Cell cultures

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 produced a dose-dependent increase in the activity of the enzyme choline acetyltransferase (EC 2.3.1.6) and no change in the number of acetylcholinesterase (EC 3.1.1.7)-positive cells . Maximal induction of choline acetyltransferase activity occurred around 1-2 nM thrombin and was first detected after five days of treatment . In addition, thrombin promoted neuronal cell aggregation, proliferation of the astroglia, and changes in astroglial cell morphology . Neuronal aggregation was first noted after 24 h of treatment, while the proliferative response of the astroglia was first apparent after four days of treatment, slightly prior to the increase in choline acetyltransferase enzymatic activity . In order to see if the induction of the enzyme choline acetyltransferase was dependent upon the astroglial cell response, we included the anti-mitotic agent 5-fluoro-2'-deoxyuridine, to find that astrocyte proliferation, as well as thrombin-induced increase in choline acetyltransferase, were both abolished . In contrast, the aggregation of neurons was not affected . Finally, thrombin-induced changes in choline acetyltransferase could not be antagonized by immunoneutralizing anti-nerve growth factor antibodies and when thrombin was added simultaneously with 100 ng/ml 2.5-S nerve growth factor, the increase in choline acetyltransferase activity was additive . In conclusion, it appears that thrombin affects cholinergic septal neurons indirectly via the responsive astrocytes in a manner distinct from nerve growth factor.

Autoimmunity, 1991, 10(1), 35 - 9
Analysis of the IgG subclass production from rheumatoid arthritis synovial cell cultures; Johnstone R et al.; In man there are four subclasses of IgG which differ from each other with respect to their biological properties . Some evidence suggests that the production of IgG3 is unusually high in rheumatoid synovia . In this study secretion of IgG subclasses by synovial lymphocytes in vitro was measured using sensitive subclass-specific ELISAs . It was found that, in both synovial membrane- and synovial fluid-derived cell cultures, the general pattern of IgG subclass secretion was IgG1 greater than 2 greater than 3 greater than or equal to 4, and that, in most cultures, IgG3 was a minor subclass accounting, on average, for only 8% of the total IgG . This was similar to the percentage of this subclass in normal human serum and in culture supernatants from the patients' peripheral blood lymphocytes.

J Mol Neurosci, 1991, 3(2), 75 - 83
Differential and coordinate regulation of TH and PNMT mRNAs in chromaffin cell cultures by second messenger system activation and steroid treatment; Carroll JM et al.; Primary cultures of chromaffin cells were prepared from bovine adrenal medullae and the levels of mRNA for tyrosine hydroxylase (TH) and phenylethanolamine N-methyltransferase (PNMT) determined . The cells expressed moderate levels of TH mRNA and low levels of PNMT mRNA . The latter appeared to be more sensitive than TH mRNA to variations in the culture medium . The treatment of cultures with agents that activate signal transduction pathways, forskolin or phorbol esters, dramatically enhanced the expression of both mRNAs . The forskolin-induced increases in the steady-state levels of TH and PNMT mRNAs occurred rapidly and were apparent within 5 hours . These data suggest that the TH and PNMT genes can be regulated by second messengers . In contrast, dexamethasone treatment dramatically increased PNMT mRNA with no change in TH mRNA . The increase in PNMT mRNA was apparent within 6 hours of addition of the drug to the culture medium.

Acta Histochem, 1991, 91(2), 131 - 9
Substance P- and neuron-specific enolase-like immunoreactivity of rodent Leydig cells in tissue section and cell culture; Angelova P et al.; Comparative immunocytochemical studies concerning the presence of a neurotransmitter (substance P), and a marker of neuroendocrine cells (neuron-specific enolase), in the Leydig cells of 3 mammalian species (golden hamster, guinea pig, and rat) were carried out on tissue sections and cell cultures . Substance P(SP)-like immunoreactivity (-LI) was found to be present in both fetal and adult generation of Leydig cells in hamster and guinea pig, while neuron-specific enolase (NSE)-LI was detected in Leydig cells of the 3 species at all stages studied: fetal, neonatal and adult . In primary cultures of Leydig cells isolated from adult hamster testes, SP- and NSE-LI was also established . This result was considered as an indirect evidence for the synthesis of the substances under study by the steroidogenic cells of the testis . A comparison of these results with data obtained in vivo suggests that Leydig cells may be related to the APUD- or the diffuse neuroendocrine system.

Acta Otolaryngol Suppl, 1991, 481, 153 - 7
Cell culture study of the vestibular ganglion cells . Morphology and immunohistochemical activity; Yamashita H et al.; Incubation of vestibular ganglion cells from the rat fetus and chick embryos was successfully done demonstrating bipolar cells and two types of multipolar cells, small round cells and large cells, in the cell cultures produced . Vestibular ganglion cells were found to be highly irregular in size . Furthermore, the presence of neurotransmitters (choline acetyltransferase and substance P) was confirmed immunohistochemically . Substance P positive cells had many bipolar cells and some multipolar cells . However, choline acetyl transferase positive cells had some small multipolar cells but few bipolar cells . These findings suggest that all vestibular ganglion cells do not have the same function.

Tsitologiia, 1991, 33(1), 57 - 63
{The autocrine regulation of proliferation in cell cultures . I . A study of the heparin-binding factors with growth-stimulating activity contained in media conditioned with transformed rat fibroblasts}; Goilo TA et al.; A study was made of the medium conditioned by spontaneously transformed rat embryo fibroblasts of line Rec1-sf, which are capable of unlimited reproduction in medium free of serum and of other endogenous growth factors (c-medium) . Addition of c-medium to stationary cultures of nontransformed rat embryo fibroblasts (REF), spontaneously transformed REF (line Rec1), and cells of Rec1-sf stimulated the incorporation of 3H-thymidine by 1.5-6 times . SDS-polyacrilamide-gel electrophoresis of proteins, marked by 35S-metionine of c-medium of the cell line Rec 1-sf, demonstrated that this medium had proteins with molecular mass from 10 to 110 kDa . The fractionating divisible by 100 ultra-concentrates of c-medium with utilization of heparin-sepharose allowed to isolate two types of heparin-binding proteins . The proteins of the first type took about 5% of all the proteins of c-medium; they were eluted with 1.1 M NaCl and stimulated the incorporation of 3H-thymidine in REF, Rec1 and Rec1-sf cultures by 1.3-1.9 times . The second type proteins took about 1% of all the proteins of c-medium and were eluted with 2M NaCl, and, like the main endogenic basic growth factor of fibroblasts, stimulated the incorporation of 3H-thymidine into REF and Rec1-sf, but not into the culture of Rec1 line cells . The results obtained are discussed in terms of a hypothesis of autocrine regulation of cell proliferation.

Adv Exp Med Biol, 1991, 283, 847 - 51
Selective alteration of cytokeratin intermediate filament by cyclosporine A is a lethal toxicity in PTK2 cell cultures; Vernetti LA et al.; The cytoplasm of eukaryotic cells contain a series of three filamentous structures, microtubules, microfilaments, and intermediate filaments that are termed the cytoskeleton . Cytokeratin, one type of intermediate filament, has no known physiological function, yet, can comprise up to 30% of the total cytoplasmic protein content . As there are no selective toxins to cytokeratins, it is not known if alterations to these hydrophobic filaments is a lethal event . Cyclosporine A, a novel hydrophobic immunosuppressant compound used to prevent allograft rejection, may show a selective toxicity to the cytokeratin filaments . This effect is seen in PtK2 cell cultures as a single large perinuclear aggregate of collapsed cytokeratin filaments (5 mM, 72 hr) . Microtubules and microfilaments are not affected in PtK2 cell cultures (5 mM, 72 hr) . Increased LDH levels into cell culturing media occur soon after cyclosporine exposure to PtK2 cell cultures (5 mM, 2 hr) . Cytokeratin filaments show no changes at 12 hr exposure but show thickening, decreased plasma membrane attachments and some peri-nuclear ring formations at 24 hr (5 mM, 24 hr) . Cyclosporine G, an analog of cyclosporine A, does not exhibit the cytokeratin filament collapse (5 mM, 72 hr) . The effect of cyclosporine A on DNA binding protein (Mr 64 kd), believed to be a nuclear scaffolding protein related to intermediate filaments, exhibited an early invagination and folding of the nuclear membrane (5 mM, 4 hr) . Due to a hydrophobic bonding potential between cyclosporine A and cytokeratin and cytokeratin-like intermediate filaments, cyclosporin A may be a selective cytokeratin toxin . Alteration of the cytokeratin filaments in PtK2 cell cultures may be a lethal event.

Cancer Immunol Immunother, 1991, 33(1), 33 - 8
Immunoregulatory cytokine release in rat spleen cell cultures after treatment with bleomycin and its analogues in vivo; Micallef M et al.; We have studied the immunological effects that accompany a change in the chemical structure of a group of antineoplastic antibiotics by comparing the immunoregulatory cytokine release during mitogen-stimulated spleen cell culture after in vivo drug treatment . Whereas bleomycin and peplomycin increased cytokine levels in culture supernatants when compared with supernatants from untreated control rat spleen cell cultures, liblomycin generally reduced cytokine levels under the same culture conditions . We then compared these results with the antitumor effects of equivalent doses of the three drugs against a highly antigenic rat fibrosarcoma, KMT-17, both in vivo and in vitro . The results suggest that the immunoaugmenting effects of these antitumor antibiotics are essential for an optimal antitumor effect in vivo, and that these effects can be drastically altered by modification of the chemical structure of the drugs employed.

In Vitro Cell Dev Biol, 1991 Jan, 27(1), 13 - 20
Characterization of epithelial cell cultures derived from human tracheal glands; Chopra DP et al.; Cultures of normal human tracheal gland epithelial cells that exhibit functional differentiation have been propagated in serum-free medium supplemented with insulin (5 micrograms/ml), epidermal growth factor (10 ng/ml), hydrocortisone (0.5 micrograms/ml), and bovine pituitary extract (25 micrograms/ml) . The cells retain many characteristics of epithelial cells including microvilli on cell surfaces, desmosomes between cells, and tonofilaments in the cytoplasm . In addition, they exhibit keratin-positive titers and react positively with Peanut agglutinin, which is specific for the disaccharide beta-D-galactose-(1----3)N-acetyl D-galactosamine, a major component of mucin glycoprotein . The cells also exhibit normal Cl- channel activity which was enhanced by the cAMP agonist Forskolin . The major component of the cellular secretion was hyaluronic acid; approximately 10% of the void volume material was resistant to hyaluronidase and may contain material similar to mucin glycoprotein . Some of the cell cultures have been maintained in serum-free conditions for 6 to 7 passages . This model will be important to study regulation of ion-channel activities and mucous glycoprotein secretion and to compare such regulations with the tracheal mucosal epithelial cells already established.

Teratog Carcinog Mutagen, 1991, 11(4), 195 - 202
In vitro reduction of peroxidation in UVC-irradiated cell cultures by concurrent exposure with Bowman-Birk protease inhibitor; Baturay NZ et al.; Bowman-Birk protease inhibitor (BBI), extracted from soybean flour, has been shown to protect cells from damage after UVC irradiation . BBI also reduces the number of transformed foci following exposure to the same doses of UVC irradiation . Proteins have been reported to act as antioxidants by their sheer intracellular bulk . In this study we assessed BBI's ability to attenuate UVC-irradiation-induced peroxidation . Balb c/3T3 cells, clone A31, subcloned by this laboratory were used . Treatment groups consisted of cells suspended in Hanks' Balanced Salt Solution with and without 100 micrograms/ml of a crude soybean extract containing the Bowman-Birk protease inhibitor . Cells were exposed to irradiation in suspension by using GE (G8T5) germicidal lamps which deliver predominantly 254 nm light at 0.385 J/m2/s as measured at the cell surface . Aliquots of UVC-exposed cells were homogenized in HBSS in the presence and absence of 100 micrograms/ml BBI with a motor-driven Teflon pestle and assayed for peroxidation . The results showed significant (P less than 0.0001) protection afforded by BBI against peroxidation at 1.16 (65%), 1.93 (60%, and 2.70 (48%) J/m2 of UVC irradiation . Transformation was reduced to 0.33 foci per dish at all levels of radiation exposure.

Acta Virol, 1991 Jan, 35(1), 81 - 5
Adaptation of human rotavirus to cell culture and characterization of the isolated strain; Gudkov VG et al.; Human rotavirus strain 649 was isolated from a child with clinical picture of acute intestinal infection in the West European part of the U.S.S.R . This strain was adapted to MA-104 (rhesus monkey kidney) cells . By electrophoresis and haemagglutination it differed from the human rotavirus strain Wa and from the simian strain Sa-11 . The productive reproduction of human rotavirus strain 649 may allow its use for the preparation of antigens for diagnostic, immunoprophylaxis, and treatment.

Neuroscience, 1991, 43(2-3), 585 - 91
Glutamate neurotoxicity in spinal cord cell culture; Regan RF et al.; The neurotoxicity of glutamate was investigated quantitatively in mixed neuronal and glial spinal cord cell cultures from fetal mice at 12-13 days of gestation . Five-minute exposure to 10-1000 microM glutamate produced widespread acute neuronal swelling, followed by neuronal degeneration over the next 24 h (EC50 for death about 100-200 microM); glia were not injured . Glutamate was neurotoxic in cultures as young as four days in vitro, although greater death was produced in older cultures . By 14-20 days in vitro, 80-90% of the neuronal population was destroyed by a 5-min exposure to 500 microM glutamate . Acute neuronal swelling following glutamate exposure was prevented by replacement of extracellular sodium with equimolar choline, with minimal reduction in late cell death . Removal of extracellular calcium enhanced acute neuronal swelling but attenuated late neuronal death . Both acute neuronal swelling and late degeneration were effectively blocked by the noncompetitive N-methyl-D-aspartate receptor antagonist dextrorphan and by the novel competitive antagonist CGP 37849 . Ten micromolar 7-chlorokynurenate also inhibited glutamate neurotoxicity; protection was reversed by the addition of 1 mM glycine to the bathing medium . These observations suggest that glutamate is a potent and rapidly acting neurotoxin on cultured spinal cord neurons, and support involvement of excitotoxicity in acute spinal cord injury . Similar to telencephalic neurons, spinal neurons exposed briefly to glutamate degenerate in a manner dependent on extracellular Ca2+ and the activation of N-methyl-D-aspartate receptors.

J Neurochem, 1991 Jan, 56(1), 199 - 206
Release of endogenous and newly synthesized glutamate and of other amino acids induced by non-N-methyl-D-aspartate receptor activation in cerebellar granule cell cultures; Levi G et al.; Amino acid release studies were performed by an HPLC procedure using differentiated rat cerebellar granule cell cultures . Kainic acid (KA; 50 microM) caused an increase (about threefold) in the release of endogenous glutamate and a lesser, but statistically significant, increase in the release of glutamine, glycine, threonine, taurine, and alanine . Quisqualic acid (QA) and, to a lesser degree, alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) (both 50 microM) enhanced the release of the following amino acids in the order glutamate greater than aspartate greater than or equal to taurine, whereas the release of other amino acids was either unaffected or affected in a statistically nonsignificant way . The release of glutamate induced by KA was partially (43%) Ca2+ dependent . The other release-inducing effects of KA and QA were not Ca2+ dependent . In all cases, the evoked release could be prevented by the non-N-methyl-D-aspartate (non-NMDA) receptor antagonist 6-cyano-2,3-hydroxy-7-nitroquinoxaline, and thus appeared to be receptor mediated . NMDA (5 and 50 microM) had no release-inducing activity . The KA-, QA-, and AMPA-evoked release of newly synthesized {3H}glutamate and {3H}aspartate (formed in the cells exposed to {3H}glutamine) was very similar to the evoked release of endogenous glutamate and aspartate . On the other hand, the release of preloaded D-{3H}aspartate (purified by HPLC in the various fractions analyzed, before radioactivity determination) induced by 50 microM KA was twice as high as that of endogenous glutamate . In the case of high {K+} depolarization, in contrast, the release of preloaded D-{3H}aspartate was approximately 30% lower than that of endogenous glutamate.(ABSTRACT TRUNCATED AT 250 WORDS)

Boll Ist Sieroter Milan, 1991-92, 70(1-2), 385 - 9
Supernatant of human umbilical vein endothelial cell culture can favour in vivo neutropoiesis; Ghezzo F et al.; Umbilical vein endothelial cells are known to be able to produce interleukins and colony stimulating factors . In the present work supernatant of human umbilical vein endothelial cell culture have been administered to neoplastic patients treated with chemotherapy to reduce the iatrogenic inhibition of hemopoiesis . While no undesired effect could be observed, neutrophil count was favourably influenced by endothelial cell supernatant administration . Such data can be considered useful in order to reduce collateral effect of antineoplastic therapy.

Tsitologiia, 1991, 33(4), 77 - 83
{The levels of DNA repair in the cells of diploid and tetraploid cell cultures}; Kudriavtsev BN et al.; Levels of unscheduled DNA synthesis after UV-irradiation were investigated in addition to the activities of DNA-repair enzyme uracil-DNA glycosilase in cells of both diploid (XC-D) and tetraploid (XC-T) cell cultures . It was shown that repair levels were increasing directly and proportionally to the cell ploidy.

Dev Biol Stand, 1991, 75, 177 - 81
Bovine viral diarrhea virus contamination of nutrient serum, cell cultures and viral vaccines; Levings RL et al.; Bovine viral diarrhea virus (BVDV) infection is common in the bovine population . Infection in utero leads to virus and antibody contamination of the fetal bovine serum used in cell cultures . These contaminants can interfere with diagnosis of viral infection . The high frequency of virus and antibody detection in individual animal or small pool samples suggests that any large pool of unscreened sera will be contaminated . Infection of cell cultures with BVDV can lead to interference with the growth of other viruses . Vaccine produced on contaminated cells may in turn be contaminated, leading to seroconversion or disease in the vaccine . The safety, purity, and efficacy of viral vaccines require BVDV testing of ingredients, cell substrates and final product . Methods for detection of BVDV in nutrient serum, cell cultures, seed viruses, and viral vaccines, and the frequency of their detection at the National Veterinary Services Laboratories are discussed.

Scand J Urol Nephrol, 1991, 25(4), 287 - 95
Human renal biopsies as source of cells for glomerular and tubular cell cultures; Blaehr H; Glomerular and tubular cells were obtained from normal and pathological human renal biopsies . Single nephron structures were isolated by microdissection for culture . Proximal and distal tubular cells were cultivated for 5-6 weeks (three passages), whereas outgrowth of glomerular cells was sparse and after three weeks infiltrated by mesangial cells . The morphology of cultures obtained from pathological tissue was comparable with the morphology of normal cells, although cultures were more often overgrown by fibroblasts . In culture, both proximal and distal tubular cells retained physiological responses characteristic of their origin . Epidermal growth factor induced growth of proximal tubular cells . The proximal tubular cells were furthermore characterized by cAMP response to parathyroid hormone (PTH) stimulation . The distal tubular cells showed cAMP response to both PTH and vasopressin stimulation . Twelve of 17 cultures obtained from patients with no tubular injuries showed cAMP response to PTH stimulation compared with 2 of 9 cultures from renal tissue with tubular injuries.

Basic Res Cardiol, 1991, 86 Suppl 2, 117 - 25
Interactions between nitric oxide and prostacyclin in myocardial ischemia and endothelial cell cultures; Schror K et al.; This study investigates biochemical and functional interactions between NO and PGI2 that generate pathways in two different in vitro assays: porcine aortic endothelial cells (PAEC) and reperfused ischemic Langendorff hearts of rabbits . Using cGMP as an index of NO generation and 6-oxo-PGF1 alpha as an index for PGI2 production in endothelial cells, it is demonstrated that the two metabolic pathways for NO and prostacyclin formation act independent of each other . Moreover, NO appears to have an autocrine function in endothelial cells which does not exist with PGI2, probably because of a lack of PGI2 receptors . Endothelial damage in the course of myocardial ischemia is associated with a marked increase in mediator release whose inhibition has consequences for both myocardial and coronary function: inhibition of NO formation also inhibits PGI2 release and the recovery of coronary vessel tone with only minor if any effect on myocardial contractility . In contrast, inhibition of PGI2-generation results in marked deterioration of myocardial recovery with only minor changes in coronary perfusion . It is concluded from these data that PGI2 in endothelial injury is important for preservation of myocardial function while NO might mainly be involved in control of local vessel tone.

Neirofiziologiia, 1991, 23(4), 427 - 35
{The kinetic properties of inhibitory postsynaptic currents in a cell culture of chick embryo spinal cord}; Mel'nik IV; Spontaneous glycine-mediated synaptic currents in culture of the spinal cord cells of chicks were analyzed by fitting exponential function to their decay . Some PSCs were of small amplitude . Their decay was single-exponential . Time constant of the decay increased with depolarization and decreased with a rise of temperature . The decay of the giant PSCs was double-exponential and had a weak potential dependence . Only slow component of the decay was sensitive to changes of the temperature . Time constant of the slow component increased in the presence of strychnine . The removal of the agonist from receptors was considered as an explanation for the variability and double-exponential character of the decays.

Nephron, 1991, 57(1), 94 - 8
Theophylline and magnesium inhibit the contraction elicited with ciclosporin and angiotensin II in mesangial cell cultures; Fandrey J et al.; One serious side effect of ciclosporin (CS) therapy is its acute nephrotoxicity characterized by a marked decrease in glomerular filtration rate (GFR) . A main determinant of GFR is the ultrafiltration coefficient which is thought to be regulated by the contractile state of the cells of the mesangium . CS enhances contractions of mesangial cells elicited with angiotensin II . By way of a lowered ultrafiltration coefficient this effect of CS may be partly responsible for its acute nephrotoxicity . We have therefore examined the effect of compounds that may attenuate the enhanced contractile response of the smooth-muscle-like mesangial cells . Prostaglandin E2 (10(-8)-10(-5) mol/l) and dibutyryl cyclic AMP (10(-4) mol/l) reduced the number of contracting cells from more than 50% to about 10% . The clinically used agent theophylline (10(-5) g/ml) decreased the number of contractions of CS-pretreated mesangial cells from 80.0 to 28.4% (p less than 0.001) . Additionally, a marked decrease in the percentage of contractions (83.3 to 33.3%; p less than 0.001) was observed when the concentration of magnesium chloride was elevated from 0 to 2 mmol/l . The finding that the enhanced contractile response of CS-pretreated mesangial cells can be overcome by theophylline and/or high magnesium levels may be of clinical interest with a view to acute CS nephrotoxicity.

Acta Clin Belg, 1991, 46(1), 7 - 12
Rapid diagnosis of viral respiratory infections . Comparison between immunofluorescence on clinical samples and immunofluorescence on centrifuged cell cultures; Pattyn SR et al.; We compared the technique of immunofluorescence on clinical samples (IFCS) and immunofluorescence on centrifuged cell cultures (IFCC) for the detection of respiratory viruses, using monoclonal antibodies . Four hundred fourty five nasopharyngeal aspirates were tested prospectively for RSV, influenza- (A and B), parainfluenza viruses (1, 2, 3) and adenovirus . IFCS and IFCC detected 94% and 51% of RSV respectively, 78.5% and 82% of influenza viruses, 8.3% and 100% of parainfluenzaviruses and 0 and 100% of adenoviruses . We conclude that for routine rapid virus diagnosis the IFCS procedure is acceptable for RSV, that for influenza virus the IFCC procedure can be omitted for samples positive by IFCS, and that for the detection of parainfluenza and adenoviruses the IFCS procedure is too insensitive.

Phytochemistry, 1991, 30(4), 1141 - 5
Immunologically active polysaccharides of Arnica montana cell cultures; Puhlmann J et al.; From the nutrition medium of Arnica montana cell cultures two homogeneous polysaccharides, an acidic arabino-3,6-galactan-protein with mean Mr of 100,000 and a neutral fucogalactoxyloglucan with mean Mr of 22,500 have been isolated by DEAE-Sepharose CL-6B and Sephacryl S-400 column chromatography . Their structures were elucidated mainly by methylation analysis, partial acidic and enzymatic hydrolysis and 13C NMR spectroscopy . The fucogalactoxyloglucan shows a pronounced enhancement of phagocytosis in vivo . The arabino-3,6-galactan-protein displays a strong anticomplementary effect and stimulates macrophages to excrete the tumour necrosis factor (TNF alpha).

Cytotechnology, 1991, 5 Suppl 2, S35 - 51
Development of a serum-free and heat-sterilizable medium and continuous high-density cell culture; Minamoto Y et al.; We tried to establish a new serum-free and heat-sterilizable medium, based on our serum-free medium in which many lymphoblastoid cells and hybridoma could grow as well as in a conventional serum-containing medium . As is well-known, L-glutamine (L-Gln) is one of the most heat-labile but essential components for cell growth . As a substitute for L-Gln, dipeptide such as Gly-L-Gln or L-Ala-L-Gln, which was quite stable even after autoclaving, was found to be utilizable for mammalian cell growth . The L-Gln dipeptide-containing serum-free medium was quite stable in a solution even after storing at 37 degrees C for 4 months . In the serum-free medium containing L-Ala-L-Gln, mouse hybridola could grow and produce more antibody than in RPMI 1640 + 10% FBS . It has been proved that BSA and transferrin, which are also heat-labile but essential for the growth of various cell lines, can be substituted by heat-stable alpha-cyclodextrin and cholesterol, and Fe-gluconate, respectively . Insulin has also proved to be heat stable in a solution of Fe-gluconate . We thus established a new serum-free medium, all the components of which could be heat-sterilizable . Moreover, by adding EGF and BSA but without the adhesion factor included in FBS, the serum-free medium was found to support a long-term serial culture of a human diploid fibroblast . Finally, with this auotoclavable serum-free medium in a perfusion culture apparatus, we were able to continuously cultivate a human lymphoblastoid cell line . The production rate of IgM was found to be markedly increased by feeding the serum-free medium enriched by glucose, bicarbonate, L-Cys, and approtinin . The cell density reached as high as 2 x 10(8)/ml in the serum-free medium . Although the working volume in the reactor was only 1 1, the rate of IgM production reached 480 mg/day . The new heat-sterilizable serum-free medium has several advantages, because L-Gln peptide is a heat-stable and available precursor of L-Gln.

