J Virol Methods, 1988 Oct, 22(1), 99 - 108 Enzyme immunoassay of mumps virus in cell culture with peroxidase-labelled virus specific monoclonal antibodies and its application for determination of antibodies; van Tiel FH et al.; Mumps neutralizing monoclonal antibodies (MAs) were purified and labelled with horseradish peroxidase and used to detect virus-infected Vero cells, which were seeded as monolayers in wells of 96-well plates . This direct enzyme immunoassay (EIA) in cell culture proved to be a sensitive method for detection and titration of mumps virus and it may be useful for diagnostic purposes . The EIA is also suitable for the rapid determination of neutralizing antibodies . Neutralization of mumps virus by preincubation with either monoclonal or polyclonal antibodies was indicated by inhibition of the absorbance at 450 nm as measured with a multichannelled photometer . The EIA (duration 2 days) for determination of mumps neutralizing antibodies is an attractive alternative for the plaque reduction test (duration 6 days). Proc Natl Sci Counc Repub China B, 1988 Oct, 12(4), 215 - 21 Effect of epidermal growth factor on the sheet formation of the human keratinocyte in cell culture; Lin HC et al.; Epidermal growth factor is an important element in maintaining keratinocyte proliferation and maturation . To evaluate its effect on keratinocyte growth in vitro, human foreskins were cultured . The epidermal keratinocyte growth in culture was separated into two stages by a conditional medium: the proliferation stage, in which the cells were maintained in a monolayer; and the differentiation stage, in which the cells grew to stratification and keratinization . The keratinocytes were cultured in various concentrations of epidermal growth factor, and their morphology and growth behavior were closely observed . Our results demonstrated that cultured keratinocytes grew in a confluent layer under the influence of epidermal growth factor . In contrast, in a culture without epidermal growth factor, the proliferation rate of cultured keratinocytes slowed down and eventually the cells stopped growing . During serum stimulation, with or without additional exogenous epidermal growth factor, the cultured keratinocytes grew continuously to the normal terminal differentiation . Under this two-stage culture model, the cultured keratinocytes could grow into an intact sheet of graftable epidermis. Biochem Biophys Res Commun, 1988 Sep 30, 155(3), 1154 - 60 Elevation of PMN cytosolic free calcium and locomotion stimulated by novel peptides from IL-1-treated human synovial cell cultures; Watson ML et al.; Treatment of human synovial cell cultures with human recombinant interleukin 1 (IL-1) results in the appearance of activity in the supernatant which stimulates human polymorphonuclear neutrophils (PMNs), as assessed by increased chemokinesis and elevation of intracellular free calcium concentration . IL-1 (or related cytokines) were unable to stimulate these responses . This activity chromatographed as a single peak on C8 reversed-phase HPLC and subsequent size exclusion HPLC revealed two peaks of chemokinetic activity with apparent molecular masses of approximately equal to 13kDa and 6kDa . Fractions containing the higher molecular mass material also elevated PMN cytosolic free calcium . The local production of such factors may mediate IL-1-induced PMN accumulation in vivo. Endocrinology, 1988 Sep, 123(3), 1442 - 8 Tumor necrosis factor-alpha inhibits collagen synthesis and alkaline phosphatase activity independently of its effect on deoxyribonucleic acid synthesis in osteoblast-enriched bone cell cultures; Centrella M et al.; Tumor necrosis factor-alpha (TNF alpha), a product of activated monocytes, induces tissue wasting in certain solid tumors in vivo and in in vitro model systems . Recent studies indicate that TNF alpha also regulates cell replication and expression of differentiated function in a variety of nonneoplastic cell systems . Since monocyte products could accumulate in bone with trauma, inflammation, or other disease states, bone cell activity might be altered by the presence of these pathophysiological molecules . Using cells obtained by sequential enzyme release from fetal rat parietal bone, we find that TNF alpha has acute stimulatory and inhibitory effects on bone cell macromolecular synthesis . Within 24 h of exposure, recombinant human TNF alpha at 0.3-100 nM progressively increases the rate of DNA synthesis in osteoblast-enriched cell cultures up to 3- to 4-fold, and 3-100 nM TNF alpha reduces collagen production and alkaline phosphatase activity by 20-30% . These decreases are not altered by 1 mM hydroxyurea, which blocks the mitogenic effect of TNF alpha by 85-90% . In addition, hydroxyproline levels in the culture medium do not increase relative to the control value after TNF alpha treatment, suggesting that decreased collagen production results from less synthesis rather than increased collagen degradation . Hybridization studies with cDNA encoding the alpha 1-chain of rat type I collagen show that TNF alpha increases type I collagen mRNA to an extent similar to its effect on cell replication . Therefore, TNF alpha appears to inhibit collagen synthesis and alkaline phosphatase activity in osteoblast-enriched cell cultures by mechanisms that are not related to its effects on cell replication. Neurobiol Aging, 1988 Sep-Dec, 9(5-6), 763 - 5 New approaches to the study of central nervous system function . Immune-nervous system interactions and cell culture; Morgan DG et al.; The paper by Lal and Forster is discussed with reference to future experiments which might provide insight into mechanisms regarding their exciting data that circulating brain reactive antibodies may cause learning deficits . The paper by Azmitia et al . on cell culture techniques is discussed with respect to the types of studies in which culture systems have proven most valuable in the past, and should continue to do so in the future. Exp Eye Res, 1988 Sep, 47(3), 457 - 63 Human retinal pigment epithelial cell cultures: phenotypic modulation by vitreous and macrophages; Kirchhof B et al.; In proliferative vitreoretinopathy (PVR), retinal pigment epithelial (RPE) cells migrate into the vitreous, where they may acquire a fibroblast-like morphology . Such cells may eventually form contractile periretinal membranes, resulting in traction retinal detachment . Among the environmental influences that could cause this change in RPE phenotype, exposure to vitreous and to macrophages is most obvious, as macrophages are invariably found in epiretinal membranes and precede membrane formation in experimental traction retinal detachment . We initiated studies to define modulation of cultured RPE cell morphology by exposure to vitreous or to macrophage-conditioned media . Vitreous, serum, and albumin alone had no effect on the epithelial appearance of RPE cells in vitro . However, macrophage-conditioned media and vitreous-serum or vitreous-albumin mixtures induced a reversible fibroblast-like appearance in these cells . These findings show that macrophages produce a morphoplastic substance for RPE cells, and suggest that vitreous also contains a factor(s) that affects RPE cell shape, and that requires mediation by serum components. J Cell Physiol, 1988 Sep, 136(3), 547 - 53 Transforming growth factor beta responsiveness is modulated by the extracellular collagen matrix during hepatic ito cell culture; Davis BH; Hepatic sinusoidal Ito cells have the capacity to produce interstitial collagen types I and III as well as other matrix proteins and may be involved in hepatic fibrogenesis . Transforming growth factor beta (TGF beta) responsiveness was evaluated during in vitro cell culture, since increasing evidence suggests that this ubiquitous polypeptide can stimulate the production of collagenous proteins in a variety of cell types . TGF beta induced marked inhibition of Ito cell proliferation for cells grown on either a type I or a type IV collagen matrix . In marked contrast, the collagen synthetic response was considerably different for cells grown on a type I versus a type IV collagen matrix . When cells were grown on a type I collagen matrix, TGF beta caused a significant increase in the accumulation of collagen type I and III . When Ito cells were grown on a type IV collagen matrix, there was no stimulation of collagen production . TGF beta responsiveness was also evaluated in the setting of altered vitamin A concentrations . Freshly isolated Ito cells are engorged with vitamin A, the usual physiologic storage site for hepatic vitamin A . During in vitro culture and during in vivo fibrogenesis, Ito cells lose their vitamin A stores coincident with a transformation to a collagen-producing myofibroblast-like cell . When cultured Ito cells were grown on a type I collagen matrix and re-exposed to an increased concentration of vitamin A, the production of interstitial collagen was reduced . However, when the vitamin A-enriched Ito cells were exposed to TGF beta, the production of interstitial collagen was increased, similar to cells that had not received vitamin A. J Cell Physiol, 1988 Sep, 136(3), 398 - 410 Differential expression of cell surface glycoproteins on various organ-derived microvascular endothelia and endothelial cell cultures; Belloni PN et al.; Glycoproteins expressed on the luminal surfaces of microvascular endothelium derived from various murine organs were analyzed and compared with those expressed by cultured vascular endothelial cells . Cell-surface vascular proteins were radiolabeled in situ via intracardiac perfusion with lactoperoxidase/Na125I . Autoradiography confirmed that the radiolabel was restricted to the vessel lumen in most tissues . Controls contained 125I-labeled serum proteins to identify adsorbed serum components . Glycoproteins were analyzed by western enzyme-linked lectin analysis using detergent extracts of 125I-labeled microvessels isolated from different organs . The western transfers were probed with a panel of lectin-peroxidase conjugates to determine differences in protein glycosylation . The same transfers were also screened for exposed 125I-labeled cell-surface proteins by autoradiography . This dual analysis detected glycoprotein patterns unique for each organ . At least seven major proteins (Mr approximately 180 K, 130 K, 95 K, 80 K, 75 K, 60 K, 12 K) were common to microvessels derived from each organ; however, certain glycoproteins appeared to be expressed differentially in particular organs . For example, a Mr approximately 135 K WGA-binding glycoprotein was detected in brain microvessels, whereas another WGA-binding glycoprotein of Mr approximately 40 K was detected only in kidney . In lung microvessels, a Mr approximately 140 K WGA binding glycoprotein and a Mr approximately 55 K RCA-I-binding galactoprotein were expressed preferentially, and liver microvessels displayed Mr approximately 220 K protein and a Mr approximately 35 K PNA-binding galactoprotein . The cell-surface-iodinated protein profiles from in situ labeled microvessels were similar to profiles derived from cultured bovine aortic endothelial cells and several short-term endothelial cell cultures isolated from different organs . The results from this study suggest that organ-associated endothelia express glycoprotein fingerprints unique to each organ. APMIS, 1988 Sep, 96(9), 768 - 72 The significance of 3H-thymidine degradation in cell culture experiments with special reference to rheumatoid arthritis; Nykanen PJ et al.; The degradation of 3H-thymidine under various cell culture conditions was analysed . It was found that a half of 3H-thymidine was degraded to 3H-thymine during 24 hours in PHA stimulation of blood lymphocytes . A control culture in which PHA was not added also caused 3H-thymidine degradation . 3H-thymidine degradation was prevented by adding 5-nitrouracil to the incubation medium at a concentration of 0.577 mg/ml . At the same time 5-NU increased 3H-thymidine incorporation into lymphocytes by 47% . 