Endocrinology, 1991 Mar, 128(3), 1450 - 8 Interaction of phorbol ester and adrenocorticotropin in the regulation of steroidogenic P450 genes in human fetal and adult adrenal cell cultures; Ilvesmaki V et al.; The role of protein kinase-C-dependent mechanisms in steroidogenic enzyme gene expression was studied in primary cultures of human fetal and adult adrenals . Cells were first cultured for 7-10 days and then stimulated with ACTH or 12-O-tetradecanoyl phorbol-13-acetate (TPA), a protein kinase-C activator, for 1-2 days . Cytoplasmic RNA was extracted and analyzed by Northern and dot blotting with 32P-labeled cDNA probes for P450scc (cholesterol side-chain cleavage enzyme/20,22-desmolase), P450c17 (17 alpha-hydroxylase/17,20-lyase), and P450c21 (21-hydroxylase); for P450c11 (11 beta-hydroxylase/18-hydroxylase/18-methyl oxidase), a 30-mer oligonucleotide was used as a probe . ACTH (200 ng/ml) increased the accumulation of all of the studied steroidogenic enzyme mRNAs in both fetal and adult cultures by several-fold . TPA inhibited this accumulation in a dose-dependent manner (0.01-100 ng/ml), whereas the inactive phorbol ester 4 alpha-phorbol-12,13-didecanoate was without effect . On the other hand, in the absence of ACTH, TPA slightly increased all steroidogenic P450 mRNAs in adult cultures . In fetal cultures TPA slightly increased P450scc, P450c11, and P450c21 mRNA levels, whereas it decreased P450c17 mRNA . (Bu)2cAMP and cholera toxin increased steroidogenic enzyme mRNAs such as ACTH . TPA down-regulated (Bu)2cAMP- and cholera toxin-induced P450mRNAs in the same way as ACTH-induced mRNAs . The secretion of ACTH-stimulated cortisol, dehydroepiandrosterone sulfate, and aldosterone was decreased by TPA in both fetal and adult cultures . The basal steroid production was slightly increased by TPA in both culture types . The changes in steroid production correlated well with the alterations in the steroidogenic enzyme gene expression . Our results show that the inhibitory effect of TPA on ACTH-stimulated adrenal steroidogenesis is mediated at the mRNA level of steroidogenic enzymes . Thus, it seems likely that both protein kinase-C- and cAMP-dependent mechanisms are involved in the long term maintenance of steroidogenic enzymes and hormone production in adrenocortical cells. In Vivo, 1991 Mar-Apr, 5(2), 123 - 6 Distribution of m-IBG in nude mice bearing LAN-5 neuroblastoma xenografts and in vitro cell culture; Van der Wall H et al.; BACKGROUND . Neuroblastoma is the commonest extra-cranial solid tumour of childhood and has a poor clinical outcome in patients with disseminated disease . Animal xenografts of this tumour offer a useful method of studying new diagnostic and therapeutic strategies for the tumour, prior to consideration of clinical trials . METHODS . 131I m-IBG was injected into nude mice bearing xenografts of the human neuroblastoma line LAN 5 in several sites . Animals were sacrificed at 24 (n = 6) and 48 hours (n = 5) and the biodistribution of the agent as well as uptake into the xenografted tumours was determined in a gamma well counter . In-vitro uptake of m-IBG into suspensions of LAN 5 cells was also determined in order to confirm the animal studies . RESULTS . There was no preferential accumulation of m-IBG in the xenografted tumours in any of the sites considered . Similarly, the in-vitro uptake of m-IBG showed the typical Michaelis-Menten kinetics of simple diffusion with no evidence of active uptake . CONCLUSIONS . The human neuroblastoma line LAN 5 failed to enrich m-IBG in either the xenograft or in-vitro situation . This may be related to the poor degree of differentiation of the tumour as evidenced by its unusual ease of growth in the sub-cutaneous site. Mutat Res, 1991 Mar-Nov, 256(2-6), 233 - 42 Differentiation of primary and secondary fibroblasts in cell culture systems; Bayreuther K et al.; As a function of the advancing development of Valo chicken, C3H mice, BN rats, and man in the embryonic, juvenile, adolescent, and senescent phases, stem cells and fibroblasts in the connective tissues of skin and lung differentiate along an 11-stage differentiation sequence in five compartments of the fibroblast stem cell system, when studied in primary ex vivo-in vitro systems . In the fibroblast stem cell system, three stem cells develop in the stem cell compartment along the cell lineage S1-S2-S3, three mitotic fibroblasts (MF) differentiate along the sequence MF I-MF II-MF III in the fibroblast progenitor compartment, three postmitotic fibroblasts (PMF) proceed in the fibroblast maturing compartment along the row PMF IV-PMF V-PMF VI . PMF VI is the terminally differentiated end cell of the fibroblast stem cell system . After a species- and tissue-specific period of high metabolic activity, PMF VI either dies as PMF VIIa in the fibroblast apoptosis compartment or transforms as PMF VIIb in the fibroblast transforming compartment . The reiterated appearance of the 11 cell types in primary stem cell and fibroblast populations and the reiterated age-related changes in the cell type composition of the primary stem cell and fibroblast populations make it very likely that stem cell, mitotic and postmitotic fibroblast equivalents exist in vivo and that age-related changes of the frequencies of the stem cell and fibroblast equivalents result from the progressing differentiation of stem cell, mitotic, and postmitotic fibroblast equivalents along the 11 stage differentiation sequence in the fibroblast equivalent stem cell system in vivo . Secondary fibroblast populations derived from connective tissue of prenatal and postnatal skin of Valo chicken, C3H mice, BN rats, and man, including the normal embryonic human lung fibroblast cell line WI38, were also found to develop along a terminal stem cell sequence . Thus, secondary fibroblast populations in vitro constitute a representative material for studies of general and special issues of cell biology, such as terminal differentiation, aging, apoptosis, and transformation, as long as stem cell system-specific concepts and methods are employed in such investigations. Epilepsy Res, 1991 Mar, 8(2), 134 - 41 Guanidino compounds that are increased in hyperargininemia inhibit GABA and glycine responses on mouse neurons in cell culture; De Deyn PP et al.; The effects of arginine, homoarginine, alpha-keto-delta-guanidinovaleric acid and argininic acid (guanidino compounds that were found to be increased in hyperargininemia) were evaluated on responses to gamma-aminoburtyric acid (GABA) and glycine (Gly) on mouse neurons in primary dissociated cell culture . GABA and Gly were applied iontophoretically and intracellular microelectrode recording techniques were used . The guanidino compounds rapidly and reversibly inhibited both GABA and Gly responses . The guanidino compounds inhibited GABA responses in a concentration-dependent manner and inhibited Gly responses at a concentration of 10 mM . Argininic acid was the most potent in reducing inhibitory amino acid responses, followed in decreasing potency by alpha-keto-delta-guanidinovaleric acid, homoarginine and arginine . The guanidino compounds were equally potent in decreasing Gly and GABA responses . Co-application of CGS 9896, a benzodiazepine receptor antagonist, did not antagonize the guanidino compound-induced inhibition of GABA responses . These findings suggest that the guanidino compounds inhibited responses to the inhibitory neurotransmitters GABA and Gly by blocking the chloride channel . This effect might underlie the in vivo epileptogenicity of some of the guanidino compounds and might contribute to the pathogenesis of seizures in hyperargininemia. Brain Res Mol Brain Res, 1991 Mar, 9(4), 327 - 32 Monoaminergic regulation of proopiomelanocortin messenger RNA concentrations in primary cell cultures of rat hypothalamus; L'Hereault S et al.; The effect of monoaminergic neurotransmitters on a 1.1-kb proopiomelanocortin messenger RNA (POMC mRNA) detected in rat hypothalamic cells maintained in culture has been evaluated . Serotonin caused a 15% increase in POMC mRNA levels, an effect which was blocked by the 5-HT2 receptor antagonist ketanserin . Dopamine markedly decreased POMC mRNA levels in a dose related manner . Haloperidol and the selective D2 antagonist (+)-butaclamol prevented the inhibitory effects of both dopamine and the selective D2 agonist, 2-bromo-alpha-ergocryptine . The selective dopamine D1 receptor agonist, SKF 38393, as well as norepinephrine and acetylcholine did not affect POMC mRNA levels . It is concluded that serotonin exerts a positive control and dopamine a negative control on POMC mRNA concentrations in primary cultures of rat hypothalamic neurons . The negative effect of dopamine appears to be exerted via D2 receptor-mediated mechanism. J Neurochem, 1991 Mar, 56(3), 953 - 60 Synergistic interactions between alpha 1- and alpha 2-adrenergic receptors in activating 3H-inositol phosphate formation in primary glial cell cultures; Wilson KM et al.; Norepinephrine (NE)-stimulated 3H-inositol phosphate (3H-InsP) formation in primary glial cell cultures is thought to be due to alpha 1-adrenergic receptor activation . Surprisingly, the alpha 1-selective agonists phenylephrine and methoxamine showed only 12-21% of the intrinsic activity of NE in activating this response . Although the alpha 2-selective agonist UK 14,304 was itself inactive, inclusion of UK 14,304 increased the response to the alpha 1-selective agonists by about threefold . This increase was concentration-dependent and occurred at all time points examined . 6-Fluoro-NE and alpha-methyl-NE mimicked the effect of NE in glial cultures, although with lower potencies . However, several partial agonists were ineffective in activating this response, in both the presence and absence of UK 14,304 . Synergistic interactions were not observed for alpha 1-mediated responses in slices of rat cerebral cortex, either for formation of 3H-InsPs or potentiation of isoproterenol- or adenosine-stimulated cyclic AMP accumulation . Both UK 14,304 and phenylephrine inhibited NE-stimulated 3H-InsP formation in concentrations similar to those necessary to activate this response directly . These results suggest that NE activates 3H-InsP formation in primary glial cultures by synergistic actions on both alpha 1- and alpha 2-adrenergic receptors . The agonists UK 14,304 and phenylephrine also can act to inhibit the response to NE competitively. Vopr Virusol, 1991 Mar-Apr, 36(2), 117 - 9 {The adaptation of an isolate of the hepatitis A virus to reproduction in cell cultures}; Farashian VR et al.; A HAV strain derived from a patient in Moscow multiplied in a continuous cell line PLC/PRF/5 at 32 degrees C (variant MI-1) and at 37 degrees C (variant MI-1.1) . These two variants of the strain MI retained the ability for reproduction both at 32 degrees C and 37 degrees C after passages in cell culture . The strain MI also replicates in MK cells . Negative results on HAV cultivation were obtained in HEF-240 and FRhK-4 cell cultures . The MI-1 and MI-1.1 variants are typical of hepatitis A viruses by their physicochemical properties, the results of ELISA, protein electrophoresis and dot hybridization tests. J Clin Microbiol, 1991 Mar, 29(3), 660 - 1 Comparison of flow cytometry and virus isolation in cell culture for identification of cattle persistently infected with bovine viral diarrhea virus; Qvist P et al.; Detection of bovine viral diarrhea virus in 143 blood samples by virus isolation in cell culture and flow cytometry was performed . The material included 37 samples later shown to originate from persistently infected cattle . Thirty-three samples were positive by virus isolation, and all 37 samples were positive by the flow cytometric assay. Biochim Biophys Acta, 1991 Feb 26, 1082(1), 27 - 32 The expression of lipoprotein lipase activity and mRNA in mesenchymal rat heart cell cultures is modulated by bFGF; Friedman G et al.; The effect of basic fibroblast growth factor (FGF) on lipoprotein lipase (LPL) production was studied in mesenchymal rat heart cell cultures . Addition of FGF to culture medium containing 20% serum resulted in a 3-fold increase in LPL activity . The minimal effective dose of FGF was 10 ng/ml and the increase occurred after exposure for 48 h . Addition of FGF was effective during the first week in culture, when enzyme activity was increasing, but not after 11 days when the cultures were superconfluent and the enzyme activity was high . Addition of FGF to serum-poor medium was able to replace serum required to sustain LPL activity . In FGF-treated cultures, more LPL activity was present in the functional pool, but not in the medium, than in the controls . The increase in enzymic activity was accompanied by an increase in enzyme mass and in LPL mRNA. J Biol Chem, 1991 Feb 15, 266(5), 2983 - 7 Propeptide-mediated regulation of procollagen synthesis in IMR-90 human lung fibroblast cell cultures . Evidence for transcriptional control; Wu CH et al.; We have demonstrated previously that the carboxyl- and amino-terminal propeptides of type I procollagen can inhibit procollagen synthesis by specifically decreasing procollagen mRNA levels . The objective of the present experiments was to determine the mechanism by which propeptides cause these pretranslational effects . IMR-90 fibroblasts were exposed to medium containing carboxyl-terminal propeptide of type I procollagen, and nuclear run-off assays were performed by hybridization to a specific alpha 1 chain type I procollagen cDNA probe . Specific type I procollagen transcription rates were found to be decreased by 50% in the presence of 75 nM carboxyl-terminal propeptide compared with control (untreated) cells . Total cellular transcription rates as well as beta-actin mRNA rates were not affected significantly by any concentration of carboxyl-terminal propeptide . Propeptide radiolabeled with 125I