Brain Res, 1986 Oct, 394(2), 217 - 23 Cholinergic neurons of fetal rat telencephalon in aggregating cell culture respond to NGF as well as to protein kinase C-activating tumor promoters; Honegger P et al.; Serum-free aggregating cell cultures of fetal rat telencephalon treated with the potent tumor promoter phorbol 12-myristate 13-acetate (PMA) showed a dose-dependent, persistent stimulation of the enzymes choline acetyltransferase (ChAT), glutamic acid decarboxylase and glutamine synthetase . After elimination of the proliferating cells by treatment of the cultures with Ara-C (0.4 microM) only the cholinergic marker enzyme, ChAT, could be stimulated by tumor promoters . The non-promoting phorbol ester, 4 alpha-phorbol 12,13-didecanoate proved to be inactive in these cultures, whereas the potent non-phorbol tumor promoter, mezerein, produced an even greater stimulatory effect than PMA . Since PMA and mezerein are potent and specific activators of protein kinase C, the present results suggest a role for this second messenger in the development of cholinergic telencephalon neurons . Stimulation of ChAT required prolonged exposure (48 h) of the cultures to PMA and the responsiveness of the cholinergic neurons to the tumor promoters decreased with progressive cellular maturation . The cholinergic telencephalon neurons showed the same pattern of responsiveness for tumor promoters as for nerve growth factor (NGF) . However, the combined treatment with NGF and either PMA or mezerein produced an additive stimulatory effect, suggesting somewhat different mechanisms of action. J Urol, 1986 Oct, 136(4), 953 - 9 Improved growth of human urothelial carcinoma cell cultures; Grossman HB et al.; From January, 1981 through June, 1982 specimens from 21 patients with bladder (urothelial) cancer were placed in tissue culture, and one long term cell line was established (5%) . From July, 1982 through February, 1984, using an improved culture medium, seven long term cell cultures were established from 21 patients (33%) . In addition, one long term culture from a patient with a bladder melanoma was established using the standard culture medium . The nine cell cultures were derived from the following types of tumors: transitional cell carcinoma (6), adenocarcinoma (1), squamous cell carcinoma (1) and melanoma (1) . All of the cell lines have produced tumors in athymic nude mice except for one transitional cell carcinoma . All of the cultures demonstrate aneuploidy . Homogeneously staining regions have been seen in some cell cultures . A common marker chromosome has not been identified. Eur J Cell Biol, 1986 Oct, 42(1), 27 - 34 A comparative analysis of collagen III, IV, laminin and fibronectin in Duchenne muscular dystrophy biopsies and cell cultures; Rampoldi E et al.; The role of collagen type III and IV (Coll III, IV) in Duchenne muscular dystrophy (DMD) was investigated by the indirect immunofluorescence technique (IIF) both in muscle biopsies and derived cell cultures . Ten dystrophic cases were studied and compared with twelve suitable control cases, extending the IIF analysis to two other representative non-collagenous proteins of the extracellular matrix (ECM), namely fibronectin (FN) and laminin (LM) . DMD biopsies generally displayed a thickening of endomysial and/or perimysial connective, as compared to control specimens . All the markers analyzed were found to contribute to this connective proliferation, although from a quantitative point of view the relative involvement decreases progressively from FN through Coll III and IV to LM . However, due to Coll IV selective endomysial localization, Coll IV alteration can be considered a specific indicator of a muscle cell defect . Results from DMD muscle cultures revealed no significant changes in comparison to control cultures, except for Coll IV . A well-organized, Coll IV-containing pericellular matrix was noted in a fraction of DMD cultures as a unique feature seen in no control culture . This alteration was again considered significant since Coll IV is the only marker clearly associated with the myotube component still expressed in vitro . The negative results obtained on the other marker proteins should not however be considered definitive, due to the lack in the simplified in vitro system used of humoral and/or neuronal factors which may be needed to express gene defect(s) in differentiated cells. Eur J Clin Microbiol, 1986 Oct, 5(5), 569 - 72 Comparison of cell culture with two direct Chlamydia tests using immunofluorescence or enzyme-linked immunosorbent assay; Pothier P et al.; Two direct tests for diagnosis of infection due to Chlamydia trachomatis were evaluated on 417 specimens collected from a population with a low disease prevalence of 8.1% . The intensity of positive results was graded according to the number of inclusions or elementary bodies and the optical density of the reaction . Thirty-four specimens were positive in cell culture, 39 positive with MicroTrak and 43 positive with Chlamydiazyme assay . The sensitivity of the two direct tests was 91.2% (31 of 34); the specificity was 97.9% (381 of 389) for MicroTrak and 96.9% (377 of 389) for Chlamydiazyme assay . The positive predictive values were 79.5% (31 of 39) for MicroTrak and 72.1% (31 of 43) for Chlamydiazyme assay . None of the specimens negative by the culture method were positive by the two direct methods . Discrepancies were restricted to the slightly positive specimens . The direct tests seem to be an alternative for diagnosing Chlamydia trachomatis infections, but slightly positive results require cell culture confirmation. J Clin Microbiol, 1986 Oct, 24(4), 665 - 8 Rapid determination of neutralizing antibodies to Semliki Forest virus in serum by enzyme immunoassay in cell culture with virus-specific monoclonal antibodies; van Tiel FH et al.; We describe in this study a rapid enzyme immunoassay for the titration of neutralizing antibodies in serum against Semliki Forest virus . For this assay L cells were added to preincubated virus-antiserum mixtures to form monolayers . Six hours after infection by residual, nonneutralized virus, the monolayers were fixed, and the E2 glycoprotein of Semliki Forest virus on the surface of infected cells was quantified with an E2-specific, peroxidase-labeled monoclonal antibody (UM 5.1) . The serum antibody titer was defined arbitrarily as the inverse value of that dilution of serum associated with a 25% inhibition of control absorbance values . These titers of both early and later mouse immune sera were similar to those determined in simultaneously performed 50% plaque reduction tests . The results indicate that the enzyme immunoassay (duration, 9 h) is reliable and compares favorably with the conventional plaque reduction test (duration, 25 h) in rapidity, ease of performance, and objectivity. J Invest Dermatol, 1986 Oct, 87(4), 454 - 9 Regulated synthesis of low-molecular-weight antigens in keratinocyte cell cultures; Hawley-Nelson P et al.; Proteins from mouse epidermis cytosol extracts react on immunoblots with a polyclonal rabbit antiserum raised against rat skin calcium-binding protein (SCaBP), a parvalbumin of the panniculus carnosus . Three mouse epidermal proteins with molecular weights between 10-12K, which are distinct from SCaBP, are recognized by the antiserum . The synthesis of these proteins in keratinocyte culture is modulated by Ca++, as is the differentiation of the keratinocytes . Proliferating mouse keratinocytes in medium containing 0.07 mM Ca++ (low Ca++) undergo terminal differentiation when the Ca++ concentration is elevated to 1.8 mM (high Ca++) . Synthesis of the 3 antigens can be demonstrated when soluble extracts of keratinocytes labeled with {35S}methionine in low Ca++ medium are immunoprecipitated with anti-SCaBP serum . These antigens are not synthesized in cultures of dermal fibroblasts . When keratinocytes are switched to high Ca++ medium, synthesis of these antigens is greatly diminished over the course of 48-72 h . However, the antigens persist in differentiating cells . When proliferating keratinocytes in low Ca++ medium are exposed to the tumor promoter 12-O-tetradecanoylphorbol-13-acetate (TPA), differentiation is induced in a subpopulation of cells, and specific antigen synthesis is transiently inhibited . The inhibition correlates with the time when many cells are differentiating in response to TPA . When proliferating keratinocytes are pulse-labeled with 32PO4, the 11K antigen is phosphorylated and the phosphorylation is not enhanced by TPA exposure . All 3 antigens are synthesized in a reticulocyte lysate preparation with added newborn mouse epidermis messenger RNA or mRNA from keratinocytes cultured in low Ca++ medium . Thus, these antigens are likely to represent unique proteins rather than processed or degraded ones . The coordinately regulated expression of these antigens associated with the differentiation state of the keratinocytes suggests that these proteins are important in keratinocyte proliferation and differentiation. Antibiot Med Biotekhnol, 1986 Oct, 31(10), 756 - 60 {Screening of human and animal tissue cell cultures as potential producers of plasminogen activator}; Kapina MA et al.; Twenty six normal cell cultures and 19 tumor cell cultures were subjected to screening for plasminogen activator (PA), a fibrinolytic enzyme . It was shown that the enzyme production depended on the nature, origin, type and species of the tissue culture . The primary cultures of the human and calf embryonic kidney cells, permanent cell lines and tumor cells possessed high PA activity . The suspension cell lines did not produce the PA. Eur J Immunol, 1986 Oct, 16(10), 1269 - 76 Leukemic cells arise from cloned cytotoxic lymphocytes during cell culture; Simon MM et al.; In spite of many promising attempts to apply T cell clones to questions of in vitro and in vivo function of T cells it is still unclear to what extent continuous propagation of T lymphocytes in vitro effects their original properties . This study describes the appearance of malignant cells from long-term cultured C57BL/6 (B6) cytotoxic T lymphocytes (CTL) . Four out of five T cell lines (CTLL.1,3,4,5) representing distinct stages of development of T effector cells in vitro were repeatedly cloned and all five CTLL were tested for various cellular parameters . It is shown that transformation of H-Y-specific CTLL into malignant cells in vitro was accompanied by alterations in growth characteristics, successive loss of specificity and cytolytic function and by quantitative changes in the expression of cell surface markers . Whereas growth of the H-Y-specific CTLL (CTLL.1) was dependent on antigen and concanavalin A (Con A) supernatant (Con ASN) the CTLL variants could be either maintained in Con ASN alone (CTLL.3) or in the absence of both antigen and lymphokine sources (CTLL.4,5) . CTLL.1 was cytolytic for male B6 target cells and lysed P815 tumor targets in the presence but not the absence of lectin . In contrast, CTLL.3 lost its original specificity but lysed P815 cells in the absence or presence of lectin . CTLL.2 representing an intermediary stage showed cytolytic activity on both male B6 and P815 target cells . In contrast, CTLL.4 and CTLL.5 lost the ability to lyse any of the indicated target cells . Although all CTLL expressed the surface markers Thy-1, Lyt-2, Kb, Db and interleukin 2 receptor (IL 2 R), Thy-1 and Lyt-2 markers were drastically reduced and Kb/Db and IL 2 R structures significantly increased on CTLL.4 and CTLL.5 compared to CTLL.1,2,3 . In addition, multiple karyotypic alterations including the appearance of metacentric chromosomes were observed in long-term cultured CTLL . Investigations on the expression of the alpha-, beta-, and gamma-chains of the T cell antigen receptor in CTLL.1-5 indicate that all three chains were expressed as mRNA irrespective of whether the lymphocytes expressed their original specificity and/or function . However, distinct beta variable chain genes were used by H-Y-specific CTLL and its long-term culture variants CTLL.2 and CTLL.3 suggesting that the expression of the new specificity was accompanied by the rearrangement of a new beta-chain gene in T effector cells.