Cell cultures

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 influence of cytotoxicity of a number of antiviral compounds on the cell culture functional activity was studied by evaluating the changes in the kinetic cell growth parameters . Significant differences between the values of cytotoxicity were found with ribamide in cell lines of different origins . The differences in the measured values of cytotoxicity of the four antiviral compounds under study were demonstrated using the proposed proliferation cytotoxicity criterion (PCT).

Vestn Khir Im I I Grek, 1988 May, 140(5), 83 - 6
{Transplantation of pancreatic islet cell cultures in the treatment of suppurative surgical diseases in patients with diabetes mellitus}; Rozental' RL et al.; The authors have analyzed the possibility to use transplantation of the pancreatic islet cell cultures of pig embryos as a method of preoperative preparing patients with type II diabetes mellitus having pyo-surgical diseases.

Mol Cell Endocrinol, 1988 May, 57(1-2), 61 - 7
Prolonged exposure to androgens suppresses follicle-stimulating hormone-induced aromatase activity in rat Sertoli cell cultures; Verhoeven G et al.; We studied the effects of androgens on basal and FSH-stimulated aromatase activity in Sertoli cell-enriched monolayers . Daily exposure to androgens from the first day of culture results in a time- and dose-dependent decrease in inducible aromatase activity . The inhibition is observed whether FSH, L-isoproterenol or (Bu)2cAMP is used as inducer of the aromatase activity . Basal activity is not affected by preincubation with androgens and the inhibitory effect is not observed after short-term exposure (24 h) to these hormones . The ability of different androgens to decrease inducible aromatase activity does not depend on their ability to serve as a substrate for the aromatase but parallels their ability to stimulate the production of androgen-binding protein . Moreover, the effect of testosterone is neutralized by the antiandrogen cyproterone acetate, suggesting that it is mediated by the androgen receptor . These data suggest that testicular androgens may be responsible for the decrease in FSH-inducible aromatase activity observed in intact rats between days 10 and 30 of life . Similarly, the removal of these androgens during the preparation of Sertoli cell cultures may explain the spontaneous increase in inducible aromatase activity observed when these cultures are maintained in the absence of androgens.

Trop Anim Health Prod, 1988 May, 20(2), 103 - 8
Use of the caprinised strain of rinderpest virus for potency testing an attenuated cell culture rinderpest vaccine; Guillemin F et al.; The caprinised strain of rinderpest virus was inoculated into goats to produce a challenge stock . These goats were kept with control animals (goats, sheep, calves) . In this trial the caprinised strain was shown to have a mild pathogenicity for goats and it spread to one of two contact goats but not from goats to other species . The caprinised strain was then tested on cattle where a febrile reaction was observed . The caprinised strain also did not spread between cattle . The cattle vaccinated with a freeze-dried vaccine produced from the attenuated Kabete RBKO strain on bovine kidney cells were then challenged with the caprinised strain with good results.

J Med Virol, 1988 May, 25(1), 91 - 103
Synthesis of human rotavirus polypeptides in cell culture; Heath RL et al.; The replication strategy of a human serotype 1 rotavirus, adapted to rapid growth in CV-1 cells, was investigated . A single cycle growth curve revealed eclipse and latent periods of 3 and 4 hours, respectively . Although the extent of reduction of host cell protein synthesis was directly related to the multiplicity of infection of the virus, incorporation of actinomycin D and excess NaCl into the medium resulted in significant reduction in host cell background and enabled observation of viral polypeptides as early as 2 hours post infection . Five polypeptides were found to be structural components of the virion, and a further eight appeared to be nonstructural proteins or intermediates . Five polypeptides were glycosylated during virus replication, but only one of these, VP7, was a definite structural glycoprotein . Pulse-chase experiments revealed that four low molecular weight polypeptides underwent post-translational modifications.

Endocrinology, 1988 May, 122(5), 2257 - 64
Testosterone-enhanced oxygen-mediated degradation of P-450(17) alpha in mouse Leydig cell cultures; Perkins LM et al.; The present study investigated the process by which the microsomal cytochrome P-450 17 alpha-hydroxylase enzyme (P-450(17) alpha) activity in Leydig cells is decreased by steroid products in an oxygen-dependent manner . Leydig cells were maintained in primary culture at ambient (19% O2) or reduced (1% O2) oxygen tension and treated with 8-bromo-cAMP or steroids for 48 h . The amount and activity of P-450(17) alpha present in the treated cells were measured to determine whether changes in the activity of the enzyme due to treatment correlated with changes in the amount of the enzyme protein . At ambient oxygen tension the amount of P-450(17) alpha declined in Leydig cells treated with cAMP, and this decrease could be prevented when cultures were maintained at reduced oxygen tension . Treatment of cultures with testosterone caused an oxygen-sensitive decrease in the amount of the enzyme similar in extent to the cAMP-induced decrease . Reductions in the amount of P-450(17) alpha corresponded to reductions in the activity of the enzyme . The decline in the amount of P-450(17) alpha was due to increased decay of the enzyme protein and not to a decrease in the rate of synthesis of P-450 . In contrast to testosterone, neither estradiol nor cortisol affected the amount of P-450(17) alpha . The data are consistent with the proposal that steroid products acting as pseudosubstrates cause decreases in P-450(17) alpha activity by enhancing oxygen radical-mediated damage to the enzyme . The damaged enzyme resulting from this process is more prone to degradation than is the intact protein.

Mol Biol (Mosk), 1988 May-Jun, 22(3), 802 - 6
{The effect of 3'-azido-3'-deoxynucleosides on the reproduction of AIDS virus in cell culture}; Galegov GA et al.; Action of three nucleoside analogues with azido-group on AIDS virus (human immunodeficiency virus--HIV) reproduction was studied in two cell species . Among these compounds there were 3'-azido-3'-deoxythymidine (Az-T), 3'-azido-3'-deoxyarabinothymidine (Az-AT) and 3'-azido-2',3'-dideoxy-5-methylcytidine (Az-dCMe) . All compounds prevent cells against HIV infection, but Az-T action was expressed more strongly . The reason for the lower activity of the Az-AT and Az-dCMe bases is probably stipulated by their poor conversion to the corresponding 5'-triphosphates . Az-T 5'-triphosphate blocks HIV reproduction due to the inhibition of the viral reverse transcriptase.

J Neurosci Res, 1988 May, 20(1), 54 - 63
Effect of epidermal growth factor on the labeling of the various RNA species and of nuclear proteins in primary rat astroglial cell cultures; Avola R et al.; This study investigated the effects of epidermal growth factor (EGF) on the labeling of various RNA species and of nuclear proteins in primary rat astroglial cell cultures . After 12 hours of EGF treatment in serum-free medium or chemically defined medium, significant increase in RNA labeling, and also in acid-soluble radioactivity and RNA content, was observed . The ratio RNA/DNA was significantly higher in EGF-treated cultures compared with controls . Ribosomal RNAs (28S and 18S), polyadenylated, and nonpolyadenylated RNAs showed a higher specific radioactivity in EGF-treated cultures . Among the nuclear proteins, the labeling of basic proteins was enhanced by EGF treatment, whereas that of total nuclear acidic protein (NHPs) was less modified, except for some NHPs separated by gel electrophoresis with a molecular weight (MW) approximately 95-83 and 44 kd, which were significantly more labeled in EGF-treated cultures.

J Biochem Biophys Methods, 1988 May, 16(1), 63 - 73
Evaluation of growth rate in adhering cell cultures using a simple colorimetric method; Pelletier B et al.; In this paper we present a simple colorimetric method for evaluating cell growth in adhering cell cultures . This technique is based on the staining of basophilic cellular compounds (mainly nucleic acids) with methylene blue . To check its reliability, we have compared the quantity of dye fixed by the cells with the number of cells released from the culture substratum by trypsinization, and with the total cellular protein content determined by the Lowry's method . In these experiments we have used human skin fibroblasts and human epithelial cells derived from the epithelium of the full-term umbilical cord . Like other alternative methods, the methylene blue colorimetric technique can be used in 96-well microplates and allows the rapid collection of large amounts of data . As an illustration, we report on the selection of optimal composition of culture medium for human skin fibroblasts.

Neuropharmacology, 1988 Apr, 27(4), 337 - 43
Negative feedback regulation of the content of proenkephalin mRNA in chromaffin cell cultures; Naranjo JR et al.; The content of proenkephalin messenger RNA (PEmRNA) in cultured bovine adrenal chromaffin cells was reduced in the presence of reserpine (1 nM to 0.1 microM) with a return to basal levels 3 days after removal of the drug . In these cells, the basal release of Met5-enkephalin-Arg6-Phe7 immunoreactivity (MERF-IR) into the medium was significantly decreased when the cultures were pretreated with 0.2 microM reserpine for 3 days . The addition of 0.1 microM etorphine for 3 days also decreased the basal release of MERF-IR without depleting stores of catecholamines . Neither drug modified the total (cells + medium) amount of MERF-IR . In contrast, reserpine was without effect on levels of PEmRNA or release of Met5-enkephalin immunoreactivity (ME-IR) in primary cultures of the striatum of the fetal rat . The present data establish a correlation between inhibition of the secretion of enkephalin and reduced accumulation of its specific mRNA, suggesting a negative feedback inhibition by low molecular weight enkephalins.

Brain Res, 1988 Apr 1, 467(2), 205 - 15
Morphology and electrophysiology of ventral mesencephalon nerve cell cultures; Ort CA et al.; In primary neuron cultures obtained from ventral mesencephalon of mouse fetuses, approximately 10-30% of the neurons were dopaminergic, as demonstrated by a rapid glyoxylic acid histofluorescence procedure, and another 10-30% were GABAergic as demonstrated by autoradiography . Resting membrane potentials averaged -58 mV and input resistances averaged 188 M omega . Action potential (AP) firing patterns were of 3 types: in 49% of cells, depolarizing current elicited bursts of APs of constant amplitude, duration, and interspike interval (Type 1); in 44% of cells, bursts consisted of APs of decreasing amplitude, increasing duration, and increasing interspike interval (Type 2); and in 7% of cells, bursts were initiated by a single high amplitude, short duration AP followed by a series of lower amplitude longer duration APs that progressively increased in amplitude and decreased in duration and interspike interval (Type 3) . Calcium APs of two distinct types, differing in duration and rate of rise, were observed when cultures were exposed to tetrodotoxin . Abundant postsynaptic activity was recorded . Simultaneous intracellular recording between pairs of cells demonstrated reciprocal innervation . The neurotransmitter antagonists haloperidol, bicuculline, naloxone, atropine, hexamethonium and pirenzepine affected synaptic activity and/or resting membrane potential of some of the cultured neurons.

Brain Res, 1988 Apr 1, 467(2), 193 - 204
Subsets of GABAergic neurons in dissociated cell cultures of neonatal rat cerebral cortex show co-localization with specific modulator peptides; Alho H et al.; The GABAergic properties of dissociated neurons from cerebral cortex of neonatal rats were studied in primary culture using electrophysiological, biochemical and immunohistochemical methods . Cultured neurons had a resting potential of -50 to -60 mV and exhibited spontaneous excitatory and inhibitory synaptic currents . Non-spontaneous (elicited) ionic currents were produced by direct application of GABA and glutamate . Cultures contained measurable amounts of GABA from the first day in culture; GABA content reached a plateau around the 10th day of culture, and continued, nearly unchanged, until the 21st day of culture . Immunohistochemistry showed that 45% of the total cells in culture contained glutamic acid decarboxylase (GAD) . Octadecaneuropeptide (ODN), a putative neuroregulatory peptide for benzodiazepine recognition sites, was present in approximately 28% of all neurons . Ninety-three percent of ODN-positive cells demonstrated GABAergic properties as well by displaying GAD-immunoreactivity . The peptide GABA-modulin (GM), a putative GABA receptor modulator, was found in about 75% of all neurons, with a further 65% of these cells exhibiting GAD-immunoreactivity . Cells immunopositive for neuropeptide Y (NPY), somatostatin (SRIF), and cholecystokinin-octapeptide (CCK), were found at much lower incidence (1-4%) . Double-labelling studies showed that 90-97% of the cells positive for NPY, SRIF and CCK were also positive for GAD . Cells immunoreactive with serotonin or tyrosine hydroxylase were not detected . We suggest that primary cultures of neonatal cortical neurons may provide a useful experimental model to investigate the function and the modulation of GABAergic neurotransmission in the cerebral cortex.

Toxicology, 1988 Apr, 49(1), 155 - 63
Toluene induces changes in the morphology of astroglia and neurons in striatal primary cell cultures; Hansson E et al.; Toluene (4.7-150) mumol per ml) was added for 30 or 60 min to astroglial and neuronal primary cell cultures from rat striatum and changes in cell morphology were analyzed by light microscopy . After 60 min incubation in 40 mumol toluene/ml, the cell bodies of the astrocytes appeared contracted, and their processes and nuclei were clearly visible . At higher doses of toluene the astrocytes seemed to be flattened and major cell damage was visualized by the uptake of vital dyes . The neurons, however, became affected and judged by morphological criteria only at the higher toluene doses . In conclusion, toluene induced morphological changes in primary astrocyte cultures and also in primary neuronal cultures at higher toluene concentrations.

Am J Obstet Gynecol, 1988 Apr, 158(4), 846 - 53
Physicochemical characterization and functional activity of fibroid prolactin produced in cell culture; Chapitis J et al.; Evidence from our laboratory with the use of cultured (primary and passaged) cells has extended our initial observation that human uterine fibroid is an extrapituitary source of prolactin . Fibroid prolactin antigen in conditioned medium reacted specifically in radioimmunoassay for human pituitary prolactin . Control experiments demonstrated that the radioimmunoassay results were not spurious due to degradation of tracer 125I-labeled prolactin . Immunoparallel dilution curves indicated antigenic relatedness of pituitary and fibroid prolactin . In a calibrated Sephadex G-100 column, fibroid prolactin eluted in the same region (20.3 to 20.9 kd) as purified pituitary prolactin . Glycosylated prolactin, detected by concanavalin A affinity column chromatography, appeared to constitute only a small percentage of fibroid prolactin made in culture . The ratio of fibroid prolactin bioactivity (lactogen Nb2 lymphoma bioassay) to antigen (radioimmunoassay) was 0.77 . These data indicate that human uterine fibroid tissue produces a molecule similar to or, perhaps, identical with pituitary prolactin.

Exp Cell Res, 1988 Apr, 175(2), 404 - 8
Induction of intercellular communications in epithelial cell cultures; Sharovskaja YuYu et al.; A popular criterion of cell-cell communication in tissue cultures is dye coupling: the ability of the injected fluorescent dye of low molecular weight to be transferred from one cell to another . We report about a new factor which induces cell-to-cell dye coupling in previously uncoupled epithelial sheets . Paradoxically it is the standard fluorescent microscopy itself (that is, blue light of 320- to 480-nm wavelength) which induces rapid morphological alterations of cell culture followed by the transfer of fluorescent dye from one cell to another . Thus monitoring cell-cell dye coupling by fluorescent microscopy may itself induce the dye coupling in previously uncoupled epithelial cells.

J Parasitol, 1988 Apr, 74(2), 288 - 93
Cultivation of Cryptosporidium baileyi: studies with cell cultures, avian embryos, and pathogenicity of chicken embryo-passaged oocysts; Lindsay DS et al.; Sporozoites of Cryptosporidium baileyi did not undergo development in primary cell cultures from either avian or mammalian hosts, or in mammalian cell lines . Oocysts of C . baileyi produced infections resulting in complete development to sporulated oocysts in chicken embryos and embryos of 8 other avian species examined . Inoculation of 4 X 10(5) oocysts was not pathogenic for avian embryos as evidenced by the lack of gross lesions or death . Oocysts obtained after C . baileyi had been passaged 10 times (first experiment) or 20 times (second experiment) in chicken embryos still caused clinical respiratory disease and gross airsacculitis when inoculated intratracheally into 2-day-old broiler chickens . Oocysts that had been passaged 10 times in chicken embryos were similarly pathogenic for 4-day-old turkeys after intratracheal inoculation.

Carcinogenesis, 1988 Apr, 9(4), 657 - 60
Glucolimnanthin, a plant glucosinolate, increases the metabolism and DNA binding of benzo{a}pyrene in hamster embryo cell cultures; Baird WM et al.; Glucosinolates are common components of cruciferous vegetables that can be hydrolyzed during food processing to yield isothiocyanates, some of which have been shown to inhibit the induction of mammary tumors in rats by 7,12-dimethyl-benz{a}anthracene . To determine how intact glucosinolates affect the metabolism of benzo{a}pyrene (B{a}P) in mammalian cells in culture, the effects of a series of glucosinolates on the metabolism and DNA binding of B{a}P were investigated in early passage Syrian hamster embryo cell cultures . Glucolimnanthin, a glucosinolate from Limnanthes douglasii increased the amount of B{a}P metabolized by the hamster embryo cell cultures during a 24 h exposure . The glucolimnanthin-treated cultures contained a higher proportion of B{a}P-phenol glucuronides and other water-soluble metabolites than control cultures . Cotreatment with glucolimnanthan and {3H}B{a}P for 24 h resulted in a greater than 2-fold increase in the amount of B{a}P bound to DNA and a 3-fold increase in the amount of deoxyguanosine adduct formed by reaction of 7R,8S-dihydroxy-9S,10R-epoxy-7,8,9,10-tetrahydroB{a}P {(+)-anti-B{a}PDE} . The glucolimnanthin was metabolized essentially completely within 24 h . An increase in B{a}P metabolism similar to that caused by glucolimnanthin was induced by cotreatment of hamster embryo cell cultures with m-methoxybenzyl isothiocyanate, a metabolite that can be formed from glucolimnanthin by enzymatic hydrolysis . These results indicate that the glucosinolate glucolimnanthin can increase the metabolic activation of B{a}P in mammalian cells in culture.

Zh Mikrobiol Epidemiol Immunobiol, 1988 Apr, (4), 21 - 5
{The sensitivity of a number of cell cultures to different strains of Rickettsia prowazekii}; Penkina GA et al.; The sensitivity of some cell cultures to different R . prowazekii strains (strain E with low pathogenicity, virulent strain Breinl, strains ERifRI and EVir) has been studied with a view to the selection of an adequate culture for growing these strains and the study of their biological properties . Experiments on titration in cells have revealed that 6- to 7-day primary and secondary irradiated quail fibroblasts and human amnion cells FL show the maximum sensitivity to all strains under study, comparable to that of chick embryos . The sensitivity of 6- to 7-day primary and secondary irradiated chick fibroblasts is faintly pronounced, and 24-hour chick and quail fibroblasts are still less sensitive . Cells FL have shown high sensitivity to strain E and mutant ERifRI in prolonged subculturing for 140 and 63 days (the term of observation) respectively after a single inoculation.

Diagn Microbiol Infect Dis, 1988 Apr, 9(4), 245 - 50
Comparison of the Abbott and Ortho enzyme immunoassays and cell culture for the detection of respiratory syncytial virus in nasopharyngeal specimens; Christensen ML et al.; A comparison of the Abbott Laboratories and the Ortho Diagnostic Systems Respiratory Syncytial Virus (RSV) Enzyme Immunoassays (EIA) and HEp-2 cell culture for the detection of RSV in 81 nasopharyngeal (NP) specimens from pediatric patients with lower respiratory tract infection was carried out . The sensitivity and specificity of the Abbott test compared to confirmed infection was 92.3% and 100.0%, respectively . The sensitivity and specificity of the Ortho test was 87.5% and 80.3%, respectively . We found the Abbott EIA test to be sensitive, specific, rapid, and easy to perform.

Biull Eksp Biol Med, 1988 Apr, 105(4), 481 - 3
{Regulation of hormonal secretion and DNA synthesis in the lactotrophs of the rat adenohypophysis in vitro in primary cell cultures}; Komolov IS et al.; Changes in DNA synthesis in lactotrophs of primary monolayer cultures of the rat pituitary cells were studied, using immunoperoxidase staining in combination with autoradiography . Pituitary cell cultures were treated for 3 days with thyroliberin (TRH), bromocriptine (CB154) or somatostatin (SRIF) . The proportion of lactotrophs labelled with 3H-thymidine in the total pool of labelled cells served as a criterion for the estimation of DNA synthesis in prolactin-secreting cells . Prolactin secretion by the same cultures was measured by homologous radioimmunoassay . TRH (10 ng/ml) stimulated DNA synthesis in the total population of pituitary cells, but not in lactotrophs . SRIF decreased selectively the proliferation of lactotrophs, but failed to depress or even stimulated DNA synthesis in some cell types of the rat pituitary gland in the cultures . The quantitative method of studying DNA synthesis in anterior pituitary may be used to evaluate the effects of a number of biologically active compounds on various cell systems.

Virology, 1988 Apr, 163(2), 299 - 307
Complete nucleotide sequence of a cell culture-adapted variant of hepatitis A virus: comparison with wild-type virus with restricted capacity for in vitro replication; Jansen RW et al.; To determine the molecular changes associated with adaptation of hepatitis A virus (HAV) to growth in cell culture, the genome of a cell culture-adapted variant of HM175 strain HAV (p16 HM175, 16th in vitro passage level) was molecularly cloned and the complete nucleotide sequence of the virus was determined . Compared with wild-type virus, p16 HM175 replicates efficiently in monkey kidney (BS-C-1) cells (approximately 58 RNA-containing particles per one infectious unit, compared with 2.4 x 10(5) for wild-type HM175) . The nucleotide sequence of p16 HM175 revealed a total of 19 mutations from the wild-type genome, including 5 mutations in the 5' nontranslated region, 1 mutation in the 3' nontranslated region, and 13 mutations predicting 8 changes in the amino acid sequences of HAV proteins . Only one amino acid substitution occurred among the capsid proteins (VP2), while others involved proteins 2A, 2B, 2C, VPg, and 3Dpol . When the sequence of p16 virus was compared with that reported previously for an independently isolated, cell culture-adapted variant of HM175 virus (J.I . Cohen et al., (1987) . Proc . Natl . Acad . Sci . USA 84, 2497-2501), there were three identical mutations in nontranslated regions of the RNA, and four mutations involving identical amino acids in proteins VP2, 2B, and 3Dpol . The distribution of these mutations within the genome suggests that changes in RNA replication may be of primary importance in adaptation of the virus to growth in vitro . These data are thus helpful in understanding the molecular basis of adaptation of HAV to cell culture and, since attenuation frequently accompanies adaptation of virus to growth in cell culture, may be of benefit in planning for attenuated vaccine development.

Biochemistry, 1988 Mar 22, 27(6), 2136 - 44
Structural characterization of heparan sulfate proteoglycan subclasses isolated from bovine aortic endothelial cell cultures; Kinsella MG et al.; Labeled heparan sulfate proteoglycans (HSPG) were isolated from wounded and confluent cultures of bovine aortic endothelial cells by nondegradative extraction with 4 M guanidine hydrochloride and detergent . HSPG were separated from more highly charged chondroitin or dermatan sulfate proteoglycans by ion-exchange chromatography, and subclasses of different hydrodynamic size were isolated by gel filtration . Three major subclasses of HSPG were characterized structurally with respect to the presence and relative size of protein core, the presence and amount of nonsulfated oligosaccharide, and size and structure of heparan sulfate (HS) chains . The largest (600-800-kDa) HSPG subclass (I), isolated from cell layers and media of confluent cultures, bears 38-kDa HS chains on an apparently heterogeneous class of relatively large glycoprotein cores . HSPG II (150-200 kDa), isolated from cell layer or media, has 22-kDa HS chains and smaller core glycoproteins (less than 50 kDa) . HSPG III, the subclass of smallest hydrodynamic size, has 13-kDa HS chains and a glycopeptide core of less than 15 kDa . All subclasses bear varying proportions of non-sulfated oligosaccharides of similar sizes . Comparisons of HS chain structure indicated that the different subclasses have similar proportions (49-55%) of N-sulfate, with both O-sulfate and highly N-sulfated blocks of disaccharide distributed similarly along HS chains . In addition, HS chains from subclasses II and III contain sequences that are insensitive to periodate oxidation or heparitinase digestion, suggesting that they contain increased proportions of iduronate.(ABSTRACT TRUNCATED AT 250 WORDS)

Lancet, 1988 Mar 19, 1(8586), 613 - 7
Fetal blood sampling in investigation of chromosome mosaicism in amniotic fluid cell culture; Gosden C et al.; 41 pregnancies in which mosaicism had been found on amniotic fluid culture (AFC) were investigated by fetal blood and repeat AFC karyotyping . Results of both investigations, and the infants, were normal in 15 of 16 cases of autosomal trisomy (including trisomies 8, 9, 13, 18, and 21) and in the 8 cases of sex chromosome mosaicism . In 1 case of trisomy 20 mosaicism the fetal blood karyotype and the infant were normal but the repeat AFC showed mosaicism . Abnormality was confirmed in 5 of 11 cases of mosaicism for structural chromosomal rearrangements and in 4 of 6 cases with de-novo supernumerary marker mosaicism . These findings indicate the potential danger of terminating pregnancies because of trisomic mosaicism in AFC, and the importance of identifying the clinical significance of certain chromosome rearrangements which, even in mosaic form, might lead to severe mental and developmental handicap.