was found to be taken up by cultured cells . Furthermore, exogenous carboxyl-terminal propeptide levels increased in the cytosolic compartment and eventually reached a steady-state level of 18 +/- 2 pmol/g cell protein by 30 min . Of particular interest was the finding that levels of radiolabeled carboxyl-terminal propeptide were also detected in the nuclear fraction and increased with time, reaching a plateau after 60 min of incubation . Incubation of nuclei from IMR-90 cells in medium containing varying concentrations of carboxyl-terminal propeptide resulted in nuclear transcription rates that were decreased by 40% compared with untreated controls . beta-Actin nuclear message levels remained unchanged under identical conditions . We conclude that carboxyl-terminal propeptide of type I procollagen can be internalized and become associated with the nuclear compartment . This suggests a feedback regulatory role on procollagen synthesis by a direct effect on procollagen gene transcription. Arerugi, 1991 Feb, 40(2), 108 - 16 {Eosinophil chemotactic activity in the supernatant of mononuclear cell culture stimulated with specific antigen}; Fukushima Y et al.; Peripheral blood mononuclear cells (PBMC) separated from patients with asthma who were sensitive to Dermatophagoides farinae (Df) were cultured in alpha-medium for 5 days at 37 degrees C in 5% CO2, in the presence or absence of 10 ng/ml of Df antigen . Eosinophils were purified from the peripheral blood of patients with eosinophilia who were not sensitive to Df . Eosinophil chemotactic activity (ECA) was tested using a modified Boyden chamber method . ECA in the supernatant of PBMC stimulated with Df antigen was detectable after 24 hrs, peaked at 72 hrs and continued throughout the experiment . ECA was not observed in the supernatant of PBMC culture from subjects who were not sensitive to Df, and negligible activity was also observed when PBMC were stimulated with an unrelated antigen . The activity was unchanged by heating at 56 degrees C for 30 min, but was inactivated at 100 degrees C for 10 min . CV-6209, a specific PAF antagonist, failed to inhibit this chemotactic activity . The molecular weight of this eosinophil chemotactic factor (ECF) was greater than 30,000 daltons as determined by an ultrafiltration study . In conclusion, these data suggest that in asthmatic patients sensitive to Dermatophagoides farinae mononuclear cells stimulated with a related antigen produce one of cytokine(s) which possess(es) ECA, and may play an important role in the recruitment of eosinophils in chronic asthma. J Neurocytol, 1991 Feb, 20(2), 124 - 32 Transitional elements with characteristics of both growth cones and presynaptic terminals observed in cell cultures of cerebellar neurons; Burry RW; As growth cones interact with targets, they become presynaptic terminals by losing growth cone characteristics and acquiring presynaptic characteristics . Results presented here show that transitional elements can be identified in cell cultures of rat cerebellum, which have some characteristics of both growth cones and presynaptic terminals . During the first week in culture, slender growth cones have fine filopodia . Subsequently, many growth cones in contact with the polylysine substrate spontaneously enlarge and become non-motile . In transitional elements, the synaptic vesicle protein p65 extends into the peripheral domain and in some cases, extends into filopodia . Many of these transitional elements have active filopodia but show no movement over the substrate for periods of up to nine days . These transitional elements have lost the actin-rich peripheral domain of the growth cone but retain actin labelling in the filopodia . With electron microscopy, transitional elements were seen to contain accumulations of synaptic vesicles at the site of contact with the substrate . Electron microscopic immunocytochemistry showed these synaptic vesicles labelled for p65 with silver-developed gold particles . Thus, transitional elements have characteristics of both growth cones and presynaptic terminals, suggesting that they may also have functional attributes of both growth cones and presynaptic elements. Cell Mol Neurobiol, 1991 Feb, 11(1), 191 - 201 Two types of asymmetric acetylcholinesterase in chick hindlimb muscle: developmental profiles, in vivo and in cell culture, and recovery after inactivation; Busquets X et al.; 1 . We have analyzed the behavior of two types of asymmetric molecular forms (A forms) of acetylcholinesterase (AChE) during development of chick hindlimb muscle, in vivo and in cell culture, and upon irreversible inactivation of peroneal muscle AChE with diisopropylfluorophosphate (DFP) in vivo . 2 . In agreement with previous developmental studies on chick muscle, globular forms of AChE (G forms) are predominant in chick hindlimb at early embryonic ages, being gradually replaced by A forms as hatching (and, therefore, onset of locomotion) approaches . Of the two A-form types, AI appears and accumulates significantly earlier than AII, so that A/G and II/I ratios higher than 1 are attained only at about hatching time . 3 . Cultures prepared from 11-day chick embryo hindlimb myoblasts express both types of A forms, with a combined activity of 27% of total AChE after 12 days in culture . AI forms appear again earlier and are much more abundant than type II asymmetric species through the life span of cultures . 4 . All AChE activity in the peroneal muscle is irreversibly inactivated by injection of DFP in vivo . The recovery of A forms follows the same sequence described for normal development, with a delayed and slower recovery of AII forms as compared with AI . 5 . Several hypotheses involving tail polypeptides or tissue target molecules, or posttranslational interconversion, are proposed to help explain the earlier appearance and accumulation of AI forms in chick muscle. Am J Trop Med Hyg, 1991 Feb, 44(2), 131 - 4 Studies on Japanese-produced chick embryo cell culture rabies vaccines; Arai YT et al.; We studied the potency, antibody response, and side reactions of commercial Japanese chick embryo cell (CEC) rabies vaccines for humans . The CEC rabies vaccines had indexes of 10(5.1) and 10(6.0) in Habel tests, and have had potencies higher than those of the International Reference Vaccine II by National Institutes of Health (NIH) tests . Thirty healthy adults received 1 ml of the CEC rabies vaccines subcutaneously as primary immunization on days 0 and 7 . Between six and 12 months after the primary immunization, 22 of the 30 subjects showed neutralizing antibody levels greater than or equal to 1:40 by the rapid fluorescent focus inhibition test (RFFIT) . The 30 subjects had been given booster immunizations of the CEC rabies vaccines at 8-14 months in addition to the primary immunization . Six to 12 months after the booster immunizations, 27 of the 30 subjects showed antibody levels greater than or equal to 1:40 . No severe side reactions were reported during the course of vaccination . Thus we conclude that CEC rabies vaccine is effective and safe for pre-exposure immunization. Br J Haematol, 1991 Feb, 77(2), 195 - 200 A fibroblast cell culture model to study vitamin K metabolism and the inhibition of vitamin K epoxide reductase by known and suspected antagonists; Ross PJ et al.; The metabolism and antagonism of vitamin K has been studied in cultured fibroblasts . Monolayers of 3T3 mouse fibroblasts (grown in the absence or presence of warfarin or other putative antagonists) were incubated for 24 h with {1',2'-3H2}phylloquinone (K1) or {1',2'-3H2}phylloquinone epoxide (K1O), the cells harvested and lipid extracts fractionated by high performance liquid chromatography . {3H}K1 was converted to {3H}K1O (about 20% of {3H} lipids) and to unidentified polar metabolites (30%) . {3H}K1O was converted to {3H}K1 (3%) and to polar metabolites (50%) . Cells grown with warfarin showed a marked increase in the {3H}K1O:K1 ratio and in the proportion of polar metabolites . The metabolic interconversion of K1 and K1O and inhibitory response to warfarin provide evidence for a fibroblast pathway analogous to the vitamin K-epoxide cycle in the liver . From the K1O:K1 ratios it was possible to grade the antagonism of vitamin K epoxide reductase activity by known and suspected inhibitors . Inhibitory ratios were seen for racemic warfarin down to 10(-8) M . S-warfarin was a more potent antagonist than the R-enantiomer . Consistently low K1O:K1 ratios were observed for N-methyl-thiotetrazole and antibiotics with (moxalactam) or without (cefotaxime) this side chain suggesting that none of these compounds are direct inhibitors of vitamin K epoxide reductase . Fibroblasts grown in cell culture provide a useful model to study the extrahepatic role of vitamin K and the mode of action of vitamin K antagonists. Mutat Res, 1991 Feb, 246(2), 285 - 300 Recommended protocols based on a survey of current practice in genotoxicity testing laboratories: III . Cell transformation in C3H/10T1/2 mouse embryo cell, BALB/c 3T3 mouse fibroblast and Syrian hamster embryo cell cultures; Dunkel VC et al.; A standardized protocol and guidelines for the performance of cell transformation testing in mouse embryo (C3H/10T1/2), mouse fibroblast (BALB/c 3T3) and Syrian hamster embryo (SHE) cells have been developed . The protocol is based primarily on current laboratory practices as determined by responses to a detailed questionnaire completed by North American and European governmental, university and contract laboratories involved with cell transformation experimentation . This report identifies those modifications to previously described methodologies which are being used on a regular basis and also serves to clarify confusing or inconsistent practices. J Parasitol, 1991 Feb, 77(1), 126 - 32 Examination of tissue cyst formation by Toxoplasma gondii in cell cultures using bradyzoites, tachyzoites, and sporozoites; Lindsay DS et al.; Tissue cyst formation by a goat isolate (GT-1) of Toxoplasma gondii was examined in bovine monocyte, human fetal lung, and Madin-Darby bovine kidney cell cultures . Transmission electron microscopy (TEM) and cat feeding studies indicated that tissue cysts were present in all 3 cell lines examined . Tissue cysts were first seen 3 days postinoculation (PI) using TEM . Standard cell culture procedures were used and no additional condition was needed to induce tissue cyst formation . Cats fed cell cultures excreted T . gondii oocysts in their feces 5-7 days PI . These oocysts caused lethal infections in mice . Tissue cysts were produced in cell cultures regardless if the initiating inoculum consisted of bradyzoites, sporozoites, or a mixture of bradyzoites and tachyzoites . Tissue cyst formation has been followed through 40 subpassages of infected cells . By TEM tissue cysts still were present after 40 passages, but when 40th-passaged cultures were fed to cats, oocytsts were not excreted . This indicates that the parasite had become oocystless after repeated passage in vitro. J Cell Biol, 1991 Feb, 112(3), 501 - 13 Gene expression and extracellular matrix ultrastructure of a mineralizing chondrocyte cell culture system; Gerstenfeld LC et al.; Conditions were defined for promoting cell growth, hypertrophy, and extracellular matrix mineralization of a culture system derived from embryonic chick vertebral chondrocytes . Ascorbic acid supplementation by itself led to the hypertrophic phenotype as assessed by respective 10- and 15-fold increases in alkaline phosphatase enzyme activity and type X synthesis . Maximal extracellular matrix mineralization was obtained, however, when cultures were grown in a nutrient-enriched medium supplemented with both ascorbic acid and 20 mM beta-glycerophosphate . Temporal studies over a 3-wk period showed a 3-4-fold increase in DNA accompanied by a nearly constant DNA to protein ratio . In this period, total collagen increased from 3 to 20% of the cell layer protein; total calcium and phosphorus contents increased 15-20-fold . Proteoglycan synthesis was maximal until day 12 but thereafter showed a fourfold decrease . In contrast, total collagen synthesis showed a greater than 10-fold increase until day 18, a result suggesting that collagen synthesis was replacing proteoglycan synthesis during cellular hypertrophy . Separate analysis of individual collagen types demonstrated a low level of type I collagen synthesis throughout the 21-d time course . Collagen types II and X synthesis increased during the first 2 wk of culture; thereafter, collagen type II synthesis decreased while collagen type X synthesis continued to rise . Type IX synthesis remained at undetectable levels throughout the time course . The levels of collagen types I, II, IX, and X mRNA and the large proteoglycan core protein mRNA paralleled their levels of synthesis, data indicating pretranslational control of synthesis . Ultrastructural examination revealed cellular and extracellular morphology similar to that for a developing hypertrophic phenotype in vivo . Chondrocytes in lacunae were surrounded by a well-formed extracellular matrix of randomly distributed collagen type II fibrils (approximately 20-nm diam) and extensive proteoglycan . Numerous vesicular structures could be detected . Cultures mineralized reproducibly and crystals were located in extracellular matrices, principally associated with collagen fibrils . There was no clear evidence of mineral association with extracellular vesicles . The mineral was composed of calcium and phosphorus on electron probe microanalysis and was identified as a very poorly crystalline hydroxyapatite on electron diffraction . In summary, these data suggest that this culture system consists of chondrocytes which undergo differentiation in vitro as assessed by their elevated levels of alkaline phosphatase and type X collagen and their ultrastructural appearance.