5-NU also eliminated the inhibitory effect of rheumatoid arthritis synovial tissue eluate on PHA stimulation . In addition 5-NU and nonradioactive thymine increased the 3H-thymidine labelling index of fresh rheumatoid arthritis synovial membrane biopsies, and also more of the isotope was accumulated in the individual cells of the membrane . These studies demonstrate that 3H-thymidine degradation is an important phenomenon in cell cultures and that it can be prevented effectively by using 5-nitrouracil with 3H-thymidine. J Cell Biochem, 1988 Sep, 38(1), 13 - 21 (2'-5')An-dependent endoribonuclease: enzyme levels are regulated by IFN beta, IFN gamma, and cell culture conditions; Floyd-Smith G; The levels of a (2'-5')An-dependent endonuclease (RNase L) were determined in extracts prepared from murine L cells and Ehrlich ascites tumor (EAT) cells by measuring specific binding of protein to a labeled derivative of (2'-5')An, (2'-5')A3{32P}pCp . RNase L levels were found to depend both on interferon (IFN) treatment and on cell growth conditions . Treatment of murine L cells and EAT cells with 100-2,000 IRU IFN beta or IFN gamma resulted in a similar 2-4-fold increase in the levels of RNase L when cells were present at low density . The levels of RNase L were also shown to increase 2-3-fold as cells approached saturation density . Serum-starved cells also displayed relatively high levels of RNase L . RNase L levels in cells maintained at high cell density did not change appreciably following treatment with IFN beta or IFN gamma . Regulation of RNase L levels by cell growth conditions as well as by IFN beta or IFN gamma treatment suggests that RNase L may play an important role in regulating the levels of cellular mRNAs as well as acting to degrade viral RNAs. J Exp Med, 1988 Sep 1, 168(3), 853 - 62 Interleukin 4 causes isotype switching to IgE in T cell-stimulated clonal B cell cultures; Lebman DA et al.; Although it has been established that IL-4 enhances both IgG1 and IgE secretion in LPS-stimulated B cell cultures, these studies failed to determine whether IL-4 preferentially induces isotype switching or preferentially allows for the maturation of precommitted precursor cells . To distinguish between these possibilities, it is necessary to ascertain the effect of IL-4 on the isotypes secreted by individual precursor cells during clonal expansion . Therefore, clonal cultures of B cells stimulated with a Th2 helper cell line specific for rabbit Ig and rabbit anti-mouse IgM were established . The majority of B cells are capable of undergoing clonal expansion under these conditions . To vary the level of IL-4 present, either IL-4 or anti-IL-4 was added to cultures . In the presence of IL-4 there was an increase in the proportion of clones that secreted IgE and a decrease in the proportion of clones that secreted IgM . The addition of IL-4 to cultures also increased the amount of IgE secreted by individual clones . Thus, these experiments definitively prove that IL-4 causes specific heavy chain class switching to IgE in Th2-stimulated B cell cultures . In contrast, IL-4 does not affect the proportion of clones secreting IgG1, suggesting that other consequences of Th cell-B cell interactions play a role in the generation of an IgG1 response. J Gen Virol, 1988 Sep, 69 ( Pt 9), 2155 - 64 Multiplication of influenza A viruses with cleavable and non-cleavable haemagglutinin in chicken embryo membranes or organs, and cell cultures derived therefrom; Scholtissek C et al.; Pathogenic properties of influenza A viruses introduced into embryonated chicken eggs via the allantoic cavity, the amniotic cavity or the yolk sac were studied using viruses with cleavable or non-cleavable haemagglutinin (HA), or reassortants derived from the highly pathogenic fowl plague virus (FPV) which has a cleavable HA . The various organs, membranes and fluids were analysed for virus yields, and by immunohistochemistry for production of viral nucleoprotein . Virus replication in primary tissue cultures derived from various embryonic organs was also studied . Some of the reassortants, which were previously found to be non-pathogenic for chickens and were temperature-sensitive (ts) at 40 degrees C, multiplied at 37 degrees C to the same extent as the highly pathogenic FPV . The spread of other non-pathogenic reassortants in the embryo was restricted . For example, 81/Ho was able to multiply to a reasonable extent only in the chorioallantoic and the allantoamniotic membranes . After inoculation of the A/PR/8/34 strain containing a non-cleavable HA into the amniotic cavity, virus multipled only in the inner epithelial layer of the amnion, in superficial epidermal cells and in superficial epithelia of the oropharyngeal cavities, the nasal and paranasal sinuses and the oesophagus . Kidneys were free of virus antigen, although the virus multiplied to high titres in primary tissue cultures derived from embryonic kidneys . Thus influenza A viruses can be non-pathogenic for chickens because they are ts and unable to multiply at the body temperature of chickens (41 degrees C), or because their spread in the animal is impaired per se. Br J Pharmacol, 1988 Sep, 95(1), 109 - 20 Effects of non-sedative anxiolytic drugs on responses to GABA and on diazepam-induced enhancement of these responses on mouse neurones in cell culture; De Deyn PP et al.; 1 . Intracellular microelectrode recording techniques were performed on mouse spinal cord and cerebral hemisphere neurones grown in primary dissociated cell culture . The effects of several anxiolytics applied by local pressure ejection on responses to gamma-aminobutyric acid (GABA) evoked by iontophoresis were investigated . Responses to GABA were depolarizing since intracellular chloride ion concentration was increased by injection from potassium chloride (3M)-filled recording micropipettes and neurones were held at large negative membrane potentials (-70 to -90 mV) . The agents studied were six 'non-sedative anxiolytics', CL 218,872 (3-methyl-6-(3-trifluoromethyl-phenyl)1,2,4-triazolo(4,3-b) pyridazine), PK 8165 (2-phenyl-4-(2-(4-piperidinyl)ethyl)-quinoline), PK 9084 (2-phenyl-4-(2-(3-piperidinyl)ethyl)-quinoline), CGS 9896 (2-(4-chlorophenyl)-2,5-dihydropyrazolo(4,3-c)quinoline-3(3H)-one) , ZK 91296 (ethyl 5-benzyloxy-4-methoxymethyl-beta-carboline-3-beta-carboxylate), buspirone (8-4-{4-(2-pyrimidinyl)-1-piperazinyl}butyl-8-azaspiro{4.5}decane- 7,9- dione), and two sedative anxiolytics, diazepam and zopiclone {( 6-(5-chloro-2-pyridyl)-6,7-dihydro-7-oxo-5H-pyrrolo{3,4-b}pyrazin- 5- yl}4-methyl-1-piperazinecarboxylate) . 2 . Direct effects on responses to GABA were studied for all drugs applied in varying concentrations . For the drugs which significantly altered responses to GABA, the effects of the benzodiazepine receptor antagonists Ro 15-1788 (ethyl-8-fluoro-5,6-dihydro-5-methyl-6-oxo-4H-imidazo(1,5a)-(1,4)benzodi azepine - 3-carboxylate) and CGS 8216 (2-phenylpyrazolo(4,3-c)-quinolin-3(5H)-one) were evaluated . For the drugs devoid of significant direct effect on responses to GABA, the influence on diazepam-induced enhancement of responses to GABA was evaluated . 3 . Diazepam, zopiclone and CL 218,872 concentration-dependently and reversibly enhanced responses to GABA . Maximal enhancement was 82% for diazepam (500 nM), 64% for zopiclone (10 microM) and 20% for CL 218,872 (10 microM) . PK 8165 effects varied with concentration, enhancing responses to GABA (up to 18%) at nM concentrations and reducing responses to GABA (up to 90%) at microM concentrations . CGS 9896, ZK 9126, PK 9084 and buspirone, in concentrations ranging from 1 nM to 10 microM, lacked significant direct effects on responses to GABA.(ABSTRACT TRUNCATED AT 400 WORDS) Vet Microbiol, 1988 Sep, 18(1), 15 - 25 Comparison of DNA-spot hybridization, cell culture and direct immunofluorescence staining for the diagnosis of avian chlamydiae; Timms P et al.; DNA-spot hybridization, cell culture and direct immunofluorescence staining were compared for the detection of avian Chlamydia psittaci strains in cell culture dilutions and in routine samples submitted for diagnosis . With dilutions of infected cell culture material, growth in BGM cells was by far the most sensitive technique, detecting 0.01 infected cells (20 elementary bodies) ml-1 . DNA-spot hybridization and direct immunofluorescence staining were of approximately equal sensitivity, both detecting 16 infected cells (3.2 x 10(4) elementary bodies) per ml-1 . When 27 avian liver and spleen samples were assayed, all 3 tests performed similarly (13 positive and 12 negative by all 3 tests) . This suggests that in most avian samples presented for diagnosis, sufficient numbers of chlamydiae are present to allow any of the test to the be used . Thus, the direct immunofluorescence staining method is currently the test of choice for routine diagnosis since it is available in kit form, is relatively simple and quick to perform, and like DNA-spot hybridization, detects non-viable as well as viable organisms . However, if low levels of chlamydiae are to be effectively detected, such as in carrier birds or birds with recently acquired infections, then cell culture should be used. J Gen Virol, 1988 Sep, 69 ( Pt 9), 2199 - 207 Phenotypes of St Louis encephalitis virus mutants produced in persistently infected mosquito cell cultures; Randolph VB et al.; Viral mutants that appeared during long-term persistent infections of mosquito cell cultures (Aedes albopictus, A . dorsalis and Culex tarsalis) with St Louis encephalitis virus were characterized . Evidence was obtained for the presence of temperature-sensitive mutants in the A . dorsalis and C . tarsalis persistently infected cultures, and small plaque mutants were predominant in all cultures except one of two cell cultures of C . tarsalis . Virus from persistently infected A . albopictus cell cultures was growth-restricted in Vero and C . tarsalis cells . One of two persistently infected A . dorsalis cell cultures also produced viral mutants that were growth-restricted in C . tarsalis cells . Further, Western blots of persistently infected A . albopictus cell extracts showed an overproduction of capsid (C) and envelope (E) structural proteins and reduced production of an Mr 27K protein (p27) which was immunologically related to the E protein . In contrast, the production of E and C proteins in persistently infected C . tarsalis cultures was consistent with the amount of infectious virus present, whereas p27 was relatively overproduced . These observations suggest that the host cell has an important influence on both the types and relative quantities of viral mutants that accumulate during long-term persistent infections. Neuron, 1988 Sep, 1(7), 557 - 68 Analysis of the development of cellular subsets present in the neural crest using cell sorting and cell culture; Maxwell GD et al.; We have tested the hypothesis that developmentally significant cellular subsets are present in the early stages of neural crest ontogenesis . Cultured quail trunk neural crest cells probed with the monoclonal antibodies HNK-1 and R24 exhibited heterogeneous staining patterns . Fluorescence-activated cell sorting was used to isolate the HNK-1+ and HNK-1- cell populations at 2 days in vitro . When these cell populations were cultured, the HNK-1+ sorted cells differentiated into melanocytes, unpigmented cells, and numerous catecholamine-positive (CA+) cells . In contrast, the HNK-1- sorted cells gave rise to melanocytes and unpigmented cells, but few, if any, CA+ cells . When neural crest cells at 2 days in vitro were labeled with R24 and sorted, both the R24+ the R24- sorted cell populations produced numerous CA+ cell, melanocytes, and unpigmented cells . These results provide evidence for the existence of developmental preferences in some subsets of neural crest cells early in embryogenesis. Cell Biol Toxicol, 1988 Sep, 4(3), 333 - 48 A cell culture model of chemically and spontaneously derived mouse lung alveologenic carcinoma; Smith GJ et al.; Malignant cell lines related to mouse lung alveologenic carcinoma have been established from urethane-induced tumors and after in vitro spontaneous transformation of preneoplastic cell lines . Both the chemically and spontaneously transformed cell lines formed invasive, poorly differentiated carcinomas with secondary lung deposits when implanted subcutaneously in immune-suppressed mice . They differed from the related preneoplastic cell line in coordinately exhibiting anchorage-independent growth, reduced epidermal growth factor receptor activity and absence of pericellular fibronectin . These data suggest that similar molecular events may occur in type 2 pneumocyte-related cells in order to generate mouse lung alveologenic adenomas and carcinomas by both spontaneous and chemical carcinogen induction mechanisms . A reduced level of pericellular fibronectin was also demonstrated in an in situ compressive urethane-induced mouse lung adenoma . Loss of pericellular fibronectin may therefore be an early and persistent phenotypic alteration during transformation to the alveologenic adenoma and carcinoma. Vopr Virusol, 1988 Sep-Oct, 33(5), 595 - 600 {Epitope analysis of mumps virus proteins in a persistent cell culture infection: changes in the hemagglutinin-neuraminidase polypeptide}; Boriskin IuS et al.; Monoclonal antibodies (MABs) against major structural mumps virus proteins were used for epitope analysis in primarily and persistently infected HEp-2 cells by means of immunofluorescence and radioimmunoprecipitation techniques . Qualitatively, no differences were found in MAB binding between corresponding proteins of the original and persistent viruses, whereas quantitative differences observed might be explained in terms of weakened viral protein synthesis in persistent infection . Limited proteolysis of MAB-bound antigen has revealed alterations in certain epitopes on persistent virus HN polypeptide . Despite the inability of HEp-2 infected cells for hemadsorption, HN protein was expressed on the surface of these cells to the same extent as in the hemadsorbing system of mumps virus-infected Vero cells. J Virol Methods, 1988 Sep, 21(1-4), 209 - 22 Techniques for establishing human epithelial cell cultures: sensitivity of cell lines for propagation of herpesviruses; Rhim JS et al.; The development of tissue culture systems for propagation of human epithelial cells has aided the investigation of events that lead normal epithelial cells to become neoplastic . The establishment of human epidermal keratinocyte and human bronchial epithelial cell lines and their usefulness for oncogenic virus assay systems are described . In addition, cultivation of primary epithelial culture from oral hairy leukoplakia and normal salivary gland biopsies in a serum-free medium is discussed. Neurosci Lett, 1988 Aug 31, 91(2), 199 - 203 Non-competitive antagonists of N-methyl-D-aspartate receptors protect cortical and hippocampal cell cultures against glutamate neurotoxicity; Rondouin G et al.; Brief exposure of cortical or hippocampal cell cultures to glutamate (500 microM) resulted in a progressive neuronal necrosis which is maximal 18-24 h later . Pretreatment of the cultures by a phencyclidine derivative: thienylphencyclidine (TCP), or the TCP precursor: GK86, or MK801 protected against glutamate toxicity . Non-competitive antagonists of N-methyl-D-aspartate receptors appear as potent tools for the in vitro protection of neuronal cells against excitatory amino acid toxicity. J Biol Chem, 1988 Aug 15, 263(23), 11504 - 10 Effect of amino acid analogs on the processing of the pancreatic polypeptide precursor in primary cell cultures; Schwartz TW; Amino acid analogs, which can be incorporated into nascent peptide chains were used in cultures of endocrine cells from canine