Culture medium

Anal Biochem, 1988 May 1, 170(2), 456 - 62
Heparin requirement for the quantitation of fibrinogen production by primary hepatocyte cultures; Dang CV et al.; We have reported a rapid method for the quantitation of proteins secreted in culture media (F.M . LaDuca, C.V . Dang, and W.R . Bell (1986) Anal . Biochem . 158, 262-267) . Using the same method, we observe that serum-free rat hepatocyte cultures exhibited a 100% increase in detectable secreted fibrinogen-antigen in the presence of 1 unit/ml heparin or greater at 24 h of culture . The amount of transferrin, haptoglobin, and albumin detected was unaltered by the presence of heparin . Since heparin is known to affect certain cellular functions, the fates of {35S}methonine-labeled fibrinogen in cell extracts and culture media were examined employing pulse-chase experiments . Labeled intracellular fibrinogen disappeared at similar rates and was initially released into the media in similar amounts in the presence or absence of heparin . At 8 h during the chase, there was a 40-50% reduction in fibrinogen-antigen in spent culture medium lacking heparin . The presence of heparin did not alter the proteolytic degradation of secreted fibrinogen as determined by immunoblotting of spent culture media proteins separated by polyacrylamide gel electrophoresis . In vitro experiments indicate that clotting of fibrinogen by thrombin reduces the amount of immunodetectable fibrinogen . The results indicate that heparin increases the amount of detectable fibrinogen secreted by cultured hepatocytes by preventing clotting and not by stimulating synthesis or secretion or by inhibiting degradation . Hence, it is critically important to include heparin when secreted fibrinogen is quantitated by the method that we have developed.

Acta Anaesthesiol Scand, 1988 May, 32(4), 308 - 9
The Venflon cannula as a sideport of infection; Cozanitis DA et al.; The possible role of infection through the valved sideport of Venflon cannulae was evaluated by one individual who followed an identical procedure of placing a syringe into the sideport and injecting saline solution . Basic aseptic techniques were used in a contaminated, busy environment . No bacterial growth was found in either anaerobic or aerobic culture media following a total of 1500 injections . The experiment shows that if the measures used could be applied, infection through the sideport would be minimal.

J Endocrinol, 1988 May, 117(2), R5 - 8
Development of a radioimmunoassay for ovine trophoblast protein-1, the antiluteolytic protein from the sheep conceptus; Vallet JL et al.; A radioimmunoassay has been developed for quantitation of ovine trophoblast protein-1 (oTP-1), a sheep conceptus secretory protein which allows for maintenance of the corpus luteum during early pregnancy . The assay was validated for dialysed and undialysed culture medium and pregnant uterine flushings ranging from no dilution (neat) to dilutions of 1:2500 for dialysed media, 1:100-1:1000 for undialysed media and 1:50-1:1000 for pregnant uterine flushings . The assay accurately measured oTP-1 added to undiluted and diluted dialysed and undialysed culture media and pregnant uterine flushings . No cross-reaction was detectable for bovine alpha or gamma interferon, bovine calmodulin, feline conceptus secretory proteins, equine conceptus secretory proteins, porcine conceptus secretory proteins, bovine conceptus secretory proteins and proteins in a uterine flushing collected from a non-pregnant ewe . Immunoreactivity in the assay matched that for oTP-1 throughout oTP-1 purification . This assay is the first validated assay which may be used to quantitate production of oTP-1 in culture or content of oTP-1 in uterine flushings.

In Vitro Cell Dev Biol, 1988 May, 24(5), 457 - 63
Characterization of extended primary and secondary cultures of hamster tracheal epithelial cells; Niles R et al.; Studies on the regulation of differentiation in airway epithelial cells have been hampered by the lack of cell culture systems that differentiate in vitro . One such system that does exhibit differentiation is hamster tracheal epithelial cells (HTE) . A major problem with this system, however, is that at the time cells differentiate, they lyze the collagen gel upon which they grow, resulting in termination of the culture . Here we report that by growing the HTE cells at 32 degrees instead of 37 degrees C we can totally prevent lysis of the collagen gel . Cells grown at this lower temperature maintain their differentiated phenotype as evidenced by abundant mucus granules and the secretion of authentic mucus glycoproteins into the culture media . We have also developed a method for subculturing the primary cells which allows growth and differentiation in secondary culture . The HTE cells were capable of being passaged at least three times and did not become transformed as judged by their inability to grow in soft agar and to produce tumors in syngeneic animals . This improved HTE cell culture system will allow detailed studies on the mechanisms which regulate growth, differentiation, and mucus secretion in surface airway epithelial cells.

Virology, 1988 May, 164(1), 91 - 8
Binding of cowpea chlorotic mottle virus to cowpea protoplasts and relation of binding to virus entry and infection; Roenhorst JW et al.; Cowpea chlorotic mottle virus (CCMV) and cowpea protoplasts were used to study initial interactions between virus and protoplast . Protoplasts and virus were incubated under varying conditions of temperature, pH, ionic strength, and the presence of added compounds . Both the amount of 35S-labeled virus bound to protoplasts and the percentage of infected cells were determined . At 0 and 25 degrees the amount of virus associated with protoplasts increased with the amount of virus added . With inoculum of 25 x 10(6) virus particles per protoplast, 4 x 10(3) and 14 x 10(3) particles per protoplast were bound at 0 and 25 degrees, respectively . In the presence of polyethylene glycol, 85 x 10(3) associated particles per protoplast were bound at both temperatures and ca . 50% of the protoplasts became infected . No infection occurred in the absence of PEG . Variation of pH or ionic strength in the absence of PEG caused little to no change in binding and no infection . In the presence of PEG, increase of pH resulted in lower binding, but infectivity was not affected . Increasing ionic strength, however, increased both binding and infectivity . The presence of unlabeled CCMV, tobacco mosaic virus coat protein, bovine serum albumin, and polycations during inoculation in the absence of PEG decreased the amount of bound CCMV . In contrast, CCMV coat protein, which has a positively charged N-terminal arm, increased binding . In the presence of PEG the effects were similar, although larger amounts of virus were bound . The percentage of infection was reduced by all additives to 5-25% . Addition of ammonium chloride, which inhibits endocytotic virus uptake in animal cells, during inoculation as well as in culture media, did not reduce infectivity . These data do not support a specific receptor-mediated endocytotic uptake of virus but favor a nonspecific mechanism of entry, possibly through membrane lesions . Observations in the electron microscope support the latter mechanism.

Am J Clin Pathol, 1988 May, 89(5), 671 - 4
Evaluation of broth media for routine culture of cerebrospinal and joint fluid specimens; Reinhold CE et al.; Broth cultures of cerebrospinal and joint fluids are important components in the culture detection of meningitis and septic arthritis . The authors examined 121 strains of bacteria isolated from clinical specimens representing 13 species or groups that cause meningitis and arthritis for growth in supplemented Thioglycolate broth (THIO), Supplemented Peptone Broth (SPB), and minced beef heart (MBH) media each alone or with added IsoVitaleX . Both SPB and MBH with IsoVitaleX performed better as broth culture media than the media without IsoVitaleX or THIO with or without IsoVitaleX.

J Vasc Surg, 1988 May, 7(5), 697 - 705
Polyglactin 910/polydioxanone bicomponent totally resorbable vascular prostheses; Greisler HP et al.; Previous studies from our laboratory have shown that bioresorbable vascular prostheses woven from lactide-glycolide copolymers and implanted into arteries of several animal models become replaced by cellular tissues; the rate of replacement parallels the kinetics of prosthetic resorption . This study evaluates the efficacy of bicomponent resorbable prostheses as a method of augmenting resistance to dilatation during the resorption period of the more rapidly resorbed component . Bicomponent prostheses (n = 37) were woven from compound yarns containing 74% polyglactin 910 (PG910) and 26% polydioxanone (PDS) and were interposed into adult white New Zealand rabbit infrarenal aortas . Resultant prosthesis-tissue complexes were harvested after 2 weeks to 12 months . Specimens were photographed and sectioned for light, scanning, and transmission electron microscopy . Randomly selected fresh explants at 1 and 3 months and control aortic segments from the same rabbits were simultaneously perfused with culture media (37 degrees C, 100/80 mm Hg, 60 ml/min) and perfusates assayed by means of tritiated radioimmunoassay techniques for the stable prostacyclin metabolite 6-keto-PGF1 alpha before and after the addition of sodium arachidonate (10 micrograms/ml) to the media . Results showed 100% patency, no aneurysms, and stenosis in 1 of 37 prostheses (3%) . PG910 was totally resorbed by 2 months and PDS by 6 months . By 1 month inner capsule thickness was 303 +/- 30 microns . In contrast to previous reports this was significantly thicker than that within 100% PDS (230 +/- 40 microns) and significantly less thick than in 100% PG910 (530 +/- 62 microns) . Inner capsules in all three groups stabilized at similar thicknesses (417 to 502 microns).(ABSTRACT TRUNCATED AT 250 WORDS)

Int J Food Microbiol, 1988 May, 6(3), 263 - 8
Inhibitory effects of selected Turkish spices and oregano components on some foodborne fungi; Akgul A et al.; The inhibitory effects of 10 selected Turkish spices, oregano essential oil, thymol and carvacrol towards growth of 9 foodborne fungi were investigated in culture media with pH 3.5 and 5.5 . The antifungal effects of sodium chloride, sorbic acid and sodium benzoate and the combined use of oregano with sodium chloride were also tested under the same conditions for comparison . Of the spices tested, only sodium chloride were also tested under the same conditions for comparison . Of the spices tested, only oregano at 1.0, 1.5, 2.0% (w/v) levels showed effect on all fungi . 8% (w/v) sodium chloride was less effective than oregano . Oregano essential oil, thymol or carvacrol at concentrations of 0.025% and 0.05% completely inhibited the growth of all fungi, showing greater inhibition than sorbic acid at the same concentrations . The combined use of oregano and sodium chloride exhibited a synergistic antifungal effect.

In Vitro Cell Dev Biol, 1988 May, 24(5), 401 - 12
A quantitative analysis of lectin binding to adult rat hepatocyte cell surfaces; Jauregui HO et al.; A quantitative evaluation of lectin binding to adult rat hepatocyte cell surfaces was done using cells isolated by two different collagenase perfusion methodologies and cultured as monolayers with two different tissue culture media formulations (protocol I vs . protocol II) . The presence of alpha-D-mannosyl and alpha-D-glucosyl groups was detected by the binding of Concanavalin A (Con A), Lens culinaris agglutinin (LCA), and Pisum sativum agglutinin (PSA) to freshly isolated cells . Furthermore, beta-D-galactose {Ricinus communis agglutinin (RCA)} and sialic acid residues {wheat germ (WGA)} were also found . Protocols I and II served as models for evaluation of: a) the stripping effect of collagenase separation procedures, b) the restoration in culture of collagenase-stripped sugar residues, c) the effect of the culture environment on cell viability {as measured by lactic acid dehydrogenase (LDH) leakage} and the protein content of hepatocytes, and d) the presence of cell surface sugar residues as a function of culture duration . The ultrastructural morphology of freshly isolated and cultured hepatocytes was also evaluated . These studies indicated that a decline in lectin binding invariably occurred earlier than a massive leakage of LDH and a decrease in the protein content of the cells in culture . Ultrastructurally, autophagocytosis was an early phenomenon in cells isolated and cultured by protocol I, which was also inferior to protocol II regarding the preservation of hepatocyte glycocalyces . Sugar residues lost due to the collagenase-stripping effect were restored, as shown by lectin binding, within the first 24 h of culture . This stripping effect was confirmed by quantitative evaluations of lectin binding to hepatocytes in culture after an incubation with collagenase . This study shows that the binding of peroxidase-labeled lectins is a useful tool for quantitative evaluation of the sugar composition of hepatocyte cultures.