(ABSTRACT TRUNCATED AT 400 WORDS) Int J Radiat Oncol Biol Phys, 1991 Feb, 20(2), 213 - 6 Radiation induction of drug resistance in RIF-1: correlation of tumor and cell culture results; Moulder JE et al.; The RIF-1 tumor line contains cells that are resistant to various anti-neoplastic drugs, including 5-fluorouracil (5FU), methotrexate (MTX), adriamycin (ADR), and etoposide (VP16) . The frequency of these drug-resistant cells is increased after irradiation . The frequency of drug-resistant cells and the magnitude of radiation-induced drug resistance are different in cell culture than in tumors . The dose-response and expression time relationships for radiation induction of drug resistance observed in RIF-1 tumors are unusual.We hypothesize that at high radiation doses in vivo, we are selecting for cells that are both drug resistant and radiation resistant due to microenvironmental factors, whereas at low radiation doses in vivo and all radiation doses in vitro, we are observing true mutants . These studies indicate that there can be significant differences in drug-resistance frequencies between tumors and their cell lines of origin, and that radiation induction of drug resistance depends significantly on whether the induction is done in tumors or in cell culture . These results imply that theories about the induction of drug resistance that are based on cell culture studies may be inapplicable to the induction of drug resistance in tumors. Dev Biol, 1991 Feb, 143(2), 320 - 34 Skeletal muscle satellite cell diversity: satellite cells form fibers of different types in cell culture; Feldman JL et al.; Following skeletal muscle injury, new fibers form from resident satellite cells which reestablish the fiber composition of the original muscle . We have used a cell culture system to analyze satellite cells isolated from adult chicken and quail pectoralis major (PM; a fast muscle) and anterior latissimus dorsi (ALD; a slow muscle) to determine if satellite cells isolated from fast or slow muscles produce one or several types of fibers when they form new fibers in vitro in the absence of innervation or a specific extracellular milieu . The types of fibers formed in satellite cell cultures were determined using immunoblotting and immunocytochemistry with monoclonal antibodies specific for avian fast and slow myosin heavy chain (MHC) isoforms . We found that satellite cells were of different types and that fast and slow muscles differed in the percentage of each type they contained . Primary satellite cells isolated from the PM formed only fast fibers, while up to 25% of those isolated from ALD formed fibers that were both fast and slow (fast/slow fibers), the remainder being fast only . Fast/slow fibers formed from chicken satellite cells expressed slow MHC1, while slow MHC2 predominated in fast/slow fibers formed from quail satellite cells . Prolonged primary culture did not alter the relative proportions of fast to fast/slow fibers in high density cultures of either chicken or quail satellite cells . No change in commitment was observed in fibers formed from chicken satellite cell progeny repeatedly subcultured at high density, while fibers formed from subcultured quail satellite cell progeny demonstrated increasing commitment to fast/slow fiber type formation . Quail satellite cells cloned from high density cultures formed colonies that demonstrated a similar change in commitment from fast to fast/slow, as did serially subcloned individual satellite cell progeny, indicating that the observed change from fast to fast/slow differentiation resulted from intrinsic changes within a satellite cell . Thus satellite cells freshly isolated from adult chicken and quail are committed to form fibers of at least two types, satellite cells of these two types are found in different proportions in fast and slow muscles, and repeated cell proliferation of quail satellite cell progeny may alter satellite cell progeny to increasingly form fibers of a single type. Endocrinology, 1991 Feb, 128(2), 937 - 43 Growth hormone regulation in primary fetal and neonatal rat pituitary cell cultures: the role of thyroid hormone; Ezzat S et al.; GH is first detectable in the fetal rat pituitary between gestational days 18 and 19 . The reasons for the GH surge soon after birth and subsequent postnatal decline to adult levels remain unclear . We therefore determined whether GH gene regulation in the developing pituitary could be distinguished from adult rat somatotroph function . In primary cultures of fetal and neonatal rat pituitary cells, GH secretion was detected by the 20th gestational day . These cells were stimulated by GH-releasing hormone (GHRH), but not by T3 or the morphogen retinoic acid . The stimulatory effect of T3 (0.25 mM) on GH secretion was detected only on the 2nd neonatal day and was similar to that seen in mature rat pituitary cell cultures . GHRH (10 nM) treatment for 24 h caused a 5-fold induction of GH secretion in pituitary cells derived from 2-, 5-, and 12-day-old neonatal rats . The presence or absence of T3 in the culture medium did not alter the response to GHRH . In contrast, only 2-fold induction of GH was observed in adult male pituitary cells during the same time course . Insulin-like growth factor-I (IGF-I; 6.5 nM), the peripheral target hormone for GH, resulted in a modest (20%) attenuation of GH secretion from pituitary cells derived from 20-day-old fetuses . IGF-I, however, produced a 70% reduction in GH levels in adult male pituitary cells grown under similar conditions . The effects of IGF-I on adult pituitary cells grown in T3-depleted medium were blunted . Addition of T3 partially restored the responsiveness of these cells to IGF-I . The results suggest that the high circulating GH levels in the fetal and neonatal rat may be secondary to relative insensitivity of the immature somatotroph to the inhibitory actions of IGF-I in addition to enhanced responsiveness to GHRH compared with the adult rat pituitary . Relative thyroid hormone deficiency in the immature rat may be contributory to this early transient state of pituitary IGF-I resistance. Zentralbl Veterinarmed A, 1991 Feb, 38(1), 49 - 53 Concentrations and patterns of released pterins of various animal cell cultures; Goldberg M et al.; Neopterin, biopterin, pterin, 6-hydroxymethylpterin and isoxanthopterin were determined in the supernatants of various animal cell cultures . The different cell lines showed distinct variations in the concentrations as well as in the pattern and total amount of the released pterins . In cells from the same organ but from diverse species differences in pattern and release were found . Cell lines derived from primates had a high release of neopterin . A further difference resulted from the origin of fetal or adult organs . Neoplastic cell lines showed different patterns of pterins, dependent on the type of tumour . Primary cultures from embryonic bovine lung had the highest total amount of released pterins in all cell lines examined. Biotechnol Appl Biochem, 1991 Feb, 13(1), 120 - 6 Purification of factor VIII:c coagulant activity from rat liver nonparenchymal cell culture medium by immobilized metal ion affinity chromatography; Mantovaara T et al.; The purification of factor VIII:c coagulant activity on the basis of its affinity for calcium is described . For this purpose, use was made of a recently introduced chelating matrix, i.e., carboxymethylated aspartic acid agarose, coupled with calcium--thereby creating a gel with specificity comparable with biospecific affinity chromatography . In a single step factor VIII:c activity was purified from rat liver nonparenchymal cell culture medium with a purification factor of 85-fold . The material exhibits a single band on polyacrylamide gel electrophoresis. Mol Pharmacol, 1991 Feb, 39(2), 177 - 83 Stable expression of two human UDP-glucuronosyltransferase cDNAs in V79 cell cultures; Fournel-Gigleux S et al.; Two human liver UDP-glucuronosyltransferase cDNA clones (HLUGP1 and HLUG25) were individually inserted into the eukaryotic expression vector pKCRH2 . Each recombinant plasmid was cotransfected with a SFVneo vector, thereby allowing establishment of several V79 cell lines retaining the exogenous UDP-glucuronosyltransferase cDNA after selection with G418 (Geneticin) . Southern blot analysis suggested that the cDNAs were integrated into the host cell genome . Northern blot and immunoblot analyses indicated that the cDNAs were correctly transcribed and translated for the production of functional enzymes . The established recombinant V79 cell lines stably expressed the UDP-glucuronosyltransferase activities towards 1-naphthol (HLUGP1) and hyodeoxycholic acid (HLUG25) at levels 10-20-fold higher than with transient expression, and in the range found in human liver . These high levels of expression of UDP-glucuronosyltransferase activity allowed the determination of apparent kinetic constants and substrate specificities of glucuronidation in the genetically engineered cell lines . HLUG25 cDNA encoded an isoform with restricted specificity towards the 6-OH group of the bile acid hyodeoxycholic acid . The other steroids, bile acids, endobiotics, and xenobiotics tested as substrates were glucuronidated in various samples of human liver microsomes, but not by this isoenzyme . This study, allowing the expression of individual UDP-glucuronosyltransferases in heterologous cells with no endogenous transferases, offered a unique solution for the characterization of UDP-glucuronosyltransferase functional heterogeneity. J Virol, 1991 Feb, 65(2), 938 - 44 The open reading frames UL3, UL4, UL10, and UL16 are dispensable for the replication of herpes simplex virus 1 in cell culture; Baines JD et al.; By means of insertion and deletion mutagenesis, we have constructed four herpes simplex virus 1 recombinants, each lacking most sequences encoding a different open reading frame . The deleted genes are located in the unique sequences of the long component and include those designated UL3, UL4, UL10, and UL16 . The recombinant virus R7211 lacks 579 of the 696 bp of UL3 . The recombinant virus R7217 lacks 307 of the 597 bp of the UL4 open reading frame . R7216 contains a 972-bp deletion within the 1,419-bp open reading frame of UL10, whereas R7210 lacks 988 bp of the 1,119-bp UL16 open reading frame . Growth curves indicated that the yields of these viruses in Vero and BHK cell cultures were only slightly reduced from or in some instances equivalent to that of the parent virus . The function of the gene products is not known . It is of interest to note that (i) the UL16 open reading frame maps entirely within the single intron of UL15 and (ii) on the basis of the extent and size of hydrophobic domains, the UL3 and UL10 gene products were predicted to be membrane proteins. Am J Med Genet, 1991 Feb-Mar, 38(2-3), 447 - 52 Improved prenatal detection of fra(X)(q27.3): methods for prevention of false negatives in chorionic villus and amniotic fluid cell cultures; Jenkins EC et al.; The reliable detection of fra(X)(q27.3) in prenatal samples is important for providing genetic counseling . We have identified 5 new cases of prenatal fragile X {fra(X)} detection in 3 chorionic villus sample (CVS) and 2 amniotic fluid (AF) cell cultures . In 4 of the 5 cases, either excess thymidine (THY) or a combination of THY and 5-fluorodeoxyuridine (FUdR) was clearly superior to FUdR alone as fra(X) inducers . Amniocytes from one case were cultured only in RPMI-1640 and later exposed to FUdR or THY separately . They showed only 2% fra(X) while parallel cultures initiated in Chang medium and incubated in RPMI for at least 7 days (recovery) before fra(X) induction exhibited strikingly increased fra(X) frequencies . Chang medium alone will not allow fra(X) induction in AF (Jenkins EC, Brown WT {1986}: "Genetic Disorders and the Fetus: Diagnosis, Prevention and Treatment." New York: Plenum Press, pp 185-204) . Now, using CVS cells, we report that only 1% and 0% fra(X) were detected using FUdR or THY in cells cultured in RPMI for 4 days after removal from Chang medium . Cells with 7 days "recovery" in RPMI exhibited increases from 2 to 6% . Therefore, we have found that Chang medium is very helpful when the appropriate recovery time in another medium is allowed before fra(X) induction . Some false negative reports can be attributed to: induction in Chang medium alone; lack of sufficient recovery time after initiating cells in Chang before induction; and unavailability of the excess THY fra(X) induction system.