Cytotechnology, 1991, 5 Suppl 2, S17 - 34
Optimization of cell culture conditions for production of biologically active proteins; Hosoi S et al.; We investigated the basic technology of cell culture conditions for production of useful substances such as cytokines, and related proteins produced by Namalwa cells . Namalwa cells (Klein, 1972), human B lymphoblastoid cells, were used for large scale production of alpha-interferon (Klein, 1979) . Namalwa KJM-1, a subline of Namalwa cells, adapted to serum- and albumin-free medium, can grow at a high density above 1 x 10(7) cells/ml in suspension mode by the use of a perfusion culture system, Biofermenter, containing a cone-type cell-sedimentation column as cell separator (Sato, 1983) . Several kinds of cytokine cDNA can be introduced and expressed in Namalwa KJM-1 cells (Miyaji, 1990a,b,c) . Some of these were produced in large quantities by use of a gene amplification method with dhfr (Miyaji, 1990c), even though the Namalwa KJM-1 cells contained endogenous dhfr genes . For stable production of the target protein, Namalwa KJM-1 cells are very useful host cells, because they have no effective endogenous protease activity in the conditioned medium . Using Biofermenter with micro-silicone fibers and a dialysis system, the specific productivity of the target proteins was not depressed at a high cell density.

J Biol Chem, 1990 Dec 25, 265(36), 22204 - 9
Synthesis of mitochondrial uncoupling protein in brown adipocytes differentiated in cell culture; Kopecky J et al.; In order to characterize the biogenesis of unique thermogenic mitochondria of brown adipose tissue, differentiation of precursor cells isolated from mouse brown adipose tissue was studied in cell culture . Synthesis of mitochondrial uncoupling protein (UCP), F1-ATPase, and cytochrome oxidase was examined by L-{35S}methionine labeling and immunoblotting . For the first time, synthesis of physiological amounts of the UCP, a key and tissue-specific component of thermogenic mitochondria, was observed in cultures at about confluence (day 6), indicating that a complete differentiation of brown adipocytes was achieved in vitro . In postconfluent cells (day 8) the content of UCP decreased rapidly, in contrast to some other mitochondrial proteins (beta subunit of F1-ATPase, cytochrome oxidase) . In these cells, it was possible, by using norepinephrine, to induce specifically the synthesis of the UCP but not of F1-ATPase or cytochrome oxidase . The maximal response was observed at 0.1 microM norepinephrine and the synthesis of UCP remained activated for at least 24 h . Detailed analysis revealed a major role of the beta-adrenergic receptors and elevated intracellular concentration of cAMP in stimulation of UCP synthesis . A quantitative recovery of the newly synthesized UCP in the mitochondrial fraction indicated completed biogenesis of functionally competent thermogenic mitochondria.

Brain Res, 1990 Dec 17, 536(1-2), 23 - 9
High oxygen atmosphere for neuronal cell culture with nerve growth factor . II . Survival and growth of clonal rat pheochromocytoma PC12h cells; Enokido Y et al.; When clonal rat pheochromocytoma PC12h cells were cultured in a 50% O2 atmosphere, cells gradually died during the cultivation . On the other hand, the addition of NGF at the final concentration of 50 ng/ml could rescue the cells from death . The culture in a 40% O2 atmosphere had little effect on the growth of PC12h cells, as compared with the culture in a normal 20% O2 condition . A very high O2 concentration, as 60%, caused severe damage to PC12h cell growth, and the restoration of cell growth by NGF seemed to be insufficient . PC12h cells were fully differentiated and extended dense long neurites by NGF even in a 50% O2 atmosphere . However, the neurite extension in the culture in a 60% O2 atmosphere was suppressed . The cell-saving effect of NGF on cell death in culture under a 50% O2 atmosphere was dose-dependent, and the ED50 value of NGF was 5 ng/ml . Basic fibroblast growth factor and epidermal growth factor also had a potent effect to rescue the cell death in the high O2 culture, but insulin had no effect . Since the differentiation effects of NGF on PC12h cells are thought to offer a model system to investigate the effect of NGF on neurons, the present observations suggest that a protection machinery for high O2 toxicity to neurons may exist in the neuronal differentiated PC12h cells by NGF, but not in the undifferentiated cells.

Brain Res, 1990 Dec 17, 536(1-2), 16 - 22
High oxygen atmosphere for neuronal cell culture with nerve growth factor . I . Primary culture of basal forebrain cholinergic neurons from fetal and postnatal rats; Kushima Y et al.; Cholinergic neurons cultured from postnatal days 11-13 (P11-P13) rat basal forebrain showed better survival in the culture condition using a 50% O2 atmosphere with and without nerve growth factor (NGF) than in a low (10 or 20%) O2 atmosphere . Except for the culture at a low cell density, the beneficial effect of the highly oxidized culture condition was found in the culture from P3 neurons, but not from embryonic day 18 neurons . The survival of microtubule-associated protein 2 (MAP2)-positive neurons in culture from P3 basal forebrain regions was more enhanced in a 50% O2 atmosphere than in 20% and also 10% O2 atmosphere . The viable number of the MAP2-positive neurons in a 10% O2 condition was about half of that in a 20% condition . These results suggest that the response of the cultured neurons to an incubator O2 concentration changes during the neuronal development in CNS from fetal to postnatal stages.

Biochem J, 1990 Dec 15, 272(3), 781 - 5
Stimulation by defined parathyroid hormone fragments of cell proliferation in skeletal-derived cell cultures; Somjen D et al.; We have reported previously that parathyroid hormone (PTH) acts on cultured bone cells to stimulate creatine kinase (CK) activity and {3H}thymidine incorporation into DNA via phosphoinositide turnover, in addition to its other actions via increased cyclic AMP production . We also found that mid-region fragments of PTH stimulate {3H}thymidine incorporation into avian chondrocytes . In the present study of mammalian systems, we demonstrate differential effects of defined synthetic PTH fragments on CK activity and DNA synthesis, as compared with cyclic AMP production, in osteoblast-enriched embryonic rat calvaria cell cultures, in an osteoblast-like clone of rat osteosarcoma cells (ROS 17/2.8) and in chondroblasts from rat epiphysial cartilage cell cultures . Unlike full-length bovine (b)PTH-(1-84) or the fully effective shorter fragment human (h)PTH-(1-34), fragments lacking the N-terminal region of the hormone did not increase cyclic AMP formation, whereas they did stimulate increases in both DNA synthesis and CK activity . Moreover, the PTH fragment hPTH-(28-48) at 10 microM inhibited the increase in cyclic AMP caused by 10 nM-bPTH-(1-84) . The increase of CK activity in ROS 17/2.8 cells caused by bPTH-(1-84) or hPTH-(28-48) was completely inhibited by either cycloheximide or actinomycin D, as was shown previously for rat calvaria cell cultures . These results indicated the presence of a functional domain of PTH in the central part of the molecule which exerts its mitogenic-related effects on osteoblast- and chondroblast-like cells in a cyclic AMP-independent manner . Since cyclic AMP formation by PTH leads to bone resorption, specific mid-region fragments of PTH might prove suitable for use in vivo to induce bone formation without concomitant resorption.

Int J Cancer, 1990 Dec 15, 46(6), 1041 - 7
Expression and modulation by cytokines of the intercellular adhesion molecule-1 (ICAM-1) in human central nervous system tumor cell cultures; Guarini L et al.; The intercellular adhesion molecule (ICAM-1) has been shown to be important in interactions involving cells of the immune system and to be upregulated in a number of cell culture systems by cytokines, including immune interferon (IFN-gamma) and tumor necrosis factor-alpha (TNF-alpha) . In the present study, we have determined by fluorescence-activated cell sorter (FACS) analysis and the anti-ICAM-1 monoclonal antibody (MAb) CL203.4 the base-line expression of ICAM-1 and its modulation by recombinant IFN-beta, IFN-gamma and TNF-alpha in early passage (less than 15) human central nervous system (CNS) tumor-derived cell cultures . These cultures were established from various malignancies, including glioblastoma multiforme (GBM), astrocytoma (AST), ganglioglioma, medulloblastoma, meningioma and a pineal tumor . ICAM-1 expression was highest in the GBM- and AST-derived cell cultures and was lowest in the ganglioglioma and normal pineal cell cultures . Variable ICAM-1 expression was found, however, in tumors of the same histological group . In several cell cultures the variable expression observed by FACS was substantiated by the intensity of the molecular species immunoprecipitated by the anti-ICAM-1 MAb CL203.4 from these cells . All the cell cultures displayed variable but consistent increases in ICAM-1 expression following treatment with IFN-gamma or TNF-alpha . In general, the degree of increase in ICAM-1 expression was greatest in cultures exposed to TNF-alpha . Upregulation of ICAM-1 expression in an established glioblastoma multiforme cell line was of greater magnitude and more rapid following TNF-alpha treatment (within 2 to 3 hr) than exposure to IFN-gamma (by 24 hr) . In several cultures, IFN-beta also increased ICAM-1 expression and enhanced the increase induced by TNF-alpha . The results of the present study indicate that variable expression of ICAM-1 is a common property of early passage cultures of CNS tumors and recombinant interferons and TNF-alpha can differentially upregulate ICAM-1 expression in these CNS tumor cell cultures.

Neurology, 1990 Dec, 40(12), 1854 - 8
Defective dystrophin in Duchenne and Becker dystrophy myotubes in cell culture; Sklar RM et al.; We examined normal and dystrophic human myotubes in cell culture for expression of dystrophin, the protein product of the Duchenne muscular dystrophy locus . Dystrophin levels in developing myotubes detected by Western blotting increased after 24 hours and reached maximum levels after 10 days in fusion medium . We did not detect dystrophin in myotubes cultured from Duchenne myoblasts (7 cases) . Myotubes from a Becker muscular dystrophy patient's biopsy produced a lower molecular weight (approximately 408 kd) dystrophin, which was the same size in a whole muscle preparation from the same biopsy . This 408-kd dystrophin was the expected size for this Becker patient whose DNA was deleted for exons 45-48 of the Duchenne gene . This cell culture system will allow a detailed analysis of the effects of potential pharmacologic agents on steady-state dystrophin levels.

Clin Mater, 1991, 7(4), 335 - 40
Biocompatibility evaluation of glass ionomer cement using cell culture techniques; Doherty PJ; Cell culture techniques have been employed to carry out a preliminary investigation into the biocompatibility of a glass ionomer cement (GIC) . A modification of the commonly used Agar Overlay test and a rapid, colorimetric assay based on the reduction of tetrazolium salt were used . The GIC is shown to leach a cytotoxic agent, possibly fluoride . This agent is effectively removed by an extraction procedure, and re-examination of the GIC demonstrates its cytocompatibility.

Cell Differ Dev, 1990 Dec 2, 32(3), 417 - 23
Differential expression of tenascin splicing variants in the chick gizzard and in cell cultures; Matsuoka Y et al.; Tenascin is a large disulfide-linked hexameric extracellular matrix glycoprotein . It is a multidomain protein containing many repeated structural units such as heptad-, EGF-like-, and fibronectin type III repeats, as well as a homology to the globular domains of beta- and gamma-fibrinogen . In the chick embryo three major tenascin variants exist . They arise from one gene by alternative splicing of three of its 11 fibronectin type III repeats . Monoclonal antibodies against the alternatively spliced domains allowed us to study the expression of tenascin variants in tissue sections and in cell cultures . In the gizzard, the largest tenascin variant was only detected in the smooth muscle layer and the connective tissue below the epithelium of the villi, whereas the shortest tenascin variant was predominant in the tendons and the intramuscular connective tissue . Differential expression of tenascin variants was also obtained in cell cultures of chick embryo fibroblasts . Fetal calf serum equally stimulated the accumulation of all three tenascin variants, whereas after transformation with polyomavirus middle-T only the secretion of the largest tenascin variant was greatly enhanced.

Scand J Immunol, 1990 Dec, 32(6), 595 - 600
Thymic epithelial cells . I . Expression of strong suppressive (veto) activity in mouse thymic epithelial cell cultures; Claesson MH et al.; We show that thymic epithelial cells grown under serum-free conditions in a chemically defined culture medium can act as veto cells in vitro . The veto activity of thymic epithelial cells results in inactivation of specific alloreactive cytotoxic T-cell precursors at the clonal level . It is concluded that the epithelial stromal cells of the thymus, by acting as veto cells, may be responsible for the negative intrathymic selection of self-reactive thymocytes leading to elimination of the vast majority of immature thymic lymphocytes.

EMBO J, 1990 Dec, 9(13), 4259 - 65
Elucidation of amidating reaction mechanism by frog amidating enzyme, peptidylglycine alpha-hydroxylating monooxygenase, expressed in insect cell culture; Suzuki K et al.; A frog 'peptidylglycine alpha-amidating monooxygenase (PAM, EC 1.14.17.3)' was expressed in cultured insect cells by using the baculovirus expression vector system . The enzyme, recovered in the culture medium, was purified to homogeneity . Its apparent molecular mass (43 kd), estimated by both SDS-PAGE and molecular sieving, was higher than the value (39 kd) for the 'PAM' (AE-I) purified from frog skin . N-terminal sequence analysis indicated that cleavage of signal sequence had occurred but the propeptide still remained at the N terminus . The glycine-extended model peptide X-Gly (mean = Ala-Ile-Gly-Val-Gly-Ala-Pro) was used as substrate for the purified enzyme . The reaction product formed at pH 5.4 was isolated and characterized by amino acid sequence analysis, FAB-MASS and 1H-NMR . It was shown that the purified enzyme had converted the model peptide to the C-terminal alpha-hydroxyglycine-extended peptide {X-Gly(OH)} instead of the amidated product (X-NH2), indicating that the enzyme widely known as 'PAM' should be called 'peptidylglycine alpha-hydroxylating monooxygenase' . A novel enzyme, present in the insect cell culture medium and separable from the expressed monooxygenase, could convert the alpha-hydroxyglycine-extended peptide to the amidated product at physiological pH values . It is concluded that the alpha-amidation of glycine-extended peptides is a two-step process catalyzed by the monooxygenase and the novel enzyme.

Br J Cancer, 1990 Dec, 62(6), 935 - 41
Growth and radiosensitivity testing of human tumour cells using the adhesive tumour cell culture system; Parkins CS et al.; The radiosensitivity of human tumour cell lines and cells cultured from xenografts or biopsy specimens was measured using the adhesive tumour cell culture system (ATCCS) . For cell lines the derived surviving fractions at 2 Gy were in good agreement with values obtained by clonogenic assay . However, the assay tended to overestimate survival at higher radiation doses, and thus to give a false impression of radioresistance . When cells taken from xenografts or tumour biopsies were cultured there was no evidence for selective growth of tumour cells: fibroblast-like cells commonly grew . Immunohistochemical staining against the intermediate filament, vimentin, supported the mesenchymal origin of the fibroblast-like cells . In cultures of artificial mixtures of tumour cells and fibroblasts, low proportions of fibroblasts were not excluded by the assay and consequently modified the radiation response . The majority of cultures grown from bladder carcinoma biopsy specimens appeared fibroblast-like, although in some cases clearly distinguishable colonies of tumour cells were also grown . In such tumour types the reliable measurement of radiosensitivity in cells taken from biopsies will require further development of techniques that allow the selective growth of tumour cells.

J Parasitol, 1990 Dec, 76(6), 926 - 8
Growth of Trichomonas vaginalis in a serum-free McCoy cell culture system; Meysick KC et al.; Axenic cultures of Trichomonas vaginalis normally require serum for proliferation, yet serum-containing medium may interfere with the detection of T . vaginalis-secreted virulence factors . Trichomonas vaginalis can, however, grow in coculture with a McCoy cell monolayer in both the presence and absence of serum . For 6 T . vaginalis isolates examined, growth in this serum-free system shows lower peak concentrations of T . vaginalis and longer doubling times than those apparent in a serum-containing McCoy cell system . McCoy cells employed in the system did not appear to secrete soluble growth factors for T . vaginalis . The presence of McCoy cells was required for serum-free proliferation of T . vaginalis possibly indicating that eukaryotic cell membrane components may be important in supporting serum-free growth in this system.

J Electron Microsc Tech, 1990 Dec, 16(4), 347 - 50
Use of microwave fixation in the preparation of cell cultures for observation with the scanning electron microscope; Argall K et al.; This paper describes a method for primary fixation of cultured cells for scanning (SEM) and transmission (TEM) electron microscopy using microwaves alone . This method of fixation takes 8 seconds and is therefore quicker and less expensive than conventional fixation techniques . The preservation of cell morphology is excellent and cultures of mammalian immune system cells and peripheral nervous tissue have been examined using this fixation.

J Clin Microbiol, 1990 Dec, 28(12), 2806 - 7
Rapid isolation of herpes simplex virus by using mink lung and rhabdomyosarcoma cell cultures; Johnston SL et al.; Highly sensitive and rapid results can be obtained by isolating herpes simplex virus from clinical specimens in simple cell culture with rhabdomyosarcoma (RD) cells . In this study, 3,186 clinical specimens were inoculated into locally produced, equivalent-age RD and mink lung (ML) cells . Of 727 positive isolates, all (100%) were isolated from RD cells and only 691 (95%) were isolated from ML cells . Furthermore, 162 of the positive isolates (22%) were isolated in RD cells earlier than in ML cells . RD cells are continuous and can be cultivated in house without decreasing sensitivity as the passage number increases . They produce a highly distinguishable cytopathic effect in response to herpes simplex virus and maintain intense confirmatory staining patterns.

Appl Biochem Biotechnol, 1990 Dec, 26(3), 217 - 29
Performance characteristics of mammalian cell culture process operating continuously with protein-free medium; Bliem R et al.; In this communication we briefly describe a continuous industrial process for the production of Tissue Plasminogen Activator (tPA), operating with a standard, basal medium (DME/F12) . Process data of two culture runs has been chosen to present a performance profile over a production period of more than 3 mo . Developmental aspects of glucose utilization (as a process parameter) are briefly discussed.

Biol Reprod, 1990 Dec, 43(6), 946 - 55
Secreted metalloproteinases in testicular cell culture; Sang QX et al.; It is well known that cultured Sertoli cells secrete plasminogen activators (Lacroix et al., Mol Cell Endocrinol 1977; 9:227-236; Hettle et al., Biol Reprod 1986; 34:895-904) . We now show that testicular cells in culture also secrete gelatinolytic metalloproteinases . Gelatin zymographic analysis of concentrated culture medium proteins reveals that Sertoli cells secrete gelatinases of 185 kDa, 110 kDa, 83 kDa, 76 kDa, and 72 kDa in addition to plasminogen activators (PAs) . Gelatinase 185 kDa is induced by FSH . Media from Sertoli (epithelial)/peritubular (mesenchymal) cell cocultures contain the Sertoli cell gelatinases and one FSH-stimulated gelatinase of 50 kDa, indicating that gelatinase 50 kDa is regulated by both FSH and cell-cell interactions . A 50-kDa fibronectinolytic activity is also present in the coculture medium from cells grown in the presence of FSH . Casein zymography demonstrates a prominent 30-kDa protease only in media from cocultures . Peritubular cells secrete urokinase-type plasminogen activator (u-PA) and exhibit slight degrading activity at 86 kDa and 74 kDa . The gelatinases are most active in the pH range 7.3-8.5 and are completely or partially inhibited by metal ion chelators indicating that they are metalloproteinases . Our data demonstrate that testicular cells in culture secrete several gelatinases in addition to PAs, and that FSH and coculture conditions regulate some of these secreted proteases . We suggest that the highly regulated secretion of these proteases may well be of physiological importance during testicular development and spermatogenesis.

Biol Reprod, 1990 Dec, 43(6), 939 - 45
Kinetics of inhibin secretion in static and superfused Sertoli cell cultures in response to follicle-stimulating hormone; Jakubowiak A et al.; It is generally accepted that inhibin secretion in the testis is regulated by FSH; however, the kinetics of inhibin secretion have not been well defined in vivo and in vitro . We investigated the kinetics of inhibin secretion in response to FSH stimulation in static and superfused Sertoli cell cultures . Sertoli cells from 18-day-old rats were cultured in chemically defined medium for 3 days and were then stimulated for different time periods with FSH (0.1 microgram/ml) . In static cultures, media were changed every 2, 4, or 8 h, and the superfusion was carried out at a steady rate of 3 ml/h . Inhibin in the culture media was measured by RIA, using antiserum against synthetic replicate {30Tyr}inhibin alpha-chain-(1-30) and, in some experiments, also by bioassay . The dynamics of inhibin secretion were similar in static and superfused Sertoli cell cultures . A significant increase (p less than 0.01) of inhibin secretion was noted after 5-6 h of FSH exposure . After 8-12 h of continuous FSH presence, the secretion of inhibin reached a maximal level, 5-10-fold higher than basal secretion (no FSH) . In the continuous presence of FSH, inhibin secretion remained stable at the high level for up to 54 h . FSH removal caused a delayed (8-h) decrease (p less than 0.01) of inhibin secretion, with return to control basal values after approximately 30 h . When FSH was removed 4 h after its addition, inhibin secretion again increased 5-10-fold between 4 and 12 h, then returned to basal values within 30 h.(ABSTRACT TRUNCATED AT 250 WORDS)

Nippon Eiseigaku Zasshi, 1990 Dec, 45(5), 1000 - 6
{Effects of cultural conditions on hexavalent chromium uptake and the cytotoxicity thereof in KB cell culture}; Okamoto M et al.; To reveal the effects of cultural conditions on the cytotoxicity of hexavalent chromium, the uptake of sodium chromate (Na2CrO4) by KB cells and the colony-forming efficiency of the cells were examined under various cultural conditions . The results were summarized as follows: 1) The chromium uptake by the cells after a certain period of incubation with hexavalent chromium was inhibited with the decrease of the temperature (3 degrees, 20 degrees, 37 degrees C), increase of the serum concentration (0, 10, 20, 30%) and increase of pH (6.8-8.2) of the medium . In particular, low temperatures inhibited the chromium uptake by the cells remarkably . However, in relation to the serum addition, no marked effect was found . 2) The chromium uptake by the cells increased with the volume of the medium containing an identical concentration of chromium (2 ppm) and then reached saturation when it was about 0.23 microgram per 10(6) cells . On the other hand, the chromium uptake positively correlated with the concentration of chromium and the total chromium in the medium . 3) The difference of chromium uptake by the cells in different culture media was more marked at acidic pH than that at alkaline pH . However, there was no effect of calcium chloride and glucose concentrations on the uptake of chromium . The chromium uptake by the cells in Ca-Mg-free phosphate-buffered solution (PBS(-} was higher than that in other culture media . Consequently, the above results suggested that the chromium uptake by the KB cells might be affected by the various cultural conditions, especially by temperature, pH and medium volume.

J Lipid Res, 1990 Dec, 31(12), 2179 - 85
Oxygenation of desmosterol and cholesterol in cell cultures; Saucier SE et al.; In order to determine whether hydration of the delta 24 bond of desmosterol contributes to the formation of the regulatory oxysterol, 25-hydroxycholesterol, {3H}desmosterol was incubated with two cultured cell lines and the labeled products were analyzed . Small amounts of 25-hydroxycholesterol were formed with Chinese hamster lung (Dede) cell cultures, but not with mouse fibroblast (L) cell cultures . Apparently, desmosterol was converted into cholesterol, a process that does not occur in L cells, before 25-hydroxycholesterol takes place . No reliable evidence could be obtained for hydration of the delta 24 bond or for the reverse reaction upon incubation of {3H}25-hydroxycholesterol . Oxygenation of desmosterol occurred in both Dede and L cell cultures to give a mixture of 24(R)- and 24(S)-25-epoxy-cholesterol . This reaction, along with the production of 7-oxygenated sterols, may account for low levels of HMG-CoA reductase repressor activity previously found to be associated with delta 24 sterols.

Differentiation, 1990 Dec, 45(3), 185 - 91
Characterization of myosin isoforms in satellite cell cultures from adult rat diaphragm, soleus and tibialis anterior muscles; Dusterhoft S et al.; Satellite cells were isolated by enzymatic dissociation and Percoll gradient centrifugation from adult rat diaphragm, soleus, and tibialis anterior muscles with fairly reproducible yields . Diaphragm and soleus muscle yielded approximately five times more satellite cells than tibialis anterior muscle . According to light microscopic criteria, no morphological differences existed between the satellite cell cultures of different origin . Contrary to the donor muscles, myotubes from the 10-day-cultured satellite cells contained a uniform myosin heavy chain (MHC) pattern with predominance of an immunochemically identified embryonic heavy chain . The three types of cultures displayed a typical embryonic light chain (LC) pattern with LC1emb, LC1f, LC2f, and traces of LC3f . The isomyosin pattern was characterized by four embryonic isomyosins, eM1-eM4, with similar distributions in the three cultures . In summary, these myosin analyses provide no evidence for the existence of satellite cell diversity among three rat muscles of different fiber-type composition, at least not under the applied in vitro conditions.

Biull Eksp Biol Med, 1990 Dec, 110(12), 659 - 61
{Method of radioautographic study of cell cultures}; Pal'tsyn AA et al.; The method of radioautographic study of cell cultures is suggested . It is based on culture embedding into epoxy resin in situ . After this manipulation the photolayer on the lower surface of the polymerized epoxy unit with the cell culture in it is applied . The advantages of this method are discussed.

Rev Sci Tech, 1990 Dec, 9(4), 1131 - 8
Cell culture propagation of calf rotavirus and detection of rotavirus specific antibody in colostrum and milk of cows and buffaloes; Khattar S et al.; A bovine rotavirus was adapted to serial propagation in Madin Darby bovine kidney (MDBK) cells . Virus of cell culture origin when examined by electron microscopy exhibited characteristic rotavirus morphology and had a typical RNA electropherotype on polyacrylamide gel electrophoresis . Mean IgG concentration in the first colostral whey was 62.80 +/- 6.56 mg/ml in cows and 104.40 +/- 31.12 mg/ml and 2.52 +/- 0.28 mg/ml respectively . The geometric mean neutralising antibody titre against bovine rotavirus in the first colostral whey was 279 in cows and 139 in buffaloes . It declined to 10 and 13 (respectively) by the tenth day.

Virus Res, 1990 Dec, 18(1), 3 - 7
Foot-and-mouth disease virus strains isolated from persistently infected cell cultures are attenuated for mice and cattle; Diez J et al.; It was previously reported that during serial passage of a BHK-derived cell line persistently infected with foot-and-mouth disease virus, (termed C1-BHK-Rc1) the virus became increasingly more virulent for BHK-21 cells (de la Torre et al 1988) . Virus strains isolated from different cell passage levels were tested for virulence in mice and cattle . The results showed that in the course of persistence in BHK cells, FMDV became progressively less virulent for mice and cattle.