Vopr Virusol, 1988 Mar-Apr, 33(2), 206 - 11
{Electron microscopy study of the nucleocapsid structure of the measles virus isolated from cell cultures with primary and chronic infections}; Lotte VD et al.; Electron microscopic examination of isolated intracellular measles virus nucleocapsids (NC) revealed a relationship between their structure, cell system, and the type of infection . Acute virus infection of Vero or Japanese quail embryo cells gave rise to the formation of linear NC strands with regularly and tightly stacked turns . Acutely infected L-41 or HEp-2 cells contained heteromorphous viral NC populations which included both typical and loosely packed NC . Persistently infected L-41 and Hep-2 cells predominantly contained NC of the latter type with the appearance of a "strings of beads".

Zentralbl Bakteriol Mikrobiol Hyg {A}, 1988 Mar, 267(4), 510 - 8
{Experiences with the demonstration of Mycoplasma in cell cultures}; Nicklas W et al.; Over an eight years period about 6200 cell cultures, sera, cell culture media and supernatants were routinely monitored for contamination with mycoplasmas, bacteria and fungi . Mycoplasmas were detected in 24.0% of 4443 samples which were checked for possible contamination . In 1742 samples from a laboratory, known to have only mycoplasma free cultures, 2 were positive, both samples having an external origin . The value of routine monitoring to prevent the introduction of mycoplasma was confirmed . Culture and direct fluorescent assay using the fluorochrome bisbenzimide (Hoechst 33258) yielded comparable results . The applicability and significance of both methods is discussed . In spite of a few disadvantages the culture method is considered to be superior to the fluorescence assay, but both methods should be employed in order to obtain sufficiently reliable results . The importance of appropriate methods for the detection of mycoplasmas is stressed because of their potential influence on experimental results . The probable sources of cell culture contamination are also discussed.

J Neurosci, 1988 Mar, 8(3), 746 - 53
Development and survival of neurons in dissociated fetal mesencephalic serum-free cell cultures: II . Modulatory effects of gangliosides; Leon A et al.; This paper analyzes the effects of exogenously supplied GM1 on the development, i.e., specific neurotransmitter uptake capability and survival, of the dopaminergic neurons present in fetal mouse-dissociated mesencephalic cells . Exogenous GM1, but not asialo-GM1, sialic acid, or the oligosaccharide chain of GM1, enhances in a time- and concentration-dependent manner the specific 3H-dopamine uptake (increase of the apparent Vmax and decrease of the apparent Km value) and the long-term survival of the dopaminergic neurons . The GM1 effects on the behavior of the dopaminergic neurons require the presence of cell-derived neuronotrophic influences present within the culture system and are associated with an increase in the response of the cells to the trophic influences . GM1 effects are not limited to dopaminergic neurons, and depend on the stable association of the ganglioside molecule with the cells . It is suggested that GM1 is not a trophic agent per se, but rather potentiates neuronotrophic activities and/or exerts independent influences to which neurons respond only if appropriately supported.

J Neurosci, 1988 Mar, 8(3), 733 - 45
Development and survival of neurons in dissociated fetal mesencephalic serum-free cell cultures: I . Effects of cell density and of an adult mammalian striatal-derived neuronotrophic factor (SDNF); Dal Toso R et al.; The use of CNS cultures for detection and quantification of neuronotrophic activity in the CNS has been analyzed . In particular the development, i.e., neurotransmitter uptake characteristics, and survival of dopaminergic and GABAergic neurons in fetal mouse (E13)-dissociated mesencephalic cells cultured in serum-free, hormone-supplemented medium have been assessed as a function of culture time and cell density . At all times, more than 98% of the cells were classified as neurons on the basis of immunocytochemical criteria . Results indicate that the increase of cell density in vitro significantly enhances specific high-affinity dopamine uptake per dopaminergic cell and cell survival . This effect is not limited to the dopaminergic cells and suggests that the development of neurotransmitter-related traits and cell survival are influenced by cell density-derived trophic signals . The above-mentioned cultures and parameters have also been used to detect neuronotrophic activity in adult mammalian brain extracts or more purified preparations . In particular, bovine striatal extracts contain activity capable of increasing high-affinity neurotransmitter uptake parameters and cell survival of at least the dopaminergic and GABAergic neurons present in the culture system . The neuronotrophic activity from bovine striatum has been partially purified and is associated with a fraction whose main component is a basic protein of approximately 14 kDa.

Diagn Microbiol Infect Dis, 1988 Mar, 9(3), 187 - 92
Comparison of cell culture and three enzyme-linked immunosorbent assays for the rapid diagnosis of respiratory syncytial virus from nasopharyngeal aspirate and tracheal secretion specimens; Ahluwalia GS et al.; A total of 211 specimens, including 144 nasopharyngeal aspirates and 67 tracheal secretions, were evaluated for the rapid detection of Respiratory Syncytial Virus (RSV) antigen by three commercial enzyme-linked immunosorbent assays (ELISA; Kallestad Diagnostic, Austin, TX; Ortho Diagnostics, Raritan, NJ, and Abbott Laboratories, North Chicago, IL) and by isolation of RSV in cell culture . Of the 61 RSV culture positive specimens, Kallestad ELISA, Ortho ELISA, and Abbott ELISA detected RSV antigen in 80%, 95%, and 92% of the specimens, respectively . An additional 28 specimens were found to be positive only by one or more RSV ELISA tests . A blocking assay confirmed the specificity of ELISA in 71% (20/28) of the RSV ELISA positive and the culture-negative specimens, and 29% were found to be false positive . Through the use of cell culture, with the resolution of ELISA positive and culture negative specimens by blocking assay, 81 specimens (61 + 20) were considered to be true positive . The sensitivity, specificity, and diagnostic accuracy were, for cell culture, 75%, 100%, and 91%; for Kallestad ELISA, 79%, 98%, and 91%; for Ortho ELISA, 95%, 99%, and 98%; and for Abbott ELISA, 93%, 96%, and 95% respectively . In our study, commercial ELISA tests have been shown to be rapid, reliable tests for the detection of RSV . Ortho ELISA and Abbott ELISA showed better sensitivity than the Kallestad ELISA for RSV detection directly in the clinical specimens.

Mutat Res, 1988 Mar, 195(2), 151 - 213
A comparative analysis of data on the clastogenicity of 951 chemical substances tested in mammalian cell cultures; Ishidate M Jr et al.; A literature review was conducted using original papers published during 1964-1985 on the in vitro clastogenicity of chemical substances . Results of tests on 951 chemical substances were abstracted from over 240 reports to form the database . The evaluation of these data relied on each author's original conclusion on a positive or negative outcome . Of these 951 substances, 447 (47%) were consistently positive either with or without activation; 417 (44%) were negative in the direct test but not tested with metabolic activation systems; 4 were negative but tested only with activation; and 30 (3%) were clearly negative both with and without activation . The remaining 53 substances gave variable results when tested under different experimental protocols or in different cell types, but were positive in at least one test . Although discrepant results were found associated with some cell types, the addition of metabolic activation systems tended to eliminate such variability . No one cell appeared to be superior in response to all clastogens . For screening purposes, the choice of cell may thus depend more on the general usefulness and reliability of a cell type than on a strong response to a particular chemical . However, the use of a suitable metabolic activation system does appear to be of critical importance . The concentration at which clastogenic effects were detected varied extensively for different test substances, ranging from a minimum of 4.3 X 10(-8) to 6.9 X 10(2) mM . Possible mechanisms of action for substances active at only high levels are discussed, but no satisfactory explanation is available at this time . The relevance of tests conducted at concentrations high enough to alter significantly the osmolarity and other culture conditions is considered, and caution urged in the interpretation of test results obtained under physiologically stressful conditions . The clastogenic potential was compared quantitatively using an index of effective concentration (D20) and one which estimates the number of cells with exchange aberrations expected per mg/ml (TR) for data obtained by using a uniform protocol and cultures of Chinese hamster lung (CHL) cells . Both values were distributed over a wide range, demonstrating the variety of genotoxic potential in chemicals . In general, a substance which was active at only high concentrations produced fewer exchange-type aberrations . In vivo activity, as measured by tumourigenic effect and formation of micronuclei in bone marrow, tended to be greater for substances with a D20 below 10(-2) mg/ml and a TR value over 10(3).(ABSTRACT TRUNCATED AT 400 WORDS)

J Steroid Biochem, 1988 Mar, 29(3), 285 - 91
Gonadotropin induction of a regulatory mechanism of steroidogenesis in fetal Leydig cell cultures; Tsai-Morris CH et al.; Studies were conducted to define further the development of the gonadotropin induced, E2 mediated steroidogenic lesion (17-alpha-hydroxylase/17,20-desmolase) in fetal Leydig cell cultures . Analysis of dispersed fetal testes purified by centrifugal elutriation demonstrated a group of cells with sedimentation velocity 12 less than to less than 16.8 mm/h.g containing a small population of adult like "transitional" Leydig cells and homogeneous "fetal" Leydig cell population collected at greater than 19.3 mm/h.g . After cells were cultured for 3 days with addition of 1 microgram oLH at 3 day intervals, the transitional cells showed testosterone accumulation comparable to the fetal cells . In contrast, transitional cells had 10-fold higher basal and hCG-stimulated aromatase activity than fetal cells, and a lack of testosterone response to acute (3 h) hCG stimulation . At day 6, transitional cells steroidogenic ability declined markedly . The fetal population maintained in culture with LH additions every 3 days, showed typical immature Leydig cell response, with enhancement of acute testosterone response to hCG at 3 day (1-fold) and at 6 day of culture (5-fold) . Higher doses of LH (5 micrograms/day) or daily treatment of 1 microgram to fetal cultures, induced a lesion of 17 alpha-hydroxylase/17,20-desmolase with reduction of enzymatic activities (P less than 0.01) and impaired testosterone production (P less than 0.01) in response to acute hCG stimulation . Also aromatase was stimulated by hCG + 140% and 50% and E2 receptors were increased by 100 and 180% at 3 days and 6 days of cultures with daily or high dose LH addition, findings consistent with the observation of the E2-mediated lesion during LH action . In conclusion, the cultured fetal Leydig cell provides a useful model to elucidate molecular mechanisms involved in the development of gonadotropin-induced estradiol-mediated desensitization . Treatment of fetal Leydig cell cultures with multiple or frequent doses of LH elevate aromatase activity to necessary levels for the induction of desensitization . We have isolated small population of transitional Leydig cells with morphological characteristics of cells found in 15 day post-natal testis but functional capabilities of adult cells . We have also demonstrated the emergence of a functional adult-like population from the fetal Leydig cell.

J Neurosci, 1988 Mar, 8(3), 1063 - 73
Differentiation and maturation of embryonal carcinoma-derived neurons in cell culture; McBurney MW et al.; We have previously shown that retinoic acid-treated cultures of the P19 line of embryonal carcinoma cells differentiate into neurons, glia, and fibroblast-like cells (Jones-Villeneuve et al., 1982) . We report here that the monoclonal antibody HNK-1 reacts with the neurons at a very early stage of their differentiation and is, therefore, an early marker of the neuronal lineage . Cells in differentiated P 19 cultures synthesized acetylcholine but not catecholamines, suggesting that at least some of the neurons are cholinergic . The neurons also carry high-affinity uptake sites for GABA but not for serotonin . In long-term cultures, neuronal processes differentiated into axons and dendrites, which formed synapses . This biological system should prove valuable for examining the development and maturation of cholinergic neurons, since their differentiation occurs in cell culture.

Am J Otolaryngol, 1988 Mar-Apr, 9(2), 68 - 78
Study on normal and otosclerotic bone cell cultures: an advance in understanding the pathogenesis of otosclerosis; Maurizi M et al.; The authors first reviewed the main theories concerning the pathogenesis of otosclerosis and studied the morphologic and functional characteristics of cell cultures derived from normal and otosclerotic bones . Light transmission and scanning electron microscopy did not permit definite identification of the cultured cells as predominantly osteoblasts, nor did these techniques show significant differences between cultured cells derived from normal and pathologic bone . Functional tests of the cell cultures proved more interesting . First, the bony nature of the cultured cells was demonstrated by studying the intracellular 45Ca++ uptake after stimulation with calcitonin and dybutryl-cAMP . Second, cell cultures derived from otosclerotic bone behaved differently from those derived from normal bone . Their peak uptake of calcium appeared later, and post-stimulatory values were higher, suggesting that cells derived from otosclerotic bone store a greater quantity of 45Ca++ . Furthermore, after stimulation with calcitonin and propranolol, we observed an inhibition of the calcium uptake and decreased intracellular cAMP levels in normal bone cell cultures . In contrast, the cell cultures derived from otosclerotic bone exhibited an initial inhibition of calcium absorption followed by massive calcium penetration . The response of adenylate cyclase to the action of Mg++, Ca++, and F- ions was evaluated in cultures derived from normal bone, otosclerotic bone, and normal skin fibroblasts . The resulting data show that activation due to Mg++ is much lower in cultured cells derived from otosclerotic bone than in those from either normal bone or skin fibroblasts . No significant differences were found after Ca++ inhibition in any of the cell cultures . Moreover, in cell cultures derived from normal bone, F- ions induced a strong activation that was lower than the levels observed in cultures of otosclerotic bone or in normal fibroblasts . We hypothesize that an alteration at the calcitonin receptor site is responsible for the difference in calcium uptake and cAMP levels observed in the cells derived from otosclerotic bone as compared to those cultured from normal cells.

Zentralbl Bakteriol Mikrobiol Hyg {B}, 1988 Mar, 186(1), 67 - 72
Immune electron microscopy in the detection of viruses other than enteroviruses on cell culture in untreated sewage; Pietri C et al.; Following the demonstration of enteroviruses in samples of untreated sewage by inoculation of cell cultures with an eluate fraction, immune electron microscopy (IEM) was employed in two stages to detect adenoviruses and the hepatitis A virus (HAV) . 6/12 samples contained adenoviruses and 5/15 other samples contained viruses having characteristics of HAV . Cell culture can thus be usefully allied with IEM in the more precise determination of the potential health hazards of waste water.

J Virol Methods, 1988 Mar-Apr, 19(3-4), 319 - 24
Detection of cytopathic and noncytopathic bovine viral diarrhea virus in cell culture with an immunoperoxidase test; Smith GH et al.; Bovine viral diarrhea virus (BVDV) antigen was detected in cell culture with an indirect immunoperoxidase (IP) procedure using a specific monoclonal antibody, and an avidin-biotin-peroxidase complex . Cytopathic and noncytopathic strains of the virus showed similar patterns of IP staining until 3 days post-infection . At six days post-infection, intensity of staining decreased in cell cultures infected with noncytopathic virus, but not with cytopathic virus . The IP procedure detected BVDV antigen in cells used to isolate virus from tissues of aborted bovine fetuses and peripheral blood lymphocytes of adult cattle . Immunoperoxidase detected BVDV isolates from 10 of 44 cases of abortion of which seven of these were noncytopathic . Noncytopathic BVDV isolates from the peripheral blood lymphocytes of 7 of 65 animals were identified.

J Clin Microbiol, 1988 Mar, 26(3), 570 - 2
Conventional tube cell culture compared with centrifugal inoculation of MRC-5 cells and staining with monoclonal antibodies for detection of herpes simplex virus in clinical specimens; Woods GL et al.; During a 15-month period, two methods for detection of herpes simplex virus (HSV) in 699 clinical specimens were compared: (i) 24-well-plate centrifugation (24WPC) with MRC-5 cells and staining with type-specific monoclonal antibodies (Syva Co., Palo Alto, Calif.) after incubation for 16 to 18 h and (ii) conventional tube cell culture with primary rabbit kidney and A549 cells . HSV was identified by conventional tube cell culture in 165 (24%) of 699 specimens and by the 24WPC method in 116 (17%) of 699 specimens . One specimen was positive for HSV by the 24WPC method alone, compared with 50 specimens positive only by conventional cell culture (P less than 0.0001) . The sensitivity, specificity, and positive and negative predictive values of the 24WPC technique with MRC-5 cells for detection of HSV in clinical specimens were 70, 99.8, 99, and 91%, respectively . Centrifugal inoculation of MRC-5 cells in 24-well plates and staining with monoclonal antibodies after incubation for 16 to 18 h is an insensitive means of detecting HSV in clinical specimens and should not replace conventional tube cell culture with primary rabbit kidney cells.

In Vitro Cell Dev Biol, 1988 Mar, 24(3), 175 - 82
Asbestos-mediated transfection of mammalian cell cultures; Dubes GR et al.; The capacity of asbestos to mediate transfection was tested in a rapid and relatively simple system: picornavirus RNAs and mammalian cells in vitro . Thirteen asbestos samples, including amosite, anthophyllite, chrysotile, and crocidolite, 4 picornaviruses (poliovirus 1 and 2, echovirus 7, and encephalomyocarditis virus), and 4 cell lines (CLI, chimpanzee liver; KB, human carcinoma; eta, monkey kidney; NIH 3T3, mouse embryo) were tested . The results showed that all asbestos samples mediated transfection and that all cell lines were transfectible by viral RNA with asbestos . Transfection was much greater with asbestos added to the viral RNA inoculum than to the cells before or after the RNA . Transfection was directly proportional to asbestos concentration . Initiation of transfection events was rapid, with half becoming irreversible by washing 2 min postinoculation . DNA in the inoculum strongly interfered with asbestos-mediated transfection by the RNA but was ineffective when added, with or without asbestos, to the cells before or after the inoculum . Asbestos compared with six classical "insoluble" facilitators (bentonite, calcium phosphate, chromic oxide, ferric oxide, kaolin, talc) was of intermediate rank in transfection mediation . It is hypothesized that the prominence of asbestos in carcinogenesis is due to a combination of properties, including transfection mediation as well as chromosome mutagenicity, fiber dimensions, biological durability, hydrocarbon transport, and prevalence.

J Clin Microbiol, 1988 Mar, 26(3), 453 - 8
Enzyme immunoassay for detection of human immunodeficiency virus antigens in cell cultures; Viscidi R et al.; A sensitive and specific avidin-biotin enzyme immunoassay that uses immunoreagents from naturally infected individuals was formulated for the detection of human immunodeficiency virus (HIV) antigens . A total of 500 cell culture samples from 111 cultures were tested in this assay and in a reverse transcriptase (RT) assay . Of 353 samples that were positive in the immunoassay, 174 were positive and 179 were negative in the RT assay . The specificity of the immunoassay results was supported by the failure of samples to react with nonimmune serum, by the ability of an anti-HIV type 1 monoclonal antibody to block the reactivity of selected samples, and by the appearance of RT activity in samples drawn from some cultures after a longer period of cultivation . HIV antigens were detected in 174 of 176 RT-positive samples (sensitivity, 98.9%) . A comparison of the kinetics of antigen production and RT activity revealed that detectable antigen levels frequently preceded the appearance of RT activity . Thus, 50% of virus-containing cultures were identified within 9 days by immunoassay compared with 14 days by RT assay . In addition, RT activity was often detected intermittently in cultures sampled on several days, whereas antigen levels did not decline after initial appearance . Enzyme immunoassays for HIV antigen detection that use easily obtained reagents and simple technologies could facilitate laboratory and clinical research which requires cultivation of viruses.

In Vitro Cell Dev Biol, 1988 Mar, 24(3), 188 - 94
A method for the rapid establishment of normal adult mammalian colonic epithelial cell cultures; Vidrich A et al.; Normal colonic epithelial cell cultures of mammalian origin are required to facilitate the study of both normal cellular functions as well as pathogenesis of certain (human) colonic diseases . To date, little information is available regarding the growth requirements of colonic epithelial cells in culture of either animal or human origin . Such data would enable the development of a long-term culture system for these cells . In this study, we present methodology that results in the establishment of homogeneous cultures of adult rabbit colonic epithelia reproducibly, quickly, and in quantity . The epithelial nature of the cultures is unambiguously established by intermediate filament typing using antikeratin antibodies . Such cultures can now be used for a variety of functional studies as well as to investigate the growth requirements of colonic epithelia in culture.

Somat Cell Mol Genet, 1988 Mar, 14(2), 113 - 21
Inactivation and reactivation of sex-linked steroid sulfatase gene in murine cell culture; Schorderet DF et al.; The murine X-linked steroid sulfatase gene (Sts) normally escapes X inactivation . However, we have observed that most long-term murine cell cultures are deficient in STS activity even though only the L cells are known to be derived from an STS- mouse strain . To investigate this phenomenon, we developed a selective system whereby STS+ cells could be selected from STS- populations . The system is based on making cells dependent on cholesterol-sulfate as the sole source of cholesterol, allowing only STS+ cells to grow . Two STS- cell lines, after treatment with either 5-azacytidine (5AC) or ethyl methane sulfonate (EMS), yielded STS+ revertants, suggesting that their STS- phenotype was due to hypermethylation . To study the evolution of STS- cell lines, we established XO and XX primary lines from STS+ strains; the XX cell line remained STS+ after more than 200 cell doublings whereas the XO became STS- after about 100 doublings . Treatment of this STS- XO cell line with 5AC produced clones with restored STS activity . All the revertants showed a growth disadvantage compared to their STS- counterparts . It would appear that aberrant methylation is the basis for much of the STS deficiency observed in established murine lines and that its propagation is due to the growth advantage of STS- over STS+ cells.

Nucleic Acids Res, 1988 Feb 25, 16(4), 1251 - 71
Functional activity of the two promoters of the myosin alkali light chain gene in primary muscle cell cultures: comparison with other muscle gene promoters and other culture systems; Daubas P et al.; Proximal upstream flanking sequences of the mouse myosin alkali light chain gene encoding MLC1F and MLC3F, the mouse alpha-cardiac actin gene and the chicken gene for the alpha-subunit of the acetylcholine receptor were linked to the bacterial chloramphenicol acetyl transferase (CAT) gene and transfected into primary cultures derived from mouse skeletal muscle or into myogenic cell lines . We demonstrate that the mouse MLC1F/MLC3F gene has two functional promoters . In primary muscle cultures, a 1200 bp sequence flanking exon 1 (MLC1F) and a 438 bp sequence flanking exon 2 (MLC3F) direct CAT activity in myotubes, but not in myoblasts or in non myogenic 3T6 and CV1 cells . Developmentally regulated expression is also seen with the alpha-cardiac actin (320 bp) and acetylcholine receptor alpha-subunit (850 bp) upstream sequences in the primary culture system . Transfection experiments with myogenic cell lines show different results with a given promoter construct, reflecting possible differences in the levels of regulatory factors between lines . Different muscle gene promoters behave differently in a given cell line, suggesting different regulatory factor requirements between these promoters.

Nature, 1988 Feb 18, 331(6157), 635 - 8
Expression of the putative Duchenne muscular dystrophy gene in differentiated myogenic cell cultures and in the brain; Nudel U et al.; Duchenne muscular dystrophy (DMD), a sex-linked degenerative disorder of the muscle, is one of the most common lethal genetic diseases in man . It affects about one male in 3,500, with an estimated one-third of cases being caused by new mutations . A less severe disease, Becker's muscular dystrophy (BMD), maps to the same chromosomal locus and is most probably an allelic form of DMD . Both diseases are sometimes associated with various degrees of mental retardation; the molecular basis of these phenotypes is unknown (for review, see ref . 1) . The giant DMD gene spans approximately 2,000 kilobases (kb) (0.05% of the human genome) and encodes a 14-kb mRNA . The tissue-specificity of its expression has not been precisely determined . Monaco et al., using Northern blots, reported expression of the gene in human fetal skeletal muscle and small intestine but not in human fetal brain, or in human cultured myoblasts and transformed B and T cells . More recently, expression was detected in mouse skeletal and cardiac muscle, but not in mouse brain . Here we show, using a ribonuclease protection assay, that the DMD gene is developmentally regulated in rat and mouse myogenic cell cultures, and that it is expressed in rat and mouse striated muscle, in mouse smooth muscle and in rat, mouse and rabbit brain . We could not detect transcripts in other non-muscle tissues.

FEBS Lett, 1988 Feb 8, 228(1), 131 - 4
Monoclonal antibody to human endothelial cell surface internalization and liposome delivery in cell culture; Trubetskaya OV et al.; A monoclonal antibody (mAb), E25, is described that binds to the surface of cultured human endothelial cells . Upon binding E25 is rapidly internalized and digested intracellularly . Selective liposome targeting to the surface of the cells is performed using a biotinylated E25 antibody and an avidin-biotin system . Up to 30% of the cell-adherent liposomal lipid is internalized.