(ABSTRACT TRUNCATED AT 400 WORDS) J Clin Microbiol, 1986 Oct, 24(4), 657 - 60 Elimination of toxicity and enhanced cytomegalovirus detection in cell cultures inoculated with semen from patients with acquired immunodeficiency syndrome; Howell CL et al.; Although semen is a particularly rich source of cytomegalovirus (CMV) and is useful for monitoring CMV shedding, its culturing is associated with extensive monolayer toxicity, isolation failures, and lengthy detection times . Inoculation of fractionated semen with immunoperoxidase staining of monolayers eliminated virtually all toxicity, increased isolation rates and monolayer infectivity, and greatly reduced detection times . Semen specimens (n = 73) were processed conventionally (C) or separated into supernatant (SF) and cellular pellet (PF) fractions, and 35% of C and SF inocula produced extensive toxicity . In contrast, virtually no toxicity was observed in monolayers inoculated with PF . C and SF isolation rates were 41 of 73 and 38 of 73, respectively, whereas that for PF was 51 of 73 . Although monolayer infectivity at initial CMV detection was often less than 10% for C and SF, it was as much as 25% for PF . Average detection times were reduced from 13 days for C and SF to 6 days with PF and were further reduced to 3 days when PF inoculation was combined with immunoperoxidase staining . Thirty percent of specimens negative by C were positive by PF. Am J Physiol, 1986 Oct, 251(4 Pt 2), R818 - 22 Cystic fibrosis and beta-adrenergic response of airway epithelial cell cultures; Widdicombe JH; Confluent cell sheets were cultured from the tracheal epithelium of normal humans or from tracheal and nasal epithelia of patients with cystic fibrosis (CF) . Changes in short-circuit current (Isc) or cyclic AMP (cAMP) levels in response to 10(-5) M isoproterenol were measured . In CF tracheal cells the response to isoproterenol was transient, and the maximal increase in Isc was one-tenth normal . In CF nasal cells, isoproterenol or epinephrine caused only small transient increases in Isc . However, in both CF nasal and tracheal cells, the Ca ionophore, A23187, caused relatively large increases in Isc that were inhibited by the Cl transport blocker, bumetanide, suggesting that Cl secretion can be induced by raising intracellular levels of Ca . In normal tracheal cell sheets, cAMP levels increased within 15 s of isoproterenol addition and continued to increase for up to 20 min . Resting levels of cAMP in CF tracheal cells were not statistically different from those of normal cells and showed linear increases for up to 4 min after addition of isoproterenol . Changes in cAMP in CF nasal cells were similar to the changes in CF tracheal cells . After 2 min, all three cell types showed cAMP levels elevated approximately equal to 10-fold . These results suggest that receptor-activated stimulation of adenylate cyclase is normal in CF . However, though raised cAMP levels stimulate Cl secretion in normal, they are unable to do so in CF airway epithelial cells. Endocrinology, 1986 Oct, 119(4), 1427 - 31 Role of arachidonic acid in the regulation of adrenocorticotropin release from rat anterior pituitary cell cultures; Abou-Samra AB et al.; In addition to cAMP-dependent mechanisms, stimulation of pituitary ACTH secretion by various stimuli, including CRF, may involve phospholipid and arachidonic acid turnover . To determine the role of phospholipase A2 activation in corticotroph function, we studied the effect of exogenous arachidonic acid, phospholipase A2, and the phospholipase A2 activator melittin on ACTH release in cultured rat anterior pituitary cells . Incubation with 1-100 micron arachidonic acid, 0.01-1 micron melittin, 0.1-10 U/ml phospholipase A2, and 0.01-10 nM CRF caused dose-dependent increases in ACTH release to 8.1 +/- 1.1- (+/- SE), 16.2 +/- 0.9-, 13.6 +/- 1.2-, and 2.9 +/- 0.3-fold; respectively . The participation of the major pathways of arachidonic acid metabolism in the control of ACTH release was analyzed in cells treated with nordihydroguaiaretic acid, a lipoxygenase inhibitor; indomethacin, a cycloxygenase inhibitor; and 5,8,11,14-eicosatetraynoic acid, an inhibitor of both pathways . The effects of arachidonic acid, melittin, and CRF were partially blocked by 10 micron nordihydroguaiaretic acid and 5,8,11,14-eicosatetraynoic acid, but were significantly enhanced by 10 micron indomethacin . These results suggest that arachidonic acid is mainly metabolized through the lipoxygenase pathway to a stimulatory metabolite and, to a lesser extent, through the cycloxygenase pathway to an inhibitory metabolite . Arachidonic acid release from anterior pituitary cells labeled with {3H}arachidonic was analyzed during cell column perifusion and stimulation by CRF and other secretagogues . Two-minute pulses of CRF (10 nM), vasopressin (10 nM) and phorbol 12-myristate 13-acetate (100 nM) caused immediate 1.5- to 2-fold increases in {3H}arachidonic acid release, and melittin (100 nM) caused a 5-fold increase in {3H}arachidonic acid release . The ability of both exogenously added and endogenously generated arachidonic acid to stimulate ACTH secretion, together with the stimulation of arachidonic acid release by ACTH secretagogues and the attenuation of stimulated ACTH release by lipoxygenase blockers, indicate that lipoxygenase products of arachidonic acid metabolism participate in the control of ACTH secretion. Funct Neurol, 1986 Oct-Dec, 1(4), 345 - 9 Excitatory amino acid signal transduction in cerebellar cell cultures; Nicoletti F et al.; In primary cultures of cerebellar granule cells excitatory amino acid recognition sites are coupled with the stimulation of inositol phospholipid (PI) hydrolysis, cGMP formation and 45Ca2+ uptake . Mg2+, 2-amino-5-phosphonovalerate (APV) and phencyclidine (PCP) potently inhibit signal transduction in response to N-methyl-D-aspartate (NMDA), glutamate (GLU) and aspartate (ASP) . Activation by quisqualate (QUIS) is transduced selectively into stimulation of PI hydrolysis and this response is not sensitive to inhibition by Mg2+, APV and PCP . Activation by kainate (KA) is transduced into potent stimulation of cGMP formation and 45Ca2+ uptake . Transduction of KA signal is not affected by Mg2+ and is relatively insensitive to PCP and APV. J Biol Chem, 1986 Sep 25, 261(27), 12675 - 9 Transcriptional regulation of thyrotropin subunit genes by thyrotropin-releasing hormone and dopamine in pituitary cell culture; Shupnik MA et al.; Thyrotropin (TSH) is an anterior pituitary glycoprotein hormone which modulates thyroid hormone production by the thyroid gland . TSH and its two subunits, TSH beta and alpha-subunit, are in turn regulated by the thyroid hormones and several stimulatory and inhibitory factors produced by the hypothalamus . Hypothalamic hormones such as thyrotropin-releasing hormone (TRH) and dopamine have profound effects on TSH secretion and may also modulate TSH synthesis . We investigated the ability of these two hormones to directly influence the expression of the TSH subunit genes by measuring subunit mRNA synthesis rates in treated and untreated pituitary cell cultures from hypothyroid rats . In this system, basal rates of mRNA synthesis were similar for the two subunits, with 95 ppm for alpha-subunit and 85 ppm for TSH beta . Treatment with 10(-5) M dopamine, an inhibitor of TSH secretion, decreased both alpha-subunit and TSH beta mRNA synthesis within 15 min, and maximal decreases of approximately 75% for both subunits were obtained after 30 min . The inhibitory effects of dopamine were overcome by co-incubation of the cells with dopamine plus 5 X 10(-4) M dibutyryl cyclic AMP (cAMP), indicating that the actions of dopamine may have been mediated by decreasing intracellular cAMP levels . Treatment with 10(-7) M TRH, a potent stimulator of TSH secretion, increased transcription of both TSH subunit genes 3- to 5-fold . Maximal stimulation occurred after 30 min of treatment; however, 2-fold increases were maintained for up to 4 h and at 48 h . Cytoplasmic levels of TSH beta and alpha-subunit mRNA after 48 h were also depressed by dopamine (50-70%) and increased by TRH (200-280%) treatment . TRH-stimulated TSH beta and alpha-subunit mRNA synthesis was partially inhibited by dopamine . In a 4-h treatment period, TSH beta mRNA synthesis was decreased from 250 to 73% of control and alpha-subunit was decreased from 333 to 182% of control values . Thus, both dopamine and TRH can act to modulate TSH subunit gene transcription, although they differ in the direction of the response and the relative effects on TSH beta and alpha-subunit . The magnitude of the transcriptional response for either TRH (200 to 500%) or dopamine (75%) is similar to the observed changes in cytoplasmic levels of TSH subunit mRNAs and indicates that transcriptional control of the TSH genes may be an important component of hypothalamic regulation of TSH. Int J Cancer, 1986 Sep 15, 38(3), 361 - 7 Cytomorphologic evaluation of the neoplastic potential of 28 cell culture lines by a panel of diagnostic cytopathologists; Boone CW et al.; A panel of 7 diagnostic cytopathologists, i.e., physicians trained to diagnose the malignant potential of human cells in Papanicolaou-stained smears, was asked to evaluate two sets of microscope slides of stained coverslip preparations of 28 cell culture lines, 15 of which were neoplastic . Slide Set I consisted of 13 pairs of cell lines, one member of each pair being nontumorigenic and the other tumorigenic; the lines were of mouse (9 pairs), rat (3 pairs), and human (1 pair) origin . Slide Set II contained 4 human lines: one lung cancer, one melanoma, and two fibroblast lines . Of a total of 114 diagnostic decisions by the panel, 88 were correct (66/86, 77%) in choosing which member of a pair was neoplastic and 22 were correct (22/28, 79%) in choosing whether a given individual human line was or was not neoplastic . Two members of the panel were correct more frequently, with 16/17 (94%) correct diagnoses, each . Five nuclear morphologic criteria of malignancy used by cytopathologists were prominent in the tumorigenic lines: altered chromatin pattern characterized by increasing size of chromatin granules and chromatin clumping, sharp angularity of large nucleolar and/or chromocenter borders with spicule formation (pointed projection), irregular parachromatin clearing (increase in the clarity of the clear spaces between chromatin threads, granules and clumps), uneven thickness of chromatin at the nuclear border, and variability in nuclear size and shape from cell to cell . These markers of neoplastic transformation, when added to those previously reported, should increase overall accuracy in the diagnosis of neoplastic transformation of mammalian cells in culture. J Chromatogr, 1986 Sep 12, 364, 11 - 24 Electrophoretic separation and analysis of living cells from solid tissues by several methods . Human embryonic kidney cell cultures as a model; Todd P et al.; Preparative electrophoresis of living cells has been considered for some time as a potential tool for isolating, from heterogeneous mixtures, subpopulations of cells according to function . Such a purification depends upon the retention of electrophoretic heterogeneity and the retention of function . Human embryonic kidney cells that had been in monolayer culture for 1-5 subcultivations were resuspended by treatment with trypsin and/or EDTA and suspended in a variety of electrophoresis buffers, ranging in ionic strength from 0.0015 to 0.15 M . Analytical electrophoresis with a Zeiss Cytopherometer or Pen Kem 3000 automated light-scattering electrophoretic analyzer indicated that electrophoretic heterogeneity was retained under the full range of conditions tested . Preparative electrophoresis by three methods--in a density gradient, with continuous flow, and in microgravity--indicated that electrophoretic heterogeneity coincided with functional heterogeneity; for example, some electrophoretically isolated subpopulations produced increased levels of urokinase while others produced increased level of tissue plasminogen activator. Exp Eye Res, 1986 Sep, 43(3), 365 - 74 Factors influencing glycosaminoglycan synthesis by calf trabecular meshwork cell cultures; Crean EV et al.; When cell or explant cultures were established from excised calf aqueous outflow pathway tissue and subsequently labeled with radioactive precursors of glycosaminoglycans {( 3H}glucosamine and {35S}sulfate), hyaluronic acid was the predominant glycosaminoglycan produced initially . As the cultures reached confluence, sulfated glycosaminoglycans became more prominent products . Further investigation revealed that the cultured trabecular cells could synthesize all of the glycosaminoglycans found in isolated calf trabecular tissue once a critical cell density had been attained . Increasing concentrations of fetal bovine serum in the culture medium stimulated glycosaminoglycan accumulation without altering the quantitative distribution of the various glycosaminoglycan products . Treatment of confluent monolayers with enzymes capable of degrading extracellular matrix molecules caused marked changes in the distribution of glycosaminoglycans produced by calf trabecular-cell cultures . The results suggest that calf trabecular cell cultures can be used to study the dynamics of extracellular matrix production and turnover under controlled experimental conditions. Environ Health Perspect, 1986 Sep, 68, 45 - 52 Metabolic activation of benzo(a)pyrene in SENCAR and BALB/c mouse embryo cell cultures; Baird WM et al.; The metabolism and DNA binding of benzo(a)pyrene {B(a)P} were compared in early passage mouse embryo cell cultures prepared from SENCAR mice, a strain especially susceptible to two-stage tumorigenesis, and BALB/c mice, a strain relatively resistant to two-stage tumorigenesis . Cultures from both strains metabolized similar amounts of B(a)P; however, the proportion of water-soluble metabolites formed was higher in the BALB/c cultures than in the SENCAR cultures . The major metabolites formed in cultures from both strains were B(a)P-9,10-diol, B(a)P-7,8-diol and the glucuronic acid conjugate of 3-hydroxy-B(a)P . The level of binding of B(a)P to DNA was greater in the SENCAR mouse embryo cell cultures than in the BALB/c cultures after 5, 24, and 48 hr exposure . The major B(a)P-DNA adduct formed in B(a)P-treated cultures from both strains was the adduct formed by reaction of (+)anti-B(a)P-7,8-diol-9,10-epoxide {anti-B(a)PDE, the isomer with the epoxide and the benzylic hydroxyl on opposite faces of the molecule} with the exocyclic amino group of deoxyguanosine . Immobilized boronate chromatography followed by high-performance liquid chromatography demonstrated the presence of small amounts of syn-B(a)PDE (the isomer with the epoxide and benzylic hydroxyl on the same face of the molecule)-DNA adducts . The proportions of these amounts were similar in cultures from both strains . The results suggest that SENCAR mouse embryo cell cultures may convert less B(a)P to water-soluble metabolites and more to DNA-binding metabolites than BALB/c mouse embryo cells.