In Vitro Cell Dev Biol, 1990 Dec, 26(12), 1144 - 50
Characteristics of human arterial smooth muscle cell cultures infected with cytomegalovirus; Tumilowicz JJ; Distinct, sequential events occurring during the destruction and simultaneous regrowth of human arterial smooth muscle cell (SMC) cultures infected with cytomegalovirus (CMV, AD169 strain) were characterized . The events were influenced by the typical phenotypic diversity reflecting relative states of differentiation of the SMC cultures . Progenitors of regeneration were a surviving population of small, undifferentiated or relatively undifferentiated SMCs . As these cells reached confluence focally, the number of cells reactive with antismooth muscle serum, i.e . differentiating, increased, and in some postconfluent foci the organization of SMCs resembled the topography of uninfected cultures . Thus, infected SMC cultures had a limited capacity to repopulate, to organize typically, and to differentiate . However, continuing cytopathic effects gradually destroyed much of the regrowth, and a relatively large, nondividing SMC with prominent cytoplasmic filaments, similar to SMCs in terminal, uninfected cultures, predominated . Infected cultures consisting overwhelmingly of the large terminal phenotype were far less productive of infectious CMV than cultures populated by SMCs with continuing capacity to divide . Gradually, cultures consisting of the terminal phenotype deteriorated as a result of sporadic cytopathic effects of CMV and an effect resembling "senescent" degeneration in uninfected, nondividing cultures in late passage . The infected, terminal phenotype could be a latent or persistent source of CMV antigen or nucleic acid-positive cells detected by different investigators in normal and in atheromatous, human tissue, assuming that it exists and survives for an extended period in vivo after infection of vascular SMC.

Biol Reprod, 1990 Dec, 43(6), 956 - 64
Identification of type IV collagenase in rat testicular cell culture: influence of peritubular-Sertoli cell interactions; Sang QX et al.; In the testis, interactions between peritubular cells (mesenchyme) and Sertoli cells (epithelium), together with proteolytic remodeling of the extracellular matrix, may play a central role in testicular development, morphogenesis, and spermatogenesis . In this study we demonstrate that a metalloproteinase of 72 kDa present in rat Sertoli cell and Sertoli-peritubular cell coculture medium is activated by p-aminophenylmercuric acetate (p-APMA) to a lower molecular mass form, indicating that it is likely to be a latent collagenase . Immunoblots using antibodies to three different domains of type IV collagenase show that the 72-kDa protease and a 76-kDa protease are type IV pro-collagenases . Sertoli cells cultured alone produce basal levels of type IV collagenase that can be immunolocalized in the cytoplasm of cultured cells . Peritubular cells cultured alone produce much less type IV collagenase . However, Sertoli and peritubular cells in coculture do produce type IV pro-collagenase, and in cultures consisting predominantly of peritubular cells, the activated form of type IV collagenase was detected by both zymography and immunoblotting . Cells growing during the transitional phase (from cell attachment to confluence) secrete more metalloproteinases than during the confluent phase . In contrast, plasminogen activator levels are unaffected by time in culture . These results show that rat testicular cells in culture produce and secrete type IV collagenase, and that the secretion and activation of this enzyme and other metalloproteases is regulated by the ratio of mesenchymal cells to epithelial cells and time in culture.

Invest Ophthalmol Vis Sci, 1990 Dec, 31(12), 2572 - 8
Evaluation of antiproliferative agents using a cell-culture model; Senderoff RI et al.; Chinese hamster ovarian (CHO) cells in culture were used to evaluate the relative antiproliferative potential of drugs . These agents have been used to improve the clinical response after glaucoma filtering surgery . The following drugs were evaluated: 5-fluorouracil (5-FU) as the benchmark, 5-fluorouridine (FUR), 5-fluorodeoxyuridine (FUDR), 5'-deoxy-5-fluorouridine (DFUR), bleomycin, and cytarabine (ARA-C) . In addition to cell counting, a colorimetric assay based on the tetrazolium salt, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) was used to follow growth response . The MTT assay was found to be extremely convenient and an indirect measure of cell activity, offering an alternate or addition to a measure of cell number . All agents tested were shown to inhibit cellular proliferation . Dose-response curves for each agent indicate the following absolute potency: FUDR greater than FUR greater than ARA-C greater than 5-FU = bleomycin greater than DFUR . Besides absolute potency, an evaluation of the effects of equivalent inhibitory concentrations of each drug on growth rate was assessed . Several agents affected the proliferation rate patterns differently . Based on these studies, it is suggested that the in vitro model can identify potential agents through an assessment of their overall activity profile in CHO cells, which includes not only their potency based on dose response, but their onset of activity, duration of effect, and potential for toxicity.

J Biol Chem, 1990 Nov 25, 265(33), 20443 - 8
Oxygen radicals generated during anoxia followed by reoxygenation reduce the synthesis of tissue-type plasminogen activator and plasminogen activator inhibitor-1 in human endothelial cell culture; Shatos MA et al.; The effect of anoxia and reoxygenation on the synthesis and secretion of tissue-type plasminogen activator (t-PA) and plasminogen activator inhibitor-1 (PAI-1) was studied in primary cultures of human umbilical vein endothelial cells . Sublethal anoxia, determined by trypan blue dye exclusion and lactate dehydrogenase release, was produced by cell culture under a 95% N2, 5% CO2 atmosphere for 2-24 h and was followed by reoxygenation with 95% air, 5% CO2 for 24 or 48 h . Anoxia did not alter the levels of mRNA for t-PA or PAI-1 in the cells or the secretion of t-PA or PAI-1 into the medium . At 24 h, t-PA secreted into conditioned medium was 7.0 +/- 1.4 ng/2 x 10(6) cells (n = 9) and PAI-1 was 300 +/- 13 IU/2 x 10(6) cells (n = 9), whereas the content of t-PA mRNA was 2.2 pg/micrograms of RNA and PAI-1 mRNA was 180 pg/micrograms of RNA . During reoxygenation, however, t-PA antigen and PAI-1 activity as well as mRNA for PAI-1 decreased proportionally to the duration of anoxia, to reach 27 +/- 1.0, 49 +/- 2.0, and 47 +/- 14% of control values, respectively, within 24 h of anoxia . t-PA mRNA also decreased significantly during reoxygenation following anoxia, but the extent could not be accurately quantitated . Addition, during anoxia, of a 200 micrograms/ml concentration of the superoxide anion radical scavenger superoxide dismutase or of a 5 mM concentration of the iron chelator deferoxamine mesylate prevented the subsequent decrease of t-PA antigen during reoxygenation; addition of these compounds during reoxygenation had no effect . Superoxide dismutase, but not deferoxamine mesylate, when added during anoxia prevented the subsequent decrease in PAI-1 activity . These studies suggest that the marked alteration of endothelial cell fibrinolysis during anoxia followed by reoxygenation is most likely mediated by a mechanism dependent on oxygen radicals . Impaired endothelial cell fibrinolysis may contribute to the pathophysiology of ischemia/reperfusion injury.

Biochem Biophys Res Commun, 1990 Nov 15, 172(3), 1109 - 15
Expression of the chloramphenicol acetyltransferase (CAT) reporter gene by the murine Cyp1a-2 (cytochrome P3(450)) promoter in hepatoma cell cultures; Owens RA et al.; In C57BL/6 mouse liver, both murine Cypla-1 (cytochrome P1(450} and Cypla-2 (P3(450} genes are inducible by 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD; dioxin), and Cypla-2 is constitutively expressed at high levels . Although the Cypla-1 gene is constitutively expressed and TCDD-inducible in mouse hepatoma Hepa-1 cell cultures, Cypla-2 gene expression is absent in these cultures . We show here that the 5' flanking region of Cyp1a-2 from - 1843 to +52 (base pairs relative to the Transcription initiation site) linked to the chloramphenicol acetyltransferase (CAT) reporter gene in stable Hepa-1 transformants produces no basal or TCDD- or cycloheximide-inducible CAT activity . On the other hand, the Cyp1a-2 promoter from -63 to +52 driving the CAT gene is inducible by cycloheximide . A chimeric plasmid containing the Cyp1a-1 TCDD-responsive enhancer (-1646 to -245) ligated to a Cyp1a-2 promoter region (-129 to +52) supports TCDD-inducible CAT expression in Hepa-1 cells and in rat 7777 cells . These data suggest that, although sequences between - 1843 and +52 +52 are not sufficient for Cyp1a-2 gene expression, the murine Cyp1a-2 promoter is functional in cell cultures.

FEBS Lett, 1990 Nov 12, 274(1-2), 185 - 8
Induction of type II iodothyronine 5'-deiodinase and mitochondrial uncoupling protein in brown adipocytes differentiated in cell culture; Houstek J et al.; Brown adipocytes differentiated in primary cell culture were found to contain a type II iodothyronine 5'-deiodinase (5'D) . Incubation of confluent cells with norepinephrine or dibutyryl-cAMP caused up to 17-fold increase in 5'D activity with a maximum after 8 h . Activation of 5'D required mRNA and protein synthesis and was accompanied by parallel, up to 5.8-fold increase in the amount of mitochondrial uncoupling protein with a maximum after 24 h . Analysis of adrenergic stimulation of 5'D suggested predominant involvement of the beta-receptors and increased intracellular cAMP levels, while the contribution of alpha 1-receptors was small.

Brain Res, 1990 Nov 5, 532(1-2), 76 - 81
Corticotropin-releasing hormone and dexamethasone do not alter secretion of immunoreactive beta-endorphin from dissociated fetal hypothalamic cell cultures; Kapcala LP et al.; Corticotropin-releasing hormone (CRH) and glucocorticoids are major regulators of the hypothalamic-pituitary-adrenal (HPA) axis controlling secretion of beta-endorphin and other pro-opiomelanocortin (POMC)-derived peptides from pituitary . Although previous work has shown that CRH stimulates secretion of beta-endorphin from adult hypothalamic explants, and that glucocorticoids can inhibit basal and stimulated secretion of POMC-derived peptides from pituitary, the role of glucocorticoids on hypothalamic beta-endorphin secretion is not known . Studies were performed to assess the effects of CRH and dexamethasone, a potent glucocorticoid, on secretion of immunoreactive (IR) beta-endorphin from dissociated fetal hypothalamic cell cultures . CRH (10(-9)-10(-6) M) did not stimulate secretion of IR-beta-endorphin from hypothalamic cells which did release IR-beta-endorphin upon potassium-induced depolarization . However, CRH did stimulate IR-beta-endorphin secretion from fetal hypothalamic explants which were similar to hypothalamic tissue from which dissociated hypothalamic cell cultures were derived . Exposure of cells to dexamethasone (10(-6) M) did not inhibit basal or potassium-stimulated release of IR-beta-endorphin . These results indicate that: (1) dissociated fetal hypothalamic cells in culture do not exhibit a functional CRH receptor coupled to stimulation of IR-beta-endorphin secretion; (2) exposure of hypothalamic cells to dexamethasone does not inhibit basal nor depolarization-induced release of IR-beta-endorphin; and (3) dissociated fetal hypothalamic cells may have limited utility in elucidating specific regulatory relationships because of in vitro conditions and/or cytoarchitectural relationships.

Clin Chim Acta, 1990 Nov 5, 191(3), 221 - 32
Gamma-glutamyltransferase from human hepatoma cell lines: purification and cell culture of HepG2 on microcarriers; Visvikis A et al.; After screening different human hepatoma cell lines, we observed that both HepG2 and PLC/PRF/5 naturally produced large amounts of gamma-glutamyltransferase . We optimized HepG2 cell culture conditions and observed that higher cell densities were obtained when cells were cultured on microcarriers, particularly when Cytodex 3 was used and that cell growth was optimal when DMEM, the basic medium, was supplemented with 5% fetal calf serum and 6 mmol/l glutamine . These culture conditions allowed us to produce the highest amounts of GGT after about 150 h of culture . The GGT obtained from HepG2 cells was partially purified and some of its physico-chemical properties characterized . Successive Con A gel chromatography separated the activity into two peaks, suggesting that GGT from HepG2 is not uniformly glycosylated . Papain-treated HepG2 GGT showed a Mr of about 120 kDa and migrated as a single-chain protein in SDS-PAGE . Immunological and kinetic properties of the GGT were similar to other human GGTs (liver, kidney and serum) . It appears that HepG2 GGT could be a source for the preparation of a human enzyme reference material.

Pneumonol Pol, 1990 Nov-Dec, 58(11-12), 604 - 9
{Effect of indomethacin on bronchial reactivity to supernatant of mononuclear cell cultures from patients with bronchial asthma}; Krasnowska M et al.; In 27 asthmatic patients a cell culture was made from mononuclear cell stimulated by PHA . Indomethacin was added to part of the cultures . After 2 days the culture was terminated and the supernatant was prepared for bronchoprovocation studies . Patients underwent baseline respiratory function studies, after which bronchial provocation was carried out with different concentrations of the supernatant . Bronchial provocation using supernatant from cells cultured with indomethacin produced significant respiratory function alterations in comparison with tests carried out with supernatant from cells stimulated with PHA.

Zentralbl Veterinarmed B, 1990 Nov, 37(9), 696 - 700
{Adaptation of permanent bovine kidney cells to serum free hormone supplemented cell culture media}; Binder S et al.; The examinations show, that it is possible to adapt MDBK-cells (ATCC CCL 22) to serum-poor (0.1% fetal calf serum) and to serum-free, adequate supplemented media . Morphology of the cells remained unchanged . The components of the serum-free medium are a 50:50 mixture of Dulbecco's modified Eagle's medium and HAM's F12 medium, supplemented with insulin (5 micrograms ml-1), transferrin (10 micrograms ml-1), thyroglobulin (10 micrograms ml-1), prostaglandin E1 (50-100 ng ml-1) . Seeding cells at a density of 26.10(3) cm-2 at day 0 resulted in better multiplication than a higher seeding density.

Zentralbl Veterinarmed B, 1990 Nov, 37(9), 641 - 50
Morbillivirus infections of seals during the 1988 epidemic in the Bay of Heligoland: III . Transmission studies of cell culture-propagated phocine distemper virus in harbour seals (Phoca vitulina) and a grey seal (Halichoerus grypus): clinical, virological and serological results; Harder T et al.; Eight harbour seals (Phoca vitulina), two of them seronegative, six seropositive against PDV and a seronegative grey seal (Halichoerus grypus) were exposed to a low doses of a cell culture-propagated phocine distemper virus isolate (PDV 2558/Han 88) . An intranasal route of inoculation was chosen . Clinical signs, resembling those of 1988's seal disease and seroconversion were observed in both seronegative harbour seals . One of them succumbed to the infection . The virus was not transmitted to another susceptible harbour seal which served as in-contact animal . Virus could be recovered from leucocytes of the diseased seals . Viremia was also present in a seropositive harbour seal that developed mild clinical signs; other seropositive seals were protected from clinical disease . The grey seal showed seroconversion upon inoculation, but did not develop any signs of disease . The humoral immune response of the seals plainly discriminated between homologous (PDV) and heterologous (canine distemper virus, CDV) virus as shown by virus neutralization tests and an antibody-binding assay (PLA).

Pediatr Res, 1990 Nov, 28(5), 473 - 6
Psychosine cytotoxicity in rat neural cell cultures and protection by phorbol ester and dimethyl sulfoxide; Sugama S et al.; In Krabbe's disease (globoid cell leukodystrophy), galactosylsphingosine (psychosine) is considered to be a causative agent of the pathology found in the nervous system of the patients . In our study, we examined the cytotoxic effect of psychosine in neural cell cultures derived from the rat nervous system . The concentration of toxic thresholds varied from cell type to cell type . The 50% of toxic doses for oligodendrocytes, astrocytes, and the sensory neurons of the dorsal root ganglia were 8, 20, and 30 micrograms/mL, respectively . Oligodendrocytes, therefore, appeared to show a higher sensitivity to psychosine than did astrocytes or neurons . When phorbol ester or DMSO was applied simultaneously with psychosine as protective agents in enriched cultures of rat oligodendrocytes, the total number of live cells and galactocerebroside-positive cells and the 2'3'-cyclic nucleotide 3'-phosphohydrolase activity in these cultures were considerably higher as compared with their levels in the experimental cultures treated with psychosine alone . These results indicate that phorbol ester and DMSO could serve as protective agents for psychosine neurotoxicity.

J Clin Microbiol, 1990 Nov, 28(11), 2482 - 4
Isolation of 16 strains of Coxiella burnetii from patients by using a sensitive centrifugation cell culture system and establishment of the strains in HEL cells; Raoult D et al.; Q fever, caused by Coxiella burnetii, may be acute or chronic . Only a few strains of C . burnetii have been isolated due to the difficulty and hazard of isolation . We report here the isolation using a centrifugation shell vial technique of 16 new strains from patients suffering chronic Q fever . Twenty-four samples were inoculated onto human embryonic lung (HEL) fibroblast cell monolayers growing in shell vials . C . burnetii was detected 6 days later by using immunofluorescence . Samples from valves (n = 10), arterial prostheses (n = 2), bone (n = 3), skin biopsy (n = 1), bone marrow (n = 1), and blood (n = 5) from 16 patients were successfully cultured . Two cerebrospinal fluid samples from two patients were negative . The strains were subcultured in HEL cells and are now established . The technique is sensitive and less hazardous than animal inoculation . We recommend the shell vial technique for isolation of C . burnetii.

J Cell Physiol, 1990 Nov, 145(2), 200 - 6
Stimulation of plasma membrane and matrix vesicle enzyme activity by transforming growth factor-beta in osteosarcoma cell cultures; Bonewald LF et al.; Transforming growth factor-beta (TGF beta) serves an important role in extracellular matrix formation by stimulating the production of numerous extracellular matrix proteins by connective tissue cells and by osteoblasts or bone-forming cells . TGF beta has been shown to stimulate alkaline phosphatase (ALPase) activity in the rat osteoblast-like osteosarcoma cell line ROS 17/2.8 . Previous studies have shown that this enzyme is elevated during calcification of bone and that it is enriched in matrix vesicles, an extracellular organelle associated with initial hydroxyapatite formation . To test the hypothesis that TGF beta plays a role in regulating mineral deposition in the matrix, the effects of TGF beta on ALPase and phospholipase A2, two enzymes associated with mineralization, were examined . ROS 17/2.8 cells were cultured at high and low density with recombinant human TGF beta (0.1-10 ng/ml) to examine the influence of cell maturation on response to TGF beta . Maximal stimulation of ALPase activity in the low density cultures was seen at 5 ng/ml; in high-density cultures, there was further stimulation at 10 ng/ml . There was a dose-dependent increase in ALPase activity seen in the matrix vesicles and plasma membranes in both types of cultures . Matrix vesicle ALPase exhibited a greater response to factor than did the plasma membrane enzyme . However, in low-density cultures, the two membrane fractions exhibited a parallel response with greatest activity consistently in the matrix vesicles . There was a dose-dependent increase in phospholipase A2-specific activity in the plasma membranes and matrix vesicles of both high- and low-density cultures . In agreement with previous studies, TGF beta inhibited cellular proliferation 50% . The results show that addition of TGF beta stimulates the activity of enzymes associated with calcification . The effect of TGF beta is dependent on the stage of maturation of the cell . This study indicates that TGF beta may play an important role in induced bone formation, calcification, and fracture repair in addition to its role in promoting chondrogenesis.

Toxicol Appl Pharmacol, 1990 Nov, 106(2), 334 - 40
Inhibition of FSH-stimulated cAMP accumulation and progesterone production by mono(2-ethylhexyl) phthalate in rat granulosa cell cultures; Treinen KA et al.; Several phthalate esters are male and female reproductive toxicants in vivo . In the male, mono(2-ethylhexyl) phthalate (MEHP), the active metabolite of di(2-ethylhexyl) phthalate (DEHP), inhibits follicle stimulating hormone (FSH)-stimulated cAMP accumulation in the Sertoli cell in vitro . Since granulosa and Sertoli cells share several structural and functional characteristics, the effect of MEHP on granulosa cell intracellular cAMP accumulation was examined to elucidate a possible mechanism for DEHP reproductive toxicity in females . MEHP (100 microM) reduced FSH-stimulated cAMP accumulation in granulosa cells by 40% after a 24-hr preincubation . Significant inhibition of cAMP accumulation by MEHP occurred by 15 hr and MEHP did not affect the dose of FSH which resulted in half-maximal stimulation . Detailed investigations regarding the mechanism of MEHP inhibition were conducted using cholera toxin, forskolin, and isoproterenol . In contrast to FSH, MEHP did not affect the ability of these compounds to stimulate cAMP accumulation . In addition, a functional endpoint of granulosa cell function, progesterone production, was inhibited in a dose-dependent manner by MEHP . Further experiments will be necessary to determine the significance of these findings to in vivo toxicity, but these experiments describe a specific site of action of MEHP in vitro which may be related to the in vivo female reproductive toxicity of phthalate esters.

Endocrinology, 1990 Nov, 127(5), 2393 - 9
Gonadotropin-releasing hormone modulation of protein kinase-C activity in perifused anterior pituitary cell cultures; Andrews WV et al.; The ability of GnRH to modulate protein kinase-C (PKC) activity was examined in perifused rat pituitary cell cultures . Under these conditions, LH release and GnRH receptor number remained unchanged after repeated pulses of 1 nM GnRM, whereas PKC (measured both enzymatically and by radioligand assay) showed an initial increase in kinase activity after the first pulse of GnRH (approximately 2-fold), followed by down-regulation of PKC activity with subsequent pulses of the releasing hormone . It was also observed that the GnRH-stimulated down-regulation of PKC was dependent on the presence of extracellular calcium, which was not the case for the initial up-regulation of PKC . These findings are consistent with a modulating role of the GnRH receptor on PKC activity through a Ca2(+)-dependent process . This study also provides further evidence that GnRH-stimulated LH release and PKC activity can be uncoupled.

Endocrinology, 1990 Nov, 127(5), 2387 - 92
Regulation of the synthetic rate of gonadotropin-releasing hormone receptors in rat pituitary cell cultures by inhibin; Braden TD et al.; Regulation of steady state levels of plasma membrane receptors for GnRH is the arithmetic result of processes that contribute to the appearance of receptors (synthesis, recycling, and unmasking) less those that contribute to the loss of receptors (degradation, internalization, and inactivation) . We have adapted the density shift technique to evaluate specifically the rate of synthesis of GnRH receptors in rat pituitary cell cultures . Recently, it has been shown that inhibin can decrease the steady state levels of GnRH receptors in rat pituitary cell cultures and can block homologous up-regulation of GnRH receptors . In the present study we have evaluated the ability of purified inhibin to affect the synthesis rate of GnRH receptors under basal conditions and after exposure of cultured gonadotropes (from female weanling rats) to GnRH . Cells were exposed to inhibin alone (4 or 12 ng/ml) or to GnRH (10(-10) M) plus inhibin (0.4, 4, or 12 ng/ml) in the presence of densely labeled amino acids . GnRH was administered as a 20-min pulse, but inhibin treatment was continued for up to 2 days . After these treatments, GnRH receptors were covalently linked to a radio-labeled photoaffinity probe (125I- Tyr5-{azido-benzoyl-D-Lys6} GnRH) and solubilized with 1% sodium dodecyl sulfate . Newly synthesized GnRH receptors (those that had incorporated the dense amino acids) were separated from previously synthesized receptors (those containing normal amino acids) by velocity sedimentation through sucrose gradients (O-20% sucrose, 1% sodium dodecyl sulfate, and 10 mM Tris-HCl, pH 7.0; centrifuged at 156,000 x g for 24 h) . After velocity sedimentation, gradients were fractionated, and the radioactivity in each fraction was quantified . Treatment with inhibin alone had no effect on the synthesis rate of GnRH receptors compared to that of control cultures (t1/2, 23.5 +/- 0.3 vs . 23.3 +/- 0.3 vs . 22.9 +/- 0.9 h for control, 4 ng/ml inhibin, and 12 ng/ml inhibin, respectively) . In contrast, inhibin blocked the stimulation of homologous receptor synthesis by GnRH in a dose-dependent manner (t1/2, 12.2 +/- 0.7 vs . 14.0 +/- 0.7 vs . 19.2 +/- 1.5 vs . 20.0 +/- 2.9 h for GnRH alone and GnRH plus 0.4, 4, or 12 ng/ml inhibin, respectively) . These data indicate that in rat pituitary cell cultures, inhibin does not decrease basal levels of GnRH receptors by affecting the synthesis rate of receptors, but prevents up-regulation of GnRH receptors by blocking stimulation of GnRH receptor synthesis by homologous hormone.

J Virol, 1990 Nov, 64(11), 5519 - 28
Unique amino acid substitutions in the capsid proteins of foot-and-mouth disease virus from a persistent infection in cell culture; Diez J et al.; Maintenance of a persistent foot-and-mouth disease virus (FMDV) infection in BHK-21 cells involves a coevolution of cells and virus (J . C . de la Torre, E . Martinez-Salas, J . Diez, A . Villaverde, F . Gebauer, E . Rocha, M . Davila, and E . Domingo, J . Virol . 62:2050-2058, 1988) . The resident FMDV undergoes a number of phenotypic changes, including a gradual decrease in virion stability . Here we report the nucleotide sequence of the P1 genomic segment of the virus rescued after 100 passages of the carrier cells (R100) . Only 5 of 15 mutations in P1 of R100 were silent . Nine amino acid substitutions were fixed on the viral capsid during persistence, and three of the variant amino acids are not represented in the corresponding position of any picornavirus sequenced to date . Cysteine at position 7 of VP3, that provides disulfide bridges at the FMDV fivefold axis, was substituted by valine, as determined by RNA, cDNA, and protein sequencing . The modified virus shows high buoyant density in cesium chloride and depicts the same sensitivity to photoinactivation by intercalating dyes as the parental FMDV C-S8c1 . Amino acid substitutions fixed in VP1 resulted in altered antigenicity, as revealed by reactivity with monoclonal antibodies . In addition to defining at the molecular level the alterations the FMDV capsid underwent during persistence, the results show that positions which are highly invariant in an RNA genome may change when viral replication occurs in a modified environment.

Ann Neurol, 1990 Nov, 28(5), 627 - 33
Guanidino compounds that are increased in cerebrospinal fluid and brain of uremic patients inhibit GABA and glycine responses on mouse neurons in cell culture; De Deyn PP et al.; Four guanidino compounds that have been found to be markedly increased in cerebrospinal fluid and brain tissue of uremic patients, namely, guanidine, methylguanidine, creatinine, and guanidinosuccinic acid, were applied to mouse spinal cord neurons in primary dissociated cell culture to evaluate their effects on postsynaptic responses to gamma-aminobutyric acid (GABA) and glycine . Intracellular microelectrode recording techniques were used . Guanidine, methylguanidine, creatine, and guanidinosuccinic acid reversibly and in a dose-dependent manner inhibited both GABA and glycine responses . Guanidinosuccinic acid was the most potent inhibitor of the amino acid responses, followed in decreasing potency by methylguanidine, guanidine, and creatinine . Guanidinosuccinic acid inhibited responses to GABA and glycine, at concentrations similar to those found in cerebrospinal fluid and brain tissue of patients with terminal renal insufficiency . The other guanidino compounds tested exerted their effects only at concentrations higher than those found in uremic biological fluids and tissues . The inhibitory effect of guanidine and methylguanidine on responses to GABA was additive . The effect of the guanidino compounds on GABA responses was not antagonized by coapplication of the benzodiazepine-receptor antagonist CGS 9896 . The results suggest that guanidine, methylguanidine, creatinine, and guanidinosuccinic acid inhibited responses to the inhibitory neurotransmitters GABA and glycine by blocking the chloride channel . The observed action of the studied guanidino compounds might contribute to the pathogenesis of the complex neurological symptomatology encountered in uremia.