J Biol Chem, 1988 Apr 25, 263(12), 5846 - 52
Expression and dexamethasone regulation of the human corticotropin-releasing hormone gene in a mouse anterior pituitary cell line; Adler GK et al.; The factors controlling the expression of corticotropin-releasing hormone (CRH), a hypothalamic neuropeptide involved in the regulation of ACTH secretion, are poorly understood partly because a suitable in vitro model is lacking . To study the regulation of CRH gene expression, an 8-kilobase (kb) DNA fragment containing the entire human CRH gene as well as approximately 6 kb of 5' sequence and 0.8 kb of 3' sequence was isolated from a lambda Charon 4A human genomic library and introduced into a mouse anterior pituitary cell line, AtT-20, by CaPO4 transfection with a neomycin-selectable marker . Approximately 10% of the neomycin-resistant lines stably expressed the CRH gene and secreted radioimmunoassay-detectable CRH into culture media at levels greater than 100 pg/ml . By Southern blot analysis the 8-kb DNA fragment containing the CRH gene had been incorporated intact into the AtT-20 genome . In each CRH-producing strain, but not in the parent AtT-20 cell line, we detected by Northern blot analysis an RNA species that hybridized to two radioactive cRNA probes specific for either the 5' or 3' portion of CRH mRNA, and that co-migrated with placental CRH mRNA . Dexamethasone treatment for 24-96 h caused a specific decrease in CRH mRNA and peptide levels of 40-50% in the five CRH-producing cell lines with half-maximal suppression at approximately 10(-9) M dexamethasone, indicating that CRH gene expression is negatively regulated by glucocorticoids . Thus, we have established an in vitro model suitable for studying in detail those cis- and trans-acting factors which regulate CRH gene expression.

Biochim Biophys Acta, 1988 Apr 15, 959(3), 220 - 8
Membrane-bound lipoprotein lipase on human monocyte-derived macrophages: localization by immunocolloidal gold technique; Goldberg IJ et al.; Macrophages from both rodent and human sources have been shown to produce lipoprotein lipase (LPL), the enzyme activity of which can be measured in culture media and in cellular homogenates . The studies reported here show the presence of LPL on the surface of human monocyte-derived macrophages . An inhibitory monoclonal antibody to human LPL was used for cellular and immunoelectron microscopy studies . This antibody is a competitive inhibitor of LPL hydrolysis of triacylglycerol but does not inhibit LPL hydrolysis of a water-soluble substrate, p-nitrophenyl acetate . Furthermore, when postheparin plasma was mixed with monoclonal antibody prior to gel filtration on 6% agarose, the LPL activity eluted with the lipoproteins and was not inhibited by the antibody . These studies suggest that the antibody recognized the lipid/lipoprotein binding site of the LPL molecule . Membrane-bound LPL was demonstrated on human monocyte-derived macrophages using colloidal gold-protein A to detect the monoclonal antibody to LPL . The surface colloidal gold was randomly distributed with a surface density of 56,700 gold particles per cell . Control cells cultured in heparin-containing media (10 units/ml) or cells reacted with anti-hepatic triacylglycerol lipase monoclonal IgG or nonimmune mouse IgG did not exhibit membrane binding of protein A-gold . Macrophages were incubated with control and monoclonal anti-LPL IgGs and 125I-labeled anti-mouse IgG F(ab')2 . Heparin-releasable membrane-bound anti-LPL antibody was demonstrated . These studies demonstrate the presence of LPL on the surface of human monocyte-derived macrophages, such that the LPL is oriented with its lipid-binding portion (recognized by this antibody) exposed . Membrane-associated LPL may be important in the interaction and subsequent uptake of lipid and lipoproteins by macrophages and in the generation of atherosclerotic foam cells.

J In Vitro Fert Embryo Transf, 1988 Apr, 5(2), 76 - 80
Deterioration of stored culture media as monitored by a sperm motility bioassay; Stewart-Savage J et al.; A hamster sperm motility bioassay was used to monitor medium quality during storage . The media studied were (1) TL-PVA, a modified Tyrode's solution; (2) TLP-PVA, a defined medium that supports hamster sperm motility but does not support capacitation; and (3) TALP-PVA (TLP-PVA + serum albumin), which supports hamster sperm survival and capacitation . Each medium was stored at 5 and -20 degrees C and tested every two weeks . All of the media deteriorated with increased storage time, but at different rates (TLP-PVA greater than TL-PVA greater than TALP-PVA) . The deterioration of the media correlated with an increase in pH during storage, probably due to a loss of CO2 and bicarbonate . Albumin, added after storage, was able to rescue frozen TL-PVA.

J In Vitro Fert Embryo Transf, 1988 Apr, 5(2), 67 - 75
A rapid sperm motility bioassay procedure for quality-control testing of water and culture media; Bavister BD et al.; A rapid bioassay procedure is described for quality-control testing water and apparatus used in the preparation of media for gamete and embryo culture . This bioassay is based on the sensitivity of hamster epididymal spermatozoa to contaminants present in water and/or in the culture apparatus . The bioassay is usually performed using a modified Tyrode's solution as the sperm culture medium, although complex media can be used . The sensitivity of this test is greatly enhanced by the absence of protein in the medium . The bioassay has been used to detect impurities in water prepared by a standard cartridge filtration system and to verify that reverse-osmosis pretreatment of water could eliminate the problem . It has also detected toxic contaminants leached from syringe filters during medium sterilization . The bioassay is simple to perform and can be completed in 1 working day . It may be a useful alternative to the conventional mouse embryo tests that are in widespread use in human in vitro fertilization (IVF) laboratories.

J In Vitro Fert Embryo Transf, 1988 Apr, 5(2), 61 - 6
Influences of prolactin upon spermatogenesis and spermatozoa during in vitro fertilization in mice; Mori C et al.; Hyperprolactinemia, induced by pituitary isografts for 20 weeks in male mice and confirmed by radioimmunoassay using anti-mouse prolactin serum, did not impair spermatogenesis in the testis and maturing processes of spermatozoa in the epididymis . Incubation of freshly obtained epididymal spermatozoa for 90 min in culture media containing various levels of mouse prolactin did not yield any adverse effects on percentage motility rates of epididymal spermatozoa . When the level of mouse prolactin in the preincubation medium for epididymal spermatozoa was 100 ng/ml, the rate of fertilization by these preincubated spermatozoa in the subsequent in vitro fertilization experiment was significantly lowered compared with that observed in controls . However, when the level of prolactin in preincubation media was 10 ng/ml, no significant reduction in the rate of fertilization occurred . The present experiments seem to indicate the existence of some differences in the effects of prolactin on male germ cells until they reach the tail of the epididymis and on the processes of capacitation and/or fertilization by epididymal spermatozoa after they leave the epididymis.

Mol Cell Biol, 1988 Apr, 8(4), 1845 - 8
Retinoic acid increases the sensitivity of the rat embryo fibroblast transformation assay; Halazonetis TD et al.; The rat embryo fibroblast focus assay is used to evaluate the transforming potential of several oncogenes . The sensitivity of this assay increased fivefold when retinoic acid was added to tissue culture media . Retinoic acid probably acts by selectively inhibiting the proliferation of nontransformed cells.

Hum Reprod, 1988 Apr, 3(3), 367 - 76
Assessment of heterospecific zona-free ovum penetration under fully defined conditions; Tomkins PT et al.; The prognostic value of the sperm penetration assay (SPA) using zona-free hamster ova has not been universally accepted . This is partly due to technical problems with the assay, resulting in 5-17% false negative results . The advantage of replacing albumin with defined synthetic polymers in the SPA culture media, increasing the calcium gradient and incorporating potential active tract agents and antioxidants, has been investigated . These studies have resulted in the development of a procedure that has produced no negative results among known fertile donors (n = 55) . Application of this technique to a non-specific group of patients with suspected sub-fertility produced a different pattern of sperm penetration compared with the 'standard' SPA in 27% of cases . Improved sperm velocity, motile survival, enhanced hyaluronidase release and rate of acrosome induction were noted using the synthetic media.

J Biomed Mater Res, 1988 Apr, 22(4), 271 - 85
Endothelialization of polymer surfaces; Absolom DR et al.; The role that substrate surface properties play in influencing the extent of endothelialization of polymer surfaces has been investigated . For a wide range of polymer surfaces, the degree of endothelialization for both porcine and bovine endothelial cells is directly related to polymer surface tension: increased endothelialization occurring with increasing substrate surface tension . As a result of adsorption of the proteins in the culture media, the surface properties of the polymers are altered considerably . The protein-coated polymers were characterized by means of liquid-liquid contact angle measurements under non-denaturing conditions . A striking correlation is observed between the degree of endothelialization and the measured dextran contact angle . The degree of endothelial cell spreading is not related to polymer surface tension . Cell morphology and extracellular matrix production, however, are influenced by substrate surface properties.

Fertil Steril, 1988 Apr, 49(4), 676 - 9
High potassium concentration improves the rate of acrosome reaction in human spermatozoa; Roblero L et al.; Progressively motile spermatozoa recovered by swim-up method from semen of two fertile men were incubated for 24 hours in culture media containing either 4.7, 15, or 25 mM of potassium (K) . Aliquots of each culture condition were obtained at 0, 1, 5, 10, and 24 hours of incubation for the assessment of progressive motility, percentage of dead spermatozoa, and percentage of acrosome reaction (AR), as measured by triple-stain technique . A total of ten experiments including each K concentration were analyzed . The results of this study showed no effect of K concentration on the percentage of progressively motile spermatozoa, irrespective of the time of incubation . The percentage of live spermatozoa was significantly greater in culture medium containing 25 mM K (P less than 0.05) . There was a greater percentage of reacted spermatozoa with 25 mM K, as compared with 4.7 and 15 mM (P less than 0.05) . Furthermore, the time taken to achieve 20% of AR was 2 hours at 25 mM K compared with 10.9 hours at 4.7 mM K.

Cell Biochem Funct, 1988 Apr, 6(2), 101 - 5
Effects of insulin and dexamethasone on adenine nucleotide levels in cultured hepatocytes from adult rat; Gallo G et al.; Insulin and dexamethasone, usually added to culture media, play an important role in maintaining the survival of functional hepatocytes . Adenine nucleotide concentrations and energy charge values of cultured hepatocytes were determined to investigate the relationship between the beneficial effects of these hormones and the energy status of the cells . The results indicate that insulin and dexamethasone are essential in maintaining the metabolic competence of cultured hepatocytes and that this correlates with the absolute concentration of ATP rather than with the energy charge.

Mech Ageing Dev, 1988 Apr, 43(1), 25 - 44
The aging of the endocrine pancreas of the rat . II . Cytoplasmic parameters of the B-cell, including insulin synthesis and secretion; de Clercq L et al.; Comparative ultrastructural stereology of 6 and 24-month-old rat B-cell cytoplasm revealed an increase with age in secondary lysosomes and a decrease in the volume density of RER and Golgi apparatus . The reduction of RER observed in freshly isolated islets could affect (pro)insulin biosynthesis in vitro: if the initial mobilization of precursor molecules for protein synthesis was the same, a delay was noted in their transit to the Golgi apparatus in B-cells of old islets . No further differences were seen in the autoradiographic distribution of radioactive amino-acids . More, the stock of insulin granules was similar in all age groups in both in vivo and in vitro conditions . Neither were any differences observed in the insulin secretion into culture media as well as during a subsequent incubation in supraphysiological glucose concentrations.

In Vitro Cell Dev Biol, 1988 Apr, 24(4), 299 - 303
Growth of H-35 rat hepatoma cells in unsupplemented serum-free media: effect of transferrin, insulin and cell density; Shapiro LE et al.; Serum-free tissue culture medium consisting of a 1:1 mixture of Dulbecco's modified Eagle's medium (DMEM) and Ham's F12 medium is herein shown to support growth of Reuber H-35 cells over several days in culture . Cells were initially plated in serum containing DMEM medium for 3 h . After cell attachment, serum is removed and replaced with a serum-free 1:1 mixture of these two commercially available tissue culture media . The doubling time of cell growth in this unsupplemented serum-free medium was 46 h in lightly plated cultures over the first 5 d . The presence of transferrin (5 micrograms/ml) and insulin (3.3 nM) results in a cell doubling time of 17 h, which equaled the growth rate in medium containing 10% fetal bovine serum . In the absence of transferrin, growth rates in serum-free medium were correlated with the cell density of cultures . Conditioned medium from dense, serum-free cultures has growth-stimulating activity in recipient lightly plated cultures . This simple, serum-free culture medium will facilitate studies on the growth regulation of H-35 rat hepatoma cells.