(ABSTRACT TRUNCATED AT 250 WORDS) Br J Haematol, 1986 Sep, 64(1), 161 - 8 Bone marrow purging procedure in Burkitt lymphoma with monoclonal antibodies and complement: quantification by a liquid cell culture monitoring system; Favrot MC et al.; Using B1, Y29/55 and AL2 monoclonal antibodies (MoAbs) to target Burkitt lymphoma (BL) cell lines, we defined optimal conditions to lyse, in the presence of baby rabbit complement, BL cells in excess bone marrow (BM) . After the purging procedure, down to one residual BL cell in 10(6) normal ones was detectable with a liquid cell culture assay . Using a cocktail of three MoAbs, on five different cell lines were observed more than 4 log BL cell depletion in samples contaminated with 1% BL cells and only one failure of the procedure on 17 experiments . However, a sixth line was constantly resistant to the procedure. Immunol Lett, 1986 Sep, 13(3), 127 - 31 Suppression of monocyte mediated anti U562 cytotoxicity by soluble factors in the serum of cancer patients and in the supernatant of K562 cell culture; Kiczka W et al.; Anti U562 cytotoxicity of monocytes isolated from blood of patients with alimentary tract cancer was shown to be depressed compared to monocytes of healthy individuals . A serum factor was demonstrated in the patients which depressed the cytotoxicity of healthy donor monocytes . Such suppressive factor was also produced by the K562 cell line and its production was blocked by indomethacin, a prostaglandin synthesis inhibitor, as well as by thymus preparations TFX and Thymex L. Cancer Res, 1986 Sep, 46(9), 4631 - 41 Morphological characterization of transformed colonies in rat tracheal epithelial cell cultures exposed to carcinogen; Kitamura H et al.; The purpose of the studies described in this and the accompanying paper (D.J . Fitzgerald et al., Cancer Res., 46:4642-4649, 1986) was to define the transformed colonies of the rat tracheal epithelial cell transformation system (see P . Nettesheim and J.C . Barrett, CRC Crit . Rev . Toxicol., 12: 215-239, 1984) in terms of differentiation and growth characteristics . Exposure of low-density primary rat tracheal epithelial cell cultures to N-methyl-N'-nitro-N-nitrosoguanidine results in the development of growth-altered colonies of different sizes and morphologies, which can be readily scored 5 wk after treatment . These colonies were classified into four morphological types (I to IV) based on their light microscopic and ultrastructural features . Colonies designated as types I and II were small, had a low cell density, and were composed principally of large, pale-staining (Giemsa) flattened cells with a low nuclear/cytoplasmic ratio . Examination of the fine structure of these colonies revealed a monolayer of extremely attenuated cells containing well-developed Golgi complexes, endoplasmic reticulum, numerous secondary lysosomes, but few tonofilaments and desmosomes . Colonies of types III and IV were large, basophilic (Giemsa), high-density colonies, consisting primarily of closely packed, small, round cells with high nuclear/cytoplasmic ratio . Analysis of the fine structure of these colonies revealed two to four stratified layers of poorly differentiated cells and cells having features of keratinocyte differentiation, as evidence by abundant tonofilament bundles, well-developed desmosomes, and occasional keratohyaline granules . Differential cell counts were carried out at the ultrastructural level on pooled cells from each colony type (except type I); approximately 90% of the cells from type II colonies were large, electron-lucent cells, while this cell type comprised only 20 to 30% of the cells of colonies of types III and IV . The predominant cell type (approximately 60%) in colonies of types III and IV showed clear signs of keratinocyte differentiation . Type IV colonies contained a significantly larger proportion (greater than 20%) of small, poorly differentiated cells than type III (6%) and type II (1%) colonies . A combined autoradiographic and ultrastructural study revealed that the small, poorly differentiated cells were most active in synthesizing DNA and had a labeling index of 60% . Other cell types were far less frequently labeled, particularly the large, electron-lucent cells which had a labeling index of only 10% . Morphometric measurements on fixed and stained colonies disclosed that each colony type differs markedly in cell population size.(ABSTRACT TRUNCATED AT 400 WORDS) Proc Natl Acad Sci U S A, 1986 Sep, 83(18), 6771 - 5 A single nucleotide change in the E2 glycoprotein gene of Sindbis virus affects penetration rate in cell culture and virulence in neonatal mice; Davis NL et al.; The nucleotide sequence of the glycoprotein genes of fully virulent Sindbis virus and derived mutants that have reduced neurovirulence for neonatal mice (attenuated mutants) has been determined . A single amino acid difference, arginine instead of serine at position 114 of the mature E2 glycoprotein, distinguished the prototype attenuated mutant from its virulent wild-type parent . Virulent revertants of the attenuated mutant showed same-site reversion to the wild-type sequence . An identical single amino acid substitution, an arginine for the serine at E2 position 114, was found in a second independently selected attenuated mutant . The strains are characterized by genetic linkage between attenuation, accelerated penetration of baby hamster kidney cells, and efficient neutralization by the E2-specific monoclonal antibodies R6 and R13; selection for change in one property simultaneously selected for change in the other two (Olmsted, R . A., Baric, R . S., Sawyer, B . A . & Johnston, R . E . (1984) Science 225, 424-427 and Olmsted, R . A., Meyer, W . J . & Johnston, R . E . (1986) Virology 148, 1-10) . The nucleotide sequence data suggest that a single mutation in the E2 gene is sufficient to cause these coordinate phenotypic changes . These findings identify a single locus in a Sindbis virus surface glycoprotein gene that determines both efficiency of interaction with cultured baby hamster kidney cells and degree of virulence in neonatal mice. Vopr Virusol, 1986 Sep-Oct, 31(5), 623 - 9 {Stimulation of arbovirus reproduction in cell cultures by hormones}; Kokorev VS et al.; The possibility of stimulation of reproduction and antigen production of arboviruses (tick-borne encephalitis, Japanese encephalitis, western equine encephalomyelitis) in cell cultures (SPEV, chick embryo fibroblasts) by using various hormones (insulin, ACTH, hydrocortisone) and optimal temperatures was demonstrated . The stimulating effect depended upon the type of cell culture, virus, method of cell treatment, and manifested by a significant increase of the hemagglutinating and infectious activity of the replicating viruses . Differences in the structure of populations, biophysical and antigenic properties of the viruses associated with the conditions of their reproduction in vitro were observed. Am J Vet Res, 1986 Sep, 47(9), 1892 - 5 Antiviral effectiveness of butylated hydroxytoluene against pseudorabies (Aujeszky's disease) virus in cell culture, mice, and swine; Pirtle EC et al.; Butylated hydroxytoluene (BHT) was evaluated for antiviral effectiveness on pseudorabies virus (PRV) in cell culture, mice, and swine . When relatively small amounts of BHT were mixed with PRV and incubated at 37 C for 30 or 60 minutes before inoculation into cell cultures, the cell cultures did not become infected with virus . The PRV was not infectious when the virus was treated with BHT and then inoculated intraperitoneally into mice, but was infectious when BHT and PRV were inoculated simultaneously or when BHT was inoculated either 30 or 60 minutes before PRV . Swine fed BHT-medicated feed for 10 days before they were intranasally exposed with virulent PRV did not have overt signs of pseudorabies, had a lower concentration of PRV in nasal mucus than did control swine, and had acceptable blood enzyme and cholesterol concentrations during the experiment . The BHT was detected in tissues of 2 swine after they were fed BHT-medicated feed for 10 days, and higher concentrations of BHT were detected in tissues of 3 swine given BHT feed for 29 days. J Clin Microbiol, 1986 Sep, 24(3), 487 - 9 Viral isolation versus immune staining of infected cell cultures for the laboratory diagnosis of herpes simplex virus infections; Hughes JH et al.; The Difco Laboratories Cellmatics kit, an immune staining method for the detection of herpes simplex virus after amplification in culture, was found to be 94.6% (138 of 146) sensitive and 100% (302 of 302) specific . However, conventional culturing detected 10 (6.4%) additional viruses that were not herpes simplex . For a full-service diagnostic laboratory, viral isolation is important. Exp Cell Res, 1986 Sep, 166(1), 47 - 62 Cleavage of vimentin in dense cell cultures . Inhibition upon transformation by type 5 adenovirus; Ben-Ze'ev A et al.; The analysis on two-dimensional isoelectric focusing and SDS polyacrylamide gels (2D gels) of the Triton X-100 and high salt-insoluble fraction of fibroblast cell lines, certain epithelial cell lines and granulosa cells revealed various amounts of a vimetin cleavage product, with a more basic pI and with a MW (1,500-2,000) lower than that of intact vimentin . This cleavage product of vimentin which constituted as much as 30% of the total vimentin in an established rat embryo fibroblast cell line (CREF), was detected by a monoclonal antivimentin antibody in whole cell and Triton-insoluble extracts, and it has a phosphorylated variant which can be degraded to form the "staircase pattern" on 2D gels similarly to intact vimentin . This processing of vimentin occurred mainly in dense cell cultures and it could not be induced in sparse cell cultures by inhibiting DNA synthesis with ara C, or by arresting cell growth in medium containing 0.1% serum . Transformation of CREF cells with intact wild-type (H5wt) and host-range cold-sensitive mutants (H5hr1 or H5d1101) of type 5 adenovirus (Ad5), or transformation of CREF cells by Ca2+-mediated DNA transfection with the transforming E1a (0-4.5 map units) or E1a + E1b (0-11.5 map units) region of Ad5 inhibits the cleavage of vimentin in dense cultures only at temperatures which are permissive for expression of the transformed phenotype . The transformation of cells with bovine papilloma virus type 1, with T24 ras oncogene, or with RSV does not interfere with the cleavage of vimentin . The organization of the vimentin network in dense cultures, where the vimentin cleavage occurs, is very different from that of sparse untransformed and sparse or dense Ad5-transformed cells . The possibility that the acidic amino acid-rich C-terminus of vimentin is cleaved in dense cell cultures in conjunction with the reorganization of the vimentin network and the inhibition of this cleavage by transformation with Ad5, are discussed. J Neurochem, 1986 Sep, 47(3), 912 - 9 Fate of cyclic nucleotides in PC12 cell cultures: uptake, metabolism, and effects of metabolites on nerve growth factor-induced neurite outgrowth; Braumann T et al.; The fate of cyclic AMP (cAMP), dibutyryl-cAMP (Bt2-cAMP), and the (Sp)-isomer of adenosine 3',5'-monophosphorothioate {(Sp)-cAMPS} was studied in the PC12 culture medium by means of HPLC . In the absence of PC12 cells, cAMP and Bt2-cAMP were rapidly degraded by nonspecific esterases and cyclic nucleotide phosphodiesterase both originating from the serum commonly used as a culture medium ingredient, whereas (Sp)-cAMPS was completely stable . Since 5'-AMP, adenosine, inosine, and hypoxanthine appeared in the culture medium after incubation with cAMP or Bt2-cAMP, we have determined their effect on nerve growth factor (NGF)-induced neurite outgrowth . 5'-AMP, adenosine, and inosine were indeed potent agents in producing a potentiating effect on NGF-induced early neurite outgrowth at a concentration of 1 mM . Thus, cAMP metabolites had the capacity to induce an effect that has been described as cAMP-specific . In serum-free culture medium and in the presence of cells, all cyclic nucleotides were taken up by PC12 cells . Uptake was highly correlated with the hydrophobic nature of the compounds, and was accompanied by a simultaneous excretion of metabolites . On incubation with cAMP, NGF had a pronounced effect on the metabolic pattern found in the culture medium . In particular, dephosphorylation of 5'-AMP was specifically enhanced . This effect of NGF on the degradation of cAMP was also apparent when cAMP metabolites were incubated with PC12 cells . Whereas 5'-AMP degradation was greatly increased, NGF had no effect on the metabolism of the other purine compounds. Biull Eksp Biol Med, 1986 Sep, 102(9), 327 - 9 {Stimulating effect of polyion on the production of cytolytic T lymphocytes in a cell culture in vitro}; Derevianchenko IG et al.; Introduction of polyion "vegetan" at the concentrations varying from 20 to 100 micrograms/ml into mixed lymphocyte culture (MLC) with insufficient antigenic stimulus, low immunogenic MLC, for the whole of the incubation period, leads to 30-70% increase in T-lymphocyte cytolytic activity . The effect of vegetan on the cytolytic activity of T-lymphocytes from normal MLC is less marked . In the in vivo model of primary synthesis of antibodies against heterologous erythrocyte antigens, vegetant causes 20-50-fold enhancement of a weak immune response compared to 1.5-2-fold when the latter approaches the highest levels . These findings indicate an inverse relationship between the immunostimulating activity of vegetan and the levels of the immune response, the latter being the target of activation . Vegetan was shown to induce more than 2-fold increase in the proliferation in both types of MLC as well as in mouse spleen lymphocyte monoculture . It is reasonable to propose that the preparation may stimulate the proliferation of both T-killers and other lymphocyte subpopulations. Acta Virol, 1986 Sep, 30(5), 436 - 9 Peculiarities of Rickettsia prowazekii in the cell culture as revealed by cryoultramicrotomy; Smirnova NS et al.; Ultrastructure of Rickettsia prowazekii has been followed in L-929 cells 4 days post-infection (p.i.) by cryoultramicrotomy . Groups of rickettsiae were present in the cytoplasm outside of vacuoles forming microcolonies . The size of rickettsiae amounted to 400 X 700 nm, the average thickness of the cell wall was 5 nm, that of periplasmic space and cytoplasmic membrane 14 and 6 nm, respectively . Within intracytoplasmic colonies the