Cytotechnology, 1990 Nov, 4(3), 243 - 50
Utilization and stability of vitamins in serum-containing and serum-free media in CHO cell culture; Kurano S et al.; The concentrations of four vitamins, ascorbic acid, nicotinamide, choline and thiamine were evaluated in the culture supernatant of Chinese hamster ovary (CHO) cells . The media used were alpha-modified Eagle's minimum essential medium (MEM-alpha) supplemented with 10% fetal calf serum, and a 1:1 mixture of Ham's F12 and Dulbecco's modified Eagle's medium (DME/F12), containing neither serum nor protein . The reference experiment without cells revealed instability of ascorbic acid and thiamine . Moreover, a significant amount of each vitamin decreased in the culture supernatant . The possibility of growth limitation by vitamin depletion is strongly suggested.

Brain Res, 1990 Oct 29, 531(1-2), 183 - 8
The effect of NMDA receptor glycine site antagonists on hypoxia-induced neurodegeneration of rat cortical cell cultures; Priestley T et al.; The neuroprotective potential of an antagonist (7-chlorokynurenic acid (7-CIKYNA)) and a low efficacy partial agonist (HA-966) for the glycine modulatory site on the N-methyl-D-aspartate (NMDA) receptor complex has been examined using a neuronal cell culture/hypoxia model of neurodegeneration . Their effects were compared to those of the potent uncompetitive NMDA antagonist, MK-801 . Hypoxic cell injury was assessed visually and quantified by measuring the appearance of two cytosolic enzymes, lactate dehydrogenase (LDH) and neurone specific enolase (NSE), in the culture medium . MK-801 prevented the hypoxia-induced cell mortality in a concentration-related manner with an IC50 of 15 nM against increases in LDH levels . HA-966 and 7-CIKYNA also produced concentration-related protective effects with IC50s of 175 and 18 microM, respectively . Although both glycine antagonists were considerably weaker than MK-801 their maximum neuroprotective effects were comparable to that produced by MK-801, i.e . complete protection . This indicates that the level of NMDA receptor activation which can take place in the presence of the partial agonist HA-966 is insufficient to cause permanent neuronal damage . Concentration-effect curves were similar when NSE was used as the marker enzyme, supporting previous observations that the increases in LDH levels accurately and specifically reflect neuronal cell death . These results provide further evidence that hypoxia-induced injury to cortical neuronal cultures is mediated by an excessive stimulation of NMDA receptors and that glycine-site antagonists and partial agonists may have therapeutic potential in conditions where pathologically high levels of NMDA receptor activation are thought to occur.

J Biol Chem, 1990 Oct 15, 265(29), 17891 - 8
Formation of high molecular weight dermatan sulfate proteoglycan in bovine aortic endothelial cell cultures . Evidence for transglutaminase-catalyzed cross-linking to fibronectin; Kinsella MG et al.; Three glucuronate-rich dermatan sulfate proteoglycan (DS-PG) subclasses were isolated and previously characterized from bovine aortic endothelial cell cultures (Kinsella, M . G., and Wight, T . N . (1988) J . Biol . Chem . 263, 19222-19231) . In the present study, pulse-chase experiments indicate that the DS-PG of highest apparent Mr (approximately 1 x 10(6)), denoted previously as HMW-DS, is a relatively stable component of the endothelial extracellular matrix and is formed at the expense of lower Mr DS-PG species . The formation of HMW-DS is reduced in a dose-dependent manner in the presence of dansylcadaverine, an inhibitor of transglutaminase-catalyzed protein cross-linking, but not when the activity of other cross-linking enzymes such as lysyl oxidase is inhibited . The putative DS-PG precursor to HMW-DS accumulates during inhibition of cross-linking only when lysosomal degradation is also inhibited by ammonium chloride, suggesting that the precursor is degraded rapidly in the absence of cross-linking . HMW-DS is precipitable from endothelial cell monolayer extracts with antibodies against fibronectin, a known transglutaminase substrate . Thus, we conclude that the stability of HMW-DS in the subendothelial matrix in culture depends upon the cross-linking of a low Mr DS-PG precursor to matrical protein(s), including fibronectin, resulting in the formation of a DS-PG subclass of high apparent molecular mass.

Endocrinology, 1990 Oct, 127(4), 1561 - 7
Possible role of transforming growth factor-beta as a mediator of luteotropic action of prolactin in rat luteal cell cultures; Matsuyama S et al.; PRL is known to be a hormone carrying luteotropic action in rats and enhances progesterone secretion by suppressing 20 alpha-hydroxysteroid dehydrogenase (20 alpha HSD) activity in the corpus luteum . In this study, we investigated the effects of PRL and transforming growth factor-beta (TGF beta) on the 20 alpha HSD activity of rat luteal cells in vitro and examined whether TGF beta is involved in the luteotropic action of PRL . 20 alpha HSD activity of luteal cells (from midpseudopregnant rats), which had been suppressed by PRL in vivo, increased when the cells were cultured for 48 h without PRL addition . TGF beta (0.01, 0.1, 1.0, and 10.0 ng/ml) as well as PRL (2, 20, 200, and 2000 ng/ml) suppressed this increase in a dose-dependent manner . Furthermore, the suppressive effect of PRL on 20 alpha HSD activity was significantly attenuated by anti-TGF beta antibody . Activin, having homology with TGF beta in its chemical structure, also suppressed the increase in enzyme activity, although the effect was much less marked than that of TGF beta . TGF beta or PRL did not affect total progestin (progesterone plus 20 alpha-dihydroprogesterone) secretion, but induced reduction in 20 alpha-dihydroprogesterone secretion during a 48-h culture period, without any alteration of DNA or protein content per culture dish . These results indicate that TGF beta, like PRL, can suppress luteal 20 alpha HSD activity without producing nonspecific cell damage, and that the luteotropic action of PRL is at least in part mediated by TGF beta or an immunoreactive TGF beta-like substance(s).

Mikrobiyol Bul, 1990 Oct, 24(4), 321 - 6
{Use of various cell culture antigens in the indirect fluorescent antibody test for the detection of antinuclear antibodies}; Kocabeyoglu O et al.; Cell culture antigens were prepared from Vero, BHK-21 and Hep-2 cells which were propagated on slide, for detection antinuclear antibody (ANA) in sera with indirect fluorescent antibody test (IFAT) . ANA were investigated in 55 sera which were positive at 1/20 titer with KB IFAT ANA test kits (Virgo), by using these cell culture antigens . 50 sera (91%) with Vero antigens, 46 sera (83%) with BHK-21 antigens and 44 sera (80%) with Hep-2 antigens were found positive at 1/20 titer . As conclusion Vero cell culture which is propagated on slide can be used as antigen for detection of ANA with IFAT.

Biologicals, 1990 Oct, 18(4), 309 - 13
Development of a cell culture assay for Bordetella parapertussis heat-labile toxin; Endoh M et al.; A cell culture assay for heat-labile toxin isolated from Bordetella parapertussis has been developed . In this assay, the ability of heat-labile toxin to induce contraction of vascular smooth muscle cells is measured . The method allows for detection of as little as 0.6 ng/ml of the toxin . The results obtained from this in vitro assay correlated well with those obtained with in vivo assays indicating that the cell culture assay may be a useful alternative to animal assays.

J Biomed Mater Res, 1990 Oct, 24(10), 1355 - 67
In vitro toxicity test of 2-cyanoacrylate polymers by cell culture method; Tseng YC et al.; The inhibition of Swiss 3T3 cell growth by the microspheres prepared from various 2-cyanoacrylate polymers was investigated to assess their cell toxicity . Poly(ethoxy-ethyl 2-cyanoacrylate) and poly(methyl 2-cyanoacrylate) microspheres inhibited cell growth in a smaller amount than poly-(isobutyl 2-cyanoacrylate) and poly (ethyl 2-cyanoacrylate) microspheres . The extent of cell growth inhibition by the microspheres decreased with the increasing molecular weight, regardless of the kind of polymers used . Every kind of the microspheres was degraded releasing formaldehyde in the culture medium . The cell growth inhibition by the medium containing the microspheres was observed within 24 h for poly(ethoxyethyl 2-cyanoacrylate) and poly(methyl 2-cyanoacrylate) . The extent of inhibition was in a linear proportion with the amount of formaldehyde released . It is concluded that the cell toxicity of 2-cyanoacrylate polymers is attributed to formaldehyde released upon polymer degradation.

Cell Biol Int Rep, 1990 Oct, 14(10), 877 - 85
The effect of transforming growth factor-beta on the alkaline phosphatase activity in rabbit renal cortical tubular cell cultures; Kanda S et al.; The effect of various growth regulators including epidermal growth factor and transforming growth factor-beta on the alkaline phosphatase activity of rabbit renal cortical tubular cells has been investigated in a serum-free culture . As a result, it was found that transforming growth factor-beta, known to be a growth inhibitor of renal tubular cells, increased the alkaline phosphatase activity of the tubular cells dose-dependently and that cycloheximide blocked any increase in the activity of this factor . In contrast, epidermal growth factor decreased the alkaline phosphatase activity in the tubular cells.

Hum Genet, 1990 Oct, 85(5), 551 - 4
In situ chromosome preparation technique for simultaneous cytogenetic and immunocytochemical studies on cell cultures of solid tumors; Henn W et al.; Immunophenotyping of cultured cancer cells requires intact antigenic structures; these are mostly destroyed by conventional chromosome preparation techniques . Thus, the simultaneous cytogenetic and immunocytochemical characterization of solid tumor cells appears unfeasible . Here, we describe a novel method that allows in situ chromosome preparation from monolayer cultures of solid tumor cells without affecting their immunological features . Using this technique, it is possible to achieve detailed cytogenetic data including chromosome banding together with the demonstration of cytoplasmic and nuclear antigens within the same tumor cells.

J Parasitol, 1990 Oct, 76(5), 631 - 8
Growth characteristics in axenic and cell cultures, protein profiles, and zymodeme typing of three Trypanosoma cruzi isolates from Louisiana mammals; Barr SC et al.; Rates of trypomastigote adherence, interiorization, amastigote division in, and trypomastigote release from Vero cells were measured for Trypanosoma cruzi isolates from a dog (Tc-D), opossum (Tc-O), and an armadillo (Tc-A) from Louisiana . Because the Tc-O and Tc-A (wild isolates) trypomastigotes became interiorized rapidly, the media were quickly depleted of trypomastigotes thus reducing the numbers available to adhere to cells . In contrast, the Tc-D trypomastigote interiorization rate was slower . Intracellular amastigote division rate was slower for the Tc-D than the wild isolates . The Tc-D trypomastigotes were released from cells approximately 2 days later than wild isolate trypomastigotes, but twice the number were released . Growth rate for Tc-D epimastigotes in liver infusion tryptose media was faster than that of wild isolates . The doubling times for Tc-D, Tc-O, and Tc-A were 48.0, 69.0, and 67.4 hr, respectively . Soluble parasite extract was produced from epimastigotes of each isolate by freeze/thawing, sonication, and high-speed centrifugation . Proteins were separated on an SDS-PAGE slab gel and stained with Coomassie blue . Although similar bands were present in each preparation, the general pattern of staining was similar only between the Tc-O and Tc-A preparations, which showed some differences from the Tc-D preparation . Each isolate was zymodeme typed using 5 enzymes in lysates produced from epimastigotes of each isolate . Enzymes were separated electrophoretically and stained . Wild isolates showed similar patterns as zymodeme 1 reference stock, whereas the Tc-D isolate produced a pattern that did not resemble any of the reference stocks examined.

J Cell Physiol, 1990 Oct, 145(1), 102 - 9
Comparison of bone and parathyroid hormone as stimulators of osteoclast development and activity in calvarial cell cultures from normal and osteopetrotic (mi/mi) mice; Graves L 3rd et al.; Osteoclast development was studied in cell cultures prepared from calvaria of neonatal osteopetrotic (mi/mi) mice or their normal littermates, using tartrate-resistant acid phosphatase (TRAPase), as an osteoclast marker . In cultures from normal mice, treatment with 10 nM PTH for 4-5 days stimulated the formation of osteoclasts . However in cultures from mi/mi mice, this response was only 7% +/- 5% that of normal mice and they were significantly smaller than osteoclasts of normal mice . Mineralized bone particles elicited osteoclast development in cultures from both normal and mi/mi mice, and osteoclast size was identical for both genotypes . Seventy-eight to 96% of the TRAPase-positive cells bound 125I-CT, as demonstrated by autoradiography . 125I-CT binding characteristics were identical in cultures from both genotypes treated with bone particles, exhibiting a Kd of 3.3-3.6 x 10(-10) M . Addition of PTH stimulated 45Ca release from the added bone particles only in the case of cultures prepared from normal mice, and CT inhibited this response . Cells from normal mice were capable of excavating bone from the surface of smooth cortical bone wafers, but such excavations were rarely seen in the case of calvarial cells from mi/mi mice . Thus, PTH-driven differentiation of osteoclasts is arrested in calvarial cell cultures from mi/mi mice, but mi/mi preosteoclasts retain the ability to express certain osteoclast markers in response to bone derived signals . We hypothesize that the lack of activity of mi/mi osteoclasts is due to the failure of mi/mi preosteoclasts to respond appropriately to resorptive agents, or to cytokines elicited by these agents.

J Exp Biol, 1990 Oct, 153, 129 - 40
The neuromuscular junction revisited: Ca2+ channels and transmitter release in cholinergic neurones in Xenopus nerve and muscle cell culture; Feng TP et al.; Although the entry of calcium ions into the presynaptic nerve terminals though voltage-gated Ca2+ channels is now universally recognized as playing an essential role in evoked transmitter release at the neuromuscular junction (NMJ), and indeed in chemical synapses generally, we have as yet very little direct knowledge of the Ca2+ channels of the presynaptic terminals . In this work, making use of co-cultured nerve and muscle cells from Xenopus embryos, we studied the NMJ formed between the soma of identified cholinergic neurones and myoball, which allowed the use of patch-clamps on both the pre- and postsynaptic components . Both whole-cell and single-channel recordings of Ca2+ channels in the presynaptic cell were made . We found only one type of voltage-gated Ca2+ channel with high-voltage activation and slow inactivation characteristics, allowing its classification either as the L or the N type . The channels were susceptible to block by metenkephalin but not to block by nifedipine or to enhancement by Bay K 8644 . This combination of pharmacological properties favours their classificaiton as the N type . Preliminary observations on the correlation between calcium currents and transmitter release disclosed a strikingly rapid run-down of the evoked release with unchanged calcium currents and spontaneous release during whole-cell recording, indicating a specific wash-out effect on some link between calcium entry and evoked transmitter release.

J Cell Sci, 1990 Oct, 97 ( Pt 2), 373 - 83
The pattern of metalloproteinase expression by corneal fibroblasts is altered by passage in cell culture; Fini ME et al.; We have examined the pattern of expression of four different matrix metalloproteinases (MMPs), collagenase, stromelysin, 92 kD gelatinase, and 72 kD gelatinase, by primary and passaged cultures of rabbit corneal fibroblasts . Primary cultures of this cell type have previously been shown to reproduce the normal tissue regulation of collagenase expression . We demonstrate qualitative and quantitative changes in the pattern of MMP expression as the cells are passaged in culture . Only a single MMP, 72 kD gelatinase, is constitutively expressed by primary fibroblast cultures . Phorbol myristate acetate (PMA) treatment upregulates expression of 72 kD gelatinase and turns on the expression of collagenase and stromelysin, as well as 92 kD gelatinase . However, the degree to which MMP expression is induced is minimal . Cells subcultured but a single time constitutively produce not only 72 kD gelatinase, but also collagenase and stromelysin . In addition, PMA treatment upregulates expression of collagenase, stromelysin and 92 kD gelatinase to high levels . In contrast, the expression of 72 kD gelatinase is repressed by treatment of passaged cell cultures with PMA . Our data indicate that the cell does not simply turn the MMP genes on or off, as a group, in response to various agents, but that it has the capacity for fine control over which MMPs are expressed and the degree to which each is expressed . Changes in MMP protein expression induced by PMA treatment are correlated with changes in specific mRNA levels in passaged cultures . The kinetics of mRNA accumulation suggest that the MMP genes can respond to multiple intracellular signals initiated in a temporal cascade by PMA . It is the combined effects of the individual signals on the accumulation of specific mRNAs that must determine the ultimate pattern of MMP protein expression . The distinct patterns of MMP expression produced by primary and passaged cell cultures may be analogous to patterns of expression that might occur under particular in vivo conditions.

Neuropharmacology, 1990 Oct, 29(10), 969 - 72
Baclofen inhibits with high affinity an L-type-like voltage-dependent calcium channel in cerebellar granule cell cultures; Wojcik WJ et al.; In primary cultures of cerebellar granule cells, D,L baclofen (p-chlorophenyl-GABA) inhibited approximately 50% of the calcium-45 influx induced with cell depolarization . The half maximal effective concentration for baclofen was 4 nM . Basal calcium influx was not influenced by baclofen thus suggesting that its inhibitory action could be exerted via a voltage dependent calcium channel (VDCC) . Whole-cell recordings by patch-clamp technique showed a calcium current that appeared to be similar to the reported L-type VDCC . Nanomolar concentrations of baclofen also inhibited this calcium current by about 60% . However, in order for baclofen to be active, it needed to be placed into the incubation buffer at least five minutes before patching a cell raising the possibility that baclofen may be acting to inhibit the VDCC via a second messenger system.

J Clin Microbiol, 1990 Oct, 28(10), 2362 - 4
Human herpesvirus 6 inhibits human immunodeficiency virus type 1 replication in cell culture; Levy JA et al.; The SF strain of human herpesvirus 6 (HHV-6SF) isolated from the saliva of a human immunodeficiency virus (HIV)-infected individual was shown to inhibit HIV type 1 (HIV-1) replication in both peripheral blood mononuclear cells and purified CD4+ lymphocytes . This suppression of HIV-1 replication led to decreased cytopathic effects of HIV-1 and prolonged survival of CD4+ cells in culture . Even low levels of HHV-6 added to peripheral blood mononuclear cells showed an inhibitory effect on HIV-1 replication . These results differ from those previously reported showing enhanced HIV-1 production following infection with another strain of HHV-6.

Exp Eye Res, 1990 Oct, 51(4), 435 - 46
The distribution of Na+,K(+)-ATPase in the retinal pigmented epithelium from chicken embryo is polarized in vivo but not in primary cell culture; Rizzolo LJ; The polarity of retinal pigmented epithelia (RPE) from chicken embryos was studied in primary cell culture . Since cultured RPE approximates the morphological polarity of RPE in vivo, we investigated whether this polarity extends to the distribution of plasma membrane proteins that are peculiar to RPE . In contrast to other epithelia, the Na+,K(+)-ATPase of RPE is located in the apical rather than basolateral plasma membrane . To examine this property, we cultured RPE on extracellular matrix-coated filters . Primary cultures were compared to embryonic RPE in situ using electron microscopy and indirect immunofluorescence of frozen sections . The viability and morphology of RPE was improved by using a serum-free medium containing a bovine pituitary extract in conjunction with an extracellular matrix coating derived from Engelbreth-Holm-Swarm tumors . Cultured RPE mimicked the morphology of RPE in vivo with microvilli and junctional complexes on the apical pole and infoldings along the basolateral plasma membrane . Functional tight junctions formed as demonstrated by an EDTA-sensitive, transepithelial electrical resistance, and by the retention of {3H}inulin added to the apical chamber . In 2 hr, only 4-6% of the {3H}inulin crossed the monolayer, compared to 24% in control filters . Despite these features of polarity, the Na+,K(+)-ATPase was detected in both apical and basolateral membranes by immunofluorescence . In embryonic eyes in which the neural retina was removed, the Na+,K(+)-ATPase was confined to the apical membrane . In addition, the polarity of cultured RPE was probed with vesicular stomatitis virus . In contrast to other epithelia, budding virus particles were observed emerging from the apical, as well as basolateral, domain further suggesting the cultured cells were only partially polarized . These data indicate that structural criteria are inadequate to determine if cultured RPE have become polarized in the same manner as the epithelium in vivo.

Differentiation, 1990 Oct, 45(1), 61 - 9
Expression of HOX homeogenes in human neuroblastoma cell culture lines; Peverali FA et al.; Mammalian genes containing a class-I homeobox (HOX genes) are highly expressed in the embryonic nervous system . As a first step towards the molecular analysis of the role these genes play in neural cells, we studied the expression of four human HOX genes in five neuroblastoma (NB) cell lines - SK-N-BE, CHP-134, IMR-32, SK-N-SH and LAN-1 - during the process of differentiation induced by treatment with retinoic acid (RA) . The four genes, HOX1D, 2F, 3E and 4B, located at corresponding positions in the four HOX loci, share a high degree of sequence similarity with the Drosophila Deformed homeotic gene and constitute a homology group, group 10 . One of these genes, HOX1D, is not expressed in the cells used, whereas the other three are highly expressed in untreated and RA-induced NB cells, even though the expression pattern in the various lines is slightly different for the three genes . Our analysis reveals a complex and specific expression pattern in these lines, paving the way to an identification of different NB-cell populations by means of specific HOX gene expression schemes . On the other hand, in every line studied, morphological maturation toward a neuronal differentiated phenotype appears to be associated with increased HOX gene expression.

Zhongguo Yi Xue Ke Xue Yuan Xue Bao, 1990 Oct, 12(5), 330 - 4
{Effect of dopamine and bromocriptine on secretion of growth hormone by pituitary growth hormone secreting tumor in cell culture}; Deng J; This article reports the effect of dopamine (DA) and its agonist bromocriptine (CB154) on the secretion of growth hormone (GH) by pure GH-secreting pituitary tumor in cell culture as well as a comparison of the effects of these dopaminergic drugs and SMS201-295 (SMS) . DA of 10(-8) and 10(-7) mol/L reduced GH secretion to 50.6 and 44.4% of the control, respectively, in 1 out of 6 tumors . CB154 of 10(-7) and 10(-6) mol/L suppressed GH secretion to 59.0 +/- 8.9% of the control in 3 out of 4 tumors . CB154 was at least 10 times less potent than SMS vis a vis GH secretion . CB154 of 10(-6) mol/L inhibited GH secretion to 63.3 +/- 13 . 8% (n = 4), but SMS of 10(-7) mol/L induced GH secretion to 45.5 +/- 13.1% (n = 4), the concentration difference between CB154 and SMS was 10 times . CB154 suppressed not only GH secretion, but also GH synthesis in two tumor cell cultures . The major role of SMS in GH secretion was inhibition . The results suggest that DA and CB154 have direct inhibitory effects on GH secretion, at least in some pure pituitary GH secreting tumors . The activities of DA and CB154 are not entirely the same as that of SMS.

J Neurosci Methods, 1990 Oct, 35(1), 79 - 88
Automatic quantification of fast axonal transport in neuronal cell cultures; Cornelissen F et al.; A method is presented which allows the automatic quantification of the fast axonal transport of endogenous organelles in neurites of cultured neuronal cells . Stretches of videotape recordings from Allen video enhanced contrast (AVEC) microscopy are digitized by currently available image processor hardware and analysed off-line on a MicroVAX II . Movements along the axon are calculated in great detail, allowing statistically significant changes to be detected . Interaction from the operator is minimised, thereby bypassing tedious manual analysis . This paper further reports the application of this system to the effect of vanadate treatment on axonal transport in cultures of rat embryonic hippocampal neurons.

J Neurochem, 1990 Oct, 55(4), 1418 - 26
Differential phosphorylation of myelin-associated glycoprotein isoforms in cell culture; Afar DE et al.; The alternative splicing of myelin-associated glycoprotein (MAG) mRNA generates two isoforms that harbor distinct potential phosphorylation sites in their cytoplasmic tails . Here we characterize the in vivo phosphorylation of MAG isoforms in NIH 3T3 cells transfected with the cDNAs encoding the two isoforms of MAG . Our results demonstrate that the longer isoform, L-MAG, is phosphorylated constitutively mainly on serine, but also on threonine and tyrosine residues . This phosphorylation is subject to change by 12-O-tetradecanoylphorbol 13-acetate (TPA) and ammonium vanadate, but not by dibutyryl-cyclic AMP . The shorter isoform, S-MAG, is constitutively phosphorylated only on serine residues . While TPA and dibutyryl-cyclic AMP have no detectable effect, ammonium vanadate induces tyrosine and threonine phosphorylation in S-MAG . 32P labeling of v-src-transformed NIH 3T3 cells that express L-MAG also show that L-MAG is likely to be an in vivo substrate for pp60v-src tyrosine kinase activity . These results demonstrate that both MAG isoforms are phosphorylated in a heterologous cell system and that this phosphorylation is subject to pharmacological manipulation.

J Biotechnol, 1990 Oct, 16(1-2), 67 - 85
Mechanisms and kinetics of monoclonal antibody synthesis and secretion in synchronous and asynchronous hybridoma cell cultures; al-Rubeai M et al.; The kinetics of monoclonal antibody synthesis and secretion have been studied in synchronous and asynchronous mouse hybridoma cell cultures . Pulse-labelling of IgG followed by immunoprecipitation and quantitation of synthesized and secreted IgG in synchronous cultures show maximum production during G1/S phases . Secretion takes place through exocytotic release of vesicle contents . Pulse-chase experiments show that 71% of the synthesized IgG is secreted within 8 h of the pulsing period and only a further 4% is secreted by 22 h . Higher specific antibody production (QA) is obtained if (a) cells are arrested and then maintained in G1/S phases, (b) viability is decreased during the death phase of batch culture, (c) the dilution rate is decreased in continuous culture or (d) cells are subjected to hydrodynamically induced stress . The increase in QA in all these cases is mainly due to the passive release of the accumulated intracellular antibody . DNA and protein synthetic activity peak during the early exponential phase and decline rapidly during mid and late exponential and death phases . Metabolic activity however peaks up to 20 h after the peak in DNA synthesis, and declines similarly during the death phase . The data are consistent with the idea that slow growth and higher death rates increase QA and that Ig secretion is probably subject to complex intracellular control.