(ABSTRACT TRUNCATED AT 250 WORDS) Rev Med Chil, 1991 Feb, 119(2), 164 - 8 {Comparison of immunofluorescence, enzyme immunoassay and cell culture for the diagnosis of Chlamydia trachomatis in urogenital infections}; Martinez A et al.; We evaluated the usefulness of the direct immunofluorescence test with monoclonal antibodies and the enzyme immunoassay in comparison with isolation in cell cultures for the diagnosis of Chlamydia trachomatis in 55 endocervical specimens from female prostitutes and 21 urethral specimens from men with diagnosis of nongonococcal urethritis . In comparison with culture, the enzyme immunoassay had a sensitivity of 100% and a specificity of 95% . The immunofluorescence test had a sensitivity of 92% and a specificity of 98% . The positive and negative predictive values for the enzyme immunoassay were 81% and 100% and for immunofluorescence 92% and 98% respectively . The immunologic methods appear to be satisfactory alternatives to culture for detecting C trachomatis in genital specimens in the studied populations. J Biochem (Tokyo), 1991 Feb, 109(2), 217 - 22 Characterization of monoclonal antibodies against human tissue plasminogen activator (tPA): quantitation of free tPA in human cell cultures by an ELISA; Kurokawa T et al.; Seven murine monoclonal antibodies produced against tissue plasminogen activator (tPA) were evaluated by means of enzyme-linked immunosorbent assays (ELISAs), and their effects on the enzymatic activities of tPA towards a synthetic substrate (S-2288) and plasminogen were investigated . One of the antibodies, TPA1-70, strongly inhibited the enzymatic activity of tPA in a fibrin agarose plate assay, while it did not affect the enzymatic activity towards the synthetic substrate or plasminogen . The antibody is directed to an epitope on the B-chain of tPA, which is necessary for the formation of a ternary complex of tPA, fibrin and plasminogen, but probably not to the active site . Another antibody, TPA2-14, partially inhibited the enzymatic activities of tPA towards the synthetic substrate and plasminogen, but it was not able to bind to the inactive tPA complexed with plasminogen activator inhibitor-1 (PAI-1) . The antibody is directed to an epitope on the second kringle region, which is probably one of the PAI-1 binding sites . This property of the antibody enabled us to develop an ELISA for selective quantitation of free tPA in culture media conditioned with several human cell lines . The results indicate that tPA in these media exists either partially or almost entirely in a complex with PAI-1. FEBS Lett, 1991 Jan 28, 278(2), 229 - 33 K+ channel expression in primary cell cultures mediated by vaccinia virus; Karschin A et al.; A recombinant vaccinia virus (VV) was used to express functional Drosophila Shaker H4 K+ channels in primary cell cultures from rat heart (atrial and ventricular myocytes, fibroblasts), autonomic ganglia (SCG neurons) and CNS (hippocampal neurons, cerebral astroglia) . In most cells the expressed currents possessed the typical characteristics of the native Drosophila muscle A currents; a few cells showed evidence of hetero-oligomers with new properties . The maximum current density corresponded to a channel density of 2-3/microns 2 . Voltage recordings in heart cells showed altered action potential waveforms after successful infection . VV vectors thus are useful for studying altered excitability and cell-specific processing of ion channel proteins. Brain Res Dev Brain Res, 1991 Jan 15, 58(1), 43 - 9 Thyroid hormone action: induction of morphological changes and protein secretion in astroglial cell cultures; Gavaret JM et al.; The effects of triiodothyronine (T3) on cell morphology and protein secretion were examined in astrocytes cultured in a chemically defined medium devoid of other hormones and growth factors . The flat polygonal astrocytic cells treated with T3 (1-50 nM) and maintained in non-renewed medium cultures were progressively transformed into process-bearing cells . These changes were initially observed 3 days after the end of T3 treatment and accounted for more than 50% of the cells 7-8 days thereafter . The proteins secreted by the T3-stimulated cells were analyzed on SDS-PAGE after cell labeling for 4.5 h with {35S}methionine . The effect of T3 on protein secretion was dose-dependent . Half-maximal stimulation was reached with 0.2-0.5 nM hormone and the proteins of 46, 59, 67, 78, 85 and 140 kDa were over-secreted (greater than 300% of control) . These results were only obtained when the cell medium was not renewed after T3 treatment. Biochem Biophys Res Commun, 1991 Jan 15, 174(1), 338 - 43 Endothelin-3 inhibits prolactin and stimulates LH, FSH and TSH secretion from pituitary cell culture; Kanyicska B et al.; The influence of endothelin-3 (ET-3) on anterior pituitary hormone secretion was investigated over a wide range of concentrations (from 10(-14) to 10(-6) M) and incubation times (from 4 to 48 hours) . ET-3 elicited a concentration-dependent inhibition of prolactin (PRL) secretion and stimulated the release of luteinizing hormone (LH), follicle stimulating hormone (FSH) and thyroid stimulating hormone (TSH) from primary monolayer cultures of anterior pituitary cells derived from female rats . The responsiveness of different pituitary cells to ET-3 differs markedly in terms of onset and duration: the maximal inhibition of PRL secretion occurred after 12 hours and the stimulation of LH, FSH and TSH reached the maximum after 4, 48 and 48 hours of incubation, respectively . These data corroborate the concept that ET-3 has an important role as a neuroendocrine modulator . Moreover, the data presented suggest different intracellular mechanisms underlying ET-3 actions. Cancer Res, 1991 Jan 15, 51(2), 696 - 706 Rat ovarian granulosa cell culture: a model system for the study of cell-cell communication during multistep transformation; Stein LS et al.; A spontaneously immortalized clonal granulosa cell line (SIGC) derived from primary rat ovarian granulosa cell cultures was developed as a model system to explore the process of transformation using an epithelial cell type . SIGC has an epithelial morphology and grows in culture without undergoing luteinization . The cell line is thought to represent an intermediate step in carcinogenesis because it seems to grow indefinitely in culture but does not form clones in soft agar or tumors in nude mice . Indirect immunofluorescence and Western blot analysis verified the constitutive expression of the recessive oncogene product p53 in the cell line, thereby suggesting a possible mechanism of immortalization . Ultrastructural studies indicated that SIGC cells are characterized by an undifferentiated phenotype with prominent intermediate filaments, desmosomes, and gap junctions . The identification of cytokeratin by indirect immunofluorescence and Western blot analysis suggests that SIGC functions as an epithelial cell type . Functional studies of cell-cell communication by a dye transfer technique (fluorescence recovery after photobleaching) showed reduced communication compared to normal primary granulosa cells in culture . SIGC cells were transfected with early region genes of SV40 virus in an attempt to generate fully transformed cell lines . The resulting cell line SV-SIGC expressed T-antigen, was anchorage independent, formed tumors in nude mice, and had reduced intercellular communication as compared to SIGC cells . Explants from the tumors in nude mice were used to generate another cell line (T-SV-SIGC), which exhibited further reduction in both the incidence and the rate of communication . These results clearly demonstrated a progressive loss of functional communication during multistep transformation of an ovarian cell type . These data demonstrate that this assay system based on an epithelioid cell type can be used to study the relationship between intercellular communication and the multistep process of carcinogenesis. Bone, 1991, 12(2), 81 - 7 Tartrate-resistant acid phosphatase is not an exclusive marker for mouse osteoclasts in cell culture; Modderman WE et al.; The method of Barka and Anderson was used for the demonstration of tartrate-resistant acid phosphatase (TRAcP) in cultures of bone marrow, spleen, lung, and peritoneal cells of the mouse . The staining was performed either in the usual way by adding both substrate (naphthol-AS-BI-phosphate) and coupler (hexazonium pararosanilin) together (the simultaneous-coupling technique) or by adding first the substrate and then the coupler (the post-coupling technique) . We measured TRAcP-activity fluorometrically after extraction of the product naphthol-AS-BI, using the same staining solution as in cytochemical method, but without the coupler . In bone marrow, spleen, lung, and peritoneal cell cultures a biochemically measurable TRAcP-activity was detected . Post-coupling generally gave a higher level of staining and larger numbers of TRAcP-positive cells than simultaneous-coupling . In bone marrow cultures macrophages, identifiable by their ability to phagocytose microspheres, became TRAcP-positive during culture . In lung cell cultures cells capable of phagocytosis of bacteria were shown to be TRAcP-positive . Peritoneal macrophages remained TRAcP-negative in the simultaneous-coupling technique . Using the post-coupling technique a small number stained TRAcP-positive . In spleen cell cultures TRAcP-positive cells containing hemosiderin were visible . In cultures of all four cell types, F4/80 positive cells staining also for TRAcP were present . F4/80 is a well known marker for macrophages, whereas osteoclasts are negative . In conclusion, mouse macrophages originating from various tissues can become TRAcP-positive in vitro . TRAcP activity alone is not a reliable marker for osteoclasts in bone marrow cultures. Zhonghua Yan Ke Za Zhi, 1991 Jan, 27(1), 16 - 8 {Gelatin matrix for corneal epithelial cell cultures}; Zhang L et al.; A simple and convenient gelatin matrix was prepared for epithelial cell cultures . Addition of fibronectin, laminin or fibrinogen enhanced the attachment and growth of corneal epithelial cells in vitro. Arch Anat Cytol Pathol, 1991, 39(1-2), 47 - 54 {Our experience in the use of epidermal cell cultures in the reconstructive surgery}; Abbes M et al.; The plastic surgery team of the Antoine-Lacassagne cancer center (Nice, France) presents the results of 18 grafts (13 patients) of epidermal cells grown in culture . Cell culture techniques and histologic features are analyzed . The possibilities of this method are discussed on the basis of study results and comparison with other publications on the repair of cutaneous wounds other than burns . The favorable rate of successful grafts (50%) and the indications for the technique are discussed . Used as a complementary procedure, epidermal cell grafts permit better quality scarring, comparable to that obtained with thin skin grafts . In addition, disfiguration of donor sites is eliminated. J Biomater Sci Polym Ed, 1991, 2(2), 81 - 9 Synthesis of protein-coated gelatin microspheres and their use as microcarriers for cell culture . Part I . Derivatization with native collagen; Altankov G et al.; A novel technique for the synthesis of native collagen (type I)-coated gelatin microspheres was developed using the emulsion-polymerization principle, which is realized in four steps: emulsification, separation, stabilization, and protein modification . A 20% gelatin solution was emulsified in sunflower oil and the resulting beads were polymerized with glutaraldehyde . The novelty of our technique consists in the protein modification step, where we derivatized the beads by saturation of the free aldehyde groups on the cross-linked gelatin in the native collagen solution (0.3 mg/ml in 0.05 M Tris, pH 8.6) and further stabilized the beads with sodium borohydride . The resulting microspheres exhibited excellent properties as microcarriers for in vitro cell culturing using Vero cells as the model system . In comparison with pure gelatin beads, the cells attached more rapidly and grew faster on native collagen-coated beads . Approximately 90% of the Vero cells attached to the microcarrier after 1 h . After a lag period of nearly 24 h, the cells began to grow rapidly and reached confluence for 4-5 days, whereas the cells on pure gelatin beads reached the same density about 1 or 2 days later. Life Sci, 1991, 48(20), 1945 - 51 Cytotoxic and aryl hydrocarbon hydroxylase-inducing effects of laboratory rodent diets . A cell culture study; Torronen R et al.; Extracts of several rodent diets were studied for their cytotoxic and aryl hydrocarbon hydroxylase-inducing properties by an in vitro method . The cell culture system based on a mouse hepatoma cell line (Hepa-1) was shown to be a convenient and sensitive method for screening of diets for these parameters implying the presence of compounds potentially harmful in vivo . Considerable differences among diets and batches were detected . Smallest effects were observed with a semipurified diet and with the unrefined diet which - contrary to other four unrefined diets - contained no fish. Prog Clin Biol Res, 1991, 364, 299 - 308 Cell culture model systems to study HDV expression, replication and pathogenesis; Gowans EJ et al.; Due to the transient nature of HDV replication in natural and experimental hosts and to the inability of the virus to replicate in easily handled cell lines, studies of the replication and expression of HDV have been impeded . These difficulties have been overcome by the development of the cell lines described in this paper. Neurotoxicology, 1991 Spring, 12(1), 33 - 46 Metabolism and toxicity of methyl iodide in primary dissociated neural cell cultures; Bonnefoi MS et al.; The metabolism and the toxicity of methyl iodide (Mel) has been studied in primary dissociated neuronal and glial murine cell cultures to further characterize the mechanisms of monohalomethane neurotoxicity . Measurement of intracellular glutathione (GSH) concentrations in cerebellar and cerebral cultures revealed GSH levels (21.6 +/- 1.9 and 29.1 +/- 1.9 nmol/mg protein, respectively) close to brain GSH levels measured in vivo . A GSH-depleting effect of Mel was demonstrated, with an ED50 for a 5 min exposure of 0.2 and 0.5 mM for glial and mixed (neurons + glia) cultures, respectively . Mel-induced GSH depletion was correlated with its neurotoxicity as the two powerful protective agents of monohalomethane toxicity, 3-amino-1-{m-(trifluoromethyl) phenyl}-2-pyrazoline (BW 755C, 1 mM) and nordihydroguaiaretic acid (NDGA, 10 microM) provided a 20-fold protection against depletion of GSH levels following Mel exposure . When glia and neurons from cerebral cultures were exposed in suspension to increasing concentrations of Mel for 30 min at 37 degrees C, a concentration-dependent increase in the production of formaldehyde resulted . Formaldehyde appeared to be an indicator of Mel metabolism as its production was decreased by sulfasalazine, a compound which was shown to be an inhibitor of the glutathione-S-transferases in this culture system . Since BW 755C and NDGA had no effect on formaldehyde production, while sulfasalazine as well as semicarbazide, a protective agent against