rickettsiae were tightly packed and their cell walls were closely adjacent to each other . No halo or capsule-like coating around them was detected . No ultrastructural details were observed in the light translucent spaces between cells . Marginal rickettsiae of the microcolonies were often in close contact with the host cell mitochondria. Vopr Virusol, 1986 Sep-Oct, 31(5), 567 - 72 {Characteristics of the ribonucleoprotein of the measles virus persisting in a human cell culture}; Andzhaparidze OG et al.; The properties of virus-specific RNPs recovered from human HEp-2 and L-41 cells chronically infected with measles virus were studied in comparison with those of RNPs formed in acute infection of L-41 cells . The persisting RNP was shown to contain nucleoprotein not differing in the electrophoretic mobility from the same protein of measles virus virions, and RNA in the persisting RNP was found to be insensitive to the action of RN-ase . RNP from chronically infected cells had a changed ultrastructure and conformation as compared with RNP of the original virus and, unlike the latter had no infectivity upon transfection of the sensitive cells by calcium-phosphate precipitation . No differences in relationships of RNP with the cytoskeleton of the infected cells in the acute and chronic infection were observed. Acta Virol, 1986 Sep, 30(5), 432 - 5 Substance P, a neuropeptide, inhibits measles virus replication in cell culture; Schroeder C; Substance P, a neuropeptide of the tachykinin group, inhibits measles virus replication in cell culture and partially blocks viral fusion activity assayed in the haemolysis system . The ID50 for the inhibition of measles virus single-cycle replication is 0.6 mumol/l, and the effect is fully reversible . The antiviral activity of substance P corresponds to that of previously described synthetic tri-to heptapeptides . Tachykinins and these oligopeptides share a short homology with the N-terminus of paramyxovirus fusion proteins. Metabolism, 1986 Sep, 35(9), 843 - 6 Divergent dopaminergic regulation of TSH, free alpha-subunit, and TSH-beta in pituitary cell culture; Greenspan SL et al.; TSH is a glycoprotein hormone composed of two nonidentical, noncovalently associated subunits, alpha and beta . We have previously shown in vivo that intrapituitary free alpha-subunit and intact TSH have divergent responses to hypothyroidism and thyroxine treatment, suggesting fundamental differences in their regulation . To explore this further, we exposed anterior pituitary cell cultures from rats previously rendered hypothyroid to thyrotropin releasing hormone (TRH), dopamine (DA), or TRH and DA and determined TSH, free alpha-subunit and TSH-beta responses . While positive or negative trends were noted at four hours, the most significant changes were observed at 24 and 48 hours . TRH increased media TSH at 24 hours to 180% of its basal value (P less than 0.01), with a comparable response at 48 hours . TRH also increased free alpha-subunit to 155% of the basal value (P less than 0.01) and TSH-beta to 145% of the basal value (P less than 0.01) at 24 hours . In contrast, DA produced concordant inhibition of TSH to 85% (P less than 0.05), free a-subunit to 42% (P less than 0.01), and TSH-beta to 53% (P less than 0.01) of the basal values at 24 hours . However, coincubation with both TRH and DA produced discordant responses: TSH was stimulated to 126% of the basal value at 24 hours (P less than 0.01), while both free alpha-subunit and TSH-beta fell significantly below the basal values (81% and 65% respectively, P less than 0.01).(ABSTRACT TRUNCATED AT 250 WORDS) J Neurosci, 1986 Sep, 6(9), 2635 - 43 Oligodendroglia development in cell culture as monitored with a monoclonal antibody; Collins JM et al.; A new marker for young oligodendrocytes has been identified by a monoclonal antibody (mOg-1, IgM isotype) prepared from cerebellar plasma membrane stimulated mouse lymphocytes . mOg-1 reactive cells in the mouse cerebellum first appear at day 19 of gestation . Future white matter layers of fixed sections of neonatal rat cerebellum were labeled with mOg-1 . Although EM analysis has shown cell-surface binding by presumptive oligodendroglia in neonatal cerebellum, the antibody does not bind to compact myelin . In cell cultures prepared from 6-d-old mice, 1.1% of the cells bound mOg-1 after 3 d in culture, but up to 5% of the cells bound mOg-1 after 2 weeks in culture . Of these same Og-1-positive cells, 69% bound anti-galactocerebroside and 65% bound anti-myelin basic protein . After a week in culture Og-1-positive cells often produced lamellar sheets extending a millimeter over the polylysine substratum in the absence of normal myelin formation . mOg-1 recognizes a cell-surface determinant distinct from well-characterized oligodendroglial molecules (galactocerebroside, sulfatide and myelin basic protein) that is expressed early in oligodendrocyte development . The antibody has been used to follow the maturation of oligodendrocytes in cultures of both normal and jimpy mouse cerebellum. Science, 1986 Aug 22, 233(4766), 883 - 6 Replication of the B19 parvovirus in human bone marrow cell cultures; Ozawa K et al.; The B19 parvovirus is responsible for at least three human diseases . The virus was successfully propagated in suspension cultures of human erythroid bone marrow from patients with hemolytic anemias; release of newly synthesized virus into the supernatants of infected cultures was observed . This culture system allowed study at a molecular level of events associated with the B19 life cycle . The B19 parvovirus replicated through high molecular weight intermediate forms, linked through a terminal hairpin structure . B19 replication in vitro was highly dependent on the erythropoietic content of cultures and on addition of the hormone erythropoietin. Clin Exp Immunol, 1986 Aug, 65(2), 434 - 42 Analysis of IgG subclass production in cell cultures from IgA deficient patients and in normal controls as a function of age; Luzi G et al.; IgA deficient individuals may also have low serum levels of IgG subclasses, especially IgG2 . In the present study we examined the development of plasma cells producing IgM, IgA or IgG, and the IgG1 and IgG2 subclasses, following lipopolysaccharide (LPS) and pokeweed mitogen (PWM) stimulation of mononuclear cells (MNC) from normal and IgA deficient individuals as a function of age . Studies of blood MNC from 38 normal donors (age range 2-44 years) revealed an age-related distribution pattern of mu, gamma, alpha, gamma 1 and gamma 2 plasma cells produced in mitogen-stimulated and control cultures . Decreased IgA responses to both LPS and PWM were consistently observed in cultures of MNC from all of the nine children with IgA deficiency . When compared with age-matched controls the IgG response was also diminished in PWM stimulated cultures, whereas the IgM responses were normal . The IgG deficit was due to reduced responses for the gamma 1 and gamma 2 subclasses, and was most pronounced for IgG2; IgG2 plasma cell differentiation was particularly depressed in LPS cultures . In contrast to normal adult cells, blood MNC from the nine children with IgA deficiency and age-matched controls (2-17 years) yielded more IgG1 than IgG2 plasma cells in both control and LPS cultures, while the pattern of response to PWM was similar in all groups (gamma 1 greater than gamma 2) . A good concordance was found between the level of secreted Ig in the culture supernatants and the relative number of IgM or, IgG and IgA plasma cells identified by immunofluorescence staining of cytoplasmic immunoglobulins. Cell Biol Int Rep, 1986 Aug, 10(8), 649 - 58 Role of albumin for the cholesterol transport and on the steroidogenic pathways in serum-free medium newborn rat adrenocortical cell cultures; Hammami M et al.; {3,4-13C}cholesterol-albumin complex incubated in serum-free medium allowed to evaluate quantitatively the transfer of cholesterol in newborn rat adrenocortical cultured cells, its accumulation as free cholesterol or cholesterol esters and its transformation into steroids which were also originated (47%) from intracellular unlabelled cholesterol . Increasing concentrations of albumin up to 5 g/l enhanced the production of total steroids but in the meantime decreased the 21-hydroxylated steroid fraction . Internalization of albumin shown by using {methyl-14C}methylated-albumin as a tracer accounted only for a minor part in the cholesterol uptake but strikingly affected the steroidogenic pathways by favoring the reductive metabolism of progesterone over the corticosteroid biosynthesis. In Vitro Cell Dev Biol, 1986 Aug, 22(8), 491 - 8 Detection and speciation of common cell culture mycoplasmas by an enzyme-linked immunosorbent assay with biotin-avidin amplification and microporous membrane solid phase; Gabridge MG et al.; An enzyme-linked immunosorbent assay (ELISA) was developed in order to serve in detecting and speciating mycoplasmas isolated from cell cultures . Its main features included a biotin-streptavidin amplification step and a solid phase consisting of a microporous membrane . Cell samples in the form of suspensions were applied to nitrocellulose or ion exchange membranes immobilized in commercially-available microtiter, multiwell manifolds . The blocking buffer contained 1% purified alpha-casein . The primary antibodies were monoclonal and the polyclonal secondary antibody was biotinylated . The enzyme utilized was streptavidin-horseradish peroxidase . The substrate-dye complex consisted of either 4-chloro-1-naphthol and hydrogen peroxide or ortho phenylene diamine (OPD) and hydrogen peroxide . The presence of homologous antiserum in the reaction sequence gave clearly visible, colored reactions on the membrane when 50 ul with approximately 10(5) or more cfu/ml were present . This new biotin-avidin microporous membrane (BAMM-ELISA) test can be used both to detect mycoplasmas and to speciate them . The BAMM-ELISA is simple, rapid, sensitive, specific and economical . As such, it has potential for aiding in the control of mycoplasma contamination in cell culture, and could prove useful in clinical diagnostic applications as well. Exp Parasitol, 1986 Aug, 62(1), 92 - 7 Anisakis simplex: larval excretory secretory protein production and cytostatic action in mammalian cell cultures; Raybourne R et al.; Excretory secretory proteins produced in vitro by Anisakis simplex larvae incubated in Medium 199 or phosphate buffered saline with dextrose are similar with respect to protein content and biological activity . Analysis by sodium dodecyl sulfate-polyacrylamide gel electrophoresis suggested that the molecular weight of the component(s) responsible for inhibition of mitogen induced lymphocyte blastogenesis is between 66,000 and 95,000 . In vitro production of excretory secretory protein, approximately 1 microgram/24 hr by a single larva, was sufficient to inhibit lymphocyte blastogenesis . Serum from a human anisakiasis patient reacted with these proteins in immunoblots, indicating that, during invasion of the gastric mucosa, enough of them are produced in vivo to induce an immune response . The excretory secretory proteins significantly inhibited proliferation of transformed mammalian cell lines of lymphoid (P3/X63-Ag8) and epithelioid (HeLa) origin . As in mitogen stimulated lymphocytes, the inhibitory effect was cytostatic rather than cytotoxic . These findings suggest that, in addition to being potent immunogens, larval excretory secretory proteins are produced in sufficient quantity to modulate the host response in anisakiasis. Kidney Int, 1986 Aug, 30(2), 246 - 54 Cell culture approaches to the analysis of glomerular inflammation; Lovett DH et al.; In this paper, we have attempted to provide an overview of the methods and findings of a large number of investigators who have dealt with an analysis of the glomerular inflammatory response using tissue culture techniques . These observations represent only a beginning . With the growing interest in this aspect of kidney disease, it is to anticipated that many further advancements in the understanding of the cell biology of the glomerulus are forthcoming . The translation of this fundamental information into new diagnostic and therapeutic modalities is an exciting challenge to investigative nephrology. J Invest Dermatol, 1986 Aug, 87(2), 217 - 20 Modulation of collagen type synthesis in organ and cell cultures of fibroblasts; Weber L et al.; Fibroblast cultures are widely used to study abnormalities of collagen metabolism in both inborn and acquired diseases . However, there is reason to question the extent to which the experimental information obtained from in vitro culture systems in fact reflects the in vivo situation . In the present study we analyzed the proportions of collagens I and III synthesized by human and mouse skin fibroblasts maintained under various culture conditions . The amount of type III collagen extracted from skin specimens was lower than that which was newly synthesized in organ culture . Cells obtained by enzymatic disintegration of skin specimens synthesized more type III collagen than fibroblasts grown from explants . However, subcultivation of the enzymatically liberated cells resulted in a continuous decline of type III collagen production which eventually reached levels similar to those observed in explant cultures. Proc Natl Acad Sci U S A, 1986 Aug, 83(16), 5958 - 62 Differentiation-inducing factor purified from conditioned medium of mitogen-treated spleen cell cultures stimulates bone resorption; Abe E et al.; Spleen cells treated with mitogens produce a potent bone-resorbing factor called osteoclast-activating factor (OAF) . To examine the relationship between the bone-resorbing factor and other protein factors produced by spleen cells, the colony-stimulating factor (CSF), the differentiation-inducing factor (DIF), the macrophage fusion factor (MFF), and the macrophage growth factor (MGF) were