Eur J Pharmacol, 1990 Sep 21, 186(2-3), 149 - 55
Memantine reduces repetitive action potential firing in spinal cord nerve cell cultures; Netzer R et al.; (1) The anticonvulsant effects of memantine were examined and compared with those of baclofen in monolayer primary cultures of murine nerve cells using conventional intracellular recordings . (2) Memantine and baclofen (each 10-100 microM) decreased spontaneous synaptic activity when action potential frequencies exceeded 6 Hz . Neurons firing action potentials at frequencies below 6 Hz (about 90% of all impaled cells), however, were not affected by the drugs . (3) Memantine reduced the duration of strychnine-elicited bursts and the firing rate of action potentials within a burst . In contrast, baclofen lowered the frequency of the bursts without reducing intra-burst firing . The duration of the bursts was increased . (4) Memantine, but not baclofen, reduced the extent of sustained repetitive firing evoked by pulses of depolarizing current . (5) In the presence of memantine, the second of two electrically evoked action potentials increasingly failed to appear as the intervals between successive stimulating pulses were shortened . Such an effect was not seen when baclofen was applied . Thus, both antispastic agents, memantine and baclofen, reduce hyperactivity of spinal cord neurons in culture, although their mechanisms of action are different.

Endocrinology, 1990 Sep, 127(3), 1517 - 25
Evidence that folliculo-stellate cells do not impede the permeability of intercellular spaces to molecular diffusion in three-dimensional aggregate cell cultures of rat anterior pituitary; Allaerts W et al.; The permeability of intercellular spaces within the anterior pituitary (AP) and the influence of folliculo-stellate (FS) cells on compartmentalization within this tissue, has become a matter of debate . In reaggregated pituitary cell cultures as well as in the AP in situ the intercellular gaps and follicle-like structures remain accessible to molecular diffusion, whereas in some studies FS cells were reported to form tight epithelia that impede macromolecular transport through the spaces between the epithelial cells . In the present study the permeability of AP cell reaggregates was examined using fluorescent BSA as a tracer . Using confocal scanning laser microscopy a direct visualization of the permeation process was achieved . Quantitative estimation of the effective diffusion coefficient (Deff) for fluorescein-BSA within the aggregates was obtained using the fluorescence photobleaching recovery technique . Deff was 1.33 +/- 0.31 x 10(-7) cm2/sec (mean +/- SD) in aggregates from 14-day-old female rats and 2.45 +/- 0.55 x 10(-7) cm2/sec in aggregates from adult female rats . These values are about three times lower than in free solution . Calculation of the time-dependent concentration distribution inside the aggregate for a Deff = 2 x 10(-7) cm2/sec revealed that the concentration of the fluorescent tracer in the center of the aggregate reaches 90% of the concentration outside the aggregate after 0.5 min for aggregates with a radius of 50 microns and 6 min for aggregates with a radius of 150 microns . Aggregates enriched in FS cells, in which we previously showed a sustained inhibition of secretory responses to stimulatory and inhibitory agents as compared to total population aggregates, showed a diffusion coefficient (Deff = 1.85 +/- 0.77 x 10(-7) cm2/sec) which was not significantly different from that in the total population aggregates . The present study shows that AP cell aggregates are fully permeable to diffusing molecules within minutes and that in a three-dimensional tissue configuration FS cells, which were reported to tonically inhibit AP hormone release in response to various secretagogues, do not impede molecular diffusion to an extent which would account for sustained inhibition of hormone release.

J Virol, 1990 Sep, 64(9), 4108 - 14
Bovine coronavirus mRNA replication continues throughout persistent infection in cell culture; Hofmann MA et al.; The existence of viral mRNA replicons was demonstrated in cells infected with the bovine coronavirus by showing a minus-strand counterpart and a corresponding replicative intermediate for each subgenomic mRNA species . mRNA replication is thus a universal property of coronaviruses, since this is now the third coronavirus for which it has been demonstrated . During the acute phase of infection (first 48 h), minus and plus strands accumulated at the same rate initially, but maximal accumulation of minus strands peaked earlier than that for plus strands, indicating that minus- and plus-strand levels are differentially regulated . In addition, packaged (input) mRNAs appeared to serve as templates for their own early replication . mRNA replication continued throughout establishment and maintenance of persistent infection (studied for 120 days), which is consistent with our hypothesis that mRNA replication contributes mechanistically to virus persistence . A replication-defective (potentially interfering) species of RNA existed transiently (beginning at day 2 and ending before day 76 postinfection), but because of its transient nature it cannot be considered essential to the long-term maintenance of virus persistence.

J Neurochem, 1990 Sep, 55(3), 922 - 9
Altered patterns of protein phosphorylation and synthesis caused by methyl mercury in cerebellar granule cell culture; Sarafian T et al.; In the preceding report we demonstrated a dose-dependent increase in 32P-phosphoprotein labeling after 24-h exposure of cultured cerebellar granule neurons to methyl mercury (MeHg), a response that was not observed in glial cultures . In the present study we have examined 32P-labeled phosphoproteins by two-dimensional sodium dodecyl sulfate polyacrylamide gel electrophoresis . At concentrations of 0.5 and 1 microM, which were not extensively cytotoxic, MeHg enhanced phosphorylation of numerous acidic proteins, particularly a cluster of proteins with Mr approximately 28,000 and pI approximately 5.7-5.9 (pp 28/5.7-5.9) and a protein with Mr approximately 58,000 and pI approximately 5.6 . The pp28 cluster displayed considerable two-dimensional pattern variability from one experiment to the next, suggesting susceptibility to subtle structural modifications . Time course studies revealed that increased 32P phospholabeling of pp28/5.7-5.9 was detectable after 12-h exposure to 3 microM MeHg and reached values of 300-500% of control by 24 h . These studies also showed that among the 21 proteins analyzed by two-dimensional densitometry, 32P phospholabeling of four proteins increased by 20-50% and of two proteins decreased by 20-50% after 24-h treatment . However, exposure to 10 microM MeHg produced stimulation of pp28/5.7-5.9 32P phospholabeling within 2 h . Under these conditions a relatively high stimulation (sevenfold) of pp28/5.7 phospholabeling occurred, while pp28/5.9 32P phospholabeling was only moderately (5-20%) enhanced . 35S and 32P double-label analysis of cells treated with 0, 0.5, and 1 microM MeHg indicated specific stimulation of 32P phospholabeling of these proteins without increased polypeptide synthesis.(ABSTRACT TRUNCATED AT 250 WORDS)

Peptides, 1990 Sep-Oct, 11(5), 1049 - 51
Comparative studies of angiotensin II and its analog {Sar1Ala8}angiotensin II on the density of cellular monolayer of cell cultures from green monkey kidney; Lambadjieva N et al.; The effects of the octapeptide angiotensin II (AT II) and its analog {Sar1Ala8}AT II on the cell density in cell culture from green monkey kidney (GMK) were studied . AT II and {Sar1Ala8}AT II provoked a decrease of the number of living cells depending on the concentration (0.001, 0.01 and 0.1 nM) and time of incubation (24, 48 and 72 hours), both peptides having a very similar activity . These data indicate that AT II and {Sar1Ala8}AT II may act on the same class of angiotensin receptors in GMK cells.

Lab Invest, 1990 Sep, 63(3), 370 - 6
Immunocytochemical and biochemical evidence of renin in human lactotrophic cell cultures; Rousselet MC et al.; Primary cell cultures from human prolactin (PRL)-secreting adenomas were used to test the ability of human lactotrophs to synthesize renin in vitro . The renin content of the culture medium and of cellular extracts was measured by enzyme-linked immunosorbent assay . The level of PRL release in the culture medium and the amount of PRL in a cellular extract were determined by radioimmunoassay . Morphologic studies included indirect immunofluorescence, pre-embedding immunoelectron microscopy using a three-layer peroxidase-antiperoxidase method and postembedding immunoelectron microscopy using protein A-gold complexes . Renin was detected in cellular extracts and was found to be absent in the culture medium, whereas PRL was extracellularly secreted . PRL and renin immunoreactivity was observed in all the cultures studied by immunofluorescence . The subcellular localization of renin was found to be similar to that of PRL and was observed in the rough endoplasmic reticulum, the Golgi apparatus, and cytoplasmic secretory granules . The results suggest that, in vitro, renin may be synthesized and intracellularly metabolized in human adenomatous lactotrophic cells rather than secreted . Cell cultures may be a useful model to further the understanding of the role of a local renin-angiotensin system in PRL secretion.

Gynecol Oncol, 1990 Sep, 38(3), 396 - 406
Regulation of epidermal growth factor and insulin-like growth factor I receptors by estradiol and progesterone in normal and neoplastic endometrial cell cultures; Reynolds RK et al.; Growth factors are polypeptides which regulate cell proliferation through binding to specific receptor proteins . Normal and neoplastic human endometrium have been shown to express epidermal growth factor (EGF) and insulin-like growth factor I (IGF-1) receptors . Endometrial cell cultures were used to test modulation of EGF and IGF-1 receptors in response to steroid hormones . Endometrial gland and stroma cells were separated by enzymatic dispersion and were incubated in medium containing estradiol (10, 100, or 1000 pg/ml) or progesterone (1, 10, or 100 ng/ml) followed by radioligand assays . Normal endometrial cultures (n = 6) treated with estradiol demonstrated 40% less EGF binding than control cultures (P less than 0.05), while IGF-1 binding was unaffected . Stromal cells treated identically decreased in only one treatment group . Progesterone treatment stimulated a significant increase in EGF and IGF-1 receptors in gland cultures . Cultures derived from adenocarcinoma (n = 2) demonstrated decreased EGF binding compared with normal endometrium (P less than 0.05) . Carcinoma cells treated with progesterone resulted in a dose-dependent increase in EGF binding over control (P less than 0.05) . These data illustrate effects of steroid hormones upon growth factor receptors in human endometrium, and suggest involvement of growth factors in the regulation of normal and neoplastic endometrial growth.

Neuroendocrinology, 1990 Sep, 52(3), 256 - 61
Adenosine 3',5'-cyclic monophosphate enhances dopamine accumulation in rat hypothalamic cell culture containing dopaminergic neurons; Kadowaki K et al.; The regulation of dopamine accumulation in cultured rat hypothalamic cells by adenosine 3',5'-cyclic monophosphate (cAMP) was investigated in cultures of newborn rat hypothalamic cells . Both dibutyryl cAMP, (Bu)2-cAMP, and forskolin enhanced {3H}dopamine accumulation in a dose-dependent manner . cAMP also enhanced {3H}dopamine accumulation, but to a lesser degree . Neither n-butyrate nor adenosine alone enhanced {3H}dopamine accumulation . (Bu)2-cAMP had no effect on basal efflux of {3H}radioactivity . The effect of (Bu)2-cAMP appeared on day 5 of culture, reached a maximum on day 6, and then rapidly decreased . These results suggest that dopamine uptake by cultured rat hypothalamic cells is regulated by intracellular cAMP.

J Gen Virol, 1990 Sep, 71 ( Pt 9), 1965 - 74
Inhibition of human immunodeficiency virus replication in cell culture by endogenously synthesized antisense RNA; Rhodes A et al.; Antisense RNA, which has a sequence complementary to mRNA, may provide the basis for antiviral therapies of high selectivity . We have explored the inhibitory effect of six antisense RNAs upon the replication of human immunodeficiency virus (HIV) in cell culture . We chose regions of the HIV genome to test whether sequences required for splicing or for translation initiation were more susceptible to antisense RNA interference . Our results suggest that inhibitory antisense RNAs contain sequences complementary to the AUG initiation codon of the tat gene and have a comparatively low tendency to form intramolecular base pairs which would interfere with intermolecular duplex formation . Inhibition can be substantial (over 70%) but is transient . Transience does not result from mutation of the input virus . Inhibition was not a consequence of the induction of interferon by antisense RNA-mRNA duplex formation . Our results suggest that at least part of the inhibitory effect is at the posttranscriptional level.

J Neurochem, 1990 Sep, 55(3), 913 - 21
Methyl mercury stimulates protein 32P phospholabeling in cerebellar granule cell culture; Sarafian T et al.; Cultures of cerebellar granule neurons have been utilized to examine morphological and biochemical consequences of methyl mercury (MeHg) . Exposure to MeHg for 24 h was found to exert toxic effects at concentrations below 1 microM characterized by neuron degeneration and neuritic varicosities . Dose-response and time course profiles for cell death were established using the 51Cr release assay, which revealed that 1 microM MeHg produced 15% cell death at 24 h, progressing to 50% at 48 h . Labeling of cultures with {32P}orthophosphate following 24-h exposure to 1 microM MeHg disclosed abnormalities in both protein and lipid phosphorylation . After 24-h exposure to 5 microM MeHg, phospholabeling of protein and lipid increased 174 and 128%, respectively, compared with controls . This stimulation of phosphorylation appeared to be neuron specific since cultures enriched in cerebellar glial cells and devoid of granule neurons displayed dose-dependent inhibition of total phosphorylation . Measurement of 32P labeling of ATP using a cyclic AMP-dependent protein kinase assay in conjunction with the firefly luciferase assay for ATP indicated no significant change in either total ATP levels or {32P}ATP specific activity at 1 or 4 h as a function of {MeHg} . Studies measuring 32P-phosphoprotein turnover indicated that MeHg had no effect on intracellular protein phosphatase activity . We conclude that one of the manifestations associated with in vitro cerebellar granule cell neurotoxicity is an abnormality in protein phosphorylation that is independent of {32P}ATP specific activity and protein phosphatase activity.

Transplantation, 1990 Sep, 50(3), 481 - 7
Failure of rat kidney nephron components to induce allogeneic lymphocytes to proliferate in mixed lymphocyte kidney cell culture; Halttunen J; Rat kidney glomerular mesangial, glomerular epithelial, and tubular epithelial cells were isolated in virtually pure form and studied in mixed lymphocyte kidney cell culture (MLKC) for their ability to induce allogeneic spleen lymphocyte proliferation . The responses were compared with proliferative responses induced by allogeneic endothelial cells or spleen lymphocytes in the same strain combination . Stimulator cells were treated or left untreated with gamma-interferon, known to increase the major histocompatibility complex class II (and class I) expression of the stimulator cells . The results demonstrate that the nephron components are not able to induce lymphoproliferation in the MLKC . In contrast, the endothelial cells of rat heart were potent inducers of lymphoproliferation in mixed lymphocyte endothelial cell cultures (MLEC), as were allogeneic spleen cells . Although the kidney parenchymal cells have been shown to be immunogenic in vivo, the present finding suggests that they are unable to function as antigen-presenting cells on their own.

Infect Immun, 1990 Sep, 58(9), 3139 - 42
Virulence of a Legionella anisa strain associated with Pontiac fever: an evaluation using protozoan, cell culture, and guinea pig models; Fields BS et al.; Legionella anisa and the amoeba Hartmannella vermiformis were isolated from an indoor fountain implicated as the infectious reservoir in an outbreak of Pontiac fever . We evaluated the ability of this strain of L . anisa to multiply in cultures of an amoeba (H . vermiformis), a ciliated protozoan (Tetrahymena pyriformis), and human mononuclear cells and to infect guinea pigs . These bacteria multiplied in the culture of H . vermiformis but failed to infect guinea pigs or the cultures of T . pyriformis and human mononuclear cells . These findings suggest that some Legionella spp . may multiply only in specific protozoan hosts . The inability of this strain of L . anisa to multiply in human phagocytic cells may be related to the development of Pontiac fever rather than pneumonic legionellosis in exposed individuals . Further studies are necessary to determine whether the ability of legionellae to infect certain host cells can be correlated to differences in human disease.

Can J Physiol Pharmacol, 1990 Sep, 68(9), 1200 - 6
Phencyclidine and related compounds evoked {3H}dopamine release from rat mesencephalic cell cultures by a mechanism independent of the phencyclidine receptor, sigma binding site, or dopamine uptake site; Mount H et al.; At concentrations greater than or equal to 100 microM, phencyclidine (PCP), N-(1-(2-thienyl)-cyclohexyl)piperidine (TCP), and MK-801 induced {3H}dopamine release from dissociated cell cultures of rat mesencephalon . This release was Ca2+ independent and tetrodotoxin insensitive . Tetrodotoxin (2 microM) itself had no effect on spontaneous release of {3H}dopamine . {3H}Dopamine release was induced by 1,3-di(2-tolyl)guanidine, a sigma ligand, and by 4-aminopyridine (1-3 mM), a K+ channel blocker . No stereoselectivity was observed for {3H}dopamine release evoked by the dioxadrol enantiomers, dexoxadrol, and levoxadrol, or by enantiomers of N-allylnormetazocine (SKF 10,047) . The selective dopamine uptake inhibitor 1-(2-{bis(4-fluorophenyl)methoxy}ethyl)-4-(3-phenylpropyl)piperazine dihydrochloride (GBR 12909) did not affect spontaneous or TCP-evoked {3H}dopamine release . Together, these data suggest that the dopamine-releasing effects of PCP-like compounds on the mesencephalic cells were not mediated by actions at the PCP receptor or sigma binding site, Ca2+, or Na+ channels, or at the high affinity dopamine uptake site . It remains conceivable that blocking actions of PCP-like compounds at voltage-regulated K+ channels may at least partly explain the response . These results are discussed in comparison with findings in intact brain.

Virus Genes, 1990 Sep, 4(3), 225 - 37
Cell culture amplification of a defective Marek's disease virus; Carter JK et al.; A highly amplified 4-kb EcoRI fragment was present in DNA isolated from high cell culture passaged stocks (greater than 93 passages) of 281MI/1, a serotype 2 Marek's disease virus (MDV) . The isolated 4-kb fragment is amplified in the presence of MDV, replicating as a high molecular weight, head-to-tail concatemer . When the 4-kb fragment was cloned into pUC18 and cotransfected with MDV DNA into chicken embryo fibroblast cells, the plasmid clone also replicated as a high molecular weight concatemer.

Cytotechnology, 1990 Sep, 4(2), 191 - 4
3-Amino-6-methoxy-9-(2-hydroxyethylamino) acridine: a new fluorescent dye to detect Mycoplasma contamination in cell cultures; Jayat-Vignoles C et al.; A new fluorescent acridine orange derivative, 3-amino-6-methoxy-9-(2-hydroxyethylamino) acridine (AMHA), has been applied to Hela cells in order to set up appropriate conditions for the detection of mycoplasma contaminations . Since AMHA staining reveals intensely fluorescent nuclei and slight fluorescent cytoplasm, we can visualize and localize mycoplasma contamination on each cell . In combination with a shortened Chen's staining method (1977), AMHA should allow a better detection of mycoplasma in animal cell cultures than the well established Hoechst dye.

Cytotechnology, 1990 Sep, 4(2), 181 - 9
A compatible support system for cell culture in biomedical research; Minuth WW et al.; The lack of a suitable system to culture epithelial cells for a long period under a luminal-antiluminal medium gradient, was the reason to develop a new system . It consists of an interchangeable sheet of permeable support material, which is set in place by two tight fitting holding rings . For special demands the supports can be coated with extracellular matrix proteins improving cellular attachment and terminal differentiation . The handling of the sheets by forceps proceeds easily and quickly, thus fastening the transfer of cultured cells without additional manipulations . The sheets can be transferred to a newly developed microperfusion chamber on which an apical and a basal perfusion over a long culture period parallel to a transepithelial electrophysiological registration becomes possible . The chamber has an extremely low amount of fluid dead space . The separate perfusion of cultured cells under isotonic, hypotonic or hypertonic conditions opens new possibilities . Thus, culture can be performed under most natural conditions e.g., that found within the kidney.

Biotechnology (N Y), 1990 Sep, 8(9), 854 - 6
Cell culture on a thermo-responsive polymer surface; Takezawa T et al.; We have used a thermo-responsive polymer, poly-N-isopropyl acrylamide (PNI-PAAm), as a substratum for the culture of human dermal fibroblasts by conjugating it with collagen . The cells attached well, spread, and grew on the substratum, indicating that the polymer has no toxicity towards the cells . PNIPAAm is insoluble in water over the lower critical solution temperature (LCST; about 32 degrees C) and reversibly solubilized below the LCST . Taking advantage of this conversion, monolayered fibroblasts cultured on the substratum containing the PNIPAAm over the LCST, were completely detachable from the substratum by simply lowering the temperature below the LCST, without the use of conventional detaching agents such as trypsin and EDTA . The detached cell sheet gradually aggregated and finally formed a multicellular spheroid . This polymer may provide a convenient and potentially useful technology for cell culture.

J Steroid Biochem, 1990 Aug 14, 36(5), 457 - 64
Altered steroidogenic pattern of human granulosa-lutein cells in relation to cumulus cell culture morphology; Gitay-Goren H et al.; It has been reported that human-oocyte complexes (COC) retrieved at a stimulated cycle manifest an asynchrony between oocyte meiotic maturation and cumulus mucification . However, when mature COC were subdivided into subtypes marked by the culture morphology of their cumulus cells following 3 days' culture, successful fertilization and cleavage were approximately 1.5-fold lower in mature COC yielding cumulus cells aggregated into clumps (type A and B COC) than in mature COC yielding homogeneously spread cells (type C-D COC) . To determine whether the existing relationship between cumulus culture morphology and oocyte functionality in the various COC types (A-D) could be extended to another follicular compartment--the granulosa-lutein (G-L) cells--basal steroid secretion by the corresponding G-L cells was evaluated within 5 days of culture . Over the first 3 days of culture, secretion of progesterone was 3-fold lower and secretion of testosterone (T) was 2.5-fold higher in cultures of G-L cells from follicles yielding type A COC than in type C-D COC . During days 4 and 5 of culture, G-L cells were incubated with or without 10(-7) M 3 beta-hydroxy-5-pregnen-20-one (pregnenolone), dehydroepiandrosterone (DHA), 4-androstene-3,17-dione (androstenedione), or T . The pattern of progesterone level noted over the first 3 days of culture was not altered in the presence of pregnanolone . DHA, androstenedione, or T . Addition of pregnenolone, DHA, androstenedione, or T increased T level 2.5-, 5.6-, 7.3-, and 17.7-fold, respectively, in cultures of G-L cells from follicles yielding type A COC, but did not significantly alter T level in cultures of G-L cells from follicles yielding type C-D COC . In cultures of G-L cells from follicles yielding type A COC, addition of androgens unsaturated at position 4 preferentially increased oestradiol-17 beta (E2) level, whereas in cultures of G-L cells of type C-D COC, DHA and androstenedione preferentially increased E2 level . Taken together, the asynchrony between oocyte and cumulus activity could be diminished when the activity of various follicular cell compartments is evaluated according to cumulus culture morphology rather than cumulus expansion and mucification . The present study suggests that follicles yielding mature COC represent a non-homogenous population in which G-L cells from follicles yielding type A-B COC manifest a less luteinized state than those from follicles yielding type C-D COC.

Calcif Tissue Int, 1990 Aug, 47(2), 92 - 104
Morphologic characterization of osteoblast-like cell cultures isolated from newborn rat calvaria; Masquelier D et al.; Two methods for harvesting osteoblast-like cell populations from newborn (10 days) rat calvaria were compared . The first one consisted in culturing the periosteum-free bones and then trypsinizing the cells on the bone surface . The second one involved the migration of the osteoblasts on glass fragments before trypsinization . Since the plating efficiency, the proportion of alkaline phosphatase-positive cells, the population doubling time, and the calcium deposition were more adequate, the second method was used to further characterize the behavior of the cultures . During the first week of culture, the cells featured shapes similar to those observed in vivo on the surface of periosteum-free calvaria . They formed multilayers and, in the presence of ascorbic acid, synthetized an organic matrix containing exclusively type I collagen . Later, small amounts of type III collagen appeared . The cells were embedded in the matrix and progressively acquired the morphologic phenotype of osteocyte-like cells . The matrix mineralized in the presence of beta-glycerophosphate . The technique of drop-inoculation (high concentration of cells in a small volume of medium) promoted the multilayer formation and the achievement of large mineralized plates (about 1 cm2) in 3 weeks of culture.

J Dent Res, 1990 Aug, 69(8), 1539 - 42
Cytotoxicity of experimental casting alloys evaluated by cell culture tests; Craig RG et al.; The cytotoxicity of a series of 29 experimental alloys and six pure metals was determined with cell culture techniques and succinic dehydrogenase histochemistry . The width of any ring of inhibition, optical density of the histochemically stained cells, and a visual ranking of the intensity of the blue color of the stained cells were compared for determination of cytotoxicity . Twenty-four of the 35 metals and alloys (approximately 70%) had the same rankings by the three methods . Of the pure metals, Au, Pd, and Ti were the least cytotoxic, followed by Ag, then Ni, and finally, Cu . Single-phase alloys with moderately high Cu and without high Pd and Au concentrations had high cytotoxicity, as did multiphase alloys, even when they were high in Au and Ag . High Pd was more effective in maintaining the biocompatibility of alloys containing Cu than was Au . Single-phase alloys with compositions typical of those to be used for porcelain-fused-to-metal restorations showed good biocompatibility, as did those base metal alloys that formed adherent oxide surface layers.

Int J Artif Organs, 1990 Aug, 13(8), 517 - 20
New approaches for biocompatibility testing using cell culture; Doillon CJ et al.; We are proposing two modified assays for biocompatibility testing which analyze the effects of cytotoxic substances leached from a biomaterial in cell culture . The biocompatibility of two vascular prostheses made of polytetrafluoroethylene was analyzed using the modified assays . One test, the "fluid medium assay" was modified by using small pieces of graft glued to a screening lid, thereby reducing the possibility of mechanical injury to cultured cells by free fragments of the tested biomaterial . Another test, the "cell inhibition assay" was modified in that the biomaterial to be tested was ground into small pieces while at very low temperature . The measure of cell toxicity used was the effect on DNA replication . Our results suggest that these modified assay methods can be used to evaluate the biocompatibility of biomaterials.

Anal Biochem, 1990 Aug 1, 188(2), 398 - 407
High-performance liquid chromatographic separation of hyaluronan and four proteoglycans produced by human bone cell cultures; Fedarko NS et al.; Four proteoglycans and hyaluronan synthesized by cultured human bone cells were isolated using a two-step high-performance liquid chromatography system involving desalting and buffer exchange with a TSK-GEL HW 40(S) column followed by ion-exchange separation on a Nucleogen 4000-10 DEAE column . The desalting of 4 M guanidinium HCl extracts by a TSK-GEL HW 40(S) column equilibrated in a formamide:KH2PO4 buffer produces greater than 95% recoveries, enables quantitation of label incorporation and requires only 40 min to complete . The Nucleogen 4000-10 DEAE column utilizes the same buffer system and requires only 100 min for the resolution of four distinct types of proteoglycans . The formamide:KH2PO4 buffer system is compatible with a previously developed polyacrylamide gel system for the electrophoretic profiling of proteoglycans . After separation by charge density, proteoglycans were further resolved by size distribution using a calibrated TSK-GEL HW 75(F) column which also enabled the estimation of the apparent Mr of hyaluronan produced by the bone cells . The same TSK-GEL HW 40(S) resin is used to exchange pooled proteoglycans into buffers for analyzing enzyme digests of glycosaminoglycan chains and core proteins . The technique has been applied to the analysis of biosynthetically labeled proteoglycans produced in culture by fetal and adult human bone cells . A distinct pattern of proteoglycan size and secretion for both cell types could be shown using this method . The method of analysis is useful for high yield and rapid screening of various cell types for both biosynthetic rate studies and analysis of patterns of proteoglycan synthesis.