formaldehyde-producing toxicants, failed to protect the cells against Mel toxicity, mechanism(s) of Mel neurotoxicity appeared independent of the GSH-mediated metabolism of this compound . It is concluded that GSH-mediated metabolic biotransformation is not necessary for the neurotoxicity of the monohalomethanes, that GSH depletion may act as a starting point in the chain of events leading to neural cell death, and that glia may be more sensitive than neurons to this primary effect . Moreover, these results demonstrate the value of primary dissociated neuronal cell cultures for studies of biochemical mechanisms of neurotoxicity. Diagn Microbiol Infect Dis, 1991 Jan-Feb, 14(1), 17 - 20 Comparison of the Kallested Pathfinder EIA, cytocentrifuged direct fluorescent antibody, and cell culture for the detection of Chlamydia trachomatis; LeBar W et al.; A total of 203 duplicate endocervical samples collected from patients at an adolescent health care center were tested for the presence of Chlamydia trachomatis by cell culture, Pathfinder enzyme immunoassay (EIA) (Kallestad) and cytocentrifuged direct fluorescent antibody (DFA) . Compared to cell culture, the Pathfinder assay demonstrated a sensitivity and specificity of 85.2% and 100%, whereas the DFA procedure demonstrated to be 92.6% sensitive and 99.4% specific. J Lipid Res, 1991 Jan, 32(1), 125 - 36 Plasma membrane sphingomyelin and the regulation of HMG-CoA reductase activity and cholesterol biosynthesis in cell cultures; Gupta AK et al.; We have examined the mechanism of the inhibition of cholesterol synthesis in cells treated with exogenous sphingomyelinase . Treatment of rat intestinal epithelial cells (IEC-6), human skin fibroblasts (GM-43), and human hepatoma (HepG2) cells in culture with sphingomyelinase resulted in a concentration- and time-dependent inhibition of the activity of HMG-CoA reductase, a key regulatory enzyme in cholesterol biosynthesis . The following observations were obtained with IEC-6 cells . Free fatty acid synthesis or general cellular protein synthesis was unaffected by the addition of sphingomyelinase . Addition of sphingomyelinase to the in vitro reductase assay had no effect on activity, suggesting that an intact cell system is required for the action of sphingomyelinase . The products of sphingomyelin hydrolysis, e.g., ceramide and phosphocholine, had no effect on reductase activity . Sphingosine, a further product of ceramide metabolism, caused a stimulation of reductase activity . Examination of the incorporation of {3H}acetate into the nonsaponifiable lipid fractions in the presence of sphingomyelinase showed no changes in the percent distribution of radioactivity in the post-mevalonate intermediates of the cholesterol biosynthetic pathway, but there was increased radioactivity associated with the polar sterol fraction . Pretreatment of cells with ketoconazole, a known inhibitor of oxysterol formation, prevented the inhibition of reductase activity by sphingomyelinase and decreased the incorporation of {3H}acetate in the polar sterol fraction . Ketoconazole had no effect on exogenous sphingomyelinase activity in vitro in the presence or absence of cells . Endogenous sphingomyelinase activity was also unaffected by ketoconazole . Addition of inhibitors of endogenous sphingomyelinase activity, e.g., chlorpromazine, desipramine, and N-(6-aminohexyl)-5-chloro-1-naphthalene sulfonamide (W-7), to the culture medium caused a dose-dependent stimulation of reductase activity . However, these agents had no effect on the inhibition of reductase activity by exogenous sphingomyelinase . Treatment of cells with small unilamellar vesicles of dioleyl phosphatidylcholine or high density lipoprotein3 resulted in increased efflux of cholesterol and stimulation of reductase activity . Under similar conditions, the inhibitory effect of exogenous sphingomyelinase on reductase activity was prevented by incubation with small unilamellar vesicles of phosphatidylcholine or high density lipoprotein . These results support the hypothesis that alteration of the ratio of sphingomyelin:cholesterol in the plasma membrane plays a modulatory role on the flow of membrane cholesterol to a site where it may be converted to a putative regulatory molecule, possibly an oxysterol. Cancer Res, 1991 Jan 1, 51(1), 282 - 7 Karyotypic changes associated with loss of prolactin dependency of rat Nb2 node lymphoma cell cultures; Horsman DE et al.; The parent line of cultured "Nb2 node" lymphoma cells is dependent on the hormone prolactin (PRL) for growth and is widely used for the in vitro bioassay of lactogenic hormones . As reported previously, PRL-independent sublines have been developed in vitro from the parental line by lactogen deprivation . The present study describes the G-banded karyotypes of the Noble (Nb) strain of rat (in which the original lymphoma developed), the PRL-dependent cell line (157th generation), and two of its PRL-independent sublines (1220th and 2372nd generations) . The karyotype of the Nb rat was determined to be the same as that of Rattus norvegicus . The stemline karyotype of the PRL-dependent cells contains a number of well-defined chromosomal abnormalities . The PRL-independent sublines examined have the same chromosomal abnormalities as the PRL-dependent cells plus a few additional changes indicative of clonal evolution from the PRL-dependent stemline . The development of PRL independence (as seen in the 1220th generation) was associated with only two karyotypic changes, i.e., loss of the Y chromosome and a translocation involving chromosomes 14 and 17 . The recently reported mapping of the rat PRL gene and other PRL-related genes to chromosome 17 suggests that rearrangement of chromosome 17 could be involved in the development of the PRL independence . The PRL-dependent and PRL-independent Nb2 cell lines provide a useful system for studying chromosomal and molecular genetic events associated with the malignant progression of polypeptide hormone-dependent cancers. Am J Pathol, 1991 Jan, 138(1), 69 - 82 Mature eosinophils stimulated to develop in human cord blood mononuclear cell cultures supplemented with recombinant human interleukin-5 . Part I . Piecemeal degranulation of specific granules and distribution of Charcot-Leyden crystal protein; Dvorak AM et al.; Human cord blood mononuclear cells were cultured for 35 days in media containing recombinant human interleukin 5 (rhIL-5) supplemented with a fraction of the culture supernatant of phytohemagglutinin (PHA)-stimulated human T lymphocytes from which interleukin 2 (IL-2) was eliminated . Cultured cells were studied by electron microscopy and an immunogold procedure to detect subcellular site(s) of Charcot-Leyden crystal (CLC) protein . The majority of cells (greater than 70%) developing in this system were mature eosinophils, with descending frequency of other cells, including macrophages, mature basophils, eosinophilic myelocytes, and mature neutrophils . Mature eosinophils were characterized by increased numbers of primary granules, small granules, tubulovesicular structures, and decreased secondary granules . These eosinophils showed extensive piecemeal degranulation (PMD) characterized by partially empty and empty secondary granule chambers in the cytoplasm . Small, smooth vesicles were evident within empty granule chambers as well as adjacent to them . Eosinophils formed close associations with phagocytic macrophages that contained both standard-shaped and irregularly shaped CLC within phagolysosomes . Subcellular sites of CLC protein were demonstrated by immunogold in eosinophils and macrophages arising in these cultures . Charcot-Leyden crystal protein was present in the nuclear matrix and extraorganellar cytoplasm of eosinophils . Primary granules and some cytoplasmic vesicles were labeled for CLC protein, but full and empty secondary granules and the extensive network of tubulovesicles were not . Charcot-Leyden crystals were absent from eosinophils, nor were they present in the extracellular space . Charcot-Leyden crystals were absent from eosinophils, nor were they present in the extracellular space . Charcot-Leyden crystals within phagosomes of macrophages were labeled by the immunogold procedure for CLC protein . These results demonstrate that rhIL-5-supplemented, PHA-stimulated, T-cell-conditioned media induced the development of mature human eosinophils from cord blood cells . These eosinophils underwent PMD of secondary granule contents . Immunogold analysis showed eosinophil CLC protein in the cytoplasm, nucleus, and primary granules of eosinophils . Macrophages also were present in these cultures . They contained CLC protein-containing crystals in their phagosomes, suggesting active sequestration of eosinophil CLC protein by macrophages in vitro. Arch Toxicol, 1991, 65(4), 344 - 7 Prostaglandin H synthase dependent metabolism of diethylstilbestrol by ram seminal vesicle cell cultures; Foth J et al.; Prostaglandin H synthase (PHS) peroxidase dependent metabolic activation has been suggested to play a role in mediating adverse effects of various carcinogens . Recently, we derived a cell line from ram seminal vesicles (SEMV cells) to conduct studies on the PHS-mediated metabolism of estrogens and xenobiotics in intact cells with the goal of relating this to an endpoint for genotoxicity inducible in this in vitro model . The present paper describes the drug-metabolizing capability of SEMV cells which has been investigated using radiolabeled diethylstilbestrol (DES) and analysing culture extracts by means of reverse phase HPLC with on-line radioactivity detection and after enzymatic hydrolysis of conjugate fractions . The synthetic estrogen DES is converted to sulfate conjugates and to the oxidative metabolite Z,Z-dienestrol (Z,Z-DIES) in a time-dependent manner . Compounds expected to modulate PHS-dependent co-oxidation of DES increased (arachidonic acid) or inhibited (indomethacin) Z,Z-DIES formation of SEMV cells in culture . A comparison of rates of arachidonic acid turnover to prostaglandins on the one hand and DES oxidation on the other reveals that DES is oxidized despite the presence of competing endogenous cosubstrates of PHS peroxidase . The results clearly indicate that SEMV cells catalyze PHS-dependent oxidation of DES as well as carrying out phase II metabolism in the absence of detectable monooxygenase activity . These features and recent data showing that DES can induce micronuclei in SEMV cells makes them an attractive model for further investigations of the role of PHS in mediating the genotoxicity of DES and other xenobiotics. Arch Insect Biochem Physiol, 1991, 18(3), 169 - 75 Stimulation of embryonic development in Microplitis croceipes (Braconidae) in cell culture media preconditioned with a fat body cell line derived from a nonpermissive host, gypsy moth, Lymantria dispar; Ferkovich SM et al.; A cell culture medium, IPL-52B, was preconditioned with host fat body and two insect cell lines to determine if they would support embryonic development of Microplitis croceipes in vitro . The medium was preconditioned with the cell line IPL-LdFB, derived from fat body of the gypsy moth, Lymantria dispar, cell line IAL-TND1, derived from imaginal discs of the cabbage looper, Trichoplusia ni, and whole fat body tissue from host Helicoverpa zea . A second cell culture medium, Excell 400, was preconditioned with only the cell line, IPL-LdFB . Pregerm band eggs were dissected from third instar host larvae and incubated in the conditioned medium for 20 h . Newly laid parasitoid eggs did not develop in unconditioned IPL-52B, but did develop to germ band stage in unconditioned Excell 400 . The IPL-52B medium conditioned with both cell lines induced germ band formation, but only the L . dispar cell line (IPL-LdFB) promoted significant development to eclosion comparable to host far body tissue . Excell 400 medium preconditioned with the cell line, IPL-LdFB also supported development to eclosion. Eur J Basic Appl Histochem, 1991, 35(3), 219 - 31 Growth and differentiation of myogenic clones from adult human muscle cell cultures; Meola G et al.; Clonal cultures only recently have been applied to normal and pathological human muscle, but detailed clonal analysis and differentiative properties of individual long term muscle subclones derived from adult normal human muscle cell (HMC) cultures have not been reported . In this study we compared the growth potential by plating efficiency (PE), muscle colony differentiation (MC) and growth cuvers and the differentiative properties by fusion index, dystrophin localization, creatine kinase (CK) total activity and isozyme electrophoresis in HMC cultures derived myogenic subclones . These properties were tested in two experimental culture systems (200 cells/dish versus single cell/well) and with two tissue culture media (standard medium--MM--versus conditioned medium--CM) . We found a significant high PE and MC in clonal cells established with single cell/well and grown in conditioned medium . In derived subclones we observed two classes of myogenic cells: one characterized by exponential growth kinetic, branched myotubes with high fusion index and predominantly sarcolemmal distribution of dystrophin and MM band at CK electrophoresis; the other with flat growth curve, low fusion index and low CK total activity . These findings demonstrate the variability of the expression of myogenic potentials in cells cloned from adult normal HMC cultures and represent an important tool for comparing the various cell types present in normal and diseased human muscle and for transplantation of normal myoblasts in Duchenne Muscular Dystrophy. Vestn Akad Med Nauk SSSR, 1991, (6), 49 - 51 {Contamination of fish tissue cell cultures by Mycoplasma}; Rakovskaia IV et al.; The inoculated and primary cell cultures of fish (carp, salmon, and sturgeon) have been studied . Acholeplasma