J Protozool, 1988 Feb, 35(1), 32 - 3
Caryocyst-like host cell formation by Caryospora duszynskii (Apicomplexa: Eimeriidae) in human fetal lung cell cultures; Lindsay DS et al.; Sporozoites of the coccidium, Caryospora duszynskii, penetrated human fetal lung cell cultures but did not undergo asexual or sexual multiplication during a 29-day observation period . Beginning three days postinoculation (PI), infected host cells lost their normal elongated fibroblast-like shape and became ellipsoidal in appearance and resembled caryocysts . These caryocyst-like infected cells were observed from 3 through 29 days PI . Sporozoites remained viable throughout the study as evidenced by motility of extracellular sporozoites in infected human fetal lung cell cultures . Results of this in vitro study suggest that some species of Caryospora may form caryocysts in secondary hosts without undergoing asexual or sexual multiplication in these hosts.

Atherosclerosis, 1988 Feb, 69(2-3), 257 - 68
Lipoprotein uptake in primary cell cultures of rabbit atherosclerotic lesions . A fluorescence microscopic and flow cytometric study; Jaakkola O et al.; To characterize the lipoprotein metabolism of lipid-filled cells of atherosclerotic lesions, uptake of 3,3'-dioctadecylindocarbocyanine (DiI)-labelled low density lipoprotein (LDL), acetylated LDL (Ac-LDL) and beta-very low density lipoprotein (beta-VLDL) was studied by fluorescence microscopy and flow cytometry in primary cultures of enzymatically dispersed aortic cells from cholesterol-fed rabbits . Most of the foam cells were identified as macrophages on the basis of Fc-receptors and high activities of nonspecific esterase and acid lipase, although cholesteryl ester (CE) inclusions were found by filipin staining also in smooth muscle cells (SMCs) . During the culture only SMCs proliferated and were confluent in about 1 week . After incubation with DiI-Ac-LDL most macrophage foam cells were brightly fluorescent, but also many SMCs accumulated fluorescence . In SMCs, an excess of LDL inhibited the uptake of DiI-beta-VLDL and DiI-LDL, indicating that these lipoproteins were taken up by the apoB,E receptor; the activity of this receptor was low 2 days after cell isolation but increased considerably during SMC proliferation . DiI-beta-VLDL was not taken up by the macrophage foam cells until after 7 days' culture, when their CE content had decreased, reflecting a feed-back regulation of these receptors as well . Our results indicate that, in primary cultures of enzyme-dispersed cells from rabbit atherosclerotic lesions, most of the foam cells have lipoprotein receptors resembling those described in macrophages and that also many SMCs accumulate Ac-LDL.

J Cell Physiol, 1988 Feb, 134(2), 200 - 10
Serum and growth factors regulate expression of a 43 kDa protein in smooth muscle cell cultures; Millis AJ et al.; Smooth muscle cells respond to injury and the presence of serum factors by modulating from a quiescent contractile cell to a motile synthetic phenotype . To evaluate the biochemical response to serum exposure, we examined the proteins synthesized and secreted in response to serum . The most prominent effect of serum was the rapid production of a protein with an apparent molecular weight of 43 kDa . Removal of serum from the culture environment led to a cessation of 43 kDa protein production . The effect of exogenous heparin on 43 kDa protein production was also evaluated . Neither the 43 kDa protein nor a previously described 38 kDa protein was induced by heparin . Further, heparin treatment did not counteract the effects of serum . These studies demonstrate that an early response of vascular smooth muscle cells to serum is the production of this previously undescribed protein and that other modifications of the culture conditions did not affect its synthesis.

Virology, 1988 Feb, 162(2), 437 - 43
Selection for accelerated penetration in cell culture coselects for attenuated mutants of Venezuelan equine encephalitis virus; Johnston RE et al.; Previous studies with Sindbis virus (SB) suggested that a single point mutation in glycoprotein E2 (serine 114 to arginine 114) conferred three phenotypic alterations: attenuation in neonatal mice, accelerated penetration of cultured cells, and efficient neutralization by two E2-specific monoclonal antibodies (Davis, Fuller, Dougherty, Olmsted, and Johnston (1986) Proc . Natl . Acad . Sci . USA 83, 6771-6775) . Moreover, selection for rapidly penetrating mutants of SB coselected for attenuation in vivo, indicating that a domain of SB E2 which influences penetration in culture overlaps an E2 domain which influences pathogenesis (Olmsted, Meyer, and Johnston (1986) Virology 148, 245-254) . To test the possibility that overlapping penetration and pathogenesis domains exist in other alphaviruses, the virulent Trinidad donkey strain of Venezuelan equine encephalitis virus (TRD-VEE) was serially passed in baby hamster kidney (BHK) cells under a stringent selective pressure for accelerated penetration . Isolates were biologically cloned from the first through the fourth passages and were characterized as to penetration time course in BHK cells and virulence in adult mice following intraperitoneal inoculation . Twenty-two of the 27 isolates segregated into two major categories: slowly penetrating and virulent (like the TRD-VEE parent) and rapidly penetrating and avirulent . Mice which received the avirulent mutants were positive for anti-VEE neutralizing antibody and were refractory to challenge with TRD-VEE . Of the seven mouse avirulent mutants, two also were attenuated in hamsters, indicating the presence of at least two genetic loci at which mutations may influence both pathogenesis and penetration.

J Gen Virol, 1988 Feb, 69 ( Pt 2), 325 - 35
Morphogenesis of yellow fever virus 17D in infected cell cultures; Ishak R et al.; The morphogenesis of yellow fever virus replication was examined in infected Vero cell cultures . Penetration and uncoating occurred by endocytosis with the formation of coated vesicles, similar to that demonstrated for other enveloped and unenveloped viruses . Inclusion bodies associated with newly formed nucleocapsids were evident in the perinuclear region during the growth cycle . No evidence of RNA synthesis in the vicinity of the inclusion bodies was obtained by autoradiography, suggesting that genome replication and assembly of viral nucleocapsids occur at separate cytoplasmic sites . An excessive proliferation of membrane-bound organelles involving both vacuoles and endoplasmic reticula was the most striking feature of virus-infected cells late in infection . No morphological changes in the appearance of nuclei or mitochondria were detected . Virus release appeared to occur by movement of nascent virions through the proliferated endoplasmic reticula followed by exocytic fusion of virus-containing vesicles with the plasmalemma . A possible mechanism whereby the internal nucleocapsid acquires an outer envelope is discussed.

J Med Virol, 1988 Feb, 24(2), 161 - 74
Morphology and development of Rift Valley fever virus in Vero cell cultures; Ellis DS et al.; Rift Valley fever virus (RVFV) grown in vero cell cultures has a completed replication cycle within 13 hours . The first signs are the appearance of intranuclear fibrillar rods, followed by aggregations of precursor viral material in host cell cytoplasm and viral nucleocapsids budding into vacuoles associated with the Golgi apparati . Mature particles, liberated by the disintegration of vero cells, contained ribosomelike structures within the nucleocapsid, which was surrounded by a typical unit membrane through which were inserted some 350-375 surface spikes whose inner ends were incorporated into the nucleocapsid structure . In the negatively stained material, the overall diameter of the virion was 90-110 nm; the spikes were 10-18 nm in length and 5 nm in diameter.

Biochimie, 1988 Feb, 70(2), 193 - 204
Cell culture of rabbit meniscal fibrochondrocytes II . Sulfated proteoglycan synthesis; Webber RJ et al.; Rabbit meniscal fibrochondrocytes were grown in vitro under culture conditions previously shown to foster growth of this cell type . Regardless of the culture regimen employed, the cells synthesized sulfated proteoglycans which could be differentiated by their solubility when dialyzed against water . The water soluble proteoglycans (WSPG) were monomeric in nature and could be separated into sub-types based on their hydrodynamic size when analyzed by gel-filtration chromatography . The water insoluble proteoglycans (WIPG) appeared to represent hyaluronic acid-dependent aggregates of the larger of the two WSPG . The proteoglycans contained approximately 87% chondroitin sulfate and 5% dermatan sulfate . Keratan sulfate could not be detected . Addition of ascorbate to the culture medium did not change the amount or the hydrodynamic size of the proteoglycan aggregates but did alter the quantity of the larger WSPG monomer synthesized depending upon the culture regimen used . Thus, these cells are capable of expressing their differentiated phenotype in short-term monolayer cell culture.

In Vitro Cell Dev Biol, 1988 Feb, 24(2), 126 - 32
Lipid peroxidation in hepatocyte cell cultures: modulation by free radical scavengers and iron; Innes GK et al.; Rat hepatocytes were isolated and then maintained in serum-free cell culture medium for 24 h . The amount of malondialdehyde (MDA) accumulated in the medium was assayed and used as a measure of lipid peroxidation . The activity of lactate dehydrogenase (LDH) and urea were measured in the medium and used as indicators of hepatocellular viability and function . The effects of iron; desferrioxamine mesylate (Desferal), an iron chelator; and mannitol, a hydroxyl free radical scavenger were investigated . The addition of iron, Fe2 resulted in a three-fold increase in the levels of MDA . Desferal inhibited the production of MDA and blocked the effect of Fe2+ . Neither iron nor Desferal had any effect on LDH or urea levels . Mannitol had no effect on MDA or urea production, but caused a 4 to 8-fold increase in the LDH levels in the medium . The results show that iron is involved in the mechanism of lipid peroxidation in hepatocyte cultures but suggest that as a pathologic event lipid peroxidation is not expressed in terms of viability during the first 24 h of hepatocyte culture.

Proc Natl Acad Sci U S A, 1988 Feb, 85(3), 934 - 8
Synaptic transmission between rat cerebellar granule and Purkinje cells in dissociated cell culture: effects of excitatory-amino acid transmitter antagonists; Hirano T et al.; Monosynaptic excitatory connections between cerebellar granule and Purkinje cells were studied in dissociated cell cultures, and identification of the transmitter and the postsynaptic receptor at this synapse was pharmacologically investigated . The presynaptic granule cell and the postsynaptic Purkinje cell were voltage- or current-clamped simultaneously, and the excitatory postsynaptic current induced by the granule cell was examined . The neurons and monosynaptic excitatory connections were identified as in our earlier study . Several pairs of granule and Purkinje cells were stained with Lucifer yellow and propidium iodide, respectively, and their morphology was examined after electrophysiological recording . The monosynaptic excitatory postsynaptic current was suppressed by 1 mM kynurenate, an antagonist for excitatory-amino acid receptors, but was little affected by 0.2 mM DL-2-amino-5-phosphonovalerate, a selective antagonist of N-methyl-D-aspartate receptors . Glutamate and aspartate induced inward current in the Purkinje cells . These currents were suppressed by kynurenate at 1 mM . DL-2-Amino-5-phosphonovalerate at 0.2 mM suppressed the inward current induced by 100 microM aspartate but did not affect the inward current induced by 10 microM glutamate . These results are consistent with the idea that glutamate, or a glutamate-like substance, but not aspartate is the transmitter released at the synapse between granule and Purkinje cells and that non-N-methyl-D-aspartate receptor channels are functioning in the postsynaptic membrane.

Cell Biol Int Rep, 1988 Feb, 12(2), 131 - 42
Effects of various substrates on human hepatoblastoma and hepatoma cell culture; Tokiwa T et al.; The effects on human hepatoblastoma (HuH-6) and hepatoma (HuH-7) cell lines of collagen type I (CI), type IV (CIV), fibronectin (FN) and laminin (LAM) were investigated . CI and CIV promoted almost equally the attachment of both cell lines more strikingly than did FN or LAM . CI and CIV were also superior to FN or LAM in supporting the growth of HuH-6 . These substrates, however, had no appreciable effect on the growth of HuH-7 . The amount of alpha-fetoprotein (AFP) and albumin secreted in HuH-6 was found to be higher on FN- and LAM- coated substrates than on the other matrix material- coated ones . HuH-7 exhibited increased levels of AFP on LAM- coated substrates 4 days after plating . These results indicate that the ability of extracellular matrix materials to enhance attachment and/or growth is different from that to enhance AFP and albumin production in HuH-6 and probably in HuH-7.

Vet Microbiol, 1988 Feb, 16(2), 145 - 58
Virus shedding and immune responses in cats inoculated with cell culture-adapted feline infectious peritonitis virus; Stoddart ME et al.; Eight specific pathogen-free cats were inoculated orally or parenterally with a cell culture-adapted strain of feline infectious peritonitis virus (FIPV) . Faeces and oropharyngeal swabs were monitored daily for infectious virus by inoculation of feline embryo lung cells . Virus was recovered from both sites for approximately 2 weeks after inoculation, before clinical signs of disease developed . Peripheral blood lymphocytes collected from these cats were tested in an in-vitro blastogenic assay using concanavalin A (con A) and FIPV antigen . All cats showed a profound suppression of the response to con A which only recovered to pre-inoculation levels in 2 cats, one of which survived . These 2 cats also responded to FIPV antigen on the 21st day after infection, the greater response being in the survivor . The other cats, surviving 16-18 days, developed no response to FIPV antigen . Antibody titres, measured by immunofluorescence and by virus neutralization, rose rapidly to very high levels in all cats, regardless of the route of inoculation.

Virus Res, 1988 Feb, 9(2-3), 133 - 44
Growth pattern of various JHM coronavirus isolates in primary rat glial cell cultures correlates with differing neurotropism in vivo; Massa PT et al.; The JHM strain of murine hepatitis coronavirus is neurotropic in rats, causing either fatal acute encephalomyelitis or subacute demyelinating encephalomyelitis . We have examined the growth properties of three JHM virus isolates in primary rat glial cultures and found a correlation with their ability to cause disease . Wild type JHM virus has the propensity to cause lytic infections in glial cultures, and a temperature-sensitive mutant designated JHM-ts43 invariably produces persistent infections with reduced cytopathic effects (CPE) as compared to the wild type . Moreover, a non-neurotropic isolate, designated JHM-Pi virus, produces either non-productive persistent infections at low multiplicity of infection (m.o.i.) or productive persistent infections at high m.o.i., with, however, no CPE . The phenotypic expression of persistence is glial cell-dependent, since all three viruses produce similarly lytic infections when grown on various susceptible cell lines . The genetic basis of JHM virus persistence can be explained at the level of direct virus-glial cell interactions.

J Clin Microbiol, 1988 Feb, 26(2), 206 - 12
Serial propagation of porcine enteric calicivirus-like virus in primary porcine kidney cell cultures; Flynn WT et al.; A porcine enteric calicivirus-like virus was adapted to serial propagation in primary porcine kidney cell cultures . Attempts to propagate this virus in primary porcine kidney cells in the presence of trypsin or pancreatin or without medium supplementation were unsuccessful . A low-pH medium (pH 6.8) was also ineffective in virus propagation . Successful serial propagation of the virus required the presence of an intestinal-content preparation, derived from uninfected gnotobiotic pigs, in the cell culture medium . The best results were obtained with six-well plate cultures which were centrifuged after virus inoculation . Infected cells were detected by immunofluorescent staining of cell monolayers or detached cells which were harvested by centrifugation . Infected cells were first detected at passage 4 (1% infected cells), and infectivity increased with successive passages, with as many as 80% of the cells infected by passage 16 . Extensive cytopathic effects were observed in inoculated cell cultures, but not in uninoculated control cell cultures, at each passage level after passage 13 . The infected cells became separated, rounded, and detached, forming holes in the cell monolayer . Only virus particles exhibiting the six-pointed star appearance or stain-filled, cup-shaped depressions characteristic of caliciviruses were detected in inoculated cell culture supernatants by immune electron microscopy . Attempts to determine the titer of the virus by a cell culture immunofluorescence assay or plaque assay were unsuccessful.

J Neurosci Res, 1988 Feb, 19(2), 230 - 8
Effect of epidermal growth factor and insulin on DNA, RNA, and cytoskeletal protein labeling in primary rat astroglial cell cultures; Avola R et al.; The effect of epidermal growth factor (EGF) and insulin on DNA, RNA, and cytoskeletal protein labeling in primary rat astroglial cell cultures was investigated . Cultures were grown for 15-30 days in vitro in 10% fetal calf serum (FCS)-supplemented medium and then maintained in serum-free basal medium (DMEM) supplemented with fatty acid-free bovine serum albumin (BSA) for a starvation period of 24 hr before the addition of factors . The effect of factors was tested at different times (4, 10, 22, and 28 hr) . At each time, {methyl-3H}thymidine or {5,6-3H}uridine was added to the control and treated cells; the incubation time after the addition of labeled precursors was 2 hr at 37 degrees C . The results obtained indicated that the addition of EGF or FCS significantly stimulated {methyl-3H}thymidine incorporation into DNA, reaching the maximum effect after 22 hr . EGF alone significantly stimulated {3H}uridine incorporation into RNA, and this effect was already maximum at 4 hr and remained constant up to 22 hr . The addition of insulin alone caused a slight increase in nucleic acid labeling for short times (4-10 hr) . In contrast with EGF, no detectable stimulation of incorporation of labeled precursors after insulin treatment for 22 hr was observed . On the other hand, the addition of insulin in the presence of EGF induced an increase of the values observed with EGF alone on macromolecular synthesis at all the times studied . Furthermore, a decrease in cell number was observed in confluent cultures maintained for 1 week in medium containing DMEM + BSA in comparison to serum-supplemented (DMEM + BSA + FCS) cultures.

J Virol Methods, 1988 Feb, 19(2), 161 - 8
Detection of human immunodeficiency virus and other retroviruses in cell culture supernatants by a reverse transcriptase microassay; Gregersen JP et al.; A micromethod for the detection of human immunodeficiency virus (HIV) and other retroviruses in cell culture supernatants is described which applies a DEAE ion exchanger for recovery of polynucleotides synthesized in vitro by the retroviral reverse transcriptase . Cell culture, sample preparation, and test performance including the washing step are adapted to microtitre plates . Compared to the standard method this technique produced less non-specific reactions, resulting in a more than 3-fold higher sensitivity, a higher reproducibility due to lower intrarun variations and allowed an increase in the daily sample accomplishment per person 3- to 4-fold at lower costs per sample.

Gen Comp Endocrinol, 1988 Feb, 69(2), 197 - 204
3,3',5-Triiodothyronine-induced differences in water-insoluble protein synthesis in primary epidermal cell cultures from the hind limb of premetamorphic Rana catesbeiana tadpoles; Ketola-Pirie CA et al.; Epidermal cells from the hind limb of premetamorphic tadpoles of the American bullfrog Rana catesbeiana (stages IX-XI) were maintained in primary culture for 120 hr . Cultures, maintained for 36 hr in medium supplemented with L-triiodothyronine (T3; 3 x 10(-10) mol T3/ml medium), synthesize water-insoluble proteins with Mrs of 59, 50, 48, and 43 . The 59 and 48 kDa proteins synthesized by T3-supplemented cultures are not detectably synthesized by 36-hr-old cell cultures without added T3, but they are synthesized by both hormone-supplemented and unsupplemented cultures after 120 hr . These two proteins have Mrs and pIs which correspond with keratins detected in stratifying mammalian epidermis and are immunoprecipitated by antibodies prepared against human keratins . These observations indicate that T3 induces epidermal cell cultures from the hind limb of premetamorphic tadpoles to precociously synthesize water-insoluble proteins which (1) have Mrs and pIs similar to keratins from differentiating amphibian epidermal tissue and (2) are biochemically and immunologically similar to keratins associated with the differentiation of mammalian epidermal tissue.

J Pharmacol Exp Ther, 1988 Feb, 244(2), 789 - 95
Benzodiazepines, but not beta carbolines, limit high frequency repetitive firing of action potentials of spinal cord neurons in cell culture; McLean MJ et al.; Effects of benzodiazepines (BDZs) and beta carbolines (beta CCs) on sustained repetitive firing at high frequency (SRF) of action potentials of mouse spinal cord neurons in cell culture were examined using intracellular recording techniques . In control medium neurons responded to depolarizing current pulses with SRF . Limitation of SRF was produced by the anticonvulsant BDZs (diazepam, clonazepam, nitrazepam and lorazepam) at low to mid nanomolar concentrations, by a convulsant BDZ which does not bind to high affinity BDZ receptors (Ro 5-4864) at high nanomolar concentrations and by a BDZ receptor weak partial agonist (Ro 15-1788) at micromolar concentrations . The limitation of SRF was accompanied by use- and voltage-dependent reduction of maximal rate of rise (Vmax) of sodium-dependent action potentials . Partial agonist and inverse agonist beta CCs did not limit SRF at concentrations up to 200 nM . The limitation of SRF by diazepam was not prevented by inverse or partial agonists at the BDZ receptor, including Ro 15-1788 and the beta CCs . These findings suggest that limitation of SRF was produced by binding of BDZs, but not beta CCs, to voltage-dependent sodium channels and not to high affinity central BDZ receptors, and that BDZs limit SRF by slowing recovery of sodium channels from inactivation . We propose that the limitation of SRF may contribute to the efficacy of BDZs against generalized tonic-clonic seizures and status epilepticus.

Biochem Biophys Res Commun, 1988 Jan 15, 150(1), 436 - 43
Ligand-dependent maintenance of ethanol-inducible cytochrome P-450 in primary rat hepatocyte cell cultures; Eliasson E et al.; Administration of ethanol, dimethylsulphoxide, 2-propanol or imidazole to rats caused 2-7-fold increases in the level of hepatic ethanol-inducible cytochrome P-450 (P-450j), without any concomitant enhancement of corresponding mRNA . All the compounds were able to stabilize P-450j in hepatocyte cultures for at least three days, whereas P-450j mRNA rapidly disappeared from the cultures . A correlation was reached between the concentration of Me2SO, ethanol and 2-propanol necessary to maintain P-450j in the cell cultures and their binding affinities to the enzyme . It is suggested that the ligand-bound form of P-450j in the hepatocytes is protected from degradation.

Cancer Res, 1988 Jan 15, 48(2), 310 - 9
Mechanism of resistance of noncycling mammalian cells to 4'-(9-acridinylamino)methanesulfon-m-anisidide: comparison of uptake, metabolism, and DNA breakage in log- and plateau-phase Chinese hamster fibroblast cell cultures; Robbie MA et al.; Resistance of noncycling cells to amsacrine (m-AMSA) has been widely reported and may limit the activity of this drug against solid tumors . The biochemical mechanism(s) for this resistance have been investigated using spontaneously transformed Chinese hamster fibroblasts (AA8 cells, a subline of Chinese hamster ovary-cells) in log- and plateau-phase spinner cultures . In early plateau phase most cells entered a growth-arrested state with a G1-G0 DNA content and showed a marked decrease in sensitivity to cytotoxicity induced by a 1-h exposure to m-AMSA or to its solid tumor-active analogue, CI-921 . Studies with radiolabeled m-AMSA established that similar levels of drug were accumulated by log- and plateau-phase cells and that there was no significant drug metabolism in either of these cultures after 1 h . However, marked differences in sensitivity to m-AMSA-induced DNA breakage were observed using a fluorescence assay for DNA unwinding (Kanter P.M., and Schwartz, H.S., Mol . Pharmacol., 22: 145-151, 1982) . Changes in sensitivity to DNA breakage occurred in parallel with changes in sensitivity to m-AMSA-induced cell killing . DNA breaks disappeared rapidly after drug removal (half-time approximately 4 min), suggesting that these lesions were probably mediated by DNA topoisomerase II . Resistance to m-AMSA may therefore be associated with changes in topoisomerase II activity in noncycling cells.

Biochim Biophys Acta, 1988 Jan 13, 937(1), 135 - 44
Comparative investigations on the uptake of phallotoxins, bile acids, bovine lactoperoxidase and horseradish peroxidase into rat hepatocytes in suspension and in cell cultures; Petzinger E et al.; Two alternative uptake mechanisms for phallotoxins by liver cells are debated: carrier-mediated uptake and receptor-mediated endocytosis . We have compared the properties of hepatocellular uptake of the phallotoxins, phalloidin and demethylphalloin, with the uptake of cholate as a substrate for carrier-mediated uptake and compared with iodinated bovine lactoperoxidase or iodinated horseradish peroxidase, as the latter are known to be taken up by vesicular endocytosis . Uptake of phallotoxins and {14C}cholate uptake into isolated hepatocytes is independent of extracellular calcium but inhibited by A23187 or by monensin . Uptake of bovine lactoperoxidase strictly depends on external Ca2+, was insensitive to A23197 and was not inhibited by monensin . No mutual uptake inhibition between phalloidin or cholate and peroxidases was seen, indicating independent permeation pathways in hepatocytes . However, high concentrations of cytochalasin B inhibited the uptake of either phalloidin, cholate or bovine lactoperoxidase . Horseradish peroxidase uptake, which was taken as an indicator for fluid pinocytosis, was low in isolated hepatocytes and could not account for the amount of phalloidin or cholate taken up . In cultured rat hepatocytes, uptake of phallotoxins decreased within 1 day to 10% of the uptake seen in freshly isolated hepatocytes . The results indicate different mechanisms for hepatocellular phallotoxin/bile-acid uptake and peroxidase internalization . As monolayer cultures of hepatocytes rapidly lost the carrier-mediated uptake of phallotoxins and bile acids, freshly isolated hepatocytes might be a more suitable experimental model than cultured cells for kinetic studies on this transport system.