pancreas to study the effect on processing of the metabolically labeled precursor for pancreatic polypeptide . Analogs for basic amino acids, canavanine, and aminoethylcysteine prevented the di-basic processing of the prohormone . The polar leucine analog, beta-hydroxyleucine, only partially perturbed the function and cleavage of the signal peptide but efficiently and unexpectedly blocked the dibasic cleavage of the prohormone . Other nonbasic amino acid analogs, beta-hydroxynorvaline and azetidine-2-carboxylic acid, which only could be incorporated into the prohormone at a distance from the processing site, also prevented dibasic cleavage of the prohormone . Although there are no phenylalanine residues in the prohormone, analogs for this amino acid, fluoro-phenylalanine and particularly phenylserine, could also block the processing of the prohormone at the dibasic site . This effect was prevented by addition of a small quantity of phenylalanine . It is concluded that amino acid analogs can interfere with precursor processing through altering both the primary and the secondary structure of the precursor but also through incorporation into cosynthesized protein(s) which are necessary for the precursor processing. Brain Res, 1988 Aug 9, 457(2), 386 - 91 A study of ionic conductances involved in plateau potential activity in putative vasopressinergic neurons in primary cell culture; Legendre P et al.; Vasopressin neurons in hypothalamic cell culture display regenerative calcium-dependent plateau potentials . The present study was undertaken to investigate the mechanisms underlying their time course and periodicity . Intracellular recordings showed that the duration of the plateaux was controlled by a progressive activation of a voltage- and calcium-dependent potassium conductance, but not by a progressive inactivation of calcium conductances . The refractory period appeared to be due to a calcium-dependent potassium conductance activated by membrane potential depolarization. Clin Orthop, 1988 Aug, (233), 274 - 82 Fluoride and bovine bone extract influence cell proliferation and phosphatase activities in human bone cell cultures; Wergedal JE et al.; The effects of fluoride (20 mumol/L) and bovine bone extract (17 micrograms/ml) were determined on cultures of human bone cells, embryonic chick bone cells, and human skin fibroblasts . The incorporation of {3H}thymidine into DNA was measured 16 hours after the addition of factors . After three to five days treatment, Triton X-100 extracts of the cells were assayed for acid phosphatase activity, in the presence and absence of tartrate, and for alkaline phosphatase activity . Fluoride stimulated {3H}thymidine incorporation and specific activity of alkaline phosphatase in human bone cells and chick bone cells but not in human skin cells . Fluoride also stimulated the cell population doubling rate of the human bone cells with an optimum of approximately 20 mumol/L . Bovine bone extract stimulated thymidine uptake into DNA several-fold and decreased alkaline phosphatase activity in all three types of cultured cells . The specific activity of tartrate-resistant acid phosphatase was increased in bone cells but not in skin fibroblasts . These results suggest that fluoride specifically stimulates the proliferation and differentiation of osteoblasts, while the growth factors in bovine bone extract primarily stimulate proliferation of bone cells . Cultures of human bone cells respond similarly to chick calvarial cells when treated with fluoride or bovine bone extract. J Bone Miner Res, 1988 Aug, 3(4), 401 - 8 Further biochemical and molecular characterization of primary rat parietal bone cell cultures; McCarthy TL et al.; Primary bone cell cultures are used widely to examine the regulation of bone metabolism by growth factors and hormones . Characterization of this model system is now being conducted at the molecular level to define modulation of gene expression . Cells were obtained from rat parietal bone by sequential collagenase digestions . Cell populations were evaluated for bone-related products, including collagen isoform expression and mRNA levels, alkaline phosphatase activity, and osteocalcin production . Serum-deprived, confluent cultures of the first and second collagenase-released populations produced a lower percentage of total protein as collagen than the third, fourth, and fifth populations, while co-culturing the third through fifth populations resulted in the highest level . Collagen typing on SDS-polyacrylamide gels revealed an abundance of mature type I collagen in all cell populations; type III collagen synthesis was undetectable by this method . This is in contrast to the presence of cytoplasmic mRNA for both type I and type III collagen in all cell populations, suggesting post-transcriptional modulation of type III collagen synthesis . The expression of alkaline phosphatase and osteocalcin was highest in cultures of later released cells, indicating that these cell populations display phenotypic characteristics associated with cells of the osteoblast lineage. Hum Reprod, 1988 Aug, 3(6), 705 - 13 Establishment of human endometrial cell cultures; Bongso A et al.; Epithelial and stromal endometrial cells from 19 patients at different phases of the menstrual cycle were enzymatically separated, isolated by successive centrifugation and primary cultures established for in-vitro studies on implantation . The behaviour of cells in vitro was evaluated using Nomarski's inverted optics, May-Grunwald-Giemsa stained coverslips and scanning electron microscopy . Epithelial and stromal cells from all patients grew successfully in Chang's medium and formed a mixed confluent monolayer of epithelioid and fibroblastic cells in 3-7 days and such monolayers could be maintained alive up to 3-4 weeks . Epithelioid cells were polyhedral and grew as islands in a whorl-like wavy pattern around glandular fragments . Fibroblasts were spindle-shaped, more long-lived and grew rapidly to form parallel bundles of cells . Significant differences were observed in the number of multinucleated cells and cells with intracytoplasmic vacuoles between endometrium from proliferative, postovulatory and secretory phases (P less than 0.01) . Scanning electron micrographs showed cells with cilia with varying densities of microvilli and apical protrusions . Endometrial cells in culture showed structural features remarkably similar to those described for cells in situ . The method described allows the propagation in vitro of separate endometrium cell types which can be used to study implantation mechanisms in unstimulated and stimulated cycles. Nippon Sanka Fujinka Gakkai Zasshi, 1988 Aug, 40(8), 1050 - 5 {Cell culture--its application in the study of hormone and endometrial carcinoma and feed-back to clinical medicine}; Kuramoto H; By statistical study on 135 patients with endometrial carcinoma, it is clarified that the most effective prognostic factor of the cancer is the histological grading . Well differentiated type is best prognostic and possesses hormone receptors . Application of cell culture is one of the most suitable choices in the study of hormone and human endometrial carcinoma . Present paper is to show usefulness of in vitro study by taking example of the above theme . 1) Binding ability of endometrial carcinoma cells to estrogen: Being explained by Gurpide et al . by using HEC-1 cells, the ability is under control of cGMP and cAMP ratio . 2) Responses to estrogen: DNA polymerase alfa of Ishikawa cells which possesses both estrogen and progesterone receptors (ER, PR) is stimulated first showing peak at 18 hours and alkaline phosphatase (ALP) is at 72 hours by E(2)10(-8)M, which is antagonized by OH-tamoxifen . PR level is also enhanced at its maximum after 3 day E2 treatment, and is analyzed by immunocytochemistry with PR mono-clonal antibody as well as biochemical assay . Gorski and Greene's theory that steroid receptor is localized in nuclei is confirmed in endometrial carcinoma . Growth of Ishikawa cells is apparently enhanced in the aspects of shortened cell cycle and unlimited saturation density . 3) Responses to progestogen: Nucleic acid syntheses of HEC-1 are immediately suppressed by progesterone (P) 2.5 microg or more . Electron microscopic findings show appearances of Golgi apparatus and lysosomal granules . Growth suppression is observed in the cell lines regardless of PR positivity . ALP activity of PR-negative HEC-50 cells J Clin Invest, 1988 Aug, 82(2), 579 - 85 The addition of endothelial cell growth factor and heparin to human umbilical vein endothelial cell cultures decreases plasminogen activator inhibitor-1 expression; Konkle BA et al.; Plasminogen activator inhibitor-1 (PAI-1) is a specific and rapid inhibitor of tissue plasminogen activator (tPA) and urokinase . Clinical studies suggest that PAI-1 may play a crucial role in the regulation of fibrinolysis . A number of factors modulate PAI-1 activity in endothelial cell culture, and the isolation of PAI-1 cDNA now allows study of PAI-1 regulation at the mRNA level . We examined the effect of endothelial cell growth factor (ECGF) and heparin on PAI-1 expression in human umbilical vein endothelial cell (HUVEC) culture . The addition of ECGF and heparin to HUVEC cultures results in a 3-10-fold decrease in the PAI-1 activity secreted into the conditioned media . This effect is mediated at the mRNA level . A decrease in PAI-1 is also seen with higher concentrations of ECGF alone, but is greatly enhanced by the addition of heparin . No significant change in tPA antigen or mRNA levels was observed. Prostaglandins, 1988 Aug, 36(2), 249 - 57 Prostaglandin E2 and atriopeptin III oppose the contractile effect of angiotensin II in rat kidney mesangial cell cultures; Fandrey J et al.; Effects of vasoconstrictory and of dilatory hormones were studied on the contractile activity of cultured rat kidney mesangial cells . By phase contrast microscopy, a rapid contraction was seen of most cells treated with angiotensin II (10(-6) - 10(-10) mol/L), which was sometimes followed by autonomous relaxation after 10 to 20 min . Prostaglandin E2 and atriopeptin III prevented the contractile effect of angiotensin II in a dose-dependent manner . Angiotensin II, but not atriopeptin III, stimulated prostaglandin E2 synthesis in mesangial cell cultures. Exp Hematol, 1988 Aug, 16(7), 613 - 9 Cell culture studies and oncogene expression in juvenile chronic myelogenous leukemia; Gualtieri RJ et al.; Juvenile chronic myelogenous leukemia (JCML) may be distinguished from adult CML based upon in vitro cell growth characteristics . We studied four untreated children with JCML and report additional unique findings . Peripheral blood (PB) and bone marrow (BM) cells were grown in soft agar . Without exogenous colony-stimulating activity (CSA) there was exuberant "spontaneous" colony formation in both PB and BM cultures . In the absence of exogenous stimulus, PB colony morphology was predominantly, but not exclusively, monocyte/macrophage . When PB was depleted of adherent cells, "spontaneous" colony formation was nearly completely abrogated . Cultures were also performed in the presence of various sources of CSA including giant cell tumor-conditioned medium (GCT-CM), a melanoma cell line-CM (LD1-CM), human placenta-CM (HPCM), and normal PB mononuclear cell (PBMC) feeder layers . Colony formation was typically increased with HPCM and PBMC, whereas in two patients GCT-CM and LD1-CM failed to stimulate additional colony growth when compared to cultures without exogenous CSA and, in fact, appeared to inhibit baseline "spontaneous" growth . The morphology of colonies in the presence of exogenous stimuli was highly variable . Because of the recent association between the c-fms protooncogene product and the receptor for the monocyte growth factor CSF-1, we analyzed the PB cells from two JCML patients for c-fms expression . Although expressed, c-fms levels were less than that in an adult with Ph1-positive CML in chronic phase . These studies indicate that in JCML, there are dramatic increases in both PB and BM colony-forming cells and that "spontaneous" growth is dependent on an accessory adherent cell fraction . Furthermore, patterns of responsiveness to various sources of CSA suggest that the colony-forming cells may not be a uniform population of malignant cells. Proc Natl Acad Sci U S A, 1988 Aug, 85(16), 5889 - 93 Parathyroid hormone modulates transforming growth factor beta activity and binding in osteoblast-enriched cell cultures from fetal rat parietal bone; Centrella M et al.; Transforming growth factor beta (TGF-beta) is produced by bone cells, is abundant in bone matrix, and regulates bone cell biochemical processes . In osteoblast-enriched fetal rat parietal bone cell cultures, low TGF-beta doses increase DNA synthesis, whereas higher levels are less mitogenic, stimulate collagen production, and decrease alkaline phosphatase activity . Parathyroid hormone by itself has minimal effects on these processes, but it opposes the effects of TGF-beta and alters TGF-beta binding to its receptors in osteoblast-enriched cultures . Some functions ascribed to parathyroid hormone in bone may therefore result from alterations in TGF-beta activity, suggesting that the local effects of TGF-beta in bone are under systemic hormonal control. J Neurosci, 1988 Aug, 8(8), 2887 - 94 Catecholamine toxicity in cerebral cortex in dissociated cell culture; Rosenberg PA; Identification of endogenous toxins and characterization of the mechanisms by which toxins produce cell injury and death may help understand both normal modeling of cell populations and connections in the CNS as well as abnormal cell loss . The toxicity of catecholamines intrinsic to the CNS was investigated using the model system of rat cerebral cortex in dissociated cell culture . All catecholamines tested, including norepinephrine (NE), dopamine, and