Cell cultures

FEBS Lett, 1985 Oct 7, 190(1), 157 - 60
Biosynthesis of abnormally glycosylated hepatoma secretory proteins in cell cultures; Alm R et al.; We studied, by electrophoretic techniques, the physiochemical properties of 4 glycoproteins, alpha 1-antitrypsin, alpha 1-antichymotrypsin, alpha 1-acid glycoprotein and transferrin synthesized by three different human hepatoma cell lines . A common feature was the export of glycoproteins with retarded electrophoretic mobility, indicating incomplete sialylation, and a predominance of atypical, highly branched carbohydrate chains . The abnormal glycosylation pattern may be specific for malignant transformation of hepatocytes and possibly related to the intracellular accumulation of some of these proteins in malignant cells.

Brain Res, 1985 Oct 7, 344(2), 369 - 72
Effects of norepinephrine on rat neocortical neurons in dissociated cell culture; Rosenberg PA et al.; Intracellular recordings were made from neurons in dissociated cell culture of neocortex during application of norepinephrine (NE) or other adrenergic agonists . In the population of neurons generally studied, greater than 18 micron in diameter, adrenergic agonists from 1 nM to 50 microM produced no change in membrane potential or input resistance 120 cells) . Adrenergic agonists increased synaptic activity impinging on the impaled cell in 25/120 neurons (21%) . In neurons in cocultures of locus coeruleus and cerebral cortex, again the same synaptic response to perfusion with NE was noted in 13/93 neurons (14%) . In addition, direct effects of NE were noted on 6/93 neurons recorded from in cocultures, all close to the explant . In these cells, NE hyperpolarized the membrane in association with a small decrease in input resistance (11%) . These responsive cells may have originated within the explant . A paradigm was used for testing the possibility of a responsive element in the cultures distinct from the impaled soma . 'Hot spots' were found using concentrations of isoproterenol as low as 10 nM.

J Neurosci Methods, 1985 Oct-Nov, 15(2), 101 - 12
Glycogen accumulation in rat cerebral cortex in dissociated cell culture; Rosenberg PA et al.; Incorporation of {3H}glucose into {3H}glycogen was demonstrated in individual coverslip cultures of dissociated rat cerebral cortex . The time course of incorporation of {3H} glucose into {3H}glycogen was investigated, and it was found that {3H}glycogen accumulation monotonically increased during at least the first hour of incubation with {3H}glucose . In order to identify which cells accumulate glycogen in these cultures we attempted to demonstrate cytochemically the localization of glycogen . We found, however, that conventional aqueous methods of glycogen cytochemistry did not reliably or consistently stain the cultures . By labelling glycogen using {3H}glucose, we were able to show that the entire {3H}glycogen compartment was extractable by water after ethanol fixation . Therefore we developed a non-aqueous technique which preserves tissue glycogen by exploiting its solubility properties . Using this technique we were able to cytochemically demonstrate glycogen in at least two different cell types in the cultures.

J Biol Stand, 1985 Oct, 13(4), 303 - 8
A simple endpoint dilution method for evaluating serum used for cell culture; Jiang SD et al.; A simple endpoint dilution method for evaluating foetal calf serum quality is described . The test uses a series of doubling dilutions of cells on microtitre trays with the test sera added to replicate dilution series . After five to six days of incubation the cells are stained with crystal violet and the end points read macroscopically . The cell growth-promoting property of serum may be expressed as a reciprocal of the cell dilution resulting in an approximately 50% coverage of cells.

J Invest Dermatol, 1985 Oct, 85(4), 381 - 4
Dermatofibrosarcoma protuberans: altered collagen metabolism in cell culture; Albini A et al.; Dermatofibrosarcoma protuberans is a low-grade malignant tumor that grows invasively but rarely forms metastases . Its origin is still controversial . We characterized the synthesis of collagen in detail in cells which were obtained from dermatofibrosarcoma protuberans tumors by enzymatic tissue disintegration . Similar to fibroblasts, all tumor cell strains produced considerable amounts of collagen . However, the rate was reduced compared to normal skin fibroblasts . Cells grown from the tumors synthesized type I collagen, but no type II could be detected . After serial passaging the cultures started to produce type III collagen, which is probably due to a slow overgrowth by normal fibroblasts.

Br J Haematol, 1985 Oct, 61(2), 293 - 302
In vitro erythropoietin assay based on erythroid colony formation in fetal mouse liver cell culture; Sakata S et al.; Critical studies were made on erythroid colony formation from cultured fetal mouse liver cells in an attempt to develop a simple and sensitive erythropoietin (Epo) assay procedure . The maximum colony formation was observed 24 h after plating of the cells when an evident dose-response relation was found for Epo added . The colony forming ability decreased steadily as the gestational age of the fetus advanced and was gradually lost by postnatal days 10-11 . By morphological and cytochemical criteria almost all the colonies were found to be erythroid . 59Fe-labelling experiments revealed a fairly good correlation between the colony number and 59Fe incorporation into both cells and haem . Dose-response curves for plasma were parallel to the Epo standard curve . Based on these findings we developed a procedure which could measure as little as 0.4 mU of Epo without requiring 59Fe . Using this method, plasma Epo titres were determined in 16 normal and 69 anaemic subjects.

Endocrinology, 1985 Oct, 117(4), 1521 - 9
Identification of androgen-binding protein from testis cytosol and sertoli cell culture medium of the cynomolgus monkey, Macaca fascicularis; Keeping HS et al.; The biochemical and immunological properties of monkey androgen-binding protein (mABP) from the cynomolgus monkey, Macaca fascicularis, have been examined in testis cytosol and medium of primary Sertoli cell-enriched cultures . mABP in testis tissue was separated from the serum protein testosterone-estradiol-binding globulin (mTeBG) by affinity chromatography on Concanavalin A-Sepharose (Con A) . mTeBG from serum extracts was completely retained by the lectin and could be displaced with buffer containing alpha-methyl-D-glucoside . In contrast, mABP from testis extracts either did not interact with the Con A and appeared in the void volume or was partially retained by the column and could be eluted with buffer alone . A third component retained by the Con A may represent mTeBG contamination, a form of mABP which binds to Con A, or both . The specific 5 alpha-dihydrotestosterone (DHT)-binding activity in the void volume of the Con A column was designated mABP and was further studied . {3H}DHT binding to mABP was saturable and of limited capacity (0.163 +/- 19 pmol/mg protein) . Scatchard analysis of the data was consistent with a single class of binding sites with an apparent dissociation constant (Kd) at 4 C of 2.6 +/- 0.2 X 10(-9) M . DHT was the most effective competitor of {3H}DHT binding to mABP, followed by 2-methoxyestradiol, testosterone, estradiol, and cyproterone acetate . Concentrated Sertoli cell culture medium subjected to steady state polyacrylamide gel electrophoresis produced a single peak of specifically bound {3H}DHT with a mobility similar to that of other androgen-binding proteins . {35S}Methionine-labeled medium proteins were immunoprecipitated with a rabbit anti-human TeBG antiserum . Two bands, corresponding to mol wt of approximately 46,000 and 48,000, were observed by fluorography, with the lighter component being more intense . After androgen affinity chromatography of radiolabeled medium proteins, these two bands were again observed on sodium dodecyl sulfate-urea-containing polyacrylamide gels . These results demonstrate that 1) mABP may be separated from mTeBG by lectin affinity chromatography, as in humans; 2) hTeBG and mABP are antigenically related; and 3) mABP consists of subunits of different mol wt in unequal ratios.

J Neurochem, 1985 Oct, 45(4), 1254 - 61
Acetylcholine synthesis by adult bovine adrenal chromaffin cell cultures; Boksa P; Adrenal chromaffin cells normally synthesize and release catecholamines . In the present study, {3H}acetylcholine synthesis and another characteristic of cholinergic neurons, {3H}choline uptake, were studied in cultures of adult bovine adrenal chromaffin cells . Chromaffin cell cultures took up {3H}choline from the medium and acetylated the {3H}choline to form {3H}acetylcholine . The rate of {3H}acetylcholine synthesis increased after 19 days in culture and continued to increase up to 28 days in culture . {3H}Acetylcholine synthesis could be increased by stimulating the cells with a depolarizing concentration of K+ . The ability for K+ to stimulate synthesis of {3H}acetylcholine developed only after 28 days in culture . {3H}Choline was taken up by the cultures through a single mechanism with a high (to intermediate) affinity for choline . {3H}Choline uptake was enhanced by Na+ omission in day-14 cultures, but was at least partially Na+-dependent in day-29 cultures . Hemicholinium-3 (IC50 less than 10 muM) inhibited {3H}choline uptake into chromaffin cell cultures . It is concluded that bovine adrenal chromaffin cells, maintained in culture, are able to exhibit cholinergic properties and this capacity is retained even by the mature adult cell.

Zh Mikrobiol Epidemiol Immunobiol, 1985 Oct, (10), 32 - 5
{Effect of proteolytic enzymes on the enhanced sensitivity of a cell culture of goat embryonic kidney to Newcastle disease virus}; Korsun LL et al.; The influence of proteolytic enzymes on the increase of the sensitivity of cell cultures to Newcastle disease virus was tested in the continuous culture of goat embryo kidney cells . In this investigation the dose of trypsin and the time of incubation for the treatment of the cell culture was selected . The preliminary treatment of the monolayer culture of goat embryo kidney cells with trypsin by incubation at a temperature of 18-20 degrees C for 30 minutes made it possible to increase the sensitivity of the culture to Newcastle disease virus 10-fold.

J Histochem Cytochem, 1985 Oct, 33(10), 1087 - 9
Identification of gastrin-producing cells in cell cultures and smears: an avidin-biotin-peroxidase complex (ABC) technique; Gower WR Jr et al.; A method is described that uses the avidin-biotin complex (ABC) immunoperoxidase technique to provide a rapid, sensitive, and specific means to quantitate isolated G cells in cultures and suspensions.

Antimicrob Agents Chemother, 1985 Oct, 28(4), 552 - 60
Antiviral activity of 5-ethyl-2'-deoxyuridine against herpes simplex viruses in cell culture, mice, and guinea pigs; Schinazi RF et al.; The susceptibility of 3 laboratory strains and 24 clinical isolates of herpes simplex virus type 1 (HSV-1) and type 2 (HSV-2) to 5-ethyl-2'-deoxyuridine was determined in plaque reduction assays in Vero cells . The median effective doses were 8.6 and 7.8 microM, respectively . The drug was less potent than acyclovir and other related antiviral drugs, but it had a high therapeutic index against both HSV-1 and HSV-2 . Drug-resistant viruses were readily produced in cell culture . These variants were cross-resistant to acyclovir, 2'-fluoro-5-iodoaracytosine, and 2'-fluoro-5-methylarauracil but were susceptible to vidarabine or phosphonoformate . These findings confirm that the selective antiviral activity of 5-ethyl-2'-deoxyuridine is mediated by the virus-induced thymidine kinase . Oral or intraperitoneal administration of the drug at nontoxic doses was ineffective in protecting mice against intracerebral challenge with virus . Using implanted osmotic minipumps or coadministering the drug with dimethyl sulfoxide failed to decrease the mortality rate . In guinea pigs infected genitally with HSV-2, topical drug treatment was more effective than placebo in reducing lesion severity and other clinical and virological variables . These effects were noted whether the drug treatment was initiated 3 or 24 h after infection (ascertained serologically) . Drug-treated animals had a significantly lower herpes antibody titer than did placebo-treated guinea pigs, suggesting that the drug can also reduce the viral antigen load . In this model, the drug appeared to be as effective as topical phosphonoformate or acyclovir.

J Comp Pathol, 1985 Oct, 95(4), 565 - 72
The alterations in pathogenicity and immunogenicity of a Kenya sheep and goat pox virus on serial passage in bovine foetal muscle cell cultures; Davies FG et al.; A Kenyan sheep and goat pox virus was attenuated by serial passage in bovine foetal muscle cell cultures . The pathogenicity of the strain was lost between the 15th and 20th passages . Serum-neutralizing antibody developed after inoculation with passages tested up to the 50th . These passages appeared to protect animals against laboratory challenge by intra-dermal titration . The 18th passage was successfully and extensively used to control the disease under field conditions.

J Neurosci, 1985 Oct, 5(10), 2690 - 5
gamma-Aminobutyric acid receptors on chick ciliary ganglion neurons in vivo and in cell culture; McEachern AE et al.; In the chick ciliary ganglion, preganglionic terminals maintain cholinergic synapses on the choroid neurons and both cholinergic and electrical synapses on the ciliary neurons . The preganglionic terminals also contain enkephalin- and substance P-like immunoreactivity, suggesting that transmission through the ganglion is more complicated than is indicated by the known synaptic connections . We report here that embryonic chick ciliary ganglion neurons also have gamma-aminobutyric acid (GABA) receptors and that GABA applied to the ganglion can block transmission elicited by preganglionic stimulation . Studies on the neurons in cell culture indicate that the GABA response is mediated by GABAA receptors: GABA activates a Cl- conductance, and the response can be mimicked by muscimol and blocked by bicuculline or picrotoxin . The GABA receptors are regulated independently from acetylcholine (ACh) receptors on the neurons since the levels of ACh and GABA sensitivity are influenced differently by culture age and by chronic exposure to GABA or elevated K+ concentrations . Application of GABA to intact ciliary ganglia increases the membrane conductance of ganglionic neurons (as in culture), reduces to subthreshold the amplitude of excitatory postsynaptic potentials in the neurons elicited by preganglionic stimulation and completely blocks transmission through the ganglion . A native source of ligand for the receptors in vivo has yet to be identified.

Antimicrob Agents Chemother, 1985 Oct, 28(4), 578 - 80
Activity of arildone with or without interferon against acute hemorrhagic conjunctivitis viruses in cell culture; Langford MP et al.; Arildone (WIN 38020; 4-{6-(2-chloro-4-methoxyphenoxy)hexyl}-3,5-heptanedione) inhibited the infectivity of acute hemorrhagic conjunctivitis viruses in tissue culture . Arildone did not inhibit interferon (IFN) production or IFN activity . Treatment of cultures with combinations of arildone and IFN resulted in an additive inhibition of acute hemorrhagic conjunctivitis virus production.

Tsitologiia, 1985 Oct, 27(10), 1156 - 63
{Dependence of ion transport across the plasma membrane on cell culture density . II . Active and passive cation transport during the growth of L cell cultures}; Marakhova II et al.; Rubidium and lithium influxes as well as intracellular potassium and sodium contents were investigated in L cells during the culture growth . In sparse culture over the cell densities 0.5-3 X 10(4) cells/cm2 ouabain-sensitive rubidium influx is small and ouabain-resistant lithium influx in high . With the increase in culture density up to 4-5 X 10(4) cells/cm2 the active rubidium influx, mediated by ouabain-sensitive component, is enhanced, and ion "leakage" tested by lithium influx is diminished . Simultaneously with the exponential growth of culture the intracellular potassium content is increased and the intracellular sodium content is decreased resulting in the higher K/Na ratio in cell . During the further transition to dense culture and in stationary state (10-17 X 10(4) cells/cm2) the sodium content and lithium influx do not change significantly, but the potassium content is decreased . The decrease in intracellular potassium is correlated with that in the portion of cells in S-phase from 27-30 to 12% . Thus, in transformed cells the density-dependent alterations in membrane cation transport are observed.

Cell Immunol, 1985 Oct 1, 95(1), 124 - 36
Synergistic interaction of a cytokine produced by embryonic fibroblasts and a lymphokine contained in Con A-stimulated spleen cell culture for murine macrophage differentiation: a model experiment using cell line cells, M1; Noro N et al.; The modulating activity of the culture supernatant of Con A-stimulated murine spleen cells for macrophages was investigated by using M-1 cells, which could differentiate into macrophage-like cells (referred to as M+1 hereafter), cocultured in a conditioned medium (CM) containing a differentiation factor (DF) obtained from the secondary culture of murine embryonic fibroblasts . DF induced Ia antigens on M-1 cells at a high rate in parallel with the appearance of Fc-receptor (FcR)-dependent phagocytic activity for erythrocytes coated with an antibody (EA) . In contrast, Con A-sup alone had no modulating effect on M-1 cells . However, the Con A-sup stimulates synergistically M-1 cells with DF . Thus, coculture of M-1 cells with Con A-sup and DF generates M++1 cells which possess higher phagocytic activity than M+1 cells . These cells also exhibited stronger accessory cell activity than M+1 cells when tested for their promoting effect on IL-2 production by Sephadex G-10-passed spleen cells . The accessory cell activity of M++1 cells was further enhanced by culture with lymphocytes in the presence of indomethacin while that of M+1 cells did not change . These findings suggest that M-1 cells probably acquire potentiating, as well as inhibitory activity at the same time when cultured with DF and Con A-sup . The functional maturation caused by Con A-sup seemed to be associated with the expression of a receptor for a lymphokine, termed phagocytosis-augmenting factor (PAF) which is present in the Con A-sup . Such a receptor appeared to be common to macrophage lineage, since PAF in Con A-sup was absorbed out with splenic adherent cells and peritoneal exudate cells (PEC) in addition to M+1 cells, but not with nonadherent splenic lymphocytes or lymphoid cell line cells, such as EL-4 and L-1210 . This fact suggests that PAF is different from interferon-gamma (IFN) which is known to modulate the function of lymphocytes . Inability of PAF to bind Cibacron Blue-Sepharose, unlike IFN, supports this notion . The molecular weight of PAF is approximately 2-3 X 10(4) . Thus, the present studies suggest the requirement of at least two signals for the full maturation of macrophages, a cytokine represented by DF and a lymphokine, by PAF.

Biochem Biophys Res Commun, 1985 Sep 16, 131(2), 593 - 9
Partial characterization of intra- and extracellular forms of the glycoprotein hemopexin in liver cell culture and cell-free translation; Katz NR et al.; Primary cultures of rat hepatocytes produce extracellular and intracellular species of hemopexin . We examined the presence of high-mannose oligosaccharides and neuraminic acid residues in these species by comparing their electrophoretic mobility on SDS-PAGE before and after digestion by endoglycosidase H and neuraminidase . The predominant intracellular form was not susceptible to digestion by neuraminidase but was sensitive to endoglycosidase treatment, which digested it to a species with a molecular weight comparable to that of the sole hemopexin species produced by tunicamycin-treated hepatocytes and that produced by in vitro translation . By contrast, both the minor intracellular and the extracellular species of hemopexin were neuraminidase-, yet not endoglycosidase H-sensitive, and may be identical . It can be concluded that the intracellular precursor contains high-mannose type oligosaccharides which are processed to complex type oligosaccharides shortly before secretion of hemopexin.

J Microsc, 1985 Sep, 139 ( Pt 3), 321 - 5
A simple re-embedding method for the preparation of testicular cell cultures for light and electron microscopy; Bowen Jones H et al.; A simple and reproducible method is described for the preparation of cell cultures for light and transmission electron microscopy . Mixed testicular cells are grown on collagen layered, PTFE-coated coverslips . Thin sheets of plastic-embedded cells are packed together and re-embedded permitting the examination of sections cut perpendicular to the plane of the culture and, thus, the intercellular relationships at different levels throughout the culture.

Prenat Diagn, 1985 Sep-Oct, 5(5), 305 - 12
Reduction of sera requirements in amniotic fluid cell culture; Chang HC et al.; Reduction in serum requirement for culture of primary human amniotic fluid cells can be achieved by the addition of 10 growth-promoting factors to the nutrient medium . This supplemented medium preserves cell types normally found in amniotic fluid cell cultures supplemented with 20-30 per cent fetal bovine serum . The volume of amniotic fluid required to initiate culture can be as little as 1 ml . Amniotic fluid samples contaminated with red blood cells with no visible clot also grow well in the low serum medium . Cell-free amniotic fluid combined with equal parts of supplemented medium is useful in initiating cell culture.

Biomaterials, 1985 Sep, 6(5), 346 - 51
Biocompatibility testing of prosthetic implant materials by cell cultures; Pizzoferrato A et al.; The main aspects of biocompatibility are discussed first then the methodology used and the results obtained . The cells used included epithelial cells, lymphocytes, fibroblasts, and macrophages . The most commonly used methods were morphological observation, radioactive tracer uptake, and chemotactic migration analysis . It is concluded that cell cultures are a reliable and sensitive method for initial screening in testing the biocompatibility of the materials used in the construction of prosthetic implants.

In Vitro Cell Dev Biol, 1985 Sep, 21(9), 505 - 8
Rat kidney epithelial cell culture for metal toxicity studies; Cherian MG; Evaluation of the potential adverse human health effects of low-level chronic exposure to heavy metals is dependent on the basic knowledge of the cellular and molecular toxicology of these metals . The use of various cell culture systems has greatly facilitated our knowledge of the cellular effects . Inasmuch as most of the acute and chronic toxic effects of metals occur primarily on the renal proximal tubules, the development of a rat kidney epithelial cell culture has provided a unique system to study the uptake and mechanism of toxicity of metals and their intracellular binding ligands . In the presence of D-valine, fibroblast growth was retarded and a primary epithelial monolayer culture was selectively grown from rat kidney cells . A distinct difference in the uptake of chemically similar divalent metals, such as Pb2+, Hg2+, Cd2+, and Zn2+, was observed in these cells . Both Pb2+ and Hg2+ were more avidly taken up by kidney cells than Cd2+ and Zn2+ salts and they also showed increased toxicity . On the other hand, the cellular uptake of Cd from cadmium-metallothionein (CdMT) was much less than from CdCl2, but CdMT was about seven times more toxic than CdCl2 when added to the renal cell culture . The cytotoxicity of CdCl2 was decreased significantly with pretreatment of the cells with CdCl2, although this had no effect on the toxicity of CdMT . The cellular toxicity of CdMT occurred probably during the process of its transport across the plasma membrane whereas that of CdCl2 occurred after it had entered the cell . Thus rat kidney epithelial cells may be a useful tool to study the mechanism of renal toxicity of environmental chemicals and drugs.

J Virol, 1985 Sep, 55(3), 760 - 7
ts1, a Paralytogenic mutant of Moloney murine leukemia virus TB, has an enhanced ability to replicate in the central nervous system and primary nerve cell culture; Wong PK et al.; A temperature-sensitive mutant of Moloney murine leukemia virus TB (MoMuLV-TB), ts1, which is defective in intracellular processing of envelope precursor protein (Pr80env), also possesses the ability to induce hind-limb paralysis in infected mice . To investigate whether ts1 has acquired neurotropism and to determine to what extent it can replicate in the central nervous system, we compared viral titers in the spleen, plasma, spinal cord, and brain throughout the course of infection of mice infected with ts1 and parental wild-type (wt) MoMuLV-TB . In both the ts1- and wt-inoculated mice, the concentrations of infectious virus recovered from the plasma and spleen increased rapidly and reached a plateau by 10 days postinfection (p.i.) . In contrast, virus concentrations in the spinal cord and brain of ts1-inoculated mice increased gradually and reached a titer comparable to that in the spleen and exceeding that in the plasma only at 25 to 30 days p.i . At this time, the virus titer was approximately 200X greater in ts1-infected spinal cord tissue and approximately 20X greater in ts1-infected brain tissue than in the same wt-infected tissues . Paralysis became evident at 25 to 30 days p.i . in ts1-inoculated mice, whereas the wt-inoculated mice were normal . In addition, a substantial amount of Pr80env was detected in the spinal cords of ts1-inoculated mice compared with that found in the spinal cords of wt-inoculated mice . The infectious virus isolated from ts1-infected nerve tissue was found to possess the characteristic phenotype of the ts1 virus . Microscopic lesions of ts1-inoculated mice at 30 days p.i . consisted of vacuolar degeneration of motor neurons and spongy change of white matter in the brain stem and spinal cord . Similar but less severe lesions were observed in wt-inoculated mice . With primary cultures of central nervous system tissue we showed that ts1 can infect and replicate in both neuron and glial cells . In contrast, although wt MoMuLV-TB replicated in glial cell-rich culture, viral replication was barely detectable in neuron-rich culture.

Endocrinology, 1985 Sep, 117(3), 939 - 46
Catecholestrogen regulation of prolactin synthesis in pituitary cell culture; Shupnik MA et al.; The 2-hydroxycatecholestrogens, 2-hydroxyestradiol {1,3,5-(10)estratriene-2,3,17 beta-triol} (2-OHE2) and 2-hydroxyestrone {2,3-dihydroxy-1,3,5-(10)-estratriene-17-one} (2-OHE1) were tested for their ability to alter PRL production and PRL messenger RNA (mRNA) levels in rat pituitary cell cultures . Treatment of cells with 10(-8) M 2-OHE1 or 2-OHE2 resulted in increased PRL secretion at 24 and 48 h (to 167% and 211% of control, respectively), but not at 4 h . Metabolism studies of radioactive 2-OHE1 and 2-OHE2 in parallel cultures demonstrated that the major metabolite at all times for either compound was the 2-methoxy derivative . After 24 h of treatment, nearly 40% of each compound was the original catecholestrogen, and at no time was there any detectable conversion to estradiol or estrone . Treatment of pituitary cells for 48 h with increasing concentrations of 2-OHE1 or 2-OHE2 resulted in a biphasic PRL dose response . PRL secretion was increased 3.6-fold for 2-OHE2 and 2.4-fold for 2-OHE1 between 10(-10) M and 10(-8) M . At concentrations above 5 X 10(-8) M, however, both compounds decreased PRL levels until, at 10(-6) M 2-OHE1 or 2-OHE2, PRL levels were 40-70% of control . Changes in PRL mRNA levels paralleled those of secretion . Treatment of pituitary cells with 10(-8) M of either 17 beta-estradiol (E2), 2-OHE1, or 2-OHE2 resulted in 2- to 5-fold increases in translatable and hybridizable PRL mRNA . The addition of 10(-7) M E2 plus 10(-8) M 2-OHE1 or 2-OHE2 resulted in PRL secretion and PRL mRNA levels equal to those resulting from E2 stimulation alone . The inhibition in PRL secretion and PRL mRNA levels caused by 10(-6) M 2-OHE1 or 2-OHE2 was partially overcome by coincubation of cultures with E2 . Thus, 2-OHE1 and 2-OHE2 at low concentrations (less than 10(-8) M) can act on the pituitary as E2 agonists to increase PRL synthesis but at high concentrations may act as inhibitors of PRL production.

Biull Eksp Biol Med, 1985 Sep, 100(9), 330 - 1
{Immunologic typing of splenic lymphocytes from human fetuses--donors of pancreatic islet cell cultures}; Krasavtseva TK et al.; To determine the HLA-phenotype of a potential donor of pancreatic islet cells, use was made of lymphocytes from 18-25-week-old human fetuses . The HLA-phenotype was clearly established in 39 out of 52 cases . In 13 cases, the authors failed to reveal histocompatibility antigens because of low viability of lymphocytes . The relationship was ascertained between the detectability of HLA-antigens in fetal donors and cytophysiological characteristics of islet cell cultures prepared from the pancreas.

Cell Immunol, 1985 Sep, 94(2), 587 - 97
Effects of thymic myoid cell culture supernatant on cells from lymphatic tissues; Kamo I et al.; Conditioned media (MCM) of cloned thymic myoid cells (IT45R92, R613Ad, and R615B2) were used to investigate their possible involvement in thymic biological events . Those myoid cells produced in a culture medium biological activities capable of stimulating the growth of thymocytes, spleen cells, and bone marrow cells of mice and rats . Surface markers detected on spleen cells proliferating in MCM were characteristic of monocyte-macrophage lineages (C3R, Fc gamma R, asialo GM1) and T-cell lineages (Thy 1) but not B cells (sIgG) . Chromatographic studies also suggested that the biological activities of MCM could be separated into two different molecular entities, such as a colony-stimulating activity and an interleukin 1-like activity which supported the growth of monocyte-macrophage lineages and T-cell lineages, respectively . These results indicate that thymic myoid cells produce cytokines important for the regulation of intrathymic interleukin cascade by which clonally differentiated thymic lymphocytes may be expanded into a sizable pool.

Mol Cell Endocrinol, 1985 Sep, 42(2), 175 - 83
Electrophoretic characterization of active renin from human kidney and inactive renin from a human chorionic cell culture; Auzan C et al.; Enzymatically inactive human renin from chorionic cells in culture is significantly distinct in polyacrylamide gel electrophoresis (pH 8.17, 0 degree C) from active human kidney renin . The inactive renin is larger and more basic than the active renin; their molecular weights derived from gel electrophoretic retardation coefficients relate as 47.5/35.3 kDa, their valences (net protons/molecule) as 2.14/1.85 . In gel electrofocusing conducted in a mixture of simple buffers, both inactive and active renins exhibit 2 components at the steady-state . The molecular size and basicity of inactive renin are consistent with the hypothesis that it may be a precursor (prorenin), although the possibility that it is an inhibitor complex cannot be ruled out.

Proc Natl Acad Sci U S A, 1985 Sep, 82(18), 6330 - 4
Transplants of Schwann cell cultures promote axonal regeneration in the adult mammalian brain; Kromer LF et al.; Transplantation of embryonic brain tissue or mature peripheral nerves into the adult mammalian central nervous system promotes axonal regrowth from axotomized central nervous system neurons; however, the cellular origin and molecular nature of the factors promoting axonal growth in vivo are unknown . To further characterize cellular environments that facilitate regeneration of central nervous system axons, we developed a methodology whereby cultured cell preparations can be transplanted into the brain of mature mammals . For this procedure, lesions are produced in the septal-hippocampal system of adult rats, and selected regions from collagen-supported Schwann cell/neuron cultures (consisting of Schwann cells, extracellular matrix, and degenerating neuronal processes and myelin but devoid of neuronal perikarya and fibroblasts) are positioned within the intracephalic cavity so that they bridge the lesion gap (approximately 3 mm) separating the septum and hippocampus . At various time up to 3 weeks after transplantation, specimens were prepared for acetylcholinesterase histochemistry and the immunocytochemical localization of laminin (an extracellular matrix protein) and C-4 (a Schwann cell membrane antigen) . All specimens (from uninjured controls and from animals with either acellular collagen or mature Schwann cell/extracellular matrix transplants) contained laminin immunoreactivity associated with the meninges, choroid plexus, ependyma, and cerebral blood vessels . All animals with transplants showed prominent laminin staining on astrocytic processes along the intracephalic cavity, but only the Schwann cell/extracellular matrix transplants exhibited dense laminin and C-4 immunoreactivity within the cellular portion of the transplants . Regeneration of acetylcholinesterase-positive septal fibers occurred only in animals containing Schwann cell/extracellular matrix transplants . By 6 days after transplantation, acetylcholinesterase-positive fibers were observed both on laminin-positive cellular tissue strands connecting the septum and the Schwann cell/extracellular matrix transplants and on the initial portions of the transplants . By day 14, acetylcholinesterase-positive fibers traversed the entire lesion cavity in intimate association with the laminin- and C-4-positive cellular layer of the transplants and reinnervated the host hippocampus . However, cholinergic fibers were not associated with all laminin-containing processes along the lesion cavity nor did they grow along acellular collagen transplants . These results indicate the presence of factors in transplants of cultured Schwann cells and their associated extracellular matrix that promote rapid regeneration of central nervous system cholinergic axons in vivo.

J Cell Physiol, 1985 Sep, 124(3), 411 - 23
Phosphate uptake by primary renal proximal tubule cell cultures grown in hormonally defined medium; Waqar MA et al.; The uptake of labeled inorganic phosphate into primary rabbit kidney proximal tubule cells has been examined . Phosphate was accumulated into the primary proximal tubule cells against a concentration gradient . This accumulation was sensitive to inhibition by metabolic inhibitors . The dependence of phosphate uptake on the extracellular phosphate concentration was examined . Similarities were observed between primary proximal tubule cells and the LLC-PK1 cell line in these regards . These phosphate uptake data were then plotted on a Lineweaver-Burke plot . A nonlinear plot was obtained, which suggested that phosphate uptake occurs by means of a Na+ dependent, carrier mediated process, as well as by another Na+ independent mechanism . The pH dependence of phosphate uptake was also examined . Unlike previous observations with LLC-PK1 cells, optimal phosphate uptake occurred at pH 6.5 . However, this difference between the two cell culture systems may possibly be explained by differences in uptake conditions . The dependence of phosphate uptake on the extracellular NaCl concentration was examined at three different pH values . The rate of phosphate uptake at pH 7.0 was observed to saturate at a lower NaCl concentration than at either pH 6.0 or pH 6.5 . Furthermore, the optimal rate of phosphate uptake at pH 7.0 was observed to be higher than at the other two pH values studied when the NaCl concentration was below 120 mM . However, when the NaCl concentration was raised to 150 mM, optimal phosphate was observed to occur at pH 6.5 rather than at pH 7.0 . These observations may be explained if the pH affects not only the rate of phosphate uptake but also the affinity of the phosphate uptake system for sodium . Phosphate uptake was also observed to be sensitive to several agents, Na2 X SO4 and NaSCN, which affect the membrane potential . As observed with phosphate uptake by LLC-PK1 (and renal brush border membrane vesicles), phosphate uptake was highly sensitive to inhibition by the phosphate analogue arsenate . Novel observations were that the phosphate analogue vanadate and its cellular metabolite vanadyl stimulated the initial rate of phosphate uptake.

Biochem Int, 1985 Sep, 11(3), 273 - 80
Monensin inhibits collagenase production in osteoblastic cell cultures and also inhibits both collagenase release and bone resorption in mouse calvaria cultures; Cowen KS et al.; Monensin, a monovalent cation ionophore, inhibited collagenase production in mouse osteoblast-rich bone cell and clonal osteogenic cell cultures . Inhibition of parathyroid hormone-stimulated bone resorption by monensin was also studied in calvaria cultures . Collagenase activity levels in the medium decreased concomitantly with the inhibition of bone resorption by monensin, indicating that monensin inhibited bone resorption by blocking collagenase secretion from osteoblasts in bone explants.

Cell Struct Funct, 1985 Sep, 10(3), 271 - 8
Myelin formation in dissociated cell cultures of rat embryonic cerebral hemispheres; Hirano S et al.; Developing cultures of dissociated cerebral hemispheres obtained from 18-day-old embryonic rats synthesized, or activated, a myelin-related enzyme, 2',3'-cyclic nucleotide 3'-phosphohydrolase (CNPase) . This increase in activity coincided with the onset of myelination . The presence of CNPase in oligodendrocytes and myelin was demonstrated using immunocytochemical staining techniques . Myelinated axons and myelin sheaths were clearly made visible by electron microscopy of 28-day-old cultures.

Brain Res, 1985 Sep, 354(1), 55 - 65
The possible modulation of the development of rat dorsal root ganglion cells by the presence of 5-HT-containing neurones of the brainstem in dissociated cell culture; Forda S et al.; Reliable methods for coculturing dissociated rat brainstem cells together with dorsal root ganglion (DRG) cells have been established . The cells were characterized using autoradiographic, morphological, immunocytochemical and electrophysiological techniques . Light-level microscopy showed the cocultures to be extensively invaded by serotonin (5-HT)-containing neuronal processes and intense clustering of 5-HT-containing varicosities to occur in the vicinity of large round phase-bright neurones thought to be DRGs . Rather extensive fine ramification of the neuronal processes throughout the culture dish were visualized using scanning electron microscopy . Intracellular recording showed the brainstem neurones to be spontaneously active, electrically excitable and sensitive to a variety of transmitter candidates including serotonin (5-HT) and gamma-aminobutyric acid . Several different responses to 5-HT have been observed . These include a depolarization accompanied by an increase in membrane resistance, a depolarization accompanied by a decrease in membrane resistance and a hyperpolarization accompanied by an increase in membrane resistance . As established by others, DRG cells grown in isolation were always quiescent . The application of 5-HT produced no effect on membrane potential, resistance or excitability . In the brainstem-DRG cocultures 52% of DRG cells exhibited synaptic activity and occasional spontaneous spiking, both of which were abolished in the presence of tetrodotoxin or low-Ca2+/high-Mg2+ solution . Transmission electron microscopy confirmed the presence of synapses on the DRG cells . The spontaneously active DRG cells were also found to be sensitive to the application of 5-HT . Thus it appears that a source of 5-HT nerve terminals may regulate the development and pharmacological sensitivity of primary afferent neurones in culture.

Blood, 1985 Sep, 66(3), 714 - 7
Effects of dibutyryl adenosine 3',5'-cyclic monophosphate on erythropoietin production in human renal carcinoma cell cultures; Hagiwara M et al.; A human renal carcinoma from a patient with 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 after the cultures reached confluency, in parallel with dome formation . Dibutyryl adenosine 3',5'-cyclic monophosphate significantly (P less than .01) stimulated Ep production and dome formation in the semiconfluent and confluent cultures of the renal carcinoma.

Probl Endokrinol (Mosk), 1985 Sep-Oct, 31(5), 67 - 70
{Results of transplantation of pancreatic islet cell cultures to patients with diabetes mellitus}; Shumakov VI et al.; Altogether 110 intramuscular pancreatic islet cell (PIC) culture transplantations were performed in diabetes mellitus patients from April 1981 to May 1984 at the Research Institute of Transplantology and Artificial Organs, USSR Ministry of Health: 65 allotransplantations of human fetal PIC and 45 xenotransplantations fo swine fetal PIC . Immunosuppressive therapy was not employed . The paper is concerned with an analysis of a therapeutic effect of the IC cell cull culture transplantation on 30 patients who were followed-up for not less than 1 year after operation . A pronounced and long-term antidiabetic effect was observed in wost of the recepients subjected to the intramuscular human fetal PIC culture transplantation: demand in exogenous insulin decreased, a durable stabilization of a course of disease in patients with labile diabetes mellitus was observed, and in patients with such diabetic complications as polyneuropathy, retinopathy and glomerulosclerosis certain regress of these complications was observed . As to the results of the intramuscular swine fetal PIC culture xenotransplantation the authors note that xenogenic transplantation as compared with IC culture allotransplantation possesses a less noticeable offect on diabetes mellitus complications.