Environ Mol Mutagen, 1988, 12(2), 179 - 83
Sister chromatid exchanges in the lymphocytes of control women, pregnant women, and women taking oral contraceptives: effects of cell culture temperature; Ghosh R et al.; The incidence of sister chromatid exchange (SCE) was investigated in the lymphocytes of control women, pregnant women, and women using oral contraceptives after culture at 37 degrees C and 40 degrees C . At 37 degrees C, the mean frequency of SCE (MEAN +/- S.E.) was found to be 7.91 +/- 0.30 in pregnant women and 8.53 +/- 0.29 in oral contraceptive users which were significantly higher than the SCE value of 5.56 +/- 0.21 found in control women . Increase in growth temperature to 40 degrees C elevated the SCE frequency to 11.86 +/- 0.44 in pregnant women, 12.76 +/- 0.46 in oral contraceptive users and 7.24 +/- 0.26 in control women . These data indicate that there is a differential induction of SCEs following increased cell culture temperature in the lymphocytes of pregnant women and oral contraceptive users, compared with control womenPIP: The incidence of sister chromatid exchange (SCE) was investigated in the lymphocytes of 44 pregnant women, 51 users of oral contraceptives (OCs), and 38 controls after culture at 37 C and 40 C . All study subjects were healthy adult females who were not occupationally exposed to mutagens, were nonsmokers, and had no recent viral infection or radiation exposure . At the normal growth temperature of 37 C, the frequency of SCEs/cell was 7.91 + or - 0.30 in pregnant women, 8.53 + or - 0.29 in OC users, and 5.56 + or - 0.21 in controls . When the growth temperature was elevated to 40 C, there was a further increase in the frequency of SCE in all 3 groups: 11.86 + or - 0.44 in pregnant women, 12.76 + or - 0.46 in OC users, and 7.24 + or - 0.26 in controls . Given the significant differences between SCE frequencies of pregnant women and OC users on one hand and controls on the other hand, it can be implied that chromosomes of pregnant women and women using hormonal contraceptives are more susceptible to the damage induced by increases in cell culture temperature . There are also indications that the higher level of hormones present in the blood of pregnant women and OC users may play an important role in increasing the sensitivity of their lymphocytes of genetic damage . The increased induction of SCEs following increased cell culture temperature in pregnant women and OC users may be attributable to the increased efficiency of such a temperature-dependent gyrase-like enzyme on the 1 hand and to the increased mutagenic activity of estrogen or its metabolites on the other hand .

Life Sci, 1988, 42(26), 2721 - 7
Renotropic stimulation in rat kidney cell culture; Yun GC et al.; A circulating renotropic factor specific for renal cells has been described in rats . The addition of sera obtained from unilaterally nephrectomized (uni) rats 24h after operation compared to sham-operated (sham) rats augments 3H-thymidine incorporation into the DNA of incubating kidney slices approximately 10%-30% . Attempting to amplify the sensitivity of the assay for this renotropic agent, we replaced slices with primary rat kidney cultures . The assay system was based on one previously used for rabbits . The cultured cells were synchronized in their growth phase by a period of protein-free starvation . Compared to sera from sham rats, sera from uni rats showed significant stimulation of thymidine incorporation into DNA, 35.5% +/- 9.3 (SEM), p less than .0001, at 16 h; 63.3% +/- 10.0 (SEM), p less than .001, at 24 h; and 19.5% +/- 6.5 (SEM), p less than .01, at 48 h post operation . Accordingly, the maximal stimulation at 24 h was greater than that previously found using the kidney slice assay . Measurable renotropic activity occurred earlier and over a shorter duration than in rabbits . Stimulation was similar when a D-valine medium, relatively specific for renal epithelial cells, replaced DME medium . We conclude that growth synchronized, primary rat renal cells in culture verify the presence of a circulating renotropin arising 24 h post uni.

Can J Microbiol, 1988 Jan, 34(1), 19 - 23
Growth characteristics in cell culture and pathogenicity in mice of two terrestrial rabies strains indigenous to Canada; Webster WA et al.; Two strains of street rabies virus from striped skunks (Mephitis mephitis) were used to infect either a murine neuroblastoma (NA 1300) or a baby hamster kidney (BHK-21/C13) cell culture and the cell infection rates were noted during 4 days postinfection . These cultures were then passaged for four consecutive passages, and the viruses obtained in the supernatant fluids of passage 4 were then treated as original isolates and used to infect both neuroblastoma and baby hamster kidney cells . The mortality period in Swiss white mice caused by the various virus suspensions was noted . The virus strain from the brain of skunks from Saskatchewan infected neuroblastoma and baby hamster kidney cells equally well, produced similar virus titres in supernatant fluids after four subcultures in both cell types, and appeared to produce similar mortality periods in mice from either the original brain tissue or from cell culture supernatant fluids . On the other hand, the virus from the brains of skunks from Ontario readily infected neuroblastoma but poorly infected baby hamster kidney cell cultures . Passage of this strain through four subcultures in both cell types produced virus titres in the supernatant fluids of equal magnitude . However, reisolation of the virus from the supernatant fluid of passage 4 in neuroblastoma cell cultures showed a similar pattern to that from the original brain, while the virus from baby hamster kidney cell passage supernatant fluid was considerably altered . Although the mortality period in mice was similar with virus from the brain and neuroblastoma cell cultures, this period was shortened when mice were inoculated with baby hamster kidney culture supernatant virus.(ABSTRACT TRUNCATED AT 250 WORDS)

Arch Dermatol Res, 1988, 280(3), 137 - 9
Defective calcium uptake in keratinocyte cell cultures from vitiliginous skin; Schallreuter KU et al.; 45Ca2+ has been used to measure kinetics for the uptake, efflux, and "steady state" of this regulatory cation in keratinocytes grown from the involved and uninvolved skin of one donor (JM) with vitiligo . Cells grown from uninvolved skin yielded a very rapid uptake and efflux of this isotope before reaching "steady state" . A similar profile has been found for keratinocytes from normal healthy adult controls . However, cells established from vitiliginous skin showed a slow uptake of 45Ca2+ before reaching the same "steady state" as the controls . 45Ca2+ efflux has not been observed in vitiliginous keratinocytes . Furthermore, vitiliginous keratinocytes yielded a higher concentration of extracellular bound 45Ca2+ compared with keratinocytes from uninvolved skin . Since Ca2+ has been found to be an allosteric inhibitor of membrane-associated thioredoxin reductase, this defect in Ca2+ transport may explain the proposed breakdown in free radical defense in vitiligo . These findings may also shed more light on the etiology of this disorder.

Poult Sci, 1988 Jan, 67(1), 135 - 40
Effects of glycerol on chicken spermatozoa incubated in vitro at 41 C in oviducal and embryonic cell cultures; Latorre JR et al.; Two experiments were conducted to determine the effects of glycerol on the quality and survival of spermatozoa in an in vitro system at 41 C in the presence of oviducal and other tissue cultures . The motility and percentages of abnormal and dead spermatozoa of glycerolized semen were significantly affected in a positive way by the presence of living cells . The most negative effect of glycerol on semen quality was observed with semen incubated in the tissue culture medium alone . Aspartate aminotransferase activity in the culture fluid revealed a harmful effect of glycerol on spermatozoa and culture cells.

Vopr Virusol, 1988 Jan-Feb, 33(1), 81 - 6
{Effect of remantadine on the morphogenesis of the Venezuelan equine encephalomyelitis virus in a Vero cell culture}; Bakanidze LG et al.; Morphogenesis of Venezuelan equine encephalomyelitis (VEE) virus was studied in Vero cell cultures with or without remantadine treatment . Remantadine was found to inhibit late stages of VEE virus morphogenesis, especially formation of virus cores in the cytoplasm of the infected cells.

Vopr Virusol, 1988 Jan-Feb, 33(1), 63 - 6
{Antiviral factor isolated from infected cell cultures}; Votiakov VI et al.; An antiviral factor of protein nature inhibiting reproduction of VEE, herpes, vaccinia VSV, fowl plague viruses was isolated from infected cell cultures . The factor has no virucidal or prophylactic effect, stable to low pH values and heating at 100 degrees C for 30 min.

Neuroscience, 1988 Jan, 24(1), 67 - 79
Zinc neurotoxicity in cortical cell culture; Choi DW et al.; Large amounts of zinc are endogenously present in synaptic vesicles of mammalian central excitatory boutons, and are likely released during synaptic activity; transient elevations in extracellular zinc concentration exceeding several hundred micromolar may accompany intense neuronal excitation . Exposure of mature cortical cell cultures, in mice, to similar concentrations of zinc for several minutes resulted in widespread neuronal injury; the extent of injury was dependent on both the concentration of zinc, and the length of exposure . Quantitative neuronal cell counts suggested an approximate neurotoxic ED50 of 600 microM for a 15 min zinc exposure, and 225 microM for an 18-24 h exposure . High zinc concentrations or long exposure times resulted in the addition of glial injury to the neuronal injury; this glial injury could also be demonstrated in neuron-free glial cell cultures, and hence likely represented a direct effect of zinc rather than a consequence of neuronal injury . Neurons in immature cultures were relatively resistant to zinc-induced injury, suggesting that neuronal vulnerability to zinc increases with maturation in vitro . An early event associated with toxic exposure to zinc was gross neuronal swelling . This swelling was dependent on the presence of extracellular sodium, and, interestingly, could be delayed by the continued presence of zinc itself . Zinc-induced neuronal cell loss, however, occurred even when both sodium and calcium were absent during the exposure to zinc . The present results provide direct evidence that zinc might be a relatively potent, rapidly acting neurotoxin, and somewhat less potent gliotoxin, in the mammalian central nervous system . We suggest that zinc should be included on the growing list of endogenous toxins which may be involved in the acute pathogenesis of central neuronal, and possibly glial, cell loss in some disease states.

Neurochem Res, 1988 Jan, 13(1), 1 - 8
The incorporation of monomethylethanolamine and dimethylethanolamine in fetal brain aggregating cell culture; Dainous F et al.; Fetal rat brain aggregating cell cultures were exposed to varying concentrations of {3H}monomethylethanolamine (MME) and {3H} dimethylethanolamine (DME) . The rate of labeling of water-soluble compounds was more rapid and the amount of radioactivity present was greater than in the lipids . After a 72 hour incubation in the presence of millimolar concentrations of these nitrogenous bases, the major water-soluble products were the phosphorylated form of the bases . Little label was associated with the free bases or their cytidyl derivative . In the phospholipids, 97% of the radioactivity was recovered in phosphatidylmonomethylethanolamine (PMME) and 3% in phosphatidyldimethylethanolamine (PDME) or 95% in PDME and 5% in phosphatidylcholine (PC) after growth in presence of {3H}MME and {3H}DME respectively . The rate of formation of the radioactive products increased as function of the concentration of the nitrogenous base added up to 4 mM, the highest concentration employed . There was no significant difference in the pattern of labeling with cells grown in media devoid of methionine or choline . The turnover of the water-soluble metabolites was more rapid than in the phospholipids where an apparent half-life of 24 hours was calculated.

Anticancer Res, 1988 Jan-Feb, 8(1), 205 - 8
Effects of hormonal modulation on cytotoxicity of chemotherapeutic agents in mouse mammary tumor cell cultures; Emerman JT; We investigated the effects of androgen modulation on the cytotoxicity of adriamycin (AD) and melphalan (MEL) on cultured cells from the androgen-responsive Shionogi mouse mammary tumor . Culture medium contained 10 ng/ml, 1 ng/ml or no dihydrotestosterone (DHT) . Growth rates of cells increased with increasing concentrations of androgen . Cells cultured in 10 ng/ml DHT rapidly reached confluence and proliferation slowed . The prolonged growth phase of cells incubated in 1 ng/ml DHT rendered the cells most sensitive to the drugs for the longest period of time . Cells cultured in the absence of DHT were least sensitive to the drugs . The results suggest that combination endocrine ablation and administration of chemotherapeutic agents may not be beneficial to the clinical management of breast cancer . In contrast, hormonal manipulations that maintain the slow growth of tumors may enhance cytotoxicity of antineoplastic agents.

Vet Microbiol, 1988 Jan, 16(1), 9 - 14
Latent infection with feline syncytial virus of cell cultures prepared from the kidneys of new-born kittens; Kukedi A et al.; Feline syncytial virus infection was detected in 4 out of 30 secondary or tertiary cultures of kidney cells from new-born kittens . Such infection was not detected in any primary cell culture . The syncytia-forming virus was readily transmissible by infected cells . One strain was transmissible to heterologous (bovine and canine) secondary cells, but did not infect MDBK and PK-15 cell lines . Based on biochemical, untrastructural and serological characteristics, the virus was designated a member of the subfamily Spumavirinae of the family Retroviridae . As endogenous infection of primary cell cultures may not be detectable by cytopathic effect, it is suggested that only well-controlled secondary cat cells or cell lines should be used in work with feline viruses.

Nippon Sanka Fujinka Gakkai Zasshi, 1988 Jan, 40(1), 87 - 9
The confirmation of norethindrone aromatization in primary human hepatocytes by the Vitafiber-II cell culture system; Yamamoto T et al.; A portion of norethindrone (17 alpha-ethynyl-19-nortestosterone) was confirmed to be aromatized to ethynyl estradiol (17 alpha-ethynyl estradiol) in primary human hepatocytes cultured using the Vitafiber-II cell culture system.

J Cancer Res Clin Oncol, 1988, 114(2), 197 - 203
Effectiveness of mitoxantrone on the proliferation of cell cultures derived from malignant mesenchymal tumors of human origin; Dietel M et al.; The antiproliferative potency of mitoxantrone (MITOX) has predominantly been established for epithelial and hematologic neoplasias . In this study the effectiveness of MITOX was investigated in vitro for 6 sarcomatous human cell lines derived from 2 synovial sarcomas, a malignant schwannoma, a malignant histiocytoma, a leiomyosarcoma, and a chondrosarcoma . The examination was performed using a proliferation assay with monolayer cell cultures . The effect of MITOX was compared with that of adriamycin (ADR) and cisplatin (CDDP) . For each drug at least 3 concentrations were tested which covered the therapeutically achievable range, i.e., for MITOX 0.2-0.002 micrograms/ml, for ADR 0.5-0.005 micrograms/ml, and for CDDP 5.0-0.05 micrograms/ml . Test incubations were performed for 3 days . Antiproliferative potency of the cytostatic drugs was assessed by counting the number of cells at the start and the end of the test period with and without drug addition . Furthermore the dose inhibiting cell growth to 50% of controls (ID50) was determined for MITOX . For comparison 4 cell lines from carcinomatous lesions were included in the study . MITOX inhibited proliferation rates of 4 sarcomatous tumor cell lines more intensively than ADR, and was less effective in 2 cell lines . However, these differences were not significant . In all mesenchymal cell lines tested the antiproliferative potency of MITOX was more pronounced than that of CDDP . In carcinomatous cell lines the MITOX-induced growth inhibition was similar to that found in response to administration of ADR and CDDP confirming the described effect on epithelial tumors . The study suggests that MITOX possesses a growth inhibitory potency for malignant soft tissue tumors in vitro . From these data it may be worthwhile to initiate clinical trials testing the treatment of sarcomatous lesions with MITOX.

Eur Urol, 1988, 14(1), 61 - 4
Common characteristics of primary cell cultures originated from invasive urothelial carcinoma; Logothetou-Rella H et al.; Ten primary cultures from invasive urothelial carcinomas were studied by light and electron microscopy . In all cultures, cells with accumulations of glycosaminoglycans were observed . Long membrane extensions with accumulations of glycosaminoglycans invaded neighboring cells and formed an extracellular matrix as well . The extracellular matrix inhibited cell proliferation and enhanced tumor nodule formation.

J Neurosci, 1988 Jan, 8(1), 22 - 30
Circadian clock in cell culture: II . In vitro photic entrainment of melatonin oscillation from dissociated chick pineal cells; Robertson LM et al.; The avian pineal gland contains circadian oscillators that regulate the rhythmic synthesis of melatonin . We have developed a flow-through cell culture system in order to begin to study the cellular and molecular basis of this vertebrate circadian oscillator . Pineal cell cultures express a circadian oscillation of melatonin release for at least 5 cycles in constant darkness with a period close to 24 hr . In all circadian systems, light regulates the rhythm by the process of entrainment that involves control of the phase and period of the circadian oscillator . In chick pineal cell cultures we have investigated the entraining effects of light in 2 ways: by shifting the light-dark cycle in vitro and by measuring the phase-shifting effects of single light pulses . A 6 hr advance or delay of a LD 12:12 light-dark cycle produced a corresponding shift in the melatonin rhythm . The phase shifts of the rhythms persisted after transfer to constant darkness, showing that the underlying circadian oscillator was entrained . Photic entrainment of the oscillator was further characterized by measuring the phase-shifting effects of single 6 hr light pulses . Single pulses of light shifted the phase of the circadian oscillator in a phase-dependent manner . Light pulses beginning early in the subjective night delayed the phase of the oscillation 8 hr relative to dark controls . Conversely, light pulses beginning late in the subjective night advanced the phase of the oscillation nearly 8 hr . Thus, photoreceptors within the cell cultures can mediate entrainment of the pineal oscillators.(ABSTRACT TRUNCATED AT 250 WORDS)

Pathol Biol (Paris), 1988 Jan, 36(1), 79 - 82
{Cell cultures in Burkitt's lymphoma . Detection of residual disease and application to autologous graft purging}; Philip I et al.; We describe a new liquid cell culture system for propagating Burkitt lymphoma (BL) cells contaminating the bone marrow (BM) . The percentage of BL cells at day 10 of culture is correlated with the number of BL cells in the initial sample, which permits the detection of as few as one BL cell in 10(6) normal cells . We currently use such an assay to detect minimal BM involvement in our patient's follow up . This has allowed to define in which clinical situations the BM collected for autologous transplantation (though cytologically normal) had to be "purged" . With the help of this modified liquid system it was possible to quantify the efficacy of various purging procedures on different models; an experimental model of allogenic irradiated BM contaminated with BL cell lines was used . Pre-clinical assays were also conducted on our patients' BM contaminated with their own tumour cells, or on invaded BM collected for autologous transplantation and for which the system had evidenced the efficacy of the purging procedure.

Pathol Biol (Paris), 1988 Jan, 36(1), 56 - 9
Cell cultures and detection of minimal residual disease: significance and limitations; Aapro M et al.; This introductory article puts into perspective the usefulness of cell cultures in the area of detection of minimal residual disease . We stress the role of single cell cultures in selective media as a means to increase the number of residual tumor cells, thereby facilitating their detection by other methods . Prior treatment may affect the growth potential of cells for a very long time . Quantitation of the event to be detected is thus highly dependent on the characteristics of the cells which are also influenced by their growth environment . This environment may be optimized by the judicious use of growth factors in defined media.

J Clin Pathol, 1988 Jan, 41(1), 89 - 92
Comparison between cell culture and serology for detecting Chlamydia trachomatis in women seeking abortion; Csango PA et al.; The efficiency of an immunoperoxidase serological assay and culture of Chlamydia trachomatis were compared in 127 women seeking first trimester abortion . Serum IgG and IgA antibodies specific for C trachomatis were detected by a single serovar (L2) inclusion immunoperoxidase assay (IPA) . Eighty (63%) women were seropositive for chlamydial IgG and 31 (24%) for IgA antibodies . C trachomatis was isolated from 21 of 127 (17%) women . Twenty of the 80 women (25%) seropositive for specific IgG antibodies and one of 47 (2%) patients without these antibodies were culture positive (p less than 0.001) . Compared with isolation, chlamydial antibodies at a titre of greater than or equal to 16 showed high sensitivity and negative predictive value (95% and 98%, respectively), but low specificity and efficiency (43% and 52%, respectively) . Chlamydial IgA antibodies at a titre of greater than or equal to 8 showed low sensitivity (52%), but a higher specificity, negative predictive value, and efficiency of 81%, 90%, and 76%, respectively . C trachomatis IgG antibodies at a titre of 16 as determined by IPA can be used as an efficient negative exclusion marker for active chlamydial infection in screening women seeking abortion.

J Neurosci, 1988 Jan, 8(1), 12 - 21
Circadian clock in cell culture: I . Oscillation of melatonin release from dissociated chick pineal cells in flow-through microcarrier culture; Robertson LM et al.; The avian pineal gland contains circadian oscillators that regulate the rhythmic release of melatonin . We have developed a dissociated chick pineal cell culture system in order to begin a cellular analysis of this vertebrate circadian oscillator . Dissociated pineal cells maintained in cyclic light conditions (LD 12:12) released melatonin rhythmically . The release of melatonin was elevated during the dark and low during the light . A circadian oscillation of melatonin release persisted for at least 5 cycles in constant darkness with a period close to 24 hr; however, there was a gradual damping of the amplitude . Analysis of the rhythm revealed that the observed damping was consistent with either desynchronization of multiple oscillators within the cultures or damping of individual oscillators . The circadian oscillation of melatonin release persisted for up to 4 cycles under conditions of constant light; however, the oscillation was strongly damped and the period of the oscillation was lengthened significantly . Thus, dissociated pineal cells express a persistent circadian oscillation of melatonin release in constant darkness, and properties of this oscillation are modulated by light treatment in vitro . This flow-through cell culture method for dissociated chick pineal cells should provide a useful model for the analysis of a vertebrate circadian system at the cellular level.

Life Sci, 1988, 42(1), 61 - 72
Superfused pituitary cell cultures: comparative responsiveness of cells derived from various stages of the estrous cycle to LHRH stimulation administered as short duration pulses; O'Conner JL et al.; We have reinvestigated the question of maintenance of differential LHRH sensitivity in culture and further investigated the role of pulsatile LHRH in the in vitro release of pulsatile LH and FSH at different stages of the estrous cycle . Pituitaries were collected on each day of the 4 day cycle at 0800 . In addition, pituitaries were also collected at 1500 and 1900 on proestrous . The cells were dispersed and exposed 48 hrs later to short duration 4 ng LHRH pulses; this dose was optimized for LH release and was applied at a frequency of 1 pulse/60 min . In terms of absolute magnitude of LH response, observed responsiveness was ranked in the following order: proestrous 1900 greater than estrous 0800 greater than diestrous 1 0800 greater than proestrous 1500 greater than diestrous 2 0800 . Responsiveness was significantly greater at proestrous 1900 (p greater than 0.01), estrous 0800 (p greater than 0.05) and diestrous 1 0800 (p greater than 0.05) when compared to either of the other stages tested . The heightened LHRH sensitivity of proestrous was therefore maintained in cell culture indicating that the system should be valid for conducting studies on the control of gonadotropin secretion during this period . FSH did not respond in pulsatile manner to the LHRH levels employed further substantiating recent evidence that LHRH seems to function somehow less directly in FSH as compared to LH secretion.

Invest Ophthalmol Vis Sci, 1988 Jan, 29(1), 78 - 89
Cell culture of the human lamina cribrosa; Hernandez MR et al.; The extracellular matrix of the lamina cribrosa may be important in the changes in the optic nerve head associated with glaucoma . To investigate the cell biology of this tissue, human lamina cribrosa was explanted in tissue culture and two cell types grown from this tissue were characterized . The most common cell type obtained was a large, flat, polygonal cell which was negative for glial fibrillar acidic protein (GFAP) and could be serially subcultured . This cell type synthesized collagens type III and type IV, fibronectin and elastin . Much less commonly grown was a cell type with conspicuous long processes and which was positive for GFAP . This presumed astrocyte synthesized collagen type IV and fibronectin . Fibroblastic cells were not obtained from this tissue but were easily grown from sclera . The cells that we have cultured from the human lamina cribrosa may produce the extracellular matrix present in the cribriform plates of this tissue and be important in the glaucomatous process.

Folia Biol (Praha), 1988, 34(4), 233 - 9
Analysis of epidermal growth factor and epidermal growth factor receptor expression in human renal carcinoma cell cultures; Sovova V et al.; In cells derived from two human renal carcinomas only the precursor form of epidermal growth factor (EGF) was found . The binding assay revealed a high level of EGF receptor expression in both cell types tested . However, these receptors are not involved in the growth activity of the cells under in vitro conditions used . The source of DNA synthesis-stimulating activity found in conditioned media of the cells tested is discussed with respect to possible participation of TGF beta.

Blood Cells, 1988, 14(2-3), 339 - 54
Inhibition of hemopoiesis in murine marrow cell cultures by recombinant murine tumor necrosis factor alpha: evidence for long-term effects on stromal cells; Eliason JF et al.; The addition of recombinant murine tumor necrosis factor alpha (rmTNF-alpha) to serum-free methylcellulose cultures inhibited macrophage colony formation stimulated by purified colony stimulating factor-1 (CSF-1), recombinant granulocyte-macrophage-CSF (rmGM-CSF), and recombinant interleukin 3 (rmIl-3) . The concentration of rmTNF-alpha inhibiting colony formation by 50% (IC50) was between 2 and 20 ng/ml . Erythroid colony formation in cultures with erythropoietin (EPO) alone or EPO, rmIl-3, and rmGM-CSF in combination were reduced to a much lesser extent . In established long-term marrow cultures (LTMC), addition of 20 and 200 ng/ml of rmTNF-alpha resulted in release of cells from the adherent layer during the first week . Treatment of cultures with rmTNF-alpha for 4 consecutive weeks led to prolonged inhibition of cell production lasting up to 8 weeks after cessation of treatment . One day after addition of a low dose of TNF (2 ng/ml), "fat" cells were no longer observed in the adherent layer . Our results indicate that TNF inhibition of hemopoiesis occurs both at the progenitor cell and stromal cell levels.