typed as A . laidlawii in terms of its biochemical properties shown in inhibited metabolism has been isolated from 19 samples . The authors consider the source of Acholeplasma-induced contamination of the cell cultures under study. Tissue Cell, 1991, 23(4), 427 - 35 Immunocytochemical localization of laminin in hamster tracheal epithelial cell cultures; Moller PC et al.; EM examination of 28 day cultures of enzymatically dissociated hamster tracheal epithelial (HTE) cells grown on collagen coated millipore filters reveals that fragments of basal lamina may be present at the basal plasmalemma . Since the basal lamina consists of several major components including type IV collagen, heparan sulfate proteoglycans, entactin/nidogen, and laminin, questions naturally arise concerning the presence of such a structure in this cell culture system . When immunocytochemical procedures utilizing anti-laminin antibody and PAP techniques are carried out with paraffin sections of HTE culture at 1,2,3, and 4 weeks in vitro, LM analysis reveals that a thin, dense line of reaction product is present between the basal surface of the HTE cells and the underlying collagen substrate . Immunoblotting evaluation carried out with supernatants of 7d HTE cell homogenates and HTE cell conditioned media also indicate that laminin is being produced by the tracheal cells . Thus, the presence of basal lamina-like fragments, the immunocytochemical localization of laminin, and immunoblot identification of laminin in hamster tracheal epithelial cell cultures, suggest that, although basal lamina components may be produced by HTE cells, at the time points tested, they are not yet being organized into a complete basal lamina. Vopr Virusol, 1991 Jan-Feb, 36(1), 37 - 40 {The formation of infectious scrapie-like structures in the persistence of the agent of amyotrophic leukospongiosis in a brain cell culture}; Poleshchuk NN et al.; Electron microscopic analysis of specimens from guinea-pig brain cell cultures infected with amyotrophic leucospongiosis agent (belonging to "unconventional" viruses) revealed accumulation in the culture fluid of abnormal filamentous structures similar to scrapie-associated fibrils (SAF) differing in morphology . Most of these SAF-like structures 10-15 nm in diameter contained helically wound protofilaments with a repeat at certain intervals (50-150 nm) . When these structures were inoculated into guinea-pig brain astrocyte cultures they produced dystrophic-destructive changes in some (25%) astrocytes, and their intracerebral inoculation to guinea pigs produced an experimental disease . The abnormal SAF-like structures were reisolated from the brains of the inoculated animals which indicated the relationship between these structures and infectivity. Viral Immunol, 1991 Spring, 4(1), 43 - 52 Stimulation of adherent cells by addition of purified proteins of viral hemorrhagic septicemia virus to trout kidney cell cultures; Estepa A et al.; Purified proteins of the virus causing viral hemorrhagic septicemia in the trout were added to cultures on semisolid medium of leukocytes obtained from either healthy or immunized rainbow trout . Adherent cells were specifically stimulated by the glycoprotein of the viral spikes and, to a lesser extent, by the nucleoproteins . In contrast, a specific memory response was associated more with the nucleoproteins than with the glycoprotein when leukocytes from trout immunized with the virus were employed . These results suggest the necessity of employing both proteins in subunit vaccination trials and the possibility of using this assay to select the proper epitopes for genetically engineered proteins during subunit vaccine development. Ann N Y Acad Sci, 1991, 625, 793 - 801 Formation of a 37 kilodalton liver protein-acetaldehyde adduct in vivo and in liver cell culture during chronic alcohol exposure; Lumeng L et al.; With the use of antibodies that can recognize acetaldehyde adducts and the application of various immunological techniques, several protein-AAs have now been shown to form in vivo during chronic alcohol ingestion . These protein-AAs include the 37-kDa liver protein-AA, the CytP450IIE1-AA, hemoglobin-AA, two serum protein-AAs with molecular weights of 50 kDa and 103 kDa, and collagen type I protein-AA in liver . If acetaldehyde is the agent responsible for alcoholic liver injury, acetaldehyde toxicity in chronic alcohol ingestion must be linked to the ability of acetaldehyde to form adducts with proteins and perhaps other macromolecules . This is at least one mechanism of acetaldehyde-mediated liver injury . For proteins that serve critical functions, acetaldehyde adduct formation may alter their functions and thereby produce organ damage . Acetaldehyde adduct formation can also elicit humoral or cytotoxic immune responses and these responses may also lead to organ injury. Ann Rech Vet, 1991, 22(2), 201 - 9 Detection of African swine fever virus by a biotinylated DNA probe: assay on cell cultures and field samples; Petit F et al.; African swine fever virus was detected in various samples using a molecular hybridization technique . A fragment located in a constant area of the viral genome was biotin-labelled . This probe, when present at a concentration of 100 ng/ml of the hybridization solution, could detect 10 pg of target DNA immobilized on nitrocellulose with cellular DNA and RNA . The virus was evidenced after being passaged on monkey kidney cells, either 8 h post-inoculation (pi) if the multiplicity of infection (MOI) was at least 1 hemadsorbing unit (HAd) per cell, or 24 h later if the inoculum was diluted up to 10(-3) HAd per cell . When passaged on pig leukocytes with a MOI of 0.1 HAd per cell, the virus was evidenced 12 h pi, or 24 h pi with a MOI of 10(-2) HAd per cell . The probe did not hybridize with another DNA virus passaged on cells, neither did it react with non-infected blood or ham, but did so if African swine fever virus was resuspended with the samples . The spleen from uninfected pig and the lymph nodes from a pig which had died from hog cholera were found to be negative, whereas the spleen from a pig which had died of African swine fever was positive . These samples were also tested with a 32P-labelled probe whose sensitivity was 10-fold higher . A non-radioactive probe could be used both for the sensitive and specific diagnosis of African swine fever and the detection of the virus in an epidemiological survey. Neirofiziologiia, 1991, 23(3), 280 - 90 {The spontaneous synaptic activity in a cell culture of chick embryo spinal cord}; Mel'nik IV; Development of spontaneous synaptic activity was investigated during first 2.5 weeks in culture by means of the "hole-cell" version of the patch-clamp technique . Glycine-mediated giant postsynaptic currents (PSCs) were the most prominent property of the synaptic activity pattern . Excitatory PSCs were not observed until 14 days in culture . The role of culture conditions for the maturation of spontaneous activity has been shown . Possible mechanisms of the burst activity generation are discussed. Klin Khir, 1991, (5), 42 - 4 {Use of allograft of pancreatic islet cell cultures in complex treatment of acute purulent process in patients with diabetes mellitus}; Pavlovskii MP et al.; The results of treatment of 170 patients with diabetes mellitus and purulent wounds which appeared after opening the abscesses and phlegmons, and as well of 270 patients with gangrene of the lower extremities are presented . Early opening the abscess, improved quality of debridement of purulent wounds, and as well allotransplantation of pancreatic islet cell cultures (45 cases) permitted to stop progressing of the purulent process, place early secondary sutures, and in patients with the sutures placed, to achieve primary healing of the wounds. Tsitologiia, 1991, 33(1), 64 - 72 {The relationship of the saturation density of multilayer cell cultures to their mass exchange with the medium}; Akatov VS et al.; Chinese hamster fibroblasts (CHF) and NIH 3T3 cells were cultured on a glass substrate at different distances from the porous membrane separating the cells from the perfusing medium . It is shown that with perfusion of medium above the membrane there is no movement of the medium near the cells . In both the types of culture, the cells grow in multilayers, however the multilayer character of growth in CHF is more pronounced than in NIH 3T3 cells . The saturation density of the cultures depends on the cell-membrane separation, and at separations of no more than 0.2 mm exceeds the saturation density in the monolayer by 8-10 fold . The dependences of the saturation density on separation are different for CHE and NIH 3T3 cells, indicating qualitative differences in the inhibition of cell growth in monolayers between these cultures . The results obtained indicate that the inhibition of cell growth in monolayer is due to mass exchange limitations, rather than to intercellular contact interactions. Vopr Virusol, 1991 Jan-Feb, 36(1), 10 - 3 {The sensitivity of a number of cell cultures to different types of influenza viruses and their reassortants}; Danlybaeva GA et al.; The study of reproductive activity of human and animal influenza A, B, and C viruses as well as influenza A virus reassortants in some cell cultures allowed one to determine the range of cells susceptible for each type (subtype) of the viruses . Differences in the range of cells were demonstrated for different strains of influenza viruses of the same antigenic subtype . It was noted that reassortants of influenza A viruses with the same hemagglutinin subtypes as the parental strains had a wider range of susceptible cell lines and a higher reproductive capacity in these cells. J Pharmacol Exp Ther, 1991 Jan, 256(1), 402 - 11 Developmental time course and ionic dependence of kainate-mediated toxicity in rat cerebellar granule cell cultures; Kato K et al.; The mechanisms associated with the neurotoxic response caused by kainate (KA) were examined in cerebellar granule cell cultures . Under the conditions studied, millimolar concentrations of quisqualate, (RS)-amino-3-hydroxy-5-methylisoxazole-4-propionic acid, glutamate and N-methyl-D-aspartate did not cause significant cytolysis . In contrast, KA induced complete cell death, which was antagonized by 6,7-dinitroquinoxaline-2,3-dione, quisqualate, (RS)-amino-3-hydroxy-5-methylisoxazole-4-propionic acid and glutamate . This neurotoxic effect was dependent on the dose of KA and the age of the cultures . Two separate components of KA-induced neurotoxicity were observed and differentiated according to morphological changes, time of onset and ionic dependence . For acute neurotoxicity, release of lactate dehydrogenase measured after 30 min of KA exposure, became apparent between 8 and 11 days in culture and was dependent on both Cl- and Na+ . However, vulnerability to acute toxicity did not correlate with {3H}KA receptor expression with receptor-mediated Cl- influx . On the other hand, delayed toxicity, as determined by lactate dehydrogenase release 24 hr after KA exposure, was dependent on Cl- . This delayed neurotoxicity induced by KA shares time course features with N-methyl-D-aspartate-mediated toxicity . Yet in contrast to studies reported for N-methyl-D-aspartate, glutamate was ineffective as an agonist, measured by its ability to elicit a neurotoxic response, and the KA delayed response did not appear to be dependent upon the presence of extracellular Ca++, during the exposure to KA. Endocrinology, 1991 Jan, 128(1), 73 - 80 Dexamethasone and 1,25-dihydroxyvitamin D3 modulation of insulin-like growth factor-binding proteins in rat osteoblast-like cell cultures; Chen TL et al.; Using the method of Western ligand blot, we have found that the major form of insulin-like growth factor-binding protein (IGF-BP) secreted by rat osteoblastic-like cells in culture is a 31-kDa protein that is immunologically identical to BP-2, the binding protein originally identified in conditioned medium of Buffalo rat liver cells (BRL-3A) . Two minor forms of IGF-BPs with apparent mol wt of 24 kDa (BP-24) and 42 kDa (BP-42) have also been identified . The amount of IGF-BPs in serum-free conditioned medium increased 3-fold on day 3 compared to the day 1 level . We also studied the modulation of IGF-BPs by dexamethasone (DEX), 1,25-dihydroxyvitamin D {1,25-(OH)2D3}, and insulin-like growth factor-I (IGF-I) . DEX coordinately reduced the level of IGF-BPs in a dose- and time-dependent manner, which resulted in less than 10% of the BP-2 remaining at 100 nM . In contrast, 1,25-(OH)2D3 at 100 nM enhanced the amount of BP-2 by 1-fold . In combined treatments, 1,25-(OH)2D3 at 10 nM was unable to antagonize the inhibitory effect of DEX in the dose range of 1-10 nM . IGF-I, at 1 and 10 nM, proved to be a potent stimulator of all IGF-BPs, and at 10 nM, it completely reversed the inhibition by 100 nM DEX . Although the roles of IGF-BPs have not been clearly defined in bone cells, they are capable of modulating the biological actions of IGFs in other cell culture systems . Modulation of the IGF-BP level by DEX, 1,25-(OH)2D3, and IGF-I suggests important roles for these binding proteins in altering IGF-I action in rat osteoblast-like cell cultures. J Infect Dis, 1991 Jan, 163(1), 23 - 8 Characterization of herpes simplex virus type 2 latency-associated transcription in human sacral ganglia and in cell culture; Croen KD et al.; The ability of herpes simplex virus type 2 (HSV-2) to establish latency in and reactivate from sacral dorsal root sensory ganglia is the basis for recurrent genital herpes . The expression of HSV-2 genes in latently infected human sacral ganglia was investigated by in situ hybridization . Hybridizations with a probe from the long repeat region of HSV-2 revealed strong nuclear signals overlying neurons in sacral ganglia from five of nine individuals . The RNA