Parasite Immunol, 1985 Sep, 7(5), 467 - 78
Giardia muris-induced depression of the primary immune response in spleen and mesenteric lymph node cell cultures to sheep red blood cells; Belosevic M et al.; Primary in vitro plaque-forming cell (PFC) responses to sheep red blood cells (SRBC) were examined for spleen and mesenteric lymph node (MLN) cell populations from susceptible (A/J) and resistant (B10.A) mice during the infection with Giardia muris . Spleen and MLN cells isolated from mice during the acute phase of the infection were less responsive to SRBC in vitro than those from uninfected mice . Depressed anti-SRBC PFC response was detected earlier and was more pronounced in MLN cell cultures when compared to the response of spleen cell cultures . Spleen and MLN cells from donors infected with G . muris for 15 days had the capacity of depressing PFC response to SRBC of cells isolated from uninfected mice . This suppressor activity was localized in the plastic-adherent fraction of spleen cell populations isolated from A/J and B10.A mice . Since G . muris is a gastro-intestinal infection of mice, lower capacity of the MLN cells to respond to an antigenic stimulation in vitro may explain, in part, the proliferation of the trophozoites during the acute phase of the infection.

Neuroscience, 1985 Sep, 16(1), 213 - 21
Neurons dissociated from rat myenteric plexus retain differentiated properties when grown in cell culture . III . Synaptic interactions and modulatory effects of neurotransmitter candidates; Willard AL et al.; We have used intracellular recordings to study synaptic interactions between myenteric neurons grown in dissociated cell culture . Intracellular stimulation of individual myenteric neurons caused several types of synaptic effects in nearby neurons: fast excitatory synaptic potentials mediated by nicotinic acetylcholine receptors; slow, non-cholinergic synaptic potentials; dual transmission having both fast cholinergic and slow non-cholinergic components and inhibition of spontaneously occurring fast nicotinic synaptic potentials . Fast nicotinic synaptic potentials were elicited by about 40% of neurons tested and often occurred spontaneously . The fast synaptic potentials were similar to those that have been studied in other autonomic neurons with respect to their estimated reversal potential and their sensitivity to cholinergic antagonists . The amplitudes of the fast synaptic potentials declined if evoked at frequencies greater than 0.5 Hz . Potentiation of the fast synaptic potentials was observed following high-frequency stimulation of presynaptic neurons . Several transmitter candidates modulated fast cholinergic transmission . Substance P and vasoactive intestinal peptide promoted nicotinic transmission by causing increased amplitudes of evoked and spontaneous fast synaptic potentials and an increased frequency of spontaneous synaptic potentials . gamma-Aminobutyrate and {Met}enkephalin both caused decreased amplitudes and frequency of nicotinic synaptic potentials . Serotonin depressed synaptic potentials in some neurons while enhancing them or having no effect in others . Slow, non-cholinergic, synaptic potentials were elicited by about 10% of neurons tested . These synaptic effects lasted 15-300s, caused depolarizations of 3-15 mv and were accompanied by increased neuronal input resistance . The transmitter(s) causing these slow synaptic potentials has not yet been identified.(ABSTRACT TRUNCATED AT 250 WORDS)

Res Vet Sci, 1985 Sep, 39(2), 241 - 6
Adherence of Moraxella bovis to cell cultures of bovine origin; Annuar BO et al.; The adherence of five strains of Moraxella bovis to cell cultures was investigated . M bovis adhered to cultures of bovine corneal epithelial and Madin-Darby bovine kidney cells but not to cell types of non-bovine origin . Both piliated and unpiliated strains adhered but piliated strains adhered to a greater extent than unpiliated strains . Antiserum against pili of one strain inhibited adherence of piliated strains but caused only slight inhibition of adherence to the unpiliated strains . Treatment of bacteria with magnesium chloride caused detachment of pili from the bacterial cell and markedly inhibited adherence of piliated strains but caused only slight inhibition of adherence by the unpiliated strains . The results suggested that adhesion of piliated strains to cell cultures was mediated via pili but that adhesins other than pili may be involved in the attachment of unpiliated strains of M bovis to cells.

Neuroscience, 1985 Sep, 16(1), 201 - 11
Neurons dissociated from rat myenteric plexus retain differentiated properties when grown in cell culture . II . Electrophysiological properties and responses to neurotransmitter candidates; Willard AL et al.; We have used intracellular recordings to study the electrophysiological and pharmacological properties of neurons that have been grown in cell cultures after having been dissociated from the myenteric plexus of the small intestine of newborn rats . Studies of action potential mechanisms revealed that all of the neurons could generate Na+-dependent action potentials in the presence of Ca2+-channel blockers and that about 70% could generate Ca2+-dependent action potentials when Na+ channels were blocked with tetrodotoxin . No neurons generated long afterhyperpolarizations after single action potentials but about 50% of neurons did so following trains of action potentials . Over 95% of the neurons tested accommodated rapidly to sustained depolarization . The effects of several enteric neurotransmitter candidates were studied by superfusing or pressure-ejecting test solutions while recording neuronal responses . All of the cultured neurons tested had nicotinic responses to acetylcholine . Subsets of neurons responded to muscarinic cholinergic agonists (slow depolarization and increased excitability), serotonin (fast depolarization or slow depolarization and increased excitability), gamma-aminobutyrate (fast depolarization), substance P (slow depolarization, biphasic fast and slow depolarization or increased excitability without a change in membrane potential), vasoactive intestinal peptide (slow depolarization and increased excitability), or {Met}enkephalin (slow hyperpolarization and/or decreased action potential duration) . We conclude that myenteric neurons grown in cell culture retain many of the physiological and pharmacological properties that they have in situ . Such cultures will permit detailed biophysical and pharmacological studies of the mechanisms of action of enteric neurotransmitter candidates.

Neuroscience, 1985 Sep, 16(1), 187 - 99
Neurons dissociated from rat myenteric plexus retain differentiated properties when grown in cell culture . I . Morphological properties and immunocytochemical localization of transmitter candidates; Nishi R et al.; We have developed procedures for dissociating neurons from the myenteric plexus of the small intestine of newborn rats and for growing those neurons in cell cultures for up to 3 months . Neurons in these cultures retain many of the differentiated properties of myenteric neurons in vivo . This is the first of a series of 3 papers describing those properties . In this paper, we describe the morphology of cultured neurons that we have observed with light and electron microscopy; we also describe the patterns of straining observed when immunocytochemical techniques were used to localize neurotransmitter candidates in the cultured neurons . Intracellular injections of a fluorescent dye, Lucifer yellow, revealed that many of the cultured neurons had morphologies similar to those of myenteric neurons in vivo . When thin sections of cultures were viewed in an electron microscope, many neurons were observed to have numerous small (40-60 nm), clear synaptic vesicles and/or large (80-150 nm), opaque-cored (p-type) vesicles . Synaptic profiles were most often observed on neuronal somata . Neurons containing immunoreactive serotonin, substance P, somatostatin, enkephalin, bombesin and gastrin/cholecystokinin were observed in about the same proportions as they occur in the intact myenteric plexus . Neurons containing immunoreactive vasoactive intestinal polypeptide were found in higher numbers than reported in vivo . Neurons containing immunoreactive neurotensin, secretin and glutamate decarboxylase were not observed . An antiserum directed against choline acetyltransferase stained 40-50% of the neurons . We conclude that myenteric neurons continue to express much of their normal differentiated properties even when they are removed from the gut, dissociated into a suspension of single cells and grown in culture . Such cultures will be useful for correlating the morphological, biophysical, pharmacological and synaptic properties of individual myenteric neurons and for testing the ability of altered environmental conditions to change those properties.

Tsitologiia, 1985 Sep, 27(9), 1011 - 20
{Dependence of ion transport across the plasma membrane on the density of the cell culture . I . Ion flows and the potassium and sodium content in 3 Chinese hamster cell lines (CHO)}; Marakhova II et al.; Cation transport has been investigated in three lines of Chinese ovary cells CHO-K1 during the cell culture growth . With the increase in the cell density potassium and sodium contents decreased from 1.2 to 0.8-0.5 and from 0.5 to 0.15-0.1 mmole/g protein, respectively . The time courses of potassium and sodium changes were different, and the increase in intracellular K/Na ratio from 1.5-2.0 to 5-10 with the increase in cell density was revealed . The rubidium influx was found to decrease during the culture growth mainly due to the decrease in ouabain-inhibitable and (ouabain + furosemide)- non-inhibitable influxes . The changes in cation fluxes and cation contents were observed in transformed cells without contact inhibition of division and were considered as a manifestation of density-dependent alterations of plasma membrane.

Proc Natl Acad Sci U S A, 1985 Sep, 82(18), 6325 - 9
Dissociated cell culture of cholinergic neurons from nucleus basalis of Meynert and other basal forebrain nuclei; Nakajima Y et al.; Degeneration of cholinergic neurons from the basal forebrain nuclei is suspected to be the cause of Alzheimer disease . We have developed dissociated cultures of cholinergic neurons from these nuclei (the nucleus basalis of Meynert, the medial septal nucleus, and the diagonal band nuclei) . Brain slices of the forebrains were made by a vibratome, and the basal forebrain nuclei were dissected out, dissociated, and cultured . Choline acetyltransferase immunocytochemistry and acetylcholinesterase cytochemistry revealed large cholinergic cells (average diameter, 20-25 micron) in these cultures . About 75% of large neurons (20 micron or larger in diameter) were cholinergic . Electrophysiological experiments were performed on these large neurons . The neurons usually did not show spontaneous firing, but steady depolarizations produced trains of action potentials, which adapted quickly . The neurons responded with depolarization to the application of L-glutamic acid . Substance P produced depolarization (sometimes hyperpolarization), and during the depolarization membrane resistance was increased.

Mol Pharmacol, 1985 Sep, 28(3), 269 - 77
Barbiturates decrease voltage-dependent calcium conductance of mouse neurons in dissociated cell culture; Werz MA et al.; Barbiturates have been shown to reduce presynaptic release of neurotransmitter . It is likely that barbiturates alter transmitter release by decreasing calcium entry since barbiturates decrease calcium influx into synaptosomes and reduce the maximal rate of rise and duration of calcium-dependent action potentials . The mechanisms of barbiturate action on neuronal calcium entry have been studied using mouse dorsal root ganglion neurons in cell culture . Dorsal root ganglion neuron action potentials have a calcium-dependent component which is decreased by the barbiturates, pentobarbital (50-500 microM) and phenobarbital (500-2000 microM) . Calcium-dependent action potential after hyperpolarization was also decreased by barbiturates . Intracellular injection of the potassium channel blocker, cesium, enhanced barbiturate actions . In voltage-clamp studies, barbiturates reduced inward calcium current and calcium chord conductance without altering the leak conductance which is present after all calcium conductance was blocked by application of cadmium ions (100 microM) . Calcium current inactivation was accelerated by barbiturates but unaffected by cadmium . We conclude that barbiturates reduce calcium conductance by enhancing calcium channel inactivation or by producing open channel block of calcium channels.

Cancer Res, 1985 Sep, 45(9 Suppl), 4595s - 4597s
Evidence for HTLV-III in T-cells from semen of AIDS patients: expression in primary cell culture, long-term mitogen-stimulated cell cultures, and cocultures with a permissive T-cell line; Zagury D et al.; The development of acquired immunodeficiency syndrome or of acquired immunodeficiency syndrome-related complex by transmission of human T-lymphotropic retrovirus III by semen has previously been implicated by epidemiological studies . In vitro investigations were performed on mononuclear cells obtained from the semen of patients with acquired immunodeficiency syndrome to identify human T-lymphotropic retrovirus III or related retrovirus . The presence of human T-lymphotropic retrovirus III was demonstrated (a) in primary cell cultures, by the detection of the Mr 24,000 protein by indirect immunofluorescence assays by Day 6; (b) in activated long-term cell culture by reverse transcriptase activity, by indirect immunofluorescence (Mr 24,000 protein); and (c) in cocultures of T-cells from semen of AIDS patients and H9 cells by reverse transcriptase activity, indirect immunofluorescence, and the presence of virus particles by electron microscopy.

Biochem Biophys Res Commun, 1985 Aug 30, 131(1), 167 - 73
Hormonal regulation of benzo{a}pyrene metabolism in human adrenocortical cell cultures; Hornsby PJ et al.; In cultured fetal human adrenocortical cells, metabolism of the carcinogen benzo{a}pyrene was found to be unresponsive to the xenobiotic inducers 3-methylcholanthrene, benz{a}anthracene and 2,3,7,8-tetrachlorodibenzo-p-dioxin . However, exposure of cultures to the hormone adrenocorticotropin (ACTH) for 48 hours stimulated benzo{a}pyrene metabolism 3-fold . The major metabolite was the 7,8-diol . Other compounds which stimulate the production of adrenocortical cell cyclic AMP (forskolin and cholera toxin) as well as monobutyryl cyclic AMP also increased benzo{a}pyrene metabolism . Human adrenocortical cells thus provide an unusual example of hormonal regulation of the metabolism of a carcinogen.

Neurosci Lett, 1985 Aug 5, 58(3), 293 - 7
Glutamate neurotoxicity in cortical cell culture is calcium dependent; Choi DW; Brief exposure to glutamate produced widespread neuronal death in mature, but not young, cortical cell cultures . Extracellular sodium replacement or addition of tetrodotoxin produced only minor reduction in this toxic neuronal loss . However, removal of extracellular calcium markedly reduced neuronal loss, and elevation of extracellular calcium accentuated neuronal loss . These observations suggest that the toxicity of glutamate on cortical neurons may depend primarily on the presence of extracellular Ca, probably through a mechanism which is distinct from simple 'excitotoxicity'.

Methods Find Exp Clin Pharmacol, 1985 Aug, 7(8), 403 - 8
The effect of 4'-deoxypyridoxine on rat heart cell cultures under oxygen deficiency; Millart H et al.; The purpose of this work was to study the consequences of an intracellular depletion of pyridoxal-phosphate (PLP) in rat heart cells in culture submitted to oxygen deficiency . Glucose (GLc) and 4'-deoxypyridoxine (DOP) effects were separately evaluated after 150 mn of partial oxygen deprivation (N2) with regard to enzyme leakage, beating rates, intracellular concentrations of PLP and the ratio glycogen phosphorylase activity (-5'-AMP/+5'-AMP) . PLP concentrations were higher in the air-GLc group than in the air group . alpha-Hydroxybutyrate dehydrogenase (alpha-HBDH) and creatine kinase (CK) leakages were observed in the N2 group, whereas 1.17 +/- 0.17 mM GLc prevented leakage . Beatings stopped in the N2 group, slightly decreased in the N2-GLc group; the ratio (-5'-AMP/+5'--AMP) increased only in the N2 group . With a 1 mM DOP pretreatment a significant decline of PLP was observed either with or without GLc in the culture medium . The ratio (-5'-AMP/+5'-AMP) was lower in the air-DOP group than in the air group, whereas beatings, alph-HBDH and CK activities were identical to the control group . ImM DOP in the N2-DOP group partially prevented alpha-HBDH and CK leakages into the medium but did not prevent the decrease in contractile activity . Surprisingly, PLP concentration increased in the N2-DOP group/air-DOP group and the increase in ratio (-5'-AMP/+5'-AMP) was higher in the N2-DOP group than in the N2 group when compared to the respective control groups.(ABSTRACT TRUNCATED AT 250 WORDS)

Xenobiotica, 1985 Aug-Sep, 15(8-9), 705 - 11
Toxicity monitored with a correlated set of cell-culture assays; Borenfreund E et al.; A set of assays for toxicity has been developed in which cell cultures serve as an alternative to toxicity testing in vivo . One test is the assessment of the highest concentration of toxicant which produces minimal morphological alterations in cell cultures, followed by the determination of the amount of neutral red dye uptake by the cells . A second test is based on 50% inhibition of uptake of {3H}uridine after incubation of the cultures with the toxicant . There is good agreement between these assays in the rank correlation of a broad spectrum of compounds tested, as well as with the data from Draize rabbit eye irritancy tests in vivo.

Xenobiotica, 1985 Aug-Sep, 15(8-9), 701 - 4
Photometric recording of cell viability using trypan blue in perfused cell cultures; Walum E et al.; A perfusion system was developed to increase the reliability of cell viability estimations by continuous measurement of the uptake of trypan blue dye . Monolayer cell cultures were perfused with buffer containing toxic substances and trypan blue, and the staining of cells was continuously recorded at 591 nm in a spectrophotometer . Using mercuric chloride and methylmercuric chloride as test substances with C6 rat glioma cells, time- and dose-dependent increases in light absorbance were obtained over a 12h recording period . Methylmercuric chloride at 10(-6) M caused a half-maximal increase in relative absorbance in 4.5 h, whereas the corresponding time for mercuric chloride was 10.5 h.

Biol Reprod, 1985 Aug, 33(1), 187 - 200
Isolation and cell culture of the epithelial cells of cauda epididymidis of the bull; Joshi MS; A simple and rapid method has been described to isolate the epithelial cells of cauda epididymidis of adult bull . Perfusion of the lumen of the cauda epididymidis with 1 mg/ml collagenase in calcium- and magnesium-free Hank's balanced salt solution and incubation of the tissue at 37 degrees C for 90 min releases the principal and basal cells into the lumen . Several individual epithelial cells and cell aggregates without the contamination of stromal or smooth muscle cells can be flushed out at the end of incubation . The isolated epithelial cells, suspended in Dulbecco's medium with 10% horse serum, attach to plastic dishes within 3 h after seeding the cells and proliferate to form a monolayer in approximately 8-12 days . The electron microscopic study and immunostaining of the cultured epithelial cells indicate that the cultured cells are principal cells . The basal cells of the intact cauda epididymidis of bull show within their cytoplasm the presence of varying amounts of "lipid-like" material often closely associated with whorls of membrane.

Brain Res, 1985 Aug, 353(2), 193 - 202
Gangliosides in postmitotic retina of chick embryo: changes in vivo and in cell cultures; Landa CA et al.; Developmental changes in ganglioside composition of postmitotic neural retina of chick embryo were analyzed by thin-layer chromatography . Gangliosides were identified by comparing their chromatographic mobilities with reference standards . The outstanding changes are decrease in the concentration of GD3L and increase in GD1a and GM1 concentrations . By depleting Muller glia cells from retina tissue of 13- and 16-day embryos (R13, R16) we determined that the bulk of the major gangliosides is associated with the neurons . Analysis of gangliosides in monolayer cultures of R13 and R16 cells highly enriched for Muller cell-derived gliocytes indicated that these cells express the same types of gangliosides as neurons, but in somewhat different concentrations and relative proportions; however, after time in culture these cells showed ganglioside types and changes in ganglioside profile that are not characteristic of normal retina . The latter observation is consistent with other evidence that the phenotype of Muller glia cells becomes altered in monolayer culture . In contrast to cultures of early embryonic retina, in organ cultures of later postmitotic retina, ganglioside composition did not continue to change as in normal development . This suggests that in postmitotic retina, normal developmental progression of ganglioside changes requires systemic and/or other conditions which are missing or altered when this tissue is isolated and cultured in vitro.

J Neurosci, 1985 Aug, 5(8), 2028 - 34
Differential synapse formation and neurite outgrowth at two branches of the metacerebral cell of Aplysia in dissociated cell culture; Schacher S; The metacerebral cell (MCC) of Aplysia californica was isolated with its bifurcate axon from the cerebral ganglion and maintained in vitro under three conditions: (a) with no targets, (b) with identified buccal ganglion neurons B1 or B2 placed near the stump of the large diameter cerebral-buccal connective (CBC) branch, and (c) with B1 or B2 placed near the stump of the small diameter posterior lip nerve (PLN) branch . After 5 days in culture, the two branches differed significantly in the formation of chemical connections and in the extent of neurite outgrowth . Chemical connections characteristic of MCC-B1(B2) connections in vivo were observed in more than 90% of the cultures in which the buccal neuron was contacted by neurites emerging from the CBC branch, but in only 20% of the cultures in which the buccal neuron was contacted by neurites extending from the PLN branch . Neurite outgrowth from the CBC stump was always greater than growth from the PLN and was not affected significantly by the presence of a buccal neuron target at either branch . In contrast, neurite outgrowth from the PLN decreased significantly when the target was contacted by neurites from the CBC branch . These results suggest that two branches of a single neuron can differ in their capacities to form chemical connections . In addition, the two branches show differential growth as a result of target interaction at one of the branches . This simple in vitro system may therefore be useful in exploring the ways in which individual neurons control neurite extension from different branches as they seek to form chemical connections with their targets.

J Lab Clin Med, 1985 Aug, 106(2), 162 - 74
Studies on the purification of thrombopoietin from kidney cell culture medium; McDonald TP et al.; A thrombocytopoiesis-stimulating factor (TSF) has been purified from human embryonic kidney (HEK) cell culture medium . In the initial purification step, crude HEK cell culture medium was fractionated with saturated ammonium sulfate (step I) . The proteins precipitated by 40% to 60% and 60% to 80% ammonium sulfate saturation increased the percent of sulfur 35 incorporation into platelets of assay mice (P less than 0.01) . The ammonium sulfate-precipitated proteins that contained significant TSF activity were further refined on Sephadex G-75 columns (step II) . The fraction containing the highest specific activity (greatest 35S incorporation into platelets of assay mice per milligram of protein) was further purified by diethylaminoethyl (DEAE)-cellulose column chromatography (step III) . TSF activity was eluted from the columns between 0.3 and 1.0 mol/L NaCl . Additional Sephadex chromatography of post-DEAE-chromatographic preparations further increased the purity of the TSF (step IV) . TSF from this four-step procedure was further processed on a DEAE-high-performance liquid chromatography (HPLC) column (step Va) or size exclusion (SE)-HPLC columns (step Vb) . After HPLC, the activity was localized in a region corresponding to a retention time of 6 to 8 minutes for the DEAE-HPLC, but longer times were found after SE-HPLC . TSF was further purified by additional sodium dodecyl sulfate-polyacrylamide gel electrophoresis and SE-HPLC (step VI) . The final product had significant TSF activity and represented a purification of approximately 500,000-fold . It was also shown that the isoelectric pH of partially purified TSF was 4.7 and the molecular weight of the more highly purified preparation was approximately 32,000 . After extraction by a combination of chromatographic procedures, a single homogeneous product was obtained.

Dev Biol, 1985 Aug, 110(2), 392 - 401
Nerve growth factor-independent development of embryonic mouse sympathetic neurons in dissociated cell culture; Coughlin MD et al.; Although ganglia from neonatal mouse sympathetic ganglia require nerve growth factor (NGF) for survival in culture, explanted sympathetic ganglia from early embryonic stages do not require added NGF for survival and growth . To determine whether the change in growth factor requirement is due to changes in the neurons themselves, to variations in neuronal populations, or to changes in nonneuronal cells, we examined the response to growth factors by dissociated sympathetic neurons at various stages of development . Results indicate that neurons from the 14-day gestational (E14) superior cervical ganglion (SCG) do not require NGF for initial survival and neurite extension, but do require the conditioned medium neurite extension factor, CMF . By 2 to 3 days thereafter, whether in vivo or in culture, most neurons have developed a requirement for NGF for survival in culture . During the same period, there is a concomitant increase in responsiveness to NGF alone as a trophic agent . Changes in response to NGF are not due to changes in NGF content of ganglia, to interactions in culture with nonneuronal cells, or to age-related differences in NGF requirements for maximum survival . The changes in growth factor requirements may be related to mechanisms regulating specificity of nerve-target connections.

J Neurochem, 1985 Aug, 45(2), 536 - 43
Cholesterol biosynthesis and its regulation in dissociated cell cultures of fetal rat brain: developmental changes and the role of 3-hydroxy-3-methylglutaryl coenzyme A reductase; Volpe JJ et al.; Primary cultures of cells dissociated from fetal rat brain were utilized to define the developmental changes in cholesterol biosynthesis and the role of 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase in the regulation of these changes . Cerebral hemispheres of fetal rats of 15-16 days of gestation were dissociated mechanically into single cells and grown in the surface-adhering system . Cholesterol biosynthesis, studied as the rate of incorporation of {14C}acetate into digitonin-precipitable sterols, was shown to exhibit two distinct increases in synthetic rates, a prominent increase after 6 days in culture and a smaller one after 14 days in culture . Parallel measurements of HMG-CoA reductase activity also demonstrated two discrete increases in enzymatic activity, and the quantitative and temporal aspects of these increases were virtually identical to those for cholesterol synthesis . These data indicate that cholesterol biosynthesis undergoes prominent alterations with maturation and suggest that these alterations are mediated by changes in HMG-CoA reductase activity . The timing of the initial prominent peak in both cholesterol biosynthesis and HMG-CoA reductase activity at 6 days was found to be the same as the timing of the peak in DNA synthesis, determined as the rate of incorporation of {3H}thymidine into DNA . The second, smaller peak in reductase activity and sterol biosynthesis at 14 days occurred at the time of the most rapid rise in activity of the oligodendroglial enzyme, 2':3'-cyclic nucleotide 3'-phosphohydrolase (CNP) . These latter observations suggest an intimate relationship of the sterol biosynthetic pathway with cellular proliferation and with oligodendroglial differentiation in developing mammalian brain.

Zentralbl Bakteriol Mikrobiol Hyg {A}, 1985 Aug, 260(1), 51 - 6
Intra-cellular and extra-cellular growth of L . monocytogenes in chick embryo fibroblast cell culture; Basher HA et al.; The growth of L . monocytogenes in isolated chick embryo fibroblast cell culture was studied . Hanks balanced salt solution and Eagles minimal essential medium were shown to support a limited growth of L . monocytogenes . Extra-cellular growth on the maintenance medium occurs for 48 h prior to the establishment of intra-cellular organisms . As the uptake of the parasite by the cell culture takes place, intra-cellular replication begins with subsequent release of the organisms into the surrounding medium . The organism continues to replicate intra-cellularly until all the cell culture is destroyed.

Lab Invest, 1985 Aug, 53(2), 122 - 31
Glomerular cell culture; Striker GE et al.; Glomerular cell culture has now become a widely used research technique . At the present time procedures are available to obtain isolated glomeruli from nearly all species . The isolation of individual cells has proven problematical . This is due to the lack of defined markers . Thus, it is not yet possible to determine the presence and relative degree of contamination by other glomerular or even nonglomerular, cell types . The importance of dealing with individual cell types, or defined mixtures, is exemplified by the variable results obtained in the assessment of prostaglandin synthesis within and between species . Several important bits of information have, nevertheless, evolved from glomerular cell culture experiments . The sites of synthesis of basement membrane components, as well as their composition, have been determined . Confirmation of the existence of a bone marrow-derived mesangial cell population and some of their properties has been obtained . The response of mesangial cells to, as well as their production of, various mediators has been shown . Finally, clear evidence for interspecies differences and similarities has been documented . Areas of controversy remain, including whether contractile mesangial cells are phagocytic, the presence of C3b receptors on epithelial cells, the amounts and types of certain extracellular matrix products synthesized by the various cell types, and the best methods for separation and culture of the individual glomerular cell types . There remain many fruitful areas for research . Fundamental questions such as the appropriate basal medium and supplements, the type of substrate, and the means to separate the individual cell types remain as unanswered or partially answered questions . When isolated cells are reliably obtained, the study of biosynthetic products in the resting and stimulated states must be again addressed . At that point, the effect of various and deliberate combinations of the glomerular cell types on the biosynthetic or proliferative responses will require further studies . For instance, although contractility mediated by receptors for angiotensin II has been assumed to be a specific property of mesangial cells, recent work shows that epithelial cells also respond to angiotensin II . In addition, the handling of immune complexes by various cells needs to be further investigated (43) . Similarly, the pharmacologic response of the diverse populations of glomerular cells represents another area of study that has just begun . Finally, these data will provide the backdrop on which the analysis of various induced and genetic diseases can be performed.(ABSTRACT TRUNCATED AT 400 WORDS)

Genitourin Med, 1985 Aug, 61(4), 255 - 7
Comparison of direct immunofluorescence and cell culture for detecting Chlamydia trachomatis; Foulkes SJ et al.; Conventional cell culture methods were compared with a direct immunofluorescence test (MicroTrak, Syva UK, Maidenhead, Berkshire) to detect Chlamydia trachomatis in 137 patients (126 women, 11 men) attending a sexually transmitted diseases (STD) clinic . Results obtained by the two tests agreed in 87.6% of cases . Of 34 positive specimens, 17 were detected by culture and fluorescence, 15 by fluorescence only, and two by culture only . The excess of specimens that were negative on culture but positive on fluorescence might be accounted for by delays in culture (up to 18 hours) . The MicroTrak test appears to be of value in peripheral hospitals that have to rely on transporting specimens to larger centres for culture.

Proc Natl Acad Sci U S A, 1985 Aug, 82(15), 5005 - 9
A contact-insensitive subpopulation in Syrian hamster cell cultures with a greater susceptibility to chemically induced neoplastic transformation; Nakano S et al.; We previously have identified a subpopulation of contact-insensitive (CS-) cells which lacks density-dependent inhibition of cell division in primary and low-passage cultures of Syrian hamster embryonic (SHE) fibroblastic cells . Further, we have shown that the proportion of these CS- cells declines as a result of the stable phenotypic conversion of the CS- cells to contact-sensitive (CS+) cells . To determine whether these transient CS- cells are more sensitive to carcinogenic/mutagenic perturbation, the susceptibility to neoplastic transformation and somatic mutation induced by N-methyl-N'-nitro-N-nitrosoguanidine (MNNG) was examined in clonally isolated cell cultures containing various proportions of CS- cells (0.02-4%) . The frequencies of morphological transformation, focus formation, and neoplastic transformation showed a positive correlation to the proportion of CS- cells in the treated cultures . In contrast, the frequency of MNNG-induced somatic mutation at the Na+,K+-ATPase locus was similar among cultures varying in their proportion of CS- cells . Thus, there is a transient subpopulation of CS- cells in primary SHE cell cultures that is more susceptible to neoplastic transformation although equally susceptible to induced point mutation . This dissociation between somatic point mutation and neoplastic transformation indicates a fundamental difference in the nature of these two phenomena . A possible relationship between the propensity of CS- cells (versus CS+ cells) to carcinogen-induced neoplastic transformation and the state of differentiation of the CS- cells is discussed.

J Clin Endocrinol Metab, 1985 Aug, 61(2), 303 - 5
Increased content of neuropeptide Y in human pheochromocytoma cell cultures; Tischler AS et al.; Cells from three human pheochromocytomas grown in monolayer culture for 21 days contained markedly greater amounts of neuropeptide Y (NPY) than freshly dissociated cells from the same tumors . The content of NPY did not consistently correlate with the extent of neurite outgrowth in the cultures and was not further increased in the presence of nerve growth factor . The content of NPY was decreased in the presence of 10(-5) M dexamethasone in two cultures . The findings suggest that human pheochromocytoma cultures may be useful in studies of cellular and molecular mechanisms regulating NPY production, and that these mechanisms may differ somewhat from those regulating production of other regulatory peptides in cultures of the same tumors.

Appl Environ Microbiol, 1985 Aug, 50(2), 523 - 6
Membrane-associated viral complexes observed in stools and cell culture; Williams FP Jr; Viral complexes observed to be membrane associated rather than clumped by antibody were detected in a rotavirus-containing stool specimen by negative-stain electron microscopy . These "viral packets" were also observed in cell culture fluids after repeated passaging and contained up to 100 virions . Other stool specimens have been observed to contain similar packets of parvovirus-like particles . Such complexes must be expected in fecally contaminated water.

Otolaryngol Head Neck Surg, 1985 Aug, 93(4), 492 - 9
Interferon sensitivity of fibroblast cell cultures derived from patients with neoplasms of the head and neck; Richtsmeier WJ et al.; Interferon (IFN) is a protein with antiviral activity that has been shown to inhibit the growth of many different types of cells . We have measured the IFN sensitivity of nine cell cultures isolated from patients with squamous cell carcinoma and one with malignant melanoma of the head and neck . Normal-appearing fibroblast cultures isolated from these tissues appear quite sensitive to the antiviral effects of IFN . When the encephalomyocarditis virus yield reduction assay is used, these diploid cells are as sensitive to IFN-alpha as are newborn foreskin fibroblast cultures . A similar antiviral effect is seen with IFN-gamma . These cells are relatively insensitive to the antigrowth effect of both IFN preparations as measured by 3H thymidine incorporation and direct observations of cell growth . This is the same relative sensitivity as fibroblasts derived from normal patients . Since these cells are at least 100 times less sensitive to the antigrowth action of the IFNs, it appears unlikely that the IFNs play a significant role in the control of normal fibroblast growth, in contrast to the sensitivity of malignant cell lines.

J Lipid Res, 1985 Aug, 26(8), 950 - 4
Cholesterol metabolism: use of D2O for determination of synthesis rate in cell culture; Esterman AL et al.; Cholesterol synthesis in cell culture in the presence of D2O yields a spectrum of enriched molecules having a relative abundance that indicates random substitution of deuterium for hydrogen . Quantitation of the absolute rate of cholesterol synthesis is obtained by isotope ratio mass spectrometry . Mevinolin and 26-hydroxycholesterol both decrease cholesterol synthesis rate but have a discordant effect on HMG-CoA reductase activity.

J Clin Microbiol, 1985 Aug, 22(2), 199 - 204
Isolation and differentiation of herpes simplex virus and Trichomonas vaginalis in cell culture; Gentry GA et al.; During the period January 1982 to January 1985, 2,234 specimens were cultured for isolation of herpes simplex virus (HSV) . HSV was isolated from 23% of these, Trichomonas vaginalis was isolated from 1.6%, and 75.3% were negative . In 0.2% of these, HSV and T . vaginalis were isolated from the same specimen . Cytopathic effects produced by HSV were identified by their sensitivity to arabinosylthymine, whereas those produced by T . vaginalis were identified by their lack of sensitivity to arabinosylthymine and by observation of motility . Cytopathic effects produced by T . vaginalis were reproduced by trophozoites from axenic cultures of T . vaginalis as well as by lysates of T . vaginalis added to serum-free BHK cells.

Endocrinology, 1985 Aug, 117(2), 488 - 91
Effects of adenosine analogs on glucagon-stimulated adenosine 3',5'-monophosphate formation in Sertoli cell cultures from immature rats; Eikvar L et al.; In the present study, we have examined the effects of two adenosine analogs, (-)N6-(R)phenyl-isopropyl-adenosine (PIA) and 2-chloro-adenosine, on glucagon- and FSH-stimulated cAMP production in Sertoli cell cultures isolated from immature (19-day-old) rats . Both FSH and glucagon caused a 5- to 10-fold stimulation of cAMP levels in the spent media from Sertoli cell cultures during an 18-h incubation . Addition of 1 microM PIA significantly inhibited both FSH- and glucagon-stimulated cAMP levels . In the presence of a maximal concentration of glucagon (2.5 micrograms/ml), PIA caused a concentration-dependent inhibition of cAMP formation, and the concentration of PIA causing half-maximal inhibition of cAMP formation (IC50) ranged from 0.5-1 nM . When Sertoli cells were incubated with increasing concentrations of glucagon (1.28 ng/ml to 4.00 micrograms/ml) in the absence and presence of either PIA (1.0 microM) or 2-chloro-adenosine (10.0 microM), the responses to glucagon, measured as cAMP formation, were almost completely abolished . 1-Methyl-3-isobutylxanthine (MIX), a well known inhibitor of cAMP phosphodiesterase activity, is also an inhibitor of adenosine binding to receptors on the cell membrane . When Sertoli cells stimulated with glucagon (2.5 micrograms/ml) were incubated in the absence and presence of MIX (0.1 mM) and increasing concentrations of PIA (0.025-10,000 nM), the presence of MIX reduced the inhibitory activity of PIA by almost 2 orders of magnitude (IC50 without MIX, 0.5 nM; IC50 with MIX, 20 nM) . Thus, the present study shows that adenosine analogs inhibit agonist-stimulated cAMP formation in cultured Sertoli cells, and that MIX reduces this effect . This indicates that cultured Sertoli cells from immature rats contain A1-receptors for adenosine mediating inhibitory effects on adenylate cyclase.

Exp Cell Res, 1985 Aug, 159(2), 313 - 22
Morphological and functional effects of extracellular matrix on pancreatic islet cell cultures; Thivolet CH et al.; Extracellular matrix (ECM) has been reported to enhance epithelial cell attachment and proliferation as well as to induce differentiation in vitro . In an attempt to determine the benefits of culturing pancreatic islet cells on ECM, we studied the morphological and functional patterns of rat islet cells and an insulin-secreting tumor cell line . ECM enhanced islet cell attachment and proliferation when compared to plastic, as suggested by a higher specific activity of DNA synthesis and a higher mitotic index . Cells on ECM were heterogeneous in size and insulin content . They showed extended areas of confluence . Cultures on plastic demonstrated an organisation in clusters and low mitotic activity . However, ECM did not allow for reconstitution of an islet-like structure . When compared to plastic, an initial decrease in basal and stimulated insulin secretion per million cells was observed on ECM, but B-cell activity was restored after 6 days of culture . Glucagon and somatostatin secretion were similar on both substrates . These data suggest that ECM enhances markedly islet cells attachment and proliferation, as well as long-term culture maintenance.

