGF I and IGF II bind with relatively high affinity to the insulin specific binding site . The present results support the hypothesis that both insulin and IGF stimulate mitotic activity by interacting with specific somatomedin receptors and suggest a physiological role of IGF in the developing brain. Br J Pharmacol, 1983 Apr, 78(4), 717 - 23 Comparison of the chronotropic effect and the cyclic AMP accumulation induced by beta 2-agonists in rat heart cell culture; Freyss-Beguin M et al.; 1 The chronotropic response and the variation in cyclic adenosine 3',5'-monophosphate (cyclic AMP) accumulation induced by isoprenaline and six beta 2-selective agonists (fenoterol, salmefamol, soterenol, zinterol, salbutamol and formoterol) were analyzed on cultured heart cells of the rat . 2 The compounds elicited an enhancement of the frequency, but the time course of the variation of the beating rate was not identical for all of them . A rapid onset was observed for isoprenaline, zinterol and formoterol while it was slower for fenoterol, salmefamol and salbutamol . 3 In contrast with isoprenaline, the beta 2-selective agonists gave concentration-beating frequency curves which were not sigmoidal . Their effects extended up to a concentration of 5 to 6 orders of magnitude . Nevertheless, the concentration at which the maximal effect occurred and the intrinsic activities of the various compounds agrees better with the responses observed on guinea-pig atria than with those on trachea . 4 All the beta 2-selective agonists increased the accumulation of cyclic AMP in rat heart cells with a maximal effect at 10(-5)M or less . The effects of beta 2-agonists on cyclic AMP production showed some analogies with those on beating frequency of the heart cells . The increase in cyclic AMP accumulation induced by beta 2-agonists also corresponded to their chronotropic effects on guinea-pig atria . Thus, the correlation coefficient between the inverse of the log of the concentration producing the half maximal cyclic AMP accumulation in cultured heart cells and the pD2 values on guinea-pig atria was 0.93 . 5 It is concluded that, in contrast to what was observed in other models, the beta 2-selective agonists induce an increase in the production of cyclic AMP in rat heart cells . Furthermore, the effects of the beta 2-agonists on cyclic AMP accumulation and on beating rate in the heart cells may correspond with their beta 1-adrenoceptor potencies. Life Sci, 1983 Mar 28, 32(13), 1427 - 35 Cerebral endothelial cell culture . II . Adenylate cyclase response to prostaglandins and their interaction with the adrenergic system; Karnushina IL et al.; The response of endothelial adenylate cyclase (AC) to prostaglandins (PGE1, PGE2, PGF1 alpha, PGF2 alpha, PGD2 and PGI2) and the relationship of PGE2 to adrenergic systems were investigated in cerebrovascular endothelial cultures . E-type prostaglandins and PGI2 were more effective in stimulating endothelial AC (EC50 = 3 x 10(-7)M, and 3 x 10(-6)M, respectively) than prostaglandins of the F-series and PGD2 which activated AC at high doses only . A modulation of endothelial AC response to either PGE2 or norepinephrine (NE) was observed in the presence of both agents in the system . It was manifested by a dose-dependent NE inhibition of the PGE2-stimulated formation of cAMP, which was partially restored by phentolamine . Alpha and beta-adrenergic agonists (alpha, clonidine and 6-fluoronorepinephrine; beta, isoproterenol) also partly blocked while forskolin and PGE2 synergistically stimulated the production of cAMP in the endothelial cultures . These findings strongly suggest that the interaction of prostaglandins and alpha- and beta-adrenergic agonists with the AC system in cerebrovascular endothelium may play a role in the regulation of the cerebral microcirculation and/or blood pressure. Biochem Biophys Res Commun, 1983 Mar 16, 111(2), 750 - 9 Enhanced chondrocytic differentiation in chick limb bud cell cultures by inhibitors of poly(ADP-ribose) synthetase; Nishio A et al.; Inhibitors of poly(ADP-ribose) synthetase, namely nicotinamide, benzamide, m-methoxybenzamide and 3-aminobenzamide, augmented chondrocytic differentiation chick embryo limb bud mesenchymal cells, in culture . These inhibitors stimulated early appearance and massive formation of cartilage nodules in micromass cultures stage 23-24 