purified from 2.68 liters of conditioned medium of mouse spleen cell cultures treated with concanavalin A . Purification was performed successively by DEAE-cellulose, Blue Sepharose, and Sephadex G-150 column chromatography and high-pressure liquid chromatography (HPLC) . The DIF was successfully separated from CSF and MGF on HPLC . CSF coincided with MGF on HPLC, but MFF disappeared before application to HPLC . Only the DIF exhibited bone-resorbing activity, whereas CSF and MGF did not . The DIFs purified from L929 cells and Ehrlich ascites tumors similarly exhibited bone-resorbing activity . The DIFs purified from spleen cells and Ehrlich ascites tumor cells exhibited neither interleukin 1 (IL-1) activity nor tumor necrosis factor (TNF) activity, though the unfractionated conditioned medium from spleen cells did exhibit them . In the light of recent reports that IL-1 beta and TNF also stimulate bone resorption, the term OAF should refer to a generic activity rather than a single factor. Jpn J Med Sci Biol, 1986 Aug, 39(4), 163 - 7 Use of microplate cell culture and enzyme immunoassay in titration of serum neutralizing antibody against Hochi strain of serotype 4 human rotavirus; Urasawa T et al.; The tube neutralization test read by enzyme immunoassay developed by Wyatt et al . (1983) for serotype determination of human rotavirus was modified so as to use stationary cultures of MA104 cells in a microtiter plate instead of roller tube cultures . Sera obtained from different age groups were titrated for neutralizing antibody against serotype 4 human rotavirus Hochi strain by this test and the results were compared with those obtained by the plaque neutralization test . There was a good correlation between the titers obtained by the two tests and the age distribution pattern of serotype 4 neutralizing antibody was similar to those of serotype 1 and 3 antibodies previously reported. J Biol Response Mod, 1986 Aug, 5(4), 351 - 61 Cytotoxicity of autologous Epstein-Barr virus-transformed cells mediated by interleukin-2 dependent, long-term T cell cultures is augmented by beta, but not alpha, recombinant interferon; Chen BP et al.; The immunomodulatory effect of two highly purified recombinant human interferons (IFN) on T cell-mediated cytotoxicity of autologous Epstein-Barr virus-transformed B cells (EBV-LCL) was assessed . Interferon beta ser, but not alpha 76, potentiated the destruction mediated by interleukin-2 (IL-2) dependent, long-term T cell cultures . The degree of potentiation of specific cytotoxicity was dependent on the concentration of the exogenously added IFN beta ser . In contrast to the potentiation of T cell-mediated cytotoxicity, IFN beta ser and alpha 76 did not modulate the cytotoxic activity of cytotoxic T cell clones that are specifically reactive to autologous EBV-LCL . These results indicate that the selective potentiation of EBV-LCL-reactive T cell-mediated cytotoxicity by IFN beta ser may not be due to a direct IFN effect on the cytotoxic T cells (CTLs) themselves . Rather, our results are consistent with IFN beta ser augmenting cytotoxic T cell reactivity indirectly, possibly via its influence on other immunoregulatory cells . Although the precise mechanism whereby IFN beta ser may potentiate cytotoxicity is not yet defined, the synergistic action of IFN beta ser and IL-2 on long-term CTL cultures offers an alternate means to selectively enhance a desirable cytotoxic response. J Pharmacol Exp Ther, 1986 Aug, 238(2), 727 - 38 Carbamazepine and 10,11-epoxycarbamazepine produce use- and voltage-dependent limitation of rapidly firing action potentials of mouse central neurons in cell culture; McLean MJ et al.; Effects of the anticonvulsant, carbamazepine (CBZ), on mouse central neurons in cell culture were examined using intracellular recording techniques . Spinal cord and cortical neurons demonstrated sustained repetitive firing (SRF) of action potentials (APs) at high frequency in response to depolarizing current pulses . Hippocampal neurons fired rapidly only in the pressure of the calcium channel blocker verapamil (1 microM) . Concentrations of CBZ equivalent to therapeutic anticonvulsant levels in cerebrospinal fluid (greater than 4.2 microM) limited firing to a few action potentials in all three cell types in parallel with use-dependent reduction of AP maximal rate of rise (Vmax) . Resting membrane properties and postsynaptic responses to ionophoretically applied gamma-aminobutyric acid and glutamate were unaffected . The CBZ metabolite, 10,11-epoxycarbamazepine, limited SRF at a concentration (4 microM) encountered at therapeutic CBZ levels . Another major CBZ metabolite, the 10,11-diol, limited SRF only at concentrations greater than those encountered with therapeutic serum CBZ levels . In control medium, APs could be evoked at a maximal rate of 450 Hz while in CBZ (10.6 microM) AP frequency was limited to 100 to 200 Hz . Limitation of SRF was voltage-dependent and could be partially reversed, or prevented, by membrane hyperpolarization . In addition, recovery of Vmax of sodium-dependent action potentials from inactivation was slowed, suggesting an effect on voltage-dependent sodium channels . CBZ and phenytoin effects of SRF were similar . We suggest that limitation of sustained high frequency repetitive firing may be a significant anticonvulsant action of carbamazepine . The 10,11-epoxy derivative of CBZ, but not the 10,11-diol, may contribute to anticonvulsant efficacy by the same mechanism. Clin Endocrinol (Oxf), 1986 Aug, 25(2), 173 - 9 Growth hormone secreting pituitary adenomas are heterogeneous in cell culture and commonly secrete glycoprotein hormone alpha-subunit; White MC et al.; Cell culture methods were used to assess whether human pituitary adenomas secreting GH and associated with clinical acromegaly also secreted the structurally unrelated glycoprotein hormone alpha-subunit . Thirty-two tumours, together with peri-adenomatous tissue from two of them and three normal pituitaries were studied . Anterior pituitary hormones were measured by radioimmunoassay and included PRL, TSH, LH, FSH, and ACTH, as well as GH and alpha-subunit . Normal pituitary tissues secreted all hormones assayed . All 32 tumours secreted GH ranging from 241 to 5556 ng/2 X 10(5) cells/24 h and 12 (37.5%) secreted alpha-subunit in amounts which could not be accounted for by cross-reaction of other hormones or contamination by normal pituitary tissue, and which ranged from 10.3 to 73.5 ng/2 X 10(5) cells/24 h . Ten other tumours also secreted alpha-subunit but in very small amounts, not exceeding 1.8 ng/2 X 10(5) cells/24 h . PRL was secreted from 21 tumours (66%), and small amounts of other hormones, chiefly LH and TSH, were occasionally secreted from tumours . These cell culture studies would suggest that pituitary adenomas causing acromegaly are hormonally heterogeneous and that PRL and glycoprotein alpha-subunit are commonly detected in addition to GH. Biochem Pharmacol, 1986 Jul 15, 35(14), 2373 - 9 Effect of trifluoperazine and other drugs on matrix vesicle formation by chicken growth plate chondrocytes in primary cell culture; Schalk EM et al.; Growth plate chondrocytes in primary culture release alkaline phosphatase (AP)-rich matrix vesicles (MV) into the culture medium . While these are thought to derive from the plasma membrane by a membrane fusion-dependent process, the mechanism is not fully understood . Recently, a cytosolic protein, synexin, has been shown to promote membrane fusion in a number of systems, and thus may be involved in MV formation . Since the action of synexin is selectively inhibited by trifluoperazine (TFP) and other phenothiazines, we examined the effects of these drugs, and imipramine, on cellular AP production and formation of AP-containing MV by cultured chondrocytes . In addition, we studied the effect on cell division, protein biosynthesis, and incorporation of palmitate into cellular and MV lipids . All of the drugs reduced cellular AP in a concentration-dependent manner; however, at the higher levels, chlorpromazine (CPZ) and TFP caused a transient sharp rise in MV AP activity . At IC50 levels, the drugs were more inhibitory to cellular AP than to MV AP, appearing to enhance significantly the transfer of available cellular AP into MV . In contrast, the drugs stimulated incorporation of {3H}palmitate into cellular lipids, but either had no effect on, or actually inhibited, incorporation of the fatty acid into MV . At these levels, the drugs had little effect on cell division and protein biosynthesis . The inhibitory effect of IC50 levels of CPZ on palmitate incorporation into MV appears to have resulted from impairment of vesicle formation per se, since at these levels the drug stimulated incorporation of the fatty acid into the cells . The transient stimulatory effect of higher levels of CPZ on MV AP levels, and the enhanced transfer of AP into MV by the drugs generally, may result from the effect of the drugs on membrane structure . Since TFP was not inhibitory to MV formation, it is doubtful that synexin was directly involved. Brain Res, 1986 Jul 2, 377(1), 54 - 62 Characterization of dopamine receptors on neurons grown in primary dissociated cell culture from ventral mesencephalon of mouse; Heyer EJ et al.; Mammalian neurons from ventral mesencephalon were grown in primary dissociated cell culture . These cultures were examined for dopamine sensitive adenylate cyclase activity and specific ligand binding of {3H}spiroperidol and {3H}flupenthixol . No stimulation of adenylate cyclase activity by 10 microM dopamine was demonstrable in cell culture homogenates . {3H}Spiroperidol bound to cell culture homogenates with high affinity and was displaced by (+)-butaclamol but not by 5-hydroxytryptamine, suggesting that the {3H}spiroperidol was bound to dopamine receptors . While {3H}flupenthixol binding was also present, it could be displaced by spiroperidol indicating that the dopamine receptor was probably of the D2 subtype . Binding of spiroperidol was proportional to the amount of cell culture homogenate, and was saturable . Are these receptors autoreceptors? The toxin 1-methyl-4-phenylpyridine (MPP+) was used to destroy dopaminergic neurons; spiroperidol binding in these cultures was found to be increased, demonstrating that most of these D2 receptors are not autoreceptors. Vopr Virusol, 1986 Jul-Aug, 31(4), 430 - 5 {Synthesis of measles virus proteins in chronically infected human cell cultures}; Andzhaparidze OG et al.; Synthesis of measles virus proteins in primary and chronic infection of L-41 and HEP-2 cells was studied by radioimmunoprecipitation test and polyacrylamide gel electrophoresis . Synthesis of measles virus main structural proteins, H, P, NP, and M, was found to occur in chronically infected cells . Persistence of measles virus in the systems under study was shown not to be accompanied by changes in electrophoretic mobility of virus polypeptides as compared with proteins synthesized in primary infection . NP protein of the original virus, however, differed significantly from NP protein of the persisting virus in its charge which indicated aminoacid replacements apparently due to mutations of the appropriate region of the virus genome . The mechanism of measles virus persistence in these systems was shown not to be associated with disorders in matrix protein synthesis . Use of monoclonal antibodies in fluorescent antibody technique and radioimmunoprecipitation revealed the stability of a number of antigenic determinants in persisting measles virus in L-41 and HEP-2 cell cultures. EMBO J, 1986 Jul, 5(7), 1465 - 72 Evidence from molecular cloning that SPARC, a major product of mouse embryo parietal endoderm, is related to an endothelial cell 'culture shock' glycoprotein of Mr 43,000; Mason IJ et al.; We describe the molecular cloning and characterization of a secreted, acidic, cysteine-rich glycoprotein (SPARC) of apparent Mr 43,000 which is a major product of mouse embryo parietal endoderm . These cells are specialized for the synthesis of a rapidly expanding basement membrane, but SPARC is not itself an integral matrix component . We show that SPARC is related structurally and antigenically to an Mr 43,000 glycoprotein secreted in large amounts by bovine aortic endothelial cells as part of a 'culture shock' response to in vitro conditions promoting their proliferation and migration. Semin Hematol, 1986 Jul, 23(3), 218 - 22 Human leukemia: cell culture; Messner HA; A number of culture systems are now in use to explore control mechanisms in normal and abnormal hemopoiesis . The combined use of these culture techniques with monoclonal antibodies, molecular probes, and growth-promoting activities have the potential to provide some insight into the mystery of leukemic proliferation. J Submicrosc Cytol, 1986 Jul, 18(3), 625 - 7 Ultrastructure of brown adipocytes mitochondria in cell culture from explants; Cinti S et al.; Brown adipocytes lose in culture their typical mitochondria, which are replaced by others of non typical morphology . During studies aimed at clarifying this phenomenon we found that better preservation of the 'typical' mitochondrial morphology is obtained in vitro after a long period of time, when cells from small fragments (explants) of brown adipose tissue (BAT) are cultured instead of collagenase-isolated brown adipocytes . These results suggest that the explant technique could be better suited to study brown adipocytes in culture than other methods employing collagenase-isolated cells. Endocrinology, 1986 Jul, 119(1), 25 - 35 Development of brain insulin receptors: structural and functional studies of insulin receptors from whole brain and primary cell cultures; Lowe WL Jr et al.; We studied the structural and functional characteristics of insulin receptors from rat brain and liver from late gestation through adulthood as well as from cultured neuronal and glial cells from neonatal rats . Specific insulin binding was present on membrane preparations from brain and liver at all stages of development studied, with maximal binding in neonates greater than 19-day-old fetuses greater than adults for both brain and liver . Maximal specific binding to cultured neuronal and glial cell membranes was similar (6.2% vs . 