Brain Res, 1985 Jul 22, 339(1), 1 - 7
Effects of the synthetic glucocorticoid betamethasone on the survival of neurons in fetal rabbit brain cell culture; Kari B et al.; Dissociated fetal rabbit brain cells were grown on petri dishes coated with collagen . Culture medium consisted of Dulbecco's Modified Eagles Medium plus 10% serum . The mitotic inhibitor 1-beta-D-arabinofuranosylcytosine was added at 6 days for a 2 day period to inhibit over-growth by glial cells and fibroblasts . In some cases cultures were chronically exposed to 0.5, 1.0 or 2.0 microM betamethasone . Examination of cultures by phase microscopy and acetylcholinesterase (AChE) staining demonstrated that cultures incubated with 1.0 and 2.0 microM betamethasone contained 2-2.5 times as many neurons as compared to control cultures . Furthermore, there was an increase in the specific activities of both AChE and choline acetyltransferase (ChAT) which were proportional to the increase in neuronal cell numbers obtained from phase microscopy and AChE staining . These results suggested that betamethasone enhanced survival of cholinergic neurons . Cultures were also examined for neuron specific gamma-aminobutyric acid (GABA) uptake . Again GABA uptake was approximately 2-2.5 times as great in cultures incubated with 1-2 microM betamethasone when compared to controls . Thus, the increase in GABA uptake paralleled the increase in neurons observed by phase microscopy and AChE staining, suggesting that the survival effect of betamethasone was not specific to cholinergic neurons . While betamethasone treated cultures always contained greater numbers of neurons the percentage of neurons lost from all cultures after 2 weeks was the same.(ABSTRACT TRUNCATED AT 250 WORDS)

Biochem Biophys Res Commun, 1985 Jul 16, 130(1), 440 - 6
Extracellular matrix (ECM) synthesis in muscle cell cultures: quantitative and qualitative studies during myogenesis; Rao JS et al.; When the synthesis of extracellular matrix components was examined in G8-1 murine skeletal muscle cells as a function of differentiation, non-collagen and to an even greater extent collagen synthesis was increased . Specifically, collagen types I, III, IV, laminin and fibronectin were identified by SDS-PAGE . Immunoprecipitation, with specific antibodies revealed that both the cell layer and medium of differentiated multinucleated myotubes contained increased levels of type IV collagen and laminin, decreased levels of type III collagen and fibronectin and equivalent levels of type I collagen compared to mononuclear myoblasts.

Experientia, 1985 Jul 15, 41(7), 930 - 1
Inhibition of virus multiplication and alteration of cyclic AMP level in cell cultures by flavonoids; Mucsi I et al.; The inhibitory effect of four flavonoid compounds on virus multiplication and their influence on the intracellular cyclic AMP (cAMP) level were studied in cell cultures . Quercetin and quercitrin reduced the yields of Human (alpha) herpesvirus 1 (HSV-1) and Suid (alpha) herpesvirus 1 (pseudorabies virus), but hesperidin and rutin had no effect . Further, quercetin and quercitrin elevated the intracellular level of cAMP, whereas hesperidin and rutin did not alter the cAMP level . Both antiviral activity and cAMP-enhancing effect were dependent on the concentrations of the flavonoids, and these effects turned out to be parallel . This study suggests that a relation exists between the antiviral effect and the cAMP-enhancing activity of flavonoids.

Avian Dis, 1985 Jul-Sep, 29(3), 768 - 77
Field vaccination against hemorrhagic enteritis of turkeys by a cell-culture live-virus vaccine; Fadly AM et al.; A cell-culture-propagated (CC) live-virus hemorrhagic enteritis (HE) vaccine was evaluated for efficacy and safety in two field trials conducted in North Carolina (NC) and Minnesota (MN) . At 4 or 5 1/2 weeks of age, 9,839 poults in NC and 15,857 poults in MN were vaccinated with a CC HE vaccine administered via the drinking water . A comparable number of poults were maintained as unvaccinated controls . Vaccinated and unvaccinated poults were compared for seroconversion, response to laboratory challenge with a virulent HE virus at 3 weeks postvaccination, livability, percentage graded A, and average weight at marketing . In both trials, vaccination with the CC HE vaccine resulted in immunity against HE as indicated by seroconversion and by resistance to HE lesions following laboratory challenge with virulent HE virus . Compared with unvaccinated groups, vaccinated groups had a significantly higher percentage of turkeys graded A in the NC trial and in two of three flocks in the MN trial (P less than 0.005) . Further, in the NC trial, livability was significantly higher (P less than 0.005) in vaccinated turkeys than in unvaccinated turkeys . These data indicate that the CC HE vaccine is efficacious and safe to use in the field.

Tsitol Genet, 1985 Jul-Aug, 19(4), 250 - 4
{Mitotic cycle of a HeLa cell culture exposed to the maximum permissible concentrations of trace elements}; Strochkova LS et al.; It is shown that administration of certain trace elements in maximum allowable concentrations induces changes in metabolism and functionation of cells in the culture . Zinc, nickel, cobalt, cadmium and fluorine are stated to inhibit mitotic activity of HeLa cells by the end of 24 hours of their action . Parallel with this they promote a decrease in H3-thymidine incorporation into DNA . Besides these substances delay various stages of the mitotic cycle for cells.

Toxicol Appl Pharmacol, 1985 Jul, 79(3), 490 - 501
Studies on the toxicity of some glycol ethers and alkoxyacetic acids in primary testicular cell cultures; Gray TJ et al.; Primary mixed cultures of Sertoli and germ cells were prepared from testes of immature rats and their response to the known testicular toxicants ethylene glycol monomethyl ether (EGM) and ethylene glycol monoethyl ether (EGE) was studied . Neither EGM nor EGE produced any morphological evidence of toxicity when added to the culture medium at up to 50 mM for 72 hr . In contrast, their metabolites methoxyacetic acid (MAA) and ethoxyacetic acid (EAA) at 2 to 10 mM for 24 to 72 hr caused degeneration of the pachytene and dividing spermatocytes, the target cells of the parent ethers in vivo . As in vivo, earlier spermatocytes, spermatogonia, and Sertoli cells appeared unaffected . EAA was less potent than MAA whereas n-propoxy- and n-butoxyacetic acid, and methoxyacetylglycine, a further metabolite of MAA, produced no morphological changes under these conditions . The same order of toxicity was observed in concurrent studies with the four acids in rats . In culture, the severity of the morphological changes was paralleled by decreases in the activity of carnitine acetyltransferase and lactate dehydrogenase-X in the attached germ cell fraction . Analysis of culture medium provided no evidence for the conversion of EGM to MAA or other metabolites or for the further metabolism of MAA . The close correspondence between the testicular toxicity of alkoxyacetic acids in culture and in vivo suggests a similar mode of action in both cases and points to the potential value of these cultures for mechanistic studies and for screening purposes . The results also emphasize the role of metabolism in the testicular toxicity of glycol ethers and indicate that MAA is an active metabolite of EGM.

In Vitro Cell Dev Biol, 1985 Jul, 21(7), 368 - 72
Human smooth muscle cell cultures of the stomach . Morphologic and biochemical studies; Ionasescu R et al.; Smooth muscle cell cultures were prepared from stomach explants obtained surgically from 10 patients with duodenal ulcer . The cultured cells grew in either overlapping layers in "hills and valleys" or in parallel arrays . The ultrastructure studies showed plasmalemmal vesicles, bundles of myofilaments associated with dense bodies, and gap junctions . The synthesis of contractile proteins illustrated the preponderance of actin on myosin and tropomyosin . The synthesis of contractile proteins in stomach smooth muscle cell cultures is significantly higher than in skin fibroblast cultures, i.e . 20 X higher for myosin, 10 X higher for actin, and 30 X higher for tropomyosin.

Am J Trop Med Hyg, 1985 Jul, 34(4), 774 - 80
Continuous propagation of Ehrlichia sennetsu in murine macrophage cell cultures; Cole AI et al.; Ehrlichia sennetsu, the etiologic agent of human sennetsu rickettsiosis was successfully propagated in a continuous cell culture using murine cell lines P388D1 and Raw 264 . Pleomorphic cytoplasmic inclusion bodies similar to Ehrlichia canis morulae were observed 3-4 days after second post-inoculation split . In the Raw 264 cell line E . sennetsu was not seen until the third passage . Relatively heavier infection was observed in P388D1 than in Raw cell line . The latter reached a maximum of 15% infection whereas P388D1 cell line attained saturation . Structural details of the organism were confirmed by electron microscopy . A unique rippled cell mass surrounding the plasma membrane was observed . Supernatants of cultures were shown to contain infectious organisms . The advantages of propagating E . sennetsu in continuous cell lines are discussed with respect to future physiochemical and immunochemical studies of this organism.

Cancer Res, 1985 Jul, 45(7), 3322 - 31
Changes in stem cell populations of rat tracheal epithelial cell cultures at an early stage in neoplastic progression; Thomassen D et al.; The development of transformed colonies and concomitant changes in proliferative and nonproliferative cell compartments were studied in rat tracheal epithelial (RTE) cell cultures following exposure to N-methyl-N'-nitro-N-nitrosoguanidine (MNNG) . Primary RTE cells were plated onto 3T3 feeder layers and treated with MNNG (0.25 micrograms/ml) or solvent . Seven days later, the feeder cells were removed to select for enhanced growth variants, which are the transformants of the RTE cell system, usually scored 5 weeks after carcinogen exposure . Most of the RTE cell colonies, which originally formed during the first 7 days of culture, disappeared within 2 weeks after feeder cell removal in control and MNNG-treated cultures . In control cultures, about 3% of the original colonies persisted, while in MNNG-treated cultures, a larger percentage (approximately 9%) of the colonies persisted . These percentages remained constant from 3 to 7 weeks . Based on colony size, cell density, and cell morphology, the persistent colonies were classified into transformed colonies (large colony size, high cell density, high nuclear:cytoplasmic ratio) and untransformed colonies (small size, low cell density, low nuclear:cytoplasmic ratio) . In the MNNG-treated cultures, about 50% of all persistent colonies showed transformed morphology . Their frequency remained unchanged between 3 and 7 weeks of culture . In contrast, only 10 to 15% of the persistent colonies in control cultures showed transformed morphology at 3 weeks, but that proportion increased steadily between 3 and 7 weeks . These data suggest that, in control cultures, transformed colonies developed spontaneously as a function of time within untransformed colonies . Autoradiographic studies with {3H}thymidine showed that labeling indices in the early "normal" RTE cell colonies between Days 4 and 7 of culture were very high, ranging between 75 and 90% . In contrast, the labeling indices of persistent colonies, both those without and those with transformed morphology, were low, i.e., between 18 and 25%, indicating that a major proportion of cells was either noncycling or cycling very slowly . The relative compartment sizes of cells with stem cell characteristics and of cells with characteristics of transformed stem cells were estimated before and after transformed colonies appeared.(ABSTRACT TRUNCATED AT 400 WORDS)

Pediatr Neurol, 1985 Jul-Aug, 1(4), 232 - 7
Differential neurochemical effects of chronic exposure of cerebral cortical cell culture to valproic acid, diazepam, or ethosuximide; Sher PK et al.; We have assessed the relative neurochemical effects of valproic acid, ethosuximide, and diazepam on dissociated cultures of mouse cerebral cortex . Cultures were exposed chronically (11 days) to each antiepileptic drug and assayed for number of neurons, total protein, tetanus toxin fixation, high-affinity uptake of gamma-aminobutyric acid and beta-alanine, choline acetyltransferase activity, and specific and clonazepam-displaceable benzodiazepine binding . Ethosuximide-exposed cultures did not evidence neuronal toxicity; exposure to valproic acid and diazepam resulted in modest neuronal toxicity . However, exposure to each of these drugs resulted in a marked reduction in benzodiazepine binding . This effect may relate to a common mechanism of action of drugs used to treat absence seizures.

Endocrinologie, 1985 Jul-Sep, 23(3), 155 - 67
Reactivity of cold thyroid nodule under hormonal influences: study on cell cultures; Ghinea E et al.; The authors have studied the reactivity of cold thyroid nodule cells (CN) by comparison with the reactivity of normal thyroid cells (Ty), hot nodules cells (N), Graves' disease cells (G) and cells from the area proximal to cold nodules (TyC), under the influence of increased amounts of T3, TSH, STH, insulin, estradiol and KI present in the culture medium . The experiment lasted for 9-10 days . The study was carried out on monolayer cultures of cells obtained by tripsinization and on organocultures . Proteic synthesis, cytochemical and cytoenzymatic activities in the culture under the influence of the above substances were examined comparatively . After interruption of the treatment, the monolayer cultures were stained with Giemsa and examined by light microscopy . Proteic synthesis and release of Tg, T3 and T4 into the culture medium, were also examined . The organocultures were used in evaluating 125I uptake from the medium under the influence of the treatment . The results showed variation in relation to the tissue and hormone dose applied . It was especially found that TSH and estradiol cause enlargement of CN cell nuclei and an increased number of mitoses and polyploidia which might be premises for possible malignization.

Prostaglandins, 1985 Jul, 30(1), 109 - 18
Hypoxia enhances prostaglandin synthesis in renal mesangial cell cultures; Jelkmann W et al.; In view of recent findings which suggest that renal prostaglandins mediate the effect of hypoxia on erythropoietin production, we have studied whether hypoxia is a stimulus for in vitro prostaglandin synthesis . Studies were carried out in rat renal mesangial cell cultures which produce erythropoietin in an oxygen-dependent manner . Production rates of PGE2 and in specified samples also of 6-keto-PGF1 alpha, as a measure of PGI2, and PGF2 alpha were determined by radioimmunoassay after incubation at either 20% O2 (normoxic) or 2% O2 (hypoxic) in gas permeable dishes for 24 hrs . Considerable variation in PGE2 production was noted among independent cell lines . PGE2 production appeared to be inversely correlated to the cellular density of the cultures . In addition, PGE2 production was enhanced in hypoxic cell cultures . The mean increase was 50 to 60% . PGF2 alpha and 6-keto-PGF1 alpha increased by about the same rate . These results indicate that hypoxia is a stimulus for in vitro prostaglandin production.

Cell Biochem Funct, 1985 Jul, 3(3), 179 - 84
Human bone cell cultures: a new model for studying the mechanism of action of calcitonin; Fano G et al.; An investigation on cell cultures obtained from temporal human bone fragments showed that they provide a suitable model for studying the mechanism involved in calcitonin action on bone cells . Furthermore they demonstrated: a transitory increase in 45Ca uptake that returned to control values ten minutes after the hormone was added; a relation between 45Ca uptake and increased cAMP concentrations when these were measured at the same time intervals; a reproduction of the salmon calcitonin (sCT) effect after incubation of the cultures with either db-cAMP or db-cGMP and inhibition of 45Ca uptake and parallel decrease in cAMP levels with propanol . These results suggest that in human bone cell cultures, sCT acts as a temporary promoter of 45Ca uptake, probably by activating an adenylate-cyclase system through a beta-receptor.

Avian Dis, 1985 Jul-Sep, 29(3), 672 - 80
Cell-culture virus-neutralization test and enzyme-linked immunosorbent assay for evaluation of immunity in chickens against fowlpox; Buscaglia C et al.; Two serological tests--the virus-neutralization (VN) test in chicken embryo fibroblasts (CEF) using a cell-culture-adapted virus, and the enzyme-linked immunosorbent assay (ELISA)--were used for evaluating the immune response in chickens against fowlpox virus . The VN test was conducted in 96-well tissue-culture plates using a fowlpox virus that was adapted to induce cytopathic effects (CPE) in CEF in 48 hr . The ELISA was carried out with an antigen prepared by precipitation of a cell-culture-propagated virus suspension with ammonium sulfate and concentration by centrifugation . A 0.1 M acetate buffer, pH 5, was used as the sensitizing solution for maximum specific binding of the antigen to the microplate plastic well . No antibodies were detected by the VN test in 228 serum samples taken from chickens at irregular intervals between 1 and 39 weeks of age, even though the birds were vaccinated against fowlpox at 13 weeks of age . However, in sera collected 4 weeks after a sample of laying hens was challenged with fowlpox virus, VN titers of 1/10 to 1/40 were detectable . On the other hand, significant antibody reactions were detected by the ELISA on sera from chickens during the growing period, following vaccination and challenge . Although no maternal antibodies were found at 1 week of age, a continuous increase in the mean ELISA titers to fowlpox was demonstrated during the entire experimental period . This study showed that the ELISA was considerably more sensitive and practical than the VN test.

Mol Cell Endocrinol, 1985 Jul, 41(2-3), 129 - 36
Electrofocusing fractionation of follicle stimulating hormone in pituitary cell culture extracts from male and female rats; Foulds LM et al.; Three-day pituitary cell cultures from adult male and female rats were incubated for 4 h in the presence of 10 nM LHRH and the molecular heterogeneity of FSH was assessed in the media of LHRH-stimulated cells and in cell extracts from unstimulated cells using an electrofocusing technique . The pI distribution of FSH showed a high degree of similarity between cell media and cell extracts of each sex although differences were observed between sexes . Pituitary cell cultures from male rats were also incubated in the presence of 10(-8) M testosterone and 10(-8) M estradiol and the pI distribution of FSH from media after LHRH stimulation was determined . No significant differences in the pI profiles were observed . Incubation with charcoal-treated bovine follicular fluid (an inhibin source) resulted in a significant reduction in recovered FSH activity in the pH region 3.61-3.92 although this decrease did not markedly alter the pI profile of FSH . Close similarities were observed in the pI distribution of FSH of pituitary cell culture extracts and pituitary gland extracts from intact animals of both sexes, however, differences in pI distribution were noted in pituitary extracts in the male but not the female following gonadectomy . It is concluded that (1) stored FSH is released from the pituitary without major modification to its structure as assessed from its pI profile, (2) sex differences in the pI profile of FSH in pituitary extracts are retained in culture and following LHRH-stimulated release, (3) the pI distribution of FSH is not affected by testosterone or estradiol and only minimally by inhibin in short-term cultures.(ABSTRACT TRUNCATED AT 250 WORDS)

Acta Virol, 1985 Jul, 29(4), 312 - 7
Ecology of the murine alphaherpesvirus and its isolation from lungs of rodents in cell culture; Mistrikova J et al.; Together 381 sera of murine rodents (Apodemus flavicollis, Clethrionomys glareolus, Microtus arvalis and Mus musculus) trapped in different localities of Czechoslovakia were examined for the presence of antibodies to a murine alphaherpesvirus (MHV) and to murine cytomegalovirus (CMV) . Positivity of rodent sera to the two MHV and one murine CMV strains varied from none to 12.5% and from none up to 11.3%, respectively, depending on the locality under study . From the lungs of A . flavicollis showing serum antibodies to MHV, a further murine herpes virus strain was isolated in rabbit embryo fibroblasts (REF) . The latter MHV reached a titre of 10(10) TCID50/ml in the 13th passage, causing typical cytopathic effect from the 2nd passage on.

Biosci Rep, 1985 Jul, 5(7), 601 - 8
Developmental study on the regulation of neurotransmitter-sensitive adenylate cyclase systems in primary cerebral cell cultures from embryonic mice; Shanker G et al.; An ontogenetic study of the effect of various neurohormones and other activators on adenylate cyclase systems was carried out using cultures of cells from 15-d-old embryonic mouse brain . Dopamine stimulated the enzyme activity at earlier culture ages (i.e . 4 and 10 d) but had little stimulatory effect at later ages (i.e . 20 and 33 d) . Further, this stimulation at the earlier ages was blocked by the dopaminergic blocker, fluphenazine, but not by alpha and beta-adrenergic antagonists . In contrast to dopamine, isoproterenol (a beta-adrenergic agonist) had little stimulatory effect at earlier ages, but its ability to stimulate cyclase activity increased with age . This increase in all age groups was blocked by propranolol (a beta-adrenergic antagonist) . Epinephrine-sensitive enzyme activity showed a steady increase with age, which could be blocked with propranolol except in 4-d-old cultures, where it was blocked instead by fluphenazine . Because the cultures are relatively enriched in neurons at earlier ages and in glia in later ages, the results suggest a predominantly neuronal localization for the dopamine sensitive adenylate cyclases and a glial localization of the isoproterenol and epinephrine sensitive adenylate cyclases . Histamine, serotonin, calcium/calmodulin and chloroadenosine were either only slightly or not at all stimulatory.

Am J Pathol, 1985 Jul, 120(1), 67 - 78
Characterization of a primary bile ductular cell culture from the livers of rats during extrahepatic cholestasis; Sirica AE et al.; The establishment of novel bile ductular cell cultures was accomplished with the use of explants of a hyperplastic bile ductular tissue preparation obtained from rat livers at 10 to 15 weeks after bile duct ligation or a bile ductular cell fraction isolated from this tissue preparation by a procedure involving Percoll density gradient centrifugation . Observations made on these primary explant and monolayer bile ductular cell cultures were limited to the first 3 days of culture where the morphologic features of the bile ductular epithelium remained fairly well preserved, while fibroblast contamination was found to be very low . These cultured cells also retained over this period a high specific activity for the bile ductular cell marker enzyme gamma-glutamyl transpeptidase, as well as possessed measurable but decreasing specific activities for leucine aminopeptidase and alkaline phosphatase . Karyotypic analysis of the cultured monolayer cells further showed them to be diploid . In addition, preliminary transplantation studies demonstrated the presence of well-differentiated bile ductular-like structures following inoculation of the freshly isolated bile ductular cell fraction into the interscapular fat pads of recipient rats.

J Biomed Mater Res, 1985 Jul-Aug, 19(6), 653 - 61
A modification of the cell culture agar diffusion test using fluoresceindiacetate staining; Schmalz G et al.; In order to reduce the uncertainties involved in the morphologic evaluation of the cell culture agar diffusion test, the originally recommended neutral red stain was replaced by fluoresceindiacetate (FDA) . This stain proved to be nontoxic in the concentrations used in this study (0.002%) . Toxicity tests with phenol, formalin, and methylmethacrylate monomer revealed results corresponding to literature evaluations by other methods . Since FDA makes cell metabolism visible and easily accessible to automated evaluation techniques, it presents certain advantages over the morphologic evaluation using the neutral red stain.

Diagn Microbiol Infect Dis, 1985 Jul, 3(4), 353 - 8
Immunoenzymatic staining of viral and chlamydial antigens in cell culture; Richman DD et al.; Methods are described for the conjugation of antibodies with biotin and for the use of these reagents in an immunoperoxidase staining procedure for infected cell cultures . This technique provides a simple, rapid, and specific approach to the identification and characterization of a number of viral and chlamydial isolates in the diagnostic laboratory.

J Cardiovasc Pharmacol, 1985 Jul-Aug, 7(4), 799 - 804
Comparative metabolic effects of halothane and enflurane in rat heart cell culture; Albrecht RF et al.; The effects of halothane and enflurane on the oxygen consumption rates and substrate utilization by beating and nonbeating rat heart myocytes in cell culture were compared . Halothane, on an equal dose and equal MAC (minimum alveolar concentration producing immobilization of 50% of subjects) basis, was significantly more effective than enflurane in reducing total myocyte oxygen consumption and contractile rate . The greater effect of halothane on oxygen consumption was not due entirely to its effect on myocyte contractile rate, since quiescent (nonbeating) cells and cells rendered nonbeating by large doses of halothane also showed greater reductions in oxygen consumption than with large doses of enflurane . Both halothane and enflurane reduced glucose and palmitic acid metabolism by myocytes when compared with controls . However, there were no significant differences between halothane or enflurane with regard to glucose metabolism . Halothane was significantly more effective than enflurane in reducing cellular palmitic acid metabolism . Although palmitic acid uptake by myocytes was reduced to the same extent by both anesthetics when compared with control uptake values, halothane reduced myocyte uptake of glucose to a greater degree than enflurane . The results of this study indicate that halothane is a more potent myocardial metabolic depressant than enflurane.

Diabetes, 1985 Jul, 34(7), 696 - 702
Neonatal rat islet cell cultures synthesize insulin-like growth factor I; Romanus JA et al.; Monolayer cultures of islet B-cells were established from neonatal rat pancreas . Serum-free media conditioned by these cultures for 72 h were concentrated and fractionated on Sephadex G-50 at acid pH into a high-molecular-weight pool containing binding protein for insulin-like growth factors (IGFs) and a low-molecular-weight pool containing IGFs . IGF activity in the IGF pool was demonstrated by a specific radioreceptor assay using rat liver plasma membranes and 125I-labeled rat IGF-II . The IGF in islet cell media was characterized further by radioimmunoassays specific for human IGF-I and for rat IGF-II . Islet cell IGF was identified as predominantly IGF-I or a closely related species and not IGF-II . Levels of approximately 15-50 ng IGF-I (based on human IGF-I standard)/10(6) islet cells accumulated in media after 72 h, and presumably represented synthesis by the islet cells . Concentrations of IGF-I attained in culture media, approximately 0.1 ng/ml, were sufficient to stimulate {3H}thymidine incorporation into B-cells . Growth hormone did not consistently increase IGF-I synthesis, suggesting that the previously described effects of growth hormone on islet cell replication do not result from stimulation of IGF-I synthesis by islet cells . Thus, although the IGF-I synthesized by islet cells may be a physiologically relevant growth factor for these cells, the mitogenic effects of growth hormone in islet cells appear to be independent and not mediated by IGF-I.

Brain Res, 1985 Jun 24, 337(1), 138 - 41
Establishment of Muller cell cultures from adult rat retina; Sarthy PV; During a recent study of the effects of photoreceptor degeneration on Muller cells, we observed that unlike glia from normal adult retinas, glia from degenerating rat retinas could be cultured and maintained in vitro for several weeks . Using a variety of cell type specific antisera, we show that at least 70% of cells in these cultures are derived from Muller cells.

Brain Res, 1985 Jun 10, 336(1), 67 - 72
Vasoactive intestinal peptide and PHI stimulate somatostatin release from rat cerebral cortical and diencephalic cells in dispersed cell culture; Tapia-Arancibia L et al.; To determine the effect of vasoactive intestinal peptide (VIP) on the secretion of somatostatin by neurons, dispersed fetal cerebral cortical and diencephalic cells grown in culture were exposed on day 10 or 11 of culture to various concentrations of VIP, and for comparison to the structurally related peptides PHI (Peptide Histidine Isoleucine-27), growth hormone (GRH1-44-NH2) and secretin and to cholecystokinin . VIP elicited a dose-dependent release of somatostatin from both cortical and diencephalic cells, the lowest effective concentration being 6 X 10(-9) M . PHI also brought about release of somatostatin, but was between 0.06 and 0.1 times as potent as VIP . Placed together in a concentration of 10(-7) M, the two peptides did not have an additive effect . In this system GRH1-44-NH2, secretin and CCK octapeptide were without effect.

J Anim Sci, 1985 Jun, 60(6), 1553 - 61
Adipocyte development in primary rat cell cultures, effect of cell density and serum source; Hausman GJ et al.; The development of adipocytes was studied in primary cultures of rat adipose tissue stromal-vascular cells on collagen-coated glass coverslips . The effects of cell density and serum source on lipoprotein lipase (LPL), esterase and lipid histochemistry were evaluated . With a mixture of fetal calf serum (FCS; 2%), horse serum (2%) and pig serum (PS; 10%), large and loosely arranged clusters of adipocytes developed with time through an increase in cell number and size . An inverse relationship was observed between cell size and the number of adipocytes in a cluster . Lower cell densities were associated with large cells and the densest areas contained smaller cells . Unilocular adipocytes were observed by d 13 after plating and were generally absent from the densest area of the coverslips . Histochemically detectable LPL activity was demonstrable before lipid deposition in adipocyte clusters . A comparison of FCS (10%) and PS (10%) as the only serum sources indicated higher level of adipocyte esterase activity and lipid deposition in PS cultures . Cultures of cells from weanling (21 to 28 d old) and old (18 mo) rats were similar, whereas cells from younger rats (2 to 4 d old) formed denser cultures that contained fewer adipocytes . These adipocytes were small (less than 30 micron) and morphologically homogeneous (all multilocular) . When cells from the very young rats (2 to 4 d old) were plated at low densities, an inverse relationship between cell size and number of cells in a cluster was also observed.(ABSTRACT TRUNCATED AT 250 WORDS)

Cell Differ, 1985 Jun, 16(4), 251 - 7
Oculopotency of embryonic quail pineals as revealed by cell culture studies; Watanabe K et al.; Pineal bodies from 8-day-old quail embryos were dissociated and cultured in order to examine their potency for differentiation under in vitro conditions . Polygonal pigment cells and lentoid bodies started to differentiate after about 2 and 4 weeks, respectively . Lentoid bodies were shown immunologically to contain all classes of crystallins . The results indicate that embryonic pineal cells of avian species retain 'oculopotency' to differentiate into several types of ocular cells.

Dev Biol, 1985 Jun, 109(2), 274 - 87
Identification of early neuronal subpopulations in avian neural crest cell cultures; Girdlestone J et al.; Gangliosides were employed as early differentiation markers to investigate how phenotypic diversity is generated in the vertebrate neutral crest cell population . Chromatographic analysis of metabolically labeled glycolipids from neural crest derivatives revealed that glia cell precursors synthesize a characteristic subset of the ganglioside types produced by dorsal root ganglion neurons . The ganglioside synthesis pattern of neural crest cultures is similar to that of glial precursors, but gangliosides characteristic of neurons are also detectable . To determine whether neuronal gangliosides are expressed by every cell in neural crest cultures, or by discrete cell subpopulations, crest cultures were stained immunocytochemically with monoclonal antibody A2B5 which recognizes a neuron-specific ganglioside in the GQ fraction . A2B5 was found to bind to about 1% of migratory stage neural crest cells isolated from neural tube explants after 1 day in culture . These A2B5+ cells were postmitotic and exhibited a uni- or bipolar neuronal morphology . A second, larger (10-20%) population of A2B5+ cells appears after culturing cells from 1-day crest cell clusters an additional 1-2 days . These cells initially have the typical small, stellate morphology of crest cells but later extend one or more processes . {3H}Thymidine incorporation and cell counting studies show that the precursors to these cells had divided at least once in culture before becoming postmitotic and expressing A2B5 immunoreactivity . The second A2B5+ population does not appear in secondary cultures of crest cell clusters isolated from 2-day-old explants of neural tubes . Another monoclonal antibody, R24, which recognizes ganglioside GD3, binds to subpopulations of both neurons and nonneurons in sensory ganglion cultures . In neural crest cultures R24 binds to a large subpopulation of cells, but not to A2B5+ ones . The significance of this immunostaining pattern is not yet understood . The early appearance of subpopulations, and the presence of heterogeneity in neural crest cultured under a variety of conditions suggest that intrinsic cellular mechanisms might generate subpopulations within the neural crest upon which environmental factors act.

Cell Biol Toxicol, 1985 Jun, 1(3), 133 - 43
Mycotoxin induction of somatic mosaicism in Drosophila and DNA repair in mammalian liver cell cultures; Belitsky GA et al.; The genotoxic activity of four mycotoxins has been studied . A high level of somatic mutagenesis in imaginal disk of Drosophila melanogaster larvae and DNA repair synthesis in human embryo and adult rat liver cells cultures was induced only by the strong carcinogen aflatoxin B1 . Patulin somewhat elevated the level of somatic mutations in D . melanogaster, but did not elicit DNA repair synthesis . Citrinin and stachybotryotoxin were inactive in both systems.

Exp Cell Res, 1985 Jun, 158(2), 321 - 32
Spatial interrelationships between proteoglycans and extracellular matrix proteins in cell cultures; Avnur Z et al.; Immunolabelling of cultured cells for chondroitin-sulfate proteoglycan (CSPG), in conjunction with antibodies to fibronectin, collagen and laminin, revealed the spatial interrelationships between the different matrix components . CSPG was organized in two major forms . Fibronectin-independent dotted patterns of CSPG were detected on the substrate and cell surfaces at early stages after plating . At later stages, however, significant overlapping was found between the two extracellular matrix components . Immunoelectron microscopic examination indicated that the CSPG was organized as granules of varying sizes which were associated with the cell surface, the substrate, or with the periphery of the fibronectin network.

Brain Res, 1985 Jun, 352(2), 235 - 9
Quail neural crest cells transformed by Rous sarcoma virus can be established into differentiating permanent cell cultures; Pessac B et al.; Quail neural crest cells derived from the truncal neural primordium, infected in vitro by Rous sarcoma virus (RSV) in January 1978, were induced to multiply and have been established into permanent cultures . These cultures contain cells that differentiate into melanocytes, neuron-like cells and flat cells . About 50% of these different cell types are tetanus-toxin positive . Electrophysiological studies have shown that some cells can generate action potentials similar to those reported in quail neural crest primary cultures . Taken together these data show that the RSV-transformed quail neural crest permanent cultures are composed of stem cells which can differentiate into cell types specific for neural crest.

Environ Res, 1985 Jun, 37(1), 228 - 38
Inhibition of viral interferon induction in mammalian cell cultures by azo dyes and derivatives activated with rat liver S9 fraction; Hahon N; Four azo dyes (Benzopurpurine 4B, trypan blue, Direct Blue 15, and Congo red) and their derivatives (o-tolidine, o-dianisidine, and benzidine) were studied for their effect on induction of interferon by influenza virus in mammalian, Rhesus monkey kidney (LLC-MK2), cell monolayers . Whereas Benzopurpurine 4B, Direct Blue 15, and Congo red inhibited viral interferon induction from approximately 35 to 60%, negligible inhibition was noted with trypan blue and other derivative compounds . By comparison, when rat S9 fraction was used for enzymatic activation of azo dyes and derivatives, interferon inhibition was significantly depressed moreover (P less than 0.01 to less than 0.0001) by all chemical compounds . The concentration of rat S9 (0.5%) used for metabolic activation of dyes and congeners was critical because concentrations greater than 0.5% of S9 per se inhibited the interferon induction process . Uninduced hamster S9 and both Aroclor 1254-induced hamster and rat S9 fractions were all comparable in their ability to activate the chemical compounds tested . That potential mutagenic and carcinogenic chemicals which require metabolic activation can be discriminated on the basis of interferon induction inhibition in eukaryotic cell cultures augurs for the usefulness and credibility of this system.

J Virol Methods, 1985 Jun, 11(2), 93 - 103
A double labeling technique for performing immunocytochemistry and in situ hybridization in virus infected cell cultures and tissues; Gendelman HE et al.; This report describes a combined immunocytochemical and in situ hybridization procedure which allows visualization of cellular or viral antigens and viral RNA in the same cell . Cultures infected with visna or measles virus were fixed in periodate-lysine-paraformaldehyde-glutaraldehyde, stained by the avidin-biotin-peroxidase technique using antibodies to viral or cellular proteins and then incubated with radiolabeled specific DNA probes (in situ hybridization) . The immunoperoxidase stain was preserved through the hybridization procedure . Nonspecific 'sticking' of probes over peroxidase stained cells was prevented by incorporation of 0.1% Triton X-100 into the hybridization solution and the post-hybridization washes . The in situ hybridization signal (silver grains/cell) on peroxidase-stained cells was reduced relative to hybridization with unstained cells . The double labeling technique was also applied to sections of paraffin-embedded tissues from a sheep infected with visna virus and mice infected with the HNT strain of measles virus . Visna virus RNA was detected in immunocytochemically identified macrophages in the synovium . A greater number of these cells had viral RNA than had viral protein . In measles virus-infected brains viral RNA was detected only in cells with viral protein . This technique provides a new approach to the study of viral pathogenesis by: identifying the types of cells which are infected in the host and identifying points of blockade in the virus life cycle during persistent infections.

Biochim Biophys Acta, 1985 May 17, 834(3), 336 - 45
Putative role of cholesteryl ester transfer protein in removal of cholesteryl ester from vascular interstitium, studied in a model system in cell culture; Stein Y et al.; A model system to study the putative role of cholesteryl ester transfer protein in the egress of interstitial cholesteryl ester is described . Confluent cultures of bovine aortic smooth muscle cells were labeled for 24 h with {3H}cholesteryl linoleyl ether and {14C}cholesteryl linoleate by incubation with bovine milk lipoprotein lipase . This method of labeling results in the transfer of cholesteryl linoleyl ether and cholesteryl ester to three compartments: a trypsin-releasable, trypsin-resistant and catabolic compartment (Stein, O., Halperin, G., Leitersdorf, E., Olivecrona, T . and Stein, Y . (1984) Biochim . Biophys . Acta 795, 47-59) . The efflux of labeled cholesteryl linoleyl ether and cholesteryl ester from the extracellular and cell-surface related compartments into a serum-free culture medium containing 1% bovine serum albumin was studied during 24 h of postincubation . The efflux was expressed as a percentage of pulse value, i.e., radioactivity retained by the cell culture at the end of the labeling period . The efflux of {3H}cholesteryl linoleyl ether, {14C}cholesteryl ester and 14C-labeled free cholesterol (formed by cellular hydrolysis of cholesterol ester) into the culture medium with 1% bovine serum albumin was about 5% of the pulse value . Addition of human lipoprotein-deficient serum resulted in a 3-10-fold increase in the efflux of {3H}cholesteryl linoleyl ether and {14C}cholesteryl ester, but did not change markedly the efflux of 14C-labeled free cholesterol . Rat lipoprotein-deficient serum which does not contain cholesteryl ester transfer protein did not increase the efflux of {3H}cholesteryl linoleyl ether or {14C}cholesteryl ester . The rate of cholesteryl ester efflux in the presence of human lipoprotein-deficient serum was linear for about 6 h and increased further up to 24 h . Addition of Intralipid to medium containing human lipoprotein-deficient serum further enhanced the efflux of {3H}cholesteryl linoleyl ether and, to a lesser extent, that of cholesteryl ester . A similar effect was observed also by addition of rat VLDL to medium containing human lipoprotein-deficient serum . Inhibition of cholesteryl linoleyl ether and cholesteryl ester efflux and marked enhancement of free cholesterol efflux occurred when rat HDL was added to medium containing human lipoprotein-deficient serum, while human HDL was only slightly inhibitory . The results obtained with human lipoprotein-deficient serum were reproduced with partially purified cholesteryl ester transfer protein . Using the partially purified cholesteryl ester transfer protein, the efflux of cholesteryl linoleate was compared to that of cholesteryl oleate and was found to be the same.