Endocrinology, 1988 Apr, 122(4), 1639 - 45
The in vitro synthesis and release of proteins by the human oviduct; Verhage HG et al.; The purpose of this study was to identify major proteins synthesized by the human oviduct and to determine if the ability of the human oviduct to synthesize these proteins was correlated with a specific stage(s) of the menstrual cycle . Oviducts, obtained from normal menstrual and immediately postpartum women, were minced and cultured in the presence of labeled precursors . The culture medium was analyzed for newly synthesized proteins by electrophoresis, followed by fluorography . One-dimensional gel electrophoretic analysis of culture medium of oviducts obtained at midcycle revealed a major diffuse band in the 120,000-130,000 mol wt (Mr) region which was greatly diminished in intensity or nondetectable in the media of oviducts obtained at all other stages of the cycle . Two-dimensional gel electrophoresis resolved the 120,000-130,000 Mr region into two major proteins, one basic and one acidic . Both proteins were intensely labeled with glucosamine, and the basic protein also incorporated leucine and methionine . Western blots of culture media incubated with antibodies against human placental proteins (PP) demonstrated that PP4 and PP7 were released throughout the cycle, while PP14 was present only at the late luteal stage of the cycle . This study demonstrates that human oviducts continue to synthesize and release macromolecules during organ culture . Additionally, the synthesis of some of these proteins appeared to be stage specific . The Mr 120,000-130,000 glycoproteins are of particular interest since they were observed in medium from midcycle oviducts, a period when the oviduct participates in gamete transport and embryo development.

Endocrinology, 1988 Apr, 122(4), 1603 - 12
Somatomedin-C/insulin-like growth factor I as an enhancer of androgen biosynthesis by cultured rat ovarian cells; Hernandez ER et al.; The ovarian granulosa cell has recently been shown to be a site of somatomedin-C/insulin-like growth factor I (Sm-C/IGF-I) production, reception, and action . These observations have generally been interpreted to suggest the existence of an autocrine loop concerned with granulosa cell physiology . It is the objective of the in vitro studies reported herein to extend these observations by evaluating the interaction of Sm-C/IGF-I with the adjacent thecal-interstitial cell . Treatment of collagenase-processed whole ovarian dispersates or highly enriched (greater than 90%) thecal-interstitial cells from immature rats with Sm-C/IGF-I (50 ng/ml) or hCG (1 ng/ml), resulted in 2.1- and 4.0-fold increments in the accumulation of androsterone (3 alpha-hydroxy-5 alpha-androstane-17-one), the main androgenic steroid identified in culture media . However, combined treatment with both agents unmasked a synergistic interaction producing a 3.3-fold increase in the hCG-stimulated accumulation of androsterone, an effect consequent to enhanced androgen biosynthesis rather than diminished degradation . Unaccounted for by an increase in viable ovarian cell numbers and independent of the hCG dose (0.1-10 ng/ml) used, the Sm-C/IGF-I effect proved time and dose dependent, with a projected minimal effective dose of 3 ng/ml and a minimal time requirement of 72 h . {125I}Iodo-Sm-C/IGF-I binding to untreated highly enriched thecal-interstitial cells proved saturable, with a single class (Hill coefficient = 0.98 +/- 0.01) of high affinity (Kd = 3.0 nM), low capacity (maximum binding = 10,840 +/- 2,108 sites/cell) binding sites . Limited specificity studies using related peptides produced a rank order of competitive potency of: Sm-C/IGF-I greater than multiplication stimulating activity greater than insulin, a pattern compatible with the presence of type I IGF receptors . Other related peptides, such as porcine proinsulin and porcine desoctapeptide insulin, proved weakly effective in inhibiting Sm-C/IGF-I binding to its receptor; unrelated peptides such as porcine relaxin and erythropoietin were without effect . Taken together, these findings suggest that 1) the thecal-interstitial cell, like the granulosa cell, may be a site of Sm-C/IGF-I reception and action, and 2) the ability of high dose insulin to stimulate ovarian androgen biosynthesis may be due to its capacity to act as a Sm-C/IGF-I surrogate, its high dose requirements reflecting cross-interaction with the type I receptor.(ABSTRACT TRUNCATED AT 400 WORDS)

J Immunol, 1988 Apr 1, 140(7), 2274 - 8
The expression and modulation of IL-1 alpha in murine keratinocytes; Ansel JC et al.; Murine and human keratinocytes produce an IL-1-like factor that appears to be similar if not identical to monocyte-derived IL-1 . IL-1 may be an important mediator in cutaneous inflammatory responses, however, little is currently known concerning factors that may modulate IL-1 expression in keratinocytes . To address this issue we examined the effect of LPS, UV, and the cell differentiation state on murine keratinocyte IL-1 mRNA expression . Our results indicated that as with the murine P388D1 monocyte cell line, PAM 212 keratinocytes constitutively express abundant amounts of IL-1 alpha mRNA . On exposure to LPS (100 micrograms/ml) for 8 h there was more than 10 times the increase in PAM 212 IL-1 alpha mRNA which was accompanied by a sixfold increase in supernatant IL-1 activity . Similarly UV irradiation had a significant effect on keratinocyte IL-1 alpha expression . High dose UV (300 mJ/cm2) inhibited PAM 212 IL-1 alpha expression at 4, 8, 24, 48 h post-UV whereas a lower dose of UV (100 mJ/cm2) inhibited UV at 4 and 8 h post-UV, but induced IL-1 expression at 24 and 48 h post-UV . The expression of IL-1 alpha varied with the differentiation state of the keratinocytes . Freshly removed newborn murine keratinocytes were found to constitutively express IL-1 alpha mRNA . Keratinocytes grown in low {Ca2+} tissue culture media (0.05 mM) for 6 days, functionally and phenotypically become undifferentiated and express increased quantities of IL-1 alpha mRNA, whereas cells grown in high {Ca2+} media (1.2 mM) for 6 days become terminally differentiated and IL-1 expression ceased . Keratinocytes cultured for 3 days in low {Ca2+} conditions expressed an intermediate level of IL-1 alpha . In contrast, little or no IL-1 beta mRNA was detected in either the PAM 212 cells or newborn murine keratinocytes . Thus LPS, UV, and cell differentiation state have a significant effect on expression of IL-1 alpha in murine keratinocytes.

Prostaglandins, 1988 Apr, 35(4), 535 - 48
Role of phospholipase C in mediating oxytocin-induced release of prostaglandin F2 alpha from ovine endometrial tissue; Silvia WJ et al.; Two experiments were conducted to determine if the ability of oxytocin to stimulate release of prostaglandin (PG)F2 alpha from ovine uterine tissue involved activation of phospholipase C (PLC) . In the first experiment, 9 ewes were injected with progesterone for 11 d (12 mg/d, im) . On days 11 and 12, ewes received an injection of estradiol (100 micrograms, im) . Caruncular endometrial tissue was collected on d 13 and incubated in the presence or absence of oxytocin (10(-6) M) . Concentrations of PGF2 alpha and its metabolite, 13,14-dihydro-15-keto-PGF2 alpha (PGFM), in culture media were determined by radioimmunoassay . PLC activity was determined by measuring the intracellular accumulation of 3H-inositol phosphates after preincubation with 3H-inositol . Concentrations of PGF2 alpha and total PGF (PGF2 alpha + PGFM) in culture media were greater for explants treated with oxytocin than for controls (p . less than .02, p less than .06, respectively) . A similar effect of oxytocin on intracellular concentrations of 3H-inositol phosphates was observed (p less than .01) . A second experiment was conducted to determine if agonists of second messengers, produced by activation of PLC, could stimulate release of PGF2 alpha from ovine endometrial tissue . Seven ewes were treated with progesterone and estradiol as in experiment 1 . Explants of caruncular tissue from each ewe were incubated with 1) control medium, 2) A23187 (10(-5) M), 3) oxytocin (10(-6) M), 4) phorbol 12-myristate 13-acetate (PMA, 10(-7) M), 5) PMA + A23187 and 6) PMA + oxytocin . Significant stimulatory effects of oxytocin, PMA and A23187 on concentrations of PGF2 alpha and total PGF in culture media were observed (p . less than .05, p less than .1, p less than .1, respectively) . In conclusion, oxytocin stimulated release of PGF2 alpha and activity of PLC in explants of ovine endometrial tissue in vitro . Second messengers associated with activation of PLC enhanced release of PGF2 alpha from ovine endometrial tissue.

Blood, 1988 Apr, 71(4), 899 - 906
Enhanced expression of transforming growth factor beta during megakaryoblastic differentiation of K562 leukemia cells; Alitalo R et al.; Platelet alpha granules contain several growth factors such as the transforming growth factor beta (TGF-beta) that are released during blood clotting and are thought to participate in the repair of tissue injury; however, the site of synthesis of platelet TGF-beta has not been demonstrated . We studied TGF-beta expression during megakaryoblastic differentiation of the chronic myeloid leukemia cell line K562 in vitro . These cells have mainly erythroid characteristics but acquire several megakaryoblastic properties when treated with the phorbol diester 12-0-tetradecanoyl-13-phorbolacetate (TPA) . During four subsequent days of megakaryoblastic differentiation the amount of the 2.5-kilobase (kb) TGF-beta mRNA increased about eightfold, and a novel 2.3-kb mRNA species was induced in the K562 cells . This occurred concomitantly with distinct induction patterns of platelet-derived growth factor A (PDGF-A) and c-sis (PDGF-B chain) RNAs and several platelet antigens . The expression of erythroid markers such as glycophorin A decreased . Culture media of TPA-differentiated K562 cells also contained TGF-beta polypeptides as shown by a sensitive radioreceptor assay and by immunoprecipitation after metabolic labeling of the cells . These polypeptides were not seen in culture media from dimethyl sulfoxide- or sodium butyrate-treated cells . Unlike in several other cells, exogenously added TGF-beta 1 or 2 affected neither TGF-beta nor PDGF RNA expression in K562 cells.

Eur J Pediatr, 1988 Apr, 147(3), 321 - 7
A variant of mucolipidosis . II . Clinical, biochemical and pathological investigations; Poenaru L et al.; We present in this paper a patient with a clinically intermediate form of mucolipidosis (ML) . Lysosomal hydrolase activity in fibroblasts was normal and levels of these enzymes in culture media were not elevated . There was a striking elevation of several hydrolases in serum and a deficiency (15% of normal) of N-acetyl-glucosamine phosphotransferase in fibroblasts . Atypical electron microscopic findings were also observed . There was no evidence of increased synthesis, slower turnover, unbalanced distribution or further changes in lysosomal enzymes . Phosphotransferase deficiency against endogenous beta-glucosaminidase and the fact that the electrophoretic mobility of lysosomal enzymes was identical to that of MLII suggest that these enzymes are not phosphorylated . Hypotheses that could explain this atypical pathology are discussed.

J Biol Chem, 1988 Mar 25, 263(9), 4259 - 62
Affinity-based chromatography utilizing genetically engineered proteins . Interaction of Bordetella pertussis adenylate cyclase with calmodulin; Haiech J et al.; An engineered calmodulin differs from vertebrate calmodulin in its ability to activate Bordetella pertussis adenylate cyclase, and this difference has been utilized as the basis for a new purification protocol for the adenylate cyclase . VU-8 calmodulin, in which 3 glutamic acid residues (residues 82-84) have been substituted with 3 lysine residues, has a 1000-fold lower apparent affinity for the adenylate cyclase, compared to vertebrate calmodulin, and decreased maximal activity . Because of the relatively calcium-independent nature of the interaction between calmodulin and the cyclase, the use of calmodulin-Sepharose conjugates in the purification of the cyclase requires the use of chaotropic agents for elution . However, when immobilized VU-8 calmodulin was tested as a calcium-dependent, affinity-based, adsorption chromatography step in the purification of the cyclase from culture media or bacterial extracts, the enzyme bound to the column in a calcium-dependent manner, and a nearly homogeneous enzyme was obtained in high yield . These results demonstrate the feasibility of using engineered calmodulins that have selective differences in activity for the rational design of rapid purification protocols for calmodulin-binding proteins as well as indicate the importance of the conserved negative charge cluster at residues 82-84 of calmodulin for activation of this cyclase.