Am J Nephrol, 1988, 8(6), 463 - 70
Production of interleukin 1 in glomerular cell cultures from patients with rapidly progressive crescentic glomerulonephritis; Matsumoto K et al.; Interleukin 1 (IL-1) activity was measured in glomerular culture supernatants from 3 patients with rapidly progressive crescentic glomerulonephritis (RPGN) . Macrophages were present in both capillary tufts and cellular crescents as identified by OKM1-positive cells on immunoperoxidase labelling . Glomeruli from 4 rejecting renal cadaver allografts were used as a disease control, in addition to glomeruli from a normal kidney . IL-1 activity as measured by the thymocyte proliferation assay was greater in the supernatants from cultured glomerular outgrowths of patients with crescentic GN than in those from rejected renal allografts and glomeruli isolated from the normal tissue . IL-1 production from cultured glomerular cells from patients with RPGN was detectable in the serum-free conditioned media harvested after 3 days of culture and increased in a stepwise fashion over 28 days of culture . The prominent feature of the glomerular outgrowth of the glomeruli in the RPGN patients was the presence of large numbers of macrophages, which were not present in cultured control glomeruli . These findings indicate that the immunoregulatory aberration in patients with RPGN may in part be due to IL-1 production by activated macrophages.

Cancer Immunol Immunother, 1988, 26(2), 121 - 4
Response of primary human mammary tumor cell cultures to a monoclonal antibody-recombinant ricin A chain immunotoxin; Bjorn MJ et al.; Malignant epithelial tumor cells were isolated and cultured from ten human mammary specimens of cancerous origin . The 260F9 monoclonal antibody (MAB) bound to frozen sections of all of the human breast tumors tested and to primary cultured cells from the tumors . Cultured cells from all ten breast tumors were sensitive to the clonal inhibitory effects of immunotoxin 260F9 MAB-recombinant ricin A chain . At an immunotoxin concentration of 200 ng/ml (about 1 nM), inhibition of colony formation was greater than 99% for all ten tumors.

Mem Inst Oswaldo Cruz, 1988 Jan-Mar, 83(1), 63 - 6
Rapid detection by transmission electron microscopy of mycoplasma contamination in sera and cell cultures; Barth OM et al.; Transmission electron microscopy has been employed for the rapid detection of mycoplasma in sera and cell cultures . High speed centrifugation of sera or low speed centrifugation of cell debris, followed by negative staining of the resuspended pellet, detected mycoplasma contamination more frequently than a culture method followed by direct fluorescence (DAPI), which was used as a control procedure . The appearance of the mycoplasma cell border and content gives some information about particle viability.

Res Exp Med (Berl), 1988, 188(6), 411 - 23
Effects of intralipid and hydrocortisone upon human fetal lung cell cultures; Kunkelmann H et al.; Organotypic cell culture systems of human fetal lungs of 15, 18, and 26 weeks' gestational age were treated with Intralipid, a phosphatidylcholine-containing lipid mixture, and with hydrocortisone of varying concentrations . The lamellar bodies found in the pneumocytes type II were ultrastructurally identified . Their amount was quantitated by point-counting, a morphometrical method . Intralipid had a stimulating effect upon the surfactant production depending on the concentration admitted . This effect was quantitatively compared to the known effect of hydrocortisone . Intralipid at a concentration of 10(-2%) produced a significant increase of the relative volume of lamellar bodies (P = 0.05) at a gestational age of 18 weeks . This effect is comparable to hydrocortisone treatment at a concentration of 10(-1%) (P = 0.05) and 10(-3%) (P = 0.01) . At a gestational age of 26 weeks, Intralipid at a concentration of 10(-1%) (P = 0.01) stimulated lamellar body production . Hydrocortisone had a similar effect at a concentration of 10(-1%) (P = 0.01) . Intralipid does not pass the placenta-barrier and is locally applied by amniocentesis . Therefore, complications to the maternal organism and probably to the fetuses are negligible . The application of Intralipid represents an alternative method to accelerate antenatal surfactant production and to improve the rate of survival of preterm infants.

Trans R Soc Trop Med Hyg, 1988, 82(3), 433 - 6
Giardia duodenalis: enhanced growth in cell culture; Bowie WR et al.; Growth of Giardia duodenalis in broth and in animals has been studied in considerable detail . In contrast, the kinetics of growth in cell culture have been little evaluated . In this study, in vitro growth of G . duodenalis was evaluated in cell culture, primarily using mouse McCoy cells in vials . The media used were Giardia broth (TYI-G), Trichomonas vaginalis broth (TYI-T), and standard cell culture media (CMGA) alone and in combination (2 parts by volume CMGA to one part of TYI broth) . Addition of cell culture enhanced the sensitivity of the systems in detecting low numbers of G . duodenalis . Growth was identified consistently with inocula less than or equal to 10/ml, and often with a calculated 10-1/ml inoculum with CMGA/TYI-T and CMGA/TYI-G with cells, and with TYI-G with and without cells . The 2 preferred systems for sensitivity and growth were CMGA/TYI-G with cells and TYI-G with cells . The pH fell minimally in the growth systems and, if CMGA was in the media, cell monolayers remained intact and viable throughout the experiment . In preliminary experiments, cell cultures did not allow growth of one strain of G . muris . These cell culture systems may be useful for detection of low numbers of non-laboratory adapted trophozoites, and should be useful in evaluating the interaction of G . dudodenalis with cells in culture.

Exp Cell Biol, 1988, 56(3), 113 - 30
Ultrastructural and autoradiographic investigations of cell cultures derived from tendons or ligamentous material from patients with fibromatous disorders; Neumuller J et al.; Cell cultures were derived from tendons or ligamentous material from patients with carpal tunnel syndrome (CTS), Dupuytren's contracture (DP), tendopathia nodosa (TN) and hallux valgus (HV) . The ultrastructure of the operation specimens as well as of the cell monolayers was investigated, using a floating sheet method in order to preserve both cell-to-cell contacts and the orientation of the monolayers . The histologic features of the tissues obtained in the operations were correlated with the ultrastructure of the cells in culture derived from these specimens . In DP, above all in the nodules, an activation of the capillary endothelium in the vicinity of myofibroblasts and mast cells was observed . In CTS the collagen fibrils varied extremely in diameter . In DP and TN biopsies a splicing process of helicoidly arranged fibrils could be seen . A disintegration of elastic fibers in the fibrillar and amorphous components was found in DP nodules, HV and TN tissues . Transitional forms between fibroblasts and myofibroblasts were observed not only in DP but also-though in a smaller percentage--in the cultures derived from the other patients . The cells showed organelles for active protein synthesis and transport . Autophagocytosis and the formation of multilamellated bodies took place in TN and HV cultures . In CTS, DP and TN cultures cells were connected via gap junctions . In some cultures, above all in those derived from CTS, monocilia were found . In CTS cultures the formation of intracellular collagen occurred . Growth parameters were rather low in HV cultures . PLmax (maximal pulse labelling index) values were higher in TN cultures than in DP and HV cultures . Plating efficiency (PE) values were higher in cultures derived from cell-rich and capillarized tissues than in biopsies with few cells.

Scand J Gastroenterol Suppl, 1988, 151, 70 - 8
Extracellular matrix proteins in small-intestinal cell cultures; Hahn U; Intestinal cell cultures offer unique possibilities to study the effect of extracellular matrix components on epithelial proliferation and differentiation . We have investigated the specific affinity for distinct matrix proteins, including laminin, collagen types I, III, and IV, and fibronectin, versus neutral control proteins in various small-intestine epithelial cell cultures . Both primary cells and intestinal epithelial cell lines display enhanced affinity for basement membrane constituents compared with interstitial collagens . Only the very undifferentiated, proliferative intestinal epithelial cells also synthesize these proteins, as determined by immunofluorescence and radioimmunoassay . On polarization and maturation, biosynthesis of basement membrane proteins is markedly reduced . Differentiation of intestinal epithelial cells is promoted only by laminin . Fibronectin and collagen type IV have no effect . Putative cell membrane receptors for individual basement membrane proteins are discussed.

Res Exp Med (Berl), 1988, 188(5), 391 - 6
Effects of pantothenic acid on fibroblastic cell cultures; Lacroix B et al.; To evaluate the effects of pantothenic acid during wound healing processes, fibroblastic cell cultures originating from foreskin were established and subcultured by trypsinization . PA (40 micrograms/ml) was added to the basal culture medium . The cell proliferation was estimated by cell count and determination of 3H-thymidine incorporation . The protein synthesis and secretion were determined by dosage in the cells and in the culture medium . When PA was added to the medium, a significant increase of cell proliferation and of 3H-thymidine incorporation was observed mainly during the first few days . PA also stimulated intracellular protein synthesis, but did not induce a release of proteins in the culture medium . The exact mechanism involved in this phenomenon remains unclear at this time.

Exp Pathol, 1988, 35(3), 159 - 76
Proteoglycan biosynthesis is stimulated by D-penicillamine in chondrifying high density cell cultures; Modis L et al.; Chondrifying high density cell cultures of stage 22-24 chick embryo limb bud mesenchyme were treated with 5, 10 and 15 mmol/l D-penicillamine (DPA) for 4 and 6 days . The cultures were analyzed with morphological and biochemical techniques to learn more about the effect of DPA on the metabolism of cartilage glycosaminoglycans (GAGs) . Using light and electron microscopic histochemical reactions for GAG, a considerable increase in the intensity of staining of the cartilage matrix could be detected in cultures treated with DPA as compared to the untreated controls . The uronic acid content of the treated cultures was higher than that of the controls . Liquid scintillation measurements and autoradiography revealed that DPA treatment increased the 35S-sulfate into the cultures . These data suggest that DPA - besides its well known inhibitory effect on collagen crosslink formation - alters the metabolism of sulfated GAGs in differentiating cartilage . It is supposed that DPA stimulates the biosynthesis of these macromolecules.

Prostate, 1988, 13(4), 289 - 97
Effect of a 4-methyl-4-aza steroid on androgen metabolism by rat ventral prostate epithelial and stromal cell cultures: selective inhibition of 5 alpha-reductase activity; Orlowski J et al.; The effect of a potent steroid metabolic inhibitor, 17 beta-N,N-diethylcarbamoyl-4-methyl-4-aza-5 alpha-androstan-3-one (DMAA), on androgen metabolism was investigated in primary monolayer cultures of rat ventral prostate epithelial and stromal cells . Using testosterone (T) as substrate, 5 alpha-reductase (5 alpha-R) activity in both cell types was inhibited by greater than 98% at an inhibitor concentration of 1000 nM . The concentrations required to produce a 50% inhibition (IC50) were 7.4 and 9.0 nM for epithelial and stromal cells, respectively . To examine the specificity of this compound, its effect on other steroid-metabolic enzymes was examined . DMAA at a concentration of 1,000 nM had no effect on 3 alpha-hydroxysteroid oxidase (3 alpha-HSORox), 3-ketosteroid reductase (3 alpha-HSORred), and 6/7-hydroxylase (6/7-HSH) activities in both cell types; 17 beta-hydroxysteroid oxidase (17 beta-HSORox) activity, located primarily in epithelial cells, also was not influenced by DMAA . In contrast, epithelial 3 beta-hydroxysteroid oxidase (3 beta-HSORox) and 3-ketosteroid reductase (3 beta-HSORred) activities were inhibited by 65% (P less than .001) and 58% (P greater than .05), respectively, albeit the latter result was not statistically significant . Stromal 3 beta-HSORox and 3 beta-HSORred activities were negligible; hence the effect of the inhibitor of these enzymes could not be assessed . In conclusion, DMAA is a relatively selective and potent inhibitor of 5 alpha-R activity in primary cultures of rat ventral prostate epithelial and stromal cells and should be a useful compound for antagonizing androgen-mediated actions in the prostate and other androgen target tissues.

Eur Urol, 1988, 15(3-4), 259 - 63
Growth characteristics of cultured human normal bladder epithelial cells: a comparison with urothelial carcinoma cell cultures; Logothetou-Rella H et al.; We present the in vitro growth characteristics and morphology of the first established long-term culture of human normal bladder epithelium . Normal bladder cells were grown with a mean generation time of 44 h and a life span of 33 population doublings in 12 weeks . The cells exhibited contact inhibition of growth . The cultures were morphologically examined and compared with transitional cell carcinoma in culture.

J Craniofac Genet Dev Biol, 1988, 8(1), 21 - 33
Alterations in craniofacial growth induced by isotretinoin (13-cis-retinoic acid) in mouse whole embryo and primary mesenchymal cell culture; Watanabe T et al.; Recent evidence has demonstrated that 13-cis-retinoic acid (13-cis-RA, or isotretinoin) is responsible for various craniofacial malformations in the rodent and human embryo . Our studies have been directed toward understanding this effect using mouse whole embryo and primary cell cultures . In whole embryo culture, 13-cis-RA caused significant overall embryonic growth retardation, especially in the primary and secondary palatal processes . In embryos explanted on day 10 of gestation and exposed for 24 or 48 hr, the mesenchyme beneath the epithelium of the nasal and maxillary processes contained pyknotic nuclei as well as a dramatically reduced number of nuclei incorporating 3H-thymidine . The secondary palatal processes and the roof of the oral-nasal cavity had fewer mesenchymal cells than control embryos . The incorporation of 3H-thymidine into TCA-insoluble macromolecules was 30% less in the retinoid-treated heads . In primary cell cultures from day-12 mouse secondary palatal mesenchyme, subsequent cell growth was decreased at concentrations of 13-cis-RA greater than 1 X 10(-5) M . After a 40-hr treatment period, labeling indices in retinoid-treated cells were significantly lower than control values (25% compared with 40%) . Retinoic acid also caused a significant, concentration-dependent decrease in 3H-thymidine incorporation . The inhibitory effect of 13-cis-RA on proliferation of oral-nasal mesenchymal cells appears to be related to the production of craniofacial malformations.

Acta Neuropathol (Berl), 1988, 76(5), 433 - 40
Correlation of DNA ploidy and morphological features of human glioma cell cultures with the establishment of cell lines; Onda K et al.; Eleven gliomas were serially cultivated and examined for DNA distribution by flow cytometry and simultaneously for morphological features by light microscopy at the various passage levels until passage 50 at most . Seven gliomas (four low-grade gliomas, three anaplastic gliomas) showed a similar DNA distribution pattern with a main diploid and small tetraploid peaks at various passages . In this group, only one culture formed a permanent cell line, whereas six cultures showed a limited growth ranging from 6 to 24 passages . In contrast, the other four gliomas (each an anaplastic glioma) showed a marked change of DNA distribution through passages and finally a single DNA aneuploid population prospered . Each of these four gliomas yielded established cell lines . Thus, it is suggested that the change of DNA ploidy and prosperity of DNA aneuploid populations in flow cytometry might be used as early and reliable indices for the later establishment of glioma-derived permanent cell lines . Since the changes of DNA distribution are frequently associated with the morphological changes, as seen in the latter group, careful tracing of morphological features is valuable in determining of the fate of cultures, especially in the absence of a flow cytometer . The correlation between the potential to become established cell lines and histology of the original gliomas is also discussed.

Neurofibromatosis, 1988, 1(2), 85 - 92
Cell culture studies on neurofibromatosis (von Recklinghausen) . VI . No increased stimulation of cell proliferation by sera from neurofibromatosis patients; Binder K et al.; The growth-stimulating activity of sera from 10 patients with von Recklinghausen neurofibromatosis (NF-1) was compared with that of sera from 10 age- and sex-matched healthy donors . Cell cultures derived from peripheral neurofibromas and from skin biopsies of healthy persons were used as indicator cells . Stimulation of 3H-thymidine incorporation increased with the clotting time of the blood samples, reaching a plateau at about 35 h with no decline of activity during the following 48 h . There was no difference between the stimulating activity of sera from the NF-1 patients and that of sera from the healthy donors with either type of indicator cells . This held true when the indicator cells were stimulated during the exponential growth phase or when serum-starved confluent cells were exposed to the various sera . Three NF-1 strains used as indicator cells during this investigation showed a significantly less vigorous response to both types of human sera and to fetal calf serum than the respective control strains . All human sera collected after 42 h clotting time were superior to fetal calf serum with both types of indicator cells.

Acta Biol Hung, 1988, 39(1), 39 - 47
Binding overlap and internalization of gonadotropin and thyrotropin in neonatal rat testicle and ovary cell cultures and the Chinese hamster ovary (CHO) cell line; Gruszczynska M et al.; Colloidal-gold-labeled gonadotropin and colloidal-gold-labeled thyrotropin were bound by Chinese hamster ovary (CHO) and primary neonatal rat ovary and testicle cell cultures in the same sites and in the same quantities . The conditions of internalization and the intracellular fate of the bound gold-labeled hormones were also similar in every respect . Pretreatment with either hormone imprinted the cells also for the related hormone, as judged from the increased binding and internalizing capacity of the pretreated cells for either hormone, and from identical patterns of post-binding receptor aggregation.

J Perinat Med, 1988, 16(5-6), 477 - 84
Hormone release by primary amniotic fluid cell cultures; Medina-Gomez P et al.; Amniotic fluid cells have been widely used in prenatal diagnosis; however, there is great heterogeneity of the cells and their origin . In this study we analyze the karyotype and release of human chorionic gonadotropin (hCG), human chorionic somatomammotropin (hCS), free estriol (E 3), prolactin (PRL) and progesterone (P) of amniotic fluid cells from primary cultures of six normal and two anencephalic fetuses . In all the amniotic fluid samples there was release of hCG; in one amniotic fluid, in which several tetraploid colonies were found . PRL and P were also released . The heterogeneity of amniotic fluid cell morphology and their hormone release in culture was confirmed . The presence of hormones like hCG supports the trophoblastic origin of some amniotic fluid cells from normal and anencephalic fetuses . Other hormones, such as PRL and P could be used in the differential diagnosis between the karyotype of fetal membranes and the true fetal karyotype . Amniotic fluid cell cultures used in prenatal diagnosis yielded second trimester placental cells without any elaborate methods that could be used as cell models for hormone studies.

Acta Physiol Hung, 1988, 72(1), 35 - 40
Role of the time factor in the development of hormonal imprinting of insulin receptors in Chang liver cell cultures; Csaba G et al.; Chang liver cells were pretreated with insulin for 5, 10, 20 and 60 minutes, and 4 and 24 hours after the treatment the binding of FITC-labelled hormone was measured . Hormone binding decreased after 5 minutes treatment, while longer treatments the binding increased considerably . The binding of hormone, at 4 hours investigation, to the nuclear membrane increased more markedly than in plasma membrane, indicating the important role of the imprinting of nuclear membrane . At 24 hours, the increased binding capacity is more characteristic for the plasma membrane.

Mol Endocrinol, 1988 Jan, 2(1), 55 - 61
Follicle-stimulating hormone induces transient expression of the protooncogene c-fos in primary Sertoli cell cultures; Hall SH et al.; The expression of the protooncogene c-fos has been associated with the transduction of cell surface stimuli into changes in nuclear function . To evaluate the possibility that this protooncogene plays a role in the gonadotropin-dependent gene regulation, the effect of FSH on the expression of c-fos was studied in primary Sertoli cell cultures . Sertoli cells were stimulated for different time intervals with FSH and c-fos mRNA levels measured by Northern RNA blot analysis . FSH treatment increased c-fos mRNA transiently with a maximal stimulation reached in 1 h . The level of c-fos mRNA returned to basal level within 4-6 h . The induction of c-fos mRNA was dependent on the concentration of FSH used with an ED50 of 3-5 ng/ml ovine FSH-16 . A similar increase in c-fos expression was induced with highly purified hFSH . The c-fos mRNA was also elevated after treatment of the Sertoli cell with (Bu)2cAMP and forskolin . (Bu)2cAMP treatment led to a sustained induction of c-fos mRNA, with increased mRNA levels being maintained after 12 h . The FSH-dependent induction of c-fos mRNA was still present in cells treated for 3 h with cycloheximide, but it was greatly reduced by actinomycin D pretreatment . These data indicate that FSH induces a transient expression of c-fos in cultured Sertoli cells . This induction is probably mediated by cAMP and likely involves an increased transcription of the c-fos gene . Early expression of this gene might be an intermediate step required for gonadotropin-dependent regulation of expression of other genes.

Atherosclerosis, 1988 Jan, 69(1), 61 - 8
Glycosaminoglycan content in neonatal rat aortic smooth muscle cell cultures; Ohishi H et al.; In the present study the biosynthesis of glycosaminoglycans (GAGs) by neonatal rat aortic smooth muscle cells in culture was studied . Heparan sulfate (HS) was the predominant GAG of the cell layer accounting for 32-49% of the total GAGs depending on the time in culture . The presence of low sulfated chondroitin sulfate (LSC) in aortic smooth muscle cell cultures is reported here for the first time . The effect of ascorbate on the synthesis and accumulation of these macromolecules resulted in a relative increase of C4S and DS in the cell layer . In contrast, the distribution of the GAGs which were secreted into the medium was not significantly effected by the addition of ascorbate . While HS was always found to be a minor component, the other GAGs were present in about equal concentrations . The total GAG accumulation in the medium was much greater (91-97%) than that of the cell layer (3-9%) indicating that the cells are synthesizing relatively large amounts of GAGs, although incorporation of these macromolecules into the extracellular matrix was consistently low.

Life Sci, 1988, 42(1), 47 - 60
Superfused pituitary cell cultures: effects of culture conditions on apparent responsiveness to LHRH stimulation administered as short duration pulses; O'Conner JL et al.; We wished to study estrous cycle related differences in LH and FSH responsiveness to pulsatile LHRH . Such studies are very difficult to perform in vivo under controlled conditions; therefore, an in vitro superfused anterior pituitary cell culture system was evaluated for its capacity to support differences in estrous stage associated LHRH responsiveness . Three vital culture system parameters were evaluated; these parameters were (1) culture medium composition, (2) duration allowed for cell attachment to microcarrier beads and (3) superfusion flow rate utilized during pulsatile LHRH stimulation . It was found that a culture system which utilized 10% Nu Serum in DMEM (final protein concentration of 1.8 mg/ml; final serum concentration of 2.5%), an attachment time of 48 hrs and a flow rate of 0.125 ml/min most successfully maximized LH responsiveness at the lowest serum concentration . These studies indicated that although one may be able to observe LHRH responsiveness under a wide range of culture conditions, responsiveness may nonetheless be maximized by judicious adjustment of culture conditions.

J Cell Physiol, 1988 Jan, 134(1), 149 - 54
Comparison of tyrosinase levels in amelanotic and melanotic melanoma cell cultures by a competitive enzyme-linked immunoadsorbent assay and by immunotitration analysis; Fuller BB et al.; Melanogenesis in mammalian pigment cells is regulated by changes in the activity of tyrosinase, the rate-limiting enzyme for melanin synthesis . Because recent evidence suggests that this enzyme may exist in pigment cells in both active and inactive stages, a competitive enzyme-linked immunoadsorbent assay (ELISA) was developed to compare tyrosinase levels in amelanotic and melanotic melanoma cell clones . The melanotic cell line used for this study, MEL-11A, had basal tyrosinase levels approximately 40 times that of the amelanotic cell line, AM-7 . Both cell lines responded to melanocyte-stimulating hormone by demonstrating large increases in tyrosinase activity . For competitive ELISA analysis of tyrosinase levels in these two clones, microtiter plates were coated with purified tyrosinase, and trypsinized cell extracts were tested for their ability to compete with bound tyrosinase for antibody binding . Although tyrosinase activity in the amelanotic clone was 1/40 that of the melanotic clone, immunoreactive tyrosinase levels in AM-7 cells were found to be approximately one-half that present in the melanotic clone . Additional evidence for the presence of an inactive (or at least, catalytically less active) enzyme in AM-7 cells was obtained from immunotitration analysis of tyrosinase in cell extracts from both cell lines . These results suggest that at least some amelanotic melanoma cells may contain significant levels of catalytically inactive tyrosinase molecules and that the level of pigmentation in mammalian melanocytes may be regulated by a tyrosinase activation process.