Endocrinology, 1985 May, 116(5), 1835 - 44
Estrogen responsiveness of normal mouse mammary cells in primary cell culture: association of mammary fibroblasts with estrogenic regulation of progesterone receptors; Haslam SZ et al.; Estrogens enhance proliferation of normal mouse mammary cells in vivo . However, when cultured alone, normal mouse mammary epithelial cells fail to exhibit a proliferative response to estrogen in vitro; the basis for this lack of in vitro responsiveness to estrogen is not known . The purpose of the present study is to determine if cultured normal mouse mammary cells possess estrogen receptors (ER) and/or progesterone receptors (PgR) and if the ER mechanism is functional, as measured by the ability of estrogens to regulate PgR . Recent findings that mammary fibroblasts can influence the behavior of mammary epithelial cells in vitro led us to investigate their effect on epithelial cell responsiveness to estrogen . In these studies, collagenase-dissociated mammary glands of midpregnant BALB/c mice were the source of mixed cultures (containing both epithelial cells and fibroblasts) and epithelial or fibroblast cultures . The purity of epithelial or fibroblast cultures was quantified immunocytochemically using antivimentin antibody as a fibroblast marker . Steroid hormone binding was quantified in intact cultured cells using {3H}R5020 and 17 beta-{3H}estradiol as the ligands . Specific high affinity binding sites for estrogen (Kd = 3.1 +/- 0.8 X 10(-10} and progestins (Kd = 3.3 +/- 1.2 X 10(-9) M) were detected in mixed cultures . To assess the possible role of mammary fibroblasts, we investigated cultures containing only fibroblasts which were derived by differential centrifugation . When 17 beta-estradiol was added to the culture medium, a significant (P less than 0.01) increase in PgR concentration was observed in mixed cultures . While mixed cultures maintain responsiveness to estrogen in vitro, as measured herein, the epithelial cultures, derived by differential centrifugation and Percoll gradient sedimentation, did not . However, estrogenic regulation of PgR appears to be specific to epithelial cells in mixed cultures, since fibroblast cultures neither contained PgR nor displayed estrogen-inducible PgR . The lack of responsiveness of epithelial cultures is not due to a loss or decrease in the ER concentration . Thus, the presence of mammary fibroblasts appears to be associated with epithelial cell responsiveness to estrogen in vitro.

Cancer, 1985 May 1, 55(9), 1918 - 23
Production of erythropoietin-like activity by human renal and hepatic carcinomas in cell culture; Okabe T et al.; Two types of human cancers, a renal cell carcinoma and a hepatocellular carcinoma, were investigated in vitro; both produced a marked erythrocytosis in each patient . These tumors, when transplanted into athymic nude mice, produced a remarkable erythrocytosis in the host mice . To analyze this phenomenon, the primary cultures from these xenotransplanted tumors were performed . To obtain pure tumor cell cultures, cells derived from host nude mice were eliminated by the treatment with the antiserum raised against nude mouse cells . Epithelial cells derived from each tumor attached and grew in the cultures . The conditioned media from both tumor cells revealed high erythropoietic stimulatory activities . We have characterized these erythropoietin-like activities by size-exclusion high-performance liquid chromatography . Three peaks of erythropoietin-like activities were noted after bovine serum albumin region . The molecular weights were estimated at about 55,000, 40,000, and 33,000, respectively . The results suggested that the human renal cell and hepatocellular carcinomas produced erythropoietin-like activities in vitro in culture and that erythrocytosis found in patients with cancer and in nude mice transplanted with the tumors was attributable to production of the erythropoietin-like activities by the tumor cells themselves.

Zentralbl Bakteriol Mikrobiol Hyg {B}, 1985 May, 180(5-6), 480 - 504
{Use of fish cell cultures for toxicity determination in order to reduce and replace the fish tests}; Ahne W; Dispersion of liver tissue from rainbow trout at 10 and 25 degrees C resultet in cells which were cultivated in vitro at 20 degrees C regularly . Using this method a cell line (R1) has been established useful for testing the toxicity of chemical substances and waste water . The toxicity of samples influenced the morphology, growth and the living duration of the cells . These parameters gave a clear answer about the toxicity of samples testet in 1-12 h . The tests on the vitality of cells (using trypan blue) and the release of LDH did not show an agreement with the cytotoxicity in all cases . From 106 samples of waste water were 53.8% cytotoxic but only 35% showed a toxicity for fish . 93.44% of 61 toxic samples of waste water showed cytotoxicity, only 60.65% of the samples were fish-toxic . According to the results obtained it is concluded that fish cell tissue cultures are useful tools for determination of the toxicity of chemical substances and waste water . Compared with the fish test the cytotoxicity test ist more sensitive and it reduces the material, time and money needed.

Biull Eksp Biol Med, 1985 May, 99(5), 565 - 8
{Changes in the protein-synthesizing system of a HeLa cell culture exposed to a number of trace elements}; Strochkova LS et al.; The authors investigated the cytopathic action of maximal allowable concentrations (MAC) of zinc, nickel, cobalt, cadmium and fluorine on cell culture . The most significant changes in RNA synthesis were noticed after exposure to zinc MAC . After exposure to the MAC of zinc, nickel and fluorine considerable modifications of protein synthesis were recorded . It was established that the content of rRNA increased after incubation with all trace elements under study for 24 hours . The amount of protein experienced significant changes after nickel introduction into the incubation medium . It is concluded that exposure to some of trace elements entails considerable changes in cell metabolism.

Antimicrob Agents Chemother, 1985 May, 27(5), 802 - 5
Determination of inhibitory concentrations of antiviral agents in cell culture by use of an enzyme immunoassay with virus-specific, peroxidase-labeled monoclonal antibodies; van Tiel FH et al.; An enzyme immunoassay (EIA) to determine 50% inhibitory concentrations of drugs which suppress Semliki Forest virus replication is described . Inhibition of virus replication was measured in L-cells, seeded as monolayers in 96-well plates by use of horseradish peroxidase-labeled monoclonal antibodies directed against the E1 glycoprotein of Semliki Forest virus . The antiviral agents tested were cycloheximide, tunicamycin, NH4Cl, and disodium cromoglycate . The 50% inhibitory concentration of these antiviral agents was arbitrarily defined as the concentration of drug, in culture medium, associated with 50% reduction of the control absorbance value measured on Semliki Forest virus-infected cells without drug in the culture fluid . Twenty-two hours after infection the 50% inhibitory concentrations of the drugs were 0.2 microgram/ml for cycloheximide, 0.8 microgram/ml for tunicamycin, 0.3 mg/ml for NH4Cl, and 4.9 mg/ml for disodium cromoglycate . These values are similar to those determined by others with conventional methods of virus quantification . This test is sensitive and easy to perform and therefore is suited for large-scale experiments.

Brain Res Bull, 1985 May, 14(5), 415 - 21
Light and electron microscopic analysis of insulin binding sites on neurons in dissociated brain cell cultures; Weyhenmeyer JA et al.; The distribution of insulin binding sites on primary cultured neurons and glia from the fetal rat was examined by the immunoperoxidase method using a specific insulin receptor antiserum . Light and electron microscopic analysis revealed a homogenous distribution of insulin binding sites on selective neuron-like cells of the dissociated cell culture system . To determine the influence of medium insulin on the distribution of insulin binding sites, dissociated cell cultures were maintained in the presence or absence of porcine insulin for varying time periods . We observed a significant increase in the number of insulin stained neuron-like cells maintained in insulin free defined medium compared to neuron-like cells maintained in insulin supplemented defined medium . Further, we examined the distribution of insulin binding sites after incubation with the antibody, which has agonistic properties in peripheral tissues, for varying time periods prior to fixation . Under these conditions, the light microscopic analysis revealed a heterogeneous (patchy) distribution of immunoreactive insulin binding sites, suggesting that the ligand receptor complex migrates . These results demonstrate the presence and distribution of insulin binding sites on neurons maintained in vitro, and provide morphological evidence to support a functional role for insulin in CNS tissues.

Exp Cell Res, 1985 May, 158(1), 177 - 91
Monoclonal antibodies to intermediate filaments in chick muscle cell cultures; Morris GE et al.; Two monoclonal antibodies, FIFI and PHIL, have been prepared using detergent-washed myogenic cells as immunogen . On Western blots of total protein extracts of muscle cells, both antibodies bind to vimentin (52 kD) and its degradation products (major band at 42 kD), but do not bind to mouse proteins or to actin (42 kD) . Specificity for a determinant common to vimentin and desmin was confirmed by 2-D gel electrophoresis of muscle cell extracts and purified desmin . Western blots with FIFI reveal particularly well the extreme sensitivity of intermediate filaments (IFs) to proteolysis, which was preventable in brain tissue only by boiling in 1% SDS, although it could be reduced in both brain and muscle by less extreme methods . Western blots suggest a large increase in IF content of differentiating myoblast cell cultures at the time of cell fusion and an increase of at least 4-fold is confirmed by a quantitative immunoassay using a direct ELISA method . Immunofluorescence microscopy shows that this increase is due to the appearance of high concentrations of the intermediate filament antigen at the ends of early myotubes, preceding the appearance of cross-striations in myofibrils . Furthermore, whereas the polar filaments detected by FIFI run right to the ends of the early myotubes and only sparingly penetrate the central area, cross-striated myofibrils (as detected by the monoclonal antibody, SAM) run the length of the myotube but do not reach the ends . Colcemid and colchicine cause the vimentin filaments in fibroblasts to collapse into perinuclear rings or caps, but do not have this effect on the polar fluorescence in early myotubes . Heat shock (2 h at 45 degrees C) has a similar differential effect . The results suggest that early in muscle differentiation intermediate filament proteins accumulate rapidly at myotube ends, where they are organized differently from those in fibroblasts.

Dev Biol, 1985 May, 109(1), 184 - 92
Differentiation of primary embryonic neuroblasts in purified neural cell cultures from Drosophila; Furst A et al.; Embryonic neuroblasts of Drosophila are undifferentiated precursor cells that give rise to the central nervous system . Centrifugal elutriation has been employed to fractionate embryonic cells on the basis of size . A fraction of large cells was found to be greatly enriched for neuroblasts, whereas mesodermal precursor cells were completely excluded . This allowed a second step of purification, based upon adhesion to glass, to provide virtually pure cultures of neural cells . The cells in these cultures had the properties of neurons of the Drosophila CNS: They gave rise to ganglion-like clusters from which neurites extended on the culture substrate, and they expressed the enzyme, acetylcholinesterase, and the cell surface antigens recognized by antisera raised against horseradish peroxidase . The production of large-scale neuronal cell cultures will be useful for immunological and molecular studies of neural cell differentiation.

Pediatr Neurol, 1985 May-Jun, 1(3), 143 - 50
Differential toxicity of chronic exposure to phenytoin, phenobarbital, or carbamazepine in cerebral cortical cell cultures; Neale EA et al.; The effects of phenytoin (30 micrograms/ml), phenobarbital (64 micrograms/ml), and carbamazepine (24 micrograms/ml) were assessed in cerebral cortical cell cultures . After antiepileptic drug exposure for eleven days, cultures were assayed for total protein, number of neurons, tetanus toxin fixation, high-affinity uptake of gamma-aminobutyric acid and beta-alanine, activity of choline acetyltransferase, and benzodiazepine binding . Carbamazepine-exposed cultures demonstrated minimal effects, whereas highly significant deficits related to generalized toxicity were observed in cultures exposed to phenytoin or phenobarbital.

Jpn J Cancer Res, 1985 May, 76(5), 414 - 9
Augmentation of the cell-mediated cytotoxic response induced in mixed cell culture by adriamycin; Arinaga S et al.; The effects of adriamycin (AM) on the generation of cell-mediated cytotoxicity in primary stimulation cultures of human peripheral blood mononuclear cells (PBM) with B-lymphoblastoid cell line Raji were assessed . When AM was added to the culture on day 1 for 24 hr at a concentration of 10(-8)M, the cell-mediated cytotoxic response was significantly augmented as compared to that in untreated culture . The augmented cytotoxic response was significantly depressed when unfractionated cells harvested from untreated culture were added to the effector cells from AM-treated culture . The response was slightly more reduced by addition of cells from untreated culture after treatment with OKT3 . However, the addition of cells from untreated culture after removal of adherent cells resulted rather in an increase of the cytotoxic response . When adherent cell fraction was removed from the effector cells, the cytotoxic response in nonadherent fraction from AM-treated culture was similar to that observed in unfractionated cells from the same culture . On the other hand, nonadherent fraction from untreated culture gave an increased cytotoxic response, which was of almost the same magnitude as that of cells from the augmented, AM-treated culture . These results suggested that the augmentation of cytotoxic response observed in AM-treated culture might be due to inhibition by AM of the generation of suppressive activity in the adherent cell population during PBM-Raji cell culture.

Clin Endocrinol (Oxf), 1985 May, 22(5), 631 - 8
Effect of nivazol on ACTH secretion by human pituitary corticotrophic tumours in cell culture; Adams EF et al.; The effects of nivazol, a novel steroid which lacks glucocorticoid activity, on ACTH secretion by cell cultures of human pituitary corticotrophic tumours has been investigated . Nivazol (0.002-20 mumol/l) inhibited ACTH secretion by 50-80%, after 4 and 24 h of incubation . (Trilostane (0.3-30 mumol/l) did not affect basal or stimulated ACTH secretion.) The potency of nivazol was comparable with that of hydrocortisone, and less than dexamethasone . The stimulatory effects of oCRF and AVP were reduced or completely blocked by nivazol in a similar manner to that previously described for hydrocortisone . It is concluded that nivazol may be of benefit in the direct treatment of Cushing's disease, particularly since it lacks glucocorticoid activity.

Antimicrob Agents Chemother, 1985 May, 27(5), 846 - 50
Studies on 44 081 R.P., a new antirhinovirus compound, in cell cultures and in volunteers; Zerial A et al.; A synthetic compound, 2-{(1,5,10,10a-tetrahydro-3H-thiazolo{3,4b}isoquinolin-3-ylidene) amino}-4-thiazoleacetic acid (S), 44 081 R.P., inhibits the multiplication of rhinoviruses in cell cultures . Of the 69 rhinovirus strains and serotypes that have been studied, 39% were inhibited at a concentration of 7 micrograms/ml, far below that which affects cellular metabolism (250 micrograms/ml) . Preliminary data indicate that the compound inhibits some early events of virus replication but that some cellular functions are also involved in its mechanism of action . Despite its antiviral activity in vitro, the compound, when self-administered intranasally as a 0.2% solution to volunteers from the day before to 5 days after inoculation with a human rhinovirus strain, had no significant effect on rhinorrhea, clinical score, or laboratory evidence of infection.

Poult Sci, 1985 May, 64(5), 841 - 3
Vaccination against Marek's disease and infectious bursal disease III . Growth rate of both turkey herpesvirus and infectious bursal disease virus in coinfected cell cultures; Chang JD et al.; Titers of the turkey herpesvirus (HVT) and infectious bursal disease virus (IBDV) in coinfected cell cultures, used to produce a live bivalent HVT/IBDV vaccine, were determined daily . Based on the daily titers, cells harvested either 2 or 3 days after inoculating the second virus (IBDV) into HVT infected cell cultures yielded the maximum titers for both viruses . Harvesting coinfected cell cultures on either day provided the most suitable source of the live HVT/IBDV vaccine against Marek's disease and infectious bursal disease.

J Neurosci, 1985 May, 5(5), 1176 - 9
Nerve growth factor treatment enhances nicotine-stimulated dopamine release and increases in cyclic adenosine 3':5'-monophosphate levels in PC12 cell cultures; Baizer L et al.; In order to examine the relationship between cyclic AMP (cAMP) levels and evoked neurotransmitter release, experiments have been performed with cultures of clonal rat PC12 pheochromocytoma cells . Stimulation of the release of endogenous dopamine by nicotine in these cultures is calcium-dependent and blocked by d-tubocurarine, a specific nicotinic cholinergic antagonist . Similarly, nicotine causes increases in cAMP levels in PC12 cell cultures that are calcium-dependent and blocked by d-tubocurarine . Cultures treated for 6 days or longer with 2 X 10(-9) M nerve growth factor (NGF) release a 3- to 4-fold greater amount of dopamine than do control cultures in response to a maximal concentration of nicotine . Correspondingly, nicotine causes a 3-fold greater increase in cAMP levels in the NGF-treated cultures than in the controls . These results suggest that stimulation of the nicotinic cholinergic receptor in PC12 cells results in some manner in the activation of adenylate cyclase and further support the notion that cAMP is involved in the process of neurotransmitter release.

Exp Cell Res, 1985 May, 158(1), 111 - 8
Further characterization of the inhibition of casein production in a primary mouse mammary epithelial cell culture by epidermal growth factor . Antagonism by cyclic AMP; Arakawa M et al.; Epidermal growth factor (EGF) inhibited casein production and the accumulation of casein mRNA activity induced by insulin (I), cortisol (F) and prolactin (P) in a primary culture of mammary epithelial cells from pregnant mice . The inhibitory effects of EGF were blocked by 8-bromo cyclic AMP (8-br-cAMP) in a dose-dependent manner . The effect of 8-br-cAMP was observed at a concentration as low as 20 microM and was maximal at 500 microM . Dibutyryl cyclic AMP (db-cAMP), cAMP, and 3-isobutylmethylxanthine (IBMX), an inhibitor of phosphodiesterase, also antagonized the inhibitory effect of EGF on casein production . 8-Br-cAMP had, however, no effect on the mitogenic activity of EGF in this system . These results suggest a possible modulatory role of cAMP in EGF-induced inhibition of casein production in cultured mammary epithelial cells.

Infect Immun, 1985 May, 48(2), 366 - 71
Adaptation of Ehrlichia sennetsu to canine blood monocytes: preliminary structural and serological studies with cell culture-derived Ehrlichia sennetsu; Holland CJ et al.; Ehrlichia sennetsu, the causative agent of human sennetsu rickettsiosis, was successfully propagated in primary canine blood monocyte cultures . The growth cycle of this organism appears to be similar to that of Ehrlichia canis . The antigen derived from our E . sennetsu cultures was used to develop an indirect fluorescent antibody test for detection and titration of serum antibodies to the organism . Using this test system, we found that five human serum samples obtained from patients clinically diagnosed as having sennetsu rickettsiosis were positive for anti-E . sennetsu antibodies . In addition, 29% of the serum samples obtained from 200 patients having a fever of unknown origin and residing in various regions of Malaysia were also serologically positive . All sera from apparently healthy individuals were negative in the test . Dogs inoculated with cell culture-adapted E . sennetsu developed a significant specific antibody titer to E . sennetsu, and the organism was subsequently isolated from their blood . These animals showed no clinical evidence of disease . The possibility of a higher prevalence of human sennetsu rickettsiosis in Southeast Asia and the potential usefulness of the canine model for studies of human sennetsu rickettsiosis are discussed.

J Clin Microbiol, 1985 May, 21(5), 669 - 73
Rapid detection of cytomegalovirus in cell culture by indirect immunoperoxidase staining with monoclonal antibody to an early nuclear antigen; Swenson PD et al.; A method for the rapid detection of cytomegalovirus (CMV) in MRC-5 cells 48 h after inoculation with clinical specimens was developed . A commercially available monoclonal antibody to a CMV early nuclear antigen was used in an indirect immunoperoxidase (IPA) staining procedure performed directly on acetone-fixed cell monolayers in standard tubes (16 by 125 mm) . Of 190 clinical specimens tested, 30 specimens produced CMV cytopathic effect in tissue culture (TC-CPE) within 14 days after inoculation and, of these 30, 28 were positive for CMV after 48 h by the IPA staining procedure (sensitivity, 93%) . Of the remaining 160 clinical specimens negative by TC-CPE within 14 days, 7 were positive by the IPA stain (specificity, 96%) . However, three of these seven specimens were positive by TC-CPE upon subculture after the initial 14-day incubation period, and one specimen was overgrown by herpes simplex virus type 2 before CMV cytopathic effect could develop . The mean time to appearance of cytopathic effect for the 30 specimens positive by TC-CPE within 14 days was 6.7 days . These findings indicate that this IPA staining is a useful method for the rapid detection of CMV in cell monolayers inoculated with clinical specimens.

Mol Pharmacol, 1985 May, 27(5), 584 - 94
Metabolic channeling of 5-fluoro-2'-deoxycytidine utilizing inhibitors of its deamination in cell culture; Boothman DA et al.; The metabolism of 5-fluoro-2'-deoxycytidine (FdC) with and without tetrahydrouridine (H4U) or 2'-deoxytetrahydrouridine (dH4U) was examined in log phase HEp-2 cells using HPLC and TLC methods which quantified: the incorporation of FdC-related antimetabolites into RNA and DNA and pool size levels of FdC-related antimetabolites . {3H}-FdC administered to log phase HEp-2 cells at a concentration of 0.01 microM for 24 hr resulted in the incorporation of 5.22 X 10(-8) mol of FdC/mol of DNA phosphate, a 0.021% substitution of FdC for dC . Coadministration of 1.0 mM H4U or dH4U resulted in 2- and 25-fold increases in the incorporation of FdC, respectively . No detectable incorporation of 5-fluoro-2'-deoxyuridine (FdU) into HEp-2 DNA resulted (detection limit, approximately 5 fmol) . In contrast, treatment of HEp-2 cells with 0.1 microM FdU resulted in the incorporation of 1.83 X 10(-9) mol of FdU (74.7 fmol detected)/mol of DNA phosphate . A linear incorporation of FdC into the DNA of HEp-2 cells was found with increasing concentrations of FdC and 1.0 mM dH4U . 0.1 microM FdC resulted in the incorporation of 2.39 X 10(-6) mol of FUMP/mol of cytoplasmic RNA phosphate and 2.23 X 10(-5) mol of FUMP/mol of nuclear RNA phosphate . Similarly, HEp-2 cells treated with 0.1 microM FdU resulted in the incorporation of 1.10 X 10(-5) mol of FUMP/mol of nuclear RNA phosphate and 9.44 X 10(-7) mol of FUMP/mol of cytoplasmic RNA phosphate . In contrast, no detectable FUMP incorporation into either nuclear or cytoplasmic RNAs of HEp-2 cells resulted when H4U or dH4U was coadministered with 0.1 microM FdC . Pool size analyses of log phase HEp-2 cells following a 30-min exposure to FdU or FdC with and without H4U or dH4U were also performed; 0.1 microM FdC treatment resulted in the formation of 169 fmol of FUMP/1.0 X 10(6) viable HEp-2 cells . Treatment with 0.1 microM FdU produced 253 fmol of FUMP/1.0 X 10(6) viable HEp-2 cells . In contrast, no detectable FUMP pools were formed when H4U or dH4U was coadministered with 0.1 microM FdC (detection limit, approximately 5 fmol) . Pool levels of FdUMP, the inhibitor of thymidylate synthetase, were also assayed; 36.9 fmol of FdUMP/1.0 X 10(6) viable HEp-2 cells were detected upon administration of 0.1 microM FdC.(ABSTRACT TRUNCATED AT 400 WORDS)

J Immunol, 1985 May, 134(5), 2847 - 52
Synthetic Fc peptide-mediated regulation of the immune response . II . Analysis of secreted immunoglobulin classes, accessory cell contribution, and lymphocyte proliferation in p23-stimulated spleen cell cultures; Hobbs MV et al.; The synthetic peptide p23 representing residues 335 to 357 in the CH3 domain of human IgG1 was able to increase levels of secreted Ig in murine spleen cell cultures . This in vitro response was optimal in the presence of between 10(-4) and 10(-3) micron p23/ml and the levels of secreted Ig reached a maximum on day 4 or day 5 of culture . Supernatants from p23-treated cell cultures generally contained more IgM than IgG and undetectable levels of IgA . Induction of Ig secretion by p23 was macrophage-independent but T cell-dependent and, with respect to the latter case, removal of T cells from spleen cells reduced the levels of both IgM and IgG . Although maintaining the B cell differentiation-inducing quality of its progenitor molecule, the Fc gamma fragment, p23 appeared to have lost the ability to induce B cell proliferation . Evidence is presented that a sequence functionally similar to p23 is extant in mouse IgG by showing that murine Fc gamma fragments were also able to induce increases in Ig-secreting cells in murine spleen cell cultures.

Virology, 1985 Apr 30, 142(2), 270 - 7
Synthesis in cell culture of the gapped linear duplex DNA of the slow virus visna; Blum HE et al.; Visna virus is a nontransforming retrovirus that causes slow infections in animals and a rapidly progressive-lytic infection in cell culture . The results of an analysis of the synthesis of viral DNA in cell culture are reported . Region- and strand-specific probes cloned in M13 have been used to define the dynamics of DNA synthesis and the major nucleic acid species formed . It is shown that (i) within the first hours of infection, a full-length copy of the viral RNA genome is synthesized by reverse transcription, (ii) early in infection a major species of DNA is formed that extends from a site near the center of the molecule to the 3' end, (iii) somewhat later a second major species of plus-strand DNA is generated that extends from the 5' end to the middle of the genome . As a consequence, most viral DNA molecules consist of a full-length minus strand, and two plus strands separated by a gap or nick in the center of the molecule (J . D . Harris, J . V . Scott, B . Traynor, M . Brahic, L . Stowring, P . Ventura, A . T . Haase, and R . Peluso (1981) . Virology 113, 573-583) . The implications of this viral DNA structure for one unusual aspect of the lentivirus life cycle, the production of viral RNA, and virions from extrachromosomal DNA are discussed (J . D . Harris, H . Blum, J . Scott, B . Traynor, P . Ventura, and A . T . Haase (1984) . Proc . Natl . Acad . Sci . USA 81, 7212-7215).

Tsitologiia, 1985 Apr, 27(4), 451 - 7
{Amine inhibition of serum-induced DNA synthesis in 3T3 and 3T6 cell cultures}; Nikol'skii NN et al.; Several amines were shown to inhibit growth stimulation in quiescent confluent and sparse cultures of Swiss 3T3 and 3T6 cells by changing for a fresh medium containing 10-20% serum . Proliferation was inhibited by dansylcadaverine (0.1 mM), chloroquine (0.05 mM), 5-methoxytryptamine (0.1 mM), cystamine (0.1 mM), dimethylurea (100 mM), methylurea (100 mM), and in some experiments--by urea (100 mM) . The inhibitory activity was not associated with a direct influence of amines on DNA synthesis or thymidine phosphorylation . The findings suggest that amines may influence the process of clustering of growth factor-receptor complexes, or the fusion of plasma membrane and intracellular vesicles containing some components required for cell proliferation.

Biol Reprod, 1985 Apr, 32(3), 631 - 43
Characterization of deciduoma marker proteins in hamster uterus: detection in decidual cell cultures; Leavitt WW et al.; The objective of this study was first, to identify the proteins associated with decidualization of the hamster uterus by comparing the protein maps of decidualized and nondecidualized endometrium in vivo, and second, to determine whether decidual cell cultures produced these characteristic proteins . Decidualization was induced in one uterine horn, and the contralateral horn was not stimulated (control tissue) . Animals were ovariectomized and a subcutaneous progesterone implant was used to maintain decidualization . Uterine proteins from nuclear and cytosol fractions were analyzed by two-dimensional electrophoresis using a highly sensitive protein staining technique . Analysis of nuclear extract and cytosol from decidualized and nondecidualized endometrium from Days 6, 7, and 8 of pseudopregnancy demonstrated the presence of 11 nuclear and five cytosolic deciduoma-associated proteins . Serum and erythrocyte proteins were identified by two-dimensional electrophoresis, and none of the 16 deciduoma-associated proteins was a serum or erythrocyte contaminant . Forty-eight-hour cultures of decidual cells harvested from Day 5 of pseudopregnancy produced all 16 of the deciduoma-associated proteins found in whole tissue in situ . Culture conditions minimized serum and erthrocyte contamination, enhancing the detection of deciduomal cell proteins . Four nuclear and two cytosolic proteins were considered deciduoma specific, i.e., they were not associated with cellular proliferation, as evidenced by their absence from cultures of rapidly dividing fetal hamster fibroblasts . Thus, these studies show that the detection of deciduomal proteins may be a useful criterion for the assessment of decidualization in vitro and in vivo.

Arch Biochem Biophys, 1985 Apr, 238(1), 237 - 46
Novel features of prephenate aminotransferase from cell cultures of Nicotiana silvestris; Bonner CA et al.; A prephenate aminotransferase enzyme that produces L-arogenate was demonstrated in extracts from cultured-cell populations of Nicotiana silvestris . The enzyme was very active with low concentrations of prephenate, but required high concentrations of phenylpyruvate or 4-hydroxyphenylpyruvate to produce activity levels that were detectable . It is the most specific prephenate aminotransferase described to date from any source . Only L-glutamate and L-aspartate were effective amino-donor substrates . Prephenate concentrations greater than 1 mM produced substrate inhibition, an effect antagonized by increasing concentrations of L-glutamate cosubstrate . The enzyme was stable to storage for at least a month in the presence of pyridoxal 5'-phosphate, EDTA, and glycerol, and exhibited an unusually high temperature optimum of 70 degrees C . The identity of L-arogenate formed during catalysis was verified by high-performance liquid chromatography . DEAE-cellulose chromatography revealed two aromatic aminotransferase activities that were distinct from prephenate aminotransferase and which did not require the three protectants for stability . The aromatic aminotransferases were active with phenylpyruvate or 4-hydroxyphenylpyruvate as substrates, but not with prephenate . Both of the latter enzymes were similar in substrate specificity, and each exhibited a temperature optimum of 50 degrees C for catalysis . The primary in vivo function of the two aromatic aminotransferases is probably to transaminate between the aspartate/2-ketoglutarate and glutamate/oxaloacetate couples, since activities with the latter substrate combinations were an order of magnitude greater than with aromatic substrates . The demonstrated existence of a specific prephenate aminotransferase in N . silvestris meshes with other evidence supporting an important role for L-arogenate in tyrosine and phenylalanine biosynthesis in higher plants.

Am J Physiol, 1985 Apr, 248(4 Pt 2), F536 - 44
Biochemical characterization of renal epithelial cell cultures (LLC-PK1 and MDCK); Gstraunthaler G et al.; The expression of enzymes in LLC-PK1 and MDCK cells was used to study the retention of differentiated properties of the renal epithelial cell lines by a biochemical approach . Activities of marker enzymes, for which intracellular and intranephron localization is known, were determined from crude cell homogenates of LLC-PK1 and MDCK monolayer cultures . The activity patterns of the particular enzymes found were then compared with the in vivo distribution of the enzymes along the rat nephron . LLC-PK1 cells exhibit high activities of apical membrane enzymes when compared with MDCK cells, whereas in the latter high activity of Na-K-ATPase could be detected . The activities of lysosomal enzymes, mitochondrial enzymes, and transaminases were higher in LLC-PK1 than in MDCK cells . Glycolytic enzymes, however, displayed identical activity levels in both the LLC-PK1 and MDCK cells, which may be due to the fact that these are continuous cell lines and to the culture conditions used, since glucose is a major energy source in the culture media.

J Cell Biol, 1985 Apr, 100(4), 1050 - 5
Identification of link proteins in canine synovial cell cultures and canine articular cartilage; Fife RS et al.; Link proteins are glycoproteins in cartilage that are involved in the stabilization of aggregates of proteoglycans and hyaluronic acid . We have identified link proteins in synovial cell cultures form normal canine synovium using sodium dodecyl sulfate-polyacrylamide gel electrophoresis, immunofluorescence, and immunolocation with specific antibodies by electrophoretic transfer . We have also found evidence for the synthesis of link proteins in these cultures by fluorography of radiolabeled synovial cell extracts . We have identified a 70,000 mol-wt protein in canine synovial cell culture extracts that has antigenic cross-reactivity with the 48,000-mol-wt link protein . Three link proteins were identified in normal canine articular cartilage . These results indicate that link proteins are more widely distributed in connective tissues than previously recognized and may have biological functions other than aggregate stabilization.

Cancer Res, 1985 Apr, 45(4), 1594 - 600
Species- and length of exposure-dependent differences in the benzo(a)pyrene:DNA adducts formed in embryo cell cultures from mice, rats, and hamsters; Sebti SM et al.; The activation of benzo(a)pyrene (BaP) to DNA-binding metabolites in early-passage embryo cell cultures prepared from various species of rodents was investigated by exposing cells from mice (BALB/c and Sencar), rats (Wistar and Fischer 344), and Syrian hamsters to {3H}BaP for various lengths of time . The BaP:DNA adducts containing cis-vicinal hydroxyl groups such as those formed from 7 beta,8 alpha-dihydroxy-9 alpha,10 alpha-epoxy-7,8,9,10-tetrahydrobenzo(a)pyrene (anti-BaPDE) were separated from the other types of BaP:DNA adducts by immobilized boronate chromatography, and the individual adducts were analyzed by high-performance liquid chromatography . A number of BaP:DNA adducts were present in the DNA from the cultures from all three species after 5 h of BaP treatment . After a 24-h exposure to BaP, the mouse and hamster embryo cell DNA contained a large amount of the adduct formed by reaction of (+)-anti-BaPDE with the 2-amino group of deoxyguanosine (dGuo) and a small amount of a 7 beta,8 alpha-dihydroxy-9 beta,10 beta-epoxy-7,8,9,10-tetrahydrobenzo(a)pyrene:dGuo adduct . A large number of BaP:DNA adducts derived from 7 beta, 8 alpha-dihydroxy-9 beta,10 beta-epoxy-7,8,9,10-tetrahydrobenzo(a)pyrene and other unidentified BaP metabolites were present in rat embryo cell cultures at all times . Neither the Fischer 344 nor the Wistar rat embryo cell cultures had a significant amount of (+)-anti-BaPDE:dGuo adduct after 5 h of BaP treatment, and in the Wistar rat cells larger amounts of other adducts were present even after a 96-h exposure to BaP . In cell cultures from all three species the proportion of (+)-anti-BaPDE:dGuo adduct increased as the length of time of exposure to BaP increased . There are major differences in the metabolic activation of BaP to DNA binding metabolites in embryo cells from various species of rodents . However, the variations between cell cultures from different strains of rats or mice are not as great as the variations between cell cultures from different species . The time-dependent alterations in the BaP:DNA adducts indicate that analysis after various lengths of time of exposure to BaP is essential to characterize accurately the pathways of metabolic activation of BaP in cells from various species and tissues.

J Rheumatol, 1985 Apr, 12(2), 237 - 41
Rheumatoid arthritis and osteoarthritis synovial fluid effects on primary human endothelial cell cultures; Semble EL et al.; Since vascular proliferation may be important in the pathogenesis of rheumatoid arthritis (RA) and/or osteoarthritis (OA), this study examined the induction of angiogenesis by these synovial fluids (SF) . Four of 11 (36%) RA and 2 of 6 (33%) OA SF caused early morphological changes in human endothelial cell cultures . SF from 7 of 11 (63%) RA and 4 of 8 (50%) OA patients resulted in the late formation of tabular networks morphologically resembling capillaries observed in vivo . Early morphological changes in cultures were associated with a significantly (p less than 0.05) longer duration of disease in patients with RA . Factors present in the SF of RA and OA patients may play a role in the excessive vascularization which often occurs in these arthropathies.