epinephrine, were toxic to neurons as well as glia at a concentration of 25 microM when added to cultures 24 hr after plating . Toxicity was evident after 48 hr exposure to NE, as monitored by loss of cells from the cultures . Toxicity did not seem to be mediated by adrenergic receptors because, although the beta-adrenergic agonist isoproterenol (but not the alpha-adrenergic agonist phenylephrine) was similar in its toxic effect to NE, the beta-adrenergic antagonist atenolol did not block the toxic effect of NE . Toxicity could be mimicked by hydrogen peroxide, a product of the oxidative degradation of catecholamines . Toxicity of NE was blocked by catalase . The neurotoxin 6-hydroxydopamine (6-OHDA), supposedly selective for catecholaminergic neurons, was found to be toxic over the same concentration range as NE . These results suggest that endogenous catecholamines may play a role in normal and abnormal cell death, and suggest that caution be used in relying on the specificity of 6-OHDA and other supposedly selective neurotoxins. In Vitro Cell Dev Biol, 1988 Aug, 24(8), 778 - 86 Proliferation of epithelial cells derived from rat dorsolateral prostate in serum-free primary cell culture and their response to androgen; Nishi N et al.; Primary cultured epithelial cells derived from the rat dorsolateral prostate proliferated in serum-free nutrient medium WAJC 404 supplemented with mitogens: insulin (650 nM), cholera toxin (120 pM), epidermal growth factor (EGF) (2.5 nM), dexamethasone (300 nM), and bovine pituitary extract (25 micrograms/ml) . The culture consisted of two types of epithelial cell colonies: one originated from single cells or small cell aggregates and the other was epithelial cell outgrowth from small tissue fragments attached to a substratum . There were differences in requirements for the mitogens between the two types of colonies . Requirements for cholera toxin, bovine pituitary extract, and dexamethasone were higher in the former type of colonies, and those for EGF were higher in the latter type of colonies . Proliferation of the epithelial cells in either type of colony was suppressed more than 50% by 1 nM dihydrotestosterone . This suppressive effect was not mediated by stromal component in the tissue fragments, and was counteracted by cyproterone acetate, indicating specific and direct action of the androgen on prostate epithelial cells . The results suggest that there is discrete participation of polypeptide growth factors and androgen in proliferation and differentiation, respectively, of prostate epithelial cells in vivo. J Gen Virol, 1988 Aug, 69 ( Pt 8), 2129 - 34 Large scale production of hepatitis A virus in cell culture: effect of type of infection on virus yield and cell integrity; Robertson BH et al.; Approaches to cell culture propagation of hepatitis A virus (HAV) have used either acute infection by passage of infected cell lysates or supernatants into uninfected cells or the passage of persistently infected cells . The findings presented here demonstrate that the growth and recovery of purified virus from foetal rhesus monkey kidney (FRhK4) cells persistently infected with HAV isolate HAS-15 decreased over a 2 to 3 month period . In contrast, high multiplicity acute infection of FRhK4 cells with purified HAS-15 HAV resulted in degeneration of the cell monolayer 2 to 3 weeks later . Large scale propagation of acutely infected cells followed by traditional picornavirus purification procedures reproducibly yielded milligram amounts of purified virus. Zh Mikrobiol Epidemiol Immunobiol, 1988 Aug, (8), 19 - 23 {Molecular hybridization of nucleic acids as a method for the laboratory diagnosis of influenza in model experiments on cell cultures and in research on nasopharyngeal smears obtained from patients}; Pliusnin AZ et al.; DNA probes containing the nucleotide sequences of the conservative genes of influenza A virus (matrix, nucleoprotein and acidic polymerase genes) show their specificity with respect to the RNA of influenza A viruses in mammal tissue cell cultures (continuous spaniel kidney cell culture and primary calf kidney cell culture) . The minimal amount of infected monolayer cells, permitting the detection of viral RNA, is 10(3) . The results obtained in the study of nasopharyngeal washings make it possible to recommend the method of molecular hybridization for use in the epidemiological analysis in addition to virological and serological tests . The method of hybridization permits the detection of virus-specific RNA in the allantoic fluid of chick embryos in subculturing the materials under study even in those cases when hemagglutinating influenza virus cannot be isolated. J Immunol Methods, 1988 Jul 22, 111(2), 179 - 88 In vitro production of monoclonal antibodies under serum-free conditions using a compact and inexpensive hollow fibre cell culture unit; Klerx JP et al.; A compact and easily portable hollow fibre cell culture system using commercially available components is described . The construction is relatively cheap and simple . As the hollow fibre cell culture cartridge we chose an inexpensive haemodialyser . Though not specially developed for this purpose this performed excellently in our system . Using a serum-free medium supplemented with ethanolamine, selenium and transferrin, an average antibody production of 30-200 mg per cartridge per day could be achieved, depending on the cell line . Because a serum-free medium was used, monoclonal antibodies could readily be purified on a large scale. Brain Res, 1988 Jul 19, 456(1), 159 - 67 Characterization of corticotropin-releasing factor receptors in dissociated brain cell cultures; Kapcala LP et al.; Although corticotropin-releasing factor (CRF) receptors have been identified throughout the brain, relatively little is known about the regulation of CRF receptors . Recent investigations aimed at developing an in vitro model for studying the regulation of CRF receptors demonstrated CRF binding in brain cell cultures . To test the hypothesis that dissociated brain cell cultures contain CRF receptors and may provide a model for studying their regulation, studies characterizing binding of labeled CRF were performed . Dissociated cells derived from hypothalamus and extrahypothalamic forebrain (predominantly cortex) of day 17 fetal rats were maintained in chemically defined medium . We used a stable 125I-labeled analog of ovine CRF, 125I-Tyro-ovine CRF (125I-oCRF), to identify and characterize CRF receptors . Although specific binding of 125I-oCRF was demonstrated in both hypothalamic and extrahypothalamic cell cultures, the concentration of CRF receptors was much greater (3-5 fold) in extrahypothalamic cells . Binding of 125I-oCRF in extrahypothalamic cells was saturable and was composed of high affinity (Kd = 0.51 nM) and low affinity (Kd = 17.25 nM) sites . Pharmacological displacement of labeled CRF from cells with a variety of CRF fragments and analogs was similar to that in studies of pituitary and brain homogenates . Extrahypothalamic cells studied at several times between 4 and 13 days in culture revealed an increase in the number of CRF receptors; the concentration of CRF receptors at 13 days was 3.5 times that observed at 4 days . Studies directed toward determining whether CRF receptor concentration could be modulated by CRF, adrenocorticotropic hormone, atropine or a CRF antagonist showed a change (36% decrease) only in response to chronic exposure with CRF . Conclusions: (1) dissociated fetal rat brain cell cultures derived from extrahypothalamic forebrain and hypothalamus contain CRF receptors; (2) CRF receptors in brain cells exhibit a differential distribution and characteristics similar to those previously reported in brain and pituitary; (3) dissociated fetal rat brain cell cultures may provide a relatively simplified in vitro model for studying the regulation of CRF receptors; and (4) CRF down-regulates its own receptor in extrahypothalamic forebrain cells. Eur J Biochem, 1988 Jul 15, 175(1), 125 - 33 Phorbol ester prevents the thyroid-stimulating-hormone-induced but not the forskolin-induced decrease of cAMP-dependent protein kinase activity in thyroid cell cultures; Omri B et al.; The potent tumor promoter 12-O-tetradecanoyl-phorbol 13-acetate (TPA) affects several thyroid cell functions and interacts with thyroid-stimulating hormone (TSH) either by inhibiting or potentiating its action on different cellular parameters . Since phorbol ester acts mainly through the activation of protein kinase C, which is its receptor, we studied this activation and its interaction with TSH and forskolin in suspension cultures of porcine thyroid cells . In thyroid cell cultures, TPA has a dual effect on protein kinase C activity: immediately (2-5 min) after exposure of cells to TPA, it began to be translocated from the cytosol to the particulate fraction . The transfer of the cytosolic enzyme was total and could occur with or without a loss of activity . The translocated enzyme still needed Ca2+ and phospholipids for its activation . The basal activity increased transiently (2-4 h) in both the cytosol and particulate fractions during translocation . The peak activity in the particulate fraction was reached 10-30 min after exposure of cells to TPA, and was followed by down-regulation of protein kinase C and almost complete disappearance of its activity . The residual activity was about 13% of control after a 2-day exposure to TPA . It was unequally distributed between cytosol (4%) and particulate fraction (9%) . Prolonged exposure of cells to TPA did not affect either the activity or the subcellular distribution of the cAMP-dependent protein kinase activity . TPA interacted with TSH and prevented the decrease of this activity induced by prolonged exposure of cells to the hormone not only when it was introduced simultaneously with TSH, but also when it was added 24 h after TSH . However, the forskolin-induced decrease in cAMP-dependent protein kinase activity was not prevented by the presence of TPA . TPA also affected the increases in cAMP accumulation mediated by TSH and forskolin . The TSH-induced increase was significantly stimulated by TPA after short contacts (5-15 min), while longer preincubations of cells with TPA provoked a very strong inhibition of the TSH action . However, the forskolin-induced stimulation of the cAMP accumulation was maintained and even further increased in the presence of TPA . Consequently, the actions of TSH and TPA are apparently interdependent, while those of forskolin and TPA seem to be parallel and independent . Neither TSH nor forskolin prevented the TPA-induced down regulation of protein kinase C . The biologically inactive phorbol ester analogue 4 alpha-phorbol 12,13-didecanoate had no effect on protein kinase C activity, and did not interact with either TSH or forskolin.(ABSTRACT TRUNCATED AT 400 WORDS) Am J Physiol, 1988 Jul, 255(1 Pt 1), C19 - 27 Attachment and maintenance of adult rabbit cardiac myocytes in primary cell culture; Haddad J et al.; The present observations demonstrate that quiescent calcium-tolerant adult rabbit cardiac myocytes can be isolated by collagenase-hyaluronidase perfusion and maintained in primary culture for at least 2 wk . Culturing large numbers of myocytes requires that the freshly isolated cells be attached to a suitable substratum such as laminin, type IV collagen, or fetal bovine serum . The cultured myocytes retain their rod-like morphology for approximately 7 days before gradually spreading into a flattened conformation by 14 days . During the 1st wk of culture, contaminating interstitial cells rapidly proliferate, making cultures unsuitable for long-term study . Pure myocyte populations can be established and maintained if freshly isolated cells are cultured in the presence of cytosine arabinoside (Ara-C, 10 microM) . This antimetabolite does not appear to adversely affect high-energy phosphates, since ATP and creatine phosphate (CrP) content of the myocytes is maintained at levels normally found in biopsy samples of rabbit myocardium . These results illustrate that an energetically stable population of adult cardiac myocytes can be maintained in primary culture in sufficient numbers to make them useful for future investigations of myocyte function. Cancer Res, 1988 Jul 1, 48(13), 3586 - 90 Effect of testosterone on imidazole-induced tyrosinase expression in B16 melanoma cell culture; Kline EL et al.; To assess the effect of androgens on tyrosinase activity in B16/C3 melanoma cell cultures, proliferating cultures were treated with testosterone (50 nM) or one of several other androgenic analogues and metabolites . None of these compounds influenced basal enzyme activity . Imidazole (10 mM), however, is a potent inducer of tyrosinase in this cell line . Testosterone blocked induction of tyrosinase by imidazole almost completely . This effect was dose dependent, being maximal at 10 nM and half-maximal at approximately 3 nM, and was rapid, occurring within 15 min . When cultures treated with both imidazole and testosterone were shifted to medium containing only imidazole, enzyme activity approximated that seen in cultures never receiving testosterone within 10 h of the shift . The other steroids tested failed to influence imidazole induction of the enzyme . This action of testosterone could not be demonstrated in broken cell preparations . Results of studies involving inhibitors of protein and RNA synthesis, as well as those quantitating mRNA hybridizable to a synthesized 20-base pair deoxyoligonucleotide tyrosinase probe, suggest that testosterone is blocking imidazole induction at a pretranslational level. Vopr Virusol, 1988 Jul-Aug, 33(4), 465 - 9 {Effect of magnesium sulfate on the reproduction of the measles virus in cell culture}; Shteinberg LSh et al.; Magnesium sulphate in concentrations of 25-50 mM induced reproducible increase in titers of extracellular measles virus (by 0.5-2.0 1g