J Pathol, 1990 Aug, 161(4), 293 - 9
Malignant haemangiopericytoma of lung: a cell culture and ultrastructural study; Yang AH et al.; Malignant haemangiopericytoma was grown in vitro to assess the histogenesis of the tumour . Disaggregated tumour cells maintained in serum-supplemented Waymouth's MB 752/1 medium showed monolayers of homogeneous spindle cells . Post-confluent cultures exhibited hillocks or plaques of multilayer growth within which considerable amount of basal lamina-like material and fibrillar matrix were present between cells . Delicate basal laminae were also expressed on cell surfaces facing matrix material . Other consistent features were attenuated cytoplasmic processes, desmosome-like junctions, abnormal mitochondria and a paucity of intracellular filaments . The three-dimensional organization of tumour cells with concomitant expression of differentiated phenotype in vitro has significant implications on the cell of origin and differentiation process of haemangiopericytoma.

Exp Mol Pathol, 1990 Aug, 53(1), 64 - 71
Application of X-ray microanalysis to the study of drug uptake in cell culture; Reasor MJ et al.; X-ray microanalysis has been used previously to study the accumulation of iodine in alveolar macrophages of rats treated with the iodinated drug, amiodarone . Due to metabolism of the drug in vivo, primarily to desethylamiodarone, it was not possible to identify the source of the iodine signal . In the present study we have utilized primary cell cultures of alveolar macrophages to study the intracellular accumulation of each of these drug species in vitro . Neither drug is metabolized by these cells in culture, permitting characterization of the accumulation of each independent of the other . Cells were incubated with equimolar concentrations of either amiodarone or desethylamiodarone for 42 hr, and X-ray microanalysis of freeze-dried cryosections of cells was used to quantify accumulation by monitoring the iodine signal associated with each drug . For both drug exposures, the highest iodine content was present in amorphous bodies and dense granules, consistent with the pattern following in vivo exposure . Higher levels of desethylamiodarone, compared to amiodarone, were measured in all compartments of the cells . The results of the in vitro investigation further demonstrate the utility of X-ray microanalysis in the study of the cellular response to amiodarone and desethylamiodarone.

Genitourin Med, 1990 Aug, 66(4), 267 - 9
A comparison of cytobrush and cotton swab sampling for the detection of Chlamydia trachomatis by cell culture; Lees MI et al.; A cytobrush was compared with a cotton-tipped aluminium shafted swab for the collection of 2024 paired endocervical specimens for the culture of Chlamydia trachomatis . There was no significant advantage with the use of either device with respect to the number of positive specimens detected or the number of inclusions present in positive specimens . However, the use of cytobrushes resulted in an increased level of cervical bleeding and increased collection of cervical mucus resulting in difficulties in the handling of laboratory specimens.

J Trauma, 1990 Aug, 30(8), 1037 - 42; discussion 1043
Growth of melanocytes in human epidermal cell cultures; Staiano-Coico L et al.; Epidermal cell cultures were grown in keratinocyte-conditioned medium for use as burn wound grafts; the melanocyte composition of the grafts was studied under a variety of conditions . Melanocytes were identified by immunohistochemistry based on a monoclonal antibody (MEL-5) that has previously been shown to react specifically with melanocytes . During the first 7 days of growth in primary culture, the total number of melanocytes in the epidermal cultures decreased to 10% of the number present in normal skin . Beginning on day 2 of culture, bipolar melanocytes were present at a mean cell density of 116 +/- 2/mm2; the keratinocyte to melanocyte ratio was preserved during further primary culture and through three subpassages . Moreover, exposure of cultures to mild UVB irradiation stimulated the melanocytes to proliferate, suggesting that the melanocytes growing in culture maintained their responsiveness to external stimuli . When the sheets of cultured cells were enzymatically detached from the plastic culture flasks before grafting, melanocytes remained in the basal layer of cells as part of the graft applied to the patient.

Clin Exp Immunol, 1990 Aug, 81(2), 278 - 85
Detection of transforming growth factor-beta in rheumatoid arthritis synovial tissue: lack of effect on spontaneous cytokine production in joint cell cultures; Brennan FM et al.; The presence of transforming growth factor-beta (TGF-beta) in inflammatory joint disease was investigated . Synovial fluid from patients with rheumatoid arthritis (RA) and patients with other non-autoimmune inflammatory joint diseases contained high levels of both active and latent TGF-beta . Levels of active TGF-beta did not correlate with drug regimen in either patient group or with the recovery period in the individuals with non-RA joint disease . Freshly isolated synovial cells from individuals with RA were shown by Northern blotting to express the mRNA for TGF-beta 1 and to secrete latent TGF-beta protein which could be neutralized by antibodies to TGF-beta 1 and TGF-beta 2 . Lipopolysaccharide-stimulated peripheral blood mononuclear cells from normal donors produced interleukin-1 (IL-1) and tumour necrosis factor-alpha (TNF-alpha) which was inhibited by pretreatment of these cells with recombinant TGF-beta . Cytokine production was not inhibited if the addition of TGF-beta was used after the inducing stimulus, suggesting that in activated cells cytokine production cannot be inhibited . This was confirmed by the observation that neither TGF-beta 1 or TGF-beta 2 inhibited spontaneous IL-1 or TNF-alpha production by rheumatoid synovial mononuclear cells in culture . These findings show that despite the presence of active TGF-beta in RA synovial joints and the spontaneous production of latent (potentially active) TGF-beta by RA cells in culture, additional TGF-beta did not inhibit ongoing cytokine synthesis in vitro . This suggests that TGF-beta may not inhibit cytokine production in the rheumatoid joint although it cannot be ruled out that in vivo TGF-beta already has an immunosuppressive effect which cannot be further increased in vitro by exogenous protein.

Am J Pathol, 1990 Aug, 137(2), 457 - 65
Lipoprotein degradation and cholesterol esterification in primary cell cultures of rabbit atherosclerotic lesions; Jaakkola O et al.; Lipoprotein metabolism and cholesterol accumulation in atherosclerotic lesions was studied using enzymatically isolated primary cell cultures from aortas of rabbits made atherosclerotic by cholesterol feeding . The cultures consisted of macrophages and smooth muscle cells, thus resembling, in composition, fatty streak lesions . The mean (+/- SD) cholesteryl ester content of the dispersed cells was 1059 +/- 445 micrograms/mg cell protein, but it declined steeply during 1 week in primary culture . The uptake of low-density lipoprotein (LDL), beta-migrating very low-density lipoprotein (beta-VLDL), and acetylated LDL (acetyl-LDL), labeled with 125I or with the fluorescent probe 1,1'-dioctadecyl-3,3,3',3'- tetramethylindocarbocyanine (DiI), was studied in 2-day-old primary cultures . DiI-acetyl-LDL was avidly taken up by the macrophages and, to a lesser extent, by some smooth muscle cells . The uptake of DiI-beta-VLDL by the macrophages was weaker and less homogeneous than that of DiI-acetyl-LDL . The degradation rates of 125I-labeled beta-VLDL, LDL and acetyl-LDL were 135 +/- 54, 195 +/- 20, and 697 +/- 14 ng/mg cell protein/8 hours, respectively . Incubation with unlabeled acetyl-LDL enhanced the incorporation of {3H}oleate into cholesteryl esters and increased the cellular cholesteryl ester content . These results suggest that arterial macrophages and, to some extent, smooth muscle cells from cholesterol-fed rabbits actively metabolize acetyl-LDL and are thus capable of accumulating cholesteryl esters by uptake of modified forms of LDL.

Am J Pathol, 1990 Aug, 137(2), 425 - 34
Focal toxicity of oxysterols in vascular smooth muscle cell culture . A model of the atherosclerotic core region; Guyton JR et al.; Cell necrosis and reactive cellular processes in and near the atherosclerotic core region might result from short-range interactions with toxic lipids . To model these interactions in cell culture, focal crystalline deposits of cholestane-3 beta,5 alpha,6 beta-triol, 25-OH cholesterol, and cholesterol were overlaid by a collagen gel, on which canine aortic smooth muscle cells were seeded . Oxysterols, but not cholesterol, caused focally decreased plating efficiency and cell death, leading to the formation of a persistent circular gap in the cell culture . Cholestanetriol was largely removed from the culture dishes over 3 to 4 weeks, whereas cholesterol and 25-OH cholesterol were largely retained . Smooth muscle cells were motile even in proximity to oxysterol crystals, with occasional suicidal migration toward the crystals . Chemoattraction, however, could not be demonstrated . Despite toxicity, cholestanetriol did not appear to alter the fraction of cells exhibiting 3H-thymidine uptake, even in areas close to the crystals . Thus, oxysterols may be toxic to some cells, without causing major impairment of the migration and proliferation of nearby cells . This would allow the simultaneous occurrence of cell death and proliferation evident in atherosclerosis.

J Gen Virol, 1990 Aug, 71 ( Pt 8), 1845 - 50
Low pH-induced cell fusion in flavivirus-infected Aedes albopictus cell cultures; Randolph VB et al.; Cell-to-cell fusion of Aedes albopictus (mosquito) cells infected with dengue and St Louis encephalitis (SLE) flaviviruses was induced by exposure to low pH . The parameters of this low pH-induced fusion were examined . Syncytium formation was maximal in cultures 36 to 48 h post-infection and occurred when cultures were maintained at the acid pH for 15 min at 35 degrees C . The optimal pH range for fusion was 5.0 to 6.5 for dengue virus-infected cells and 5.0 to 5.5 for SLE virus-infected cells . Syncytia were not observed in vertebrate cells (Vero and BHK) under these conditions despite similar virus yields . Fusion was shown to be ATP-dependent and could be prevented by the addition of either polyclonal antiviral antibodies or monoclonal antibody to the envelope glycoprotein . The lysosomotropic amine ammonium chloride inhibited the replication of SLE virus in both mosquito and vertebrate cells, consistent with the idea that low pH-induced fusion is necessary for virus entry into both types.

Mol Pharmacol, 1990 Aug, 38(2), 274 - 81
Pertussis toxin inhibits norepinephrine-stimulated inositol phosphate formation in primary brain cell cultures; Wilson KM et al.; Norepinephrine (NE) increased formation of {3H}inositol phosphates ( {3H}InsPs) in primary cultures of neuronal and glial cells from 1-day-old rat brain . This response appeared to be mediated by alpha 1-adrenergic receptors, because prazosin was 40-fold more potent than yohimbine in blocking it . Pretreatment with pertussis toxin (PTX) dose-dependently decreased this response by 70-80% . The IC50 for PTX (7 ng/ml) was similar to that for blocking of alpha 2-adrenergic receptor-mediated decreases in cyclic AMP accumulation in the same cells . PTX pretreatment caused only a small, not statistically significant, inhibition of the {3H}InsP response to the muscarinic cholinergic receptor agonist carbachol in these cells . Radioligand binding studies showed that both neuronal and glial cultures contained mixed populations of alpha 1a- and alpha 1b-adrenergic receptor subtypes . Selective inactivation of the alpha 1b population by chloroethylclonidine reduced NE-stimulated {3H}InsP formation by 25 +/- 6% . Pretreatment with both PTX and chloroethylclonidine caused additive decreases (90 +/- 3%) in the NE response . NE-stimulated {3H}InsP formation was partially dependent on extracellular calcium, because it was decreased 64 +/- 6% by removal of calcium and 56 +/- 13% by addition of 1 mM CdCl2, although it was not affected by 1 microM nifedipine . These results suggest that NE stimulates {3H}InsP formation in neuronal and glial cultures through a pertussis toxin-sensitive guanine nucleotide-binding protein . This response appears to be mediated primarily by the alpha 1a subtype and may be subsequent to calcium influx.

Neuron, 1990 Aug, 5(2), 121 - 6
21-Aminosteroids attenuate excitotoxic neuronal injury in cortical cell cultures; Monyer H et al.; We studied the protective efficacy of novel 21-aminosteroids against several forms of neuronal injury in murine cortical cell cultures . Concentrations of 200 nM to 20 microM partially attenuated the damage induced by glucose deprivation, combined oxygen-glucose deprivation, or exposure to NMDA; maximal protection was less than that produced by NMDA antagonists, but the combination of a 21-aminosteroid plus an NMDA antagonist produced a greater benefit than either drug alone . 21-Aminosteroid addition did not attenuate NMDA-induced whole-cell current, but did block almost all of the damage induced by exposure to iron, a protective action consistent with inhibition of free radical-mediated lipid peroxidation . Lipid peroxidation may be a downstream event mediating a portion of the injury triggered by excess stimulation of NMDA receptors.

Virology, 1990 Aug, 177(2), 684 - 91
Identification of three genes nonessential for growth in cell culture near the right terminus of the unique sequences of long component of herpes simplex virus 1; Barker DE et al.; We report the construction of herpes simplex virus 1 recombinants from which all or part of the coding sequences of four open reading frames have been deleted . In recombinants R7101 and R7108, the BamHI D' fragment containing the coding sequences of the dUTPase gene and the promoter-regulatory domain of a late gene was either replaced with a fragment containing a thymidine kinase gene or deleted, respectively . The recombinant R7107 lacks, in addition to BamHI D', 202 of a total of 244 predicted amino acids from the 3' end of the adjacent open reading frame UL51 . The deletion in recombinant R7105 encompasses two genes, i.e., the entire open reading frame of UL47 and the amino terminus of UL46 . These genes map next to that specifying the alpha trans-inducing factor . R7105, R7101, and R7108 do not exhibit demonstrable defects in viral replication . The recombinant R7107 forms minute plaques and replicates best in multiplying cells in subconfluent cultures . The results indicate that the multiplying cells supply at least some of the functions expressed by the protein encoded in UL51.

Eur J Cell Biol, 1990 Aug, 52(2), 236 - 40
pH-dependent changes of ganglioside biosynthesis in neuronal cell culture; Iber H et al.; Ganglioside biosynthesis was studied in primary cultured murine cerebellar cells after labeling with {14C}galactose . A shift in biosynthesis from "a"-series to "b"-series gangliosides was observed after lowering the pH of the culture medium from 7.4 to 6.2; this effect was fully reversible on changing back to pH 7.4 . The observed regulatory effect of pH is in accordance with a recent model of ganglioside biosynthesis . Sialyltransferase II (ST II), the first enzyme for biosynthesis of "b"-series gangliosides, is more active at pH 6.2 than Gal-NAc-transferase, the first enzyme for synthesis of "a"-series gangliosides, which is more active than sialyltransferase II at pH 7.4.

Arzneimittelforschung, 1990 Aug, 40(8), 896 - 9
Effects of proxigermanium on interferon production and 2',5'-oligoadenylate synthetase activity in the lung of influenza virus-infected mice and in virus-infected human peripheral blood mononuclear cell cultures; Ishiwata Y et al.; Proxigermanium (SK-818) is a synthesized organic germanium compound having various biological activities . The effects of proxigermanium on interferon (IFN) production in mice infected with influenza virus and virus-infected human peripheral blood mononuclear cells (hPBMC) were investigated . Proxigermanium alone did not induce IFN production in normal mice or in hPBMC without viral infection . On the other hand, proxigermanium enhanced alpha/beta IFN production in viral-infected mice and hPBMC . Since proxigermanium is known to have antiviral activity in vivo but not in vitro, it is likely that the IFN production augumenting activity of proxigermanium is involved in its antiviral activities.

J Interferon Res, 1990 Aug, 10(4), 375 - 8
Role of calmodulin/protein kinase C in interferon production by poly(rI).poly(rC) in primed human cell cultures; Lin HY et al.; Human cells treated with trifluoperazine (TFP) or K252a prior to and/or during priming with human interferon-alpha (HuIFN-alpha) produce significantly more IFN on induction with poly(rI).poly(rC) than primed cells in the absence of the drug . Results suggest that calmodulin/protein kinase activity may be involved in priming activity of HuIFN-alpha.

Gen Comp Endocrinol, 1990 Aug, 79(2), 275 - 82
Thyroid hormone-induced differential synthesis of water-insoluble proteins in epidermal cell cultures from the hind limb of Rana catesbeiana tadpoles in stages XII-XV and XVI-XIX; Ketola-Pirie CA et al.; Hind limb epidermal cell cultures from stage XII-XV and XVI-XIX tadpoles of the American bullfrog Rana catesbeiana were maintained for 36 and 120 hr in medium containing either fetal calf serum or thyroid hormone (3,3',5-triiodothyronine, T3; 3 x 10(-10) mol/ml) . T3 induces the precocious synthesis (within the first 36 hr) of a 59-kDa keratin associated with epidermal stratification in cultures from stages XII-XV . In epidermal cell cultures from stages XVI-XIX, T3 produces an overall pattern of water-insoluble proteins, including keratins, which is strikingly similar to temporally differentiated cultures (120 hr) . A 73-kDa protein is among the water-insoluble proteins precociously synthesized by 36-hr-old cultures from stages XVI-XIX treated with T3 . This protein, which is not immunoprecipitated by antibodies raised against keratins, corresponds in Mr and pI to a mammalian differentiation-specific desmosomal plaque protein . Immunoprecipitation of keratins from 120-hr-old cultures shows that many of the water-insoluble proteins synthesized are, indeed, keratins . Further, quantitation, by laser densitometry, of immunoprecipitated keratins from 120-hr cultures demonstrates that greater amounts of keratins, particularly the 65 and 59 kDa which are associated with a differentiated epidermis, are present in stages XVI-XIX . This study indicates that epidermal cell cultures from stages XVI-XIX respond more quickly to the differentiating effects of T3.

J Neurochem, 1990 Aug, 55(2), 673 - 82
Cyclic AMP-dependent induction of serotonin N-acetyltransferase activity in photoreceptor-enriched chick retinal cell cultures: characterization and inhibition by dopamine; Iuvone PM et al.; The activity of serotonin N-acetyltransferase (NAT), a key regulatory enzyme in the melatonin biosynthetic pathway, was examined in low-density monolayer cultures of chick embryo retinal cells prepared with three levels of photoreceptor enrichment . In cultures prepared from embryonic day 8 retinas (E8), photoreceptors represented approximately 30% of the total cell population, whereas in those prepared from embryonic day 6 retinas (E6), approximately 70% of the cells were photoreceptors . In E8 retinas treated with kainic acid to destroy neurons (E8K), the relative content of photoreceptors was increased to approximately 50% . NAT activity was detectable in the cultures under all conditions studied, and was markedly increased by drugs that increase intracellular cyclic AMP levels and cyclic AMP-dependent protein kinase activity: 8-bromocyclic AMP, forskolin, and 3-isobutyl-1-methylxanthine (IBMX) . Consistent with the hypothesis that NAT is localized in photoreceptors, the effects of the stimulatory treatments were significantly greater in E6 and E8K cultures than in E8 cultures . The stimulation of NAT activity in E6 cultures was inhibited by actinomycin D and cycloheximide, suggesting the involvement of RNA and protein synthesis . Dopamine inhibited the induction of NAT activity by forskolin and IBMX, but not that elicited by 8-bromocyclic AMP . The dopamine-mediated suppression of activity was significantly inhibited by pertussis toxin and by spiperone and sulpiride, both D2-dopamine receptor antagonists, but not by SCH 23390, a D1-dopamine receptor blocker, or antagonists of alpha-adrenergic, beta-adrenergic, or serotonergic receptors . Because the inhibitory effect of dopamine on E6 and E8K cultures was at least as great as that on E8 cultures, the results suggest that dopamine acts on D2-like receptors on photoreceptors . The receptors appear to be coupled to adenylate cyclase through an inhibitory GTP-binding protein and to mediate inhibition of cyclic AMP synthesis and consequent induction of NAT activity.

J Neurochem, 1990 Aug, 55(2), 583 - 7
Demyelination induced in aggregating brain cell cultures by a monoclonal antibody against myelin/oligodendrocyte glycoprotein; Kerlero de Rosbo N et al.; A monoclonal antibody (8-18C5) directed against myelin/oligodendrocyte glycoprotein (MOG) induced demyelination in aggregating brain cell cultures . With increasing doses of anti-MOG antibody in the presence of complement, myelin basic protein (MBP) concentration decreased in a dose-related manner . A similar, albeit less pronounced, effect was observed on specific activity of 2',3'-cyclic nucleotide 3'-phosphohydrolase . In the absence of complement, anti-MOG antibody did not induce detectable demyelination . In contrast to the effect of anti-MOG antibody and as expected, anti-MBP antibody did not demyelinate aggregating brain cell cultures in the presence of complement . These results provide additional support to the suggestion that MOG, a quantitatively minor myelin component located on the external side of the myelin membrane, is a good target antigen for antibody-induced demyelination . Indeed, they show that a purified anti-MOG antibody directed against a single epitope on the glycoprotein can produce demyelination, not only in vivo as previously shown, but also in cultures . Such an observation has not been made with polyclonal antisera raised against purified myelin proteins like MBP and proteolipid protein, the major protein components of the myelin membrane, or myelin-associated glycoprotein . These observations may have important implications regarding the possible role of anti-MOG antibodies in demyelinating diseases.

Enzyme Microb Technol, 1990 Aug, 12(8), 571 - 6
Flow cytometric study of hybridoma cell culture: correlation between cell surface fluorescence and IgG production rate; Sen S et al.; Flow cytometry was applied to measure the fluorescence of IgG molecules on hybridoma cell surface stained with FITC-conjugated anti-mouse IgG F(ab')2 . Using light scattering to exclude the dead cell population from the analysis, the surface fluorescence intensity allows for the differentiation between producing and nonproducing cells . A linear correlation has been found between the intensity of mean surface fluorescence of the cell population and the specific antibody production rate.

Brain Res, 1990 Jul 30, 524(1), 10 - 6
Characterization of oxytocin-binding sites in primary rat brain cell cultures; Di Scala-Guenot D et al.; Detection and characterization of oxytocin-binding sites in dissociated brain cell cultures were performed, using a highly selective iodinated oxytocin antagonist {( 125I}OTA) . Dissociated cells derived from hypothalamus and extrahypothalamic forebrain of 16-day-old fetal rats were maintained in chemically defined medium in order to enrich the cultures in neuronal cells . Specific binding of {125I}OTA, demonstrated in both hypothalamic and forebrain cell cultures, was temperature- and time-dependent; maximal binding was obtained by incubating the iodinated ligand for 60 min at 37 degrees C . The binding parameter were shown to be identical in both cell type cultures . The Scatchard plot analysis suggested the presence of a single class of binding sites of high affinity (Kd about 0.06 nM) and low binding capacity (Bmax about 4 fmol/dish) . The specificity of these binding sites tested in competition experiments revealed that the unlabelled OT antagonist was the most potent in inhibiting specific {125I}OTA binding (Ki = 0.1 nM) . A lower affinity, of the nM range was demonstrated for oxytocin (OT), arginine-vasopressin (AVP) and the V1 antagonist, whereas the V2 AVP agonist poorly competed for {125I}OTA binding sites (Ki about 250 nM) . In conclusion, the {125I}OTA binding characteristics, identical in both hypothalamic and forebrain cultures, fulfil the classical conditions required for the existence of receptor sites since the binding was reversible, saturable and specific . As these characteristics were similar to those already described in the adult rat, at the central level in hippocampus, and at the periphery in the mammary gland, it could be postulated that {125I}OTA binds to an OT receptor site.(ABSTRACT TRUNCATED AT 250 WORDS)

FEBS Lett, 1990 Jul 30, 268(1), 24 - 6
Expression of phosphoglycerate mutase mRNA in differentiating rat satellite cell cultures; Castella-Escola J et al.; Poly(A)+ mRNA was isolated from rat satellite cell cultures and analyzed by Northern blot analyses for mRNA content of phosphoglycerate mutase (PGAM) isozymes . In non-differentiating satellite cells only PGAM-B mRNA was detected, but when cells were differentiated into myotubes, which undergo spontaneous contraction, mRNA for PGAM-M muscle-specific isozyme was also detected . This finding is in perfect concordance with the transition of PGAM isozymes encountered in the same cell cultures, and strongly supports a transcriptional control of PGAM expression throughout myogenesis independently of nerve influence.

Biochim Biophys Acta, 1990 Jul 25, 1018(2-3), 243 - 7
Differentiation of brown adipose tissue and biogenesis of thermogenic mitochondria in situ and in cell culture; Houstek J et al.; Differentiation and biogenesis of mitochondria in brown adipose tissue (BAT) was studied in situ and in cell culture by Western blotting, enzyme activity measurements, {35S}methionine incorporation and immunofluorescence microscopy . In different rodent species the perinatal development of BAT thermogenic function resulted from the formation of thermogenic mitochondria which replaced the preexisting nonthermogenic mitochondria . Their biogenesis was characterized by the sudden appearance and rapid increase of the uncoupling protein (UCP), increase of cytochrome oxidase (COX) and decrease of H(+)-ATPase . In primary cell culture, differentiation of precursor cells from mouse BAT to typical multilocular adipocytes was accompanied by increasing content of COX and H(+)-ATPase . A selective synthesis of UCP was induced by activation of beta-adrenergic receptors or by elevated levels of cellular cAMP . UCP was quantitatively incorporated into mitochondria and within 24 h after stimulation reached near physiological concentration . Both in situ and in cell culture, the conditions enabling the expression of UCP gene were accompanied by activation of intracellular thyroxine 5'-deiodinase.