chick embryos . They also induced nodule formation in micromass and cartilage colonies at micromass plating densities from stage 18-19 embryo Benzamide, however, did not prevent differentiated chondrocytes from undergoing a pleiotypic change in cell type . These results are compatible with the putative regulatory function of poly(ADP-ribose) on cell differentiation. J Cell Sci, 1983 Mar, 60, 209 - 16 Metabolism of palmitate by anoxic and reoxygenated heart cell cultures; Bailey PJ et al.; Under anoxic incubation conditions heart cell cultures showed enhanced uptake of {U-14C}palmitic acid into neutral lipids, while incorporation into phospholipids was unaltered . Fractionation of the neutral lipids showed greatest incorporation of radiolabel into the triglyceride fraction . Uptake of fatty acid in normoxic cultures may be dependent upon the supply of glycerol 3-phosphate from glycolysis, as 2-deoxyglucose and L-lactate, respectively, inhibited and stimulated incorporation of fatty acid into neutral lipid fractions . When previously anoxic cultures were reoxygenated, oxidation of fatty acid was depressed and the mitochondrial function of anoxic cultures appeared to be more readily uncoupled by 2,4-dinitrophenol, in comparison with cultures maintained under normoxic conditions . Similar behaviour was seen when oxidation of endogenous lipid or oxidation of glucose was examined . Previously anoxic cultures show a preference for oxidation of endogenous rather than exogenous lipid substrates . The results suggest that anoxia-stimulated lipid accumulation may prove injurious to subsequent mitochondrial function and may be a contributory factor in the pathological processes associated with hypoxic injury of cardiac tissue. Invest Radiol, 1983 Mar-Apr, 18(2), 199 - 206 Human endothelial cell culture as an evaluation system for the toxicity of intravascular contrast media; Laerum F et al.; Endothelial cells in primary cultures from human umbilical cord veins were incubated with various radiographic contrast media in increasing concentrations up to approximately 60 mgI/ml for 24 hours in order to study their toxicity . Cell death was recorded with the chromium-51 (51Cr)-release method and controlled by dye exclusion tests, Coulter counting, and protein determination . The hyperosmolar, ionic contrast medium, meglumine metrizoate, was far more toxic to the endothelium than the nonionic media, metrizamide and iohexol, which are far less hyperosmolar . The 51Cr-release test on endothelial cultures provides a simple and useful technique in the evaluation of various intravascular contrast media and their components. Res Vet Sci, 1983 Mar, 34(2), 249 - 51 Ultrastructure of Babesia bovis sexual stages as observed in Boophilus microplus cell cultures; Droleskey RE et al.; Propagation of Babesia bovis in a Boophilus microplus cell line resulted in the appearance of the sexual stage of the parasite normally found only within tick intestine . These sexual stages, which possessed spike-like projections containing microtubules, were present in the medium and within cultured cells . Other ultrastructural characteristics of this sexual stage are described. Neurochem Res, 1983 Mar, 8(3), 333 - 40 Uptake and utilization of CDP-choline in primary brain cell cultures from fetal brain; Vecchini A et al.; The utilization of double-labeled CDP-choline by cultured brain cells has been studied . CDP-choline is demonstrated to be rapidly hydrolysed into CMP and choline phosphate . The fragments, or their hydrolysis products, penetrate into the cells and are utilized for lipid synthesis . At short times after the isotope administration a rapid labeling of phosphatidylcholine was detected, when cells were incubated with CDP-choline . The same was not seen when cells were incubated with labeled choline . From these observations it can be inferred that either CDP- choline can penetrate the cell membrane or that some mechanism involving CDP-choline and leading to phospholipid synthesis can work at the external surface of the plasma membranes. Zh Mikrobiol Epidemiol Immunobiol, 1983 Mar, (3), 87 - 92 {Evaluation of the toxic action of prophylactic and therapeutic preparations on cell cultures . III . The detection of toxic properties in medical biological preparations by the degree of cell damage in the L132 continuous cell line}; Kravchenko AT