J Immunol Methods, 1988 Mar 16, 107(2), 157 - 63
Modification and application of a simple, surface hydrophobicity detection method to immune cells; Hazen BW et al.; A simple method to assess fungal cell surface hydrophobicity involves enumeration of cell-attached, polystyrene latex microspheres . Modifications and conditions of this method for application to immune cell populations were investigated . The media used for suspending cells and microspheres appeared to influence microsphere attachment . Several tissue culture media supported high levels of microsphere attachment, but serum inhibited attachment . The concentration of microspheres also influenced the apparent level of detectable cell surface hydrophobicity . Under conditions which allow different levels of apparent cell surface hydrophobicity to be discriminated, the assay revealed that surface hydrophobicity of YAC-1 cells, which are used as standard targets in murine natural killer cell assays, varied depending on the time of harvest during growth and that phagocytic populations differed in cell surface hydrophobicity . Trypsinization experiments indicated that one hydrophobic constituent of the cell surface includes protein . These results indicate that the microsphere assay is a useful method for assessing cell surface hydrophobicity . The possibility that the assay could be used to determine quantitatively the surface free energies of immune cells and cell targets is discussed.

Cancer Res, 1988 Mar 15, 48(6), 1505 - 11
Purification and composition of a novel gastrointestinal tumor-associated glycoprotein expressing sialylated lacto-N-fucopentaose II (CA 19-9); Klug TL et al.; Monoclonal antibody 1116NS 19-9 (Mab 19-9) exhibits selective reactivity with human gastrointestinal carcinomas and recognizes a carbohydrate determinant (CA 19-9) defined as a sialylated lacto-N-fucopentaose II . A scheme was devised for the purification of a human gastrointestinal tumor-associated glycoprotein antigen expressing CA 19-9 from colorectal carcinoma cell line SW1116 culture media in high yield . The key steps in the purification were immunoaffinity column chromatography with Mab 19-9 followed by reduction and alkylation of the specifically bound proteins in the presence of 6 M guanidine hydrochloride and a second Mab 19-9 immunoaffinity fractionation . The purified CA 19-9 containing glycoprotein ran as a single band on sodium dodecyl sulfate-polyacrylamide gradient gels with an apparent molecular mass of 210 kilodaltons . In the absence of detergents, this purified glycoprotein apparently reassociated to form aggregates of 600-2000 kilodaltons molecular mass as determined by size-exclusion chromatography . Amino acid analysis of CA 19-9 containing glycoprotein revealed that serine, threonine, and proline together accounted for greater than 35% of the amino acid residues, consistent with a mucin-like structure for the protein . Carbohydrate compositional analysis, however, was in contrast to a typical mucin with a fucose:mannose:galactose:N-acetylgalactosamine: N-acetylglucosamine:N-acetylneuraminic acid molar ratio of 4:1:12:2.5:5:5 . The presence of both N-acetylgalactosamine and mannose suggested that both O- and N-linked oligosaccharides may exist on CA 19-9 containing glycoprotein . Protein and carbohydrate analyses indicated that this novel tumor-associated glycoprotein was 85% carbohydrate by weight . This purification procedure may be applicable to the isolation of other epithelial tumor-associated antigens.

Andrologia, 1988 Mar-Apr, 20(2), 169 - 72
ATP-content and kinetics of acrosome reaction in human spermatozoa: influence of various culture media and incubation time; Dolci S et al.; The ATP content and the kinetics of acrosome reaction of isolated human spermatozoa were investigated during an in vitro culture period of 3 hours in 3 serum-enriched media commonly adopted in IVF and GIFT procedures (Earle solution, Ham F10 and Menezo B2) . The ATP concentration in spermatozoa did not change between 1 and 3 hr of incubation and no differences were seen using the three media under investigation . The percentage of acrosome reactions significantly increased during culture, to a similar extent in the three media . The results suggest that each of the three media is equally well suitable for human sperm culture in preparation of IVF and GIFT procedures.

Immunol Lett, 1988 Mar, 17(3), 217 - 22
Sulfhydryl groups generated by macrophages into the culture medium; Jokay I et al.; Previous data showing that thiols can functionally replace macrophages in certain in vitro lymphocyte reactions have raised the possibility that macrophages generate SH groups in the medium . The SH activity of cell-free medium decreases considerably due to auto-oxidation . The presence of macrophages inhibited the spontaneous decrease of SH activity and even increased the number of free SH groups in the culture media . This was designated as SH generation . Resident as well as in vivo stimulated (NaIO4, thioglycollate medium, paraffin oil or BCG) macrophages have nearly the same capacity to generate SH groups when cultured in vitro . The SH-generating ability of macrophages depends on the viability and density of cells, and is greatly influenced by the serum concentration of the media . The majority of SH groups produced by macrophages could be demonstrated as albumin SH groups and proved to be more resistant to autooxidation than non-protein thiols . Moreover albumin could replace whole serum in respect of SH generation . It is suggested that macrophages by generating SH activity, may exert a non-specific helper function to lymphocytes or other cells in their environment.

Mech Ageing Dev, 1988 Mar, 42(3), 215 - 27
Culture media variation as related to in vitro aging of human fibroblasts: II . Effects on nucleolar number/cell, volume/nucleolus and total nucleolar volume/cell; Weinstein ME et al.; The relative effect of five commonly used culture media (MEM, BME, McCoy's 5A, M199 and HMEM) on the average nucleolar number/cell, the average volume/nucleolus and the total nucleolar volume/cell was examined during in vitro senescence of WI-38 human fetal fibroblasts . Statistical analyses show that cells aging in MEM show a higher number of nucleoli/cell than that of cells aging in any other medium . For cells aging in the other four media, there are no significant differences in the average number of nucleoli/cell . Linear regression analysis shows that in all cases there is a linear decrease in the average number of nucleoli/cell as a function of PDL . Statistical analyses show that the average volume/nucleolus is significantly greater for cells aging in M199 than in any other medium . Cells aging in HMEM show smaller average nucleolar volume than cells aging in M199, but display larger volumes than that of cells aged in BME, McCoy's 5A, or MEM . Cells aging in BME and McCoy's 5A media show no significant difference among each other in terms of average nucleolar volume, but a difference in this parameter is noted in cells aging in BME and MEM . A linear regression analysis shows that the average volume/nucleolus increases linearly as a function of age for cells grown in all five media . Analysis of the total nucleolar volume/cell in the five media shows that cells aging in M199 and HMEM are not significantly different from each other in terms of this variable, but show significantly larger volumes than those of cells aging in BME, McCoy's 5A and MEM . Cells aging in BME, McCoy's 5A and MEM display no significant difference with regard to this parameter . Linear regression analysis shows a positive linear relationship between the PDL and the total nucleolar volume/cell . The relative effects of all five media are not the same on the three cellular variables studied during in vitro aging of WI-38 cells . We, therefore, suggest that one should note this medium differential in order to allow meaningful comparison of results on possible changes in various morphological parameters during in vitro senescence of diploid human fibroblasts.

J Cell Biol, 1988 Mar, 106(3), 883 - 91
Mitogen activation induces the enhanced synthesis of two heat-shock proteins in human lymphocytes; Haire RN et al.; We have used mitogenic lectin (PHA) and a monoclonal antibody (OKT3) to stimulate human peripheral blood (G0) lymphocytes, in the presence of monocytes, and have found two major preferentially synthesized proteins, 73 and 95 kD, which are induced by the mitogens . The elevated synthesis of both proteins begins approximately 4-6 h after mitogen addition (early to mid G0/G1) before entry into first S phase . Maximum synthesis of both proteins is reached by 12 h after mitogen addition when P95 synthesis represents approximately 4%, and P73 approximately 2%, of the total protein synthesis, compared with less than 0.5% for each protein in cells cultured without mitogen . Thus, the proteins appear to be major components of activated cells . We find that both P73 and P95 are induced by heat stress as well as mitogenic stimulation . The induction of the proteins is not affected by either deleting glucose from the culture media or, alternatively, by supplementing it . Using polyclonal antibodies prepared to each of the proteins isolated from mitogen activated cells and monoclonal antibodies that were raised to heat shock proteins, we are able to show that P95 is electrophoretically and immunologically identical to the HSP 90 induced by heat stress . P73 is one of the 70 kD HSPs, (termed HSC 70; Pelham, H . R . B . 1986 . Cell . 46: 959-961), but is different from the most strongly heat inducible form of HSP 70 (72 kD) . The distribution of both proteins in subcellular fractions of mitogen activated lymphocytes is similar to the reported localization of the respective HSP's in other cell types . The results suggest that HSP 90 and HSC 70 may have functional roles in stress response and growth processes of human lymphocytes.

Andrologia, 1988 Mar-Apr, 20(2), 173 - 81
Paracrine factors in adult rat testis gonadotrophin control of opioids and LHRH like peptide; Saint Pol P et al.; A paracrine regulation involves agents which are produced by one cell type and act on an other one within an organ . In rodent testis, local control mechanisms modulate the actions of the gonadotrophins according to local requirements . Two groups of peptides-opioids and testicular LHRH are defined as paracrine factors and in vivo they are both modified by HCG . In vitro, after HCG exposure, we first localized an opioid like material in Sertoli cells cytoplasma by immunohistochemistry . This material is detected in freeze dried homologous culture media using a dot immunobinding technique . With a longer HCG exposure, an LHRH like material is then visualized in the basal compartment of the Sertoli cells and it is detected in freeze dried homologous culture media by the same technical procedure than for opioid material . By adding synthetic enkephalins to culture medium, we obtain the same results as with the endogenous opioid material, excreted after HCG addition . If naloxone a potent opiate antagonist, is added to the culture medium previously to HCG or enkephalins, the Sertoli cells cytoplasma are no more immunoreactives with the anti-enkephalin serum and no LHRH material is neither visualized by immunohistochemical technique neither detected in culture media . We conclude that testicular opioids, synthetized by the Leydig cells and which have specific Sertoli cells receptors are one Leydig-Sertoli paracrine communication factor . One way of response to their receptor fixation is the synthesis and excretion by Sertoli cells of testicular LHRH . This one is known to act on Leydig cells via specific receptors and it is one Sertoli-Leydig cells paracrine communication factor.(ABSTRACT TRUNCATED AT 250 WORDS)

Mol Cell Endocrinol, 1988 Mar, 56(1-2), 35 - 40
Selective control of rat granulosa cell inhibin production by FSH and LH in vitro; Zhang Z et al.; We studied the role of luteinising hormone (LH) and human chorionic gonadotropin (hCG) in regulation of rat granulosa cell inhibin production . Whereas pregnant mare serum gonadotropin (PMSG) or purified rat follicle-stimulating hormone (FSH) stimulated inhibin accumulation in culture media up to 6-fold in a dose-dependent manner, hCG or LH alone were without significant effect . Concomitant addition of hCG or LH to cultures containing half maximal or maximal stimulating concentrations of PMSG did, however, result in a dose-dependent inhibition of PMSG/FSH-induced inhibin production . However, in the 2-step culture system a biphasic effect of hCG treatment on FSH-primed granulosa cell inhibin and progesterone production was evident . hCG was only inhibitory when the cells in the 2-step system were primed with PMSG . These data indicate that granulosa cell inhibin production is under direct control of both FSH and LH, and provide a possible explanation for alterations in inhibin activity around the time of ovulation in vivo.