J Neurochem, 1990 Jul, 55(1), 311 - 20
Mechanisms of toxicity and cellular resistance to 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine and 1-methyl-4-phenylpyridinium in adrenomedullary chromaffin cell cultures; Reinhard JF Jr et al.; Bovine adrenomedullary chromaffin (BAMC) cells, cultured in a defined medium, were used to study the mechanisms of toxicity and cellular resistance to the catecholamine neuron toxicants 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) and 1-methyl-4-phenylpyridinium (MPP+) . The viability of the cells was assessed biochemically {cellular catecholamine content and the catalytic activities of tyrosine hydroxylase (TH) and lactate dehydrogenase (LDH)} and anatomically (by electron microscopy) . When cultures of BAMC cells were exposed to MPTP or MPP+ for 3 days, a marked loss of cellular catecholamines and TH activity was observed . The addition of an inhibitor of monoamine oxidase (MAO) B (Ro 19-6327), but not MAO A (clorgyline), prevented the toxicity of MPTP but not that of MPP+ . In addition, the cellular toxicity of MPP+, but not MPTP, was antagonized by desmethylimipramine, an inhibitor of cellular catecholamine uptake . The toxicity of MPP+ was time dependent, with losses of TH and the release of cellular LDH occurring after 48 h in culture . Catecholamine depletion occurred somewhat sooner, being evident after 24 h of exposure to MPP+ . The cellular toxicity of MPP+ was concentration dependent and significantly enhanced by inhibitors of catecholamine vesicular uptake (reserpine, tetrabenazine, or Ro 4-1284) . Electron microscopic examination of cells treated with either MPP+, tetrabenazine, or their combination revealed that MPP+ damaged BAMC cells and that this damage was markedly potentiated by the inhibition of vesicular uptake by tetrabenazine . The concentration of glucose in the culture media of untreated cells slowly decreased as a function of time . The rate of glucose consumption was markedly accelerated by MPP+ treatment and the losses in cell TH and the release of LDH into the media were preceded by a 99% depletion of glucose from the media . In cultures not treated with MPP+, lactate accumulated in the media as a function of time . Addition of MPP+ to the media increased the formation of lactate, in a concentration-dependent manner . Reserpine pretreatment further enhanced the production of lactate in response to MPP+ . Culturing cells in glucose-free medium greatly potentiated the effects of MPP+ on cellular TH and catecholamines . The toxicity observed after 3 days' exposure of BAMC cells to MPP+ could be prevented when the medium was replaced with fresh medium every 24 h . The effects of glucose deprivation and reserpine were observed to be additive . The ability of MPP+ to affect mitochondrial function is determined by the capacity of the storage vesicle to sequester the pyridinium, acting as a cytosolic "buffer."(ABSTRACT TRUNCATED AT 400 WORDS)

Brain Res, 1990 Jul 9, 522(2), 204 - 14
Cell culture of cryopreserved human fetal cerebral cortical and hippocampal neurons: neuronal development and responses to trophic factors; Mattson MP et al.; Past knowledge of the human brain at the cellular and molecular levels has come largely from studies of postmortem fixed tissue or by way of extrapolation from studies of lower mammalian species . The ability to study living human brain neurons would provide a new avenue for further insight into mechanisms operative in human brain development, function, and disease . The present study established procedures for the cryopreservation and culture of human fetal cerebral cortical and hippocampal neurons, and characterized the development of the cells in culture . The predominant cell type in both cortical and hippocampal cultures was pyramidal-like neurons that extended one long axon-like process and a few minor dendrite-like processes . Bipolar and stellate cells, as well as astrocyte-like glia were also present in cultures from both brain regions . Fibroblast growth factor (FGF), but not nerve growth factor (NGF), enhanced long-term neuronal survival in both cerebral cortical and hippocampal cultures . Immunocytochemistry demonstrated the presence of both FGF- and NGF-like immunoreactivities in neurons and glia, from both cerebral cortex and hippocampus, suggesting that these endogenous growth factors may play roles in human fetal brain development . The ability to cryopreserve large numbers of viable dissociated human fetal brain neurons, and subsequently study them in cell cultures, provides new opportunities to understand dynamic aspects of the human brain at the cellular and molecular levels.

J Steroid Biochem, 1990 Jul 4, 36(4), 325 - 31
Estrogen metabolism in primary kidney cell cultures from Syrian hamsters; Brueggemeier RW et al.; Estrogen metabolism was evaluated in freshly isolated kidney and liver microsomes and in primary kidney cell cultures from Syrian hamsters, a potential experimental model for examining the possible role(s) of estrogens in tumor initiation and development . Initial velocity studies of the conversion of estradiol to 2-hydroxyestradiol, as determined by the 3H2O release assay with the substrate {2-3H}estradiol, resulted in similar apparent Kms of estrogen 2-hydroxylase of 2.85 and 6.25 microM for liver and renal microsomes, respectively . The apparent Vmax for freshly prepared liver microsomes was 0.13 nmol.mg-1.min-1, while that for renal microsomes was 0.040 nmol.mg-1.min-1 . Evaluation of estrogen metabolism was also performed in primary cell cultures of hamster kidney cells, consisting of 75% epithelial cells . {6,7-3H}Estradiol (10 microM) was incubated for 0, 24 and 48 h in primary kidney cell cultures, and the organic soluble metabolites analyzed by reverse-phase HPLC . The cultures from untreated, castrated hamsters metabolize {3H}estradiol to yield small quantities of estrone and significant amounts of polar metabolites, while no catechol estrogens were isolated . Estrogen metabolism by diethylstilbestrol-treated (DES-treated) hamster kidney cell cultures also provided small quantities of estrone and no evidence of catechol estrogens . Additionally, larger amounts of additional polar metabolites were isolated in the cultures from DES-treated hamsters . Finally, levels of estrogen 2-hydroxylase were detected in these cultures using the 3H2O release assay . Thus, the short-term primary kidney cell cultures from the Syrian hamster are capable of metabolizing estrogens . Furthermore, the enzymatic processes appear to be available for the conversion of any catechol estrogens formed into more polar metabolites . These investigations in intact cells, capable of performing all biochemical processes, complement both in vivo and subcellular biochemical studies and may aid in elucidating the roles of estrogens and estrogen metabolism in the initiation and development of estrogen-induced, estrogen-dependent kidney tumors in the Syrian hamster.

Biomaterials, 1990 Jul, 11, 41 - 3
Effects of acidic fibroblast growth factor and epidermal growth factor on fetal rat calvaria cell cultures; Nicolas V et al.; An inhibitory effect of alkaline phosphatase (LP) activity on short and long term fetal rat calvaria cell cultures was recorded with both acidic fibroblast growth factor (aFGF) and epidermal growth factor (EGF) at a concentration of 30 ng ml-1 . This inhibition was well correlated with the nodule number on long term culture, except for EGF treatment in subconfluent cell culture.

J Bone Miner Res, 1990 Jul, 5(7), 769 - 74
Bilirubin as an inhibitor of cartilage metabolism: effect on avian chondrocyte proliferation in cell culture; Vassilopoulou-Sellin R et al.; Elevated levels of bilirubin and other tetrapyrroles are common to a number of chronic hematologic and liver diseases that can result in growth failure . This report establishes a cellular model system for the study of these endogenous growth inhibitors . Primary chondrocyte cultures were prepared from embryonic chick cartilage; cells were incubated (0.3 X 10(5) cells per plate) in tissue culture medium containing 10% fetal bovine serum (FBS) with or without added bilirubin, 0.01-0.10 mM . Bilirubin caused profound, dose-dependent inhibition of chondrocyte proliferation: after 7 days, control incubations contained 11.45 +/- 2.0 X 10(5) cells per plate versus 4.92 +/- 0.55 X 10(5) cells per plate for wells with added 0.01 mM bilirubin and 1.76 +/- 0.30 X 10(5) for cultures with added 0.05 mM bilirubin . In chondrocytes cultured for 3 days, the addition of 0.05 mM bilirubin was associated with inhibition of {3H}thymidine incorporation into DNA (47 +/- 5% of control), {3H}uridine incorporation into RNA (11 +/- 0.05% of control), and {14C}leucine incorporation into proteins (16 +/- 1% of control) . The inhibition of chondrocyte proliferation induced by a range of bilirubin concentrations (0.01-0.10 mM) was in no way attenuated by the addition of physiologic concentrations of albumin (4 g/dl) . After 3 days in media containing bilirubin or heme (at equimolar concentrations), chondrocytes were subsequently incubated in FBS alone for an additional 3 days; only partial reversal of the bilirubin (or heme)-induced inhibition was then observed.(ABSTRACT TRUNCATED AT 250 WORDS)

J Clin Invest, 1990 Jul, 86(1), 235 - 41
Structural and kinetic analyses of herpes simplex virus type 1 latency-associated transcripts in human trigeminal ganglia and in cell culture; Krause PR et al.; Only one herpes simplex virus type 1 (HSV-1) gene is expressed in sensory neurons of latently infected animals and humans, yielding two RNAs, called latency-associated transcripts (LATs) . The LATs appear to modulate virus reactivation . In mice and rabbits the 5' origins, kinetics of synthesis, and splicing pattern of the LATs are well established . Because these details of LAT structure and expression have not been defined in humans, we sought to do so . Using primer extension and Northern hybridization analyses, we demonstrate that in human trigeminal ganglia, the smaller (1.35 kb) HSV-1 transcript differs from the larger (1.85 kb) LAT by excision of an intron near its 5' end; they are otherwise colinear, and 5' coterminal . In infected cells only the 1.85 kb LAT is detected . Its expression is inhibited by cycloheximide or acyclovir, indicating this LAT is synthesized late in the viral replicative cycle . All of these features of the LATs in humans are consistent with those reported in rabbits and mice and further validate the animal models of human HSV-1 infection.

J Cell Biol, 1990 Jul, 111(1), 1 - 8
The permeability of the nuclear envelope in dividing and nondividing cell cultures; Feldherr CM et al.; The objective of this study was to determine whether the permeability characteristics of the nuclear envelope vary during different phases of cellular activity . Both passive diffusion and signal-mediated transport across the envelope were analyzed during the HeLa cell cycle, and also in dividing, confluent (growth-arrested), and differentiated 3T3-L1 cultures . Colloidal gold stabilized with BSA was used to study diffusion, whereas transport was investigated using gold particles coated with nucleoplasmin, a karyophilic Xenopus oocyte protein . The gold tracers were microinjected into the cytoplasm, and subsequently localized within the cells by electron microscopy . The rates of diffusion in HeLa cells were greatest during the first and fifth hours after the onset of anaphase . These results correlate directly with the known rates of pore formation, suggesting that pores are more permeable during or just after reformation . Signal-mediated transport in HeLa cells occurs through channels that are located within the pore complexes and have functional diameters up to 230-250 A . Unlike diffusion, no significant differences in transport were observed during different phases of the cell cycle . A comparison of dividing and confluent 3T3-L1 cultures revealed highly significant differences in the transport of nucleoplasmin-gold across the envelope . The nuclei of dividing cells not only incorporated larger particles (230 A versus 190 A in diameter, including the protein coat), but the relative uptake of the tracer was about seven times greater than that in growth-arrested cells . Differentiation of confluent cells to adipocytes was accompanied by an increase in the maximum diameter of the transport channel to approximately 230 A.

Obstet Gynecol, 1990 Jul, 76(1), 114 - 7
Comparison of chemiluminescent DNA probe to cell culture for the screening of Chlamydia trachomatis in a gynecology clinic population; Mercer LJ et al.; Two hundred ninety-five endocervical swab specimens were obtained from patients presenting to a gynecology clinic in order to compare a nonradioactive chemiluminescent DNA probe with cell culture for detection of Chlamydia trachomatis . Discrepancies between cell culture and DNA probe were resolved by retesting and reculturing samples . In a population with a 10.8% prevalence, the corrected sensitivity, specificity, positive predictive value, and negative predictive value for the DNA probe were 80.6, 95.8, 71.4, and 97.3%, respectively . These results compare favorably to other non-culture methods such as direct fluorescent antibody and enzyme immunoassay tests for the detection of C trachomatis in populations with similar prevalence rates.

J Protozool, 1990 Jul-Aug, 37(4), 11S - 16S
NACM, a cytopathogenic protein from Naegleria gruberi, EGs; purification, production of monoclonal antibody, and the immunoidentification of a product that develops in NACM-treated vertebrate cell cultures; Dunnebacke TH et al.; The story of NACM involves the discovery of a deleterious response of cultured vertebrate cells to a component in cell-free lysates prepared from free-living amebae of the genus Naegleria; hence the acronym NACM derived from Naegleria ameba cytopathogenic material . The cellular reaction is the basis for the biological assay that has been fundamental in the study of the action of NACM in a variety of cell cultures . It also has been used in the determination of the physical characteristics, and to monitor the behavior of NACM during isolation procedures . All findings are compatable with the conclusion that NACM is a 35 Kd protein . Recently, the use of monoclonal antibodies (MAbs) prepared to amebae-derived purified NACM have resulted in the visual display of a product that develops exclusively in NACM-treated cells . That cellular product is shown to be related to NACM by its immunostaining reaction with the MAb; the relationship of the MAb with NACM is demonstrated by its ability to neutralize the biological activity of NACM, and as an immunostain, to react with purified fractions of NACM and with whole amebae . The combination of these observations describes a unique set of interactions in which NACM, an amebic component, identified as a protein, has characteristics of an infectious agent when introduced into cultures of avian and mammalian cells.

J Cell Sci, 1990 Jul, 96 ( Pt 3), 509 - 17
Regulation by calcium of proliferation and morphology of normal human tracheobronchial epithelial cell cultures; Chopra DP et al.; Human tracheobronchial epithelial cells have been serially passaged in serum-free medium . This serum-free model was employed to investigate the effects of different concentrations of Ca2+ (0.1, 1.0 and 2.0 mM) on multiplication and morphology of the cells . The responses were analysed in terms of growth kinetics, histochemical and ultrastructural alterations . Culturing of the cells in high Ca2+ (1.0-2.0 mM) medium stimulated cell multiplication characterized by increased colony forming efficiency, greater number of cells per colony and cell population doublings per day . Additionally, the high Ca2+ concentrations induced proliferation in cultures grown to confluency in low Ca2+ (0.1 mM) medium . Cells propagated in low Ca2+ medium consisted of relatively heterogeneous cell populations, with most cells staining positive with periodic acid-Schiff (PAS) reagent . Ultrastructurally the cells exhibited secretory vesicles and microvilli on their surfaces, small desmosomes and intercellular interdigitation between cells and numerous large secretory vesicles in the cytoplasm . The cells grown in high Ca2+ medium acquired characteristics of a highly proliferative phenotype . The cultures consisted of closely packed, relatively homogeneous cells that did not stain with PAS reagent . Their characteristic features were: absence of surface secretory vesicles, reductions of microvilli and intercellular interdigitations, and increases in size and number of desmosomal junctions . The results show that low Ca2+ in the culture medium inhibits cell multiplication and favors the secretory cell phenotype, while high Ca2+ levels stimulate cell multiplication and inhibit the secretory cell phenotype.

Fiziol Zh, 1990 Jul-Aug, 36(4), 80 - 4
{The cytological and hormonal characteristics of cell cultures from the testes of newborn piglets}; Potikha OP et al.; A cell culture of neonatal pig testes has been studied . Leydig cells, Sertoli cells, sex cells and fibroblasts have been identified by cytologic and cytochemical research methods, Gonadotropin concentration (0.1 U/ml) of nutritional medium, increases proliferation of the Leydig cells and increases their 3-beta-ol-steroiddehydrogenase activity as well as increases amount of lipid inclusions and stimulates testosterone production . The cell culture can be used to treat some forms of hypogonadism.

APMIS, 1990 Jul, 98(7), 605 - 8
Proliferation of adherent synovial fluid cell cultures is modified by eicosanoids; Andreasen A et al.; Synovial fluid cells obtained from a carrageenan-induced chronic arthritis in the juvenile dog knee were allowed to adhere and proliferate in culture flasks . After twelve days secondary cultures were made and either 10(-8)M leukotriene B4 (LTB4), 25 microM 15-hydroxy-eicosatetraenoic acid (15-HETE), or both, were added and the cells were cultured for another 6 days . LTB4 is generated via the 5-lipoxygenase pathway of arachidonic acid metabolism and stimulates a number of phagocyte functions . Compared to control cells LTB4 increased proliferation in 9 out of 10 cell cultures (p less than 0.05) . The 15-lipoxygenase product, 15-HETE, is not proinflammatory and is an endogenous inhibitor of 5-lipoxygenase . Addition of 15-HETE decreased proliferation of cell cultures by 23% (p less than 0.01) . It is speculated that LTB4 in addition to its effect on phagocytes may play a role in synovial hyperproliferation observed in arthritis.

J Virol, 1990 Jul, 64(7), 3212 - 8
Characterization of human parainfluenza virus type 3 persistent infection in cell culture; Moscona A et al.; Three cell lines persistently infected with human parainfluenza virus type 3 were characterized on a molecular level in this study . All six structural protein genes were transcribed into monocistronic RNAs in the persistently infected cells . In both acutely and persistently infected cells, polycistronic transcripts were abundant, although the ratio of polycistronic to monocistronic transcripts was reduced in the persistently infected cells . Each of the persistently infected cell lines contained a distinct subgenomic RNA species . The subgenomic RNAs were present in purified nucleocapsid cores, indicating that they represent viral genome RNA, were far more abundant than full-length RNA, and were stably maintained through at least 36 cell passages . Nucleotide sequence analysis of the subgenomic RNAs from two of the persistently infected cell lines revealed that the 5' ends are identical to that of the standard genome . Hybridization experiments with oligonucleotide probes showed that both fragments retain sequences from the 5' end of the standard genome and contain approximately 1,200 nucleotides (cell line 1) and 1,500 nucleotides (cell line 2) of the polymerase gene sequence . The demonstration of several alterations in viral gene expression in persistently infected cells offers insight into the factors associated with persistence of parainfluenza virus 3.

J Pharm Sci, 1990 Jul, 79(7), 595 - 600
Epithelial transport of drugs in cell culture . II: Effect of extracellular calcium concentration on the paracellular transport of drugs of different lipophilicities across monolayers of intestinal epithelial (Caco-2) cells; Artursson P et al.; A human intestinal cell line, Caco-2, was used as a model to study the passive diffusion of a homologous series of drugs (beta-blocking agents) of different lipophilicity across intestinal epithelium . The permeability of the Caco-2 monolayers was modulated by the use of a calcium switch assay . The transmembrane resistance could be reversibly decreased from approximately 280 ohms.cm2 (a resistance similar to that of colon epithelium) to approximately 60 ohms.cm2 (a resistance similar to that of small intestine epithelium) . Transmission electron microscopy showed that the increased electrical permeability was caused by a reversible separation of the components of the junctional complex and not by cell detachment . In general, the increased paracellular permeability resulted in a 2- to 9-fold increase in the apparent permeability coefficients for the more hydrophilic drugs (e.g., from 0.20 +/- 0.010 x 10(-6) to 1.43 +/- 0.185 x 10(-6) cm/s for atenolol), while the transport parameters for the more lipophilic drugs remained unchanged (e.g., 43.03 +/- 3.64 x 10(-6) and 46.10 +/- 3.25 x 10(-6) cm/s for propranolol) . These findings indicate that it is possible to study the contribution of the paracellular pathway to the transport of drugs in the Caco-2 model.

J Cell Sci, 1990 Jul, 96 ( Pt 3), 501 - 8
Adaptation to non-ammoniagenic medium and selective substrate feeding lead to enhanced yields in animal cell cultures; Hassell T et al.; Methods for the adaptation of three animal cell lines to media in which glutamine is replaced with either glutamate or 2-oxoglutarate are described . The cell lines differ in their rate of adaptation . The consequences of the adaptation of the McCoy cell line to a glutamate-based medium was measured in terms of: cell yield (increased by 17%), ammonia accumulation (reduced by 70%), glucose consumption (decreased by greater than 70%) and lactate accumulation (decreased by greater than 75%) . The value of such adaptation and concomitant changes in energy metabolism lies in the potential for increased cell yields . Batch feeding of adapted cells in a microcarrier culture with a cocktail of glucose and amino acids resulted in increases in cell yields of 80% compared to unfed controls.

Medicina (Firenze), 1990 Jul-Sep, 10(3), 298 - 302
{The use of continuous cell cultures for the study of tumor heterogeneity and drug sensitivity}; Del Bufalo D et al.; Intratumoral heterogeneity was observed in two tumor lines (SbC11 and SbC12) derived from a single biopsy of a melanoma patient . Differences in drug sensitivity were observed in three cell lines of small cell lung carcinoma derived from the same patient, before (AE1), and after (AE2 and AE3) therapy with Adriamycin (ADM) and Cisplatinum (DDP) . Moreover, heterogeneity in biological features and in drug sensitivity was observed in three continuous human glioma derived cell lines (LI, DF, and DP) . The results show the importance of continuous cell lines for studying tumor heterogeneity and evaluating the effectiveness of antineoplastic agents.

J Physiol, 1990 Jul, 426, 453 - 71
Substance P mediates synaptic transmission between rat myenteric neurones in cell culture; Willard AL; 1 . Whole-cell patch-clamp recordings were made from pairs of neurones in cell cultures of rat myenteric neurones . In some pairs, action potentials evoked in the first neurone evoked a slow excitatory postsynaptic potential (EPSP) in the second neurone . 2 . Action potentials at a frequency of at least 5 Hz were required to evoked slow EPSPs . In one group of cells, the slow EPSP followed a series of nicotinic fast EPSPs; in another group, fast EPSPs did not precede the slow EPSP . 3 . The slow EPSPs were 2-16 mV in amplitude and were accompanied by decreased resting potassium conductance . 4 . Most (17/28) neurones in which action potentials evoked only slow EPSPs in a follower cell contained substance P (SP)-like immunoreactivity; they were not immunoreactive for 5-hydroxytryptamine (0/15) or vasoactive intestinal peptide (0/22) . 5 . Postsynaptic responses to SP, neurokinin A and a synthetic tachykinin {( pGlu6, Pro9}SP6-11) mimicked the slow EPSPs . The non-tachykinin peptide vasoactive intestinal polypeptide (VIP), which was not found in neurones that evoked only slow EPSPs, also mimicked the slow EPSPs . Responsiveness to SP decreased significantly during slow EPSPs . 6 . Desensitization to either SP or VIP reduced or prevented the slow EPSPs and also responses to each other . Two proposed antagonists of SP receptors, {D-Arg1, D-Pro2,D-Trp7,9,Leu11}substance P and {D-Arg1,D-Trp7,9,Leu11}substance P, did not affect the slow EPSPs significantly . 7 . Antisera against SP reversibly blocked or reduced slow EPSPs evoked by eight of thirteen presynaptic neurones that evoked slow EPSPs without evoking fast EPSPs . All eight of the presynaptic neurones that evoked anti-SP-sensitive slow EPSPs contained SP-like immunoreactivity . None of the presynaptic neurones that evoked anti-SP-insensitive slow EPSPs contained detectable SP-like immunoreactivity . Normal sera and anti-VIP antisera did not alter the slow EPSPs detectably . 8 . It is concluded that subsets of myenteric neurones release an SP-like transmitter to evoke slow EPSPs . These neurones appear to lack a 'classical' neurotransmitter that evokes fast EPSPs.

J Clin Microbiol, 1990 Jul, 28(7), 1597 - 9
Isolation by a sensitive centrifugation cell culture system of 52 strains of spotted fever group rickettsiae from ticks collected in France; Peter O et al.; Boutonneuse fever caused by Rickettsia conorii is transmitted mainly by the brown dog tick, Rhipicephalus sanguineus . We collected 540 ticks in Marseille, France, and tried to isolate as many strains of rickettsia as possible . Ticks were evaluated for the presence of rickettsia by the hemolymph test and by a new culture system, the centrifugation-shell vial technique . We avoided contamination in the culture system . Prior to ticks being submitted to the hemolymph test, they were disinfected . Only 5.6% (27 of 478) of the cultures were contaminated . A drop of hemolymph from each of 478 R . sanguineus ticks was cultured in two shell vials, and another drop was stained by the Gimenez method or indirect immunofluorescence . Since Gimenez staining in our hands was not satisfactory, comparison of the hemolymph test and culture is based on the results of indirect immunofluorescence . Thus, 50 of 369 (13.5%) examined ticks were hemolymph test positive, and 44 (11.9%) cultures were positive . After disinfection, another pool of 62 ticks were examined by the hemolymph test . The ticks were kept individually in a sterile environment . A few days later, the hemolymph of these ticks was collected again and cultured . The contamination rate was not significantly higher (6.4%) than in the above-described conditions . It allowed us to isolate eight more strains . Thus, we recommend screening ticks with the hemolymph test and culturing only the hemolymph test-positive ticks.

EMBO J, 1990 Jul, 9(7), 2079 - 84
Identification and characterization of C-terminal fragments of the beta-amyloid precursor produced in cell culture; Wolf D et al.; The mechanism of amyloid formation in Alzheimer's disease is unknown but appears to involve proteolytic processing of the amyloidogenic peptide from a larger precursor . When the C-terminus containing the amyloid-forming and cytoplasmic domains of the precursor was recombinantly expressed in cultured mammalian cells, a 16 kd protein accumulated which had a tendency to aggregate and form deposits . These deposits had physical characteristics resembling those of preamyloid . Recombinant expression of the full-length precursor was found to produce a similar, cell-associated 16 kd C-terminal fragment as well as a 12 kd fragment, neither of which formed detectable aggregates suggesting efficient catabolism of these fragments . Identification of these two naturally occurring metabolic products of the beta-amyloid precursor provides a system permitting the characterization of the proteolytic processing events of the amyloid precursor protein.

J Biotechnol, 1990 Jul, 15(1-2), 71 - 89
Methods for increasing monoclonal antibody production in suspension and entrapped cell cultures: biochemical and flow cytometric analysis as a function of medium serum content; Heath C et al.; The growth and antibody production of the SP2/0-derived hybridoma HB124 (ATCC) grown in media containing varying amounts of fetal bovine serum (FBS) were monitored using biochemical and flow cytometric methods . Hybridomas grown in 100 ml spinner flasks with RPMI-1640 containing varying amounts of serum demonstrated that cell growth, viability and IgG production show significant changes when serum content is decreased from 10.0 to 5.5 to 1.0 and 0.5% . A longer lag phase resulted when the lower serum content media were used . Cellular rates of glucose uptake showed a significant increase as serum levels were lowered . Similarly, exponential phase IgG production rates increased as the amount of serum was decreased, probably as a result of the decreased rate of exponential growth . Flow cytometric analysis showed a similar increase in cellular IgG content as medium serum levels declined . In contrast, the maximum IgG concentrations were found in flasks containing 1% FBS or above with the lowest concentration in the 0.5% FBS flask being due to the lower numbers of viable cells . Cells grown in microporous hollow fiber reactors were fed with medium containing serum which was decreased stepwise with time . Decreasing medium serum content stepwise from 10 to 2.5% resulted in increased antibody production . However, complete removal of serum from the medium resulted in a significant drop in antibody productivity . Cumulative antibody production was equivalent for cells grown entirely in medium containing 10% FBS and for those which experienced a drop to 2.5% FBS . To compare a defined serum-free medium preparation with medium containing 10% FBS, cells were again grown in batch suspension culture and analyzed . The growth rates were similar but there was a significant difference in IgG production rates . The serum-free culture exhibited both higher cellular production rates and higher IgG concentrations . These results indicate that decreasing medium serum content can adversely affect antibody yield because of lower cell viabilities, not because of lower production rates . Use of a defined serum-free medium, as done in this study, results in higher yields because of a higher IgG production rate as well as good cell growth and viability.