7.1%, respectively) . {125I}Iodoinsulin cross-linking to the insulin receptor demonstrated that the mol wt (Mr) of the brain alpha-subunit was less than that of the liver alpha-subunit at all stages . {125I}Iodoinsulin cross-linking also demonstrated that the glial cell alpha-subunit (Mr, 130,000) migrated on sodium dodecyl sulfate-polyacrylamide gel electrophoresis to a position intermediate between the liver (Mr, 135,000) and brain (Mr, 119,000), whereas the neuronal cell alpha-subunit (Mr, 118,000) comigrated with the brain alpha-subunit . In solubilized lectin-purified preparations from brain and liver during development as well as from neuronal and glial cells, insulin stimulated phosphorylation of the beta-subunit . The Mr of the brain beta-subunit, as determined by migration on sodium dodecyl sulfate-polyacrylamide gel electrophoresis, was less than that of the liver beta-subunit . The neuronal cell beta-subunit comigrated with the brain beta-subunit while the glial cell beta-subunit migrated to a position intermediate between the brain and liver beta-subunit . Solubilized lectin-purified preparations from all tissues demonstrated insulin-stimulable phosphorylation of exogenous substrates . From these studies we conclude that 1) functional insulin receptors are present in the brain during development in the rat; and 2) the structural differences demonstrated between neuronal and glial cell and between brain and nonneuronal insulin receptors taken together with previously demonstrated functional differences of the insulin receptor on these tissues suggest a unique function for insulin receptors on neuronal tissues. Biull Eksp Biol Med, 1986 Jul, 102(7), 116 - 9 {Bilayer agar cell cultures for studying cellular growth factors}; Cherepantseva EA et al.; The method for biological testing of growth factors (GF) produced by transformed cells is described . The method is suitable for studying the ability of donor cells to release GF that stimulate colony formation of test-cells . Donor cells and test-cells are placed into different semisolid agar layers and separated by intermediate agar layer . The method provides a much more efficient testing of GF biological activity than the use of conditioned cultural fluids of transformed cells . It permits the assessment of GF dialyzation and the role of donor cell proliferation in the production of GF. Semin Thromb Hemost, 1986 Jul, 12(3), 228 - 30 Rapid release and deactivation of plasminogen activators in human endothelial cell cultures in the presence of thrombin and ionophore A23187; Booyse FM et al.; alpha-Thrombin, DFP-thrombin, and ionophore A23187 induce the rapid release (less than 5 minutes) of a variety of proteins, including t-PA forms (Mr 110 and 70 k, after SDS-PAGE) from primary cultures, and both t-PA and u-PA (Mr 90 and 54 k) from subcultured human HUVECs . All PA activity forms are rapidly decreased in the releasates by some unknown mechanism . gamma-Thrombin does not induce the release of PAs from cultured HUVECs. Res Vet Sci, 1986 Jul, 41(1), 133 - 4 Passage of duck hepatitis virus in cell cultures derived from avian embryos of different species; Davis D et al.; An egg-attenuated strain of duck hepatitis virus was successfully passaged through cell cultures of avian embryos derived from goose, turkey, guinea fowl, Japanese quail, pheasant and chicken . Two field strains of the virus were passaged in a more limited range of species. Vet Med (Praha), 1986 Jul, 31(7), 385 - 92 {Immunoenzyme detection of enzootic bovine leukemia viruses (BLV) in cell cultures}; Hofirek B et al.; There is a description of the method of immunoenzymic test used for detection of enzootic bovine leukosis virus (BLV) on the cells of foetal lamb spleen (FLS) permanently producing BLV and on the lymphocytes of infected animals . In both cases (in FLS cells and lymphocytes) the presence of BLV was demonstrated by the immunoperoxidase test . Using the defined serums from the animals after experimental and natural infection, BLV was detected by the above-mentioned test in FLS cells in all cases of the use of positive serums with anti-BLV antibodies . The specificity of the antibodies was also demonstrated in this way . The presence of BLV was analogically detected in the lymphocytes of the infected animals where the infection time was precisely defined . The method of immunoperoxidase test on lymphocytes is recommended in indicated cases for the demonstration of the presence of BLV in infected animals. Endocrinology, 1986 Jul, 119(1), 429 - 31 Estrogens directly down-regulate receptors for angiotensin II in anterior pituitary cell culture without decreasing target cell responsiveness; Carriere PD et al.; Chronic estradiol treatment in vivo has been shown to reduce the density of receptors for angiotensin II (ANG II) in the anterior pituitary lobe (AP) . We studied whether estradiol is directly involved in the down-regulation of ANG II receptors, using AP cells in culture . Binding affinity and density of ANG II receptors were measured in disrupted AP cells with the radiolabeled antagonist {125I}Sar1, Ile8-ANG II ({125I}SARILE) . Estradiol treatment (10 nM) for either 48 or 96 h caused a marked reduction (approximately 70%) in the density of receptors for ANG II in cultured AP cells, with no change in the dissociation constant of {125I}SARILE (Kd, 0.5 +/- (SE) 0.1 nM) . In the AP, specific binding sites for ANG II are present in lactotrophs and ANG II has been shown to release prolactin (PRL) . In AP cells treated with estradiol for 48 h, dose-response curves revealed that ANG II still increased PRL release (P less than 0.01) . The average net PRL release (ANG II-stimulated minus basal) was greater in estradiol-treated cells than in controls, whereas the half-maximal stimulation dose (ED50) of ANG II was the same (0.07 +/- 0.04 nM) . These results suggest that estrogens are directly involved in the modulation of ANG II receptors in the AP, causing marked receptor down-regulation without decreasing target cell responsiveness. Diagn Microbiol Infect Dis, 1986 Jul, 5(2), 135 - 41 Relative sensitivity of cell culture systems in the detection of herpes simplex viruses; Chang RS et al.; Guinea pig embryonic fibroblasts were more sensitive and McCoy and Hep-2 cells were less sensitive than human foreskin fibroblasts in parallel titrations of herpes simplex virus . No difference in sensitivity was found for five lines of human fetal lung fibroblasts (including WI-38 cells), two lines of human embryo fibroblasts, one line of human foreskin fibroblasts, cells from a human fetal kidney, amnion cells from a human placenta, the Chang liver cell, the HeLa cell, and a line of mink cells . The cell doubling level of human or guinea pig fibroblast lines did not affect their sensitivity . One hundred ninety-five clinical specimens submitted for herpes simplex virus isolations were tested in parallel in primary rabbit kidney, guinea pig embryo, and human fetal lung fibroblast cultures . The percentages of positive or false-negative cultures were essentially the same for the three types of cells. Probl Endokrinol (Mosk), 1986 Jul-Aug, 32(4), 63 - 6 {Properties of (Ala5, Orn9)-somatostatin . The inhibition of pancreatic and hypophyseal hormone secretion in cell culture}; Sadovnikova NV et al.; Ability of Ala5, Orn9-somatostatin to inhibit the secretion of insulin, glucagon, somatotropic hormone (STH) and prolactin was tested on the model of primary monolayer culture of isolated insular pancreatic and adenohypophyseal cells . Chemical substitution performed between positions 5 and 9 in somatostatin molecule failed to change essentially hormone's biologic activity in suppression of insulin and glucagon in the culture of pancreatic insular cells isolated from newborn rats . Somatostatin analog revealed its natural hormonal ability to inhibit STH secretion but failed to affect that of prolactin . The results are suggestive of the principal possibility to produce somatostatin biologically active analog through lyzine residue substitution in position 9 with obligatory simultaneous substitution amino acid residue in position 5. Vopr Virusol, 1986 Jul-Aug, 31(4), 435 - 8 {Effect of elevated temperature on the persistence of measles virus vaccinal strain L-16 in a human cell culture}; Koptiaeva IB et al.; The influence of high temperature (40 degrees C) on the virus carrier state and synthesis of virus-specific macromolecules in the HEp-2 cells--measles virus system was studied . The fluorescent antibody technique showed that cultivation at this high temperature led to changes in virus antigen morphology, a decrease in the number and then complete disappearance of the cells producing virus-specific antigen . Simultaneously, gradual cessation of synthesis of all kinds of virus-specific RNA was observed in chronically infected cells . The method of radioimmunoprecipitation showed the incubation at 40 degrees C to affect first of all the synthesis of virus nucleocapsid protein . The temperature-sensitive nature of synthesis of virus macromolecules may be explained by the existence of its mutations in the genome of the persisting virus. Biochem Pharmacol, 1986 Jul 1, 35(13), 2107 - 14 Methylation differences in the murine P1450 and P3450 genes in wild-type and mutant hepatoma cell culture; Peterson TC et al.; The murine P1450 and P3450 genes and flanking regions contain 14 and 15 Msp I sites (C-C-G-G-), respectively, designated M1 through M14 or M15 . These two genes from mouse Hepa-1 wild-type (wt) parent and three mutant cell lines were studied for methylation differences with use of the isoschizomers Msp I and Hpa II . The mutant lines included: c1, having high constitutive P1450 mRNA and believed to carry a mutation in the P1450 structural gene; c2, having negligible levels of Ah receptor; and c4, having a defect in nuclear translocation of the inducer-receptor complex . The P3450 gene was not expressed constitutively or after treatment of these four cell lines with the P1450 inducer 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) and, correspondingly, the P3450 Msp I sites remained methylated . Treatment of all four cell lines with TCDD did not alter the P1450 methylation pattern, nor was there any evidence of P1450 gene amplification . Treatment of all four lines with 5-azacytidine caused demethylation of the P1450 Msp I sites but did not change the usual P1450 catalytic activity pattern found in each of the lines . The only detectable difference in the P1450 gene among the four lines was hypomethylation of the M9 site in c1 that was not seen in wt, c2 and c4 cells . The M9 site is part of a 9-bp box (5'-C-C-G-G-G-A-C-A-T-3'), located near the beginning of exon 3 . It is of interest that the same nine bases are found in intron 2 about 80 bp upstream from the 5' end of exon 3 in the homologous P3450 gene. J Biol Chem, 1986 Jun 25, 261(18), 8505 - 13 Preparation and use of synthetic cell culture surfaces . A new reagent for the covalent immobilization of proteins and glycoproteins on a nonionic inert matrix; Raja RH et al.; To study cell interactions with external molecules immobilized on a chemically defined nonionic, inert matrix, we have prepared flat polyacrylamide matrices containing covalently attached carbohydrate or protein . A new acrylamide derivative, containing a terminal 1,2-dihydroxy group, was synthesized and then copolymerized with acrylamide and bisacrylamide to make 20% polyacrylamide matrices, which could be oxidized with NaIO4 to generate reactive aldehyde groups . Molecules containing a free amine (e.g . proteins or glycopeptides) can be coupled to the aldehyde-activated matrix by formation of a Schiff base and reduction with NaCNBH3 to form a stable -CH2-NH-bond . Unreacted aldehyde groups are reduced to hydroxyl groups with NaBH4 . In order to immobilize polysaccharides on the activated surfaces, these molecules are first modified to contain a free amine . We have described a procedure to convert purified hyaluronic acid oligosaccharides to a reactive alkylamine derivative uniquely modified at the reducing end (Raja, R . H., LeBoeuf, R . D., Stone, G . W., and Weigel, P . H . (1984) Anal . Biochem . 