Br J Cancer, 1985 Apr, 51(4), 505 - 14
The effects of 6-mercaptopurine nucleotide derivatives on the growth and survival of 6-mercaptopurine-sensitive and -resistant cell culture lines; Johnston HP et al.; 6-Mercaptopurine (MP)-sensitive and -resistant cell culture lines were used to further characterize the apparent ability of MP nucleotide derivatives to overcome resistance to the parent drug . 6-Mercaptopurine-9-beta-D-ribofuranoside 5'-monophosphate {MPRP}, bis(6-mercaptopurine-9-beta-D-ribofuranoside)-5', 5"'-monophosphate {bis(MPR)P}, bis(O2',O3'-dibutyryl-6-mercaptopurine-9-beta-D-ribofuranoside)-5', 5"'-monophosphate {bis(dibut.MPR)P}, and O2',O3'-dibutyryl-6-mercaptopurine-9-beta-D-ribofuranoside 5'-monophosphate {dibut.MPRP} were tested for cytotoxic and/or growth inhibitory effects against MP-resistant sublines of V79 Chinese hamster lung fibroblasts (CH/TG) and L1210 mouse leukaemia cells (L1210/MPR) in which deficiencies of hypoxanthine-guanine phosphoribosyltransferase, and hence drug nucleotide forming capacity were the basis of resistance . L1210/MPR cells were totally resistant to 1 mM 6-mercaptopurine-9-beta-D-ribofuranoside {MPR} and 2 mM MPRP, but were inhibited by high concentrations (greater than 0.25 mM) of bis(MPR)P . These results suggested that bis(MPR)P was taken up by cells as the intact molecule since MPR and MPRP were its extracellular breakdown products . L1210/MPR cells were much more sensitive to the lipophilic bis(dibut.MPR)P derivative which had a predominantly cytotoxic action as judged by trypan blue staining and the ability of treated cells to produce macroscopic colonies in soft agar medium . However, cells killed by bis(dibut.MPR)P did not disintegrate appreciably over periods of up to 10 days . The effects of bis(dibut.MPR)P were probably the result of cellular uptake of the intact molecule . Dibut.MPRP showed minimal ability to inhibit L1210/MPR cells although this compound was a possible breakdown product of bis(dibut.MPR)P and a source of the same extracellular degradation products . The median cell size decreased in L1210/MPR cultures during exposure to both bis(MPR)P and bis(dibut.MPR)P . This effect was elicited more rapidly and at lower concentration by bis(dibut.MPR)P than by bis(MPR)P . In contrast, sodium butyrate, a breakdown product of bis(dibut.MPR)P induced increases in cell size at high concentration . Bis (dibut.MPR)P was also cytotoxic to MP-resistant CH/TG cells and was approximately 300 times more effective than bis(MRP)P and MPR which exhibited similar activity against this cell line . Bis(dibut.MPR)P and dibut.MPRP were equivalent and less active than MPR in their effects on MP-sensitive L1210/0 cells where their predominant mechanism of action was via degradation to release MPR.(ABSTRACT TRUNCATED AT 400 WORDS)

Tohoku J Exp Med, 1985 Apr, 145(4), 437 - 45
Comparative enzymology of eleven acid hydrolases in cultured amniotic fluid cells, skin fibroblasts and embryonic lung fibroblasts, and the respective changes with the increasing age of the cell cultures; Minami R et al.; Assay conditions were studied for eleven lysosomal enzymes (beta-D-galactosidase, alpha-D-mannosidase, beta-hexosaminidase, beta-D-glucuronidase, alpha-D-galactosidase, alpha-D-glucosidase, arylsulfatase, beta-D-glucosidase, alpha-L-fucosidase, alpha-D-neuraminidase and alpha-L-iduronidase) in cultured amniotic fluid cells (CAFC), cultured skin fibroblasts (CSF) and cultured embryonic lung fibroblasts (CELF), and the properties of the enzymes were compared among these cultured cells . In addition, changes in these enzymes from the three cell types were investigated between 4-6 earlier passages and 24-26 later passages . With the exception of alpha-D-glucosidase, alpha-D-neuraminidase and alpha-L-fucosidase, all enzymes assayed for the 4-6 earlier passages and the 24-26 later passages had the same Km values and the same pH optima, and were also unchanged with the increasing age of cell cultures, with regard to their points . The specific activities of beta-D-glucuronidase, arylsulfatase, alpha-D-glucosidase and beta-D-glucosidase for the 4-6 earlier passages increased significantly with development, though no change was observed with development in the specific activities of other enzymes . Variations were observed between the levels of these enzymes in the three cell types with the increasing age of cell cultures, such as increases in some, decreases in others and no change in still others.

Zh Mikrobiol Epidemiol Immunobiol, 1985 Apr, (4), 57 - 60
{Standardization of the conditions for measuring the specific fluorescence intensity in continuous cell cultures infected with variants of the Japanese encephalitis virus}; Cherednichenko IuN; The use of modernized photometric accessories to a model microscope manufactured by the Leningrad Optico-Mechanical Amalgamation (USSR), as well as the practical application of innovations facilitating and standardizing research work, has made it possible to obtain objective data indicating the presence of significant, direct, linear correlation between infectious activity and the intensity of fluorescence emitted by fluorescent antibodies bound with the antigen of 11 studied variants of Japanese encephalitis virus in continuous cell lines . The study of the dynamics of fluorescence intensity permitted the objective evaluation of the previously revealed regularity in the increase of the intensity of the induced fluorescence of Japanese encephalitis antigen in continuous cell cultures.

J Steroid Biochem, 1985 Apr, 22(4), 507 - 12
Relationships between unconjugated and sulphated steroids in porcine primary Leydig cell culture; Orava M et al.; Steroidogenesis in immature porcine Leydig cells was investigated in primary culture at 48-84 h under basal conditions and in the presence of hCG . The basal accumulation of unconjugated steroids was close to linear only during the first 4 h of study, whereas the sulphate-conjugated steroids accumulated essentially linearly over the 36 h experimental period . At the last time point, 95% of the steroids measured were sulphated . Stimulation with hCG (1 ng/ml) led to a still more pronounced sulphate conjugation, and approx 99% of the steroids measured were sulphated at 36 h . Under maximal stimulation with hCG (100 ng/ml) the sulphates accounted for 74% of the total steroids measured at 36 h . Testosterone, androstenedione, dehydroepiandrosterone, 5-androstene-3 beta, 17 beta-diol and estrone were usually quantitatively the most important unconjugated steroids, and sulphated dehydroepiandrosterone, estrone, testosterone and 5-androstene-3 beta, 17 beta-diol were the most important steroid sulphates, especially following maximal stimulation of the cultures . These data emphasize the importance of steroid sulphates in porcine testicular steroid metabolism . Under stimulation with hCG, there was a rapid response in testicular steroidogenesis, initially seen as a rapid increase in the secretion of unconjugated and sulphated steroids . At approx 4-12 h, the rate of sulphate conjugation appeared to reach or even to exceed that of steroid biosynthesis, which lead to stabilisation or a decrease in the concentrations of unconjugated steroids . Only high doses of hCG, 10-100 ng/ml, were then able to lead to a net accumulation of unconjugated steroids, at 24-36 h of incubation with hCG.

J Virol, 1985 Apr, 54(1), 78 - 85
Infectious hepatitis A virus particles produced in cell culture consist of three distinct types with different buoyant densities in CsCl; Lemon SM et al.; Although hepatitis A virus (HAV) released by infected BS-C-1 cells banded predominantly at 1.325 g/cm3 (major component) in CsCl, smaller proportions of infectious virions banded at 1.42 g/cm3 (dense HAV particles) and at 1.27 g/cm3 (previously unrecognized light HAV particles) . cDNA-RNA hybridization confirmed the banding of viral RNA at each density, and immune electron microscopy demonstrated apparently complete viral particles in each peak fraction . The ratio of the infectivity (radioimmunofocus assay) titer to the antigen (radioimmunoassay) titer of the major component was approximately 15-fold greater than that of dense HAV particles and 4-fold that of light HAV particles . After extraction with chloroform, the buoyant density of light and major component HAV particles remained unchanged, indicating that the lower density of the light particles was not due to association with lipids . Light particles also banded at a lower density (1.21 g/cm3) in metrizamide than did the major component (1.31 g/cm3) . Dense HAV particles, detected by subsequent centrifugation in CsCl, were indistinguishable from the major component when first banded in metrizamide (1.31 g/cm3) . However, dense HAV particles recovered from CsCl subsequently banded at 1.37 g/cm3 in metrizamide . Electrophoresis of virion RNA under denaturing conditions demonstrated that dense, major-component, and light HAV particles all contained RNA of similar length . Thus, infectious HAV particles released by BS-C-1 cells in vitro consist of three distinct types which band at substantially different densities in CsC1, suggesting different capsid structures with varied permeability to cesium or different degrees of hydration.

J Pathol, 1985 Apr, 145(4), 341 - 54
Monoclonal antibodies specific for subsets of epidermal keratins: biochemical and immunocytochemical characterization--applications in pathology and cell culture; Knight J et al.; Keratin composition has been widely used as a biochemical marker of differentiation in normal epithelia, cell culture systems and tumours of epithelial tissues . We have been developing a model system for the study of human squamous epithelial cell differentiation, and among a panel of monoclonal antibodies we have generated for analysing this system are two antibodies recognizing subsets of epidermal keratins . The two antibodies, designated LICR-LON-16a and LICR-LON-29b, were raised to the human squamous carcinoma cell line LICR-LON-HN-5, and we describe here their biochemical and immunocytochemical characterization . Antibody 16a reacts with only epidermal basal cells in normal human skin and shows specificity for the 45 and 46 kdalton keratins . Antibody 29b stains all living layers of the epidermis, and reacts with a broad range of ketain polypeptides, (45-56 kdaltons) in immunoblotting analyses . We have investigated the alterations of cellular staining that occur in chronic hyperproliferative skin diseases and carcinomas and compared this with the staining of multilayered cultures of normal keratinocytes and the HN-5 cell line . We show that in squamous cell carcinomas and in HN-5 cell xenografts 16a and 29b stain only the well-differentiated cell types . Furthermore we found that the basal cell specificity of 16a was lost in all of the hyperproliferative skin lesions examined including psoriasis and eczema . This transition to suprabasal staining pattern was also seen in the cultures of normal keratinocytes and HN-5 cells . We conclude that aberrant keratin synthesis or abnormal post-translational processing of keratins associated with an increased rate of cell turnover could account for the altered expression of the epitope recognized by antibody 16a.

Exp Cell Res, 1985 Apr, 157(2), 520 - 32
Cell density and cell shape-related regulation of vimentin and cytokeratin synthesis . Inhibition of vimentin synthesis and appearance of a new 45 kD cytokeratin in dense epithelial cell cultures; Ben-Ze'ev A; The pattern of the intermediate type filament protein synthesis was examined in cultured bovine mammary gland epithelial (BMGE) cells under conditions of varied cell shape and cell-cell contact . In dense monolayer and suspension cultures BMGE cells expressed a new cytokeratin of 45 kD identified as a member of the acidic subfamily of cytokeratins . This polypeptide has a phosphorylated component and is dissociated from the cytokeratins complex in the presence of 6.5 M urea . The mRNA of the new cytokeratin accumulated in dense cell cultures, as revealed by in vitro translation in a cell-free system . In BMGE-H cells that express also vimentin, the synthesis of vimentin decreased dramatically in dense cell cultures, while the synthesis of the 45 kD cytokeratin was maximal under these conditions . The results suggest that the expression of certain cytokeratins and that of vimentin can be coordinately regulated by factors in the cellular environment that effect cell shape and cell surface contacts.

Biochim Biophys Acta, 1985 Mar 21, 844(3), 273 - 9
Stimulation of skeletal-derived cell cultures by different electric field intensities is cell-specific; Binderman I et al.; Pulsed electric stimulation, coupled capacitively to different cell cultures of skeletal origin, caused immediate changes in the cellular levels of cyclic AMP and a later enhanced DNA synthesis . Changes both in cyclic AMP level and DNA synthesis were correlated with the strength of the applied electric field . Cultures of calvaria bone cells which contain mainly two cell types, parathyroid hormone responsive cells (osteoblast-like) and prostaglandin E2 responsive cells (fibroblast-like), respond to both low (13 V/cm) and to high (54 V/cm) electric field strength, with no response at intermediate (24 V/cm) field strength . Rat epiphyseal cartilage responded like bone cells both to low and high field intensities, while rat condylar cartilage responded only to the intermediate field strength . Moreover, subcultures of calvaria bone cells, which lost their osteoblastic phenotype expression during subculturing, were responsive only to low field strength . On the other hand, osteoblast-enriched cultures, derived from calvaria bone grown in low calcium, were responsive only to the high field strength . These findings suggest that the response to various electric field intensities is cell-specific and might be used as an additional parameter to characterize cell types . Our study points to the possibility that when exposing a whole organ to an electrical stimulation it is possible to affect specifically only one cell population out of the many cell types existing in the organ.

Biochem J, 1985 Mar 15, 226(3), 631 - 6
Human tissue-type plasminogen activator . Production in continuous serum-free cell culture and rapid purification; Kruithof EK et al.; A simplified procedure for the production and purification of human tissue-type plasminogen activator (t-PA) is described . Bowes-melanoma cells were maintained in continuous serum-free culture . The cell nutrient consisted of Dulbecco's modified Eagle's medium (DMEM) supplemented with insulin (5 mg/litre), transferrin (5 mg/litre), progesterone (1 nM), cortisol (10 nM), aprotinin (2 X 10(4) units/litre) and a mixture of trace elements . t-PA accumulated in the culture medium at a rate of 40 units/day per ml and was harvested every third day . Cell losses during each harvest, leading to a steady decline of enzyme yields, were compensated for by treating the cells with 5% (v/v) fetal-bovine serum in DMEM every 6-8 weeks . t-PA was rapidly purified by a combination of cation-exchange chromatography and gel filtration . The procedure yielded mainly single-chain t-PA of a specific activity of 80 000 to 100 000 units/mg.

Int J Cancer, 1985 Mar 15, 35(3), 367 - 75
Alterations in the phosphoprotein composition of tumor cell clones induced by cell culture techniques used routinely in assessing metastatic behavior in vivo; Greig RG et al.; To investigate whether the cell dispersion techniques commonly employed to harvest monolayer cultures of tumor cells for injection into experimental animals might induce alterations in cellular biochemistry, we compared the phosphoprotein profiles of 6 B16 melanoma clones of distinct metastatic potential using 2-D gel electrophoresis after growth in monolayer culture, after suspension by treatment, with trypsin/EDTA and after injection of suspended cells into syngeneic mouse plasma . Trypsin/EDTA treatment and subsequent exposure to syngeneic mouse plasma induced significant alterations in phosphoprotein composition in all clones . Most alterations were quantitative, involving either enhanced or diminished expression of specific phosphoproteins, but qualitative changes involving expression of novel phosphoproteins were also observed . None of the changes in phosphoprotein composition correlated with metastatic potential . The principle alteration induced in all clones by trypsin/EDTA involved enhanced phosphorylation of an NP-40-soluble component with a molecular weight of 79,000 and an isoelectric point of 6.3 {pp79 (6.3)} . This determinant was detected in extracts of B16 monolayer cultures but its level of phosphorylation was enhanced significantly by trypsin/EDTA treatment and by exposure of the harvested cells to syngeneic mouse plasma . These data indicate that procedures commonly employed to harvest tumor cells for assay of tumorigenic and metastatic potential may provide extensive alterations in phosphoprotein composition and that biochemical investigations of tumor cells grown in monolayer culture may not accurately reflect the metabolic status of the same cells immediately prior to and following i.v . injection into experimental animals.

Vopr Virusol, 1985 Mar-Apr, 30(2), 219 - 23
{Characteristics of a cell-culture infection with the tick-borne encephalitis virus with periodic replacement of the culture medium}; Dzhivanian TI et al.; The following features were revealed in pig embryo kidney cell cultures infected with tick-borne encephalitis (TBE) virus in which the maintenance medium was changed periodically: more rapid accumulation of TBE virus-specific proteins, active proliferation of spherical vesicular structures and membrane elements of the endoplasmic reticulum, transformation of the latter into regularly arrayed complexes, a statistically significant increase in the yield of the infectious virus.

Cell Struct Funct, 1985 Mar, 10(1), 17 - 27
Molecular intactness of transferrin recycled in a myogenic chicken cell culture; Kimura I et al.; Evidence has been accumulating that transferrin (Tf), an iron-binding glycoprotein, is a unique ligand in that it is recycled intact . We examined the properties of chicken Tf molecules recycled in a myogenic chicken cell culture . No difference in the isoelectric focusing pattern before and after recycling, nor any marked change in the total Tf concentration in the culture medium was found during culture . And, although recycled Tf had no myotrophic activity in vitro, it regained its original activity when reloaded with iron . These results are evidence of the molecular intactness of recycled Tf.

Prenat Diagn, 1985 Mar-Apr, 5(2), 159 - 62
Further examples of trisomy-20 mosaicism in amniotic cell cultures; Grewal MS et al.; Amniotic fluid cultures from two patients showed trisomy-20 mosaicism . No trisomy-20 cells were found in a normal full term infant and in multiple tissue biopsies and fetal blood from a fetus after a termination of pregnancy . No definitive advice is yet possible for parents where trisomy-20 amniotic cell mosaicism is detected . Fetoscopy and fetal blood sampling are of no value and termination of pregnancy is not indicated by empirical evidence . Preferential trophoblastic non-disjunction (Kalousek and Dill, 1983) is discussed as a possible partial explanation for the variable occurrence and distribution of this type of mosaicism.

Eur J Biochem, 1985 Mar 1, 147(2), 263 - 72
Thyrotropin modifies the synthesis of actin and other proteins during thyroid cell culture; Passareiro H et al.; Primary cultures of dog thyroid cells have been used to study the effects of thyrotropin on the synthesis of proteins . The cells were cultured for 4 days in serum-free and thyrotropin-free conditions . Thyrotropin was then added for varying periods of time (6-96 h) . In the absence of thyrotropin, the cells have an elongated flattened aspect . Exposure to thyrotropin for 6-24 h produces retraction and rounding up of cells whereas cells incubated with thyrotropin for longer periods of time have an epithelial cuboidal shape . After varying periods of culture the cells were labelled with {35S}methionine for 6 h and then analyzed by one- and two-dimensional gel electrophoresis, followed by autoradiography . The results were as follows . After exposure to thyrotropin for 32 h and 48 h, the synthesis of about 18 proteins was increased while that of about 14 others was decreased . After 6 h the labelling of three and five of these proteins was already increased or decreased, respectively . Some of the proteins whose synthesis is modified in the presence of thyrotropin were identified . Actin synthesis was markedly decreased with a maximum 24-48 h after the addition of thyrotropin . A modification in the ratio between alpha and beta tubulins was also observed together with very large changes in a group of proteins having both the relative molecular mass (30 000-40 000) and the isoelectric points of tropomyosins . Forskolin and cholera toxin caused the same qualitative and quantitative changes as thyrotropin; this suggests that the regulation by thyrotropin of the synthesis of several thyroid cell proteins is mediated by cAMP . In conclusion, the data obtained in this work might help to explain the molecular mechanisms by which thyrotropin (and cAMP) triggers the changes in cell shape which occur during thyroid cell culture . They also indicate that one of the main effects of thyrotropin takes place at the level of several proteins which belong to the cytoskeleton and which are involved in the definition of the cytostructure of the thyroid cells.

J Cell Physiol, 1985 Mar, 122(3), 424 - 34
Human endothelial cell cultures: phenotypic modulation by leukocyte interleukins; Montesano R et al.; We report here that soluble factors from activated mononuclear leukocytes have a dramatic effect on cultured endothelial cells . While human umbilical vein endothelial cells grown under standard conditions show a polygonal, epithelial-like morphology, cells exposed to culture media conditioned by lectin-activated human mononuclear leukocytes become extremely elongated and/or send out numerous cytoplasmic processes, assuming a dendritic configuration . This effect cannot be mimicked by exogenous cyclic AMP, is reversible upon interruption of the treatment, and appears specific for endothelial cells, since it has not been observed so far with other cell types . The shape changes are accompanied by a reorganization of the endothelial cell cytoskeleton: actin microfilament bundles tend to be disposed in parallel arrays, while intermediate filaments and microtubules penetrate up to the extremity of the cytoplasmic processes . Colchicine prevents endothelial cell elongation but only slightly impairs the formation of lateral cell processes ("dendritic configuration") . Purified interleukins were tested for their ability to induce these changes of cell shape . Escherichia coli-recombinant human interleukin 2 had no effect, and gamma-interferon only a slight effect on endothelial cell morphology . Interleukin 1 induced moderate cell elongation, while combined treatment with both interleukin 1 and gamma-interferon resulted in shape changes indistinguishable from those elicited by supernatants of activated mononuclear leukocytes . The possible relevance of the observed endothelial cell changes to the reported angiogenic activity of mononuclear cell products is discussed.

In Vitro Cell Dev Biol, 1985 Mar, 21(3 Pt 1), 189 - 94
Cross-linked collagen surface for cell culture that is stable, uniform, and optically superior to conventional surfaces; Macklis JD et al.; A new type of collagen surface for use with cultures of peripheral nervous system cells is described . Collagen is derivatized to plastic culture dishes by a cross-linking reagent, l-cyclohexyl-3-(2-morpholinoethyl)-carbodiimide-metho-p-toluene sulfonate (carbodiimide), to form a uniform and durable surface for cell attachment and growth that allows dry storage, long-term culture, and improved microscopy . Surfaces of collagen derivatized to plastic were compared to surfaces of adsorbed or ammonia-polymerized collagen in terms of collagen binding and detachment, growth by dorsal root ganglion cells, and electron microscopy appearances . Derivatized collagen surfaces retained more collagen and showed much less evidence of degradation and cellular damage over periods of many weeks than did conventional adsorbed surfaces . Long-term survival of cells on derivatized collagen was far superior to that on the other surfaces, with almost 90% of cultures still viable after 10 wk . Transmission electron microscopy showed an organized layer of single fibrils that supported cell growth well, and scanning electron microscopy demonstrated an increased uniformity of derivatized collagen surfaces compared to ammoniated collagen surfaces . Applications for this improved substrate surface are discussed.

Stain Technol, 1985 Mar, 60(2), 81 - 7
Intensifier for Bodian staining of tissue sections and cell cultures; Katz MJ et al.; Intensification of the standard Bodian silver stain with a poststaining photographic enhancer produces high resolution of fine cell processes such as axonal growth cones . This technique can be used on tissue sections and is especially useful for visualizing individual cells fixed in tissue cultures.

Biochem Biophys Res Commun, 1985 Feb 28, 127(1), 232 - 8
Estrogen synthetase stimulation by hemin in human choriocarcinoma cell culture; Bellino FL et al.; The ability of hemin to stimulate estrogen synthetase (aromatase) in cultured human trophoblast cells and in cellular homogenates was investigated and compared with aromatase stimulation by dibutyryl cAMP {(Bu)2 cAMP} . Cells grown with hemin for 24 h, or homogenates incubated for 45 min with hemin, showed maximal aromatase stimulation (150 to 200% of activities in the absence of hemin) at 25 microM and 0.1 microM, respectively . Aromatase stimulation in culture by 25 microM hemin was observed within 4 h after hemin addition, while (Bu)2 cAMP required more than 6 h . Intracellular heme and porphyrin levels were higher (160 to 185%) in 96 h (Bu)2 cAMP-grown cells than control cells.

J Biol Chem, 1985 Feb 25, 260(4), 2092 - 9
Steroid product-induced, oxygen-mediated damage of microsomal cytochrome P-450 enzymes in Leydig cell cultures . Relationship to desensitization; Quinn PG et al.; Treatment of Leydig cells with 1 mM 8-Br-cAMP for 48 h decreased microsomal cytochrome P-450 activities, 17 alpha-hydroxylase and C17-20 lyase, by 60-75% and resulted in desensitization of the steroidogenic response . Reduction of the oxygen tension from 19 to 1% O2 prevented the decrease in P-450 activities but not the reduction in steroidogenic capacity . The decrease in activity was also prevented by blocking steroid synthesis with aminoglutethimide . Treatment of cultures with steroid products, androstenedione or testosterone, or product analogs, epitestosterone or 17 alpha-methyltestosterone, at a concentration of 2 microM, which is equivalent to the concentration of testosterone resulting from stimulation with cAMP, also caused oxygen tension-sensitive decreases in hydroxylase activity . Treatment of cultures with 2 microM cortisol, estradiol, or methyltrienolone, an androgen receptor agonist, did not decrease hydroxylase activity, nor did treatment with an androgen receptor antagonist prevent the cAMP- or testosterone-induced decreases in hydroxylase activity . Reductions in hydroxylase and lyase activities resulting from testosterone- or epitestosterone-treatment had little or no effect on acute cAMP-stimulated testosterone production, whereas desensitization with cAMP caused an 80-90% reduction in steroidogenic capacity . 22R-Hydroxycholesterol-supported testosterone synthesis was decreased by both cAMP- and steroid-treatment at 19% O2, but not below the cAMP-stimulated level . Reduction of the oxygen tension partially prevented this decrease . These data are consistent with the hypothesis that the decline in microsomal P-450 enzymes in desensitized Leydig cells results from product (pseudosubstrate)-induced, oxygen-derived, free-radical damage rather than a steroid receptor-mediated process.

J Pediatr Gastroenterol Nutr, 1985 Feb, 4(1), 107 - 17
Cell culture of embryonic chick duodenal cells: preparation of epithelial-fibroblast bilayers and homotypic cultures of fibroblasts and epithelial cells; Zinman HM et al.; A method for the primary cell culture of trypsin-dissociated embryonic chick duodenum is described . Both heterotypic (epithelial cells and fibroblasts together) and homotypic (highly enriched cultures of epithelial cells or fibroblasts alone) cell cultures were established . Dispersed duodenal epithelial cells and fibroblasts grown in 10% fetal bovine serum (FBS) spontaneously aggregated and proliferated as a bilayer of cells with the epithelial cells growing on top of the fibroblasts . Changing the serum supplement to 6% chicken serum (CS) and 4% FBS when the fibroblast monolayer reached confluence resulted in epithelial cell proliferation . Homotypic cultures of epithelial cells and fibroblasts were prepared and analyzed by scanning electron microscopy and transmission electron microscopy . Fibroblasts, isolated by differential adhesion and grown in 10% FBS, did not demonstrate measurable alkaline phosphatase activity . Homotypic epithelial cell cultures, isolated by floating them off the fibroblasts with collagenase, and maintained on collagen in 6% CS/4% FBS, demonstrated higher alkaline phosphatase-specific activity (16.1 +/- 2.3 U/mg protein) compared with epithelial-fibroblast bilayer cell cultures (12.1 +/- 1.3 U/mg protein).

J Antimicrob Chemother, 1985 Feb, 15(2), 209 - 17
Ultrastructural analysis of the effect of trimethoprim and sulphamethoxazole on the development of Chlamydia trachomatis in cell culture; Hammerschlag MR et al.; Folic acid antagonists have been demonstrated to cause gross changes in the morphology of chlamydial inclusions in cell culture . The ultrastructural changes were examined by electron microscopy in cultures of Chlamydia trachomatis treated with trimethoprim and sulphamethoxazole . Examination of these cultures demonstrated a failure of the reticulate bodies to develop into normal elementary bodies . These changes were seen in cultures treated with either compound, although sulphamethoxazole was more active by weight, and suggest that both drugs ultimately interfere with nucleic acid synthesis.

Mol Pharmacol, 1985 Feb, 27(2), 200 - 9
Rapid reciprocal changes in adrenergic receptors in intact isolated hepatocytes during primary cell culture; Schwarz KR et al.; In hepatocytes freshly isolated from adult female rat livers, catecholamine-stimulated glycogenolysis is mediated predominantly by alpha 1-adrenergic receptors, and to only a minimal extent by beta 2 receptors . Primary cell culture of these hepatocytes results in a switch in the adrenergic control of glycogenolysis from an alpha 1 to a predominant beta 2 type of response . To investigate whether this switch is due to an alteration in the plasma membrane receptor composition, we characterized alpha 1 and beta 2-adrenergic receptors in both freshly isolated and cultured hepatocytes, using radioligand-binding techniques . Binding of the selective alpha 1-adrenergic antagonist {3H}prazosin and the beta-adrenergic antagonist {125I}pindolol to intact freshly isolated hepatocytes was of high affinity, saturable, and of appropriate specificity for an alpha 1- and beta 2-adrenergic receptor, respectively . Equilibrium binding studies evaluated by a computer-assisted curve-fitting procedure indicated interaction with a single class of high affinity sites for radiolabeled prazosin (KD = 126 +/- 10 pM; Bmax = 93,000 +/- 5,500 sites/cell) and pindolol (KD = 66 +/- 6 pM; Bmax = 2,000 +/- 700 sites/cell) . In intact hepatocytes and in membranes prepared from these hepatocytes, competitive inhibition curves revealed the coexistence of two different sites with high and low affinities for agonists at both alpha 1- and beta 2-adrenergic receptors . When isolated hepatocytes were kept in monolayer cell culture for up to 72 hr, the switch in adrenergic control of glycogenolysis (phosphorylase a activation) from an alpha to a beta pathway was confirmed and was associated with a progressive decrease in the number of alpha 1 receptors and an increase in beta 2-adrenergic receptor density, without marked change in the affinity of agonists or antagonists . To investigate the mechanism(s) of this reciprocal change, a number of perturbations were examined including alterations in the composition of the culture medium and the influence of various hormones and inhibitors of cellular function . De novo protein synthesis is implicated in both receptor alterations as the inhibitors cycloheximide and actinomycin D prevented the increase in beta- and attenuated the decrease in alpha-adrenergic sites . The other perturbations were without effect . Thus, these studies provide evidence for a coupling of the functional alteration in glycogenolysis to changes at the receptor level per se . The mechanism underlying the reciprocal changes in hepatocyte adrenergic receptors during culture remains undefined.(ABSTRACT TRUNCATED AT 400 WORDS)

Exp Cell Res, 1985 Feb, 156(2), 439 - 49
Communication compartments in mixed cell cultures; Pitts JD et al.; Mixed cultures of epithelial (BRL) cells and fibroblasts (BHK), which sort themselves out into separate domains of each cell type, form communication compartments . Electrical coupling, dye coupling and metabolic coupling measurements have been used to show that small ions and molecules can move freely via intercellular junctions between all the cells in a domain, while their movement across the boundaries between domains is severely restricted . Metabolic coupling is the most sensitive method for detecting trans-boundary communication but the results obtained from all three methods are compatible . The data suggest the reduced transfer across the boundaries is due to fewer channels, resulting from a lower frequency of junction formation between heterologous cells, rather than to channels of smaller diameter . Concentration gradients of small cytoplasmic molecules can be established within these communication compartments which are similar to those predicted to explain pattern formation in developing systems . It is suggested that the cell surface features which cause this sorting out are also responsible for the reduced frequency of heterologous junction formation and hence for compartmentalization.

Environ Res, 1985 Feb, 36(1), 138 - 43
Clastogenicity of a male contraceptive, gossypol, in mammalian cell cultures with and without the metabolic activation by S9 mix; Liang JC et al.; The clastogenicity of a potential male contraceptive, gossypol, was examined in cultured Chinese hamster cells with and without the presence of a metabolic activation system (rat liver S9 mix) . Gossypol at concentrations of 1, 5, and 10 micrograms/ml did not induce chromosome breakage either in the presence or absence of the S9 mix . The ability of this compound to induce chromosome breakage and polyploidy was further examined in human lymphocyte cultures . Neither increased frequencies of chromosome breakage nor polyploidy was found in lymphocyte cultures from two healthy donors . The present study indicates that gossypol does not cause genetic damage at the chromosomal level . This is consistent with previously reported findings that it does not produce mutations in the Ames test . Gossypol has been under clinical trial in China for years and shown to be effective in 99.9% of over 10,000 men tested with no or mild side effects . If this compound can be further proven to be "safe" and approved for world-wide use as a male contraceptive, it would be for the benefit of all mankind.

Proc Natl Acad Sci U S A, 1985 Feb, 82(3), 930 - 4
Effects of phorbol ester on catecholamine secretion and protein phosphorylation in adrenal medullary cell cultures; Pocotte SL et al.; The effects of phorbol 12-myristate 13-acetate (PMA) on catecholamine secretion and protein phosphorylation from intact and digitonin-treated chromaffin cells were investigated . PMA (10-300 nM), an activator of protein kinase C, caused a slow Ca2+-dependent release of catecholamine from intact chromaffin cells that was potentiated by the Ca2+ ionophore ionomycin . PMA also enhanced secretion induced by Ba2+ . In cells with plasma membranes rendered permeable by digitonin to Ca2+, ATP, and protein, PMA (100 nM) enhanced Ca2+-dependent secretion approximately 70% at 0.5 microM Ca2+ and 30% at 10 microM Ca2+ . PMA enhanced the maximal response to Ca2+ approximately 25% and decreased the Ca2+ concentration required for half-maximal secretion approximately 30% . The effects of PMA on chromaffin cells were associated with a 2- to 3-fold increase in the phosphorylation of a 56-kDa protein that may be tyrosine hydroxylase . Other proteins were phosphorylated to a lesser extent . The experiments suggest that PMA increases protein kinase activity and secretion in chromaffin cells and raise the possibility that protein kinase C modulates catecholamine secretion in chromaffin cells.

Mol Cell Endocrinol, 1985 Feb, 39(2), 107 - 13
Glucagon-stimulated cyclic AMP production and formation of estradiol in Sertoli cell cultures from immature rats; Eikvar L et al.; Addition of glucagon to the incubation medium of cultured Sertoli cells isolated from immature (19-day-old) rats resulted in a time- and concentration-dependent stimulation of cAMP accumulation measured both in the cells and in the medium . Maximal intracellular levels of cAMP were reached after 30 min, after which the levels decreased . In the medium cAMP levels reached a plateau after 6 h . The magnitude and kinetics of the responses were comparable to those observed with FSH in the same culture preparations . 1-Methyl-3-isobutylxanthine (MIX), a phosphodiesterase inhibitor, greatly potentiated the magnitude of the effects of glucagon and FSH . Glucagon stimulated adenylate cyclase activity in isolated membrane preparations from similar cultures, and the concentration causing half-maximal stimulation (EC50) was approximately 300 ng/ml . Glucagon also stimulated aromatization in cultured Sertoli cells to the same extent as FSH . It is concluded that cultured Sertoli cells isolated from immature rats contain receptors for glucagon, coupled to adenylate cyclase, and that glucagon also stimulates aromatization of testosterone to estradiol.

J Neurochem, 1985 Feb, 44(2), 495 - 501
Regulation of dopamine release from PC12 pheochromocytoma cell cultures during stimulation with elevated potassium or carbachol; Baizer L et al.; To examine the role of cyclic AMP in the process of catecholamine release experiments have been performed with cultures of PC12 pheochromocytoma cells . Elevated potassium (56 mM) and carbamylcholine (carbachol, 10(-4) M) cause rapid increases in cyclic AMP levels in the cultures that show a time course similar to that of evoked dopamine release . These secretogogue-induced increases in cyclic AMP levels are well correlated with release in terms of relative magnitude and calcium dependence . Forskolin (a direct activator of adenylate cyclase) causes dose-related increases in cyclic AMP levels in PC12 cell cultures that are synergistic with those caused by either elevated potassium or carbachol . At low concentrations forskolin significantly increases evoked release, whereas at higher concentrations it increases both spontaneous and evoked release . These results suggest that cyclic AMP may be involved in the process of dopamine release from PC12 cells in culture.

J Embryol Exp Morphol, 1985 Feb, 85, 163 - 75
Benzamide on chondrocytic differentiation in chick limb bud cell culture; Nakanishi S et al.; Benzamide, an inhibitor of (ADP-ribose) transferase, augmented chondrocytic differentiation of chick limb bud mesenchymal cells in micromass cultures; the incorporation of 35SO4(2-) into the trichloroacetic-acid-insoluble constituents of cell masses as well as the formation of cartilage nodules (Nishio, Nakanishi, Doull & Uyeki, 1983) occurred about 24 h earlier than in untreated cultures and continued to be enhanced in benzamide-treated cultures of stage 23- to 24-chick limb bud cells . Benzamide also significantly increased cell proliferation . However, benzamide did not affect DNA and RNA syntheses except for one period: 24 to 30 h after the start of culture, RNA synthesis was stimulated . From 48 h of culture, (ADP-ribose) transferase activity decreased daily in untreated cultures, whereas benzamide treatment diminished (ADP-ribose) transferase activity 24 h earlier . On the other hand, intracellular NAD levels increased daily in untreated cultures, and benzamide significantly increased the NAD levels above untreated cultures . ATP levels did not differ significantly during the culture period, and benzamide did not affect ATP levels.

Vet Med Nauki, 1985, 22(10), 16 - 9
{Demonstration of the swine fever virus by the infection of cell cultures of lymphocytes and lysed red blood cells}; Chenchev I et al.; A method was worked out for the diagnosing of swine fever by means of infecting sensitive cell cultures on lamellae with lyzed red blood cells and lymphocytes, the demonstration being performed through immunofluorescence . The new method made it possible to avoid killing of suspected animals for diagnostic purposes (in order to prepare suspensions of parenchymal organs and isolate the virus) while the characteristic morphologic changes were still not well manifested.

J Hirnforsch, 1985, 26(5), 555 - 64
Development of embryonic rat brain cells in aggregating cell cultures with special reference to cholinergic functions; Bruckner M; Mechanically dissociated cells from embryonic rat brain regions (telencephalon and metencephalon 15-16 days gestation) reassociate in a rotation culture to form aggregates . These reaggregate cultures show a variety of developmental phenomena, expression of specific cell types and the formation of synapses as the most prominent ultrastructural change . The biochemical maturation is evident in increasing specific activities of choline acetyltransferase (ChAT, EC 2.3.1.6.) and acetylcholinesterase (AChE, EC 3.1.1.7.) . The binding of 3H-quinuclidinylbenzilate(3H-QNB) represents the presence of the muscarinic acetylcholine receptor (mAChR) in these cultures . The expression of the cholinergic character was also shown by histochemical demonstration of specific AChE activity in cryostat sections of reaggregates.