Am J Pathol, 1988 Mar, 130(3), 427 - 30
Tumor necrosis factor enhances interferon-induced Ia antigen expression on murine islet parenchymal cells; Wright JR Jr et al.; Islets from male B10.BR mice (H-2k) were isolated by the collagenase technique, hand-picked with a Pasteur pipette, and incubated in tissue culture media supplemented with recombinant murine interferon-gamma (rIFN-gamma), recombinant murine tumor necrosis factor (rTNF), or both . IAk-molecules could be identified on the surface of islets incubated for 5 days with a combination of rIFN-gamma (1, 10, or 100 ng/ml) and rTNF (10 or 50 U/ml) by indirect immunofluorescence . Optimal concentrations of rIFN-gamma and rTNF, 10 ng/ml and 50 U/ml, respectively, were used in all subsequent experiments . Weak Ia-positivity could be identified on the surface of islets cultured with both cytokines for as little as 48 hours; however, the staining appeared most intense after 5 days of culture . Intensely Ia-positive islets were then carefully washed and cultured in media without either cytokine; Ia positivity could be identified on the surface of these islets for up to 1 week . Dispersed islet cells were cultured with rIFN-gamma alone (10 ng/ml), rTNF alone (50 U/ml), or both cytokines for 5 or 10 days . After either 5 or 10 days of culture with both cytokines, intense immunofluorescent staining for Ia could be identified on the surface of greater than 80-90% of the viable islet cells . Culture with IFN alone for 10 days resulted in 15-20% Ia positivity; culture with TNF alone did not cause Ia expression.

Fertil Steril, 1988 Mar, 49(3), 516 - 21
Mouse embryo culture as quality control for human in vitro fertilization: the one-cell versus the two-cell model; Davidson A et al.; Female mice were superovulated with pregnant mare serum gonadotropin (PMSG) and human chorionic gonadotropin (hCG) and mated with male mice . One-cell (n = 429) and 2-cell (n = 450) embryos were collected 20 and 42 hours after hCG and cultured in Ham's F-10 medium (Gibco, Grand Island, NY) (282 mOsm/l, pH 7.4) and in media of altered osmolality (260, 300, 316 mOsm/l), altered pH (7.0, 7.8, 8.0) or various dilutions of Cidex (Surgikos, Arlington, TX) (1:1000, 1:10,000, 1:100,000) . Stages of development were observed for 4 days . The development of embryos in the 1-cell system was significantly impaired under all studied conditions by the 4-cell stage of development . The 2-cell system failed to detect trace amounts of Cidex in the culture media and an increase in osmolality to 300 mOsm/l . Other changes in osmolality (260 mOsm/l) and pH (7.8) were detected by the 2-cell system only at the blastocyst stage . The authors conclude that the 1-cell system is more sensitive than the 2-cell system to mild changes in in vitro fertilization culture media.

J Neuroimmunol, 1988 Mar, 17(4), 323 - 30
Activated suppressor cell function in multiple sclerosis--clinical correlations; Antel J et al.; Activated suppressor cell function mediated by either freshly isolated peripheral blood mononuclear cells (MNCs), freshly isolated CD8+ lymphocytes or by CD8+ cell lines, has previously been found to be reduced compared to controls in multiple sclerosis (MS) patients with progressive disease (MS-P) . In this study, we found that suppressor activity mediated by CD8+ cell lines, derived from MS patients with stable disease (MS-S) patients and maintained in culture for 14 days, was significantly greater (45 +/- 6%) compared to that mediated by MS-P patients' CD8+ cells (11 +/- 4%, P less than 0.005) . The MS-S suppressor values were, however, suggestively reduced compared to controls (60 +/- 6%, P less than 0.05) . MNC-mediated suppressor values for the MS-S group (61 +/- 5%) did not differ from the control group (67 +/- 6%) . Values for the MS-P group (7 +/- 6%) were significantly reduced compared to MS-S and control groups . Cytotoxic activity mediated by CD8+ cell lines showing defective suppressor function did not differ from control values . The cell lines in MS and control did not differ with respect to their rate of proliferation in the presence of IL-2 and OKT3 . Suppressor function in this assay was ablated if exogenous IL-2 was removed from the culture media . These data suggest that defective activated suppressor function is characteristic of the progressive form of MS, although a suppressor defect is also partially expressed in stable MS patients when CD8+ cell lines are studied.

Acta Trop, 1988 Mar, 45(1), 67 - 76
Moulting and exsheathment of the infective larvae of Onchocerca lienalis (Filarioidea) in vitro; Pudney M et al.; Fourth-stage larvae (L4) of the cattle filarial parasite Onchocerca lienalis were produced from third stage forms (L3) and maintained in vitro using a variety of culture media and feeder cell layers . Up to 50% of the L3 larvae moulted to the fourth stage in the presence of two jird (Meriones unguiculatus) cell lines, a monkey kidney cell line and also with Vero cells . Moulting took place 2-5 days after of the initiation of the cultures: These L4 parasites could be maintained in a healthy condition for up to 88 days, although no significant increases in size of these larvae were observed during the culture period . The medium giving the most promising results, both in terms of moulting and survival, was 199 supplemented with heat inactivated foetal bovine serum (20%) and glucose (2 mg/ml) . The feeder layer cells could be replaced by fresh jird red blood cells . Moulting of parasites occurred in medium alone but the viability was reduced under these conditions.

Am J Pathol, 1988 Mar, 130(3), 489 - 95
Macrophage-derived cytokines amplify immune complex-triggered O2- . responses by rat alveolar macrophages; Warren JS et al.; Interleukin-1 (IL-1) and tumor necrosis factor (TNF) are monocyte macrophage-derived hormonelike regulatory proteins that participate in many physiologic and pathophysiologic processes . Several proinflammatory activities have been attributed to these cytokines, but their importance in anatomically compartmentalized inflammatory processes is unclear . The current in vitro studies have been designed to examine modulatory influences of these cytokines on O2- . responses of rat phagocytes implicated as effector cells in immune complex mediated lung injury . Purified human IL-1, recombinant human TNF (rTNF), and culture supernatant from zymosan-activated alveolar macrophages significantly amplified O2- . responses of immune complex-stimulated alveolar macrophages but did not enhance the responses of neutrophils . Equivalent concentrations of IL-1, rTNF, and alveolar macrophage culture supernatant had no direct stimulatory effect on alveolar macrophages as measured by O2- . production . Culture media from unstimulated alveolar macrophages exerted negligible effects on O2- . generation by immune complex-activated alveolar macrophages . These data indicate that O2- responses of immune complex alveolar macrophages can be enhanced by the presence of IL-1, TNF, or media from activated macrophages . It is possible that macrophage products may greatly amplify tissue injury through the enhancement of oxygen radical production.

J Chromatogr, 1988 Feb 26, 424(2), 315 - 23
Simple isocratic high-performance liquid chromatographic method for the separation of the six vitamers of vitamin B6; Argoudelis CJ; A simple, sensitive and fast isocratic high-performance liquid chromatographic method has been developed for the separation of all six biologically active forms (vitamers) of vitamin B6 . The separation is accomplished using a strong cation-exchange column and a mobile phase of 0.1 M ammonium dihydrogenphosphate adjusted to pH 4.0 . All six vitamers are separated within 20 min at a flowrate of 1 ml/min . The concentration of the vitamers is determined with a fluorescence detector (excitation 290 nm; emission 389 nm) . The within-run precision of the method expressed as the coefficient of variation is below 5% at the 25 pmol level . Pyridoxal 5'-phosphate can be determined using either pre- or post-column derivatization with sodium bisulfite . Application of the method to cell-free yeast culture media is presented.

JAMA, 1988 Feb 26, 259(8), 1223 - 7
Diagnosis of trichomoniasis . Comparison of conventional wet-mount examination with cytologic studies, cultures, and monoclonal antibody staining of direct specimens; Krieger JN et al.; The accuracy of (1) conventional wet-mount examination, (2) Papanicolaou-stained gynecologic smears, (3) a direct slide test using fluorescein-conjugated monoclonal antibodies against Trichomonas vaginalis, and (4) two different culture media for the diagnosis of trichomoniasis in a high-risk population of 600 women was compared . Use of Feinberg-Whittington or Diamond's culture medium resulted in a diagnosis of 82 and 78 cases, respectively, and the combination of two cultures identified 88 infected women . In comparison, wet-mount examination detected only 53 (60%) of the cases . Cytologic smears were interpreted as positive for T vaginalis in 49 (56%) of the 88 cases but also resulted in seven false-positive smears, and specimens from 18 women with negative cultures were interpreted as "suspicious" for trichomoniasis . Monoclonal antibody staining detected 76 (86%) of the 88 positive specimens, including 27 (77%) of the 35 cases missed by wet-mount examination . In summary, wet-mount and cytologic studies were insensitive, and cytology study was the least specific method for diagnosis of trichomoniasis . Direct immunofluorescence with monoclonal antibodies holds promise as a sensitive and specific alternative to cultures for rapid detection of T vaginalis in clinical specimens.

Cancer Res, 1988 Feb 15, 48(4), 822 - 5
Role of oxygen radicals in 12-O-tetradecanoylphorbol-13-acetate-induced squamous differentiation of cultured normal human bronchial epithelial cells; Gabrielson EW et al.; The tumor promoter 12-O-tetradecanoylphorbol-13-acetate (TPA) inhibits growth and induces terminal squamous differentiation of normal human bronchial cells when added to the culture media {J . C . Willey, A . J . Saladino, C . Ozanne, J . F . Lechner, and C . C . Harris, Carcinogenesis (lond.), 5: 209-215, 1984} . We have investigated the possibility of oxygen free radicals being involved as intermediates in this process . Electron paramagnetic resonance measurements using the spin-trapping agent 5,5-dimethyl-1-pyrroline-1-oxide failed to detect oxygen free radicals in bronchial epithelial cells exposed to TPA, although oxy radicals were detected in bronchial epithelial cells after a nontoxic exposure to menadione, and in human neutrophils after exposure to TPA . Addition to the culture media of free radical scavenger, i.e., reduced glutathione, N-acetylcysteine, D-alpha-tocopherol, copper (II) (3,5-diisopropylsalicyclic acid)2, or the combination of superoxide dismutase and catalase did not affect the dose-dependent growth inhibition of TPA on the bronchial epithelial cells . Moreover, exposure of the bronchial epithelial cells to TPA did not result in increased DNA single strand breaks measured by alkaline elution, as would be expected with a free radical mediated mechanism . Thus, our results argue against the importance of oxygen free radicals in the inhibition of growth and the induction of squamous differentiation by TPA in normal human bronchial epithelial cells.

Hybridoma, 1988 Feb, 7(1), 69 - 77
Effects of culture media on murine hybridomas: definition of optimal conditions for hybridoma viability, cellular proliferation, and antibody production; Long WJ et al.; Different cell culture media were compared for their ability to support and promote the growth of stable hybridoma cell lines derived from three commonly used parental murine myelomas . Supplemented Dulbecco's modified Eagle's media (DMEM) and RPMI 1640 media were studied . The DMEM-based media were found to support greater numbers of cells for longer time periods than were the RPMI 1640-based media . Aminopterin supplemented medium was shown to be significantly less effective in supporting hybridoma reproduction and viability than medium without aminopterin . Antibody levels were directly related to cell concentration and viability regardless of the medium used for the hybridoma culture . An optimally formulated DMEM-based medium is suggested as the medium of choice for hybridoma propagation and maintenance.

J In Vitro Fert Embryo Transf, 1988 Feb, 5(1), 25 - 30
Effects of prolactin on gametes and zygotes during in vitro fertilization in mice; Fukuda A et al.; Hyperprolactinemia is one of the major causative factors of infertility . However, the effect of prolactin on gametes during in vitro fertilization has not been elucidated . In the present study, the effects of mouse prolactin on the motility of spermatozoa, in vitro fertilization, and in vitro development of the zygote were investigated in mice using media containing three different concentrations (10, 50, and 100 ng/ml) of mouse prolactin . The development of unfertilized and fertilized oocytes (zygotes) was not affected in vitro by prolactin regardless of the amount of prolactin added to culture media . However, the fertilizing capacity of the spermatozoa was suppressed when they were preincubated for 90 min in culture media containing prolactin at concentrations of 50 and 100 ng/ml . The motility of spermatozoa was not affected by prolactin regardless of the concentration of prolactin used for preincubation . The present study indicates that prolactin may have some effects on the capacitation process of spermatozoa in vitro . This result should be taken into consideration in in vitro fertilization and embryo transfer procedures in humans.