detected overlaps with the transcript for infected cell protein O but in the opposite, or "anti-sense," orientation . These observations mimic those made previously with HSV-1 in human trigeminal ganglia and confirm the recent findings during latency in HSV-2-infected mice and guinea pigs . Northern hybridization of RNA from infected Vero cells showed that an HSV-2 latency-associated transcript was similar in size to the larger (1.85 kb) latency transcript of HSV-1 . Thus, HSV-1 and HSV-2 latency in human sensory ganglia are similar, if not identical, in terms of their cellular localization and pattern of transcription. Folia Morphol (Warsz), 1991, 50(1-2), 13 - 26 Influence of 3-indoleacetic acid on cytochemical changes in nuclei and cytoplasm of human fibroblasts in the cell culture; Kowalska E; The performed studies covered the action of 3-indoleacetic acid (3IAA) on human fibroblasts cultured in vitro . The 3IAA implemented in the studies was provided by Chemapol Firm (Czechoslovakia) . All the investigations were carried out on I passage fibroblasts, taken from the human skin in form of monolayer culture . Feulgen's method and autographic one were resorted to in the studies . The optimal time of acid hydrolysis was 10 min . Quantitative measurements of DNA in nuclei of fibroblasts were done on integrating cytophotometer . Incubation with labelled 3H uridine as RNA precursor was employed for demonstrating the activity of cell nucleus transcription . As concerns the quantitative changes in nucleic acids, it was disclosed that DNA and RNA synthesis was intensified . Nuclei were seen to appear with polydiploidal DNA amount in cultures being influenced by 3IAA action . The smallest number of mitoses was revealed in the cultures exposed to the action of the highest dose of the said compound . Stronger 3H uridine incorporation was recorded in cultures 24 and 72 hours after the administration of 10 mg/1000 ml of 3IAA . The results were presented in the form of diagrams, nucleinogram and tables. Acta Biol Hung, 1991, 42(1-3), 161 - 74 Degradation of intracellular endogenous proteins following serum deprivation in mammalian cell cultures: theoretical considerations of the role of very-fast turnover proteins in growth regulation; Wheatley DN et al.; This paper discusses the way in which serum deprivation affects the turnover of nascent or newly synthesised proteins in mammalian cells . A theoretical treatment of their turnover relative to changes in rate of protein synthesis and the turnover of existing or "resident" proteins is presented . Previous experimental work has not seriously addressed this question because the pulses of radiolabelling of proteins have been too long to identify the very-fast turnover population (Wheatley et al., 1980; Bohley, 1987) . Logically one would expect cell growth rate to be regulated by the rate at which new proteins become incorporated into the cell within the first 30 min of their existence . This requires their successful integration at what we will refer to as the "growing point", recognizing that at any time there may be thousands of such sites . Growth is a simple term betraying the complexity of the processes involved--synthesis, processing, sorting, targeting, and stabilization of macromolecules, and their successful integration into functional assemblies at appropriate locations . Turnover of the truly short-lived, very-fast turnover proteins at the "growing point" is affected by serum adjustment, but it is not the only change since synthetic rate quickly responds, as also does the turnover rate of long-lived proteins . Our theoretical discussion will relate to recent findings in 3T3 and HeLa cell cultures after serum modulation, lines with quite different dependencies on serum growth factors. Vasa Suppl, 1991, 33, 140 - 1 {Cell culture as a prescreening system for drug prevention of restenosis?}; Voisard R et al.; Migration and proliferation of smooth muscle cells (SMC) from the media into the subendothelial space are important steps in the development of restenosing events after angioplasty; therefore a medical inhibition of this activity seems to be of clinical interest . Primary stenosing plaque material of 20 patients and restenosing plaque material of 6 patients was removed by atherectomy (Prof . Hofling, Dr . Bauriedel, Munich) . For the isolation of plaque cells a mixture of Collagenase/Elastase was used . The vast majority of plaque cells was identified as smooth muscle cells by positive reaction with monoclonal antibodies against smooth muscle alpha-Actin . Propranolol (10(-4) mol/l to 10(-9) mol/l), Prednisolone (10(-3) mol/l to 10(-8) mol/l) and Etoposide (10(-4) mol/l to 10(-9) mol/l) were added to the cultures one day after seeding . After 3 days cell number and cell size distribution were analysed in a cell counter (Casy I, Scharfe System, Reutlingen) . While Propranolol didn't change proliferative activity of SMC, Prednisolone caused a slight, but dose dependent inhibition of SMC-proliferation . Etoposid inhibited SMC-proliferation even below clinical concentrations to 50% . The local application of steroid or cytostatic agents might improve long term results after angioplasty . The clinical relevance of this 'Prescreening System' has to be evaluated by experimental and clinical studies. J Clin Invest, 1991 Jan, 87(1), 208 - 12 Synthesis and secretion of an atriopeptin-like protein in rat kidney cell culture; Ritter D et al.; The synthesis and secretion of an atriopeptin(AP)-like prohormone (AP126ir) has been demonstrated in rat neonatal renal cell cultures . AP126ir could be detected in the cellular extract and the medium from cultured kidney cells of neonatal and adult rats using an enzyme immunoassay specific for cardiac AP prohormone . On reverse-phase high-performance liquid chromatography, the AP obtained from the extract and the medium comigrated with cardiac AP prohormone . Incubation of the renal AP in the medium with thrombin resulted in the generation of a single low molecular mass peak which migrated with the cardiac carboxy-terminal 28-amino acid AP . Neonatal kidney cells pulsed with {35S}methionine secreted radiolabeled AP126ir, which was detected by immunoprecipitation and sodium dodecyl sulfate-polyacrylamide gel electrophoresis chromatography . Incubation of neonatal kidney cell cultures with the protein synthesis inhibitor cycloheximide resulted in a significant decrease in both the cellular and media AP . No decrease in cellular and media AP was detected when neonatal atrial cultures were treated with cycloheximide . These data demonstrate the de novo synthesis of an AP prohormone-like protein in neonatal rat kidney cultures . Furthermore, unlike the atria, kidney cells appear to secrete AP solely by constitutive means . In primary adult rat kidney cultures, most of AP126ir was detected in the cortical tubule fraction demonstrating that these cells secrete AP126ir in the adult rat kidney . We hypothesize that the renal AP may be important as an autocrine or paracrine regulator of renal function. Yao Xue Xue Bao, 1991, 26(11), 876 - 80 {Separation and identification of main medicinal saponin components from mass cell cultures of Panax notoginseng (Burk) F . H . Chen}; Zhou LG et al.; Five main saponins were separated from the cell cultures of Panax notoginseng (Burk) F . H . Chen . They were sanchinoside Rb1, Re, R1, Rg1 and Rh1 identified by TLC, HPLC, IR, M . P., 13CNMR and EI-MS . Saponin components of the cell cultures were almost the same as those of the cultivated plants . But the content of saponins was different between the cell cultures and the cultivated plants . Saponin extraction and separation procedure suitable for the cell cultures has to be different from that for the cultivated plants. Folia Microbiol (Praha), 1991, 36(4), 387 - 90 Sterilization of sparingly soluble compounds for cell culture use; Alam A; A method is presented for the successful decontamination of sparingly soluble pteridine derivatives by microwave irradiation . The method is nondestructive, rapid and effective in eliminating contamination. Scanning Microsc Suppl, 1991, 5(4), S53 - 73 High voltage electron microscopy and low voltage scanning electron microscopy of human neoplastic cell culture; Malecki M; Improved procedures were developed to correlate cell culture data with the images provided by advanced ultrastructural technologies . These procedures were compatible with the two main types of cellular behavior: adherent, spreading (melanomas, rhabdomyosarcomas) and non-adherent in suspension (leukemias) . The ultrastructure and function of spreading neoplastic cells primarily depend on surface properties of the attaching substrates . Therefore, the films used for cultured cell whole-mount ultrastructural analysis must have adherence features identical to those of standard cell culture vessels . Improved procedures were developed to produce the polystyrene films of required qualities . These films allowed processing of cells for electron microscopy including chemical fixation, cryo-immobilization, and immunolabelling . Furthermore, these polystyrene films permitted observations of the same cell in the high voltage electron microscope to reveal the internal organization and in the low voltage scanning electron microscope to reveal the surface topography . Neoplastic cells in suspension may dramatically change their ultrastructure as a result of interactions with substrates or other cells . Therefore, immobilization of cellular processes must occur rapidly while cells remain in suspension . These processes were cryo-immobilized by high pressure freezing through the use of the newly designed specimen carrier . Procedures allowing high yield attachment of cryo-fixed neoplastic cells to amino-propyl-derived glass carriers enabled observations of cell surface topography . Furthermore, freeze-substitution and drying of freeze-fractured cells revealed their three-dimensional internal organization in the low voltage scanning electron microscope. Dtsch Z Mund Kiefer Gesichtschir, 1991 Jan-Feb, 15(1), 64 - 8 {Porous hydroxylapatite ceramics with homologous osteoblasts from cell cultures for bone replacement}; Lang H et al.; In an animal model using 24 inbred Lewis rats the effect of the simultaneous implantation of in vitro cultivated osteoblasts and a porous hydroxyl apatite ceramic material on bone regenerations was studied . Bone tissue was harvested from 2 inbred rats and a cell line of osteoblasts was established . The osteoblasts were multiplied in cell cultures and after 3 passages inserted along with granular HA into monocortical femur defects in 24 rats of the same inbred line . After various times of observation the femurs were examined radiographically and bone growth was assessed histologically . No significant increase in bone growth rate was observed, although earlier studies in the same model showed a marked qualitative effect of reimplanted in vitro cultured osteoblasts on new bone formation . Considering that pre-incubation largely reduces the toxic effect of HA ceramics, the results of this study indicate that the activity of the implanted osteoblasts in the defect is prevented by the simultaneous implantation of replacement material and cells. Acta Derm Venereol Suppl (Stockh), 1991, 170, 3 - 12 Establishment of gerbil epidermal cell culture and comparative analysis of the behaviour of these cells with cultured mouse epidermal cells; Folly LM et al.; The culturing of gerbil epidermal cells is described for the first time . Cells grew first as clumps, formed monolayers of proliferating basal cells and then differentiated, giving rise to a stratified epithelium . To regulate the process of proliferation and differentiation, two kinds of medium were used: the standard one and a low-Ca++ medium . Under these two conditions, cultured gerbil cells were compared with mouse epidermal cell cultures . The cultures maintained in the low-Ca++ medium showed greater differences between the two species, the gerbil epidermal cells displaying a greater proliferative capacity and maintained capacity to differentiate, than did the murine cells . It is believed that this in vitro model may lead to a better understanding of the difference between the two species in vivo, and might explain their differing susceptibility to cutaneous tumour induction. Z Kardiol, 1991, 80 Suppl 9, 7 - 13 {Arteriosclerosis research in the animal model and cell cultures}; Betz E; Feeding animals a diet of increased cholesterol content gave rise to the feeding hypothesis, and balloon experiments were the basis of the injury hypothesis of atherogenesis . When analyzing the sequence of events in arterial walls during atherogenesis, it turned out that in spite of complex causes and courses of stenosing processes in arterial walls the final results were relatively uniform: atheromas of fibromuscular proliferates or a mixture of both develop . Calcification of the thickenings of arterial walls occurs frequently in the progression of the disease . In animal experiments, the development of arteriosclerosis can be speeded up with various experimental techniques and enables study of the action of drugs to inhibit atherogenesis, as well as therapies for restenoses which frequently occur after angioplasty or bypass operations . In studies of drug effects the limitations of animal experiments become obvious when trying to transfer results obtained in animal experiments to the situation in humans . Cultures of cells from human arteries are, therefore, necessary supplements . Mass cultures and clone cultures of arterial walls are, however, very artificial systems . Therefore, co-cultures of endothelial cells, smooth muscle cells, and adventitial tissue have been established which imitate the morphology of arterial walls . With transfilter co-cultures, it has become possible to produce fibromuscular proliferates