TCD50/0.5 ml) in Japanese quail embryo cells . MgSO4 effect was observed with all methods of cell cultivation: stationary, roller, or on microcarriers . Its effect was associated not with its stabilizing influence on the extracellular virus but rather with the stimulation of the synthesis of intracellular viral proteins. Vet Parasitol, 1988 Jul, 29(1), 9 - 17 The use of amphotericin B in prevention of Trypanosoma theileri growth in bovine cell culture; Lostrie-Trussart N et al.; Trypanosoma theileri is an ubiquitous blood parasite of cattle . This parasite grows easily in RPMI medium and perturbs lymphocyte tests based on {3H}thymidine uptake . This paper reports that 0.5 microgram ml-1 amphotericin B in the medium is adequate to get Trypanosoma growth inhibition without alteration of either mitogenic or allogenic stimulation of bovine lymphoid cells in vitro. J Virol Methods, 1988 Jul, 20(3), 195 - 202 A microtiter cell-culture assay for the determination of anti-human immunodeficiency virus neutralizing antibody activity; Robertson GA et al.; A microtiter cell-culture assay is described for measuring neutralizing antibody activity directed against the human immunodeficiency virus (HIV) . The assay relies upon inhibition of HIV-mediated cell killing of infected MT-4 lymphoid cells . The assay exhibits comparable sensitivity to two other methods used for such measurements, is relatively rapid, may be adapted for screening large numbers of samples and involves minimal handling of infectious virus. J Neuroimmunol, 1988 Jul, 18(4), 333 - 40 Transferrin, carbonic anhydrase C and ferritin in dissociated murine brain cell cultures; Griot C et al.; It is shown here that transferrin (Tf), the iron transport protein and carbonic anhydrase C (CA C) are specifically located within oligodendrocytes in murine brain cell cultures . Ferritin (F), the major iron storage protein, was demonstrated in oligodendrocytes, as well as in astrocytes and microglial cells and was more prominent in the former . CA C and Tf were seen first after 6-7 days in culture . CA C and F positivity increased rapidly and at day 20, 80-85% of galactocerebroside + oligodendrocytes were positive for both proteins . Only a small number of oligodendrocytes was Tf+ up to day 14, after which their numbers increased rapidly until day 20, when 67% of the oligodendrocytes were Tf+ . Because of the presence of Tf and F in oligodendrocytes it is suggested that these cells may play an important role in the metabolism of iron within the central nervous system. Eur J Obstet Gynecol Reprod Biol, 1988 Jul, 28(3), 221 - 8 Effect of progesterone in long-term endometrial cell culture: study of the synthesis of prostaglandins, thromboxane, prostacyclin and prolactin; Nemme R et al.; Cultured monolayers of endometrial cells synthesized PGE2, PGF2 alpha, 13,14-dihydro-15-keto-PGF2 alpha (DHKF2 alpha), TXB2 and 6-keto-PGF1 alpha . Synthesis increased with the duration of culture, reaching a maximum at approximately the 20th day for PGF2 alpha and PGE2; PGF2 alpha levels always exceeded those of PGE2 . Transformation of PGF2 alpha into DHKF2 alpha was minimal . Maximal levels of TXB2 and 6-keto-PGF1 alpha were noted after 7 and 14 days respectively . After three weeks, traces of DHKF2 alpha, TXB2 and 6-keto-PGF1 alpha were noted, whereas PGF2 alpha levels were marked after 30 days of culture . Addition of progesterone (0.32 ng/ml) reduced PG production, notably for PGF2 alpha, but promoted prolactin synthesis, indicating that this progesterone concentration induced decidualization of endometrial cells during the first days of culture. J Electron Microsc Tech, 1988 Jul, 9(3), 235 - 63 Renal cell culture; Kreisberg JI et al.; Methods for the establishment and growth of renal cell types in culture are reviewed, with emphasis on current trends . General techniques available for the isolation and culture of glomerular cells have progressed from explant to enzyme dissociation and cloning techniques . The growth characteristics and properties of cultured glomerular endothelial, epithelial, mesangial, and bone-marrow-derived cells are discussed . Studies are described in which cultures of contractile mesangial cells have led to an elucidation of their role both in normally functioning glomeruli and in disease states . Renal tubule culture techniques also have progressed from mixed tissue explants and cell isolates to fractionation of enriched tubule populations and growth of specific, individually microdissected proximal convoluted, proximal straight, thick ascending limb of Henle's loop, and collecting tubules . The differentiated tubule epithelial-specific properties of such primary cultures are discussed in relation to those of permanently growing cell lines such as MDCK and LLC-PK1 . Renal tubule cultures will be invaluable for the study of the role of hormones and extracellular matrix in epithelial growth and polarity of normal structure and function . In addition, in vitro models of cultured renal tubules have been established to study the effects of age, nephrotoxins, and anoxic injury. J Cell Biol, 1988 Jul, 107(1), 333 - 40 Comparison of the effects of NGF, activators of protein kinase C, and a calcium ionophore on the expression of Thy-1 and N-CAM in PC12 cell cultures; Doherty P et al.; The addition of nerve growth factor (NGF) to PC12 cells induces an approximate doubling in the cell surface expression of the Thy-1 glycoprotein and the neural cell adhesion molecule (N-CAM) after 24 h of culture . Although both responses are measured at the same time point, their sensitivity to NGF differed with half-maximal induction of Thy-1 apparent at NGF concentrations (approximately 0.1 ng/ml NGF) that had little effect on N-CAM expression . Phorbol ester derivatives capable of activating Ca2+/phospholipid-dependent protein kinase (protein kinase C) and the calcium ionophore A23187 were found to mimic the NGF induction of Thy-1, but not N-CAM . Similar results were observed when a synthetic diacylglycerol was added to PC12 cell cultures . Increased expression of Thy-1 consequent to phorbol ester, calcium ionophore, or NGF treatment was associated with an increase in the expression of the mRNA species that encodes Thy-1 . Increased expression of Thy-1 consequent to all three treatments was also reduced by treatment with the transcription inhibitor cordycepin . Treatment of PC12 cells with high concentrations of phorbol esters was found to inhibit the NGF induction of Thy-1, but not N-CAM . Whereas the above results are consistent with activation of protein kinase C underlying the NGF induction of Thy-1, the same data are not consistent with this pathway being important in the N-CAM response. Avian Dis, 1988 Jul-Sep, 32(3), 548 - 52 Some observations on the response of ring-necked pheasants to inoculation with various strains of cell-culture-propagated type II avian adenovirus; Fadly AM et al.; The response of ring-necked pheasants to inoculation with three strains of cell-culture-propagated type II avian adenovirus was examined . Marble spleen disease (MSD) virus of pheasants and both avirulent and virulent strains of hemorrhagic enteritis virus (HEV) of turkeys all induced typical gross and microscopic splenic lesions of MSD; neither MSD-associated lung lesions nor mortality were noted in inoculated pheasants, regardless of strain of virus used . Pheasants inoculated with a cell-culture-propagated avirulent strain of HEV were properly immunized against challenge with virulent HEV, as indicated by seroconversion and by protection against virus-induced splenic lesions . We conclude that these strains of type II avian adenovirus are comparable in pathogenicity for pheasants and cannot be distinguished . Further, absence of MSD-associated lung lesions and mortality in pheasants maintained under controlled laboratory conditions suggest that other environmental factors are probably involved in induction of such lesions and mortality in field cases of MSD. Anal Biochem, 1988 Jul, 172(1), 78 - 81 Fluorometric analysis of DNA in cell cultures; Janakidevi K et al.; A simple, reproducible fluorescent assay has been described which can be used confidently at microgram and submicrogram levels . The method is most suitable for cell cycle and cell growth analysis using {3H}thymidine as the radioactive tracer . A modified method is also described for preparing diaminobenzoic acid hydrochloride. J Clin Microbiol, 1988 Jul, 26(7), 1298 - 303 Sensitivity of rhabdomyosarcoma and guinea pig embryo cell cultures to field isolates of difficult-to-cultivate group A coxsackieviruses; Lipson SM et al.; Forty-two difficult-to-cultivate group A coxsackieviruses (i.e., group A types other than A7, A9, and A16), collected primarily from throat swab specimens of patients suffering from fever, pharyngitis, lymphadenopathy, and cough during the 1986 enterovirus season, were isolated in less than 24-h-old suckling mice . Thirty-six moribund mice were sacrificed and autopsied, and then their brains and back musculature were inoculated into rhabdomyosarcoma (RD), guinea pig embryo (GPE), rhesus monkey kidney (RhMk) and human carcinoma of the larynx (HEp-2) cell cultures . Twelve of the 36 suckling mice isolates were adapted to grow in RD and GPE cells after two passes and have been identified in RD cells by type-specific antisera as group A coxsackievirus types A2, A4, and A8 . Three passes in RhMk or HEp-2 cell cultures were insufficient to affect a discernible cytopathic effect . Coxsackievirus types A1, A19, and A22, unable to grow in any of the four cell cultures tested, were identified by virus neutralization in suckling mice . These data denote the efficacy of suckling mice for the isolation of difficult-to-cultivate group A coxsackieviruses. Hum Genet, 1988 Jul, 79(3), 286 - 8 Increased amounts of small polydisperse circular DNA (spcDNA) in angiofibroma-derived cell cultures from patients with tuberous sclerosis (TS) Neidlinger C, Assum G, Krone W, Dietrich C, Hochsattel R, Klotz G. Much greater amounts of small polydisperse circular DNA (spcDNA) have been detected, in cell cultures derived from angiofibromas of six patients with tuberous sclerosis (TS) than in those from the skin of these patients or from the skin of 11 healthy donors . This observation could be confirmed by spreading the DNA of appropriate fractions from CsCl density gradients . The findings suggest the existence of a relationship between the chromosomal instability observed in angiofibroma cultures and the mobilization of spcDNA. Endocrinology, 1988 Jul, 123(1), 277 - 83 A pertussis toxin-sensitive G-protein mediates the alpha 2-adrenergic receptor inhibition of melatonin release in photoreceptive chick pineal cell cultures; Pratt BL et al.; The avian pineal gland is a photoreceptive organ that has been shown to contain postjunctional alpha 2-adrenoceptors that inhibit melatonin synthesis and/or release upon receptor activation . Physiological response and {32P}ADP ribosylation experiments were performed to investigate whether pertussis toxin-sensitive guanine nucleotide-binding proteins (G-proteins) were involved in the transduction of the alpha 2-adrenergic signal . For physiological response studies, the effects of pertussis toxin on melatonin release in dissociated cell cultures exposed to norepinephrine were assessed . Pertussis toxin blocked alpha 2-adrenergic receptor-mediated inhibition in a dose-dependent manner . Pertussis toxin-induced blockade appeared to be noncompetitive . One and 10 ng/ml doses of pertussis toxin partially blocked and a 100 ng/ml dose completely blocked norepinephrine-induced inhibition . Pertussis toxin-catalyzed {32P}ADP ribosylation of G-proteins in chick pineal cell membranes was assessed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and autoradiography . Membranes were prepared from cells that had been pretreated with 0, 1, 10, or 100 ng/ml pertussis toxin . In the absence of pertussis toxin pretreatment, two major proteins of 40K and 41K mol wt (Mr) were labeled by {32P}NAD . Pertussis toxin pretreatment of pineal cells abolished {32P} radiolabeling of the 40K Mr G-protein in a dose-dependent manner . The norepinephrine-induced inhibition of both cAMP efflux and melatonin release, as assessed by RIA of medium samples collected before membrane preparation, was also blocked in a dose-dependent manner by pertussis toxin . Collectively, these results suggest that a pertussis toxin-sensitive 40K Mr G-protein labeled by {32P}NAD may be functionally associated with alpha 2-adrenergic signal transduction in chick pineal cells. Cancer Res, 1988 Jul 1, 48(13), 3751 - 9 Cell culture of the mucinous variant of human colorectal carcinoma; Tibbetts LM et al.; Two cell lines, RW-2982 and RW-7213, have been established for the first time from the mucinous variant of human colorectal carcinoma, which is a distinctive and important subtype that has a worse prognosis than the more common nonmucogenic large bowel carcinoma . Methods of establishment and observations made during 7 and 3 years, respectively, of continuous culture are described . These cell lines required 4-9 months of adaptation to tissue culture conditions before noticeable growth occurred . Both cell lines have the following unique properties: (a) growth in vitro as delicate branching three-dimensional tumor particles within a wide gel of insoluble, often translucent mucus (proteoglycan); (b) production of large quantities of carcinoembryonic antigen; (c) ability to survive or adapt to growth in media free of serum, hormones, growth factors, and all protein; and (d) tumorigenicity in multiple sites in nude mice, including liver, with especially rapid growth in the peritoneal cavity as gelatinous material that is nonadherent and noninvasive and thus resembles pseudomyxoma peritonei . Unlike other reported colorectal cell lines, these mucus-coated particulate cell lines will not readily grow