139, 168-177) . The covalent attachment of {3H}hyaluronate-amine, {14C}ethanolamine, or 125I-bovine serum albumin, to activated surfaces was complete within 5-24 h . The amount immobilized was directly proportional to the amine concentration and to the aldehyde content of the matrix and inversely proportional to the molecular weight of the amine . About 90% of the available aldehyde groups reacted with ethanolamine, whereas less than 0.01% reacted with albumin . Molecules larger than 3300 Da were excluded from the interior of the matrix and could therefore only be attached to the surface of the matrix . These synthetic surfaces can be used in long term culture experiments to study cellular interactions with virtually any type of immobilized molecule. Thromb Res, 1986 Jun 15, 42(6), 749 - 60 Characterization and fibrin-binding properties of different molecular forms of pro-urokinase from a monkey kidney cell culture; Wijngaards G et al.; Culture fluid of a monkey kidney cell culture was harvested every two days, for a two week period, in order to obtain urokinase in the zymogen form . Pro-urokinase was isolated by immunoadsorption chromatography and gel filtered on Sephadex G-150, which resulted in three peaks with pro-urokinase activity . SDS-polyacrylamide gel electrophoresis showed that the first peak contained 55 kd pro-urokinase, aggregated with high molecular weight contaminants, whereas the second and third peaks consisted of almost pure 55 kd and 30 kd pro-urokinase, respectively . The latter form represented a relatively unknown and inactive precursor of low molecular weight urokinase, which was, like 55 kd pro-urokinase, activatable with plasmin . In comparison with tissue-type plasminogen activator, 55 kd and 30 kd pro-urokinase only bound weakly to purified fibrin clots and fibrin-sepharose columns . The extent of binding of the two pro-urokinases and their plasmin-activated forms to fibrin-sepharose decreased in the following order: 55 kd pro-urokinase 30 kd pro-urokinase 55 kd urokinase 30 kd urokinase . These results indicate that the two precursors exhibited stronger binding to fibrin-sepharose than the corresponding active enzymes, and the two 55 kd forms exhibited stronger binding than the corresponding 30 kd forms . This indicates the importance of both the zymogen nature and an intact NH2-terminal part of the molecules for binding to fibrin. Thromb Res, 1986 Jun 15, 42(6), 761 - 8 Specific fibrinolytic properties of different molecular forms of pro-urokinase from a monkey kidney cell culture; Rijken DC et al.; The specific fibrinolytic properties of both high molecular weight (55 kd) and low molecular weight (30 kd) pro-urokinase from a monkey kidney cell culture were evaluated in a plasma clot lysis system and compared with those of human urokinase . The system was composed of a radiolabelled plasma clot immersed in plasma containing the fibrinolytic agent . On unit base, 55 kd pro-urokinase was approximately 1.5 times more effective in lysing the clot than 30 kd pro-urokinase and equally effective as urokinase . In contrast to urokinase, both pro-urokinase forms induced clot lysis without degrading fibrinogen in the surrounding plasma . However, a considerable activation of the fibrinolytic system in the plasma occurred as a large amount of alpha 2-antiplasmin was consumed, indicating that pro-urokinase was not fully fibrin-specific . Quenching antibodies against tissue-type plasminogen activator (t-PA) added to the plasma clot lysis system retarded but did not prevent pro-urokinase-induced clot lysis . This indicated that not only was t-PA in plasma involved in the activation of pro-urokinase (probably via plasmin), but that an additional mechanism also existed. Science, 1986 Jun 13, 232(4756), 1390 - 5 Large-scale cell culture in biotechnology; Arathoon WR et al.; The purpose of this article is to review the current state of large-scale cell culture in terms of its applications, problems, and potential . Because of the commercial and proprietary nature of most large-scale cell culture processes, this review does not contain many detailed scientific results although an attempt is made to address some key issues and findings . Much of this summary deals with processes having an established, commercial track record but some attention is given to more recent innovations with interesting potential applications . Reference is made to plant cell culture but the main emphasis is on mammalian cells. Food Chem Toxicol, 1986 Jun-Jul, 24(6-7), 789 - 93 Phototoxicity testing in vitro: evaluation of mammalian cell culture techniques; Lock SO et al.; We have studied the effects of ultraviolet light on cells, and the phototoxic actions of a range of UV- or visible light-absorbing compounds in three separate in vitro tests (photolysis of red blood cells and phototoxicity to macrophages and to Chinese hamster CHV79 cells) . Exposure to UV light for short periods was non-toxic to primary macrophages and red blood cells, but produced extensive killing of CHV79 cells . CHV79 cells can repair a considerable amount of UV-induced damage by unscheduled DNA synthesis but scheduled DNA synthesis is seriously disrupted . In vitro phototoxic compounds selectively damage discrete cellular components, such as the nucleus, plasma membrane or cytoplasmic organelles . Cellular damage can be shown to occur via two distinct photochemical pathways, either oxygen dependent or oxygen independent. Food Chem Toxicol, 1986 Jun-Jul, 24(6-7), 557 - 62 Assessment of thyrotoxicity using in vitro cell culture systems; Brown CG et al.; The potential thyrotoxic effect of SK&F 93479 (an H2-receptor antagonist), aminoglutethimide (AG) and glutethimide were studied by examining their effects on 125iodide uptake and organification (incorporation into thyroglobulin) in cultured thyroid cells from the pig and rat . These effects were compared with those of positive controls (3-isobutyl-1-methylxanthine (IBMX), methimazole (MTI), propylthiouracil (PTU) and NaClO4) . SK&F 93479 had no effect on 125I uptake in rat FRTL-5 cells or on 125I uptake and organification in porcine thyroid cells . In contrast, MTI, PTU and NaClO4 all depressed 125I uptake and organification and IBMX potentiated 125I uptake . It is concluded, therefore, that SK&F 93479 does not increase 125I uptake in rats in vivo by a direct action on the thyroidal iodide trapping mechanism . AG, but not glutethimide, depressed 125I organification by cultured porcine thyrocytes without affecting uptake . These results suggest that AG is thyrotoxic in rats and man probably as a result of its ability to inhibit iodide organification in thyroid cells. Pathol Res Pract, 1986 Jun, 181(3), 273 - 9 Effects of estradiol and promegestone on human breast cell cultures . An ultrastructural study; Chomette G et al.; Effects of estradiol (E2) and promegestone (R 5020) were tested on primary cultures of epithelial human breast cells prepared from surgical specimens of reduction mammoplasty . Comparative morphological studies were performed by means of transmission and scanning electron microscopy in cells without any hormonal adjunction and in those previously treated with E2 or E2 + R 5020 . The results were as follows: In basal medium used as control the majority of epithelial cells, although well-differentiated, remained flat with few microvilli, well-developed Golgi apparatus and rough endoplasmic reticulum, was obvious . R 5020 added with E2 increased differentiation of epithelial cells covered with scarce long and branched microvilli, but reduced the turn-over of young cells . E2 seemed to generate more active proliferation of cells . R 5020 inhibited this effect and induced a higher differentiation of mammary epithelial cells. Biochem Cell Biol, 1986 Jun, 64(6), 583 - 93 Androgen metabolism and actions in rat ventral prostate epithelial and stromal cell cultures; Orlowski J et al.; The rat ventral prostate requires androgens for normal development, growth, and function . To investigate the relationship between androgen metabolism and its effects in the prostate and to examine differences between the epithelial and stromal cells, we have established a system of primary cell cultures of immature rat ventral prostate cells . Cultures of both cell types after reaching confluency (6-7 days) actively metabolized 3H-labelled testosterone (T), 5 alpha-dihydrotestosterone (5 alpha-DHT), 5 alpha-androstane-3 alpha,17 beta-diol, and 5 alpha-androstane-3 beta,17 beta-diol . The epithelial cells actively reduced T to 5 alpha-DHT and formed significant amounts of 5 alpha-androstane-3,17-dione from T, 5 alpha-DHT, and 5 alpha-androstane-3 alpha,17 beta-diol . All substrates were converted to significant amounts of C19O3 metabolites . The stromal cells also metabolized all substrates, but very little 5 alpha-androstane-3,17-dione was formed . The metabolism studies indicate that both cell types have delta 4-5 alpha-reductase, 3 alpha- and 3 beta-hydroxysteroid oxidoreductase and hydroxylase activities . The epithelial cells have significant 17 beta-hydroxysteroid oxidoreductase activity . The epithelial cells cultures grown in the presence of T have higher acid phosphatase (AP) contents (demonstrated histochemically and by biochemical assay) . Tartrate inhibition studies indicate that the epithelial cells grown in the presence of T are making secretory AP . Stromal cell AP is not influenced by T . The results indicate that the cultured cells maintain differentiated prostatic functions: ability to metabolize androgens and, in the case of the epithelial cells, synthesize secretory AP. Neurochem Res, 1986 Jun, 11(6), 789 - 800 Nuclear and mitochondrial DNA synthesis and energy metabolism in primary rat glial cell cultures; Avola R et al.; DNA synthesis in nuclei and mitochondria purified from serum-supplemented rat glial cell cultures at different days after plating was studied . Furthermore in mitochondria, some enzymatic activities related to energy transduction (citrate synthase, malate dehydrogenase, total NADH-cytochrome c reductase, cytochrome oxidase and glutamate dehydrogenase) were measured . For DNA labeling {methyl-3H}thymidine was added to the culture medium at different days after plating . During the culture times studied the specific activity of total, nuclear, and mitochondrial DNA decreased from 8 days in vitro (DIV) to 21 DIV and increased at 30 DIV . The specific activity of nuclear DNA was always higher than that of mitochondrial DNA . The specific activity of the above mentioned mitochondrial enzymes increased from 8 DIV up to 21 DIV and decreased at 30 DIV, suggesting a relationship between the energy metabolism and the differentiation of glial cells in culture. Anal Quant Cytol Histol, 1986 Jun, 8(2), 138 - 47 Automatic digital image analysis for identification of mitotic cells in synchronous mammalian cell cultures; Eccles BA et al.; Mitotic frequency in a synchronous culture of mammalian cells was determined fully automatically and in real time using low-intensity phase-contrast microscopy and a newvicon video camera connected to an EyeCom III image processor . Image samples, at a frequency of one per minute for 50 hours, were analyzed by first extracting the high-frequency picture components, then thresholding and probing for annular objects indicative of putative mitotic cells . Both the extraction of high-frequency components and the recognition of rings of varying radii and discontinuities employed novel algorithms . Spatial and temporal relationships between annuli were examined to discern the occurrences of mitoses, and such events were recorded in a computer data file . At present, the automatic analysis is suited for random cell proliferation rate measurements or cell cycle studies . The automatic identification of mitotic cells as described here provides a measure of the average proliferative activity of the cell population as a whole and eliminates more than eight hours of manual review per time-lapse video recording. J Neurosci, 1986 Jun, 6(6), 1719 - 25 Cooperative regulation of neurotensin content in PC12 pheochromocytoma cell cultures: effects of nerve growth factor, dexamethasone, and activators of adenylate cyclase; Tischler AS et al.; Nerve growth factor, dexamethasone, and forskolin or cholera toxin (CT) act cooperatively to increase the content of neurotensin (NT) in PC12 pheochromocytoma cells . Relatively small increases in NT content occur in the presence of NGF alone or dexamethasone alone, but not of forskolin or CT alone . Increases of 10- to 100-fold occur in the presence of NGF plus dexamethasone or NGF plus forskolin, and up to 600-fold increases occur in the presence of all three agents . These increases are extremely stable and persist for at least 2 weeks after removal of dexamethasone or forskolin . The complex regulation of NT stores in PC12 cells might reflect mechanisms that regulate NT content of normal chromaffin cells and/or neurons during development or in adult life . A small amount of stored NT is released in response to stimulation with 52 mM K+ . This release is blocked in the presence of 2 mM Co2+, suggesting that it occurs via Ca2+-mediated exocytosis. J Allergy Clin Immunol, 1986 Jun, 77(6), 880 - 90 Variability of IgE protein measurement in cell-culture supernatants: results from a multicenter collaborative study; Helm RM et al.; The sensitivity, specificity, and precision of immunoassays for quantitation of IgE in cell-culture supernatants were tested in a multicenter trial involving 22 laboratories . Fourteen coded test samples included cell-culture medium alone, culture medium with varying concentrations of polyclonal or myeloma IgE, and medium from unstimulated or pokeweed mitogen (PWM)-stimulated peripheral blood mononuclear cell (PBMC)-culture supernatants . Two laboratories reported measurable IgE in non-IgE-containing control samples . Although the IgE content of a single 0.50 ng/ml polyclonal IgE sample should have been measured easily by the claims of all laboratories, only 13 laboratories measured IgE in this sample in 22 of the 48 assays . Most laboratories could measure both polyclonal and myeloma IgE at 5.0 ng/ml; however, the IgE determinations for the myeloma proteins were nearer the predicted value . Although 15 laboratories found measurable IgE in the PWM-stimulated PBMC-culture supernatants from a single nonatopic donor, the levels did not differ significantly from that measured in unstimulated PBMC-culture supernatants . Three laboratories reported considerably higher IgE levels in the PWM-stimulated PBMC-culture supernatants than in other PBMC-culture supernatants . Only 13 laboratories could quantitate IgE in each of coded duplicate samples containing 0.50 ng/ml polyclonal IgE . These findings indicate a wide variability in the sensitivity, specificity, and precision of assays used to quantitate low levels of IgE protein . Investigators should be encouraged to make greater use of existing national and international reference materials in the standardization and performance of their IgE immunoassays. Genitourin Med, 1986 Jun, 62(3), 175 - 6 Comparison of enzyme immunoassays and cell culture for detecting Chlamydia trachomatis; Mohanty KC et al.; Endourethral or endocervical swabs were taken from 403 patients to detect Chlamydia trachomatis by four different methods including standard tissue culture . Two immunoenzyme assays, Chlamydiazyme and