in vitro . When oxidized LDL-particles and monocytes are added to the culture medium, lipid-containing proliferates develop. Tsitologiia, 1991, 33(5), 18 - 26 {A stereoscopic analysis of the centrosome structure in the cells of continuous and primary cell cultures}; Alieva IB et al.; A 3D reconstruction of the centrosome region was made based on series of semithick sections in tissue culture cells . It was shown that: 1) the total number of microtubules attached to the centrosome is about 30-50 of which only 20% or less run farther than 2 microns away from the centrosome; 2) a certain number of short microtubules (less than 1 micron length) is present in the vicinity of the centrosome, the majority of them are attached to the centrosome; 3) many microtubules around the centrosome have no direct contact with either centrioles, or other microtubule-convergent structures; 4) the majority of free microtubules are comparatively long (more than 1 micron length); 5) almost all the microtubules running closer than 2 microns to the centrosome are oriented towards it with their proximal ends . The radial distribution of free microtubules around the centrosome support the supposition that they may appear as a result of their detachment from the microtubule-nucleating centres. Dev Biol Stand, 1991, 75, 183 - 9 Virus zoonoses and their potential for contamination of cell cultures; Mahy BW et al.; Silent virus infections of laboratory animals present a human health hazard, from direct exposure and from contamination of biological products for human use . Here we report two recent examples . In 1989, an outbreak of lymphocytic choriomeningitis virus (LCMV) infections was recognized among workers at a cancer research center after an animal caretaker developed viral meningitis . Investigation revealed that multiple tumor cell lines at the facility were infected with LCMV, as were research animals injected with these cell lines . Of 82 workers tested, eight (10%) were found to have been infected . The infected workers were more likely than other animal handlers to report handling athymic (nude) mice (p less than .0.007) . The number of nude mice used in this facilty had increased five-fold in the previous year, possibly explaining the timing of the outbreak . This is the first reported LCMV outbreak since 1975, and the first to implicate nude mice as a source of human LCMV infections . In November 1989 and January 1990, infections caused by two distinct Ebola-like filoviruses were discovered in non-human primates at quarantine facilities in Virginia and Pennsylvania . Although 22 persons were considered to have high- or medium-risk exposures for Ebola infection, no Ebola-compatible illnesses occurred . One of the medium-risk persons had Ebola IgG antibodies confirmed by IFA and Western blot . Rigorous use of barrier precautions may have limited exposure and infection with these filoviruses . In February 1990, new groups of filovirus-infected monkeys were identified in Virginia and in Texas . Seroconversion occurred in four animal handlers, including one to very high titer, but again no illness was observed.(ABSTRACT TRUNCATED AT 250 WORDS) Bone, 1991, 12(4), 277 - 82 Requirement of vitamin C for cartilage calcification in a differentiating chick limb-bud mesenchymal cell culture; Boskey AL et al.; Mesenchymal cells isolated from stage 21-24 chick limb-buds plated in a micro-mass culture differentiate to form chondrocytes and synthesize a calcifiable matrix . In the presence of inorganic phosphate (4 mM), hydroxyapatite mineral deposits around cartilage nodules . Ascorbic acid is, in general, an essential co-factor for extracellular matrix synthesis in culture, since it is required for collagen synthesis . In this study we demonstrate that in the absence of ascorbic acid supplementation in the mesenchymal cell cultures, mineral deposition (indicated by X-ray diffraction, measurement of Ca:hydroxyproline ratio, and 45Ca uptake) does not occur . Concentrations of 10-50 micrograms/ml ascorbate were compared to find the "optimal" concentration for cell mediated mineralization; 25 micrograms/ml was selected as optimal based on matrix appearance at the EM level and the rate of 45Ca uptake . High concentrations of ascorbic acid (greater than 75 micrograms/ml), while increasing the amount of hydroxyproline in the matrix synthesized, caused some cell death and hence less cell-mediated mineralization . This study demonstrates both the need for viable cells and a proper matrix for in vitro cell-mediated mineralization, and shows that varying the concentration of L-ascorbate (vitamin C) in the medium can have a marked effect on mineralization in vitro. Biosens Bioelectron, 1991, 6(8), 653 - 61 Application of cell culture toxicity tests to the development of implantable biosensors; Zhang Y et al.; Cell culture toxicity testing methods were modified and applied to the development of implantable glucose microsensors, and positive and negative control materials suitable for the microsensor assessment were established . The location, source and degree of the toxic effect in a multi-component biosensor was spatially visualized with cell monolayers . A freshly prepared sensor showed moderate toxicity, mainly as a result of the presence of glutaraldehyde and the residual solvents in the polymer layers . However, it was possible to reduce the toxicity by removing the leachable toxic substances through extraction in phosphate buffer, and a non-toxic sensor was readily obtained. Acta Anat (Basel), 1991, 142(2), 97 - 104 Ultrastructure of mineralized nodules formed in rat bone marrow stromal cell culture in vitro; Satomura K et al.; Rat bone marrow stromal cells were cultured in vitro . At days 14-15 of culture, dense clusters of polygonal cells were formed, and they mineralized 2-3 days later . The cells resembling osteoblasts or young osteocytes were histologically observed to be embedded in mineralized or unmineralized extracellular matrices of the nodules . Next, these mineralized nodules were electron-microscopically examined . The osteoblastic cells associated with the nodules had a well-developed rough endoplasmic reticulum, an evident Golgi apparatus and some mitochondria as their intracellular organellae . Some lysosomes and microfilaments were also visible in the cytoplasms . Moreover, some cells protruded cell processes toward the neighboring cells through the extracellular matrix . The extracellular matrix consisted of numerous collagen fibrils which were striated with 60-70 nm axial periodicity and which was similar to bone tissue collagen . A large number of matrix vesicles were scattered among the collagen fibrils in the unmineralized area of the nodules . In contrast, in the mineralized area, numerous matrix vesicles at different stages of maturation and many calcified spherules were observed . That is the mineralization in this culture system was considered to be initiated in association with the matrix vesicles and to progress along the collagen fibrils . From these findings, it was confirmed by the present study that the mineralized nodules formed in this bone marrow stromal cell culture were ultrastructurally similar to bone and that the mineralization also proceeded by going through the normal calcification process . This culture system is considered to be available to study osteogenic differentiation and calcification mechanisms. Adv Exp Med Biol, 1991, 295, 415 - 37 In vitro cell cultures as a model of the basal forebrain; Wainer BH et al.; The basal forebrain has attracted considerable attention because of its putative role in complex functions such as learning, memory and behavioral state control as well as its vulnerability in neurological disorders such as Alzheimer's Disease (AD) . The finding that nerve growth factor provides trophic support for the cholinergic basal forebrain neurons has stimulated further interest in understanding trophic interactions of basal forebrain neurons as well as in possible trophic factor therapeutic strategies for disease states . Our laboratory has utilized primary cell cultures and developed immortalized central nervous system cell lines to study the trophic interactions that establish and maintain the septohippocampal pathway, a basal forebrain component which plays an essential role in cognitive function and is prominently affected in AD . The results of our primary cell culture studies have demonstrated the importance of trophic signals elaborated by the hippocampus in mediating the development of septal cholinergic neurons . Nerve growth factor plays an important role in this process, but it cannot account for all of the trophic signals elaborated by authentic hippocampal target cells . The development by this laboratory of clonal cell lines of septal and hippocampal lineage offers the prospect of investigating both the response to and elaboration of neural trophic signals at a more precise level of resolution than can be achieved with primary cultures . The technology and information that is generated from the engineering of such cell lines will also serve as a strategy to study trophic interactions in other brain circuits in future years, and to investigate possible changes or dysfunctions that occur neurological diseases. Psychopharmacol Bull, 1991, 27(3), 199 - 208 Signal transduction modulation by lithium: cell culture, cerebral microdialysis and human studies; Manji HK et al.; Considerable evidence suggests that signal transduction pathways are targets of lithium (Li) action . A number of investigators have reported that Li attenuates both adenylate cyclase (AC) activity and phosphoinositide (PI) turnover in rodents and in humans, thus "dampening" these systems . We have studied selected components of these second-messenger systems in a series of clinical and preclinical investigations . To overcome confounding effects of alterations in mood state, we examined AC activity and G-protein ribosylation in peripheral blood cells from 10 healthy volunteers, prior to and following 14 days of Li administration . Basal and postreceptor {cesium fluoride (CsF) or Gpp(NH)p} stimulated AC activity were unaffected in lymphocytes . In contrast, both basal and stimulated AC activity in platelets were significantly augmented, compatible with an attenuation of Gi function . Ribosylation of platelet Gs by cholera toxin was unchanged, whereas that of Gi by pertussis toxin (PT) was increased . Given that undissociated G protein is the preferred substrate for PT, our results suggest that Li interferes with subunit dissociation and the subsequent activation of Gi . To determine if Li has similar effects on Gi in the central nervous system, we measured extracellular (EC) cyclic adenosine monophosphate (cAMP) in rat brain by in vivo microdialysis, revealing a dose-dependent increase in cAMP by norepinephrine (NE) antagonized by propranolol . Chronic (4-week) Li doubled basal EC cAMP, while decreasing the fractional response to 100 microM NE . Thus, using in vivo microdialysis, we observed the reported reduction in NE-stimulated AC activity, but only as a function of elevated basal cAMP . Increased basal AC activity has been observed following chronic Li in both humans and rat tissues but generally has not been considered relevant . The PI generating system is another proposed major target for Li that we have studied using an in vitro cell culture model of peripheral blood cells . Chronic (6-day) exposure of neutrophil-like HL60 cells to 1 mM LiCl did not affect agonist fMet-Leu-Phe (fMLP) induced PI turnover . In contrast, Li attenuated both agonist and phorbol ester stimulated Na+/H+ exchange, suggesting reduced protein kinase C (PKC) function . Western blot analysis revealed altered levels of PKC in both membrane and cytosolic fractions . The functional consequences of these complex effects on the two major signal transduction pathways and their interactions in the intact living organism remain to be elucidated. Crit Rev Ther Drug Carrier Syst, 1991, 8(4), 305 - 30 Cell cultures as models for drug absorption across the intestinal mucosa; Artursson P; This review deals with cell culture models for studies of drug absorption across the intestinal mucosa . The selection of appropriate cells and cell culture conditions is discussed, guidelines for the characterization of the cell models are presented, and the intestinal barriers to drug absorption are discussed and compared with those in the cell culture models . Finally, recent applications of the cell culture models in drug and peptide absorption and metabolism studies are reviewed. Fetal Diagn Ther, 1991, 6(1-2), 84 - 6 Why cell culture is successful after early amniocentesis; Byrne D et al.; The current interest in early amniocentesis as a possible alternative to chorion villus sampling has driven many centres to attempt cell culture from first trimester amniotic fluid . This study examines the cellular content of 125 amniotic fluid specimens collected between 8 and 18 weeks of gestation and shows that there is a higher proportion of viable cells at the time of early amniocentesis than at traditional amniocentesis . Although there is a small increase in viable cell number from 8 to 18 weeks of gestation, this is not as dramatic as the exponential rise in total cell number seen over the same period . The similar concentration of viable cells in early pregnancy to that in the second trimester is sufficient to explain the unexpected culture success after early amniocentesis. Neuroscience, 1991, 45(1), 195 - 204 Thrombin indirectly affects cholinergic cell expression in primary septal cell cultures in a manner distinct from nerve growth factor; Mazzoni IE et al.; The effects of thrombin were examined in primary cultures of dissociated medial septal cells from fetal (embryonic day 17) rat brains . Seven days of continuous exposure of these cultures to thrombin pro