as monolayers and grow much more slowly with a doubling time of 2 weeks or more . A serially transplantable tumor from the RW-7213 surgical specimen has also been maintained in nude mice since August 8, 1984 . This tumor retains properties of the original specimen . Observations made on the tumor biology of mucogenic colorectal carcinoma using these cell lines are discussed. Am J Pathol, 1988 Jul, 132(1), 123 - 44 Cytokeratins in normal and malignant transitional epithelium . Maintenance of expression of urothelial differentiation features in transitional cell carcinomas and bladder carcinoma cell culture lines; Moll R et al.; The pattern of cytokeratins expressed in normal urothelium has been compared with that of various forms of transitional cell carcinomas (TCCs; 21 cases) and cultured bladder carcinoma cell lines, using immunolocalization and gel electrophoretic techniques . In normal urothelium, all simple-epithelium-type cytokeratins (polypeptides 7, 8, 18, 19) were detected in all cell layers, whereas antibodies to cytokeratins typical for stratified epithelia reacted with certain basal cells only or, in the case of cytokeratin 13, with cells of the basal and intermediate layers . This pattern was essentially maintained in low-grade (G1, G1/2) TCCs but was remarkably modified in G2 TCCs . In G3 TCCs simple-epithelial cytokeratins were predominant whereas the amounts of component 13 were greatly reduced . Squamous metaplasia was accompanied generally by increased or new expression of some stratified-epithelial cytokeratins . The cytokeratin patterns of cell culture lines RT-112 and RT-4 resembled those of G1 and G2 TCCs, whereas cell line T-24 was comparable to G3 carcinomas . The cell line EJ showed a markedly different pattern . The results indicate that, in the cell layers of the urothelium, the synthesis of stratification-related cytokeratins such as component 13 is inversely oriented compared with that in other stratified epithelia where these proteins are suprabasally expressed, that TCCs retain certain intrinsic cytoskeletal features of urothelium, and that different TCCs can be distinguished by their cytokeratin patterns . The potential value of these observations in histopathologic and cytologic diagnoses is discussed. Biochim Biophys Acta, 1988 Jun 15, 960(3), 455 - 7 Tunicamycin-treated rat heart cell cultures synthesize an inactive nonreleasable lipoprotein lipase; Friedman G et al.; Cells isolated from newborn rat hearts were cultured in the presence of 100 mM Hepes (4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid) . Lipoprotein lipase activity was present in an intracellular and heparin-releasable pool and was also secreted into the culture medium . Treatment of the cultures with 5 micrograms/ml tunicamycin caused almost complete loss of lipoprotein lipase activity in all three compartments . In control cultures, immunoblotting of lipoprotein lipase derived from all three pools revealed a single band of lipoprotein lipase with an apparent Mr of 56,000 . In the tunicamycin-treated cultures, the enzyme appeared only intracellularly and had an apparent Mr of 49,000 . No immunoreactive enzyme was found in the medium . Thus, glycosylation of lipoprotein lipase in heart cell cultures is mandatory for enzyme activity and translocation from an intracellular to the heparin-releasable pool and for secretion into the medium. Xenobiotica, 1988 Jun, 18(6), 649 - 56 Metabolizing systems in cell culture cytotoxicity tests; Benford DJ et al.; 1 . The addition of 9000 g supernatant of rat liver homogenate (S9) or rat liver microsomal fractions to a cytotoxicity test system using BCL-D1 cells has been investigated . 2 . The choice of culture medium influenced the intrinsic cytotoxicity of the metabolising system to the BCL-D1 cells . Use of Ham's F10 nutrient mixture resulted in greater cytotoxicity compared with several other media . 3 . Microsomal fractions provided greater cytochrome P-450 dependent activation of cyclophosphamide and were less cytotoxic than S9 . 4 . Direct-acting toxic compounds, such as p-aminophenol, were less toxic in the presence of a metabolising system . This was due to protein-binding rather than enzymic detoxification. J Virol Methods, 1988 Jun, 20(2), 109 - 14 Detection of adenovirus by rapid 24-well plate centrifugation and conventional cell culture with dexamethasone; Woods GL et al.; Two methods for rapid detection of adenovirus were tested: (i) 24-well plate centrifugation followed by staining with a monoclonal antibody after incubation for 24 h and 48 h, and (ii) pretreatment of A549 cells used in conventional cell culture and 24-well plate centrifugation with 10(-5)M dexamethasone . Twenty-seven clinical isolates of adenovirus and 12 specimens from which adenovirus had been recovered were included in the analysis . Both isolates and specimens had been frozen at -70 degrees C for up to 6 months . By 24-well plate centrifugation both with and without dexamethasone, 21 (78%) and 27 (100%) isolates were positive for adenovirus at 24 h and 48 h, respectively . Of the specimens, 6 (50%) and 8 (67%) were positive by 24-well plate centrifugation without dexamethasone at 24 h and 48 h, respectively, whereas with dexamethasone 3 (25%) were positive at 24 h and 7 (58%) were positive at 48 h . Overall, combining isolates and specimens, the sensitivity of 24-well plate centrifugation for detection of adenovirus at 24 h was 69% without dexamethasone and 62% with dexamethasone, and at 48 h the sensitivity was 90% without dexamethasone and 87% with dexamethasone . The specificity under all conditions tested was 100% . In conventional tissue culture dexamethasone inhibited recovery of adenovirus . Without dexamethasone, adenovirus was recovered from all 39 samples within 7 days after inoculation; however with dexamethasone pretreatment, the virus was detected in only 31 (79%) of the samples tested in the same period of time. Genitourin Med, 1988 Jun, 64(3), 162 - 4 Evaluation of non-radioactive in situ hybridisation method to detect Chlamydia trachomatis in cell culture; Naher H et al.; A DNA probe combined with a non-radioactive stain was used to detect inclusions of Chlamydia trachomatis in cell culture . Of 39 positive cultures detected by monoclonal antibodies in combination with direct immunofluorescence, 35 were positive by the DNA hybridisation method, the sensitivity being 89.7% . Staining with iodine showed a sensitivity of 87.2%, corresponding to 34 positive cultures . The specificity of DNA hybridisation method was 100%, as all 162 cultures that were negative by the immunofluorescence method were also negative when assessed by the DNA hybridisation method. Prenat Diagn, 1988 Jun, 8(5), 339 - 53 A study of chromosomal aberrations in amniotic fluid cell cultures; Wolstenholme J et al.; This paper represents the analysis of 1916 routine amniotic fluid specimens harvested by an in situ fixation technique in a prospective study with regard to cultural chromosome anomalies . Excluding constitutional abnormalities, 2.9 per cent of 19,432 cells analysed showed some form of chromosome anomaly, terminal deletions (57 per cent) and chromatid/chromosome breaks and gaps (18 per cent) being the most frequent, followed by interchange aberrations (13 per cent) and trisomy (5 per cent) . No case was found of more than one colony from the same culture showing the same anomaly without it being present in other cultures from the same fluid . The wholly abnormal colonies had a surplus of trisomies and from the mathematical considerations presented one may infer that these are likely to reflect the presence of abnormal cells in the amniotic fluid . Partly abnormal colonies appeared at a frequency that would correspond to virtual absence of selection against chromosomally abnormal cells when cultured in vitro . The aberrations found were similar to those seen as single cell anomalies, except for chromatid breaks and exchanges . The data suggest a basic preferential induction of trisomy for chromosomes 2, 18, 21, and the Y-chromosome . Structural aberrations showed a marked clustering of breakpoints around the centromeres . The frequency of mutant cells was low (1.4 X 10(-3)) before culture was initiated . At harvest, the frequency of abnormal cells was much higher (3 X 10(-2)) corresponding to 3 X 10(-3) mutations per cell per generation accumulating over approximately ten generations in vitro. Neuroscience, 1988 Jun, 25(3), 759 - 69 Conditioned medium alters electrophysiological and transmitter-related properties expressed by rat enteric neurons in cell culture; Nishi R et al.; We have previously shown that rat enteric neurons display many of their in vivo phenotypes when they are dissociated and grown in long-term cell culture . To assess the degree of plasticity of these phenotypes we have examined the effect of medium conditioned by rat heart cells because this treatment strongly affects transmitter properties in rat sympathetic neurons in culture . Growth of enteric neurons for 3-4 weeks in conditioned medium caused several changes that are similar to previously described effects of conditioned medium on other neuronal cell types in culture . When compared to cultures grown in control medium, cultures grown in conditioned medium: (i) contained three times as many large (greater than 25 micron) neurons; (ii) synthesized and stored 3-4 times as much acetylcholine; (iii) contained 4-5 times as many neurons with detectable 5-hydroxytryptamine immunoreactivity; and (iv) contained 10 times as many neurons that fired repetitively during sustained depolarization . Several other changes, which have not been reported in other systems, were also observed . Conditioned medium cultures: (i) contained many fewer neuronal processes with immunohistochemically detectable vasoactive intestinal polypeptide, substance P, somatostatin, and {Met}enkephalin; (ii) contained 70% fewer neuronal cell bodies with vasoactive intestinal polypeptide-like immunoreactivity; and (iii) contained four times as many neurons that had muscarinic responses to acetylcholine . None of the changes in properties described above uniformly affected all enteric neurons, even after 6 weeks of growth in conditioned medium . We conclude that the heterogeneity of enteric neuron phenotypes is established prior to birth and limits the capacity of certain subsets of neurons to respond to exogenous factors in the environment . Nevertheless, the phenotypes of other subsets of neurons displayed considerable plasticity when exposed to conditioned medium. Scanning Microsc, 1988 Jun, 2(2), 985 - 92 Fibroblast and epidermal cell-type I collagen interactions: cell culture and human studies; Doillon CJ et al.; Fibroblast and epidermal cell-type I collagen sponge interactions were studied in cell culture as well as in humans . In cell culture, fibroblasts were observed to migrate and proliferate throughout a type I collagen sponge containing either hyaluronic acid (HA) or fibronectin (FN) . Fibroblasts accumulated in the center of the pores in sponges containing HA and appeared to surround themselves with newly synthesized extracellular matrix . In sponges containing FN, fibroblasts attached to and elongated along the collagen fibers of the sponge . In the absence of FN or HA protein synthesis of fibroblasts appeared to be inhibited by the presence of the type I collagen sponge . Epidermal cells grown on plastic or on type I collagen, formed sheets . Epidermal cells grown on a collagen sponge morphologically appeared different than cells grown on plastic . The type I collagen matrix studied in cell culture was applied to dermal wounds of patients with pressure ulcers in order to evaluate its effect on dermal wound healing . The areas of ulcers treated for 6 weeks with a type I collagen sponge decreased by about 40% compared with no change in the areas of untreated controls . Preliminary results suggest that a type I collagen sponge is a biocompatible substrate with fibroblasts and epidermal cells and may be effective in enhancing healing of chronic skin ulcers. J Neurochem, 1988 Jun, 50(6), 1900 - 7 1-Methyl-4-phenyl-1,2,3,6-tetrahydropyridine metabolism and 1-methyl-4-phenylpyridinium uptake in dissociated cell cultures from the embryonic mesencephalon; Schinelli S et al.; 1-Methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP), a contaminant found in a synthetic illicit drug, can elicit in humans and monkeys a severe extrapyramidal syndrome similar to Parkinson's disease . It also induces alterations of the dopamine (DA) pathways in rodents . MPTP neurotoxicity requires its enzymatic transformation into 1-methyl-4-phenylpyridinium (MPP+) by monoamine oxidase followed by its concentration into target cells, the DA neurons . Here, we show that mesencephalic glial cells from the mouse embryo can take up MPTP in vitro, transform it into MPP+, and release it into the culture medium . MPTP is not taken up by neurons from either the mesencephalon or the striatum in vitro (8 days in serum-free conditions) . However, mesencephalic neurons in culture revealed a high-affinity uptake mechanism for the metabolite MPP+, similar to that for DA . The affinity (Km) for DA uptake is fivefold higher than that for MPP+ (0.2 and 1.1 microM, respectively), whereas the number of uptake sites for MPP+ is double (Vmax = 25 and 55 pmol/mg of protein/min for DA and MPP+, respectively) . Mazindol, a DA uptake inhibitor, blocks the uptake of DA and MPP+ equally well under these conditions . Moreover, by competition experiments, the two molecules appear to use the same carrier(s) to enter DA neurons . Small concentrations of MPP+ are also taken up by striatal neurons in vitro . The amount taken up represented less than 10% of the MPP+ uptake in mesencephalic neurons . Depolarization induced by veratridine released comparable proportions of labeled DA and MPP+ from mesencephalic cultures.