Tissue Cell, 1988, 20(4), 477 - 92
Rat heart-derived endothelial and smooth muscle cell cultures: isolation, cloning and characterization; Diglio CA et al.; This report describes the initiation, cloning and establishment of long-term serial cultures of rat heart-derived vascular endothelial (EC) and smooth muscle cells (SMC) . Populations of these cells derived from both the macro-and microcirculation were obtained utilizing isolated heart perfusion technique . Elimination of potential mesothelial cell contamination was achieved by ethanol fixation of the pericardial surface prior to perfusion . Initial outgrowths from perfusate yielded both endothelial (rapid adhering) and smooth muscle (slow adhering) appearing cell populations . Subsequent pooling of individual EC colonies resulted in maintaining, with gradual subcultivation, a stable homogeneous population which was designated RHE-parent . Upon continual subculture late passage (greater than P10) RHE-parent cell cultures expressed a marked heterogeneity in endothelial phenotypes . Cloning experiments resulted in establishing two distinct EC populations designated RHE-clone 1A and RHE-clone 2A . All RHE cell cultures exhibited the typical cobblestone growth pattern and positive immunofluorescent staining for factor VIII related antigen . In contrast, rat heart-derived smooth muscle cell (RH-SMC) cultures displayed the typical multilayered 'hill and valley' pattern and positive fluorescence for SMC-specific actin and myosin antibodies . Additional EC preparations, obtained without prior fixation of the pericardial surface, revealed cell clusters which stained positive for cytokeratin . On the other hand, RHE parent and cloned populations stained exclusively for vimentin, further confirming the absence of mesothelial cell contamination in these cultures . Cell growth studies on early (less than P10) and late (greater than P10) passage RHE-parent population revealed markedly different cell growth responses and cell morphology . Both EC cloned populations and more notably RHE-parent (less than P10) cultures were capable of significant growth when maintained in limiting serum concentration . Growth studies using serum-free RHE-parent conditioned medium demonstrated mitogenic activity when tested on RHE-parent cultures indicating the presence of an endothelial cell-derived growth factor . These studies indicate that long-term RHE and RH-SMC derived cell cultures can serve as a useful model to study the biology of vascular cells derived from different sites . In addition the demonstration of mitogenic activity in these cultures will enable us to explore further the nature of this response and compare this phenomenon with growth factors identified in large vessel cell systems.

Trans R Soc Trop Med Hyg, 1988, 82(3), 360 - 2
A septate polycarbonate cell culture unit used for Plasmodium falciparum and hybridomas; Thelu J et al.; A new material, makrolon, is used for the construction of a large-scale cell culture vessel . It is strong, light, transparent, thermostable, septate and inexpensive . Several independent vessels of 500 ml each can be stacked . It has been used for Plasmodium falciparum and hybridoma cultures, where frequent renewal of the medium and a large gas/liquid interface are required.

Comp Immunol Microbiol Infect Dis, 1988, 11(3-4), 207 - 14
{Diagnosis of rabies by cell culture}; Barrat J et al.; Mouse inoculation test (MIT) is a highly sensitive test for rabies diagnosis but slow and expensive . To detect rabies virus an in vitro technique using Neuro 2a cell culture (CC) was compared with MIT in two laboratories . In one laboratory, CC appeared to be on the whole more sensitive than MIT, nevertheless MIT was the only one to detect some positive samples . In the other laboratory, MIT was more sensitive . These results justify the use of CC for epidemiological diagnosis but emphasize the interest of MIT (the reference technique) for special cases.

J Neural Transm, 1988, 72(3), 173 - 83
Anticonvulsant drug actions on neurons in cell culture; Macdonald RL; Two actions of clinically used antiepileptic drugs have been studied using mouse neurons in primary dissociated cell culture . The antiepileptic drugs phenytoin, carbamazepine and valproic acid were demonstrated to limit sustained high frequency repetitive firing of action potentials at free serum concentrations that are achieved in patients being treated for epilepsy . Furthermore, an active metabolite of carbamazepine also limited sustained high frequency repetitive firing while inactive metabolites of phenytoin and carbamazepine did not limit sustained high frequency repetitive firing . Phenobarbital and clinically used benzodiazepines limited sustained high frequency repetitive firing of action potentials, but only at concentrations achieved during the treatment of generalized tonic-clonic status epilepticus . Ethosuximide did not limit sustained high frequency repetitive firing even at concentrations four times those achieved in the serum of patients treated for generalized absence seizures . Phenobarbital and clinically used benzodiazepines enhanced postsynaptic GABA responses at concentrations achieved free in the serum during treatment of generalized tonic-clonic or generalized absence seizures . However, phenytoin, carbamazepine, valproic acid and ethosuximide did not modify postsynaptic GABA responses at therapeutic free serum concentrations . These results suggest that the ability of antiepileptic drugs to block generalized tonic-clonic seizures and generalized tonic-clonic status epilepticus may be related to their ability to block high frequency repetitive firing of neurons . The mechanism underlying blockade of myoclonic seizures may be related to the ability of antiepileptic drugs to enhance GABAergic synaptic transmission . The mechanism underlying management of generalized absence seizures remains unclear.

Diagn Clin Immunol, 1988, 5(6), 304 - 8
Depression by Fc gamma receptor ligands of SRBC-induced IgM-PFC generation in human blood mononuclear cell cultures; Gabrielli A et al.; A tissue-culture system to stimulate human peripheral blood mononuclear cells (PBMC) has been employed in which IgM plaque-forming cell (IgM PFC) generation in response to sheep erythrocytes (SRBC) is dependent on macrophages and T suppressor and helper lymphocytes . In this system PBMC from normal subjects give IgM PFC responses ranging from 26 to 938 PFC/culture . Heat-aggregated human IgG or immune complexes present for the duration of culture induce a significant depression of PFC . Unaggregated IgG has no effect on the response or only a moderate stimulatory effect at the highest dose . The results of these experiments are compatible with previous results in a murine system, which indicated that Fc gamma receptor-positive (Fc gamma R+) cells in the suppressor subset are the target for aggregated IgG and induce depression of the PFC response . A similar mechanism may be operating in the human system described here, although the target cell has not been identified . These results may reflect a mechanism of immunomodulation dependent on interaction of Fc receptor (FcR) ligands with FcR, which may play a role in the pathogenesis of immune complex disorders.

Lung, 1988, 166(6), 339 - 46
Localization of transglutaminase activity in type II epithelial cell cultures and elevation of enzyme activity in lungs of rats instilled with quartz; Hind AL et al.; Transglutaminase activity, assessed by the incorporation of {14C}-putrescine into N-acetylated dephosphorylated beta-casein, was not detectable in sonicates of alveolar macrophages and fibroblasts but was located in all preparations of rat alveolar type II cells . Enzyme activity was induced in these cells up to 7 days in vitro but not stimulated further by the direct addition of quartz to the cultures . Transglutaminase activity in whole lung sonicates increased significantly after short-term exposure to DQ-12 quartz . An increase in the numbers of type II cells and subsequent release of activated transglutaminase, concomitant with a quartz-induced elevation in lung calcium levels and potential protein substrates, is likely to lead to an increase in protein crosslinking both in the alveolar space and interstitium.

Dev Neurosci, 1988, 10(3), 165 - 72
Developmental expression of glial fibrillary acidic protein and glutamine synthetase in serum-free aggregating cell cultures of fetal rat telencephalon; Monnet-Tschudi F et al.; Serum-free aggregating cell cultures of fetal rat telencephalon were examined by a combined biochemical and double-labeling immunocytochemical study for the developmental expression of glial fibrillary acidic protein (GFAP) and glutamine synthetase (GS) . It was found that these two astroglial markers are co-expressed at different developmental stages in vitro . During the phase of cellular maturation (i.e . between days 14 and 34), GFAP levels and GS activity increase rapidly and in parallel . At the same time, the number of immunoreactive cells increase while the long and thick processes staining in early cultures gradually disappear . The present results demonstrate that in this particular cell culture system only one type of astrocytes develops which expresses both GFAP and GS and which attains a relatively high degree of maturation.

J Neurosci, 1988 Jan, 8(1), 185 - 96
Pharmacology of glutamate neurotoxicity in cortical cell culture: attenuation by NMDA antagonists; Choi DW et al.; The antagonist pharmacology of glutamate neurotoxicity was quantitatively examined in murine cortical cell cultures . Addition of 1-3 mM DL-2-amino-5-phosphonovalerate (APV), or its active isomer D-APV, acutely to the exposure solution selectively blocked the neuroexcitation and neuronal cell selectively blocked the neuroexcitation and neuronal cell loss produced by N-methyl-D-aspartate (NMDA), with relatively little effect on that produced by either kainate or quisqualate . As expected, this selective NMDA receptor blockade only partially reduced the neuroexcitation or acute neuronal swelling produced by the broad-spectrum agonist glutamate; surprisingly, however, this blockade was sufficient to reduce glutamate-induced neuronal cell loss markedly . Lower concentrations of APV or D-APV had much less protective effect, suggesting that the blockade of a large number of NMDA receptors was required to acutely antagonize glutamate neurotoxicity . This requirement may be caused by the amplification of small amounts of acute glutamate-induced injury by subsequent release of endogenous NMDA agonists from injured neurons, as the "late" addition of 10-1000 microM APV or D-APV (after termination of glutamate exposure) also reduced resultant neuronal damage . If APV or D-APV were present both during and after glutamate exposure, a summation dose-protection relationship was obtained, showing substantial protective efficacy at low micromolar antagonist concentrations . Screening of several other excitatory amino acid antagonists confirmed that the ability to antagonize glutamate neurotoxicity might correlate with ability to block NMDA-induced neuroexcitation: The reported NMDA antagonists ketamine and DL-2-amino-7-phosphono-heptanoate, as well as the broad-spectrum antagonist kynurenate, were all found to attenuate glutamate neurotoxicity substantially; whereas gamma-D-glutamylaminomethyl sulfonate and L-glutamate diethyl ester, compounds reported to block predominantly quisqualate or kainate receptors, did not affect glutamate neurotoxicity . The present study suggests that glutamate neurotoxicity may be predominantly mediated by the activation of the NMDA subclass of glutamate receptors--occurring both directly, during exposure to exogenous compound, and indirectly, due to the subsequent release of endogenous NMDA agonists . Given other studies linking NMDA receptors to channels with unusually high calcium permeability, this suggestion is consistent with previous data showing that glutamate neurotoxicity depends heavily on extracellular calcium.

Glia, 1988, 1(1), 64 - 73
Electrical coupling between astrocytes and between oligodendrocytes studied in mammalian cell cultures; Kettenmann H et al.; The characteristics of electrical coupling between astrocytes and between oligodendrocytes were analyzed in cell cultures derived from rodent central nervous system . Experiments were carried out by impaling one member of a glial pair with separate voltage recording and current passing electrodes (cell 1) and the other cell, a measured distance from the first, with a voltage-recording electrode (cell 2) . Astrocyte pairs within 300 microns of one another were always coupled . The coupling ratio was determined for 23 astrocytic pairs various distances apart, and decreased with distance in a roughly exponential manner . The average coupling ratio of astrocytes within 100 microns of each other was 0.44 +/- 0.32 . Oligodendrocytes were less strongly coupled to each other than astrocytes . Even cells immediately adjacent to one another were often uncoupled . Among coupled oligodendrocytes within 100 microns of each other, the average coupling ratio was 0.11 +/- 0.1 . Current passage between pairs of astrocytes and pairs of oligodendrocytes was nonrectifying . Application of 0.5 mM BaCl2 or 44.6 mM CsCl (substituted for NaCl) depolarized and increased the input resistance of astrocytes and oligodendrocytes . These ions also increased the coupling ratio in astrocyte pairs and oligodendrocyte pairs; this effect was rapid in onset and completely reversible . Ba++ and Cs+ appear to block resting K+ conductance in glia and probably increase the coupling ratio by increasing the effective length constant of the glial membrane without any direct effect on junctional resistance . In three cases, oligodendrocyte pairs that appear uncoupled in normal solution exhibited coupling in the presence of BaCl2 or CsCl . This suggests that oligodendrocytes may be widely coupled by junctions that provide only weak electrical interaction; such junctions might be important for the exchange of small metabolically active molecules . The strong electrical coupling among astrocytes, in concert with their K+-selective membrane conductance, would provide for an electrical syncytium well designed to transport K+ away from areas of focal extracellular accumulation (i.e., the spatial buffer mechanism), and these cells, more than oligodendrocytes, may provide this function.

Horm Res, 1988, 29(4), 162 - 7
Cushing's syndrome due to bilateral adrenal macronodular hyperplasia with undetectable ACTH: cell culture of adenoma cells on extracellular matrix; Cheitlin RA et al.; A 59-year-old man developed Cushing's syndrome with massive bilateral adrenal macronodular hyperplasia . Plasma ACTH levels were undetectable both peripherally and in the inferior petrosal sinus . Computed tomography scans of his pituitary were normal . Hypercortisolism was not suppressed by high doses of dexamethasone . His adrenal cells were successfully isolated and grown on bovine corneal extracellular matrix . The cultured cells displayed strikingly rapid growth and hypersecretion of cortisol during incubation with control serum . Addition of the patient's serum to cultured fetal adrenal cells did not accelerate their growth . This was the first experience with our in vitro system in this rare clinical condition . The techniques described here may be used for future in vitro adrenal studies . These in vivo and in vitro data indicate that this patient's bilateral adrenal hyperfunction and growth were independent of ACTH.

Comp Immunol Microbiol Infect Dis, 1988, 11(2), 93 - 8
Propagation and quantitation of animal herpesviruses in eight cell culture systems; Peterson RB et al.; A comparative study was carried out to determine the relative sensitivities of eight different cell culture systems to six different herpesviruses of animals . The cells used were: OFL (ovine fetal lung), ML (mink lung), FK (ferret kidney), PTK-2 (potoroo kidney), TEK (turkey embryo kidney), ED (equine dermal), BT (bovine turbinate), and PK15 (porcine kidney) . The viruses tested were: PRV (pseudorabies) of swine, CPHV (caprine herpesvirus), IBRV (infectious bovine rhinotracheitis virus), DN-599 strain of bovine herpesvirus type 4, EHV-1 (equine herpesvirus), and CHV (canine herpesvirus) . On the basis of virus titers obtained and the time of appearance of CPE (cytopathic effects), ML cells were found to be the most useful because of their sensitivity to all six viruses tested . BT and OFL cells were also found to be highly sensitive to all viruses with the exception of CHV.

Bone, 1988, 9(2), 89 - 92
Forskolin inhibition of glucose transport in bone cell cultures through a cAMP-independent mechanism; van Valen F et al.; Studies are presented demonstrating inhibition of glucose transport by forskolin in human MG-63 osteosarcoma cells as well as osteoblast-like cells derived from normal human trabecular bone and chick calvaria . The cAMP-stimulators parathyroid hormone, prostaglandin E2, and isoproterenol did not influence glucose transport . Benzyl alcohol, a membrane lipid fluidity modulator, also provoked inhibition of the glucose uptake rate . Effects of forskolin and benzyl alcohol were not additive . It is suggested that cAMP is not a mediator of glucose transport in bone cells, and that forskolin inhibits glucose transport via a cAMP-independent mechanism.

Acta Physiol Hung, 1988, 71(1), 35 - 40
Imprinting with oxytocin and vasopressin in Chang liver cell cultures: hormonal overlap, binding and influence on cell division; Bohdaneczky E et al.; In Chang liver cells the administration of oxytocin and vasopressin as well as the combined application of the two hormones will result in a positive binding imprinting for oxytocin and a negative binding imprinting for vasopressin . The hormones are able to increase the mitotic capacity of the liver cells even without previous imprinting, both in the case of treatment for 4 hours and for 24 hours; the change, however, is more marked in the case of treatment for 4 hours . Treatment for 24 hours results also in some functional imprinting.

Med Microbiol Immunol (Berl), 1988, 177(2), 91 - 100
Adaptation of hepatitis A virus to high titre growth in diploid and permanent cell cultures; Gregersen JP et al.; A hepatitis A virus isolate originally obtained from the feces of a clinically ill patient and passaged in diploid human embryonic kidney and lung cells was adapted to grow in MRC-5, Cercopithecus aethiops muscle and in Vero cells . Three different adaptation methods were applied . Either method proved to be suitable to finally give high virus titres of cell-bound as well as cell-free virus in the supernatant of infected cultures during 10 to 15 passages . An easily performable immunoperoxidase staining method was used for the titration of hepatitis A virus in microtitre plates . Cytopathogenic changes in MRC-5 cell cultures infected with fully adapted virus are described.

Calcif Tissue Int, 1988 Jan, 42(1), 23 - 33
In vitro evidence that local and systemic skeletal effectors can regulate 3{H}-thymidine incorporation in chick calvarial cell cultures and modulate the stimulatory actions(s) of embryonic chick bone extract; Farley JR et al.; These investigations were intended to determine whether local and systemic skeletal effectors--3'5'-cyclic adenosine monophosphate (cAMP), prostaglandin E2 (PGE2), parathyroid hormone (PTH), 1,25-dihydroxyvitamin D (1,25(OH)2D), calcitonin, and NaF--could regulate 3{H}-thymidine incorporation (i.e., into DNA) in serum-free, monolayer cultures of embryonic chick calvarial cells, and/or modulate the activity of embryonic chick bone extracts to increase 3{H}-thymidine incorporation . In the absence of added bone extract, we found that calcitonin (0.1 U/ml), NaF (100 microM) and low-dose PTH (0.1 nM) stimulated 3{H}-thymidine incorporation, P less than .05 for each; isobutylmethylxanthine (IBMX--1 mM), 1,25OHD (10 nM), and high-dose PTH (10 nM) decreased 3{H}-thymidine incorporation; and PGE2 (1 microM) had no effect . The stimulatory actions of calcitonin, fluoride, and low-dose PTH were inductive, and the inhibitory actions of IBMX and 1,25(OH)2D were acute . PTH had complex time-dependent actions on 3{H}-thymidine incorporation, being inhibitory after 4-8 hours of exposure and stimulatory after 20-24 hours (P less than .001 for each) . The effects of calcitonin, fluoride, and low-dose PTH to increase 3{H}-thymidine incorporation were greater in calvarial cell cultures enriched for undifferentiated osteoprogenitor cells than in cultures enriched for differentiated osteoblastlike cells . PTH inhibited 3{H}-thymidine incorporation in the latter (i.e., osteoblastlike) cultures (P less than .005) . The inhibitory actions of IBMX and 1,25(OH)2D were independent of cell differentiation . Additional studies further revealed that these local and systemic skeletal effectors could also modulate the activity of embryonic chick bone extracts to increase 3{H}-thymidine incorporation in calvarial cell cultures . We found that calcitonin, fluoride, and low-dose PTH enhanced the effect of the extracts to increase 3{H}-thymidine incorporation (P less than .001 for each) . These activations were noncompetitive, indicating (1) mechanistic differences between the stimulatory actions of the effectors and the chick bone extract (i.e., different rate-limiting steps for the effects of each on 3{H}-thymidine incorporation); and (2) that neither calcitonin, fluoride, nor 0.1 nM PTH altered the apparent affinity of the cells for stimulatory activity(s) in the extract . High-dose PTH was a noncompetitive inhibitor with respect to bone extract activity, indicating that the effect of 10 nM PTH to decrease 3{H}-thymidine incorporation was mechanistically distinct from the effect of the bone extract to increase 3{H}-thymidine incorporation.(ABSTRACT TRUNCATED AT 400 WORDS)

J Clin Microbiol, 1988 Jan, 26(1), 22 - 4
Detection of herpes simplex virus in conventional tube cell cultures and in shell vials with a DNA probe kit and monoclonal antibodies; Espy MJ et al.; Specimens submitted for diagnosis of herpes simplex virus (HSV) infection were inoculated into shell vials and reacted with a commercial DNA probe kit (Pathogene; Enzo Biochem, Inc., New York, N.Y.) and an immunofluorescence assay at 16 h postinoculation . The results were compared with isolation of the virus in conventional tube cell cultures . Of 504 specimens, 105 (20.8%) were positive for HSV . Of the 105, 93 HSV-positive specimens (89%) were detected by all three assay systems . Maximum detection of HSV (100 of 105 {95%}) was obtained by probe or monoclonal antibody assay in shell vials, which had sensitivities of 98 and 97%, respectively, compared with viral recovery in conventional tube cell cultures (mean time for recognition of cytopathic effects, 2 days) . Both shell vial assays were 99% specific . The DNA probe kit may be used as an alternative to a monoclonal antibody and fluorescence assay in shell vials as a diagnostic method for rapid laboratory detection of HSV infection.

Braz J Med Biol Res, 1988, 21(5), 991 - 3
Comparison between the antigenic composition of bloodstream and cell culture-derived trypomastigotes of Trypanosoma cruzi; da Silva AM et al.; Antigens of bloodstream and cell culture-derived trypomastigotes of T . cruzi were compared by western blotting using sera of chronic chagasic patients as a source of antibodies . The immunoblots demonstrated that the two forms display extensive homology except for the 85- and 52-kDa bands . These antigens were more strongly stained in culture-derived trypomastigotes . Although the reported differences are not related to major antigens, these results might offer an explanation for previous studies showing that culture-derived trypomastigotes are more antigenic and infective in vitro than bloodstream trypomastigotes.

J Basic Microbiol, 1988, 28(8), 501 - 7
{Experimental studies of in vitro IFN induction by different polynucleotides in human cell culture systems}; Gluck B et al.; Several polynucleotides were tested for interferon induction in comparison with the standard Poly (IC) in human diploid fibroblasts and in human leukocyte suspensions . We received high IFN-titre following superinduction of the polynucleotides in human fibroblasts . These results show that under superinduction conditions partly 3 times more interferon is induced in comparison with the standard inductor Poly (IC) . The tested concentration of 10 micrograms/ml polynucleotides did not result in any IFN yields or only in very low ones in human leukocytes, which were only within the range of detectability.

Cytobios, 1988, 55(221), 113 - 23
Differential silver staining in lymphocytes and lymphoblastoid cell cultures; Martin-DeLeon PA et al.; An analysis of the patterns of silver-stained nucleolar organizer regions (AgNORs), an indication of rDNA transcriptional activity, was carried out in metaphases from peripheral lymphocytes and young lymphoblastoid cell cultures (LCL) transformed by Epstein-Barr virus . Four individuals previously shown to carry a double NOR (dNOR) were studied . The dNOR varied in staining frequency and showed intra-individual variation in appearance in both cell types . For each individual a significantly greater mean number of AgNORs was observed in LCL (7.0-8.04) when compared with lymphocytes (5.8-6.4) . To control for possible serum stimulation effects, lymphocytes from one individual were cultured in 20% serum as was the LCL of all subjects . The mean number of AgNORs in these lymphocytes remained significantly lower than in the LCL, as did the mean size of the dNOR, based on a relative score . Our results suggest that LCL have increased expression of rDNA, as evidenced by silver staining.

Adv Exp Med Biol, 1988, 239, 279 - 85
Partial characterization of suppressor factors in spleen cell culture supernatants of Mycobacterium lepraemurium-infected mice; Richard L et al.; Suppressor factors (SF) were released into the culture supernatant when spleen cells from M . lepraemurium-infected C57BL/6 mice were incubated at 37 degrees C in the absence of any inducing agents . Some of the SF appeared to be specific in that they inhibited the blastogenic response to mycobacterial antigens by opposition to the others which inhibited the blastogenic responses to PHA, Con A and LPS . All SF seemed to be produced in a cyclic manner . In mice infected 9 weeks earlier, the release of "specific" SF occurred early (4-8 h) during the incubation period whereas, the nonspecific SF were released later on (12-32 h) . Adoptive transfers of SF-containing culture supernatants depressed the expression of DTH to M.1m antigens but not its induction.

Cancer Res, 1987 Dec 15, 47(24 Pt 1), 6560 - 4
Inhibitory and stimulatory effects of glucocorticoid on androgen-induced growth of murine Shionogi carcinoma 115 in vivo and in cell culture; Hiraoka D et al.; It has been generally accepted for 20 years that the growth of Shionogi carcinoma 115 (SC115) is stimulated only by androgen in vivo and in cell culture . However, we recently found that the growth of SC115 is also stimulated by pharmacological, but not physiological, doses of glucocorticoid both in vivo and in cell culture and by pharmacological doses of estrogen only in vivo . In the present study, therefore, we investigated the effect of dexamethasone on androgen-induced growth of SC115 cells in vivo and in cell culture . In a serum-free medium {Ham's F-12:Eagle's minimum essential medium (1:1, v/v) containing 0.1% bovine serum albumin}, the proliferation of SC-3 cells (a cloned cell line from SC115 cells) estimated by cell number and DNA synthesis reached a plateau at 10(-8) M testosterone (up to 93-fold) or 10(-6) M dexamethasone (up to 7.2-fold); high stimulation induced by higher than 10(-8) M testosterone was inhibited by the addition of 10(-5)-10(-8) M dexamethasone in a concentration-dependent manner, whereas low stimulation induced by lower than 10(-10) M testosterone was significantly enhanced by the addition of dexamethasone . The presence of typical glucocorticoid and androgen receptors in SC-3 cells was also demonstrated; dexamethasone did not bind to androgen receptor and testosterone did not bind to glucocorticoid receptor . In castrated mice, the concomitant administration of dexamethasone again significantly inhibited the high growth of SC115 tumors induced by high doses of androgen but significantly enhanced the low growth induced by low doses of androgen . The present results demonstrate both inhibitory and stimulatory effects of glucocorticoid on androgen-induced proliferation of SC115 cells in cell culture and probably in vivo.