J Endocrinol, 1988 Feb, 116(2), 247 - 58
The time-course of oxytocin secretion from cultured bovine granulosa cells, stimulated by ascorbate and catecholamines; Luck MR et al.; Bovine granulosa cells secrete oxytocin when cultured in a serum-supplemented medium . The time-course of secretion is similar to that in the early corpus luteum in vivo, with a delay of 1 to 2 days followed by a peak and decline over the first 5 days of culture . We have investigated the basis of this time-course in vitro and studied the temporal characteristics of the stimulatory actions of ascorbic acid and adrenaline on this process . Cells cultured on stirred microcarriers showed a similar pattern of secretion of oxytocin to those cultured on conventional flat plates, despite continuing and rapid mitosis . This indicated that the secretion profile in conventional culture was not an artifact related to the cessation of mitosis . Furthermore, secretion of oxytocin and progesterone by cells on microcarriers was stimulated without a corresponding change in mitotic rate, showing that the secretion per cell had been increased . In conventional culture, addition of ascorbic acid to culture media (0.5 mmol/l) increased the secretion of oxytocin (up to 4.5-fold) but only if ascorbic acid was present during the first day of culture . The cells showed a progressive refractoriness to stimulation after 12 h . Since the time-course of secretion was unaltered by treatment, this resulted in a delay of 1 to 2 days before the action of the ascorbate was seen . The secretion of progesterone was similarly affected but with less stimulation and less consistency . In contrast, cells treated with adrenaline (10 mumol/l) secreted more oxytocin on the day of treatment and did so at any time during culture provided that there was sufficient basal secretion of hormone . Adrenaline also failed to alter the time-course of secretion but treated cells showed a persistent response, maintaining enhanced secretion for up to 3 days after the adrenaline had been removed . Ascorbate and adrenaline were highly synergistic in their effects, provided that the ascorbate was present from the start of culture; the response to adrenaline strongly reflected the degree of ascorbate stimulation . We conclude that granulosa cells secrete oxytocin according to an inherent time-schedule and that there is a limited period during which they can respond to ascorbate . Since ascorbate is required for the biosynthesis of oxytocin, this suggests that the availability of ascorbate during corpus luteum formation may determine the amount of oxytocin which can be released subsequently in response to catecholamines.

J Clin Microbiol, 1988 Feb, 26(2), 383 - 4
UV red fluorescence of Veillonella spp; Brazier JS et al.; A total of 34 clinical isolates and 7 type strains of Veillonella spp . were tested for their ability to fluoresce on various culture media . Fluorescence was medium dependent and varied among the species . Scanning absorption spectrophotometry of culture extracts showed that the absorption spectrum of the fluorescent pigment is typical of a metal-free porphyrin.

Radiat Res, 1988 Feb, 113(2), 243 - 51
Factors influencing the oxidation of the radioprotector WR-1065; Tahsildar HI et al.; N-(2-Mercaptoethyl)-1,3-diaminopropane (WR-1065) is the free thiol form of the radio- and chemoprotector S-2-(3-aminopropylamino)ethylphosphorothioic acid (WR-2721) . Interest currently exists in the clinical use of WR-2721 and WR-1065 as radio- and chemoprotectors of normal tissues . However, measurement of plasma levels of WR-1065 has proven difficult, due to rapid drug oxidation . Therefore, we studied factors influencing the oxidation of WR-1065, in Hepes-buffered saline as well as in tissue culture media containing 10% fetal bovine serum . The rate of oxygen consumption by WR-1065, as determined using the Clark oxygen electrode system, was faster in medium plus serum than in Hepes-buffered saline . That this effect is largely due to the presence of trace metal ions in tissue culture media and serum was indicated by the observation that addition of Cu2+ or Fe3+ to buffer stimulated oxygen consumption . Addition of KCN inhibited the reaction of WR-1065 with oxygen, and this effect was dependent on KCN concentration . That KCN blocked WR-1065 oxidation to the disulfide was verified using Ellman's reagent to quantitate the free thiol form . The rate of oxygen consumption was shown to be affected by temperature as well as concentration of WR-1065 . Catalase reduced the rate of oxygen consumption of WR-1065, indicating that peroxide is formed in this system . Superoxide dismutase had a stimulatory effect . WR-1065 was found to stimulate the hexose monophosphate shunt in A549 cells . Since this stimulation was prevented by the presence of catalase, it appeared to be due to the response of the cells to peroxide, formed as a result of WR-1065 autooxidation.

Cryobiology, 1988 Feb, 25(1), 31 - 7
Skin preservation at 4 degrees C: a species comparison; Rosenquist MD et al.; There are numerous experimental studies in the literature regarding skin storage and preservation . These studies are difficult to interpret due to the variety of storage techniques utilized and the number of different animal species used as skin donors . This study utilized a single cold storage protocol to test the effect of species variation on skin graft viability . Donor skin was obtained from five animal species and human surgical panniculectomy specimens . The skin was stored in modified Roswell Park Memorial Institute (RPMI) 1640 tissue culture media at 4 degrees C . Stored skin was transplanted to surgically created defects on athymic (nude) mice after specific storage intervals . Ten days after transplantation, the grafts were examined by gross and microscopic techniques . The viability of mouse, rat, and dog skin was significantly different from human skin, while stored rabbit and pig skin were similar to human skin . These results demonstrate the difficulty of applying the data of skin storage studies from nonhuman species to clinical practice . The data indicate that rabbit and pig skin may be used in laboratory studies of skin preservation at 4 degrees C with a strong likelihood that the results may be of clinical relevance in predicting the behavior of human skin under similar storage conditions.

Eur J Haematol, 1988 Feb, 40(2), 126 - 9
Kinetics of hemopoietic stem cells in a hypoxic culture; Ishikawa Y et al.; The influence of low oxygen tension on the clonal growth of hemopoietic stem cells in vitro was examined . The numbers of colonies of neutrophil, macrophage, and eosinophil progenitors (CFU-C), derived from human bone marrow, increased at a rate 1.7 times higher in low oxygen tension (7% O2) than in a gas phase that contained air (19% O2) . The erythroid (BFU-E) and multipotential (CFU-mix) progenitors increased about 2.4 times in 7% O2, and the increase in the composed cell type of mixed colonies showed no rate difference in either gas phase . Under atmospheric conditions, a mouse mast cell progenitor (CFU-mast) formed colonies, with the addition of 2-mercaptoethanol (2-ME) . Under low oxygen tension, the CFU-mast formed colonies without 2-ME, but a further enhancement was observed with the addition of 2-ME . Blood gas analysis of human bone marrow showed a pO2 of 51.8 +/- 14.5 mmHg, which was closed to O2 tension in a gas phase culture media containing 7% O2 . This data shows that the physiological O2 tension enhances hemopoietic stem cell proliferation in vitro, and that a part of the enhancing effect by 2-ME is due to a prevention of O2 toxicity at 19% O2.

Prostaglandins Leukot Essent Fatty Acids, 1988 Feb, 31(2), 73 - 81
Effect of phorbol ester on prostaglandin regulation of proliferation in rabbit endometrial cells; Orlicky DJ et al.; We have proposed that two of the endogenously synthesized endometrial prostaglandins, prostaglandin F2 alpha (PGF2 alpha) and prostaglandin E1 (PGE1), play a regulatory role in growth control of the endometrium . PGF2 alpha increases DNA synthesis and PGE1 inhibits that effect . Primary cultures of rabbit endometrial cells were used here to examine the effects of the tumor-promoting, diacylglycerol mimicking, phorbol ester, 12-O-tetradecanoyl phorbol-13-acetate (TPA), on the prostaglandin control of cell proliferation . TPA treatment of these cultures results in: a decrease in control levels of proliferation and complete inhibition by TPA of PGF2 alpha stimulated DNA synthesis; a reduction in {3H}PGF2 alpha binding with short term treatment but an increase to above control binding level with long term treatment; an inhibition of the normal PGF2 alpha stimulated inositol polyphosphate synthesis; and a small increase in accumulation of PGF2 alpha in the culture media . Furthermore, in this culture system, TPA does not down regulate {3H}PGE1 binding; it does not alter the normal PGE1 stimulation of cAMP synthesis; and it has no effect on the normal endogenous PGE1 synthesis by these cultures . The above results are consistent with our previous observations that PGF2 alpha works through the intracellular messengers inositol polyphosphate/diacylglycerol whereas PGE1 works through cAMP.

J Immunol Methods, 1988 Jan 21, 106(1), 57 - 61
A turbidometric assay for measuring proteins in culture media; Wojciechowicz D et al.; An immunoturbidometric assay was developed for the measurement of proteins in culture fluids of hepatocytes . The assay is simple to perform and avoids the biohazards associated with radioimmunoassays . The limit of detection of this assay exemplified by hemopexin and transferrin is 5 ng/ml protein . This degree of sensitivity is attained by incorporating into the procedure the addition of polyethylene glycol to enhance formation of primary antigen-antibody complexes and of a second antibody to further increase the immune complex size, which favors the ratio of specific to background light scattering.

Thromb Res, 1988 Jan 15, 49(2), 287 - 97
Progestogen regulation of tissue plasminogen activator in a human melanoma cell line; Kjaeldgaard A et al.; The influence of progestogens, i.e . medroxyprogesterone, levonorgestrel and norethisterone, was studied in a melanoma cell line producing tissue-type plasminogen activator (t-PA) . The cell cultures were exposed to the three progestogens by addition of the steroids dissolved in a weak alcoholic solution to the culture media, in which the released t-PA was assayed by an immunoradiometric method . Ethanol (0.76% w/v) stimulated the t-PA production, while significant inhibitory effect of the present progestogens in the concentration of 1.7 x 10(-6) M was recorded . By tenfold decrease in progestogen concentration significant reduction of t-PA levels was still seen in the cultures exposed to levonorgestrel, while medroxyprogesterone and norethisterone in this dose had no effect on t-PA release . Norethisterone differed from the other two progestogens in having weak toxic effect on melanoma cells . It was concluded that the progestogens studied, in particular norethisterone and levonorgestrel, had an inhibitory effect on the production of t-PA in melanoma cell culture.

Vopr Onkol, 1988, 34(7), 832 - 9
{Structural differences of the supramolecular DNA complex from tumor strain cells}; Blokhin DIu et al.; Elastoviscosimetric examination was carried out to study the structure of supramolecular DNA complexes obtained from transplantable tumor strain cells and those of the same strains showing pronounced growth in synthetic culture media . The supramolecular DNA complex was isolated as a nucleoid and examined by ethidium bromide elastoviscosimetric titration method . Levels of supercoiled DNA were found to be significantly lower in the chromatin of cells of in vitro growing strains, as compared to parental strain cells where the said type of DNA predominated . Two patterns of DNA compact packing--supercoiled-domain and one characterized by a faster bond between DNA and non-histone proteins, and single-stranded DNA areas preexisting in domains--were suggested.

J Hyg Epidemiol Microbiol Immunol, 1988, 32(1), 79 - 85
Investigation of hemolytic activity of leptospirae on solid culture media; Volina EG et al.; Results of investigation of hemolytic activity of leptospirae against red blood cells of various animal species on solid culture media using the technique of agar layers are presented . It has been shown that hemolytic properties of pathogenic and saprophytic leptospirae differ with respect to red blood cells of the sheep, the rabbit, the hamster, the albino rat and the albino mouse . Hemolytic activity of leptospirae regarding the erythrocytes of the cattle, the horse and the fowl depends on the strain of leptospirae . The advantage of using solid nutrient media for the determination of hemolytic properties of leptospirae in comparison with liquid media has been demonstrated.

Diagn Microbiol Infect Dis, 1988 Jan, 9(1), 7 - 10
Discrepancy between growth of Coccidioides immitis in bacterial blood culture media and a radiometric growth index; Ampel NM et al.; Spherules of Coccidioides immitis grew readily after inoculation in vented trypticase soy broth, biphasic brain heart infusion media, and aerobic tryptic soy broth bottles used in a radiometric system (BACTEC) . However, visible growth was not accompanied by a significant radiometric growth index . Growth of C . immitis can be visually detected in routine bacterial blood culture media while the radiometric growth index remains negative.