(ABSTRACT TRUNCATED AT 250 WORDS) J Microsc, 1988 Jun, 150 ( Pt 3), 225 - 31 A new method for cell culture on an electron-transparent melamine foil suitable for successive LM, TEM and SEM studies of whole cells; Westphal C et al.; A new cell culture technique is described which is based on the observation that foils cast from the melamine resin hexamethylol-melamine-ether are suitable for the cultivation of beating heart muscle cells and fibroblasts of the rat . This foil can be flamed for sterilization, is about 80 nm in thickness, homogeneous and smooth, withstands dehydration and critical point-drying, can be removed from glass and permits the imaging of whole cells successively by light microscopy, transmission and scanning electron microscopy . The method is capable of narrowing the gap between light and electron microscopy, yielding excellent whole cell preparations in various kinds of microscopic studies to be performed on one and the same cell. Arch Biol Med Exp (Santiago), 1988 Jun, 21(1), 257 - 62 {Synthesis and secretion of the surface antigen from hepatitis B virus in animal cell cultures}; De Ioannes AE et al.; Stable mammalian cell lines synthesizing and secreting Hepatitis B surface particles have been obtained through genetic engineering techniques . These particles show by electron microscopy a size of 22 nm, they are structurally and immunochemically similar to the particles present in the plasma from chronic hepatitis B patients . Therefore these particles are an excellent source for the preparation of a vaccine against the virus. Biochem J, 1988 Jun 1, 252(2), 537 - 43 Reconstitution of cytochrome P-450-dependent digitoxin 12 beta-hydroxylase from cell cultures of foxglove (Digitalis lanata EHRH.); Petersen M et al.; Cytochrome P-450-dependent digitoxin 12 beta-hydroxylase from cell cultures of foxglove (Digitalis lanata) was solubilized from microsomal membranes with CHAPS (3-{(3-cholamidopropyl)dimethylammonio}propane-1-sulphonic acid) . Cytochrome P-450 was separated from NADPH: cytochrome c (P-450) reductase by ion-exchange chromatography on DEAE-Sephacel . NADPH:cytochrome c (P-450) reductase was further purified by affinity chromatography on 2',5'-ADP-Sepharose 4B . This procedure resulted in a 248-fold purification of the enzyme; on SDS/polyacrylamide-gel electrophoresis after silver staining, only one band, corresponding to a molecular mass of 80 kDa, was present . The digitoxin 12 beta-hydroxylase activity could be reconstituted by incubating partially purified cytochrome P-450 and NADPH:cytochrome c (P-450) reductase together with naturally occurring microsomal lipids and flavin nucleotides . This procedure yielded about 10% of the original amount of digitoxin 12 beta-hydroxylase. Eur J Clin Microbiol Infect Dis, 1988 Jun, 7(3), 415 - 7 In vitro activity of sulphamethoxazole/trimethoprim and sulfadoxine/pyrimethamine against Chlamydia trachomatis SA2f in McCoy cell culture; Mumtaz G et al.; Chequerboard titrations of sulphamethoxazole/trimethoprim and sulfadoxine/pyrimethamine were performed against Chlamydia trachomatis (strain SA2f) using McCoy cell monolayers in vials . The experiments were continued for ten passages each . The mean fractional inhibitory concentration index for each combination was calculated . Results demonstrated synergistic activity between sulphamethoxazole and trimethoprim, and between sulfadoxine and pyrimethamine. J Bioenerg Biomembr, 1988 Jun, 20(3), 313 - 23 Cell culture studies on patients with mitochondrial diseases: molecular defects in pyruvate dehydrogenase; Robinson BH; There is a group of inborn errors of metabolism that result in the condition of chronic lacticacidemia of childhood . Nearly all of the defects that can be identified occur in mitochondrial proteins, and can be demonstrated in cultured skin fibroblasts from the patients concerned . One approach to the diagnosis of these defects involves a simple incubation of the fibroblast culture with glucose-containing medium followed by the measurement of accumulated lactate and pyruvate . The total amounts of lactate and pyruvate and the ratio between them is different in cells from patients with defects in the pyruvate dehydrogenase complex or the respiratory chain . Measurement of 1-14C-pyruvate oxidation to 14CO2 can also reveal defective oxidative metabolism . Localization of the defects can be achieved using individual assays for the enzymes concerned . The clinical sequelae of the different defects is discussed. Hum Cell, 1988 Jun, 1(2), 145 - 9 {Recent problems in cell culture systems using normal human cells}; Okumura H et al.; Some remarkable studies of human cell culture have contributed to many basic and applied sciences on "normal human cells"; developments of biological products or physiologically activated substances . In this reviews, some problems concerning in vitro culture systems were discussed and recent advances on the researches of normal human cells were described. Immunol Lett, 1988 Jun, 18(2), 119 - 24 Three-dimensional collagen matrices as cell culture substrata affect the generation of alloreactive cytotoxic T lymphocytes; Kubota K et al.; Primary one-way mixed lymphocyte cultures (MLC) of C3H/He responder and DBA/2 stimulator were performed in three-dimensional (3-D) collagen matrices and the generation of alloreactive cytotoxic T cell (CTL) responses was compared to those in MLC which were done on usual plastic surfaces or on collagen-coated plastic surfaces . MLC in the 3-D collagen matrices were found to generate strong CTL responses . Flow cytometric analysis of Lyt-2 and L3T4 antigen expressions on the effector cells showed that the Lyt-2/L3T4 ratios were substantially higher in the 3-D collagen matrices, and that a larger proportion of the cells in the 3-D collagen matrices were Lyt-2+ lymphoblasts . These results indicate that the milieu of the 3-D collagen matrices favors the proliferation of Lyt-2+ lymphocytes, and suggests that cell-to-matrix interactions in 3-D collagen matrices may play a regulatory role in the maturation process of alloreactive CTLs. In Vitro Cell Dev Biol, 1988 Jun, 24(6), 491 - 9 Primary liver cell cultures grown on gas permeable membrane as source for the collection of primary bile; Petzinger E et al.; Isolated rat hepatocytes maintained in primary culture on gas permeable membrane for 20 h form monolayers and establish at their cell borders a network of canaliculi (approximate diameter 3.5 micron) . In the presence of the known choleretic bile acid dehydrocholate, dilation of canaliculi occurs . When nonfluorescent carboxyfluorescein diacetate ester is added to the culture medium, fluorescent carboxyfluorescein appears in the intracanalicular space . In the dilated state, fluid containing the fluorescent compound could be collected from the canaliculi by puncture with a micropipette . The intracanalicular space shows a negative electrical potential difference of 31 mV in reference to the bath solution and is 13.5 mV more positive with reference to recordings from the cytosol of cultured rat hepatocytes . Cultured rat hepatocytes grown on gas permeable membrane are energetically stable over 3 d . On Day 4, ATP levels increase markedly, whereas Na+-K+-ATPase activity declines . Ionic composition of hepatocytes, as measured by electronprobe element analysis on cryosection samples, does not change markedly during monolayer formation . With formation of bile canaliculi, the activity of alkaline phosphatase rapidly increases within 24 h and is stable for the next 3 d . Within that time the activity of gamma-glutamyltranspeptidase, however, increases steadily, reaching a 1.6-fold higher activity than freshly isolated hepatocytes . Bile acids appear in the culture supernatant after 1 d . When unconjugated {14C}cholic acid is added to the cultures the supernatant contains also {14C}tauro- and {14C}glycocholic acid, indicating the preservation of conjugation capacity in these cultures . Total bile acid concentrations in the supernatant increase from 5 to 26 microM on Day 4 . The cultures do not secrete alpha-fetoprotein . Monolayer cultures of hepatocytes in the presence of choleretic bile acids seem to be a suitable model system to collect and to analyze the composition of primary bile . In conjunction with the electrical parameters, it is possible to describe directly properties of bile secretion at the canalicular pole of the intact hepatocyte. Diagn Microbiol Infect Dis, 1988 Jun, 10(2), 121 - 4 Detection of cytomegalovirus in clinical specimens using shell vial centrifugation and conventional cell culture; Forbes BA et al.; Shell vial centrifugation and conventional cell culture methods for detecting cytomegalovirus (CMV) were compared in clinical specimens . Our data confirm that the shell vial centrifugation method is more sensitive and rapid than conventional cell culture; however, due to the shell vial method's problems with toxicity to the fibroblast monolayer, both methods must be performed if all specimens positive for CMV are to be detected. J Clin Microbiol, 1988 Jun, 26(6), 1233 - 5 Effect of dexamethasone on detection of herpes simplex virus in clinical specimens by conventional cell culture and rapid 24-well plate centrifugation; Woods GL et al.; During a 4-month period, two methods for rapid detection of herpes simplex virus (HSV) were examined: (i) pretreatment of A549 cells with dexamethasone for conventional tissue culture (277 specimens) and (ii) 24-well plate centrifugation using A549 cells with and without dexamethasone pretreatment and staining with serotype-specific monoclonal antibodies (Syva Co., Palo Alto, Calif.) after incubation for 16 to 18 h (153 specimens) . By conventional tube cell culture, both with and without dexamethasone, HSV was identified in 88 of 277 (32%) specimens . Significantly more specimens were positive for HSV at 24 h (46 versus 27 specimens) and at 48 h (a total of 72 versus 59 specimens) (P less than 0.0001) in dexamethasone-treated A549 cells . Of the 153 specimens tested by conventional culture and 24-well plate centrifugation, HSV was detected in 44 (29%) by conventional culture, and by 24-well plate centrifugation with and without dexamethasone, HSV was detected in 32 (21%) and 30 (20%) specimens, respectively . The sensitivity, specificity, and positive and negative predictive values of 24-well plate centrifugation with A549 cells for detection of HSV were 73 (71% without dexamethasone), 100, 100, and 90%, respectively . In conventional tube cell culture, pretreatment of A549 cells with dexamethasone results in more rapid detection of HSV . Centrifugal inoculation of dexamethasone-treated and untreated A549 cells in 24-well plates and staining with monoclonal antibodies after incubation for 16 to 18 h is an insensitive means to detect HSV in clinical specimens and should not replace conventional tube cell culture. Epidemiol Infect, 1988 Jun, 100(3), 481 - 92 Isolation and characterization of rotavirus from feral pigeon in mammalian cell cultures; Minamoto N et al.; Avian rotaviruses were isolated from feral pigeon faeces treated with trypsin using roller tube cultures of mammalian cells . Two pigeon strains, designated as strains PO-8 and PO-13, produced a marked cytopathic effect (CPE), small intracytoplasmic inclusion bodies and high titres of infectious particles in infected MA-104 and MDBK cell lines without cell adaptation and roller drum apparatus . The pigeon rotaviruses shared a common group specific antigen with the Lincoln strain of bovine rotavirus by indirect immunofluorescence, but differed from both the Lincoln strain and the Wa strain of human rotavirus in neutralization tests . The RNA segment profile of this virus on polyacrylamide gel electrophoresis differed from that of group A mammalian rotaviruses . The results of a serological survey suggested that antibody to pigeon rotaviruses was widespread in avian species in Japan. Mutat Res, 1988 Jun, 199(2), 353 - 68 The study of multi-stage carcinogenesis in retinoblastoma and familial polyposis coli patient-derived skin fibroblast cell culture systems; Kinsella AR; Carcinogenesis is considered to be a multi-step process comprising 'initiation', 'promotion' and 'conversion' events . Skin fibroblasts from patients with hereditary retinoblastoma (RB) and familial polyposis coli (FPC) were chosen for study since their predisposition to the tumour may be due to an inherited 'initiation' event which is present in every cell . Experiments involving skin fibroblasts from FPC patients showed certain of these cells to grow in semi-solid medium following treatment with the complete carcinogen N-methyl-N'-nitro-N-nitrosoguanidine (MNNG) and the tumour promoter 12-O-tetradecanoylphorbol-13-acetate (TPA) alone . When tested prior to the commencement of the experiments the FPC patient cell populations had shown no strong predisposition to malignant transformation as assessed by increased saturation densities, reduced serum requirements, altered migration patterns in collagen gels, anchorage-independent growth and tumourigenicity in nude mice . Following carcinogen or promoter treatment, apart from exhibiting low-level frequencies of anchorage-independent growth, the cells appeared no more transformed than they were before . Parallel cytogenetic studies showed TPA to increase both tetraploidy and the chromosome-aberration frequency during the course of these transformation studies . However, the FPC cell clones induced by TPA to grow in semi-solid medium were, at best, considered to be only partially transformed when their properties were compared with those of tumour-derived cell lines. J Virol, 1988 Jun, 62(6), 2050 - 8 Coevolution of cells and viruses in a persistent infection of foot-and-mouth disease virus in cell culture; de la Torre JC et al.; Virus and cells evolve during serial passage of cloned BHK-21 cells persistently infected with foot-and-mouth disease virus (FMDV) . These carrier cells, termed C1-BHK-Rc1 (J.C . de la Torre, M . Davila, F . Sobrino, J . Ortin, and E . Domingo, Virology 145:24-35, 1985), become constitutively resistant to the parental FMDV C-S8c1 . Curing of late-passage C1-BHK-Rc1 cells of FMDV by ribavirin treatment (J.C . de la Torre, B . Alarcon, E . Martinez-Salas, L . Carrasco, and E . Domingo, J . Virol . 