J Am Coll Nutr, 1985, 4(6), 613 - 7
Magnesium variability of lymphocytes from cell culture; Hosseini JM et al.; We determined the magnesium content for two different Burkitt's lymphoma cell lines (EW 36 and CA 46) after transfer to new media for 7 consecutive days . Aliquots of the cell culture were washed and the cell pellet was obtained by centrifugation and lysed with distilled water with and without the addition of 0.25% lanthanum oxide . Magnesium was determined by atomic absorption spectrophotometry . The addition of lanthanum permitted the detection of between 8 and 40% more magnesium . The increased magnesium liberated by the addition of lanthanum was calculated as the "bound" magnesium . The results show that the total magnesium is inversely related and the bound magnesium directly related to the age of the cell culture . Thus, there is a decrease of lymphocyte magnesium content and an increase in the percentage of magnesium bound for these two cell lines with increasing age of the cell culture.

Carcinog Compr Surv, 1985, 10, 177 - 87
Cell culture studies on the mechanism of action of chemical carcinogens and tumor promoters; Weinstein IB; The evolution of a fully malignant tumor is a multistep process resulting from the action of multiple factors, both environmental and endogenous, and involves alterations in the function of multiple cellular genes . Chemical carcinogens that initiate this process appear to do so by damaging cellular DNA . In addition to producing simple point mutations, this damage appears to induce the synthesis of a trans acting factor that can induce asynchronous DNA replication . This response may result in gene amplification and/or gene rearrangement . This phenomenon may also play a role in synergistic interactions between chemicals and viruses in the causation of certain cancers . The primary target of the tumor promoters TPA, teleocidin, and aplysiatoxin appears to be the cell membrane . All three of these agents act, at least in part, by enhancing the activity of the phospholipid-dependent enzyme protein kinase C . We have proposed a stereochemical model to explain the interaction of these amphiphilic compounds with the PKC system . We have found that TPA and teleocidin markedly enhance the transformation of C3H 10T1/2 mouse fibroblasts when these cells are transfected with the cloned H-ras human bladder cancer oncogene . Thus, tumor promoters can act synergistically with an activated oncogene to enhance cell transformation . Furthermore, carcinogen-transformed rodent cells display aberrations in the expression of various endogenous retrovirus-related sequences . Activation of some of these sequences may lead to insertion mutations and further aberrations in gene expression . Thus, multistage carcinogenesis may involve both changes in cellular oncogenes and aberrations in the function of DNA sequences that control gene transcription.

Ultrastruct Pathol, 1985, 8(2-3), 165 - 76
Contribution of electron microscopy of cell cultures to the classification of three round cell sarcomas in children; Harb JM et al.; We have studied three round cell sarcomas from pediatric patients in tissue culture to compare the electron microscopic morphology of cells in culture to cells from original biopsy specimens . None of the original tumors displayed distinctive features by light microscopy that would allow classification of a specific tumor type, and electron microscopy was not helpful in identifying specific morphologic features that would allow further classification of tumor types . However, electron microscopy of cells in culture from the three neoplasms revealed distinctive morphologic features that did allow further classification of all three tumors . Cells from an inguinal lymph node, which were cultured in soft agar tumor colony-forming assay, revealed Z-bands and actin and myosin filaments indicative of a rhabdomyosarcomatous nature for the tumor . Cells from 5-day, 10-day, and 4-month cultures of a bone marrow metastasis of a second tumor revealed features of skeletal muscle in the young cultures and neuroblasts in the older culture, suggesting a primitive neuroectodermal neoplasm . Cultured cells from the third tumor, a neoplasm of the calf in an infant, displayed large lakes of glycogen, typical of cells of Ewing's sarcoma, which were not present in the cells examined from the original lesion . Ultrastructural studies of cells in culture have the potential to add morphologic data that may be useful to further define and classify a neoplasm, as illustrated in the 3 cases reported here.

Prog Clin Biol Res, 1985, 187, 45 - 55
Osteoclasts isolated in primary cell culture--a model to study conditional changes in vitro; Lambrecht JT et al.; Osteoclasts have been isolated out of the bone marrow of laying hens kept on a low calcium diet for eight days . After purification the cells are cultured in a DMEM suspension . Development of the incubated single cells was observed up to 34 days and morphological changes are reported . No major difference could be found if the osteoclasts were kept on acrylic dishes or on gas permeable teflon dishes as a ground substrate.

J Toxicol Clin Toxicol, 1985, 23(4-6), 299 - 308
Effects of dissolved carbon monoxide on the respiratory activity of perfused neuronal and muscle cell cultures; Walum E et al.; In order to address the question of whether small amounts of dissolved CO may inhibit cellular respiration, cultured mouse neuroblastoma cells and primary cultures of chick neurons, rat astrocytes and chick skeletal muscle and heart cells were exposed to CO containing buffer solutions in a closed perfusion system . Oxygen uptake was measured simultaneously with two polarographic oxygen electrodes as the difference in partial pressure of oxygen between the inlet and outlet of the perfusion chamber . After registration of the basal respiratory activity, perfusion solutions containing 5 ul 02/ml were bubbled with CO or N2 at a rate of 200 ml/min for 120 sec . By this procedure the partial pressure of 02 was decreased to reach a value of about 50% of the initial 02 content for both gases . Perfusion was then continued for 30 min at a rate of 0.5 ml/min . The respiratory activity of all the perfused cell cultures, except chick neurons, was found to be inhibited (13-29%) by perfusion solutions bubbled for 120 sec with CO as compared to N2 controls . Of the cells from the nervous system, astrocytes were more sensitive than neurons . Apparently, small amounts of dissolved CO can inhibit cellular respiration in the presence of a physiologically adequate amount of oxygen.

Dev Biol Stand, 1985, 60, 55 - 61
Use of plasma protein fractions as serum substitutes for in vitro cell culture; MacLeod AJ et al.; Fractions produced during routine production of human plasma protein concentrates using cold ethanol precipitation have been tested for their ability to support animal cell growth in vitro . A mouse-mouse hybridoma cell line, ES-6, which secretes a monoclonal antibody (mab) to human group A red blood cell antigen, has been used as a model . Fraction 4-1 protein has been found to be effective at 0.5-1.0 g/l in RPMI 1640 and has supported growth of ES-6 for 18 months, both growth and mab . production being comparable with that achieved in 10% foetal calf serum . Fraction 4-1 protein has been shown to contain albumin, transferrin, alpha-1 antitrypsin and immunoglobulin . It is reported to be a source of somatomedin C . The immunoglobulin content can be reduced . Other mouse and rat hybridomas, human mab . producing lymphoblastoid cells, Namalva and HEP-2 cells have grown well in medium supplemented with Fraction 4-1 protein.

Neuroscience, 1985 Jan, 14(1), 207 - 24
Distinctive structural and cytoskeletal properties of the long-surviving neurons in cell cultures of embryonic spinal cord; Debbage P; A distinctive population of neurons survives for longer than 3 months in cell cultures of chick or rat spinal cord . These neurons form a minor proportion (1%) of the neurons initially developing in the cultures, but are the only ones to survive longer than 30 days in vitro . In addition to their longevity, they share important morphological and cytoskeletal characteristics, which render them distinctive as a group even in early cultures which contain numerous other neurons of short-term viability . Each long-surviving neuron projects one neurite of great length relative to its other neurites, or to those of the shorter-lived neurons, and the length of this neurite is maintained constant for many weeks in vitro . This well-defined morphological feature may indicate the lineage(s) of these neurons . Structurally these cells are very different to the shorter-lived neurons . They are rich in neurofilaments and contain very few microtubules, whereas the shorter-lived neurons contain few neurofilaments but many microtubules . These differences in cytoskeleton coincide exactly with the distinction between limited and prolonged survival in vitro, and the possibility is considered that cytoskeletal stability in the presence of numerous small inflows of calcium might underlie "hardiness" in vitro . The state of development of the long-lived neurons is considered in the context of their shared features, and it is suggested that they may provide a model in which the regulation of development of neuronal function can be analysed.

J Steroid Biochem, 1985 Jan, 22(1), 9 - 14
Rapid method for quantitation of androgen binding protein in Sertoli cell cultures and its use for measurement of binding kinetics; Johnson AR et al.; The accurate measurement of the kinetics of binding of 5 alpha-dihydrotestosterone to the Sertoli cell specific protein, androgen binding protein (ABP), has been frustrated by the extremely rapid rate of dissociation of the ABP-dihydrotestosterone complex . We describe a rapid and highly sensitive assay suitable for ABP quantitation which utilizes DEAE Bio-Gel and {3H}dihydrotestosterone . The assay has been used to accurately measure the rate of dissociation (8.25 X 10(-4) s-1, t1/2 14 min) and the rate of association (2.04 X 10(5) M s-1) of the binding of {3H}dihydrotestosterone to rat ABP . The ratio of these rate constants is in perfect agreement with the equilibrium dissociation constant determined by Scatchard analysis (4.0 nM) . This multipoint assay is extremely rapid such that binding can be measured at equilibrium, it has high precision (coefficient of variation 3%), and is particularly useful at low protein concentrations (50 ng/ml); furthermore, the assay background of nonspecific 3H-binding is extremely low (0.2%) . Since at such low protein concentrations a 10 point Scatchard analysis can be performed on 1 ml culture medium containing as little as 3 fmol ABP, the assay is suitable for monitoring changes in ABP secretion resulting from manipulations of cells in culture . The assay which utilizes DEAE Bio-Gel A is compared to five alternative methods: the standard method of steady state gel electrophoresis, Dextran-coated charcoal assay, hydroxylapatite assay, DEAE filter assay, and radioimmunoassay . The DEAE Bio-Gel assay has advantages over all of these alternative methods . In summary, this new assay is particularly useful for monitoring temporal changes in the secretion of ABP, and the method is equally effective in quantitating ABP in rat, rabbit and hamster Sertoli cell cultures.

J Neurooncol, 1985, 3(2), 137 - 46
Differentiation markers (S-100, GFAP, NSE and D2) in fetal rat brain cells during malignant transformation in cell culture; Laerum OD et al.; Malignant cell lines obtained by ethylnitrosourea (EtNU)-induced transformation of fetal rat brain cells in culture express protein markers of different types of neural cells . These are the nervous system-characteristic S-100 protein; glial fibrillary acidic protein (GFAP); neuron-specific-enolase (NSE), and the D2-cell adhesion molecule . S-100 protein was absent in fetal brain cells in culture, but gradually appeared in the later stages of malignant transformation and further increased at onset of rapid growth of atypical cells (stage IV) . GFAP and D2 were weakly expressed in primary fetal brain cells and did not change throughout malignant transformation . NSE was present in both normal and carcinogen-treated fetal brain cells, and increased at later stages of malignant transformation . From stage III (40-100 days) some cultures were strongly positive and some negative, and the same was seen in the resulting tumorigenic cells about 100 days later . In conclusion the stepwise process of malignant transformation of brain cells in culture ended with a stable phenotype of cells capable of expressing varying types of differentiation markers . The presence of these markers in rat brain cells undergoing malignant transformation may indicate that EtNU given at 18th days of gestation is acting on multipotent neuroectodermal cells.

Vopr Onkol, 1985, 31(6), 73 - 6
{DNA reparative synthesis in liver cell cultures from mice with various susceptibilities to spontaneous hepatic carcinogenesis}; Budunova IV et al.; Ultra-violet radiation-induced unscheduled DNA synthesis in liver cell cultures from CBA, C57Bl/6j and AKR adult male mice was studied autoradiographically . Ultra-violet radiation (254 nm; 1.5-36 J/m2) triggered on an active unscheduled DNA synthesis in all cell cultures studied . The levels of unscheduled DNA synthesis in the liver cell nuclei of the spontaneous hepatocarcinogenesis-susceptible strain (CBA) and relatively, resistant ones (C57Bl/6j and AKR) were similar . It is suggested that the susceptibility to hepatocarcinogenesis in different murine strains is determined by such parameters as DNA damage and repair (stage of initiation) as well as factors peculiar to the stage of promotion.

Int Arch Allergy Appl Immunol, 1985, 77(1-2), 238 - 40
Appearance of basophilic cells/mast cells and IgE antibodies in liquid human cell cultures; Ahlstedt S et al.; Maturation of basophils/mast cells as well as formation of IgE antibodies are parts of the immune response to antigens . We have analyzed these important constituents of the allergic inflammatory response in vitro, cultivating human peripheral blood cells from allergic and normal individuals . Mesenteric lymph nodes were also cultured from two normals . In liquid cultures of peripheral blood cells, the number of small basophils initially present decreased and large basophilic cells appeared over a 2-week period . In normals, the maturation of large basophils from precursor cells could be stimulated with supernatants from peripheral blood cell cultures from atopic individuals stimulated with the relevant allergen . The IgE levels declined in cultures of peripheral blood, whereas the IgE antibodies in cultures of cells from a mesenteric lymph node from one of the nonallergic individuals increased during the 2-week period . In separate cultures, the IgE formation could not be reproducibly stimulated by antigen or mitogen . The study shows the possibility to stimulate the maturation of basophil/mast cell precursor cells present in the blood of allergic and healthy individuals with antigen-induced factors . In contrast, the IgE formation by blood cells was difficult to stimulate.

Eksp Onkol, 1985, 7(2), 9 - 15
{Problem of screening of antineoplastic drugs in tissue and cell cultures}; Dobrynin IaV; Problems on the anticancer drugs' screening in vitro are reviewed on the basis of generalized data from literature . The probability to screen in vitro the active substances is not less than the chance to screen the drugs active against the human tumours in experiments on animals . The use of special test-systems permits screening specific substances including those inducing the tumour cell differentiation.

Leuk Res, 1985, 9(3), 369 - 73
Monitoring the remission induction therapy of acute nonlymphoblastic leukemia by bone marrow cell culture criteria; Bohinjec J et al.; In 31 cases of acute nonlymphoblastic leukemia, bone marrow cells were serially cultured in semi-solid agar during the remission induction therapy . A normal in vitro cell growth pattern returned in 15 out of 22 patients up to 77 days before a complete remission was established by clinical and hematological criteria . In 6 cases the return of normal colonies coincided with clinical and hematological evidence of a complete remission . Nine patients failed to attain a remission and died from complications of bone marrow aplasia . Only one had a normal number of colonies and a normal cluster/colony ratio in cultures prepared 11 days after the completion of the first course of chemotherapy . At this time, his platelet count increased to normal level, possibly indicating a developing remission . Bone marrow cell culture criteria are useful in monitoring the remission induction therapy in patients with acute nonlymphoblastic leukemia . An early return of normal in vitro cell growth pattern suggests an approaching remission, which may be achieved several weeks later.

Basic Res Cardiol, 1985, 80 Suppl 1, 137 - 42
Anoxia in neonatal rat heart cell cultures; Van der Laarse A et al.; Monolayer cultures of heart cells are prepared by dissociation of neonatal rat hearts with collagenase . The regularly and synchronously contracting monolayer is subjected to oxygen and metabolic substrate deprivation for some time (anoxia), and is, in a number of experiments, followed by a short period of oxygen and metabolic substrate repletion (reoxygenation) . Analysed were the frequency and regularity of beating, number of nonvital cells, and enzyme activities and DNA content in the cells as well as in the extracellular medium . We observed that a correlation exists between the released activity of a cytoplasmic enzyme, alpha-hydroxybutyrate dehydrogenase (HBDH) and i) number of nonvital cells, ii) depression of beating frequency measured during reoxygenation, iii) the released activities of enzymes from sarcolemma (L-leucylnaphthylamidase), from lysosomes (N-acetyl-beta-glucosaminidase), and mitochondrial outer membrane (monoamine oxidase) . No correlation exists between the released activity of HBDH and a) the released activity of an enzyme system from the mitochondrial inner membrane (succinate: cytochrome c reductase), and b) the released amount of DNA . Furthermore, reoxygenation of anoxic heart cell cultures leads to a suddenly occurring HBDH release which phenomenon is known as "oxygen paradox".

Tsitologiia, 1985 Jan, 27(1), 99 - 103
{Growth of lymphoid cell cultures of the Raji line in a medium containing low-molecular serum components}; Kruman II et al.; The effect of fetal bovine serum ultrafiltrate, containing low-molecular components (lower than 14 000 D), on the growth of cultures of the lymphoid Raji cells and fibroblasts BHK-21 was studied . The growth of the former did not differ from that in the control (in the medium with the whole fetal serum), while the latter did not proliferate in the medium with the ultrafiltrate . Thus, the growth of the lymphoid Raji cells and fibroblasts BHK-21 is controlled by different serum components . The Raji cells were exposed to an ultrafiltrate (up to 14 000 D) of the adult animal serum whose growth stimulating activity is known to be lower than that of the fetal serum . After the removal of components with molecular weights higher than 14 000 D from the adult animal serum, the growth stimulating activity of such a serum was seen increased, but not up to the level of the fetal serum . BHK-21 cells did not proliferate in the medium with this ultrafiltrate . It is proposed that the increase in the growth stimulating activity of the whole bovine serum in respect to the Raji cells after the removal of the components with high molecular weights may be due to the removal of lymphocyte growth inhibitors whose activity depends on the age of the animal serving a donor of serum.

Res Commun Chem Pathol Pharmacol, 1985 Jan, 47(1), 35 - 47
The involvement of an oxidative mechanism in the adriamycin induced toxicity in neonatal rat heart cell cultures; Julicher RH et al.; In order to investigate the oxidative component of adriamycin-induced cardiotoxicity in the rat, we used neonatal cardiac myocytes in culture . All incubations, with or without adriamycin (ADM), were performed under normoxic circumstances and additionally under circumstances which make cells more vulnerable towards oxidative challenges: hyperoxia or treatment with 1,3-bis(2-chloroethyl)-1-nitrosourea (BCNU) . ADM (100 microM) produced a decrease in the beating rate and enzyme release of the cultures . These effects were potentiated by hyperoxia and by BCNU treatment . Cellular GSH was depleted due to ADM . However, no significant increase in GSSG could be detected, even if the O2-concentration was increased . Lipid peroxidation, measured as thiobarbituric acid reactive material, could be detected only in case ADM plus additional stress were given to the cells . It is concluded that redox-cycling of ADM occurs in rat cardiac myocytes . Formation of ADM-glutathione conjugates or mixed disulfides is strongly indicated . From this it can be inferred that ADM-toxicity in cardiac cells may involve an oxidative mechanism . An important role for the glutathione system is indicated in the detoxification of reactive intermediates . In addition the results implicate that neonatal rat heart cell cultures provide a good screening system for the evaluation of oxidative challenges in the cardiotoxic action of anthracycline analogs.

J Orthop Res, 1985, 3(1), 36 - 42
Cell culture of rabbit meniscal fibrochondrocytes: proliferative and synthetic response to growth factors and ascorbate; Webber RJ et al.; This study was undertaken to determine whether the cells of the fibrocartilaginous meniscal substance are capable of proliferation and matrix synthesis . Cells were isolated from the fibrocartilaginous menisci of young New Zealand white rabbits, and grown in two alternative culture regimens differing only in the basal nutrient medium used to initiate primary monolayer growth . Under each culture regimen, the cells attached and proliferated both initially and after passage into secondary (2 degrees) culture . Differences were noted in cell morphology and time to reach confluence in primary (1 degrees) culture . Upon passage into 2 degrees culture, the fibrochondrocytes assumed two distinct morphologies depending upon the type of medium used for 1 degree culture . These morphological changes were accompanied by differences in the population doubling time and incorporation of 35SO4 into sulfated proteoglycans . The proliferation of both fibrochondrocyte subtypes was stimulated by the addition of either pituitary fibroblast growth factor (FGF) or human platelet lysate in a dose-dependent manner . Both FGF (10 ng/ml) and ascorbate (40 micrograms/ml) decreased 35-sulfate incorporation, whereas only ascorbate was found to alter the amount of sulfated glycosaminoglycan in the pericellular coat . We conclude that the fibrochondrocytes of the meniscal substance are capable of replication and synthesis of matrix macromolecules if given the proper stimuli . Additionally, there may be two subpopulations of fibrochondrocytes that can be distinguished by their in vitro behavior.

Radiat Environ Biophys, 1985, 24(1), 27 - 44
Micronucleus induction in mammalian cell cultures treated with ionizing radiations; Bertsche U; Exponentially growing and plateau phase cultures of Ehrlich ascites tumor cells (suspension strain) were treated with either fast electrons, X-rays, fast neutrons or Am-241-alpha-particles in a dose range from about 0.02 Gy to 1 Gy and for comparison also at higher doses . After the first post-irradiation division, cells were scored for the presence of micronuclei and the micronucleus fraction as well as the number of micronuclei/cell was determined . Micronuclei were counted using the DNA specific stain H 33258 in a fluorescence microscope . A comparison with cytofluorometric measurements established that microscopic detection accounted for up to 90% of all micronuclei present within a sample, the rest probably being hidden in direct observation by the main nucleus . Dose response curves based on the micronucleus fraction as well as on the number of micronuclei/cell were found to be linear in the whole dose range tested at low and at high ionization density . Linearity was maintained also when repair of primary lesions was promoted or suppressed . The RBE of alpha-particles compared with X-rays was dependent on the time of fixation and was at a maximum immediately after the first division (RBE = 4.8 +/- 0.5) . Micronucleus distribution showed overdispersion relative to Poissonian statistics with every radiation quality used, in accordance with earlier observations on the distribution of acentric fragments in irradiated cultures.

J Cancer Res Clin Oncol, 1985, 109(1), 29 - 37
Some properties of Bomirski Ab amelanotic melanoma cells, which underwent spontaneous melanization in primary cell culture . Growth kinetics, cell morphology, melanin content and tumorigenicity; Slominski A; Four types of the Bomirski Ab amelanotic melanoma primary cell culture, differing in the presence of calf serum in the medium and in the cell number used for starting the culture, were employed in the study . In all types of cell culture, rapid melanization occurred in the cytoplasm of the cultured cells . Calf serum in the culture medium stimulated both melanization and proliferation of the Ab melanoma cells . The process of melanin synthesis occurred during the logarithmic phase of growth and was over when the cells reached the plateau phase . Heavily melanized cells changed their adhesive properties, lost the ability to divide in vitro, and showed decreasing tumorigenicity down to complete absence, though they retained some parameters of viability . The rate of melanin synthesis was lower in the cells cultured at high cell density than in those at low cell density . Highly melanized cells that did not divide in vitro but were still tumorigenic in vivo caused the growth of tumors whose morphology was typical for the amelanotic melanoma, melanin being absent . In conclusion, it may be stated that the present findings suggest the persistence of a highly anaplastic and malignant phenotype of Bomirski amelanotic melanoma, being a result of the regulatory action of the host, while the change in the phenotype in vitro does not rule out autoregulatory influences of the tumor itself on its differentiation level and malignancy.

Arch Dermatol Res, 1985, 277(6), 439 - 43
Cell-culture studies on neurofibromatosis (von Recklinghausen's disease) . III . Experiments on X-ray sensitivity; Mao R et al.; The X-ray sensitivity of strains of fibroblast-like cells derived from peripheral neurofibromas of ten patients with neurofibromatosis was compared with that of 12 strains of skin fibroblasts derived from healthy donors . Quantitative parameters of the dose-dependent reduction in colony-forming ability did not differ significantly between these two groups of strains . The cloning efficiencies of nonirradiated controls varied within the same range in strains derived from patients and from healthy donors.

Dev Biol Stand, 1985, 60, 331 - 6
Purification of arachidonic acid metabolites from cell cultures by octadecyl reversed phase cartridges; Bedetti C et al.; The use of rat basophilic leukemia (RBL-1) cell-line as a biosynthetic source of arachidonic acid metabolites has been previously described . We have developed a rapid and effective procedure for the extraction of lipoxygenase and cyclooxygenase products . The cells grown in spinner cultures were harvested and washed in phosphate buffer and stimulated by calcium ionophore A23187 . The ethanolic extract of the cells was dried, dissolved in 20% methanol acidified with acetic acid and applied to octadecyl reversed phase cartridge (Backer C18) . The cartridge was washed with 50% methanol (4 ml) . Prostaglandins were eluted with 75% methanol (4 ml) and leukotrienes and related compounds with 100% methanol (4 ml) . The identification of the fractions was carried out with pure standards . The recoveries of radiolabelled compounds ranged from 70% to 90% . This system can therefore accomplish an initial separation of major arachidonate metabolites and be applied in investigating metabolic pathways of arachidonic acid in different experimental conditions.

J Toxicol Environ Health, 1985, 15(2), 245 - 54
Aroclor 1254 increases the genotoxicity of several carcinogens to liver primary cell cultures; Mendoza-Figueroa T et al.; The genotoxicity of both direct-acting and precarcinogenic chemicals was evaluated in liver primary cell cultures (LPCC) from untreated and Aroclor 1254 (Ar) pretreated rats . Hepatocytes were isolated from partially hepatectomized rats and their DNA was labeled in vitro with {3H} dThd; the molecular weight of single-stranded DNA was determined by alkaline sucrose sedimentation . Two parameters of DNA damage were defined: the mean effective dose (ED50), i.e., the carcinogen concentration that decreased the DNA molecular weight to half the original, and the DNA breaking potency (DBP), i.e., the number of breaks per DNA molecule produced by 2 h exposure to 1 mM concentration of the chemical . Two hours exposure of LPCC from untreated rats to the direct-acting alkylating agent N-methyl-N'-nitro-N-nitrosoguanidine (MNNG) (6.8-340 microM) and to the precarcinogens benzo{a}pyrene (BaP) (0.05-0.33 mM) and dimethylnitrosamine (DMN) (0.45-16 mM) produced a concentration-dependent decrease in the molecular weight of DNA . Pretreatment of rats with Ar decreased significantly the sedimentation velocity of DNA and increased five, three, and two times the DBP of MNNG, BaP, and DMN, respectively . These results show that Ar-pretreatment of rats increases the genotoxicity of both direct-acting and precarcinogenic chemicals and suggest that Ar might increase the genotoxicity of chemical carcinogens perhaps by enhancing their metabolic activation, by producing direct genotoxic effects, or both . Our results also emphasize the carcinogenic risk that the environmental pollution by polychlorinated biphenyls might represent to humans.

Cell Biol Toxicol, 1985 Jan, 1(2), 67 - 73
Toxicity of 6-thioguanine and 8-azaguanine to non-dividing liver cell cultures; Berman JJ et al.; 8-azaguanine and 6-thioguanine were both toxic to non-dividing liver cells in primary cultures . In addition, these agents were toxic to an established line of liver-derived epithelial cells brought to growth arrest by serum deprivation . These observations demonstrate that the toxicity of 8-azaguanine and 6-thioguanine can occur at least in part through mechanisms that do not involve effects on DNA synthesis or incorporation of the analogs into DNA.

IARC Sci Publ, 1985, 67, 77 - 91
Neoplastic transformation in cell cultures: in-vitro/in-vivo correlations; Bertram JS; The behaviour of the C3H 10T1/2 C18 (10T1/2) cell line is reviewed in the context of its ability to reflect accurately events known to occur during carcinogenesis in vivo . It is concluded that, despite their embryonic fibroblastic origin and their infinite life-span in culture, 10T1/2 cells are capable of a wide range of responses to carcinogens and modulators of carcinogenesis that correspond closely to those observed in vivo, for the most part in epithelial tissues . For this and other reasons the 10T1/2 cell line has been widely employed in cancer research.

IARC Sci Publ, 1985, (58), 181 - 202
Cell culture models of multistep carcinogenesis; Barrett JC; The cellular and molecular basis for the multistep process of carcinogenesis can be studied in part by the use of cell culture models . A model system for the study of carcinogen-induced neoplastic transformation of cells in culture is described . Different stages in the neoplastic development of Syrian hamster embryo (SHE) fibroblasts can be identified and quantitated . At least two steps are required for the neoplastic progression of these cells . Morphological alterations are observed early after carcinogen treatment; such cells are preneoplastic and, after further growth in culture, acquire the ability to grow in agar and to form tumours in animals . The induction of morphological transformation of SHE cells occurs within one week after carcinogen treatment in a dose-dependent manner consistent with a one-hit mechanism . Furthermore, this change is induced at frequencies (greater than 1% of the surviving cells) that are higher than those of specific locus mutations . Carcinogens that fail to induce measurable gene mutations induce cell transformation (e.g., diethylstilboestrol, asbestos and arsenic) . All three of these carcinogens induce chromosomal changes--either numerical and/or structural aberrations--suggesting a role for such changes in the action of these carcinogens . Different preneoplastic cells vary in the rate of their progression to anchorage-independent growth and tumorigenicity . Cells with the ability to grow in agar arise at rates of 10(-4) to 10(-7) variants/cell per generation, depending on the preneoplastic cell line . Different preneoplastic cell lines also vary in their sensitivity to induction of neoplastic transformation by mutagens and by transfection with viral oncogenes . This heterogenicity of response suggests different intermediate states of neoplastic progression.

Vet Med Nauki, 1985, 22(9), 45 - 52
{Acetone-celloidin method for the cytological and immunofluorescent study of cell cultures infected with various viruses}; Mitov B; A method was worked out for the extraction of a cell monolayer from test tubes with the use of 10 per cent acetone solution of celloidin . This made it possible to carry out parallel cytologic and immunofluorescent studies of cultures infected with various viruses . In contrast to the alcohol-ether celloidin used in histology the use of the acetone solution of celloidin, suggested by us, preserved to a larger extent the viruses of different structure and composition, and, therefore, was shown to be more suitable in immunofluorescent investigations . The method was employed for the cytologic and the immunofluorescent identification of newly isolated virus agents . It was also used in the controlled replication of noncytopathogenic and slightly cytopathogenic strains of bovine rotaviruses, corona viruses, respiratory-syncytial virus, pestivirus, and others that required roller cultivation . The celloidin method makes it possible to obtain from one to four preparations with material of the cell monolayer of each test tube, and renders it unnecessary to maintain permanently cultures on glass lamellas.

Prog Clin Biol Res, 1985, 184, 215 - 22
Human megakaryocytopoiesis in cell culture; Messner HA et al.; In conclusion, a culture system is now available that reproducibly facilitates the development of megakaryocyte colonies and the emergence of megakaryocytes within multilineage colonies . The assay is dependent upon the use of human plasma and a source of exogeneous MEG-CSA . Patients with perturbed hemopoiesis plasma may contain activities able to replace exogeneous MEG-CSA . This system can therefore be used to evaluate MEG-CSA activities . In addition it is now feasible to study blast populations of patients with leukemia characterized by a megakaryocyte phenotype.

Dev Biol Stand, 1985, 60, 297 - 303
An integrated system for large scale cell culture; Berg GJ; The potential of tissue culture as a means of production of various viruses for use in vaccines, or various biomolecules such as the interferons, interleukins, various enzymes and growth factors, has been steadily increasing over the last few years . If the full potential for the production of these products is to be realized, significant technological advances in the area of large scale cell culture are required . Most cells producing the above mentioned molecules and products grow only when attached to a surface . Therefore, the ability to harness cell culture as a tool for large scale production of products is dependent on the availability of large surface areas that are biocompatible with cell attachment and growth and are presented in such a form that proper nutrient solutions are continually being offered to the cells . This presentation will describe a closed loop circumfusion system with an available surface area scaleble to 72 m2 . This system is integrated with a computer based controller providing programmable monitoring, control, and documentation of the culture . The system has been extensively tested in both in-house and field trials . This testing has shown the system to be compatible with the typical production environment rigors while highlighting the economic potentials of production using this unique system.

Dev Biol Stand, 1985, 60, 171 - 4
Production of rabies vaccine by an industrial scale BHK 21 suspension cell culture process; Pay TW et al.; A process for the production of an inactivated rabies vaccine for animal use is described, whereby the LEP Flury strain of rabies virus is grown in 1 000 L cultures of BHK 21 suspension-adapted cells . The virus is inactivated with aziridine and, after concentration and purification by hollow fibre ultrafiltration, is adsorbed onto aluminum hydroxide gel . The BHK 21 suspension-adapted Master Cell Seed used has satisfied all the requirements of the US Code of Federal Regulations, Title 9, (1977) for cell lines . Cultures prepared from it produce high yields of rabies virus antigen and vaccines with an average potency of 6 IU can be produced without difficulty . Such vaccines give at least 3 years' protection in dogs and are safe to use in all species . Experience gained with the closely analogous process for the production of FMD vaccines suggests that the process can be readily transposed and successfully operated in developing countries if and when it becomes cost-effective to do so.

Gerontology, 1985, 31(2), 65 - 75
Glucocorticoid-induced modulation of insulin-stimulated DNA synthesis: differential responsiveness in cell cultures derived from donors of different ages; Germinario RJ et al.; The replicative ability of variously 'aged' cell cultures, their insulin binding and biological responsiveness under control and glucocorticoid (i.e . hydrocortisone) amplified conditions have been studied in human fibroblast cultures . Insulin stimulation of DNA synthesis in early and late passage cultures and in cultures from young and old donors showed no age-related difference in insulin responsiveness . Hydrocortisone amplification of insulin-stimulated DNA synthesis in early and late passage cells expressed no age-related differences . Hydrocortisone affected basal DNA synthesis in cultures from in vivo young and old donors differently . Additionally, hydrocortisone amplified insulin-stimulated DNA synthesis in young donor cell cultures was observed to be higher than in old donor cell cultures . Specific 125I-insulin binding was increased by hydrocortisone in both early and late passage cultures and in cultures from young and old donors but no age-related differences in 125I-insulin binding were observed in the presence or absence of hydrocortisone . The data suggest that an age-related loss of an insulin postreceptor interaction during hydrocortisone amplification of insulin-stimulated DNA synthesis is being expressed in the cultures from old donors.

Endocrinology, 1985 Jan, 116(1), 398 - 405
Chicken egg white genes: multihormonal regulation in a primary cell culture system; Sanders MM et al.; A primary cell culture from estrogen-withdrawn chicken oviduct has been developed in which the genes for the major egg white proteins, ovalbumin and conalbumin, are induced by steroid hormones . The responsiveness of the isolated cells depends on the combination of enzymes employed to dissociate the oviduct; trypsin and pronase are essential . Administration of estradiol to cultured cells is insufficient to induce ovalbumin mRNA (mRNAov) to levels achieved in vivo . Both insulin and a second steroid (a glucocorticoid, progestin, or androgen) are required for the induction of the ovalbumin gene by estradiol to the extent reached in vivo . Although low levels of a progestin or androgen can synergize with estradiol to obtain an induction of mRNAov, we have demonstrated that a physiological concentration of corticosterone (10(-8) M) potentiates a large response to estradiol without causing an induction when added alone . When estradiol, corticosterone, and insulin are present in the culture medium, mRNAov increases from about 20 to 6500 molecules/cell by 55 h . With the same culture conditions, conalbumin mRNA increases from about 120 to 4300 molecules/cell by 52 h . After about 55 h, the level of egg white mRNAs decreases, and this reduction in mRNAov correlates with a diminished transcription of that gene . The data obtained with the cultured cells demonstrate that the induction of the ovalbumin gene requires the permissive effects of insulin and a second steroid (probably a glucocorticoid in vivo) to facilitate the response to estradiol.

J Cell Biochem, 1985, 28(1), 1 - 6
Interaction of epidermal growth factor with initiators and complete carcinogens in the C3H10T1/2 cell culture system; Herschman HR et al.; Unlike 12-O-tetradecanoylphorbol-13-acetate, epidermal growth factor (EGF) could not promote the appearance of type III foci from initiated C3H10T1/2 cells . At appropriate concentrations, EGF induced the formation of type II colonies in the absence of any initiator . At higher concentrations, EGF suppressed the induction of both type II and type III colonies elicited by methylcholanthrene.

Am J Pediatr Hematol Oncol, 1985 Winter, 7(4), 321 - 6
Serial cell culture and cytogenetic studies of marrow cells from a patient with monosomy 7 syndrome; Wang WC et al.; Serial bone marrow cell culture studies, with cytogenetic characterization, were performed in an 18-month-old boy with monosomy 7 syndrome--a condition that usually converts to nonlymphoblastic leukemia . Over a 12-month course, his marrow demonstrated: decreased colony formation and increased cluster formation in vitro with disease progression (consistent with preleukemia); persistence of only the monosomy 7 karyotype in direct marrow studies; and in contrast, proliferation of both 46,XY and monosomy 7 cells in in vitro marrow cultures . The findings indicate that certain dormant marrow cell populations may be expressed under in vitro culture conditions and suggest the need for determining factors which would allow their expression in vivo.

Acta Anthropogenet, 1985, 9(1-3), 103 - 8
A case of Menkes disease cell culture examination and elastic cartilage electronmicroscopy; Kulczycka H et al.; A boy with clinical characteristics of Menkes disease was described . Extremely low serum copper concentration, low ceruloplasmin level and increased copper accumulation in cultured fibroblasts confirmed the diagnosis . Electronmicroscopy of elastic cartilage showed abnormalities of chondrocyte function and a derangement of extracellular substance polymerization.

Am J Pediatr Hematol Oncol, 1985 Summer, 7(2), 156 - 64
Cytologic diagnosis of the acute nonlymphoid leukemias . II . Flow cytometry, surface markers, cytogenetics, and use of cell culture techniques; Altman AJ; Part I of this two-part article discussed the use of morphologic, histochemical, and ultrastructural studies for the diagnosis and classification of ANLL variants of acute nonlymphoid leukemia . However, a small proportion of acute leukemias are not amenable to definition by these techniques and have, in the past, been classified as acute undifferentiated leukemias . The use of supplemental techniques such as flow cytometry, surface marker analysis, cytogenetics, and in vitro growth patterns will often identify the correct cellular lineage for these cases.