Free Radic Biol Med, 1988, 4(2), 79 - 83
Quantitation of intracellular oxidation in a renal epithelial cell line; Scott JA et al.; We quantitated the presence of intracellular oxidizing species in response to oxidative stimuli using fluorescent cell analytic techniques . The studies were performed with a laser-activated flow cytometry system using 2',7'-dichlorofluorescin diacetate (DCFDA) as a probe for intracellular oxidation events . Oxygen radical formation was initiated by the addition of FeCl2 or xanthine oxidase to the culture media . Xanthine oxidase and FeCl2 both increased intracellular DCFDA oxidation over control (p less than .001) . Increases in intracellular DCFDA oxidation in response to xanthine oxidase exposure were inhibited by extracellular superoxide dismutase, catalase and dimethyl sulfoxide (p less than 0.001), implicating the superoxide anion, hydrogen peroxide, and the hydroxyl radical in producing the changes in intracellular dichlorofluorescein fluorescence . Increases in intracellular DCFDA oxidation in response to xanthine oxidase correlated with loss of cellular viability, as established by decreased plating efficiency . We conclude that relative intracellular oxidation can be quantitated within the cultured renal cell and that some extracellularly generated radicals may be capable of traversing the intact cell membrane to oxidize DCFDA in the cell interior.

J Orthop Res, 1988, 6(2), 259 - 64
Time-varying magnetic fields: effects of orientation on chondrocyte proliferation; Elliott JP et al.; The purpose of this study was to determine the effect of orientation of pulsed electromagnetic fields (PEMFs) on cellular proliferation and extracellular matrix synthesis . Bovine articular chondrocytes were cultured in PEMFs (repetitive pulse at 72 Hz) generated using Helmholtz coils oriented either parallel (horizontal) or perpendicular (vertical) to the plane of cell adhesion . Dissipation of signal energy in the form of heat increased the temperature of the PEMF coils by 2 degrees C and the tissue culture medium by 1 degree C . Therefore, control coils, which emitted no PEMFs, were heated to the temperature of PEMF coils by circulating water . Chondrocytes were cultured in 16-mm-well culture plates, and the data for individual wells were pooled as triplicates . Although not observed by microscopic examination of individual wells, positionally dependent electric field effects may be minimized by this approach . PEMFs generated by coils oriented vertically significantly decreased chondrocyte proliferation . The effect was dependent on the concentration of serum in the culture media . At 3% serum concentration, the total cell number attained after 10 days of culture was reduced by 50% in stimulated cultures when compared with controls . At 5% serum concentration, there was no effect . PEMFs applied by coils oriented horizontally did not alter proliferation of articular chondrocytes . PEMFs had no effect on synthesis of extracellular matrix by chondrocytes plated at high density, irrespective of orientation.

J Lab Clin Med, 1988 Jan, 111(1), 28 - 34
Complement fixation by pemphigus antibody . IV . Enhanced epidermal cell detachment in the absence of human plasminogen; Doubleday CW et al.; It has been reportedly previously that complement enhances pemphigus vulgaris antibody-induced epidermal cell detachment . The present studies were designed to eliminate the possibility of the human plasminogen-plasmin system contributing to the cell detachment observed in previous complement-mediated studies . To assay for the presence of protease contamination in the reagents, a simple, sensitive, caseinolytic-fluorometric assay was used . Confluent murine or human epidermal cell cultures were incubated with pemphigus IgG with and without complement and plasminogen . Detached epidermal cells, as determined by light microscopy and cell-counter analysis, were enumerated at 48 hours . Incubating pemphigus IgG with complement resulted in marked cell detachment, compared with minimal cell detachment when pemphigus IgG was incubated with plasminogen . Culture media were collected after 48 hours and assayed for activation of plasminogen . It was determined that the added plasminogen had not been activated, particularly in experiments where significant detachment was observed . The plasminogen could still be activated by streptokinase and urokinase . These studies suggest that epidermal cell detachment in pemphigus appears to be mediated at least in part by complement activation.

Fertil Steril, 1988 Jan, 49(1), 108 - 11
Endotoxins in culture medium for human in vitro fertilization; Fishel S et al.; Endotoxins were detected in a few batches of culture medium during the authors' human in vitro fertilization program . Two distinct levels of endotoxins were assayed: greater than 1 ng/ml and less than 1 ng/ml . The source of endotoxin was traced to culture media obtained from a reputable manufacturing company . The incidence of fertilization per patient was not significantly affected by the presence of endotoxins, but fertilization assessed on the overall number of oocytes was significantly reduced (53%) when endotoxin levels were greater than 1 ng/ml compared with an assay negative for endotoxins (66%) (P = 0.047) . The percentage of oocytes cleaving after the observation of two pronuclei was not significantly different, but the degree of fragments observed in the conceptus was significantly more severe if the endotoxin level reached 1 ng/ml . In this investigation, the incidence of pregnancy was 8% when the endotoxin level was greater than 1 ng/ml, 30% if less than 1 ng/ml, and 32% if no endotoxins were detected . This study suggests that, although endotoxins may be present in the culture medium at a deleterious level of at least 1 ng/ml, fertilization and cleavage will be obtained, but there will be a significant increase in the incidence of conceptuses with cytoplasmic fragments; this may result in a reduction in the incidence of pregnancy.

J Burn Care Rehabil, 1988 Jan-Feb, 9(1), 52 - 4
Short-term skin preservation at 4 degrees C: skin storage configuration and tissue-to-volume medium ratio; Rosenquist MD et al.; This study was designed to examine the effect of the storage configuration of skin and the ratio of tissue-to-storage medium on the viability of skin stored under refrigeration . Human skin specimens were stored in four physical configurations in RPMI 1640 tissue culture media at 4 degrees C . Skin was transferred to surgically created defects on nude mice after specific storage intervals . Grafts were examined grossly and microscopically after ten days . In the rolled configuration, on storage day 15, the viability of the outside of the roll was significantly better than the inside (P less than 0.01) . The graft viability of the outside of the skin rolls was similar for both tissue-to-media ratios as well as for both free-floating configurations (P = 0.27) . These findings suggest the optimum cold storage configuration is free floating, and 300 cm2/100 mL is an appropriate skin surface area to volume media ratio . This proportion of tissue to media is in agreement with the minimum ratio currently recommended by the Skin Council of the American Association of Tissue Banks.

Aust J Biol Sci, 1988, 41(2), 189 - 99
Development of sheep embryos in vitro in a medium supplemented with different serum fractions; Batt PA et al.; Sheep embryos will generally develop into expanded blastocysts in vitro only in culture media supplemented with serum or serum components . In order to better understand how serum supports embryo development, a batch of ovine serum was fractionated by (a) ultrafiltration into two components containing substances with molecular weights greater and less than 10 Kd (kilodaltons), and (b) gel filtration into protein fractions 1, 2 and 3, containing groups of proteins with mean molecular weights of about 500, 150 and 65 Kd, respectively . The principal protein in fraction 3 was albumin . Day 6 sheep morulae were cultured in vitro for 48 hours in a bicarbonate-buffered salt solution supplemented with various concentrations of ovine serum or of these components or protein fractions of serum . Morulae could develop to fully expanded blastocysts in medium supplemented with whole serum or with the greater than 10 Kd component or protein fraction 3 only, but could not develop in medium supplemented with the less than 10 Kd component only or with the less than 10 Kd component and protein fractions 1 or 2 . However, the proportion of embryos that developed fully in medium supplemented with the greater than 10 Kd component or protein fraction 3 was increased by adding the less than 10 Kd component of serum to the medium . The addition of protein fraction 2 decreased the proportion of embryos that developed to expanded blastocysts in medium containing protein fraction 3 and the less than 10 Kd component, but not in medium containing whole serum . Since the compositions of different sera may vary markedly, these results suggest (a) reasons why different sera vary in their ability to support embryo development in vitro, and (b) factors which may influence development of the sheep embryo in the uterus, where plasma proteins comprise nearly all the protein in the fluid bathing the embryo.

Reprod Nutr Dev, 1988, 28(6B), 1791 - 5
{Search for specific factors in culture media of embryos}; Testart J et al.; Mitogenic activity of culture media having contained either unfertilized or cleaved embryos have been studied . In these media human eggs have been cultured for one day, beginning 20 hours following in vitro insemination . B-lymphocyte proliferation was estimated by thymidine incorporation following 3 day culture in the presence of eggs or embryo culture media . No reproducible effect on B lymphocyte proliferation was observed . Therefore the viability of embryos could not be ascertained by the mitogenic activity of their culture media.

J Immunoassay, 1988, 9(2), 193 - 206
A simple radioimmunoassay of human interleukin-2; Lee TP et al.; We have developed a liquid phase radioimmunoassay of human interleukin-2 which uses inexpensive commercially available reagents exclusively . The assay is simple, reproducible and specific in detecting different batches of human interleukin-2 of natural as well as recombinant origin, but not detecting recombinant murine interleukin-2 . The assay is sensitive to a concentration of approximately 0.05 ng/ml and can be used in measurement of IL-2 in serum containing culture media.

Acta Biol Hung, 1988, 39(1), 75 - 85
Effect of heavy metals on the growth of tissue cultures (II); Maroti M et al.; The effect of toxic concentrations of three heavy metal compounds on the growth of the secondary callus tissue of Nicotiana tabacum L . and Ruta graveolens L . was studied . The metal compounds examined were ZnSO4, NiSO4, CuSO4 . The metal compounds used were placed in Murashige, Skoog (1962) and White (1943) culture medium at 10(-6) and 10(-4) M concentration, respectively, before autoclaving . The culture media containing macro- and microelements and vitamins were completed with carbon source and regulators (IAA, GA, kinetin for Nicotiana and IAA, 2, 4-D for Ruta) . The cultures were kept for 4 weeks at 25 (+2) degrees C under 16/8 n light/dark conditions . The value of pH was 5.6 before the autoclave treatment . The increase in fresh weight of the secondary callus tissue was inhibited by the metal compounds applied with both plant species (to 75-87% by zinc, 7-97% by nickel, 5-98% by copper with tobacco; to 47-69% by zinc, 5-88% by nickel, 57-90% by copper with rue) . The cell number and dry weight per g of callus tissue partly increased, partly decreased compared to the control in response to the heavy metal treatment . The growth values obtained with various concentrations of the heavy metals were different in the two plant species due to differences in metabolism and organization potential between them.

Tissue Cell, 1988, 20(4), 541 - 54
Primary culture of bovine mammary acini on a collagen matrix; Baumrucker CR et al.; Lactating bovine mammary epithelial acini were isolated and primary culture on rat tail attached collagen gels are described . Acini rapidly attach to the gels and morphologically change little over days of culture under the culture conditions described herein . Cells release lactose, alpha-lactalbumin and alpha-s1 casein over a 6-day period . A new HPLC method for measuring lactose in mammary cell culture media is described . Comparisons of acini cultures with individual cell cultures show acini to be 1.5-5 times more productive than cells in secreting lactose and casein, respectively.

Cell Tissue Res, 1988, 254(3), 611 - 5
Monoclonal antibody C1B8A8 recognizes a ventricular secretory product elaborated in the bovine subcommissural organ; Meiniel R et al.; To obtain specific immunological probes for investigation of the cellular and molecular aspects of the subcommissural organ (SCO), we produced monoclonal antibodies directed against extracts from the bovine SCO . An hybridoma cell line (C1A8B8) was isolated by screening the culture media by means of the immunofluorescence method . This clone produces an IgG1 that recognizes the ventricular secretory material of the SCO including Reissner's fiber . A competition test using C1B8A8 immunoglobulin and lectins (concanavalin A and wheat-germ agglutinin) was applied to demonstrate that both the immature and mature forms of the glycoprotein were recognized . This antibody will offer a good tool for immunocytochemical localization and immunoaffinity purification of the antigen and for isolation of cDNA clones encoding it.