61:233-235, 1987) did not restore sensitivity to FMDV C-S8c1 . The resistance of C1-BHK-Rc1 cells to FMDV C-S8c1 was not due to an impairment of attachment, penetration, or uncoating of the particles but to some intracellular block that resulted in a 100-fold decrease in the amount of FMDV RNA in the infected cells . FMDV R59, the virus isolated from late-passage carrier cells, partly overcame the cellular block and was more cytolytic than FMDV C-S8c1 for BHK-21 cells . Sequencing of the VP1 gene from nine viral clones from C1-BHK-Rc1 cells showed genetic heterogeneity of 5 X 10(-4) substitutions per nucleotide . Mutations were sequentially fixed during persistence . In addition to resistance to FMDV C-S8c1, C1-BHK-Rc1 cells showed a characteristic round cell morphology, and compared with BHK-21 cells, they grew faster in liquid culture, were less subject to contact inhibition of growth, and had an increased ability to form colonies in semisolid agar . Reconstitution of a persistent infection was readily attained with late-passage C1-BHK-Rc1 cells and FMDV C-S8c1 or FMDV R59 . The results suggest that coevolution of BHK-21 cells and FMDV contributes to the maintenance of persistence in cell culture. J Pharm Pharmacol, 1988 Jun, 40(6), 437 - 8 The stable prostacyclin-analogue, iloprost, unlike prostanoids and leukotrienes, potently stimulates cyclic adenosine monophosphate synthesis of primary astroglial cell cultures; Seregi A et al.; The effect of different eicosanoids on adenosine-3', 5'-cyclic-monophosphate (cAMP) accumulation in primary astroglial cell cultures prepared from newborn rat brain was studied . The stable prostacyclin-analogue, iloprost, effectively stimulated cAMP synthesis in a concentration-dependent, saturable manner, the EC50 being about 3 x 10(-8) M . Prostaglandin (PG) E2 was less potent, without reaching plateau even at 10(-5) M . Prostaglandins D2 and F2 alpha, and the stable thromboxane A2-analogue, U 46619, as well as leukotrienes (LT) B4, C4, D4 and E4 were not effective and did not attenuate basal or isoprenaline (10(-8) M)-stimulated astroglial cAMP formation . This is the first indication for the existence of a prostacyclin receptor coupled positively to the adenylate cyclase in astrocytes . Other eicosanoids are unlikely to be involved in receptor-mediated regulation of astroglial cAMP levels. Brain Res, 1988 May 24, 449(1-2), 54 - 60 Effects of alpha-keto-delta-guanidinovaleric acid on inhibitory amino acid responses on mouse neurons in cell culture; De Deyn PP et al.; The experimentally proven convulsant alpha-keto-delta-guanidinovaleric acid (alpha-K-delta-GVA) was applied to mouse spinal cord neurons in primary dissociated cell culture to assess its effects on postsynaptic gamma-aminobutyric acid (GABA)- and glycine (GLY)-responses . Intracellular microelectrode recording techniques were used . alpha-K-delta-GVA reversibly inhibited both GABA- and GLY-responses in a concentration-dependent manner . The effect of alpha-K-delta-GVA on GABA-responses was not antagonized by co-application of the benzodiazepine receptor antagonist CGS 9896 . The results suggest that alpha-K-delta-GVA inhibited responses to the inhibitory neurotransmitters GABA and GLY by blocking the chloride channel . This action might underlie the convulsant effect of this compound in rabbit . The possible pathophysiological importance of alpha-K-delta-GVA in hyperargininemic patients is discussed. Int J Cancer, 1988 May 15, 41(5), 762 - 6 Enzymatic elimination of O6-ethylguanine from the DNA of ethylnitrosourea-exposed normal and malignant rat brain cells grown under cell culture versus in vivo conditions; Huh NH et al.; The developing rat brain exhibits a pronounced susceptibility to the tumorigenic effect of ethylnitrosourea (EtNU) and an extremely low repair activity for the DNA alkylation product O6-ethylguanine (O6-EtGuar-) . We have recently found that a collection of malignant neural cell lines originating from prenatal BDIX-rat brain cells were all highly O6-EtGua repair-proficient (O6-EtGuar+) . Subcloned lines showed considerable variability of the repair capacity, suggesting instability of the O6-EtGua repair phenotype . Using one of the subcloned lines (BT3Caf) as a model, we show here that BT3Caf cells grown in monolayer culture repair O6-EtGua much more rapidly than those grown in the form of s.c . tumors in BDIX-rats (whereas O4-ethylthymine is not repaired under either condition) . Furthermore, normal prenatal BDIX-rat brain cells (O6-EtGuar- in vivo) gradually acquire an O6-EtGuar+ phenotype upon transfer to long-term monolayer culture . The cellular capacity for enzymatic DNA repair is of particular relevance in relation to both the malignant transformation of normal cells and the therapeutic inactivation of cancer cells by DNA-reactive drugs . Further analyses are thus required of the molecular mechanisms controlling the expression of DNA repair enzymes as a function of cell differentiation, in terms of the cellular response to altered microenvironmental conditions, and in search for possibilities to reduce the repair capacity of cancer cells. FEBS Lett, 1988 May 9, 232(1), 12 - 6 Effect of Graves' IgG on gene transcription in human thyroid cell cultures . Thyroglobulin gene activation; Kung AW et al.; In Graves' disease (GD) the presence of antibodies to the thyroid stimulating hormone (TSH) receptor leads to stimulation of the thyroid gland . The thyroid stimulating activity of Graves' IgG is normally ascertained by bioassays measuring cAMP production . We have investigated the effect of Graves' IgG on the quantitative activation of thyroglobulin (TG) gene in cultured human thyroid cells by RNA hybridisation . TG mRNA expression was activated by TSH and Graves' IgG . Nuclear transcription assays showed that the increase in cytoplasmic mRNA levels was due to increased transcription of TG specific mRNA in nuclei of thyroid cells . Whilst TSH led to a dose dependent increase in TG mRNA levels, Graves' IgG led to a variable activation of TG gene . A significant correlation between the increased TG mRNA transcription and cAMP production was observed with Graves' IgG . Thus the activation of the TG gene by Graves' IgG occurs in parallel with elevation of cAMP. Biochemistry, 1988 May 3, 27(9), 3175 - 82 Elastin mRNA levels and insoluble elastin accumulation in neonatal rat smooth muscle cell cultures; Barone LM et al.; Insoluble elastin accumulation, elastin mRNA translational efficiencies, and elastin mRNA levels were evaluated in cultures of neonatal rat aortic smooth muscle cells grown for several days in consecutive passages . When the products of in vitro translation were immunoprecipitated with an anti-alpha-elastin antibody, a single 79,000-Da protein was obtained . Northern blot analysis also indicated an elastin mRNA species corresponding to approximately 4.2 kilobases . Insoluble elastin accumulation increased in cells cultured for 7-21 days in first through fourth passages, while with one exception, relative levels and translational activity of elastin mRNA decreased with time in culture . The data indicated that a simple relationship between elastin accumulation and elastin mRNA levels was not evident. Prostaglandins Leukot Essent Fatty Acids, 1988 May, 32(2), 75 - 81 The effect of temporary hypoxia on prostaglandin synthesis in mouse brain cell cultures during development; Kaeser F et al.; Cultures of dissociated brain cells of new born mice represent a model for the study of brain development . One and two weeks old, they correspond in regard to oligodendrocyte differentiation to about the developmental stage of a human newborn and a six months old infant respectively . Such cultures were used to establish the developmental prostaglandin pattern and to study early and late recovery of prostaglandin synthesis from temporary hypoxia . Basal and bradykinin stimulated prostaglandin release were examined . Most prominently in stimulated release, the developmental prostaglandin pattern at one week showed a prevalence of PGE2 (33 +/- 4%) over PGD2 (12 +/- 5%), which in two weeks old cultures changes to an opposite distribution (PGE2 10 +/- 4%; PGD2 25 +/- 6%) . This change goes parallel with the number and differentiation of oligodendrocytes . During the first day post hypoxia, imposed at the end of one week, the production of 6 oxo PGF1 alpha, PGE2, PGD2 and TXB2 was significantly decreased in two study series and increased compared to control in another . Since the arachidonic acid uptake was the same in all three series, this differential observation indicates differential early recovery . 8 days post hypoxia (late recovery), PG release was not different from control, indicating complete recovery at that time . During early recovery from hypoxia on 14 days old cultures, basal PG release was not significantly different from control, however bradykinin stimulated release was significantly inhibited in all three series . This may indicate that mainly regulatory influences on PG release in older cultures are compromised by hypoxia.(ABSTRACT TRUNCATED AT 250 WORDS) J Virol Methods, 1988 May, 20(1), 33 - 8 A simple planimetric method to quantify cytotoxicity in cell culture monolayers; Richards GP et al.; Planimetry was shown to be a rapid, simple and reproducible method to quantify gross cytotoxicity in cell culture monolayers . A shellfish extract prepared from blue mussels (Mytilus edulis) was cytotoxic to Buffalo green monkey kidney cells . Exposure of cells to mussel extracts for 1, 2 and greater than or equal to 4 h followed by agar (plaque assay) overlays produced 34, 87, and 100% destruction of the monolayers, respectively, within 72 h . Planigraphs, prepared from tracing areas of cytotoxicity onto sheets of paper, served as permanent records of toxicity which were easily measured by planimeter experienced and nonexperienced scientists . Planimetric procedures may have utility in measuring biological and chemical toxicity as well as physiological stress in cell monolayers. Endocrinology, 1988 May, 122(5), 1786 - 800 The effect of simultaneous versus sequential estradiol and progesterone treatments on prolactin production in monkey pituitary cell cultures; Bethea CL et al.; Dispersed monkey pituitary cells were cultured in serum-free medium and on extracellular matrix for 30 days . After 10-14 days with only insulin, transferrin, and selenium (ITS), the addition of estradiol (E) significantly increased PRL secretion compared to that in vehicle-treated controls . Simultaneous addition of E plus progesterone (P) increased PRL secretion similarly to E alone . PRL secretion in cultures treated with E plus P after 10 days induction by E was also similar to that in cultures maintained continuously in E . PRL secretion declined in wells switched from E to P and in wells switched from E back to vehicle, relative to that in wells maintained continuously in E . Estrogen receptors (ER) were detected in whole pituitary tissue and in serum-free pituitary cultures with a monoclonal antiestrogen receptor antibody (H222) in a sucrose density gradient shift assay . Immunocytochemical staining for ER with the same antibody also showed positive cell nuclei in serum-free pituitary cultures . In summary, ER are maintained in monkey pituitary cells during tissue culture, and PRL production can be further increased by E treatment after long term serum-free culture . Neither simultaneous nor sequential E plus P treatment alters PRL secretion compared to E alone. J Reprod Fertil, 1988 May, 83(1), 249 - 61 Isolation, cell culture and immunocytochemical characterization of oviduct epithelial cells of the cow; Joshi MS; Incubation of cow oviducts flushed with 0.1 mg collagenase/ml, for 90 min helped to dislodge large numbers of ciliated and secretory cells . About 90-95% of the isolated epithelial cells were viable . The epithelial cells suspended in DMEM:F-12 + 10% serum attached to the plastic culture dish in 18-20 h after seeding . The ciliated cells which attached to the plastic dish lost their cilia after 4-5 days in culture . The attached cells, which proliferated to form a confluent monolayer 8-10 days after seeding in a 35-mm dish, could be subcultured at least 3 successive times . Some cell aggregates which did not attach to the culture dish proliferated into floating balls of cells . The ciliated cells in the unattached floating colonies maintained the ciliary movement for 9-10 days in the same culture medium . The primary cultures of the ciliated and the secretory cells maintained most of the histoarchitecture observed in intact epithelium . The secretory cells maintained their secretory activity of specific proteins in culture as indicated by immunocytology . The cultured cells contained keratin, a specific cytoskeletal component of epithelial cells. Vopr Virusol, 1988 May-Jun, 33(3), 338 - 42 {Analysis of the possible mechanisms for the persistence of the vaccinal strain of the measles virus in a human cell culture}; Andzhaparidze OG et al.; The mechanisms (factors) of the measles virus vaccine L-16 strain persistence in HEp-2 cell culture were analysed . Among the known mechanisms, most likely is the reduction of the cell-destroying properties of the persisting virus due to mutations in nucleoprotein gene manifested by changes of the isoelectric point of NP protein and temperature sensitivity of its synthesis. Vopr Virusol, 1988 May-Jun, 33(3), 324 - 7 {Index of the proliferative activity of a cell culture in assessing the cytotoxicity of antiviral chemical preparations}; Lymar' VT et al.; The in