et al.; The methods of the quality control of medical biological preparations, including tests on animals, do not ensure the complete absence of toxicity in a final product . The use of the method of "subcultures with the introduced preparation" makes it possible to determine the toxicity of both specific and nonspecific components of vaccines and sera from the number of dead and damaged cells . The toxic action of preparations kills and damages the cells at the site of injection, thus inducing the formation of autoantigens whose effect on the body cannot be predicted . Thus thimerosal, commonly used as preservative, has been found not only to render its primary toxic effect, but also capable of changing the properties of cells . This fact suggests that the use of thimerosal for the preservation of medical biological preparations, especially those intended for children, is inadmissible. J Neurophysiol, 1983 Mar, 49(3), 792 - 803 Ionic mechanism of muscarinic cholinergic depolarization of mouse spinal cord neurons in cell culture; Nowak LM et al.; 1 . Muscarinic cholinergic actions were investigated in a population of large multipolar spinal cord neurons in primary dissociated cell culture using conventional intracellular recording and single-microelectrode voltage-clamp techniques . 2 . Cholinergic agonists were applied to the surface of neuronal somata by pressure ejecting drug-containing bathing medium from small blunt (2-10 microns) glass micropipettes . Atropine was applied by diffusion from large (20-30 microns) blunt micropipettes positioned near the soma . 3 . Muscarine increased action-potential firing and evoked slow sustained membrane depolarization . Action potentials but not slow membrane depolarizations were eliminated by the sodium channel blocker, tetrodotoxin . Muscarine-induced depolarizing responses were unaffected by the calcium channel blocker, cadmium . 4 . Depolarizing responses evoked by selective and nonselective muscarinic cholinergic agonists were dose dependent, reversibly antagonized by atropine, and did not desensitize . 5 . Muscarine depolarized neurons and decreased membrane conductance during recording with both 3 M KCl- and 4 M KAc-filled intracellular recording micropipettes . When membrane potential was held constant using the single-electrode voltage-clamp technique (KCl-filled micropipettes), muscarine and gamma-aminobutyric acid (GABA) evoked inward currents at resting membrane potential . GABA-induced inward current responses were decreased by depolarization and had reversal potentials near -30 mV, consistent with GABA increasing chloride conductance . Muscarine-induced inward current responses were increased by depolarization and had extrapolated reversal potentials near -80 mV, consistent with muscarine decreasing a potassium conductance . 6 . Unlike GABA-induced currents, muscarine-induced currents evoked in normal Tris-buffered saline (5 mM potassium) did not vary as a linear function of membrane potential and did not reverse polarity in six of seven neurons near potassium equilibrium potential . However, in high-potassium medium (15 mM) muscarinic responses did reverse polarity and current was linearly related to membrane potential . Thus, the apparent voltage dependence of muscarine responses was probably due to voltage dependency of the potassium conductance and not due to potassium channel rectification . 7 . Preliminary evidence (37) indicates that muscarine decreases a time- and voltage-dependent potassium current in some cultured spinal cord neurons . Whether reduction of m current can completely account for muscarine postsynaptic actions in these cells remains unclear . Muscarine may also block a small population of non-voltage-dependent potassium channels in addition to reducing m current. J Cell Physiol, 1983 Mar, 114(3), 263 - 6 Transepithelial transport in cell culture: bioenergetics of Na-, D-glucose-coupled transport; Sanders MJ et al.; The renal cell line LLC-PK1 cotransports Na and D-glucose from the apical to the basolateral side of the cell monolayer, and the short-circuit current (Isc) measures the net amount of Na transported . Under conditions of maximal cotransport, the addition of phlorizin or removal of Na reversibly decreased oxygen consumption by one-half . In the absence of glycolytic substrates, alpha-methyl-D-glucoside stimulated Isc