Biochem J, 1987 Dec 15, 248(3), 625 - 33
Pyridinedicarboxylates, the first mechanism-derived inhibitors for prolyl 4-hydroxylase, selectively suppress cellular hydroxyprolyl biosynthesis . Decrease in interstitial collagen and Clq secretion in cell culture; Tschank G et al.; Two pyridinedicarboxylates, predicted {Hanauske-Abel (1983) M.D.-Ph.D . Thesis, Philipps Universitat Marburg} and later found to be potent reversible inhibitors of purified prolyl 4-hydroxylase {Majaama, Hanauske-Abel, Gunzler & Kivirikko (1984) Eur . J . Biochem . 138, 239-245} were investigated with respect to their effect on hydroxyprolyl biosynthesis in the fibroblast/collagen and the macrophage/Clq systems, and the effect was compared with that of the iron chelator 2,2'-dipyridyl, the compound usually employed to inhibit cellular hydroxyprolyl formation . Only the enzyme-mechanism-derived pyridinedicarboxylates were highly selective inhibitors, and only they lacked overt cytotoxicity . Morphologically, their effect was restricted to the site of cellular hydroxyprolyl biosynthesis, i.e . the cisternae of the rough-surfaced endoplasmic reticulum . They were equally effective in the different cell types studied, and human and guinea-pig fibroblasts showed the same sensitivity . The minimal lipophilicity of the pyridinedicarboxylates necessitated high concentrations to achieve suppression of cellular hydroxyprolyl formation, but lipophilic bio-activatable pro-inhibitors may overcome this disadvantage . For the first time, experimental evidence is presented suggesting that, in cell culture, the biosynthesis of interstitial collagens and Clq can be suppressed selectively, identifying the pyridinedicarboxylates as promising pilot compounds for experiments in vivo.

Brain Res, 1987 Dec 8, 436(1), 9 - 17
Phencyclidine blocks two potassium currents in spinal neurons in cell culture; Aguayo LG et al.; We studied the effects of phencyclidine (PCP) on the transient and delayed outward K+ currents recorded from spinal cord neurons grown (10-20 days) in cell culture . Sodium channels were blocked with tetrodotoxin (1 microM) and solutions containing low calcium concentrations in the presence of Mg2+ or Co2+ (5 mM) were used to reduce Ca2+ currents . PCP decreased the amplitude and prolonged the decay phase of the action potentials recorded at a holding potential of -70 mV . PCP (0.1-0.5 mM) was more effective than tetraethylammonium (TEA) or 4-aminopyridine (4-AP) in reducing both transient and delayed currents . The amplitude of the transient current during control experiments was always larger than that of the delayed current . It appeared that 4-AP (5 mM) was more potent in blocking the transient current, while TEA (10 mM) modified the delayed current more effectively . Both currents were also reduced by about 10% when the cell soma was perfused with Co2+ . This suggested that a small fraction of the total outward current is a Ca2+-activated K+ current . The PCP-induced blockade of K+ currents in central neurons coupled with the profound synaptic effects of the drug may provide the basis for explaining the psychopathology of this hallucinogenic agent.

Med J Aust, 1987 Dec 7-21, 147(11-12), 552 - 4
Rapid diagnosis of cytomegalovirus infection by immunofluorescent detection of early antigen in cell culture; Phillips PA et al.; The conventional culture method for the isolation of cytomegalovirus was used in parallel with the rapid method of early-antigen detection to test 485 specimens that had been collected from a total of 208 patients over a period of 11 months . Statistically, the rapid immunofluorescent method was significantly better than was the conventional test for the detection of cytomegalovirus infections . The rapid method is technically simple, sensitive and specific, and is also inexpensive when a suitable polyclonal antiserum is available . This test merits use in virology laboratories that service large hospitals.

Cell Tissue Res, 1987 Dec, 250(3), 627 - 32
Biochemical and morphological characterization of primary kidney cell cultures from beige mutant mice; Lyerla TA et al.; Primary kidney cultures from adult beige-J (bgJ/bgJ) mice were selected for epithelial cell growth using D-valine medium . After 2 weeks of attachment and proliferation in vitro, the cells form a confluent or nearly confluent monolayer that retains several phenotypic characteristics of the beige-J mutant . These include large, multilamellar inclusion bodies that are apparently dysmorphic lysosomes, and higher concentrations of neutral glycosphingolipids and dolichols than control cells . beta-Glucuronidase activity, used as a lysosomal enzyme marker, is not elevated in beige-J-cultured kidney cells compared with controls, as it is in the intact kidney . The high levels of beta-glucuronidase activity in both control and mutant cells may mask expression of this difference in vitro . The action of the beige-J mutation in kidney cells is thought to be due to a block in exocytosis that results in the accumulation of abnormal lysosomes and their components . The maintenance of the beige phenotype in vitro indicates that the mutation is not suppressed in primary kidney cell cultures . The expression of the beige phenotype in vitro should be useful for studies concerning the primary lesion of this mutation.

J Clin Oncol, 1987 Dec, 5(12), 1912 - 21
Comparison between clinical response and in vitro drug sensitivity of primary human tumors in the adhesive tumor cell culture system; Ajani JA et al.; The newly described adhesive tumor cell culture system (ATCCS) offers a distinct advantage over other assays in that it has a high plating efficiency requiring low cell inoculum, it affords workable assays in approximately 70% of specimens from the heterogenous tumor types, and it has the ability to assay up to nine drugs at four different concentrations . Clinical correlations based on the ATCCS were obtained in 65 patients undergoing 71 clinical trials . Patients with melanoma, lung cancer, and sarcoma dominated the group . The most active in vitro drug was correlated per clinical trial . Thirteen of 17 (76%) sensitive in vitro predictions and 51 of 54 (94%) resistant in vitro predictions were accurate . The assay in this study had a sensitivity of 81% and specificity of 93% . These preliminary results are encouraging and warrant prospective trials to establish the true value of this assay to patients.

Diabetes, 1987 Dec, 36(12), 1460 - 7
Regulation of vascular permeability in cell culture; King GL et al.; Although one of the earliest findings of diabetic retinopathy is altered capillary permeability, metabolic factors in diabetes that may increase the permeability of capillaries to fluorescein are unknown . We have studied the effect of a variety of vascular and retinal cells and hyperglycemia on the diffusion rate of fluorescein . These studies were performed with a cell culture system that mimics the cross-section of a capillary by having two chambers separated with a porous membrane that can support the growth of cells on either side of the membrane . The addition of a confluent layer of endothelial cells or retinal pigmented epithelial (RPE) cells inhibited fluorescein diffusion between the two chambers 20- and 300-fold, respectively, after cells were cultured for greater than 5 days . Exposure of endothelial cells to 400 mg/dl glucose for either 3 or 100 days did not affect the barrier function of these cells . The barrier function of capillary endothelial cells isolated from BB rats with chronic diabetes and from nondiabetic animals did not differ . In contrast to endothelial cells and RPE cells, arterial smooth muscle and pericytes, which are not known to form tight junctions, did not inhibit the diffusion of fluorescein more than 2-fold . Surprisingly, the dual culture of endothelial cells with either retinal pericytes or smooth muscle cells resulted in a 50-fold increase in the rate of fluorescein diffusion, showing a disruption of the endothelial barrier . In summary, the intercellular connections between endothelial and epithelial cells that are responsible for the barrier to fluorescein diffusion are not functionally affected by chronic exposure to hyperglycemia or diabetic conditions.(ABSTRACT TRUNCATED AT 250 WORDS)

Gynecol Endocrinol, 1987 Dec, 1(4), 339 - 43
Aromatase activity in monolayer cell cultures of human endometrium; Neulen J et al.; Monolayer cell cultures of proliferative human endometrial stromal cells were incubated with 100 nCi 3H-androstenedione or 100 nCi 3H-testosterone for 15 hours . After termination of the incubations 16% of the recovered radioactivity was identified as estrone by thin layer chromatography and subsequent recrystallization . 3H-testosterone was almost quantitatively converted to 3H-androstenedione . 3H-dihydrotestosterone remained unmetabolized . These findings indicate a highly active 17 beta-ol-dehydrogenase and a high aromatizing capacity of human endometrial cells.

Pharmazie, 1987 Dec, 42(12), 848 - 50
{Is the prediction of LD50 using cell cultures generally valid?}; Halle W et al.; In this article we use the linear regression model to compare the values of IC50 and LD50 p.o . (mice and rats) taken from the literature and estimated in our laboratory . By this analysis it was found that for different substance classes a similar relationship exists between IC50 and LD50, even if different cell types or cellular activities were used for estimating the IC50 on the one hand and different modes of substance application in animal toxicity tests on the other hand . The minimum and maximum values of the measured LD50 (LD50G) registered in animal experiments deviated from the theoretical LD50 values (LD50T) on the (log transformed) regression line in approximately 77 per cent of the cases by the factor FG less than or equal to log 5 only . The linear correlation between IC50 and LD50 is to be expected only for such substances which do not act specifically via higher levels of integration, cell type specific reactions or metabolites, respectively . These and previously reported results indicate a certain general validity not only for the positive linear correlation between IC50 and LD50 but also for the predicting the LD50 in a relatively narrow dosage range on the basis of in vitro estimated IC50 values . With these results new possibilities were opened for reducing animal experiments to estimate LD50.

J Inorg Biochem, 1987 Dec, 31(4), 241 - 6
Coordination chemistry in biological media: reactions of antitumor Pt(II) and Au(III) complexes with cell culture media; Bell JD et al.; Reactions of cis-PtCl2(NH3)2 (1), Pt(1,2-diaminoethane)Cl2 (2), PtCl4(2-) (3), and AuCl4- (4) with intact cell culture media have been studied by spin-echo 500 MHz proton NMR spectroscopy . This has allowed us to observe reactions of components of the media at submillimolar concentrations . Upon the addition of 400 microM 1, 2, or 3 to the media, the S-methyl peak of methionine decreases in intensity and in each case a new peak appears which we have tentatively assigned to the S-CH3 of Pt(N,S-L-Met)2 . In the spectra of the media with 2, an additional peak appears, assignable to the S-CH3 of Pt(1,2-diaminoethane)(N,S-L-Met) . Upon the addition of Au(III) to the media, the S-CH3 peak of methionine also decreases in intensity and new peaks appear in the 2.6 to 2.8 ppm region, including a peak identified as the S(O)-CH3 peak of methionine sulfoxide . The other peaks are assignable to Au(I)-S(Met) species . Practical methods of following the reactions of metal complexes in cell culture media are becoming of wider significance with the increasing use of cell cultures for drug screening instead of animal tests.

Am J Pathol, 1987 Dec, 129(3), 511 - 22
Differentiation of tracheal mucociliary epithelium in primary cell culture recapitulates normal fetal development and regeneration following injury in hamsters; McDowell EM et al.; Hamster tracheal epithelial cells were grown in primary culture for 8 days on a collagen gel substrate, in hormone-supplemented serum-free medium (Ham's F-12) . On Days 1-3 in culture, the colonies were composed of a monolayer of poorly-differentiated flattened cells . Most of these were large cells which appeared to be altered secretory (mucous) cells . On Days 4 and 5, many of the epithelial cells were cuboidal . The rough endoplasmic reticulum was moderately developed, and mucous granules were seen at the cell apices . Preciliated and newly formed ciliated cells were observed on Day 6, and a differentiated mucociliary epithelium was established by Day 7 in culture . The study shows that in the hamster tracheal epithelium, the stages of normal fetal development and regeneration following injury, which have been characterized previously in vivo, are recapitulated in vitro . Formation of a mucociliary tracheal epithelium occurs within 7 days in vito and in vitro.

Diagn Microbiol Infect Dis, 1987 Dec, 8(4), 229 - 34
Comparison of two enzyme-linked immunosorbent assay (EIA) kits with immunofluorescence and isolation in cell culture for detection of respiratory syncytial virus (RSV); Subbarao EK et al.; Rapid diagnosis of respiratory syncytial virus (RSV) infections is based upon detection of viral antigen in cells obtained from the respiratory tract and usually employs immunofluorescence (IF) reactions or enzyme-linked immunosorbent assays (EIA) . The Pathfinder EIA kit (Kallestad Diagnostics) was compared with the Abbott EIA kit by evaluating each against isolation of RSV in cell culture and detection of antigen by IF . The Pathfinder kit identified 116 of 129 culture-positive and 72 of 90 culture-negative specimens; the sensitivity was 90 percent and the specificity was 80 percent . The sensitivity of the Abbott EIA test compared to isolation of RSV in cell culture was 91% (115 of 127), and the specificity was 83% (74 of 89) . Of 165 specimens evaluated by IF, the Pathfinder kit detected 97 of 105 IF-positive and 45 of 60 IF-negative specimens, giving a sensitivity of 92% and a specificity of 75% . The Abbott EIA compared similarly with IF, showing a sensitivity of 91% (98 of 108) and a specificity (42 of 54) of 78% . Visual reading of the Kallestad test resulted in a sensitivity of 92%, a specificity of 91%, positive predictive value of 95%, and negative predictive value of 86% . The Pathfinder EIA kit compared well with IF and the Abbott EIA for detection of RSV antigen but performed faster than the Abbott test and offers the option of a visual reading.

Diabetes, 1987 Dec, 36(12), 1476 - 82
Glyburide modulates insulin-mediated locomotor response of confluent cell cultures to wounding; Mascardo RN et al.; We determined whether the sulfonylurea glyburide could modify the insulin-induced locomotor response to wounding of bovine aortic, pulmonary artery endothelial, and gerbil fibroma cells . Serum-deprived cells were preincubated in medium containing glyburide (10(-4) M) or diluent, then exposed to insulin (10(-6) to 10(-12) M) at the time of wounding, after which the centrosomal-orientation and migratory responses of the cells bordering the experimental wound were monitored by immunofluorescence microscopy and time-lapse cinemicrophotography, respectively . Insulin (10(-6) M) stimulated the centrosomal-orientation and locomotor responses, resulting in the narrowing of the linear wound . Glyburide enhanced the effect of insulin on both parameters of the locomotor response and also sensitized the cells to a previously ineffective concentration of insulin (10(-12) M) . The action of glyburide depended on a preincubation period of greater than or equal to 60 min and was blocked by cycloheximide and actinomycin D . The relevance of this novel action of glyburide to the impaired healing process observed in diabetic patients requires further study.

J Cell Biol, 1987 Dec, 105(6 Pt 2), 3097 - 104
Fibronectin receptors of human keratinocytes and their expression during cell culture; Toda K et al.; Keratinocyte attachment to fibronectin (FN) substrata was inhibited by the peptide Gly-Arg-Gly-Asp-Ser-Pro-Cys, but not by the variant peptide Gly-Arg-Gly-Glu-Ser-Pro . The RGDS-containing peptide did not inhibit keratinocyte adhesion to collagen . Keratinocyte adhesion to FN substrata also was inhibited by polyclonal anti-FN receptor antibodies originally prepared against the 140-kD FN receptors of Chinese hamster ovary (CHO) cells . Anti-CHO FN receptor antibodies did not, however, inhibit keratinocyte adhesion to collagen substrata . A monoclonal antibody designated VM-1 that was prepared against human basal keratinocytes inhibited keratinocyte adhesion to collagen but not to FN . Based on these results, we conclude that keratinocytes have distinct FN and collagen receptors . Experiments were performed to compare the expression of FN receptors on keratinocytes freshly isolated from skin and keratinocytes harvested from cell cultures . Cells harvested from keratinocyte cultures were able to neutralize the inhibitory activity of anti-CHO FN receptor antibodies and were able to attach and spread on anti-CHO FN receptor-coated substrata . Cells freshly harvested from skin, however, did not neutralize the antibodies, nor did they attach and spread on antibody-coated substrata . To learn more about the biochemical nature of the keratinocyte FN receptors, we performed immunoaffinity chromatography and immunoprecipitation experiments using the anti-CHO FN receptor antibodies . Extracts from metabolically radiolabeled, 10-d cultured keratinocytes contained FN receptors that had a 135-kD component under reducing conditions and 115- and 155-kD components under nonreducing conditions . Similar components were observed in extracts from surface-radiolabeled cells indicating that the FN receptors were expressed on keratinocyte cell surfaces . On the other hand, extracts from metabolically radiolabeled, 1-d cultured keratinocytes lacked intact FN receptors but contained a component that migrated at 48 kD under reducing conditions and 50 kD under nonreducing conditions . Because this fragment was not detected in surface-radiolabeled keratinocytes that were freshly isolated from skin, it seems likely that the fragment was located inside the cells rather than on the cell surface . A 50-kD FN receptor fragment also was observed in extracts from 10-d cultured keratinocytes if leupeptin and pepstatin were omitted from the extraction buffer . The results suggested that human keratinocytes cultured for 10 d express the 140-kD class of FN receptors, but that these receptors are not expressed on the surfaces of keratinocytes freshly isolated from skin.(ABSTRACT TRUNCATED AT 400 WORDS)

Am Rev Respir Dis, 1987 Dec, 136(6 Pt 2), S23 - 6
The electrophysiologic and neurochemical properties of paratracheal neurones in situ and in dissociated cell culture; Burnstock G et al.; Although our knowledge of the intrinsic innervation of the airways is at present rather limited, it would appear that these neurones have several features in common with other intramural ganglia . They show a level of electrophysiologic diversity that might permit considerable integration and modulation to take place . Furthermore, the localization of a wide variety of neuropeptides in the neurones and in fibers within the paratracheal ganglia indicates that they may also possess the neurochemical specialization necessary to allow these ganglia to act as sites of complex local regulation.

Antiviral Res, 1987 Dec, 8(5-6), 299 - 310
Antiviral activity of uridine 5'-diphosphate glucose analogues against some enveloped viruses in cell culture; Gil-Fernandez G et al.; Twenty five analogues of uridine 5'-diphosphate glucose were screened against herpes simplex type 2, vaccinia virus, Sindbis virus and African swine fever virus . After screening, the compound 5'-{{{{(2",3",4",6"-tetra-O-benzoyl-alpha-D- glucopyranosyl)oxi}carbonyl}amino}sulfonyl}uridine (2), the synthesis of which has been reported (Camarasa et al., J . Med . Chem . 28, 40-46, 1985), was selected for further study . This compound showed in vitro activity against all viruses tested . The replication of herpes virus type 2 and African swine fever virus was completely inhibited at 100 micrograms/ml and 150 micrograms/ml respectively; vaccinia virus and Sindbis virus were inhibited to a lesser extent . The compound may inhibit several steps in the viral replication process.

Am J Pathol, 1987 Dec, 129(3), 503 - 10
Retrovirus-induced osteopetrosis in mice . Effects of viral infection on osteogenic differentiation in skeletoblast cell cultures; Schmidt J et al.; Newborn female strain NMRI mice were injected with a mouse retrovirus (OA MuLV) known to induce osteopetrosis . Primary skeletoblast cell cultures were established from humeri and calvaria of 3-day-old, 7-day-old, and 28-day-old animals . Infectious ecotropic MuLV was found in all humerus cultures from infected animals and in 7-day and 28-day calvaria cell cultures . Levels of alkaline phosphatase activity were markedly higher in cultures of calvaria and humeri from infected mice than in those from controls . In vitro infection of undifferentiated periosteal cells was followed by a decrease in cell growth and an increase in alkaline phosphatase activity . In contrast, differentiated osteoblast-like cells were barely susceptible to OA MuLV infection, and the virus did not influence their cell growth or differentiation . Electron-microscopic studies of skeletal tissue from infected old osteopetrotic mice showed virus particles associated with and budding from osteocytes and accumulated in devitalized osteocyte lacunae . The results indicate that progenitor cells of the osteoblastic lineage represent the target cells for OA MuLV in bone tissue, that virus infection induces an increase in osteoblastic activity, and that infected cells produce virus until full development of the disease.

Mol Cell Endocrinol, 1987 Dec, 54(2-3), 203 - 11
Alpha-adrenergic stimulation of growth hormone release in perifused rat anterior pituitary reaggregate cell cultures; Maertens P et al.; It has been previously shown that beta-adrenergic agonists stimulate growth hormone (GH) release from perifused rat anterior pituitary cell aggregates cultured in serum-free defined medium for 5-6 days . The present data show that the natural beta-agonist epinephrine (EPI) stimulates GH release through an additional alpha-adrenergic mechanism . The involvement of this mechanism was suggested by the following findings . The intrinsic activity of EPI, at concentrations greater than 10 nM, was significantly higher than that of isoproterenol (ISO) in stimulating GH release . Phenylephrine (PHE), a specific agonist of alpha 1-adrenergic receptors, provoked a significant rise of GH release . The effect was concentration dependent between 100 nM and 10 microM . The stimulatory effect of EPI and PHE was lowered, respectively blocked, by low concentrations of the potent alpha 1-adrenergic antagonist prazosin . The EPI-induced GH release could be totally blocked only by administration of both alpha- and beta-receptor antagonists . Under the same conditions and using concentrations up to 1 microM, the alpha 2-agonist clonidine had no or only a slight stimulatory effect; dopamine had no effect . Administration of both PHE and ISO resulted in a more than additive stimulation of GH release . The effectiveness of PHE but not clonidine, together with the high potency of the alpha 1-blocker prazosin suggest that the alpha-adrenergic receptor is predominantly of the alpha 1-subtype . When tested on days 1, 3, 6 and 8 in culture, both alpha- and beta-adrenergic responses appeared to be higher in the presence of the glucocorticoid dexamethasone compared to the responses in the absence of dexamethasone.(ABSTRACT TRUNCATED AT 250 WORDS)

Tohoku J Exp Med, 1987 Dec, 153(4), 375 - 82
Monolayer-forming islet cell culture from neonatal pig pancreas: using sequential treatment with EDTA-dispase and monoiodoacetic acid for preparation and purification; Ohgawara H et al.; A new method for the preparation and purification of a monolayer-forming islet cell culture from the neonatal pig pancreas is described . Single cell preparations of the pig pancreas were obtained by gently stirring the chopped pancreases in a medium containing sequential EDTA-dipase . Exposure of the cells to monoiodoacetic acid (10 micron) and BSA-gradient resulted in improved preparation, highly enriched in the endocrine cells . The cells were maintained in tissue culture medium 199 containing 5.5 mM D-glucose, with or without 0.1 mM 3-isobutyl-1-methylxanthine, or 16.7 mM D-glucose and 20% fetal calf serum . During a 6-day culture period, many small cell aggregates appeared in the media . The cell clusters attached to the bottom of the dish and formed monolayers . Provocative stimulation and radioimmunoassay for insulin showed the existence of numerous and viable B cells in the cell clusters . The other three types of islet cells were also demonstrated immunohistochemically in the cell cluster . It is concluded that this improved culture technique provides a useful tool for morphological and biochemical studies of islet cells and a potential source of material for transplantation of B-cells from the pancreas.

J Immunol Methods, 1987 Nov 23, 104(1-2), 237 - 43
Purification of polymeric immunoglobulin from cell culture supernatants by affinity chromatography using secretory component; Jones CL et al.; Human secretory component bound covalently to Sepharose 4B has been used as an affinity adsorbent to isolate and purify polymeric immunoglobulin from cell culture supernatants . The method was used to isolate murine IgM isotype anti-trinitrophenol antibody and rat IgM isotype anti-lymphocyte antibody from hybridoma cell culture supernatants . Gel filtration of the eluted antibodies followed by enzyme immunoassay showed that all recovered IgM was of pentameric molecular size . Murine IgA isotype anti-dinitrophenol antibody and murine IgA anti-human rotavirus antibody were isolated from cell culture supernatants of a plasmacytoma and a hybridoma respectively . Most of the IgA recovered following affinity adsorption with secretory component was of greater molecular size than dimer . Murine IgG was not adsorbed by secretory component bound to Sepharose . Eluted antibody retained antigen binding activity . Affinity chromatography using human secretory component bound covalently to a solid phase provides an antigen-independent technique for purification of murine and rat IgA and IgM polymeric immunoglobulin from cell cultures.

J Immunol Methods, 1987 Nov 23, 104(1-2), 103 - 9
A method suitable for the isolation of monoclonal antibodies from large volumes of serum-containing hybridoma cell culture supernatants; Malm B; A method suitable for the isolation of monoclonal antibodies from large volumes of serum-containing hybridoma cell culture supernatants is described . The purification is carried out in three steps; buffer exchange by chromatography on Sephadex G-25, fractionation by cation exchange chromatography on S Sepharose Fast Flow and, as a final step, gel filtration on Sephacryl S-200 HR . Experience from purification of 11 different mouse monoclonal antibodies is reported.