Brain Res, 1990 Jun 25, 521(1-2), 131 - 7
Expression of the neuron-specific glycoprotein GP50 by granule cell cultures; Paladino T et al.; The expression and properties of the neuron-specific glycoprotein, GP50, by neonatal rat granule cells grown in primary tissue culture were studied using the monoclonal antibody MabSM-GP50 . GP50 was expressed by granule cells in culture but not by astrocytes or PC12 cells that had been induced to differentiate with nerve growth factor . Immunocytochemical staining of granule cell cultures demonstrated that immunoreactivity was concentrated in the cell body . Although the amount of GP50 increased slowly throughout the culture period the maximum level of expression in vitro was markedly lower than that observed in cerebellum of the comparable age . Radiolabelling of cell surface proteins by lactoperoxidase-catalyzed iodination demonstrated that GP50 was expressed on the surface of granule cells . Following phase-partitioning of granule cells in the presence of Triton X-114, 75% of GP50 was found to be present in the detergent phase, indicating that it is an integral membrane protein . Sucrose density gradient centrifugation of Triton X-100 extracts of granule cells resolved GP50 into two components with sedimentation coefficients of 3.6S and 7.3S . The 3.6S species (Mr 42,000 Da) accounted for greater than 80% of the total, indicating that GP50 is present predominantly in a monomeric form within the membrane . These properties are similar to those of GP50 expressed in P12 cerebellum but contrast with the behavior of GP50 from mature brain, in which the 7.3S, hydrophilic form is the predominant species . The results suggest that the mature expression of GP50 may be dependent upon the histotypic pattern of development which occurs in vivo.

FEBS Lett, 1990 Jun 18, 266(1-2), 45 - 7
Reduced levels of ADP-ribosylatable elongation factor-2 in aged and SV40-transformed human cell cultures; Riis B et al.; The elongation step is involved in the regulation of protein synthesis during the cell cycle, environmental stress, ageing and transformation . Using a diphtheria toxin-mediated assay for measuring the levels of ADP-ribosylatable elongation factor EF-2, we have observed an irreversible decrease of up to 64% in the amount of ADP-ribosylatable EF-2 in normal diploid human fibroblasts MRC-5 undergoing ageing in vitro . However, a similar decrease in low serum-associated G0/G1-arrested cells is reversible both in MRC-5 cells and in their SV40-transformed counterparts . Reduced levels of ADP-ribosylatable EF-2 could account for the slowing-down of protein synthesis during cell cycle arrest and during cellular ageing in culture.

J Comp Neurol, 1990 Jun 15, 296(3), 437 - 46
Ultrastructure of an identified molluscan neuron in organ culture and cell culture following axotomy; Berdan RC et al.; We examined the ultrastructure of neuron 5 from the buccal ganglion of the mollusc Helisoma trivolvis after axotomy and organ culture, and after isolation of the same neuron in culture . Buccal ganglia containing axotomized neurons 5 were cultured either in host snails or in Leibovitz medium conditioned with ganglia . In addition, some neurons 5 were isolated from buccal ganglia by micro-dissection and plated into culture . Neuron 5 and its processes were identified in both whole mounts and plastic sections of buccal ganglia after intracellular injection with Lucifer Yellow or horseradish peroxidase . Five days after axotomy of neuron 5, thick sections of buccal ganglia stained with toluidine blue revealed that densely staining basophilic bodies (Nissl bodies) within the cytoplasm had dispersed, i.e., they had undergone chromatolysis . Coincident with chromatolysis was an overall increase in diffuse basophilic staining within the cytoplasm of neuron 5 when maintained in organ culture . The dispersion of Nissl bodies viewed by light microscopy correlated with a more freely arranged rough endoplasmic reticulum and associated polysomes within neuron 5 as seen by electron microscopy . Isolated neurons 5 did not possess densely staining Nissl bodies when examined after 2 days in vitro, thus indicating that chromatolysis occurred earlier in isolated neurons . Furthermore, no increase in diffuse cytoplasmic basophilia was observed within isolated neurons 5 cultured in vitro . However, isolated neurons 5 exhibited a marked increase in the number of lipid-like bodies (0.5-1.5 micron in diameter) that were particularly evident in scanning electron micrographs . Scanning and transmission electron micrographs revealed that the isolated neurons were free of associated glia, but non-neuronal cells (hemocytes) would attach themselves to the somata and neurites . Glia surrounding neuron 5 within buccal ganglia exhibited a marked hypertrophy following axotomy and organ culture . Hypertrophy of glia was absent, however, if ganglia were axotomized and left within the animal or axotomized ganglia were implanted into host animals and examined 5 days later by electron microscopy . These observations indicate that, following axotomy, a molluscan neuron may exhibit different morphological features depending on its microenvironment . In addition, the hypertrophy of glia surrounding neurons in Helisoma was not associated with axotomy per se, but with organ culture.

Cancer Res, 1990 Jun 15, 50(12), 3652 - 6
Aromatase inhibition by an enzyme-activated irreversible inhibitor in human carcinoma cell cultures; Brueggemeier RW et al.; 7 alpha-(4'-Amino)phenylthio-1,4-androstadiene-3,17-dione (7 alpha-APTADD), a potent enzyme-activated irreversible inhibitor, was examined in two different human cell culture lines, MCF-7 human mammary carcinoma cells and JAr choriocarcinoma cells . Both the MCF-7 and JAr cell culture systems exhibit aromatase activity, and 7 alpha-APTADD was evaluated for its aromatase-inhibitory activity, for its ability to inactivate the enzyme complex, and for the time course of recovery of enzymatic activity . This inhibitor produced a dose-dependent inhibition of aromatase activity in MCF-7 cells and in JAr cells, with EC50 values of 91 and 7.3 nM, respectively . Two other steroidal inhibitors, 7 alpha-(4'-amino)phenylthio-4-androstene-3,17-dione and 4-hydroxyandrostenedione, produced similar dose-response curves and EC50 values, while the nonsteroidal aminoglutethimide was less effective . Both cell culture systems exhibited prolonged inhibition of aromatase activity following exposure to 7 alpha-APTADD, suggesting enzyme inactivation by this inhibitor . Thus, 7 alpha-APTADD is an effective inhibitor of aromatase in MCF-7 mammary carcinoma cells and in JAr choriocarcinoma cells . These studies encourage further development of this group of medicinal agents for the treatment of estrogen-dependent mammary carcinoma.

Science, 1990 Jun 15, 248(4961), 1349 - 56
A cell culture model for T lymphocyte clonal anergy; Schwartz RH; T lymphocytes respond to foreign antigens both by producing protein effector molecules known as lymphokines and by multiplying . Complete activation requires two signaling events, one through the antigen-specific receptor and one through the receptor for a costimulatory molecule . In the absence of the latter signal, the T cell makes only a partial response and, more importantly, enters an unresponsive state known as clonal anergy in which the T cell is incapable of producing its own growth hormone, interleukin-2, on restimulation . Our current understanding at the molecular level of this modulatory process and its relevance to T cell tolerance are reviewed.

Brain Res, 1990 Jun 4, 518(1-2), 269 - 73
Presynaptic enhancement of synaptic transmission in hippocampal cell cultures by phorbol esters; Finch DM et al.; The calcium-diacyglycerol activated protein kinase (C kinase) plays an important role in synaptic plasticity, and possibly in long-term potentiation (LTP) . We have found that phorbol esters, which activate this protein kinase, increase the frequency of miniature excitatory postsynaptic currents (mEPCs) in hippocampal cell cultures . An analysis of distributions of these miniature synaptic currents shows that amplitudes, and thus postsynaptic function, are not influenced by phorbol esters . Synaptic transmission is therefore enhanced by phorbol esters at a presynaptic locus . The method of analysis employed here is applicable to a broad range of situations involving modification of synaptic transmission in the mammalian central nervous system.

J Steroid Biochem, 1990 Jun, 36(1-2), 83 - 8
Pharmacological characterization of type II glucocorticoid binding sites in AtT20 pituitary cell culture; Gannon MN et al.; Recent evidence indicates that at least two functional glucocorticoid receptors (Type I and Type II) are present in many tissues . It has also become increasingly recognized that, as in other systems, stimulus-response relationships for steroid hormones are often nonlinear . Thus, precise pharmacological parameters are required to establish a functional relationship(s) between binding site and response characteristics . We therefore pharmacologically characterized a glucocorticoid binding site present in AtT20 mouse pituitary cells, a cell line extensively used in studying Type II glucocorticoid receptor function . By several different criteria, glucocorticoids were shown to bind to a single class of binding sites, which, in comparison to available literature, correspond to classical Type II glucocorticoid receptors . No evidence for Type I adrenal steroid binding sites was observed, under the experimental conditions used . Unambiguous Kb values for both glucocorticoid agonists and antagonists were therefore calculated . These parameters should prove of use in elucidating the relationships between glucocorticoid receptor activation and different responses in both AtT20 cells and other glucocorticoid responsive tissues.

J Neurosci, 1990 Jun, 10(6), 1745 - 52
Serotonin regulates type II corticosteroid receptor binding in hippocampal cell cultures; Mitchell JB et al.; Previous work from our laboratory has shown that early postnatal handling of rat pups permanently increases hippocampal type II, but not type I, corticosteroid receptor binding . Handling also increases hippocampal 5-HT turnover, and the effect of handling on type II corticosteroid receptor binding is blocked by concurrent administration of the 5-HT2 receptor antagonist ketanserin . In view of these findings, the present studies examined the effects of 5-HT on type I ({3H}corticosterone) and type II ({3H}RU 28362) corticosteroid receptor binding in dispersed hippocampal cell cultures derived from animals killed at E19-20 in order to verify that 5-HT can act directly on hippocampal cells to alter corticosteroid receptor binding . Both type I and type II receptors were measurable in cultured hippocampal cells and the apparent affinity (Kd) for {3H}corticosterone (0.4 +/- 0.1 nM) and {3H}RU 28362 (0.8 +/- 0.1 nM) was similar to that from studies with intact animals . 5-HT increased type II, but not type I, corticosteroid receptor binding capacity in a dose-related manner, with the maximal effect (+188%) observed at 10 nM 5-HT and no change in the affinity of the receptor for {3H}RU 28362 . The effect of 10 nM 5-HT on {3H}RU 28362 binding required a minimum of 4 d exposure and persisted for at least 7 d following the removal of 5-HT . The effect of 10 nM 5-HT on {3H}RU 28362 binding was completely blocked by the 5-HT2 receptor antagonists ketanserin and mianserin . There were no effects of the 5-HT1a antagonist, BMY 7378, or the 5-HT3 antagonist, MDL 72222.(ABSTRACT TRUNCATED AT 250 WORDS)

J Clin Microbiol, 1990 Jun, 28(6), 1447 - 8
Comparison of IDEIA III and cell culture for the detection of Chlamydia trachomatis in endocervical specimens; Lebar WD et al.; A total of 803 endocervical samples were obtained from females with clinical or epidemiological histories suggesting chlamydia infection . These specimens were tested by IDEIA III and cell culture for the presence of Chlamydia trachomatis . After resolution of discrepant results by direct fluorescent-antibody staining of pelleted cell culture transport materials, IDEIA III demonstrated sensitivity, specificity, and positive and negative predictive values of 93.8, 99, 92.9, and 99.1%, respectively.

J Clin Microbiol, 1990 Jun, 28(6), 1159 - 62
Rapid detection of respiratory syncytial virus and influenza A virus in cell cultures by immunoperoxidase staining with monoclonal antibodies; Waris M et al.; Peroxidase-labeled monoclonal antibodies against respiratory syncytial virus (RSV) and influenza A virus were used for immunoperoxidase staining (IPS) of cell cultures inoculated with nasopharyngeal aspirates . Cells were grown in 24-well plates, and specimens were inoculated by low-speed centrifugation . Cultures were incubated for 2 days at 37 degrees C and then fixed, stained, and observed by light microscopy . IPS was compared with standard virus isolation by using cultures of human diploid fibroblasts and Vero, HEp-2, and HeLa cell lines for RSV and Madin-Darby canine kidney cells for influenza A virus; these cultures were inoculated with specimens that were previously stored at -70 degrees C . Of 40 known RSV-positive specimens, 30 were found to be positive on reinoculation by both methods, and an additional 5 specimens were found to be positive by IPS only . Of 190 specimens tested for influenza A virus, 14 were positive by IPS and in tubes, and a further 8 specimens were positive by IPS only . IPS was also compared with direct detection of viral antigens in nasopharyngeal aspirates by a time-resolved fluoroimmunoassay (TR-FIA) . Fresh nasopharyngeal aspirates were inoculated into human diploid fibroblasts and Madin-Darby canine kidney cells and tested for RSV and influenza A virus, respectively, by IPS . Of 110 specimens tested for RSV, 37 were positive in total, 32 were positive by IPS, and 33 were positive by TR-FIA . Of 150 specimens tested for influenza A virus, 39 were positive in total, 35 were positive by IPS, and 34 were positive by TR-FIA . IPS of cultures inoculated by centrifugation and incubated for 2 days is a sensitive method for the diagnosis of respiratory virus infections, and 24-well plates allow for the easy processing of a large number of specimens.

J Histochem Cytochem, 1990 Jun, 38(6), 829 - 36
Insulin and insulin mRNA are detected in neuronal cell cultures maintained in an insulin-free/serum-free medium; Schechter R et al.; We investigated the effect of a serum-containing medium, exogenous insulin, and insulin-free/serum-free media in the regulation of the rabbit brain insulin-like peptide (ILP) in neuronal cell cultures . The presence of serum or insulin in the medium resulted in approximately 3-5% of neurons that were positive for the peptide by immunohistochemistry and for insulin mRNA by in situ hybridization . The absence of insulin in the medium resulted in a three- to fourfold increase (p less than 0.001) in the insulin-immunoreactive and insulin mRNA-containing neurons . Additionally, in the presence of exogenous insulin or serum, the amount of insulin present in the medium, as measured by ELISA, decreased with time (approximately 80%), the former slower than the latter when compared with their respective zero time point values . However, an increase (approximately 80% from zero time) was noted in the absence of insulin or serum, and this lasted for 24-48 hours alone . The presence of an increased insulin/ILP content in the medium and the increase in the numbers of insulin-immunoreactive neurons suggests an important role of the peptide in the brain . The observation of increased synthesis and secretion of neuronal ILP in the absence of insulin is indicative of an autocrine effect of exogenous insulin on neuronal production of ILP, which in turn may be important in the growth and maintenance of neuronal and possibly glial cells.

In Vitro Cell Dev Biol, 1990 Jun, 26(6), 647 - 56
Increase in cyclic AMP levels by relaxin in newborn rhesus monkey uterus cell culture; Kramer SM et al.; A novel relaxin sensitive cell line of apparent smooth muscle origin has been established from a newborn rhesus monkey uterus (NRMU) . NRMU cells respond to relaxin, in the presence of 1 microM forskolin, by producing intracellular adenosine 3', 5'-cyclic monophosphate (cAMP) . The increase in cAMP levels is dose, time and cell density dependent, reaching peak levels at 10 min when cells are seeded at 1 X 10(5) cells/well . Specificity was demonstrated by neutralization of the relaxin activity with anti-relaxin monoclonal and polyclonal antibodies, degradation of cAMP in the presence of phosphodiesterase, and confirmation of the absence of cGMP . Three synthetic analogs of human relaxin generated a dose-related cAMP response as did synthetic native human relaxin . Natural relaxin purified from human corpora lutea tissue also generated a response similar to synthetic human relaxin . Porcine and rat relaxins also increased levels of cAMP . Insulin, but not IGF I or IGF II, was capable of increasing cAMP levels in NRMU cells, however, 200 ng/mL were required to achieve cAMP levels comparable to 6.25 ng/ml relaxin . Combinations of relaxin with insulin, IGF I or IGF II did not increase cAMP levels above levels obtained with relaxin alone . The effect on NRMU cells of other hormones, growth factors and drugs potentially present in cell culture systems or serum samples was evaluated . In combination with relaxin, oxytocin significantly decreased the cAMP production below the levels induced by relaxin alone, whereas progesterone and prostaglandin E2 resulted in additive increases in cAMP . These data suggest that the NRMU cell line is an appropriate target tissue for studying relaxin-mediated biological responses in vitro as well as functioning as the primary component of a relaxin in vitro bioassay.

Endocrinology, 1990 Jun, 126(6), 2919 - 26
Interferon-gamma inhibits stimulated adrenocorticotropin, prolactin, and growth hormone secretion in normal rat anterior pituitary cell cultures; Vankelecom H et al.; Preincubation of rat anterior pituitary (AP) cells with homologous interferon-gamma (IFN-gamma) caused a dose-dependent inhibition of ACTH secretion stimulated by CRF . The effect was seen in both monolayer and aggregate AP cell cultures and was not due to cytotoxicity . In monolayer cultures IFN-gamma also inhibited PRL and GH release stimulated by various hypothalamic releasing factors . IFN-gamma did not affect the time kinetics of the ACTH response to CRF . The dose needed for half-maximal inhibition amounted to approximately 1 (antiviral) U/ml . The effect of IFN-gamma was abrogated by an IFN-gamma-neutralizing monoclonal antibody . Furthermore, ACTH secretion by the AP cells was not affected by the anti-IFN-gamma antibody added alone, indicating that in the culture system no endogenous IFN-gamma is operational in regulating the ACTH response studied . Of the other cytokines tested {interleukin-1 (IL-1), tumor necrosis factor-alpha (TNF-alpha), interleukin-6 (IL-6), and interferon-alpha/beta (IFN-alpha/beta)} only TNF-alpha and IL-6 were found to inhibit CRF-stimulated ACTH release, although this inhibition was less pronounced than that caused by IFN-gamma . Lipopolysaccharide, even at high doses, did not significantly inhibit the ACTH response to CRF . These results identify IFN-gamma as one of the inflammatory cytokines that, like IL-1, TNF-alpha, and IL-6, have the potential to regulate pituitary function.

J Clin Microbiol, 1990 Jun, 28(6), 1098 - 100
Amino acid requirements for growth of Chlamydia pneumoniae in cell cultures: growth enhancement by lysine or methionine depletion; Kuo CC et al.; Amino acid requirements for growth of two isolates of Chlamydia pneumoniae were studied and compared with those for one strain of Chlamydia trachomatis in a HeLa 229 cell culture . It was shown that among 13 amino acids in Eagle minimum essential medium, C . pneumoniae required all amino acids except lysine . A true requirement for arginine, isoleucine, leucine, threonine, and valine could not be determined because depletion of 100% of these amino acids caused cell detachment . Consequently, a requirement for these amino acids was based on 90% depletion . C . trachomatis biovar trachoma required all amino acids except threonine, which was indeterminable . Depletion of 100 and 90% of the lysine and 90 to 70% of the methionine was shown to enhance the growth of C . pneumoniae . This phenomenon was shown to be a property of C . pneumoniae because the effect of lysine and methionine reduction was also demonstrated in another human line, HL cells, and a mouse line, McCoy cells.

J Pharm Sci, 1990 Jun, 79(6), 476 - 82
Epithelial transport of drugs in cell culture . I: A model for studying the passive diffusion of drugs over intestinal absorptive (Caco-2) cells; Artursson P; A human intestinal cell line, Caco-2, was used as a model to study the passive diffusion of drugs across intestinal epithelium . The cells formed continuous monolayers when grown on permeable filters of polycarbonate . After 10 days in culture, the monolayers had a transmembrane resistance of approximately 260 ohms.cm2 and a cell density of 0.9 x 10(6) cells/cm2 . At this time the cells were impermeable to {14C}polyethyleneglycol (MW 4000) . These characteristics remained constant for 20 days (i.e., from day 10 to day 30) . Six beta-blocking agents with a 2000-fold range of lipophilicity were studied for their transepithelial transport properties . The transport parameters were independent of drug concentration and transport direction . The apparent permeability coefficients ranged from 41.91 +/- 4.31 x 10(-6) cm/s for the most lipophilic drug, propranolol, to 0.203 +/- 0.004 x 10(-6) cm/s for the most hydrophilic drug, atenolol . The transport parameters were compared with those published for rat ileum . The transport rates were similar for four out of five drugs . Atenolol was transported at a slower rate in the Caco-2 model, which may be explained by the fact that the Caco-2 cells form a tighter epithelium than the rat ileal enterocytes . The findings of this paper indicate that Caco-2 cells may be used to model the intestinal absorption of drugs.

Malays J Pathol, 1990 Jun, 12(1), 35 - 8
Comparison of direct immunoperoxidase and direct immunofluorescence for the detection of herpes simplex virus antigen in cell culture; Sabil D et al.; 150 specimens from suspected herpes simplex genital and skin lesions were received in virus transport medium . They were inoculated into Hep-2 cell monolayers, examined for the presence of virus by cytopathic effect (CPE), direct immunoperoxidase (DIP) and direct immunofluorescence (DIF) . Of 39 (26.0%) virus-positive specimens by CPE, 37 (24.7%) were HSV-positive by DIP and 36 (24.0%) by DIF staining . DIP staining had a sensitivity of 100%, specificity of 99.1%, positive predictive value of 97.3% and negative predictive value of 100% in relation to DIF as a standard test . Of 39 specimens positive by CPE, only 25.6% were HSV-positive within 24 h post-inoculation compared to 94% HSV-positive by DIP and DIF staining at the same time.

Biull Eksp Biol Med, 1990 Jun, 109(6), 612 - 3
{Method of total staining of monolayer cell cultures using vanadium hematoxylin}; Viktorov IV; Original method of cytological staining for monolayer cell cultures and outgrowth zone of tissue explants with vanadium hematoxylin is described . This staining method is simple, universal, reproducible and give opportunity to stain rapidly practically all cellular elements of cultivated monolayer with high degree of cytological resolution of intracellular structures (nucleus, cytoplasmic organelles etc.).

In Vitro Cell Dev Biol, 1990 Jun, 26(6), 585 - 8
Mycoplasma detection in cell culture by concomitant use of bisbenzamide and fluoresceinated antibody; Freiberg EF et al.; Conditions are presented for application of both bisbenzamide (Hoechst 33258) stain and a specific fluoresceinated anti-Mycoplasma hyorhinis IgG to a single cell culture preparation . This allows the same field on a slide to be viewed for presumptive diagnosis of any cell culture contaminant mycoplasma by bisbenzamide staining and for definitive diagnosis of M . hyorhinis strains using fluoresceinated antibody . The use of this method plus a cultural procedure will permit identification of the "noncultivable" M . hyorhinis strain DBS 1050.

Exp Cell Res, 1990 Jun, 188(2), 302 - 11
Determination of apical membrane polarity in mammary epithelial cell cultures: the role of cell-cell, cell-substratum, and membrane-cytoskeleton interactions; Parry G et al.; The membrane glycoprotein, PAS-O, is a major differentiation antigen on mammary epithelial cells and is located exclusively in the apical domain of the plasma membrane . We have used 734B cultured human mammary carcinoma cells as a model system to study the role of tight junctions, cell-substratum contacts, and submembraneous cytoskeletal elements in restricting PAS-O to the apical membrane . Immunofluorescence and immunoelectronmicroscopy experiments demonstrated that while tight junctions demarcate PAS-O distribution in confluent cultures, apical polarity could be established at low culture densities when cells could not form tight junctions with neighboring cells . In such cultures the boundary between apical and basal domains was observed at the point of cell contact with the substratum . Immunocytochemical analysis of these cell-substratum contacts revealed the absence of a characteristic basement membrane containing laminin, collagen (IV), and heparan sulfate proteoglycan . However, serum-derived vitronectin was associated with the basal cell surface and the cells were shown to express the vitronectin receptor on their basolateral membranes . Additionally, treatment of cultures with antibodies against the vitronectin receptor caused cell detachment . We suggest, then, that interactions between vitronectin and its receptor, are responsible for establishment of membrane domains in the absence of tight junctions . The role of cytoskeletal elements in restricting PAS-O distribution was examined by treating cultures with cytochalasin D, colchicine, or acrylamide . Cytochalasin D led to a redistribution of PAS-O while colchicine and acrylamide did not . We hypothesize that PAS-O is restricted to the apical membrane by interactions with a microfilament network and that the cytoskeletal organization is dependent upon cell-cell and cell-substratum interactions.

Appl Microbiol Biotechnol, 1990 Jun, 33(3), 287 - 90
Anchorage-dependent mammalian cell culture using polyurethane foam as a new substratum for cell attachment; Matsushita T et al.; Anchorage-dependent mammalian cells were cultivated at high cell density in a novel culture system using polyurethane foam (PUF) as a substratum for cell attachment . PUF has a macroporous structure giving a high surface area to volume ratio . Monkey kidney cells (Vero) and Chinese hamster ovary cells (CHO-K1) attached to the internal surface of PUF and grew to a high cell density (1.04 X 10(8) cells/cm3 PUF and 3.5 X 10(7) cells/cm3 PUF, respectively) in PUF stationary cultures . In addition, we have designed a PUF-particle packed-bed culture system for high density mass cell culture . A maximum cell density of 2.4 X 10(7) cells/cm3 culture vessel volume was obtained in a packed-bed culture of Vero cells.

J Chromatogr, 1990 May 25, 508(1), 75 - 9
Improved sample preparation method for high-performance liquid chromatography of deoxyribonucleoside triphosphates from cell culture extracts; Harmenberg J et al.; The accurate determination of deoxyribonucleoside triphosphates in cells is difficult owing to the high concentrations of interfering ribonucleoside triphosphates . The latter can be degraded to their respective bases by periodate oxidation of cell extracts . However, the large amount of bases so produced can interfere with subsequent high-performance liquid chromatographic (HPLC) analysis . The use of a weak ion-exchange cartridge to partially purify and concentrate deoxyribonucleoside triphosphates in periodate-treated cell extracts, prior to HPLC, thus allowing accurate determination is described . The recovery of the deoxyribonucleoside triphosphates is greater than 95%, and greater than 90% of the interfering bases are removed.

Cancer Lett, 1990 May 15, 51(1), 11 - 6
Chemotherapy screening assay using 3-dimensional cell culture; Kupchik HZ et al.; A new alginate culture method (ACM) was used to test the effects of vincristine and 5-fluorouracil (5-FU) on the commonly used human colon carcinoma cell line HT-29 . Colorimetric analysis of viability following treatment with physiologically tolerated levels of vincristine showed significant reduction of the surviving cell population . Hematoxylin and eosin staining of sections of treated organoid growths corroborated the colorimetric analyses . The ACM is inexpensive, rapid and extremely versatile . Many alginate beads containing "mini-tissue" growths of many cell lines can be used simultaneously to evaluate numerous chemotherapeutic agents in an "in vivo-like" environment.

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