IDEIA, were found to be satisfactory and could be valuable for large and busy sexually transmitted disease (STD) clinics. In Vitro Cell Dev Biol, 1986 Jun, 22(6), 320 - 4 Human chorion cells respond to growth factors but lose steroidogenic capacity in primary monolayer cell culture; Ferguson KA et al.; This study has defined a method for preparation and monolayer culture of cells from chorion laeve . Cell number and cell protein content are stable over 7 d in culture . The cells will divide in response to epidermal growth factor in the presence of a supplemented, enriched medium and a collagen matrix, but they lose steroidogenic activity over time in culture . This culture system can be used as the starting point for the development of a chemically defined hormone-supplemented, serum-free culture system for studies of chorion cell differentiation and fetal membrane cell interactions. J Pharmacol Exp Ther, 1986 Jun, 237(3), 1001 - 11 Sodium valproate, but not ethosuximide, produces use- and voltage-dependent limitation of high frequency repetitive firing of action potentials of mouse central neurons in cell culture; McLean MJ et al.; Effects of the anticonvulsant drugs sodium valproate (NaVP) and ethosuximide (ES) on mouse central (spinal cord and cortical) neurons in primary dissociated cell culture were studied using intracellular recording techniques . Drug effects on two properties of the neurons were assayed: the ability to sustain high frequency repetitive firing (SRF) of sodium-dependent action potentials, a voltage sensitive nonsynaptic membrane property; and the amplitude of responses to iontophoretically applied gamma-aminobutyric acid (GABA), a postsynaptic effect of this inhibitory amino acid neurotransmitter . At concentrations equivalent to the clinically useful therapeutic range in cerebrospinal fluid (6-200 microM), NaVP limited SRF to a few action potentials in both spinal cord and cortical neurons during long (450 msec) depolarizing current pulses . The limitation of SRF was paralleled by use-dependent reduction of maximal rate of rise (Vmax) of the action potentials and prolongation of recovery of Vmax from inactivation . This action was similar to limitation of SRF produced by phenytoin and carbamazepine . The 2-en-metabolite of NaVP, sodium 2-propyl, 2-pentenoate, did not limit SRF at 12 to 120 microM . However, the diphenyl analog of NaVP, sodium diphenylacetate, limited SRF at concentrations between 4.7 to 23.5 microM . ES did not affect SRF at concentrations up to 700 microM . At concentrations of 120 to 1000 microM, including the upper limit of therapeutic range, NaVP did not affect postsynaptic GABA responses in 80% of spinal cord neurons . In the remaining 20%, GABA responses were augmented less than 33% . ES reduced slightly (22%) GABA responses at a high concentration (700 microM) . These findings suggest that limitation of SRF may be an important cellular mechanism by which NaVP, but not ES, exerts anticonvulsant efficacy and that neither ES nor NaVP have anticonvulsant action by enhancing postsynaptic GABA action. J Neurosci, 1986 Jun, 6(6), 1796 - 802 Ascorbic acid increases the thyrotropin-releasing hormone content of hypothalamic cell cultures; Glombotski CC et al.; Thyrotropin-releasing hormone (TRH) is one of many COOH-terminal alpha-amidated neuropeptides . Recent work with the intermediate pituitary has indicated that ascorbate is a required cofactor for the COOH-terminal alpha-amidation of alpha-melanotropin . This is consistent with the ascorbate requirement of an enzyme found in pituitary and hypothalamus capable of converting peptides with a COOH-terminal glycine (-X-Gly) to alpha-amidated molecules (-H-NH2) . Thus, it has been proposed that COOH-terminal glycine-extended TRH (TRH-Gly) may be the direct precursor to TRH . In the present study, primary hypothalamic cultures supplemented with ascorbate for 7 d contained two- to threefold more TRH immunoactivity (amide-specific) than cultures maintained without ascorbate . A dose-response experiment indicated that 20 microM ascorbate was capable of producing 50% of the maximum observable increase in culture TRH immunoactivity; this concentration is similar to the Km value for ascorbate uptake obtained in adrenal chromaffin and pituitary cells . A stereoisomer of ascorbate, D-isoascorbate, was also capable of producing an increase in TRH immunoactivity, but oxidized ascorbate was not . Recent studies have shown that the amidation enzyme from pituitary is capable of utilizing both L-ascorbate and D-isoascorbate but is incapable of utilizing oxidized ascorbate . The culture extracts were analyzed further by reversed-phase high-performance liquid chromatography; the increased TRH immunoactivity observed in extracts of cultures maintained in ascorbate comigrated with standard synthetic TRH.(ABSTRACT TRUNCATED AT 250 WORDS) Mol Cell Endocrinol, 1986 Jun, 46(1), 29 - 36 The effects of inhibin purified from bovine follicular fluid in several in vitro pituitary cell culture systems; Robertson DM et al.; The effects of inhibin purified from bovine follicular fluid (bFF) and a charcoal-treated bFF preparation were investigated in several inhibin in vitro systems based on the use of pituitary cells in culture . FSH, LH, TSH and PRL were determined in the medium and cell extracts before and after a 4 h LHRH stimulation test . Both pure inhibin and bFF preparations markedly inhibited the basal release, cell content and LHRH-stimulated release of FSH in a parallel dose-dependent manner with minor or negligible effects on LH, TSH and PRL . Using parallel line bioassay statistics the inhibin activities of the purified inhibin preparations in the various in vitro systems were calculated with the charcoal-treated bFF as standard . Significantly higher inhibin values were obtained using the basal release procedure than with the cell content or LHRH-stimulated release procedures . This difference was influenced by the length of time the inhibin preparations were in culture . The highly purified preparations showed no signs of cytotoxicity in culture as assessed by a 51Cr release test . It is concluded that purified bFF inhibin is specific in suppressing pituitary FSH and not LH, TSH or PRL . The observation that purified bFF inhibin is more active in the basal release procedure is attributed to a loss of activity of the bFF standard in this system in contrast to that observed in either the cell content or LHRH-stimulated release procedure. Cancer Res, 1986 Jun, 46(6), 2697 - 702 Benzo(a)pyrene:DNA adduct formation in normal human mammary epithelial cell cultures and the human mammary carcinoma T47D cell line; Pruess-Schwartz D et al.; The benzo(a)pyrene (BaP):DNA adducts formed in normal human mammary epithelial cell cultures and the human mammary carcinoma T47D cell line were analyzed by chromatography and acid hydrolysis of the BaP:deoxyribonucleoside adducts to BaP:purine adducts and BaP:tetraols . Human mammary epithelial cell cultures and human mammary carcinoma T47D cells were exposed to {3H}BaP for 24 h, and the levels of binding were 81 and 182 pmol BaP/mg DNA in normal and T47D cultures, respectively . Analysis of BaP:deoxyribonucleoside adducts resolved by immobilized boronate chromatography and reversephase high-performance liquid chromatography demonstrated the presence of three BaP:deoxyribonucleoside adducts in both cells: M2, MS1, and MS2 in a ratio of 1.6:1:14 . Two adducts (MS1 and MS2) bound to the immobilized boronate column indicating the presence of cis-vicinal hydroxyl groups, a configuration which would result from reaction of 7 beta, 8 alpha-dihydroxy-9 alpha, 10 alpha-epoxy-7,8,9,10-tetrahydroBaP (anti-BaPDE) with DNA . MS2 was identified as (+)-anti-BaPDE:deoxyguanosine (dGuo) for it cochromatographed with a {14C}-(+)-anti-BaPDE:dGuo marker, the BaP:purine hydrolysis product of MS2 cochromatographed with {14C}-(+)-anti-BaPDE:guanine, and the tetraol hydrolysis products cochromatographed with (+/-)-anti-BaPDE:tetraols . MS1 was identified as (-)-anti-BaPDE:dGuo for MS1 eluted in the same relative position as a (-)-anti-BaPDE:dGuo marker, the BaP:purine hydrolysis product of MS1 cochromatographed with {14C}-(+)-anti-BaPDE:guanine, and the tetraol hydrolysis products cochromatographed with (+/-)-anti-BaPDE:tetraols . Thus, both adducts that bound to the immobilized boronate column were formed from (+/-)-anti-BaPDE . One major adduct that did not contain cis-vicinal hydroxy groups, M2, was detected in both cell types . M2 was formed from (+/-)-7 beta, 8 alpha-dihydroxy-9 beta, 10 beta-epoxy-7,8,9,10-tetrahydroBaP (syn-BaPDE) as M2 eluted in the same relative position as a syn-BaPDE:dGuo adduct marker and the tetraol hydrolysis products of M2 cochromatographed with tetraols formed from (+/-)-syn-BaPDE . The isolation of the individual BaP:DNA adducts followed by acid hydrolysis allowed the identification of the BaP:DNA adducts formed in human mammary cell cultures and demonstrated the presence of (-)-anti-BaPDE:dGuo . Thus, this work provides the first evidence, other than cochromatography, that (-)-anti-BaPDE is formed in cell systems and reacts with DNA in cells to form (-)-anti-BaPDE:dGuo.(ABSTRACT TRUNCATED AT 400 WORDS) J Cell Physiol, 1986 Jun, 127(3), 397 - 402 Collagen synthesis by murine bone marrow cell culture; Waterhouse EJ et al.; Collagen types synthesized by murine bone marrow cells were studied and the effect of lithium chloride on collagen biosynthesis in vitro was investigated . In the liquid culture system used, an adherent, mixed cell population supports hemopoiesis . Radioactive labeling of cell cultures and subsequent fractionation with ammonium sulfate, enzyme digestion, immune precipitation, and gel electrophoresis indicated that the bone marrow cells synthesized precursors to collagen types I, III, and IV, and fibronectin . A previously undescribed molecule or fragment with an apparent molecular weight of 17,000 daltons that was susceptible to bacterial collagenase and containing no interchain disulfide bonds was also identified in the culture media of both control and lithium-treated cells . Lithium treatment did not affect the types of collagen synthesized, although the relative proportions of collagen types may differ from controls . However, lithium does have an effect on the appearance of some, as yet unidentified, non-collagenous components in the cell culture media. Infect Immun, 1986 Jun, 52(3), 725 - 9 Effect of polymyxin B and colimycin on induction of plasminogen antiactivator by lipopolysaccharide in human endothelial cell culture; Dubor F et al.; The effect of lipopolysaccharide (LPS) on the production of fibrinolytic inhibitor by human endothelial cells was determined because results of previous experiments have shown us that it is possible to stimulate this synthesis with muramyl dipeptide . Treatment of these cells with LPS resulted in a marked enhancement of fibrinolytic inhibitor, as estimated in a urokinase-induced fibrinolysis assay . A dose-response curve was obtained for LPS concentrations ranging from 10 to 1,000 ng/ml, thus demonstrating the great sensitivity of these cells . This inhibitor did not reduce plasmin activity and formed complexes with high- and low-molecular-weight urokinase as visualized by fibrin enzymography on sodium dodecyl sulfate-polyacrylamide electrophoretic gels . The molecular weight of this inhibitor was estimated to be 54 to 58 kilodaltons . These findings led us to conclude that LPS stimulates formation of a plasminogen antiactivator . This LPS effect could be suppressed by polymyxin B and colimycin . The stimulatory effect of muramyl dipeptide required doses which were at least 1,000 times greater than those of LPS and was not decreased by polymyxin B . These results show the possibility of independent modulation of plasminogen antiactivator production at the endothelial level, which could be important in endotoxemia . Under these conditions colimycin might have an additional advantage for clinical use because of its ability to prevent fibrinolytic inhibition. J Virol, 1986 Jun, 58(3), 960 - 2 Susceptibility of fetal guinea pig brain cell cultures to replicating guinea pig cytomegalovirus infection is increased with increasing fetal age: infection of astrocytes; Kari B et al.; Guinea pig brain cell cultures were established from fetuses at 25, 31, and 37 days of gestation (DG) . After 7 days in vitro, the cultures were infected with guinea pig cytomegalovirus (GPCMV) . Based on cytopathic effect, immunofluorescence staining for GPCMV by using virus-specific antiserum, and the amount of virus recovered, cultures established from fetuses at 25 DG were least susceptible to replicating infection, and cultures established from fetuses at 37 DG were most susceptible . Using cell-type-specific markers, it was determined that the increase in susceptibility to replicating infection paralleled an increase in the number of differentiated cells . Astrocytes were the most abundant cell type identified and were susceptible to replicating GPCMV infection, whereas neurons were not. J Gen Virol, 1986 Jun, 67 ( Pt 6), 1211 - 4 Enhancement by carprofen or indomethacin of interferon induction by 10-carboxymethyl-9-acridanone in murine cell cultures; Storch E et al.; Non-steroidal anti-inflammatory drugs such as carprofen or indomethacin enhanced interferon (IFN) production induced by suboptimal concentrations of 10-carboxymethyl-9-acridanone (CMA) in murine cell cultures . This effect was observed in fibroblasts and in different populations of leukocytes as in peritoneal exudate and spleen cells, and was most pronounced in bone marrow-derived macrophages . Carprofen was the most effective compound causing an up to 500-fold increase of CMA-induced IFN production in pure bone marrow-derived macrophages . In these macrophage cultures the potentiating effect on CMA-induced IFN production by carprofen and indomethacin did not depend on inhibit