Dev Biol Stand, 1985, 60, 513 - 6
An appraisal of a cell culture test for revealing the presence of E . coli thermolabile enterotoxin (L.T.); Gaicu N et al.; Three methods were comparatively used for revealing the presence of thermolabile enterotoxin (L.T.) of E . coli strains causing diarrheal illness of children . From 228 patients 52 L.T.+ strains (22.8%) were identified with the classic rabbit skin test out of which 50 L.T.+ strains (21.9%) also reacted positively in a cell culture test using CHO-KI cells and only 33 L.T.+ strains (14.4%) in the rabbit intestinal loop test . The cell culture test appears to have a constant sensitivity, it is inexpensive, easy to perform and therefore adequate to be carried out in experienced field laboratories.

Dev Neurosci, 1985, 7(5-6), 340 - 50
Effect of mitogens in various organs and cell culture conditioned media on rat oligodendrocytes; Saneto RP et al.; Extracts prepared from embryonic, neonatal and adult rat brain were examined for the presence of oligodendroglial mitogens . Brain-derived mitogenic activities were found in all developmental stages with specific activity increasing during neonatal development . Extracts from postnatal day 7 contained the highest specific activity . Upon fractionation by molecular weight, each developmental stage expressed a peak of mitogenic activity corresponding to 67,000 daltons . This fraction was able to induce proliferation in cultures grown in serum, serumless chemically defined medium or serum-free medium alone . Neonatal and adult brain extracts had an additional peak of activity at 14,000 daltons . This latter activity was expressed only under serum-supplemented culture conditions . Mitogenic activity was also found in conditioned media from the clonal glioma cell line C6 and primary astrocytes and in extracts derived from neonatal rat liver . These data indicate that a limited range of brain-derived mitogens for oligodendrocytes exist during development and adulthood.

Adv Enzyme Regul, 1985, 24, 155 - 75
Potentiation of antimetabolite action by dibromodulcitol in cell culture; Olah E et al.; The postulation that the activity of key enzymes that reveal marked increases should be potential targets for anticancer chemotherapy (47) was supported by new evidence on the alterations of CDP reductase, CTP synthetase and OMP decarboxylase in hepatoma 3924A cell cultures . Inhibitors of these enzymes (VF-122, acivicin, pyrazofurin) and that of IMP dehydrogenase (tiazofurin) efficiently killed hepatoma 3924A cells in culture, as demonstrated by the clonogenic assay . Acivicin, pyrazofurin, tiazofurin and VF-122 were lethal against tumor cells in the exponential phase of growth with IC50 of 1.5, 5, 10 and 4.5 microM, respectively . All these antimetabolites exhibited cytotoxicity preponderantly against exponential-phase cultures, indicating that all the four drugs belong to Class II (phase-specific agents) in the Kinetic Classification of Anticancer Agents (38) . Dibromodulcitol, a bifunctional alkylating agent, revealed cycle-specific cytotoxicity (Class III agent) against hepatoma 3924A, yielding IC50 values of 2.3 and 5.5 microM for exponentially and stationary growing cells, respectively . Using isobologram analysis on the survival data of 3924A cells, synergistic interaction was observed when DBD in combination with acivicin, pyrazofurin and tiazofurin was examined . DBD in combination with VF-122 exhibited additive lethality against hepatoma cells in culture . The synergistic and additive cytotoxicity in combinations of DBD with these antimetabolites was accompanied by the concurrent depletion of ribonucleotide and/or deoxyribonucleotide pools . The synergistic biological results of drug combinations of acivicin with DBD can be accounted for by the action of acivicin in inhibiting CTP synthetase, resulting in a synergistic decrease in CTP content, and by inhibition of DNA synthesis caused by DBD . The synergistic and additive depletion of UTP, CTP, dTTP and dCTP pools in the combinations of DBD with pyrazofurin may be responsible for the synergistic lethality of these combinations . Synergism, in terms of pool depletion, was observed for GTP and dCTP; summation was detected for dGTP when DBD and tiazofurin were given concurrently . The synergistic cytotoxicity of this drug combination may be a consequence of these alterations . The additive lethality of DBD-VF-122 drug combinations was reflected in the additive elevations of the ribonucleoside diphosphate concentrations . These observations indicate that treatments based on the Kinetic Classification and on the biochemical targeting of the drug should have an impact on the design of in vivo chemotherapy.

Oncology, 1985, 42(5), 317 - 21
Enhanced nitrosourea cytotoxicity in cell culture by sodium butyrate; Vu VT et al.; In HeLa S3 cells, sodium butyrate was found to potentiate the cytotoxicity of chloroethylnitrosoureas and alkylating agents in vitro . Using a soft-agar colony-forming assay, 2.5 and 5.0 mM sodium butyrate pretreatment for 22 h increased the cell killing efficacy of both methyl- and chloroethylnitrosoureas by between 30 and 70% . The potentiation of cytotoxicity of bifunctional nitrogen mustards by butyrate was less than that of nitrosoureas, with a 15-30% increased cell kill at 5 mM butyrate . Sodium butyrate per se reduced plating efficiency and caused growth delay if residual levels (calculated at 100 microM for starting concentrations of 5 mM) were not removed by washing prior to plating.

Nat Immun Cell Growth Regul, 1985, 4(2), 90 - 102
Generation of natural killer-like activity in mixed lymphocyte-tumor cell cultures . I . Role of HLA-DR antigens as stimulatory molecules; Matera L et al.; A number of human lymphoid and non-lymphoid leukemic cell lines differing for expression of HLA-DR antigens were analyzed for the ability to induce natural killer(NK)-like activity and proliferation in lymphocytes from healthy donors . The ability to elicit the generation of NK-like activity in the responder lymphocytes varied greatly depending upon the type of antimitotic treatment (gamma-irradiation versus mitomycin C) received by the tumor cells prior to the start of the mixed cultures . By contrast, the induction of T-cell proliferation was positively correlated with the presence of DR molecules on the tumor cell lines . Nevertheless, DR- leukemic cells pretreated with the appropriate antimitotic agent did induce a proliferative response in the mixed cultures . T lymphocytes cultured without stimulator cells in spent medium containing high levels of cell-free DR antigens failed to undergo blastogenesis and proliferation, indicating that DR antigens can function as stimulatory molecules only when they are cell-associated.

Nat Immun Cell Growth Regul, 1985, 4(2), 103 - 12
Generation of natural killer-like activity in mixed lymphocyte-tumor cell cultures . II . Correlation between expression of HLA-DR antigens on the activated T cells and their cytotoxic capability; Santoli D et al.; In this study, we investigated the role of DR antigens in human mixed lymphocyte reactions (MLR) at the responder cell level . Upon stimulation by allogeneic lymphocytes or leukemic cell lines, a large proportion of T cells underwent blastogenesis and began expressing DR antigens . Analysis by a fluorescence-activated cell sorter revealed that both subpopulations of large activated T cell blasts and of small T lymphocytes became DR+ by synthesis and/or uptake . Depletion of DR+ responder cells from 6-day-old MLRs by treatment with anti-DR monoclonal antibodies (mAbs) plus complement (C) reduced but did not completely abrogate the natural killer (NK)-like activity of the responder lymphocytes, suggesting that the MLR-induced cytotoxic cells include both DR+ and Dr- populations . The expression of NK-like activity by the responder cells was also greatly reduced upon addition of anti-DR mAbs (without C) at the start of the mixed cultures . This effect was observed regardless of the presence of DR antigens on the stimulator cells, indicating that the anti-DR mAbs can interact with the antigens present on both the stimulator and responder populations . These data show that during an MLR, the continued presence of DR antigens on the responding population is essential for the expression and maintenance of the proliferative and cytotoxic capabilities of these cells.

C R Acad Sci III, 1985, 301(7), 361 - 3
{Adaptation to the cell line PLC/PRF/5 of hepatitis A virus released in the cell culture medium}; Crance JM et al.; The propagation of hepatitis A virus (HAV), CF53 strain, released without any cytopathic effect into the PLC/PRF/5 cells supernatant, was studied in the course of six serial passages (6th to 11th) . The decrease (from 5 to 1 week) of incubation time required to detect HAV, by RIA, in culture supernatant, the increase in Hepatitis A antigen (from 777 to 10,038 c.p.m./50 microliter) and infectivity titre (from 10(3.0) TCID 50/ml to 10(4.5) TCID 50/ml) were consistent with the adaptation of this virus to the cell line PLC/PRF/5.

Mol Gen Genet, 1985, 201(1), 136 - 9
Enhancement of genetic transformation frequencies of mammalian cell cultures by damage to the cell DNA; Postel EH; Ultraviolet (UV)-light and 5-fluorodeoxyuridine (FUdR), two known DNA damaging agents, were found to enhance the frequency of stable plasmid transformations in several different animal cell lines . Combined treatment with the two agents was more effective than treatment with either agent alone . A correlation between the transformability of a cell line in the absence of treatment and its response to damaging treatment was also observed . Southern blot analysis of transformed clones indicated that the stimulation in transformation frequency was not due to an increased number of copies of the integrated plasmid in the transformed cells.

Acta Physiol Hung, 1985, 66(2), 121 - 6
Influence of receptor formation and receptor movement inhibitors on hormonal imprinting in cell culture; Csaba G et al.; Hormonal imprinting takes place at the first interaction of the cell with the adequate hormone, and exerts a lasting influence on cellular binding capacity and functional response over many subsequent cell generations . Hormonal imprinting can also be induced in cell lines . In a Chinese hamster ovary (CHO K1) cell line, inhibitor of endocytosis and cellular protein synthesis inhibited hormone binding in themselves, and in cultures preexposed to TSH they inhibited imprinting by TSH in a dose-dependent manner . The protein synthesis inhibitor cycloheximide and the microfilament de-organizing agent cytochalasin-B inhibited imprinting by TSH to a greater degree than all other inhibitors tested, indicating that apart from cellular binding capacity, unimpaired cellular protein synthesis and microfilament activity are essential prerequisites of hormonal imprinting.

Acta Neuropathol (Berl), 1985, 66(3), 233 - 8
Astrocyte differentiation induced by Junín virus in rat brain cell cultures; Berria MI et al.; Morphological and immunocytochemical differentiation was observed in astroglial cell cultures of the rat infected with Junin virus . From days 3 to 6 postinoculation (p.i.), GFAP immunostaining was observed in both the perikaryon and processes of maturated astrocytes, whereas it was limited to the perikaryon in less differentiated cells . The rather slow spontaneous differentiation usually occurring in astroglial cell cultures was seen to be accelerated by viral infection, mimicking the astrocytic reaction formerly described in Junin virus-inoculated mice . Infected cell monolayers showed orderly development, maintenance of contact inhibition, and exhaustion of cell cultures beyond the 6th-7th passages . The morphological and immunocytochemical maturation effects of Junin virus on astroglial cells were evident, but to a significantly lesser degree than those caused by rat brain extract . The glial cell cultures proved a valuable tool for the study of virus-cell interaction, since the immune response and the structural complexity of the whole animal can thus be avoided.

Prog Clin Biol Res, 1985, 178, 289 - 94
Morphology of bluetongue virus-infected Aedes albopictus (C6/36) cell culture; King BM et al.; Morphological changes observed in bluetongue virus (BTV)-infected Aedes albopictus (C6/36) suspension cell culture at 28 C were compared to those observed in mammalian stationary cell culture at 37 C . The presence of cytoplasmic macrotubules, viroplasms and progeny virions was confirmed and appears to be the same as seen in mammalian cell culture . In addition progeny virions were observed budding from cell membranes where they appeared to acquire a lipoprotein envelope . Intranuclear macrotubules that are the same diameter as the cytoplasmic macrotubules were also seen . C6/36 cell culture is a model for gaining insight into BTV-replication in the insect host.

Jpn Heart J, 1985 Jan, 26(1), 81 - 9
Adult rat heart cell culture . The morphological study of tissue fragments on various environments in culture; Yamana K et al.; This paper reports a modification of Jacobson's method for culturing adult rat heart cells after mechanical and enzyme treatment . For heart cell dissociation, it is useful to wash tissue fragments 3 times with saline A solution after enzyme treatment, and it is important to change the medium immediately after culturing heart cells in order to prevent myocardial cells from degeneration caused by the enzyme solution and toxins in the cell debris.

J Recept Res, 1985, 5(1), 45 - 57
Relationship between hCG receptors disappearance and steroid accumulation during long term hCG stimulation in porcine Leydig cell cultures; Mombrial FC et al.; LH-hCG receptors and steroidogenesis can be maintained for more than a week in porcine Leydig cell culture (chemically defined medium) . Free receptors and steroid accumulation were measured following long time exposure to hCG . As expected, the percent of receptor disappearance increases with the hCG concentration in the incubation medium . However, the steroidogenesis was also proportional to hCG concentration for 24 hours as well as 48 hours . A 50% receptor disappearance was obtained for 0.5 ng hCG/ml which led to a steroid accumulation of 50% of the maximal accumulation obtained with 100 ng/ml . A very significative positive correlation (rho) was observed between receptor occupancy (internalized and/or irreversibly occupied receptors) and steroid accumulation (T and DHAS) during a 24 or 48 hour period (rho = 0.97 and 0.98 for T, and rho = 0.96 and 0.94 for DHAS) . Following 24 or 48 hour exposure to increasing hCG concentration, the cells were acutely stimulated by 25 ng hCG/ml for 4 hours . The steroidogenic response obtained to this acute stimulation was inversely proportional to the concentration of hCG during the previous period . A negative correlation was found between receptor occupancy and acute steroidogenic response (rho : -0.96 and -0.98 for T) . These results indicate that during long term stimulation as well as during acute restimulation the absolute number of occupied receptors conditioned the steroidogenic response . These results do not fit into the concept of spare receptors and suggest that in these immature cells all receptors are potentially active in the regulation of steroidogenesis.

J Cell Biochem, 1985, 27(4), 443 - 8
A beta-type transforming growth factor, present in conditioned cell culture medium independent of cell transformation, may derive from serum; Stromberg K et al.; An alpha-type transforming growth factor (TGF alpha) is produced at high levels by rat embryo cells transformed by the Snyder-Theilen strain of feline sarcoma virus (FeSV) . Addition of 2 ng mouse epidermal growth factor (mEGF) during purification identified the presence of a second, EGF-dependent growth factor of the TGF beta type (TGF beta) in this conditioned medium . This factor had an approximate Mr of 12,000 and eluted at 37% acetonitrile during high performance liquid chromatography . This extracellular type of TGF beta activity also was present in conditioned medium of rat cells after infection with a transformation defective strain of Abelson leukemia virus, and hence expression of this growth factor activity was independent of cell transformation . Moreover, the presence of an EGF-dependent, 12,000 Mr clonogenic activity in extracts of bovine serum alone suggests serum as an origin for the B-type transforming growth factor initially observed in conditioned medium of Snyder-Theilen FeSV transformed cells . This does not, however, preclude the possibility that TGF beta is also secreted by the transformed rat embryo cells themselves.

Microbiologica, 1985 Jan, 8(1), 17 - 22
Susceptibility of various cell culture systems to bovine viral diarrhea virus; Ferrari M; A comparative study was carried out on the susceptibility of primary bovine embryo kidney (pBEK) cell cultures and those of three other cell culture systems, i.e., primary calf testicle (pCT) cells and two cell lines, one of which originated from bovine embryonic trachea (EBTr) and the other from buffalo lung (IMR-31), to bovine viral diarrhea (BVD) virus . The virus titers obtained in the three culture systems under study did not differ significantly from those of pBEK cells . Moreover, the growth curve study demonstrated thad the pCT cells, as well as the two cell lines, were similar to the pBEK cultures in their susceptibility to the virus . Accordingly, it appeared reasonable to conclude that pCT, EBTr and IMR-31 cell cultures possess the general requirements for use in studies on the BVD virus . However, inasmuch as the cytopathic effects (CPE) produced by the EBTr and the IMR-31 cell lines did not display any distinctive characteristics typical of BVD virus-induced CPE, it is recommended that the two cell lines not be used for isolation of the virus from specimen material.

J Virol Methods, 1985 Jan, 10(1), 59 - 68
Detecting bluetongue virus RNA in cell culture by dot hybridization with a cloned genetic probe; Squire KR et al.; A 70% copy of genome segment 7 of bluetongue virus (BTV)-17 has been cloned into the plasmid pBR-322 . This cloned BTV segment when used as a radioactive probe will hybridize to BTV double-stranded RNA extracted from cell cultures and dotted onto nitrocellulose paper . This dot hybridization technique is therefore suitable for detecting and identifying BTV in cell culture . The specificity of cloned probes is discussed in relation to detecting gene sequences specific for either the bluetongue serogroup or different serotypes of BTV.

Epilepsia, 1985 Jan-Feb, 26(1), 58 - 68
Effects of phenytoin on primary glial cell cultures; White HS et al.; The activity of enzymes involved in anion and cation transport, the concentration of intracellular potassium (K+i), and the transmembrane potential (Em) were determined following acute and chronic exposure of primary astroglial cultures to micromolar concentrations of phenytoin (PHT) . Na+, K+-ATPase activity of homogenates of cultured glial cells was determined in the presence of an increasing K+ concentration (1-20 mM) . Acutely, PHT had little effect on the K+ activation pattern of Na+, K+-ATPase . In contrast, the percentage of Na+, K+-ATPase activated by elevating the K+ concentration was dose dependently increased by chronic PHT treatment . This effect was accompanied by a marked increase in K+i and a significant membrane hyperpolarization . The acute effect of PHT on the Em was biphasic, characterized by membrane hyperpolarization at concentrations of 10(-6)-10(-5) M; at concentrations between 10(-5) and 10(-4) M, the Em progressively returned to control values . These results suggest that glial cells acutely and chronically treated with therapeutic concentrations of PHT have an enhanced capacity to control elevated extracellular potassium levels . Return of the Em to control values at PHT concentrations greater than 10(-5) M suggests that these cells are less able to regulate extracellular potassium . These data can partially explain the excitatory effects of PHT at high therapeutic concentrations.

Arch Virol, 1985, 83(3-4), 305 - 10
Induction of Spodoptera littoralis nuclear polyhedrosis virus in cell cultures; Saldanha JA et al.; A nuclear polyhedrosis virus (NPV) infection was initiated in a Spodoptera littoralis cell line when haemocytes from granulosis virus (GV)-infected S . littoralis larvae and ultra-violet-inactivated Sendai virus were added.

Arch Virol, 1985, 83(1-2), 123 - 7
Isolation of ovine rotavirus in cell cultures . Brief report; Makabe T et al.; Three cytopathic rotavirus strains were isolated in MA 104 cells from intestinal contents of lambs with diarrhea . Pretreatment of virus with pancreatin and incorporation of a small amount of pancreatin in maintenance medium were important for establishment of the strains in these cells . The isolates showed marked cross reactions with bovine rotavirus in immunofluorescence, but no cross reaction with bovine, human, simian, equine, porcine and lapine rotaviruses in neutralization.

Neuroendocrinology, 1985 Jan, 40(1), 72 - 7
Beta-adrenergic stimulation of adenosine-3',5'-monophosphate (c-AMP) accumulation and of prolactin and growth hormone secretion in rat anterior pituitary cell cultures; Swennen L et al.; The beta-adrenergic agonist 1-isoproterenol (ISO) (10(-9)-10(-7) M) provoked a prompt and profound increase of intracellular c-AMP accumulation in monolayer cultures of rat anterior pituitary . In superfused reaggregate cell cultures ISO also stimulated c-AMP outflow . The effect was blocked by propranolol and the highly selective beta 2-receptor blocker ICI 118.551 . Epinephrine (E), norepinephrine (NE) and the highly selective beta 2-agonist zinterol (ZIN) also stimulated c-AMP accumulation . The order of potency ZIN greater than or equal to ISO greater than E much greater than NE together with the high potency of ICI 118.551 suggests the beta-effect is mainly through the beta 2-receptor subtype . The same concentrations of ISO strongly stimulated growth hormone (GH) release and, as previously shown, prolactin (PRL) release from superfused reaggregate cell cultures, but not luteinizing hormone or thyroid stimulating hormone release . Stimulation of PRL and GH release from these cultures was also induced by the adenylyl cyclase activator forskolin . Since interference of beta-adrenergic effects on contaminating fibroblasts or endothelial cells could be reasonably excluded, the present data suggest that beta-adrenergic stimulation of c-AMP accumulation in anterior pituitary cells elicits GH and PRL release.

Auris Nasus Larynx, 1985, 12 Suppl 2, S226 - 33
Generation of human cytotoxic T lymphocytes against autologous solid tumors by autologous mixed lymphocyte tumor cell culture with T cell growth factor; Ichino Y et al.; Autologous mixed lymphocyte tumor cell cultures (MLTC) generated cytotoxicity against autologous tumors in 5 out of 15 (33%) patients with head and neck cancer . Further cultivation of MLTC-activated lymphocytes with T cell growth factor (TCGF) resulted not only in the propagation of cytotoxic T lymphocytes (CTL) and an increase of cytotoxicity against autologous tumors, but also in the induction of killer cells against allogeneic tumor cells . The results of crisscross tests using fresh tumor cells from 17 donors and lymphocytes from 10 donors indicate that cultivation of MLTC-activated lymphocytes with TCGF generated killer cells cytotoxic for allogeneic tumors in most cases, but not for autologous or allogeneic phytohemagglutinin-induced lymphoblasts . Further, the results of a cold target inhibition test undertaken in an autologous tumor killing system suggest that at least 2 different subsets, specifically cytotoxic for autologous tumors and cytotoxic for both autologous and allogeneic tumors, were developed in the CTL induced by autologous MLTC followed by cultivation with TCGF . The phenotypes of the effector cells were shown to be OKT3+, Leu2a+, OKT8+, OKM1-, and Leu7- by blocking assay using complement and monoclonal antibody against the T cell surface marker.

Dev Biol Stand, 1985, 61, 205 - 14
Involvement of filamentous hemagglutinin in the adherence of Bordetella pertussis to human WiDr cell cultures; Urisu A et al.; Attachment of 35S-methionine-labelled Bordetella pertussis to monolayers of WiDr cells, an epithelium-like cell line from a human intestinal carcinoma, was examined . The amount of adherence was proportional to the density of the WiDr cells and to the concentration of B . pertussis in the assay . The amount of attachment of virulent strains Tohama phase I, 114 and 338 was much greater than the attachment of avirulent strains, Tohama phase III and 423 phase IV . Attachment of strain Tohama 325, a spontaneous mutant of Tohama I, and strains 353 and 354, Tn5 insertion mutants of 338 was also examined . These mutants have phenotypic properties similar to the parent strain, except the mutants are deficient in the production of the filamentous hemagglutinin (FHA) . The FHA deficient mutants adhered to WiDr cells much less efficiently than the parent strains . Goat antibody to FHA inhibited, in a dose dependent manner, the attachment of Tohama I, but not of strain 325 . At the same protein concentrations, normal goat antibody or goat antibody to the lymphocytosis-promoting factor had no effect on attachment . Preincubation of bacteria with FHA enhanced the attachment of Tohama I, Tohama 325, and Tohama III . These data support the involvement of FHA in the adherence of B . pertussis to human WiDr cell cultures.

Histochemistry, 1985, 82(5), 497 - 500
DNA synthesis in lactotrophs of primary rat anterior pituitary cell cultures . Effects of thyroliberin, bromocriptine and somatostatin; Komolov IS et al.; Prolactin immunostaining in combination with thymidine autoradiography was used to characterize changes in the DNA-synthesizing activity of lactotrophs in primary monolayer cultures of the rat anterior pituitary gland treated for 3 days with thyroliberin (TRH), somatostatin (SRIF) and bromocriptine (CB 154) . The number of lactotrophs labelled with 3H-thymidine within the total pool of labeled pituitary cells was used to estimate DNA synthesis in prolactin-producing cells . TRH (10 ng/ml) stimulated DNA synthesis in the whole population of cultured cells but not in lactotrophs . TRH only weakly counteracted the noticeable inhibitory effect of CB 154 (0.75 microM/l) on thymidine incorporation into lactotrophs . SRIF (20 ng/ml) inhibited DNA synthesis in lactotrophs to a lesser extent than CB 154 . The combination of methods used in this paper may be useful for studying the selective effects of regulators on the proliferative activity of various pituitary cell types in vivo and in culture.

Neuroscience, 1985 Jan, 14(1), 349 - 60
Regulation of acetylcholinesterase secretion from neuronal cell cultures.--1 . Actions of nerve growth factor, cytoskeletal inhibitors and tunicamycin; Lucas CA et al.; The release of enzymatically active acetylcholinesterase was measured from two types of neuronal cell cultures . Evidence is presented that primary cultures of chick optic lobe and clonal rat PC12 cells released acetylcholinesterase by similar mechanisms . The secretion was selective for the tetrameric or larger molecule forms of acetylcholinesterase . Thus the chick primary cultures released the so-called G4 isoenzyme of acetylcholinesterase . The rat PC12 cells, after pretreatment with nerve growth factor, secreted mainly G4 acetylcholinesterase and a small amount of an isoenzyme sedimenting at 16S . The rate of secretion of acetylcholinesterase was regulated by the three types of agents used . (1) Acetylcholinesterase secretion was markedly increased by nerve growth factor . (2) Microtubule inhibitors (colchicine, taxol and nocodazole) suppressed the rate of release of acetylcholinesterase . (3) Acetylcholinesterase release was also decreased by an inhibitor of protein glycosylation, tunicamycin . We provide evidence that these three types of agents regulate acetylcholinesterase secretion not by a direct effect on secretion, but by modulatory effects on the synthesis, intracellular transport and turnover of the enzyme . (1) Nerve growth factor acts on acetylcholinesterase secretion via increased synthesis of messenger RNA . (2) Microtubule inhibitors act by preventing intracellular transport . (3) Tunicamycin acts by decreasing the intracellular activity of acetylcholinesterase, possibly via enhanced proteolytic breakdown.

J Inherit Metab Dis, 1985, 8(1), 25 - 32
Inability of second trimester human amniotic fluid cell cultures to aromatize C19-steroids; O'Shannessy DJ et al.; Amniotic fluid (AF) and fibroblast (F) cell types, derived from human amniotic fluid by amniocentesis in the second trimester, were cultured using media of varying composition and their ability to transform C19-steroids to oestrogens was investigated by fluorimetry, radioimmunoassay, tritium release and measurement of cytochrome P450 . Although the cells were shown to be metabolically active by the presence of mitotic figures, the incorporation of tritiated leucine into protein and the production of the beta-subunit of hCG, no evidence of aromatization was obtained despite the inclusion in the medium of a number of steroids known to be transformed to oestrogens in several in vivo and in vitro preparations . The addition of reported stimulants of aromatase activity, viz . hCG, FSH, diethylstilbestrol, prostaglandins E2 and F2 alpha and dibutyryl cyclic AMP, was without effect.

Arzneimittelforschung, 1985, 35(8), 1209 - 15
{In vitro methods for testing of plant derived, bacterial and synthetic agents and toxins with the use of leukocyte primary cell cultures and cell lines}; Bessler WG et al.; In this article a variety of in vitro methods is presented to test bacterial, plant-derived and synthetic products on eukaryotic cells . These in vitro methods give a lot of valuable information and thus in some cases may substitute for animal experiments . The influence of plant-derived substances on the macromolecular cellular syntheses (RNA-, DNA- and protein syntheses) and on cell membranes (incorporation of oleate into membrane lecithin, histamine release, cell lysis) was investigated . The in vitro experiments were performed on primary leucocyte cultures with lima bean lectins, suzukacillin, trichotoxin, alamethicin, cianidanol, and beta-hydroxy-ethylrutoside . Also various cell activating compounds were used to test the stimulation of mitosis and the production of immunoglobulins in B-lymphocytes: lipopolysaccharide (endotoxin), lipoprotein, and the synthetic lipoprotein analogues tripalmitoyl pentapeptide (TPP) and tripalmitoyl cysteine (TPC) . Moreover we studied the effects of these substances on the incorporation of unsaturated fatty acids into the cell membrane constituents . Plant-derived and synthetic metal-complexing agents and polyethyleneglycols were also tested for synergistic or inhibitory effects on mitogen activated primary cell cultures and lymphoid cell lines . In many cases, the conclusions from the in vitro experiments may be extended to predict the results of in vivo experiments . In some cases, in vitro results are definitive, therefore experiments with animals can be avoided.

Acta Derm Venereol, 1985, 65(5), 379 - 84
Studies on fibronectins in the skin . VIII . Influence of corticosteroids on cell cultures from normal human skin; Fyrand O; Fibronectins are important glucoproteins of mesenchymal tissue . Fibronectins are also found in the human skin, and tissue cultures demonstrate the production of soluble dimers and insoluble fibrous polymers from dermal fibroblasts . Under the influence of different glucocorticoids, inhibited production of these fibronectins from cultured human skin cells is demonstrated.

Int J Cancer, 1984 Dec 15, 34(6), 781 - 8
The proliferative response of low-density human cell cultures to tumor promoters and its relevance to carcinogenic mechanisms in vitro; Kopelovich L et al.; The effects of the tumor promoter -12-O-tetradecanoyl phorbol-13-acetate, its analogues, and other tumor promoters, on the proliferation of human skin fibroblasts (SF) have been investigated . We have previously shown (Kopelovich and Bias, 1979), that TPA caused a biphasic (upward concave) dose effect in the cloning efficiency assay of normal and mutant human fibroblastic cell strains . Here we report that the biphasic dose response pattern, consisting of an inhibitory phase and a stimulatory phase, was shared by the active analogues of TPA, e.g., phorbol-12,13-dibutyrate and phorbol-12,13-dibenzoate . This biphasic dose effect relationship, however, was not seen with phorbol-12,13-diacetate or the inactive analogues of TPA such as phorbol and 4-O-methyl-12-O-tetradecanoyl phorbol-13-acetate, nor was it seen with mezerein, teleocidin, or bile-acid derivatives of humans . An analysis of the cloning efficiency data by the median-effect equation (Chou and Talalay, 1981) showed that in low-density cultures both the inhibitory phase and the stimulatory phase of the dose-effect relationships of TPA, its analogues, mezerein, and teleocidin exhibited a linear median-effect plot and thus closely followed the basic mass-action principle . The median-effect plot of these data allowed quantitative determination of growth curve characteristic such as regression coefficient, slope (a measure of sigmoidicity), median-effect concentrations such as I50 for the inhibitory effect, and A50 for the stimulatory effect (i.e., the relative potency of the analogues) and the transition point of the biphasic phenomenon in the case of the phorbol esters . In addition, we have demonstrated a relationship between the dose response effect of TPA on the proliferation of various human cells and tumor progression in vitro.

Am J Clin Oncol, 1984 Dec, 7(6), 613 - 6
Encapsulated iodine-125 in radiation oncology . II . Study of the dose rate effect on potentially lethal damage repair (PLDR) using mammalian cell cultures in plateau phase; Marchese MJ et al.; Potentially lethal damage repair (PLDR) is considered to be an important mechanism of cellular radioresistance . Studies have suggested that radioresistant human tumor cells may have increased PLDR capability . Studies have also shown that PLDR may be decreased with smaller dose fractions . PLDR occurring with low dose rate continuous irradiation (LDCI) has not been assessed . The C3H/10T1/2 murine cell line in plateau phase (15% cycling fraction) was used to study the effect of dose fraction size and LDCI on PLDR . The cells were found to have substantial PLDR capability . Uncorrected data showed increased PLDR with higher single fractions . However, when the single fraction results are extrapolated to multiple fractions to give equivalent cell survival for the large and small fractions, no difference in PLDR was observed . The data from the LDCI experiments demonstrated some survival enhancement with delayed plating after 5 and 8 hours of irradiation, but not after 16 or more hours . The results indicate that PLDR occurs during the LDCI and that the magnitude of PLDR is similar for LDCI relative to acute irradiation.

Am J Clin Oncol, 1984 Dec, 7(6), 607 - 11
Encapsulated iodine-125 in radiation oncology . I . Study of the relative biological effectiveness (RBE) using low dose rate irradiation of mammalian cell cultures; Marchese MJ et al.; The use of encapsulated iodine-125 seeds has increased considerably since 1965, due largely to their physical characteristics . The 28 keV x-ray emission offers improved radiation protection and rapid fall-off of dose outside the treatment volume . The Relative Biological Effectiveness (RBE) of the low energy I-125 x-rays has not been adequately assessed . The limited studies have found a wide range of values, but most are between 1.2-1.5 relative to hard x-rays . We used C3H/10T1/2 mouse embryo cells in contact inhibited plateau phase to assess the RBE of I-125 seed x-rays relative to Cs-137 gamma rays (660 keV) using low dose rate continuous irradiation . Replicate experiments found the RBE to be 1.2 . This did not vary with dose rate over the range of 10-76 cGy/hour . Calculations made from our cell survival data suggest that, in the case of permanent I-125 implants, where the dose is administered over a considerable period of time, the resultant surviving fraction of tumor cells is dictated largely by the length of the cell cycle . It is suggested that, for this reason, permanent I-125 implants may be less suitable for rapidly growing tumors, such as glioblastomas, than temporary I-125 implants, which are calculated to be virtually independent of the cell cycle duration.

J Appl Toxicol, 1984 Dec, 4(6), 297 - 303
Dimethylnitrosamine genotoxicity in rat liver primary cell cultures with low cytochrome P-450 levels; Mendoza-Figueroa T; Liver primary cell cultures (LPCC) with decreasing concentrations of cytochrome P-450 were used to investigate the genotoxicity of the hepatic carcinogen dimethylnitrosamine (DMN) and the correlation between DMN genotoxicity and cytochrome P-450 levels . Hepatocytes were isolated from partially hepatectomized rats and incubated with {3H}thymidine; single-strand DNA molecular weight was determined by alkaline sucrose sedimentation . The molecular weight of DNA decreased 50% in LPCC plated either 2 or 24 h before being treated for 24 h with 70 micron DMN . Cytochrome P-450 content was 188 pmol per mg protein in freshly isolated hepatocytes, whereas it was 70 and 32 pmol per mg protein in hepatocytes that had been cultured 24 and 48 h, respectively . Incorporation of 14C into acid-insoluble material was the same in LPCC exposed 24 h to {14C}DMN starting either 2 or 24 h after cell plating . At non-toxic concentrations (0.01-1 microM), SKF 525-A, an inhibitor of mixed-function oxidase enzymes, inhibited approximately 20% of the binding of 14C from {14C}DMN to acid-insoluble material in LPCC plated either 2 or 24 h before they were exposed to DMN for 24 h . Hepatocyte cultures exposed to the direct-acting alkylating agent N-methyl-N'-nitro-N-nitrosoguanidine (at concentrations ranging between 6.8 X 10(-8) and 6.8 X 10(-5) M) starting 2 and 24 h after plating, exhibited significant unscheduled DNA synthesis . These results indicate that DMN genotoxicity was similar in LPCC differing considerably in cytochrome P-450 levels, and they suggest that DMN genotoxicity in these cultures is due mainly to similar DMN activation than to decreased DNA repair.

Fed Proc, 1984 Dec, 43(15), 2991 - 3
Cell culture mutants as aminoacyl-tRNA synthetase complex probes; Hampel A et al.; A number of Chinese hamster ovary cell culture mutants have been characterized by us for their high-molecular-weight aminoacyl-tRNA synthetase (aaRS) complexes . The results all support a model of aaRS complex function wherein the high-molecular-weight complexes form aminoacyl-tRNA by utilizing extracellular amino acids immediately on their transport and before they have equilibrated with the internal pool . The low-molecular-weight aaRS forms exclusively utilize amino acids from only the intracellular pool.

Br J Urol, 1984 Dec, 56(6), 655 - 7
Choosing the right intravesical chemotherapeutic agent . Results of an in vitro monolayer cell culture assay; Whelan P et al.; The use of a monolayer cell culture technique is described . Twenty-four superficial bladder tumours have been tested against Thiotepa, Adriamycin and Mitomycin C . Cultures were set up in parallel for a clonogenic assay after the method of Salmon . In the monolayer all 24 tumours grew; 21 showed a greater than 50% inhibition to Mitomycin, 13 to Adriamycin and 6 to Thiotepa . In the clonogenic assays, only 12 grew satisfactorily . It is concluded that this monolayer assay gives a reasonable prediction of the sensitivities of the commonly used intravesical agents.