Biochem Biophys Res Commun, 1987 Nov 13, 148(3), 1118 - 23
Differential effects of cycloheximide on rat liver cytochrome P-450 gene transcription in the whole animal and hepatoma cell culture; Bhat GJ et al.; The induction of cytochrome P-450 (c+d) messenger RNAs in rat liver by 3-methyl cholanthrene follows a biphasic pattern . Administration of cycloheximide blocks the induction of cytochrome P-450 (c+d) messenger RNAs by 3-methylcholanthrene as well as cytochrome P-450 (b+e) messenger RNAs by Phenobarbitone . Transcription of these messenger RNAs in isolated nuclei is also blocked by cycloheximide administration . Thus cycloheximide not only fails to mimic the superinduction effects reported in hepatoma cell cultures, but actually blocks the specific transcription process . Exogenous hemin, while counteracting the effects of CoCl2 (heme biosynthetic inhibitor) on cytochrome (c+d) messenger RNA induction by the hydrocarbon, fails to counteract the effects of cycloheximide . It is suggested that a positive labile transcription factor is involved in the regulation of cytochrome P-450 gene expression in vivo.

J Immunol Methods, 1987 Nov 5, 103(2), 185 - 8
An improved method for elimination of mycoplasmas from cell cultures; Kreipe H et al.; Cell lines infected by different species of mycoplasma (Mycoplasma orale, Mycoplasma hominis) were decontaminated by co-culture with human blood monocyte (BM)-derived macrophages and pooled human immunoglobulin preparations . Co-cultures with BM-derived macrophages or murine peritoneal macrophages (PM) alone were not successful . The phenotype of infected cell lines did not differ from that of uninfected cell lines as revealed by morphological, enzymecytochemical, and immunocytochemical analysis.

Exp Cell Res, 1987 Nov, 173(1), 129 - 36
Inhibitory effects of tetradecanoylphorbol acetate and diacylglycerol on erythropoietin production in human renal carcinoma cell cultures; Hagiwara M et al.; A human renal carcinoma from a patient with an erythrocytosis, serially transplanted into athymic nude mice, was grown in primary monolayer cell cultures . After reaching confluency the cultured cells formed multicellular hemicysts (domes) which became more abundant as the cultures approached saturation density . Erythropoietin (Ep) production by this renal carcinoma in culture was only slightly increased at the time of semiconfluency but showed a marked increase in Ep levels in the culture medium after the cultures reached confluency, in parallel with an increase in dome formation . The phorbol ester tumor promoter 12-O-tetradecanoylphorbol-13-acetate (TPA) showed a significant dose-related inhibitory effect on Ep production and dome formation in the renal carcinoma cell cultures, suggesting an important role of protein kinase C, the only known receptor for TPA, in inhibiting the expression of differentiated phenotypes in the renal carcinoma cells . TPA also suppressed Ep secretion over a period of 96 h, indicating a time course of suppression of this differentiated function of the renal carcinoma cells in culture . This hypothesis was further supported by the observation that diacylglycerol, the endogenous activator of protein kinase C, likewise inhibited Ep production and dome formation in the renal carcinoma cell cultures . These studies suggest a role of the inositol-lipid second messenger path and protein kinase C in the regulation of Ep production.

Hum Genet, 1987 Nov, 77(3), 269 - 76
Loss of heterozygosity in hypotriploid cell cultures from testicular tumours; Parrington JM et al.; We have established cell lines with a hypotriploid chromosome number from four testicular tumours . Each line had at least one Y chromosome and most of the informative centrosome and enzyme markers were heterozygous implying that the tumours originated from germ cells before the first meiotic division . The small metacentric marker chromosome (i12p), specific for testicular tumours, was present in all tumour cell lines and up to three copies were found in some lines . Rearrangements of chromosome 1 and 11 were each found in three out of four tumours . The rearrangements of chromosome 1 all resulted in duplication of 1q and deletion of short-arm material from the same chromosome giving loss of heterozygosity for enzyme markers on 1p . Loss of satellite material from chromosome 13 and the centromere region of chromosome 9 were found in single cases . This study shows that even where the chromosome number of tumour cells is near triploid, regions of the genome can be deleted . The chromosomes most frequently involved in rearrangements, 1, 11, and 12 all contain sites of ras oncogenes and it is suggested that loss of normal alleles could result in homozygosity for mutant oncogenes which may play a part in tumour progression.

Vopr Virusol, 1987 Nov-Dec, 32(6), 701 - 9
{Dependence of the synthesis of virion structures in the tick-borne encephalitis virus on the cell culture type}; Liapustin VN et al.; Variations in synthesis of antigenic structures are observed in tick-borne encephalitis virus replication in cell cultures of different origin . In a number of cell cultures: pig and Syrian hamster embryo kidney cells, as well as in Chinese striped hamster cells and Tasmanian rat kangaroo cells, virion antigens (VA) are synthesized which differ in the direction of movement in the electric field, namely, anode and cathode VA . In other cell cultures: green monkey kidney, barking deer kidney, and human fibroblasts, only cathode VA is synthesized . In chick embryo fibroblast cultures, in addition to the above-mentioned VA, considerable amounts of a VA which does not move in the electric field are synthesized . In all the cultures, a low molecular nonvirion antigen (NA) is actively produced, the virus-containing fluids of human fibroblast cultures and Chinese striped hamster kidney cell cultures contain lower amounts of high molecular NA, while rat kangaroo and green monkey kidney cell cultures have no high-molecular NA of tick-borne encephalitis virus.

Vopr Virusol, 1987 Nov-Dec, 32(6), 696 - 701
{Electron microscopic study of a tick-borne encephalitis virus-infected cell culture exposed to tunicamycin}; Lisak VM et al.; Ultrastructural features of tick-borne encephalitis virus-infected pig embryo cell culture treated with different concentrations of tunicamycin at various intervals after infection were studied electron microscopically . The inhibition of glycosylation did not prevent virion formation in the infected cells . At the same time, treatment with tunicamycin led to marked accumulation of virus particles in cisterns and vacuoles of the Golgi complex and to a decrease in the number of virions released into the extracellular space . It is assumed that inhibition of glycosylation leads to disorders in the regulation of the final stages of virus particles transportation and release from the infected cell . The results of the study indicate an important role of the Golgi complex in realization of the final stages of flavivirus morphogenesis.

Placenta, 1987 Nov-Dec, 8(6), 639 - 46
The use of pregnancy serum to obtain trophoblastic cell cultures; Ungar L et al.; A method for obtaining trophoblastic cell cultures from first-trimester human placental villi is described . The essential feature of the method is the use of serum from a pregnant woman in the culture medium . Using this technique, pure cultures of trophoblastic cells are produced in 88 per cent of cases.

Placenta, 1987 Nov-Dec, 8(6), 573 - 85
Development of a placental cell culture system for studying the control of mouse placental lactogen II secretion; Thordarson G et al.; Fetal placental tissue from 11 days pregnant mice was dissociated in collagenase and DNase solution and then separated on a 40 per cent Percoll gradient . Trophoblast cells banded at a density of 1.05 g/ml . When cultured on rat tail collagen, these cells formed colonies of mono- and binucleate cells varying in size from 40 to 70 microns . At the time of plating, about 13 per cent of the trophoblast cells secreted mouse placental lactogen II (mPL-II) as determined by reverse haemolytic plaque assay . The ratio of mPL-II-producing cells increased significantly in culture and reached 63 per cent after 48 h . The secretion of mPL-II increased continuously during six days of culture, whereas the total protein release was highest after the first day, declined the second day and then remained relatively constant for the last four days of culture . The DNA content of the cells did not change significantly during the six-day period . When the trophoblast cells were incubated with insulin (1 ng/ml to 5 micrograms/ml), a modest but significant reduction in mPL-II secretion was observed . No change in the mPL-II secretion was seen when epidermal growth factor was administered to the culture in concentrations from 1 ng/ml to 10 micrograms/ml . It is concluded that this in vitro culture system is suitable for studying both mPL-II secretion and the differentiation of mPL-II-producing cells.

Br J Pharmacol, 1987 Nov, 92(3), 573 - 85
A patch clamp study of gamma-aminobutyric acid (GABA)-induced macroscopic currents in rat melanotrophs in cell culture; Kehl SJ et al.; 1 . The macroscopic currents induced in cultured rat melanotrophs by exogenous gamma-aminobutyric acid (GABA) were analysed using the patch clamp recording technique . 2 . Using various concentrations of intra- and extracellular chloride it was demonstrated that the conductance activated by GABA was chloride selective . Since these currents were blocked with bicuculline and enhanced with chlordiazepoxide the involvement of GABAA receptors similar to those in the CNS is indicated . 3 . When chloride was symmetrically distributed across the membrane the voltage/current relationship was linear; pronounced rectification of GABA mediated currents was evident when there was an asymmetrical distribution of chloride . 4 . With concentrations of GABA greater than 10 microM a fading of the current was seen during prolonged (5-10 s) applications . This effect appeared to be due to a decline of conductance rather than a shift of the chloride equilibrium potential . 5 . Values for the Hill coefficient derived from dose-response curves suggested that the binding of 2 molecules of GABA to the receptor is required for the activation of the chloride channel . 6 . There was no indication of a direct, GABAB receptor-mediated change of conductance.

J Immunol, 1987 Nov 1, 139(9), 3146 - 52
Immune regulation of the L5178Y murine tumor-dormant state . II . Interferon-gamma requires tumor necrosis factor to restrain tumor cell growth in peritoneal cell cultures from tumor-dormant mice; Suzuki Y et al.; L5178Y lymphoma cells are restrained from progressive growth in peritoneal cell ("in vitro tumor-regressor" PC) cultures prepared from many DBA/2 mice which harbor the tumor cells in the peritoneal cavity in a tumor-dormant state . Treatment of these PC cultures with 'antibodies to murine interferon-gamma (MuIFN-gamma) and murine tumor necrosis factor (MuTNF) but not with antibody to interleukin 2 (IL-2) receptors eliminated the restraint on tumor cell growth and permitted their progressive proliferation . L5178Y cells were found to be resistant to the direct toxic effects of large concentrations (3,000 U/ml) of MuIFN-gamma and of MuTNF, either alone or in combination . Treatment of PC cultures from tumor-dormant mice, in which tumor cells grew progressively ("in vitro tumor-progressor"), but not PC cultures from normal mice, with exogenous MuIFN-gamma resulted in a marked inhibition of tumor cell growth . The MuIFN-gamma-induced cytotoxic activity was cell-mediated since no soluble tumor-cytotoxic factors could be detected in the cultures . MuIFN-gamma induced cytotoxic activity in plastic-adherent peritoneal cell (AD-PC) cultures, but induced no cytotoxic activity in nonadherent-PC cultures unless small numbers (2%) of AD-PC were present, and inclusion of antibody to MuTNF in these mixed PC cultures blocked the development of cytotoxic activity . Antibody to MuTNF also blocked the development of cytotoxic activity in cultures of MuIFN-gamma-treated whole PC and AD-PC from tumor-dormant mice . These results indicate that MuIFN-gamma and MuTNF are both important in restraining tumor cell growth in PC cultures from tumor-dormant mice, and that MuIFN-gamma requires the presence of MuTNF to induce cytotoxic activity in these cultures.

Neuroscience, 1987 Nov, 23(2), 423 - 32
Quinolinate neurotoxicity in cortical cell culture; Kim JP et al.; The central neurotoxicity of the endogenous tryptophan metabolite, quinolinate, has been postulated to participate in the pathogenesis of the neuronal cell loss associated with several neurological disease states . In the present study, quinolinate neurotoxicity was quantitatively studied in dissociated cell cultures prepared from the fetal mouse neocortex . Sufficient exposure of cortical cultures to quinolinate was associated with considerable neuronal cell loss, but no glial cell loss; this neurotoxicity could be blocked by 2-amino-5-phosphonovalerate and kynurenate, drugs known to block N-methyl-D-aspartate receptors . The quinolinate dose-toxicity relationship showed that the potency of quinolinate as a neurotoxin is relatively low, especially with brief (20 min) exposure times, where an ED50 of 2 mM was observed . However, with longer exposure times of 24 and 96 h, quinolinate is more potent: the latter exposure was characterized by an ED50 of 250-400 microM . Ion substitution experiments suggested that quinolinate neurotoxicity can be separated into two distinct components on the basis of differences in time course and ionic dependence: an acute, sodium-dependent "excitotoxic" component, marked by early cell swelling; and a late, calcium-dependent component, marked by delayed cell degeneration . Acute neuronal swelling was seen only with exposure to quinolinate concentrations in excess of 1 mM, so under actual pathophysiological conditions, quinolinate neurotoxicity might be nearly completely related to the calcium-dependent component, with little or no "excitotoxic" contribution.

Transplantation, 1987 Nov, 44(5), 680 - 92
In vivo and in vitro induction of class II molecules on canine renal cells and their effect on the mixed lymphocyte kidney cell culture; Esquenazi V et al.; Canine renal cortical cells were obtained by collagenase extraction from allogeneic haploidentical, donor-recipient beagle littermate pairs and from unrelated mongrels . Peripheral blood lymphocytes (PBL) of the mongrels, as well as of one member of the beagle pair that exhibited high mixed lymphocyte culture (MLC) reactivity against the other were also stimulated by renal cortical cells derived from both normal and rejected transplanted kidneys in mixed lymphocyte kidney cell culture (MLKC) . A moderate autologous MLKC reactivity occurred in response to normal renal cortical cells . However, rejected kidney cortical cells were markedly more stimulatory than normal renal cortical cells in both allogeneic and autologous MLKC reactions . Lymphocytes from donor animals responded more strongly to autologous cortical cells isolated during rejection of the transplant than to cortical cells from normal allogeneic kidneys . Recipient infiltrating lymphocytes and propagated T cell lines extracted from the rejected kidney also responded more strongly than PBL to cortical cells from this kidney . Gradient purification of the stimulating cortical cells resulted in one virtually pure preparation of distal tubular epithelial cells, as demonstrated by immunohistochemical stains and electron microscopy, which caused enhanced stimulation in MLKC . Class II marker analysis of the canine renal cells from rejected kidneys revealed the presence of these molecules on tubular cells that were absent on normal kidney cells . A 16-hr coculture of normal renal cortical cells not exhibiting class II surface markers in the presence of allogeneic or autologous lymphocytes induced the expression of these molecules, associated with an increased stimulatory capacity . This also occurred to a lesser extent with MLC (and MLKC) cell culture media supernatants . However, the low level of class II expression by all the various gradient-purified fractions in the absence of rejection or coculture, and the increased but equivalent expression on all fractions after coculture did not correlate with the preferential stimulatory capacity of the purified distal tubular cell layer . We conclude that two signals are necessary for the MLKC reaction, one involving tissue (kidney)-associated epitopes (the nominal antigen demonstrated in this study to be present in normal distal tubular cells), the other involving class II molecules as costimulatory (amplification) moieties.

Immunology, 1987 Nov, 62(3), 439 - 44
A suppressor B lymphocyte inhibiting IL-2 consumption in spleen cell cultures from Mycobacterium bovis BCG-infected mice; Turcotte R; In concanavalin A (Con A)-activated spleen cell (SC) cultures from normal C57BL/6 mice, the production of IL-2 peaked at 18-20 hr after initiation of cultures and declined rapidly during the next 24 hr, the decline of IL-2 activity being due, at least in part, to its utilization by the Con A-induced IL-2 receptor cells . In Con A-activated SC from BCG-infected mice, significant levels of IL-2 activity persisted in the 48-hr and 72-hr culture supernatants, a situation which seemed to be related to the depressed capacity of infected splenocytes to acquire IL-2 receptors . In cell mixing experiments, SC from infected mice actively depressed the utilization of IL-2 by Con A-activated normal SC, thus indicating that suppressor cells can down-regulate IL-2 responsiveness . These suppressor cells may belong to the B-cell lineage since they possessed the Thy-1-, sIg+ and FcR+ phenotype.

Dev Biol, 1987 Nov, 124(1), 239 - 47
Characterization of photoreceptor cell differentiation in the rat retinal cell culture; Araki M et al.; Photoreceptor cell differentiation in the rat retina was studied in vivo and in vitro, using an immunohistochemical method to demonstrate opsin-like immunoreactivity . Cells in a dissociated monolayer culture expressed some properties characteristic of rat rod cells developing in vivo, including a ciliary structure and opsin-like immunoreactivity . Immunoblot analysis revealed that cultured retinal cells synthesize a polypeptide with the same molecular weight as that synthesized by the intact retina . Although the outer segment (OS) was not present in the culture, immunoreactive cells possessed a ciliary structure . Opsin-like immunoreactivity was found on the plasma membrane, including the cilia . The neuritic extensions were also intensely stained . In mature rod cells of the intact rat retina, opsin was detected only on the OS but, during development, it was found both in the somatic region of the rod cells and on the differentiating OS . During maturation of rod cells opsin immunoreactivity seemed to shift to the OS from other locations . However, some "displaced" photoreceptor cells, found in the inner nuclear layer and extending fibers bipolarly, retained immunoreactivity throughout their structure . The absence of polarized distribution of opsin in these cells is considered to be due to an abnormal environment, which may also be the case with cultured retinal cells . The present culture conditions will offer a useful model system to understand the cellular mechanism of the hereditary retinal dystrophy of rodent animals in which photoreceptor cells selectively degenerate.

J Neurosci, 1987 Nov, 7(11), 3665 - 74
Alpha-2 adrenergic regulation of melatonin release in chick pineal cell cultures; Pratt BL et al.; The chick pineal gland expresses a circadian rhythm of melatonin biosynthesis, with elevated levels at night and low levels during the day . The rhythm of melatonin is regulated both by circadian oscillators located within the gland itself and by adrenergic input from the sympathetic nervous system . Previous work has shown that norepinephrine administration inhibits melatonin biosynthesis, as measured by the activity of the enzyme serotonin N-acetyltransferase . As a first step toward understanding the mechanisms by which norepinephrine regulates melatonin production in the chick pineal, we have identified the adrenergic receptor involved . Dissociated chick pineal cell cultures were prepared and melatonin release was measured on days 5 and 6 of culture using radioimmunoassay . The effects of adrenergic agonists and antagonists on the nocturnal increase of melatonin release during the 12 hr dark portion of a LD12:12 light cycle were determined . Norepinephrine inhibited melatonin release in a dose-dependent manner, with an average EC50 of 19.7 nM +/- 2.23 (SEM) . Melatonin release values ranged from 100 to 4% of the level seen in control cultures, depending on the dose of norepinephrine . The physiological response to epinephrine, norepinephrine, and isoproterenol was stereospecific . The (-) stereoisomer was 6, 8, and 37 times more potent than the (+) stereoisomer, respectively . EC50 values (in nM) for adrenergic agonists were as follows: alpha-methyl-(-)-norepinephrine, 2.46; tramazoline, 3.06; guanabenz, 3.31; clonidine, 3.70; oxymetazoline, 4.29; (-)-epinephrine, 7.44; (-)-norepinephrine, 19.7; (-)-isoproterenol, 463; and (-)-phenylephrine, 659 . Schild analysis was used to determine the relative potency of adrenergic antagonists . pA2 values for adrenergic antagonists were as follows: rauwolscine, 9.55; RX78 1094, 8.32; yohimbine, 8.14; phentolamine, 7.11; prazosin, 5.93; and (-)-propranolol, less than 6 . The relative potencies of both adrenergic agonists and antagonists demonstrate that alpha-2 receptors mediate norepinephrine-induced inhibition of melatonin release in chick pineal cell cultures . The identification of alpha-2 receptors in chick pineal cells should aid in our understanding of the biochemical events initiated by receptor activation that regulate melatonin synthesis.

Zhonghua Min Guo Wei Sheng Wu Ji Mian Yi Xue Za Zhi, 1987 Nov, 20(4), 349 - 55
Susceptibility of mammalian cell cultures to infection with arbo-togaviruses; Liu TH et al.; Six alphaviruses and four flaviviruses were inoculated intracerebrally into 1 to 3 days old suckling mice . All mice developed CNS diseases and died 2-5 days post-infection . It appeared that alphaviruses were more virulent than flaviviruses since they brought death to the injected mice earlier than the flaviviruses . The susceptibilities of nine different culture cells to those viruses were also investigated using plaque assay . BHK-21 cells produced clearer plaques and were more sensitive than other cells . Plaque reduction tests using antibodies against various viruses revealed no cross-reactions between alphavirus and flavivirus . However, minor cross-reactions were found within groups . The results of this study suggest that BHK-21 cells can be used for the large scale screening of viruses in domestic animal specimens and for detecting serum neutralizing antibodies against arboviruses.

J Virol Methods, 1987 Nov, 18(2-3), 193 - 203
Continuous production of hepatitis A virus in PLC/PRF/5 cell cultures: use of antigen for serology; Crance JM et al.; The strain CF53 of hepatitis A virus (HAV) previously adapted to growth in PLC/PRF/5 cells was grown in 175 cm2 flasks, at different passages . After infection, cells were incubated at 32 degrees C in RPMI 1640 medium supplemented with 2.5% foetal calf serum (FCS) for 6-12 months . HAV which was released continuously in the culture medium was harvested weekly . Hepatitis A virus antigen (HAAg) and infectious virus production was stable during each passage . The antigen titre, determined by radioimmunoassay, was about 50 for each passage whereas the infectious virus titre increased from 10(3.7) (passage 7) to 10(6.0) TCID50/ml (passage 13) . Virus production was not influenced by the FCS concentration (0-2.5%) in the maintenance medium . The cell culture produced HAAg was used for detection of total anti-HAV antibodies, anti-HAV titration and IgM antibody capture assay and the results were identical to those obtained with commercial kits . HAAg produced by this practical and cheap method could easily replace primate derived antigen for the detection of anti-HAV antibodies.

Virology, 1987 Nov, 161(1), 181 - 9
Multiple defects in the genome of pseudorabies virus can affect virulence without detectably affecting replication in cell culture; Lomniczi B et al.; Several independently isolated vaccine strains of pseudorabies virus were studied to identify the functions that play a role in the expression of virulence of this virus . All the strains that were studied grew well in three different cell types . No differences that could be correlated with avirulence could be detected either in the virus yield produced by the cells or in the length of the eclipse phases . All the attenuated strains, however, had lost their ability to replicate efficiently in the brains of day-old chickens . The defects leading to the decrease in the virulence for day-old chickens varied in the different vaccine strains . The Tatarov vaccine strain is defective in the thymidine kinase (TK) gene; restoration of a functional TK gene restores to this strain its virulence for day-old chickens and for pigs . Three out of four different, independently isolated avirulent strains were found to be defective in different loci, as determined by their ability to generate virulent recombinants . Two strains, Bartha and Buk Z300, however, yielded few virulent recombinants, indicating that they were defective in at least one closely linked function . Furthermore, all the virulent recombinants obtained from cells coinfected with different pairwise combinations of the vaccine strains had higher LD50 values than virulent wild-type virus, indicating that the recombinants had not acquired all the functions necessary for optimum expression of virulence . Partial virulence was also restored to Buk Z900 by marker rescue with sequences originating from three different regions of the wild-type pseudorabies virus genome . All three of these regions were different from those that had previously been shown to rescue virulence of the Bartha strain (B . Lomniczi, S . Watanabe, T . Ben-Porat, and A . S . Kaplan, 1987, J . Virol . 61, 796-801) . Our results thus show that (1) defects in several different loci of the pseudorabies virus genome can affect virulence without detectably affecting growth in cell culture and (2) most vaccine strains have multiple defects contributing to their lack of virulence.

Respir Physiol, 1987 Nov, 70(2), 131 - 41
Effect of PO2 on prostaglandin E2 production in renal cell cultures; Roszinski S et al.; There is evidence both from in vivo and in vitro studies which suggests that hypoxia stimulates the synthesis of prostanoids in some tissues . In the present study, the in vitro production of prostaglandin E2 (PGE2) was studied in three different renal cell lines incubated at various PO2 values between 143 and 3 mm Hg for 24 h . In rat kidney mesangial cell cultures, PGE2 production increased up to 99 ng PGE2/mg protein at 7 mm Hg O2, compared to 52 ng/mg at 143 mm Hg O2, but was lowered at 26 ng/mg at 3 mm Hg O2 . PGE2 production by the pig kidney tubule cell lines LLC-PK1 and PK-15 was insensitive to PO2 changes . Because PGE2 production is known to be Ca2+-dependent and was indeed stimulated by the Ca-ionophore A 23187, effects of hypoxia on 45Ca2+-fluxes were also studied . In none of the 3 cell lines, net 45Ca-influx was altered after incubation at low PO2 . However, net 45Ca-efflux increased during hypoxic incubation of mesangial cells possibly as a result of intracellular Ca-mobilization . These results indicate that hypoxia stimulates PGE2 synthesis in mesangial but not in tubule cell cultures . However, at very low PO2 values, or anoxia, the formation of cyclic endoperoxides from arachidonic acid may be lowered . Since mesangiocytes are smooth muscle-like cells, the hypoxia-induced synthesis of relaxing prostanoids could play a role in the regulation of smooth muscle tone.

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