J Cell Physiol, 1984 Dec, 121(3), 558 - 68
The in vitro cell culture age and cell density of articular chondrocytes alter sulfated-proteoglycan biosynthesis; Malemud CJ et al.; The effect of cell culture age and concomitant changes in cell density on the biosynthesis of sulfated-proteoglycan by rabbit articular chondrocytes in secondary monolayer culture was studied . Low density (LD, 2 d), middle density (MD, 5-7 d), and high density (HD, 12-15 d) cultures demonstrated changes in cellular morphology and rates of DNA synthesis . DNA synthesis was highest at LD to MD densities, but HD cultures continued to incorporate {3H}-thymidine . LD cultures incorporated 35SO4 into sulfated-proteoglycans at a higher rate than MD or LD cultures . The qualitative nature of the sulfated-proteoglycans synthesized at the different culture ages were analyzed by assessing the distribution of incorporated 35SO4 in associative and dissociative CsCl density gradients and by elution profiles on Sepharose CL-2B . Chondrocytes deposited into the extracellular matrix (cell-associated fraction) 35SO4-labeled proteoglycan aggregate . More aggregated proteoglycan was found in the MD and HD cultures than at LD . A 35SO4-labeled aggregated proteoglycan of smaller hydrodynamic size than that found in the cell-associated fraction was secreted into the culture medium at each culture age . The proteoglycan monomer (A1D1) of young and older cultures had similar hydrodynamic sizes at all cell culture ages and cell densities . The glycosaminoglycan chains of A1D1 were hydrodynamically larger in the younger LD cultures than in the older HD cultures and consisted of only chondroitin 6 and 4 sulfate chains . A small amount of chondroitin 4,6 sulfate was detected, but no keratan sulfate was measured . The A1D2 fractions of young LD cultures contained measurable amounts of dermatan sulfate; no dermatan sulfate was found in older MD or HD cultures . These studies indicated that chondrocytes at LD synthesized a proteoglycan monomer with many of the characteristics of young immature articular cartilage of rabbits . These results also indicated that rapidly dividing chondrocytes were capable of synthesizing proteoglycans which form aggregates with hyaluronic acid . Culture age and cell density appears primarily to modulate the synthesis of glycosaminoglycan types and chain length . Whether or not these glycosaminoglycans are found on the same or different core proteins remains to be determined.

Endocrinol Jpn, 1984 Dec, 31(6), 749 - 53
In vitro study on release of thyroid hormone in solitary autonomously functioning thyroid nodules using cell culture method; Sugenoya A et al.; In vitro release of thyroid hormone was investigated under basal and TSH-stimulated conditions in the solitary autonomously functioning thyroid nodules (AFTN) . A small portion (0.5 g of wet weight) of the nodules and adjacent thyroid tissues removed surgically from five patients with solitary AFTN were prepared for the dispersed cell culture . In the experiment on non TSH-stimulated (basal) conditions, those culture media which were totally replaced on the 5th day after primary culture were utilized for the determination of thyroxine (T4) and triiodothyronine (T3) by radioimmunoassay . T4 and T3 levels in culture media of the functioning nodules were 1.15 +/- 0.33 microgram/dl (mean +/- SEM) and 2.72 +/- 0.68 ng/ml, contrasted with levels of 0.67 +/- 0.09 microgram/dl and 1.24 +/- 0.22 ng/ml in the paranodular tissues . The mean ratios of T3/T4 of the nodules and paranodular tissues were 0.25 +/- 0.02 and 0.19 +/- 0.02, respectively (p less than 0.05) . Meanwhile, in another experiment under TSH stimulatory conditions employing 40 and 80 microU/ml of human TSH, there were no significant differences in T4 and T3 releases when the two groups were compared.

Endocrinology, 1984 Dec, 115(6), 2311 - 7
Inhibition of luteinizing hormone (LH)-releasing hormone-induced secretion of LH in rat anterior pituitary cell culture by testosterone without conversion to 5 alpha-dihydrotestosterone; Liang T et al.; The role of 5 alpha-reduction of testosterone in the inhibition of LH secretion was investigated in rat anterior pituitary cell cultures . Pituitary cells were preincubated with testosterone or dihydrotestosterone (17 beta-hydroxy-5 alpha-androstan-3-one) for 17 h and then with LHRH for an additional 4 h . Dihydrotestosterone was 6-fold more potent than testosterone in the inhibition of LHRH-induced LH release . Basal LH secretion was not affected by either androgen . The inhibition curves of testosterone and dihydrotestosterone were not shifted by the presence of the 5 alpha-reductase inhibitors 17 beta-N,N-diethylcarbamoyl-4-methyl-4-aza-5 alpha-androstan-3-one (4-MA) and 17 beta-N,N-diisopropylcarbamoyl-4-aza-androstan-3-one (DIPA) . Neither 4-MA nor DIPA alone had an effect on either basal or LHRH-induced LH release . When pituitary cells were incubated with {3H}testosterone for 17 h, the radioactivities were found to be unmetabolized testosterone (66.9 +/- 2.4%), dihydrotestosterone (13.3 +/- 0.5%), androstenedione (15.9 +/- 1.3%), 5 alpha-androstane-3,17-dione (2.8 +/- 0.3%), and 3 alpha (beta), 17 beta-androstanediol (less than 1%) . In the presence of 4-MA or DIPA, 5 alpha-reduction of testosterone was completely inhibited; androstenedione was the only metabolite . Androstenedione was only 12% as potent as testosterone in the inhibition of LHRH stimulation of LH release, and conversion of {3H}androstenedione to testosterone and dihydrotestosterone did occur in these cells . When {3H}dihydrotestosterone was incubated with pituitary cells, the radioactivities were dihydrotestosterone (64.4 +/- 0%), 5 alpha-androstanedione (19.3 +/- 1%), 3 alpha (beta), 17 beta-androstanediol (7.7 +/- 1.7%), and unknown polar metabolites . 4-MA and DIPA had no effect on the metabolism of dihydrotestosterone . These results indicate that both testosterone and dihydrotestosterone inhibit LHRH-induced LH release and that this activity of testosterone does not depend on its 5 alpha-reduction.

Clin Endocrinol (Oxf), 1984 Dec, 21(6), 709 - 18
Effect of pancreatic growth hormone releasing factors on GH secretion by human somatotrophic pituitary tumours in cell culture; Adams EF et al.; Human pancreatic growth hormone releasing factors (hpGRF(1-40) and hpGRF(1-44) significantly stimulated GH secretion when added to cell cultures of human somatotrophic pituitary tumours for 2 h . There was little difference in potency between the two peptides, and the response of different tumours varied, ranging from 30% to 500% increases in GH secretion over control levels . This stimulatory effect was blocked by somatostatin (SRIF) and bromocriptine . The suppressive effect of bromocriptine, but not of SRIF, was overcome by high doses of hpGRF(1-44) . TRH stimulated GH secretion by one of three somatotrophic tumours in cell culture, and it was found to potentiate the stimulatory effects of hpGRF(1-44) . These results demonstrate that hpGRFs increase serum GH levels in man by a direct action at the pituitary somatotroph level.

Brain Res, 1984 Nov 19, 322(1), 17 - 31
Uptake of {3H}GABA by oligodendrocytes in dissociated brain cell culture: a combined autoradiographic and immunocytochemical study; Reynolds R et al.; Uptake of {3H}GABA by dissociated mixed cell cultures of fetal mouse brain was studied using light microscopic autoradiography . Major cell types in the cultures were identified and quantified by immunocytochemical localization of reliable cell type-specific antigenic markers . In 12 days in vitro (DIV) cultures {3H}GABA uptake was predominantly into neurons and oligodendrocytes, whilst at 28 DIV the only surface cells labeled were oligodendrocytes . This was confirmed by complement-dependent antibody-mediated cytotoxicity against galactocerebroside-positive oligodendrocytes . There was a moderate labeling of almost all flat cells, the majority of which were glial fibrillary acidic protein (GFAP)-positive astrocytes . Heavily labeled astrocytes were only occasionally observed . Oligodendrocytes accumulated {3H}GABA more rapidly than astrocytes but slower than neurons . Oligodendroglial labeling was predominantly over the cell body, whereas neuronal labeling was more uniformly distributed over cell body and processes . The uptake was inhibited by diaminobutyric acid (DABA) and nipecotic acid, but not by beta-alanine, and thus had similar characteristics to neuronal rather than astroglial uptake . Oligodendrocytes did not accumulate {3H}beta-alanine, which labeled only astrocytes . Oligodendroglial {3H}GABA uptake was Na+-dependent and sensitive to ouabain, but was only slightly enhanced by aminooxyacetic acid (AOAA), whereas astroglial uptake was not sensitive to ouabain but was markedly enhanced by AOAA . The results indicate that oligodendrocytes, in addition to astrocytes, may also be involved in the modification of neuronal function by the uptake and inactivation of neuroactive substances.

J Immunol Methods, 1984 Nov 16, 74(1), 59 - 64
The use of detergents in velocity sedimentation of cell culture IgM; McIntosh RV et al.; The biosynthetically radiolabelled secreted proteins of human lymphoblastoid cell lines have been analysed by sucrose gradient velocity sedimentation . The addition of detergent to the gradient buffer dramatically improves the recovery of immunoglobulins from cell line supernatants, allowing the gradient fractions to be analysed further both serologically and biochemically . The results of these analyses show that IgM is synthesised in the 19S form and can be clearly separated from other components which include free light chains . Immunoglobulin appears to form a much larger portion of the proteins secreted by human lymphoblastoid lines than has formerly been recognised, a finding which may be of practical importance for the development and exploitation of human monoclonal antibodies.

Lancet, 1984 Nov 10, 2(8411), 1071 - 5
Isolation of a virus from chimpanzee liver cell cultures inoculated with sera containing the agent of non-A, non-B hepatitis; Prince AM et al.; A membrane-coated virus having a diameter of 85-90 nm and containing a 40-45 nm core was found to replicate in cell cultures derived from chimpanzee liver after inoculation of serum containing infective non-A, non-B (NANB) hepatitis viruses from two independent sources . Replication of this agent was not observed when the same cells were inoculated with a chloroform-extracted inoculum or were left uninoculated . Replication involves assembly of virus cores on tubular structures similar to those seen in liver cells of chimpanzees infected with most isolates of NANB virus.

J Theor Biol, 1984 Nov 7, 111(1), 61 - 79
Influence of the existence of a resting state on the decay of synchronization in cell culture; Hirsch HR; General relationships between the distribution of cell doubling times and the growth pattern of an initially synchronized cell population are applied to the model proposed by Smith and Martin (1973) in which the mitotic cycle or "B" phase is preceded by a random-exit resting "A" state . Results show that culture synchronization decays so rapidly as to be virtually unobservable unless the time spent by a cell in the B phase is at least equal to that spent in the A state . If synchronization persists over several mitotic cycles, the growth pattern is determined to a much greater extent by variation in the duration of the B phase than by the probability of exit from the A state . Accordingly the growth pattern of a cell population, like the doubling time distribution which governs the pattern, is of limited usefulness in detecting the existence of a resting state.

Clin Genet, 1984 Nov, 26(5), 477 - 80
A supernumerary microchromosome in amniotic cell cultures and talipes equinovarus in a live born female; Muneer RS et al.; Amniocentesis for advanced maternal age resulted in the demonstration of a supernumerary microchromosome in the amniotic fluid cells . Cytogenetic analysis of peripheral blood from the female infant revealed a mosaic karyotype 46,XX/47,XX, + marker . The only anomaly noted in the infant was talipes equinovarus.

Nature, 1984 Nov 1-7, 312(5989), 63 - 5
New evidence that growth in 3T3 cell cultures is a diffusion-limited process; Dunn GA et al.; When a confluent culture of 3T3 cells is wounded, new growth occurs at the wound margins . This indicates that the suppression of growth in the intact confluent sheet is under local control, a phenomenon known as 'topoinhibition', and it has been suggested that intercellular contact is responsible . An alternative explanation for topoinhibition is possible, however, namely that a soluble factor necessary for growth is locally depleted from the medium by cells so that each cell in a confluent sheet normally receives an insufficient supply and its growth is inhibited . Here we show that the pattern of release from topoinhibition in a wounded culture can be distorted simply by inducing a gentle laminar flow of medium across the wound . Growth remains suppressed at the upstream margin of the wound despite the reduced level of intercellular contact at wound edges . We conclude that the signal for topoinhibition is carried by the flow as would be predicted if it were the local depletion of growth factor.

Cancer Res, 1984 Nov, 44(11), 5068 - 72
Enhanced induction of the anchorage-independent phenotype in initiated rat tracheal epithelial cell cultures by the tumor promoter 12-O-tetradecanoylphorbol-13-acetate; Steele VE et al.; The purpose of the studies reported here was to compare the response of noninitiated and initiated primary rat tracheal epithelial (RTE) cell cultures to the mouse skin tumor promoter 12-O-tetradecanoylphorbol-13-acetate (TPA) . The endpoints measured were number of cells per culture, colony-forming efficiency, subculturability, and colony formation in soft agarose . Primary RTE cell cultures were exposed on Day 1 to either 0.2% dimethyl sulfoxide, or to 0.1 micrograms per ml of N-methyl-N'-nitro-N-nitroso-guanidine (MNNG) . Thereafter, the same cultures were exposed twice weekly from Days 6 to 30 to either 0.2% dimethyl sulfoxide or to TPA (10 pg/ml) . Sequential exposure to MNNG and TPA did not increase the number of viable cells per culture beyond that seen in MNNG-exposed cultures . Determination of the frequency of colony-forming cells 10 days after the end of the initiation-promotion treatment (Day 40 of culture) revealed a marked enhancement in colony-forming efficiency of treated cultures compared to dimethyl sulfoxide-exposed control cultures . However, sequential exposure to MNNG and TPA had an additive or slightly more than additive effect on the colony-forming efficiency of RTE cells exposed to MNNG or TPA only . Treatment of the primary cultures with MNNG alone or TPA alone increased the subculturability of RTE cells to a similar extent . The sequential exposure to MNNG followed by TPA appeared to have an additive effect on the frequency of subculturability . The most pronounced effect of the sequential MNNG-TPA exposure as compared to single-agent exposure was a marked enhancement of the anchorage-independent (ag+) phenotype . Of the cultures treated with MNNG followed by TPA, over 50% were ag+ at 60 days . In contrast, of the cultures treated either with MNNG alone or with TPA alone, only 3% were ag+ on Day 60 . (All control cultures were ag-.) Colony-forming efficiency in soft agarose also increased disproportionately between 60 and 120 days in initiated-promoted cultures . These experiments indicate that the major effect of the tumor promoter TPA on initiated RTE cell cultures is to enhance the appearance of the late ag+-phenotype.

J Clin Endocrinol Metab, 1984 Nov, 59(5), 817 - 24
A human pituitary adenoma secreting thyrotropin and prolactin: immunohistochemical, biochemical, and cell culture studies; Jaquet P et al.; A 43-yr-old woman, who had previously had a subtotal thyroidectomy, presented with hyperthyroidism and amenorrhea-galactorrhea due to a pituitary adenoma secreting TSH, TSH-alpha, and PRL . Her serum T4 concentration was 14 micrograms/dl; T3, 5.7 ng/ml, and TSH, 19-33 microU/ml . Serum TSH was not altered by TRH stimulation or T3 suppression . Basal plasma PRL levels were 19-27 ng/ml and plasma PRL doubled after TRH stimulation . A 900-mg pituitary tumor, removed by transphenoidal surgery, was studied in cell culture . After dispersion, tumor cells were maintained on an extracellular matrix produced by bovine corneal endothelial cells in a defined serum-free medium . The hormones released in the culture medium were analyzed by high pressure gel chromatography . Three fractions of tumor TSH were found, with respective apparent mol wts of 45,000 (11%), 28,000 (70%), and 20,000 (19%) . Tumoral PRL eluted as a single peak of apparent mol wt of 24,000 . Pharmacological studies of TSH, TSH-alpha, and PRL release using thyroid hormones (T3), dopamine agonist (bromocriptine), TRH, and cholera toxin yielded the following results: 1) T3 after 3 days of incubation produced a dose-dependent inhibition of TSH, TSH-alpha, and PRL release . Maximal inhibition (81%) was obtained at 10(-9) M and half-maximal inhibition at 4-6 X 10(-11) M . 2) Bromocriptine produced rapid and partial inhibition of hormone release . Maximal inhibition (51%) was obtained at 10(-8) M and half-maximal inhibition at 5 X 10(-10) M . 3) TRH at 10(-8) M concentration significantly stimulated PRL release but it had no effect on TSH release . 4) Adenylate cyclase activation by 10(-11) M cholera toxin increased TSH (152%), TSH-alpha (150%), and PRL (220%) . Immunohistochemical analysis of serial 2 micron sections of the tumor showed that: 1) TSH-alpha immunoreactive cells were the most numerous, 2) TSH-beta positive cells were always positive for TSH-alpha, 3) PRL immunoreactivity was found either uniquely in some cells and colocalized with TSH-alpha immunoreactivity in other cells . However, by electron microscopy, the tumor cells were thyrotrophs . These data indicate that in this patient's tumor: 1) cells secreting TSH were responsive in vitro to near physiological concentrations of thyroid hormones . 2) The colocalization of PRL and TSH-alpha immunoreactivities in some cells raises the possibility either of fusion of differentiated pituitary cells synthesizing distinct hormones or of transformation of less differentiated multipotential pituitary cells.

Horm Metab Res, 1984 Nov, 16(11), 581 - 4
Stimulatory effect of Sertoli cell culture medium on the FSH release by pituitary halves in vitro; Janecki A et al.; The influence of the medium collected from cultured rat Sertoli cells on the spontaneous and LHRH-stimulated release of gonadotropins by incubated rat pituitary halves was examined . The homogeneity of the cultured population of Sertoli cells taken from 20-day-old rats ranged up to 98% . The cells in culture responded to FSH stimulation with characteristic morphological changes and with increased secretion of estradiol-17 beta . The hemi-pituitaries obtained from sexually mature male rats were incubated for 5 hours in the presence of Sertoli cell culture medium (SCCM) or its fractions obtained by use of ultrafiltration . The SCCM fraction deprived of MW less than 10 kD compounds exhibited a typical inhibin-like activity, whereas crude SCCM as well as its low-molecular-weight fraction stimulated the basal FSH release to about 150% and 175% of the control values, respectively . These fractions exerted an inhibitory effect on the LHRH-stimulated secretion of both LH and FSH . It is concluded that Sertoli cells cultured in chemically defined medium release, apart from inhibin, a non-steroidal, heat-labile substance of MW less than 10 kD which stimulates the basal secretion of FSH and LH and inhibits the LHRH-stimulated secretion of both gonadotropins from incubated rat hemi-pituitaries.

Neuropeptides, 1984 Nov, 4(6), 457 - 66
Fibronectin-regulated methionine enkephalin-like and somatostatin-like immunoreactivity in quail neural crest cell cultures; Sieber-Blum M; The presence of cells with peptidergic immunoreactivity in neural crest cell cultures and the influence of fibronectin on their in vitro differentiation were evaluated by indirect immunostaining . Met-enkephalin-like immunoreactive and somatostatin-like immunoreactive cells were observed . Met-enkephalin-like immunoreactive cells resembled endocrine cells . A variety of morphologies was observed in the cells with somatostatin-like immunoreactivity: cells without processes which looked like endocrine cells, multipolar cells with long varicose processes resembling sympathetic neurons, and bipolar cells which were similar to sensory neuroblasts . Differentiation of both types of peptidergic cells was promoted by adding fibronectin to the culture medium . The results suggest that neural crest cell cultures may become a valuable experimental system with which to study the early development of peripheral peptidergic neurons and endocrine cells.

J Neuroimmunol, 1984 Nov, 7(1), 1 - 20
Immunocytochemical characterisation of cell cultures grown from dissociated 1-2-day post-natal rat cerebral tissue . A developmental study; Walker AG et al.; A range of cell-specific markers have been employed with immunocytochemical methods to characterise and quantitate the cell types present in mixed brain cell cultures derived from dissociated 1-2-day post-natal rat cerebral hemispheres and grown in the presence of FCS . Protoplasmic astrocytes (GFAP+, A2B5-) were the major cell type to develop in culture, a confluent monolayer forming in 5-8 days . A population of smaller round cells of oligodendrocyte-like morphology appeared on this astrocyte layer . Greater than 70% of these smaller cells were GC- and thus were not oligodendrocytes . The GC- cells were A2B5+ and, in early cultures, may therefore be progenitor glial cells . Examination of GFAP and A2B5 co-expression by these smaller cells was difficult due to the dense underlying GFAP+ astrocyte layer . In less dense areas of older cultures these smaller cells with processes were GFAP+ and A2B5+: these are Type 2, fibrous astrocytes . GC+ oligodendrocytes, comprising 5-10% of the total identified cell population, were initially distributed over the astrocyte monolayer; in older cultures (after about 8 days) GC+ cells were observed in clumps over places where NF+ cells were identifiable . Such GC+ cells mostly became MBP+ . Neurones accounted for about 6% of the identifiable cells in early cultures but a lower percentage in older cultures . Minor populations of ependymal cells and macrophages were present; cells displaying fibronectin, fibroblasts, were rarely identified . Use of horse serum in place of FCS gave lower yields of GC+ cells in cultures, slowed down astrocyte development, and resulted in the formation of trunks of GFAP+ cells throughout cultures . Other sera gave lower numbers of GC+ cells.

Proc Soc Exp Biol Med, 1984 Nov, 177(2), 257 - 61
Synergism of antiviral activity in cell cultures treated with low concentrations of interferon and interferon-treated lymphocytes; Weigent DA et al.; Human T cells treated with low levels of interferon (IFN) (1-10 units/ml), and washed to remove the IFN, transferred the same level of antiviral activity to recipient WISH cells as an equivalent IFN treatment alone could induce in WISH cells . Further, when T cells pretreated with IFN (1-10 units/ml) were cocultivated with WISH cells in the presence of IFN (1-10 units/ml), a 2.5- to 5-fold greater level of protection developed than could be expected from the additive effect of each . Antibody to leukocyte, fibroblast, or immune IFN blocked the antiviral effect of the respective IFN types but had no effect on the transfer of antiviral activity initiated by leukocyte, fibroblast, or immune IFN . Also, treatment of T cells with actinomycin D blocked the transfer of antiviral activity of IFN-treated T cells . Taken together, the data suggest that the increased antiviral activity is not merely an additive effect of the IFN, but represents a synergistic amplification of protection most likely due to the combination of the separate effects of IFN and IFN-induced transfer . Such interactions would be expected to play a major role in early protection against virus infections in vivo when low levels of interferon are present and lymphocytes are migrating into the area.

Ann Neurol, 1984 Nov, 16(5), 603 - 10
Infection of neuronal cell cultures with reovirus mimics in vitro patterns of neurotropism; Dichter MA et al.; Primary neuronal cell cultures of rat fetal cerebral cortex serve as in vitro models for the study of a variety of neuronal membrane receptors . Such studies have focused primarily on receptors for neurotransmitters and drugs . In the present series of experiments, we have employed this model to study the in vitro pattern of infection with reovirus types 1 and 3, two well-characterized neurotropic viruses that show specificity for neurons (type 3) or ependymal cells (type 1) in vivo and whose specificity has been linked with surface receptors on somatic cells . We have found that in primary neural cell culture, reovirus type 3 maintained its specificity by infecting neurons whereas reovirus type 1 did not infect neurons . Both serotypes infected astrocytes in the cultures, type 1 to a greater extent than type 3 . In addition, reovirus type 3 bound to the surface of neurons whereas type 1 did not . Using recombinant viral clones, the in vitro tropism and the neuronal binding were shown to be properties of the viral hemagglutinin, a small outer capsid viral protein, as is the case with the neurotropism in vivo . It is postulated that the neurotropism of reovirus type 3 is related to the interaction of the viral hemagglutinin with an as yet undefined structure on the neuronal surface.

J Virol, 1984 Nov, 52(2), 650 - 5
Induction of antibody to foot-and-mouth disease virus in presensitized mouse spleen cell cultures; Collen T et al.; Cultures of spleen cells from immunized mice were stimulated in vitro by soluble preparations of purified foot-and-mouth disease virus . Virus-specific antibody, as detected by an enzyme-linked immunosorbent assay, was produced by immune spleen cells but not by normal, nonimmune cells . The optimal specific response was obtained with 1 microgram of virus per ml of culture; as the virus concentration was increased, the production of specific antibody was reduced . For very low concentrations of virus (less than 0.01 microgram per culture), there was tentative evidence of suppression of the specific antibody response . The levels of specific antibody induced were dependent on the source and number of plastic-adherent cells present in the cultures . We intend to use this model system to study further the basis of immunity to foot-and-mouth disease virus.

FEBS Lett, 1984 Oct 29, 176(2), 479 - 83
Enhanced casein kinase II activity in human tumour cell cultures; Prowald K et al.; Casein kinase II (CKII) activity is enhanced as much as 2-3 fold in established and 4-5-fold in transformed human cell lines when compared to that of fibroblasts and primary human tumour cell cultures where CKII activity never exceeded a basic level . The high activity of CKII in transformed cells and in established cell lines was reduced to about the same basic level after treatment with heparin, a highly specific inhibitor of CKII activity . The activity of the cAMP-dependent protein kinase was virtually the same in fibroblasts and various human tumour cell lines investigated.

Neurosci Lett, 1984 Oct 12, 51(2), 281 - 6
Morphine treatment increases clonidine binding in brain cell cultures; Plishka RJ et al.; Exposure of murine brain cells in culture to 75 microM morphine for 6 days produced an increase in the number of {3H}clonidine binding sites without affecting the apparent affinity . Similar treatment increased the binding of this alpha 2-adrenergic receptor agonist to cell cultures prepared from cerebral cortex but not to cultures of brain minus cortex or to neuroblastoma-glioma hybrid cells which possess both opiate and alpha 2-adrenergic receptors.

Exp Cell Res, 1984 Oct, 154(2), 581 - 90
Culture shock . Synthesis of heat-shock-like proteins in fresh primary cell cultures; Wolffe AP et al.; The isolation of Xenopus liver, lung and testis cells by collagenase digestion of the tissue, followed by Percoll density gradient centrifugation, was characterized by the preferential synthesis of two proteins whose size and charge were similar to 70 and 85 kD heat-shock proteins . The synthesis of these two heat-shock-like proteins, relative to that of total protein, declined gradually in the first 3-4 days after the cells were plated out for primary culture . In fresh primary cultures of liver parenchymal cells albumin mRNA concentration declined rapidly and plateaued at 3-4 days of culture . Freshly isolated male Xenopus hepatocytes were refractory to induction by estrogen of vitellogenin gene transcription but they reacquired hormonal response during the first 3 days of culture . Both of these differentiated phenotypic functions of the Xenopus hepatocytes were quantitatively associated with the decline in synthesis of hsp-like proteins in freshly prepared primary cell cultures . Freshly isolated or heat-shocked hepatocytes exhibited a rounded shape with little intercellular contacts, whereas during the recovery period of 3 days they acquired a flattened shape with a high degree of intercellular and cell-substratum interaction . These results present the first evidence for the preferential synthesis of heat-shock-like proteins by procedures for establishing primary cell cultures . They emphasize the necessity of monitoring normal and heat-shock protein synthesis and cell morphology before using primary cell cultures for studying normal regulatory and developmental processes.

Zentralbl Bakteriol Mikrobiol Hyg {B}, 1984 Oct, 179(5), 406 - 30
{Biological effects of smog . VIII . Impulse cytophotometric cell cycle analysis of synchronized Syrian hamster kidney cell cultures (line 14-b)}; Giebel P et al.; Syrian hamster kidney cultures of line 14-1b were synchronized by excess of thymidine . Thereafter in the phase of DNA synthesis cell cultures were exposed to extract and fractions of city smog, derived from a polluted area at the river Rhine and Ruhr . Using impulscytophotometry and estimation of mitotic frequency cell cycle analyses were conducted on synchronized exposed and control cultures . The total extract obtained by methanol treatment of city smog was further fractionated by organic solvents leading to fractions of cyclohexane, polyaromates and propanol . Cell cycle progression of synchronized cultures was inhibited in a dose dependent manner by increasing concentration of city smog extract and its fractions . This inhibition led to a prolongation of DNA synthetic phase and to an accumulation of cells in G2(+ M)-phase . The total cell cycle showed a prolongation of 3-5 h . The strongest effect was induced by the total extract . We have to emphasize that already amounts of city smog which were found in air volumes of 2-5 m3 exerted strong effects . With a declining sequence of toxicity followed the fractions of cyclohexane, propanol and of polyaromates . Our results show, that samples of city smog from polluted areas contain substances which induce heavy alterations in cell cycle progression of mammalian cells in vitro . These highly effective toxic substances are dangerous for human health, especially after a long-time exposition.

J Neurol Sci, 1984 Oct, 66(1), 77 - 90
Infection of rat brain primary cell cultures with an avirulent A7 strain of Semliki Forest virus; Bruce CB et al.; The ability of A7 Semliki Forest Virus (SFV) to infect primary brain cell cultures has been examined using cultures prepared from 1-2-day neonatal rat cerebral hemispheres . These cultures, characterised immunocytochemically using cell-specified markers, contain mainly GFAP+ protoplasmic astrocytes and smaller multiprocessed A2B5+ cells, probably fibrous astrocytes . 10% of the cells are GC+ oligodendrocytes and some neurones are also present . These cultures support virus growth and a cytopathic effect was observed . Using double labelling techniques with the cell-specific markers and anti-SFV antibody A7 has been shown to readily infect cells which carry either the A2B5+ antigen or galactocerebroside marker . Protoplasmic astrocytes (GFAP+/A2B5-) are not readily infected under the conditions used . The protein labelling studies using {35S}methionine show that host cell protein synthesis is not completely shut off and continues in the astrocyte protein region . These results suggest that cells derived from a common A2B5+, GFAP-, GC- progenitor glial cell, i.e . GC+ oligodendrocytes and A2B5+/GFAP+ fibrous astrocytes, are more readily infected than other brain cell types including the protoplasmic astrocytes.

Coll Relat Res, 1984 Oct, 4(5), 377 - 88
The influence of cell culture variables on human collagenase inhibitor expression; Welgus HG et al.; This study investigated the modulation of human collagenase inhibitor expression by a variety of cell culture variables . Inhibitor production was found to be largely invariant with respect to the concentration of serum incorporated into culture medium . Similarly, environmental pH failed to affect inhibitor expression over the pH range of 6.8-8.2 . When inhibitor production was examined as a function of cell culture density, synthesis of this protein per cell was greatest during logarithmic growth and early confluence, but even in late post-confluent cultures levels approaching 50% of maximal were routinely observed . When basal levels of collagenase and inhibitor were compared in 8 different normal human skin fibroblast cell lines, inhibitor production varied by only 2-fold, whereas collagenase levels displayed a range exceeding 20-fold . Thus, despite manipulations in the presence or absence of serum and even across different cell lines, inhibitor production seemed to be marked most conspicuously by its constancy . The kinetics of inhibitor secretion into culture medium were also examined . Whether cultured in the presence or absence of serum, inhibitor levels reached maximal values in the medium after 24-48 hours of incubation and remained constant thereafter . Interestingly, assessment of intracellular versus extracellular quantities of inhibitor demonstrated that there was no significant intracellular storage of this protein . Thus, the data suggest that human collagenase inhibitor is a secretory protein which is rapidly exported into the extracellular space without significant accumulation intracellularly.

Mutat Res, 1984 Oct, 130(5), 333 - 42
The use of sister-chromatid exchange in Chinese hamster primary lung cell cultures to measure genotoxicity; Shimizu RW et al.; Primary cell cultures derived from Chinese hamster lung (CHL) were established, and their response for the induction of sister-chromatid exchange (SCE) by direct- and indirect-acting mutagens was characterized . An increase in SCE frequency was induced in CHL cells by 3-methylcholanthrene (MCA), benzo{a}pyrene (BaP), and 2-aminoanthracene (2AA) . The SCE frequency increased slightly after exposure to cyclophosphamide, but did not respond to the hepatocarcinogen dimethylnitrosamine (DMN) . A slight increase in SCE frequency by DMN was observed in the CHL system with use of Aroclor-1254-induced rat liver homogenate fraction (S9) . This response to DMN in CHL cells was lower than that seen when CHO cells were the target in the presence of S9 . At low (1) and high (20) passages, the CHL cells responded with a similar dose-related increase in SCE frequency to direct- (ethyl methanesulfonate, EMS) and indirect-(MCA) acting mutagens . This response indicates that even after prolonged culturing in vitro, the cells retained the ability to metabolically activate xenobiotic promutagens . The induction of SCE by MCA occurred at concentrations that also induced macromolecular binding . SCE induction was also examined in primary lung cell cultures from animals exposed by nose-only inhalation to MCA aerosol . A significant increase in SCE frequency above controls was observed in cells from animals after a single exposure to MCA . No detectable increase in SCE frequency was observed after repeated inhalation exposures . Because CHL cells are of lung origin and showed metabolic activity, the CHL system appears to be appropriate for study of the genotoxic potential of inhaled compounds.

Isr J Med Sci, 1984 Oct, 20(10), 927 - 30
Cell culture models as ancillary tools in the isolation and characterization of mycoplasmas; Hopps HE et al.; Since the late 1960s, there have been an increasing number of reports describing the isolation and identification of fastidious strains of Mycoplasma hyorhinis in cell cultures but not in conventional mycoplasma media, i.e., agar and broth . The application of those techniques normally used in studying viruses, i.e., infection of cell cultures coupled with the subsequent use of immunological and biological test procedures, has provided a reliable alternative procedure for M . hyorhinis detection . Major isolation surveys have revealed that as many as 60 to 80% of M . hyorhinis isolates from contaminated tissue cultures failed to grow in agar medium . Efforts to elucidate the mechanisms involved in failure of the cell culture-derived M . hyorhinis strain to grow in standard cell-free mycoplasma media are ongoing . Initial data indicated that extracts prepared from tissue cultures (BHK 21) and incorporated in Macpherson's broth and agar medium would permit growth of fastidious strains . Moreover, it appears that these strains are particularly sensitive to inhibition by yeast products often found in mycoplasma media . While M . hyorhinis appears to be unique with respect to its sensitivity to medium components, these fastidious strains are isolated with such frequency that the routine use of an indicator cell system is strongly recommended.

J Biol Response Mod, 1984 Oct, 3(5), 547 - 60
Suppression by tumor growth of T cell growth factor production in mouse lymphoid cell cultures; Kedar E et al.; Splenocytes of normal (NS) and tumor-bearing (TBS) mice (Balb/c, C57BL/6, C3H) were stimulated in vitro for 24 h with concanavalin A and the amount of T cell growth factor (TCGF) generated was measured . TBS of mice carrying subcutaneous implants (greater than 1 cm tumor diameter) of several T lymphomas, pulmonary and mammary carcinomas, and a melanoma, or intraperitoneal implants (greater than 10(8) cells) of ascitic lymphomas produced (per culture) 40-90% less TCGF than that generated by NS . TBS also contained a greater proportion of phagocytic cells and a higher ratio of Lyt 2+/Lyt 1+ T cells as compared with NS . In cocultures consisting of NS and TBS (1:1), TCGF production by NS was markedly suppressed . In contrast, addition of up to 30% tumor cells to NS decreased production only slightly . Removal from TBS of either phagocytes or Lyt 2+ T cells, or treatment in vitro with indomethacin {IND; an agent inhibiting prostaglandin (PG) synthesis}, appreciably reduced their capacity to inhibit NS and improved TCGF production in TBS cultured alone . TCGF production by TBS could be completely restored by depletion of both phagocytes and Lyt 2+ T cells . Elevated quantities of TCGF (up to threefold) were generated by TBS of mice pretreated in vivo 1-4 days previously with low doses of either cyclophosphamide or X-irradiation, with or without IND . It is concluded that suppression of TCGF production in vitro by TBS is mediated by phagocyte-released PG and by Lyt 2+ T lymphocytes.

In Vitro, 1984 Oct, 20(10), 780 - 95
Rat liver epithelial cell cultures in a serum-free medium: primary cultures and derived cell lines expressing differentiated functions; Chessebeuf M et al.; Rat liver epithelial cells explanted in a serum-free medium (SFM) composed of Ham's F10 basal medium plus free fatty acids adsorbed on bovine albumin gave successful rise to primary cultures and then to long-term cell lines that expressed liver functions; induction of L-tyrosine aminotransferase by glucocorticoids, hepatic pattern of progesterone metabolism, and biosynthesis of murine primary bile acids; chenodeoxycholic and cholic acid common to higher vertebrates and alpha-muricholic acid specific of the rat bile.

What Is Amino Acid?, What Is Activated Sludge?, What Is Growth Medium?, What Is Cell Biology?, What Is Nitrification?, s, Microbe, o, Bacteria, n, Microorganism, i, Microorganisms, a, Bacteriology, o, Escherichia coli, e, Microbial, c, Streptococcal, r, Vibriosis, c, Staphylococcus, a, Microorganism, e, Bacteriophage, a, Clostridia, i, Salmonella, n, Clostridia, a, Streptococcal, c, Microorganism, a, Clostridia, o, Streptococci, s, Bacillus subtilis, s, Yeasts, a, Cell cultures, o, Bacillus anthracis, s, Salmonella, s, Escherichia coli, r, Pseudomonas aeruginosa

Scientific Publications - Work Done by Microbiology Reader Bioscreen C
Agricultural Microbiology Anaerobic Microbiology Antimicrobial Susceptibility Artificial Atmosphere Bioassay of Antibiotics Biofilm Microbiology Bioreactor Technology Biotechnology Cell Biology Clinical Microbiology Environmental Microbiology Experiments with Yeast	Fermentation Food Microbiology Functional Genomics Gene Technology Growth Media Development Growth Rate and Lag Time Industrial Microbiology Medical/Pharmaceutical Field Microbiological Assay Microbiological Research Microbiology of Cosmetics go to a specific theme...	Military Microbiology Molecular Microbiology Mutagenicity and Genotoxicity Oral Microbiology Patents Postantibiotic Studies Soil Microbiology Spore Microbiology Veterinary Microbiology Waste/Wastewater Treatment Water Microbiology Wine Microbiology

� 2005 Transgalactic Ltd (manufacturer of Bioscreen C software) | Privacy Statement | P.O. Box 1393, 00101 Helsinki, Finland, phone: +358 9 85172920, fax: +358 9 8749481, e-mail: microbiology@bionewsonline.com

Last modified: May 25, 2005