Pharmacology, 1988, 37(3), 154 - 64
Cell-catalyzed binding of 3H-(-)-trans-7,8-dihydroxy-7,8-dihydrobenzo{a}pyrene to cellular and exogenous DNA and the role of purified human liver epoxide hydrolase; Gozakara EM et al.; Cultured human monocytes, lymphocytes, Fischer rat liver (TRL-2) cells, and Buffalo rat liver (BRL) cells catalyzed the conversion of 3H(-)-trans-7,8-dihydroxy-7,8-dihydrobenzo{a}pyrene {3H(-)t-7,8-dihydrodiol BP} to r-7,t-8-dihydroxy-t-9,10-oxy-7,8,9,10-tetrahydrobenzo{a}pyrene (diol epoxide I) and r-7,t-7-8-dihydroxy-c-9,10-oxy-7,8,9,10- tetrahydrobenzo{a}pyrene (diol epoxide II; r-7 indicates that the substituent at the 7-position is the reference, and t and c indicate that the substituents trans and cis, respectively, to the reference substituent) . These appear to be the most reactive metabolites of benzo{a}pyrene (BP) and were covalently bound to both exogenous and intact cellular DNA in tissue culture media . The cells induced by benzanthracene (BA) exhibited greater levels of DNA binding than the controls and this binding was linear with increasing cell content in human monocytes, in TRL-2 cells and in Buffalo rat liver cells . The binding to DNA was greater than controls in BA-preinduced lymphocytes, but was not linear . The DNA binding in control cells showed a nonlinear increase with increasing cell concentration in all experiments . The addition of human liver epoxide hydrolase (EC 3.3.2.3) to the incubation medium reduced the amount of reactive metabolites binding to DNA by 12-15% in control and by 23-41% in BA-induced monocytes . Thus, with whole cell systems of either human monocytes or lymphocytes, the addition of purified human liver epoxide hydrolase reduced the binding of 3H(-)t-7,8-dihydrodiol BP metabolites to DNA . Human monocytes and lymphocytes also catalyzed the covalent binding of 3H(-)t-7,8-dihydrodiol BP to intact cellular DNA . The addition of 3H(-)t-7,8-dihydrodiol BA to tissue culture media caused the inhibition of covalent DNA binding in BA-preinduced monocyte by 58% and lymphocytes by 25% . Previous work has shown that BA is metabolized and converted to BA-diol epoxides by microsomes . These results indicate that BA-diol epoxides and BP diol epoxides are competing for the same binding sites on DNA . On the other hand, the addition of 10 nmol of 3H(-)t-7,8-dihydrodiol BP to the incubation of control and BA-preinduced cell homogenate and further incubation at 37 degrees C for 25 min showed that the DNA binding in BA-preinduced cell homogenates was much greater than controls . Homogenates of cells induced by BA exhibited a greater level of DNA binding than controls.(ABSTRACT TRUNCATED AT 400 WORDS)

Exp Pathol, 1988, 33(3), 173 - 7
Ethanol damage to rat gastric mucosa is unlikely to be mediated by ethanol; Sewell RB et al.; During injury to the gastric mucosa, lysosomes become more fragile and lysosomal enzymes, which are activated at acid pH, leak into the surrounding environment . It is not clear whether these changes contribute to the mechanism of damage or are merely a secondary result of it . To test whether lysosomes modulate gastric mucosal damage, we pretreated rats with a lysosomal labilizing agent, Triton WR 1339 (1.5 g/kg) and histologically assessed mucosal damage in vivo after challenge with 30% ethanol . No significant differences were found in the length or depth of eroded mucosa: mean erosion length, Triton 23.9 +/- 6.6% vs . control 19.7 +/- 5.2%; mean depth (micron), Triton 19 +/- 4 vs . control 20 +/- 7 . After a similar pretreatment regimen, rat antral mucosa was cultured, challenged with ethanol and damage assessed by release into media of previously incorporated mucosal 51chromium . With 15% ethanol challenge, no change in 51chromium release was seen: after Triton, 9.8 +/- 1.4% vs . control 10.3 +/- 1.0% . Triton pretreatment perturbed gastric lysosomes as shown in organ culture by significantly raised tissue lysosomal enzyme activities and increased lysosomal enzyme release into culture media after ethanol challenge . The lack of effect of this pretreatment regimen suggests that lysosomes do not have a major pathogenetic role in ethanol-induced gastric damage.

Life Sci, 1988, 43(13), 1069 - 77
Characterization of 1,1-dimethyl-4-phenylpiperazinium induced increased proenkephalin processing in bovine chromaffin cells; Cherdchu C et al.; Acute stimulation of bovine adrenal chromaffin cells in culture with 1,1-dimethyl-4-phenylpiperazinium (DMPP) gives rise to a significant increase in secretion of {Met5}-enkephalin immunoreactive material (ME-IRM) into the culture medium (1) . Following this secretion the cellular ME-IRM levels do not decrease, suggesting the replenishment of the peptides . The repletion of the cellular ME-IRM appears to result from an increase in processing of large molecular weight peptides containing {Met5}-enkephalin and {Leu5}-enkephalin . Gel filtration chromatography on Bio-Gel P-10 was used to fractionate the enkephalin-like peptides (ELPs) present in the culture media and chromaffin cell extracts . Fractionation was done for samples before and after nicotinic receptor stimulation by DMPP to demonstrate the secretion and repletion of the ELPs . Gel chromatographic profiles of ELPs present in the culture media after DMPP stimulation revealed the presence of 4 peaks, representing different molecular forms of these peptides (Peaks 1-4), with a selective increase in secretion of Peaks 3 and 4 . The chromatograms of ELPs extracted from cultured chromaffin cells showed similar patterns to those obtained from ELPs present in the culture medium after stimulation . Analyses of individual peaks after fractionation of cell culture extracts showed an increase in the amount of immunoreactive material found in Peak 4 with a concomitant decrease in the immunoreactivity found in the higher molecular weight peaks (Peaks 1-3) . Further purification of Peak 4 from cell extracts on reversed-phase HPLC (RP-HPLC) showed a significant amount of ELPs existed as the sulfoxide derivative of {Met5}-enkephalin . The content of {Met5}-enkephalin sulfoxide (ME-O-enk) did not decrease following DMPP stimulation . We conclude that acute stimulation of nicotinic receptors in the chromaffin cells enhances the processing of proenkephalin precursors to keep pace with the secretion of low molecular weight peptides.

Leuk Res, 1988, 12(7), 567 - 74
Haemoglobin synthesis in K562 erythroleukaemia cells is affected by intimate contact with monolayers of various human cell types; Zuhrie SR et al.; The haemoglobin content of K562 erythroleukaemia cells was affected by co-culture over monolayers of various human cell types . Haemoglobin synthesis was increased after co-culture with umbilical-cord-derived endothelial cells and most monolayers of bone-marrow-derived macrophages, and inhibited after co-culture with two fibroblast lines, blood-monocyte-derived macrophages, a neuroglial cell line (U-251 MG) and most monolayers of bone-marrow-derived stromal cells . These effects were modified when a thin layer of agar was placed over the monolayers . Cell-free culture media conditioned by all but two of the seven types of monolayer studied inhibited haemoglobin synthesis by K562 cells; those conditioned by blood-monocyte-derived macrophages and two of 11 monolayers of bone-marrow-derived macrophages stimulated haemoglobin synthesis . Thus, the haemoglobin content of K562 cells appeared to be influenced both by intimate contact between K562 cells and the cells of the monolayers and by humoral factors released by the monolayers . The data support the concept that erythroid differentiation is partly dependent on intimate contact between erythroid progenitor cells and microenvironmental cells.

Int J Biochem, 1988, 20(8), 817 - 22
Inhibition of plasminogen activators and the growth of cultured human tumour cells; Scott GK; 1 . Inhibition of a human fibroblast cell-surface proteinase with alpha-1-antitrypsin also inhibited cell proliferation, confirming earlier observations . 2 . Although a similar proteinase inhibition could be observed with human tumour cells, the growth of these cells was unaffected . 3 . Plasminogen activators on the cell surface and in culture media could be inhibited with specific antibodies, but there was no corresponding effect on the growth of any of the cell types tested.

Eur J Gynaecol Oncol, 1988, 9(3), 222 - 7
Effect of alpha-difluoromethylornithine (DFMO) on the growth of human ovarian carcinoma; Manetta A et al.; The antiproliferative effect of alpha-difluoromethylornithine (DFMO) was investigated in a human ovarian cancer cell line (NIH:OVCAR3) both in vitro and in vivo . DFMO at 5 X 10(-5) M, 5 X 10(-4) M and 1 X 10(-3) M concentrations showed growth inhibition of NIH:OVCAR3 cells in culture . Parallel determination of CA 125 in the culture media of these cells show significant decrease in the presence of DFMO compared to controls . Oral administration of DFMO (2% aqueous solution) to nude mice bearing intraperitoneal tumors resulted in a mean survival of 45 days (38-60) versus 25 days (20-35) for control . Both in vitro and in vivo results suggest that DFMO has potential value in the treatment of ovarian carcinoma and should be considered for clinical trials in appropriate cancer patients.

Am J Trop Med Hyg, 1988 Jan, 38(1), 111 - 7
Murine schistosomiasis: selective inhibition in vitro of fibroblast collagen production by mononuclear cells; Mansour MM et al.; Primary cell cultures from the livers of mice infected with Schistosoma mansoni were prepared and cells with the appearance of fibroblasts by light microscopy were isolated . Collagen synthesis was estimated by measuring incorporation of 14C-proline into collagenase-sensitive proteins for both culture media and cell layers . Coculture of splenic T cells from infected mice with these hepatic fibroblasts caused greater selective and specific reduction in collagen production than did coculture using spleen cells from normal mice . There was a parallel inhibition in collagen within the cell layer which indicates that the marked decrease in collagen production was due to inhibition of synthesis and not related to changes in solubility or secretion . Primary culture of mouse skin fibroblasts showed similar responses to coculture but an established fibroblast line, 3T3, was unresponsive . Inflammatory cells appear to influence hepatic fibroblasts isolated under our experimental condition in several ways, such as opposite effects on collagen synthesis and cell proliferation.

Ital J Surg Sci, 1988, 18(3), 201 - 5
The stimulating effect of a cytosol extract from regenerating liver on isolated hepatocytes and the positive role of insulin; Gorini P et al.; Several kinds of damage induce a regenerative response in the liver . Partial hepatectomy is a well known method, suitable for studying liver regeneration and factor/s involved in it . The presence of the so called Hepatic Stimulator Substance has been demonstrated in the cytosol obtained from regenerating hepatocytes, and it has been partially characterized, from a molecular point of view . Mainly, it stimulates liver regeneration and Tritiated Thymidine incorporation in cultured hepatocytes as demonstrated in this paper (Control Hepatocytes: 8447 +/- 660.4 cpm, (mean +/- sd), Test Hepatocytes: 16974 +/- 2720.9 cpm; Index of Stimulation: 2.01) . Furthermore, the stimulating activity of HSS is increased by the presence, in the culture media, of insulin which has been proved to have a positive role in liver regeneration (Control Hepatocytes: 8889 +/- 367.3 cpm; Test Hepatocytes: 21588.5 +/- 1232.4 cpm; Index of Stimulation: 2.42, p less than 0.05) . These data seem to suggest that liver is probably the site of production/activity and action of factor/s that stimulate/s liver regeneration; but other substances exist, such as insulin, which play a permissive role.

Biol Met, 1988, 1(1), 9 - 17
High-performance liquid chromatography of siderophores from fungi; Konetschny-Rapp S et al.; A reversed-phase HPLC separation of iron(III) chelates of 16 representative fungal siderophores including ferrichromes, coprogens and triacetylfusarinine C was established in order to investigate siderophore production of fungi . For comparison purposes, the widely used bacterial siderophore ferrioxamine B was included . Culture filtrates of the fungi Penicillium resticulosum, Fusarium dimerum, Aspergillus fumigatus and Neurospora crassa were quantitatively analyzed for the presence of known and unknown siderophores after growth in low-iron culture media and adsorption on XAD-2 columns using this HPLC separation system . Photodiode array detection allowed the distinction between siderophores and non-siderophores . According to their ultraviolet/visible spectra, a further classification of the siderophores into four types due to the number of anhydromevalonic acid residues per molecule (0-3) was possible.

J Hosp Infect, 1988 Jan, 11(1), 77 - 81
Isolation of organisms in CAPD peritonitis: use of nutrient broth cultures and Bactec blood culture media; Stokely DJ et al.; Cultures of the first cloudy dialysates from 51 consecutive episodes of peritonitis in patients undergoing continuous ambulatory peritoneal dialysis were carried out using concentrated nutrient broth and Bactec blood culture media in addition to primary culture on solid media . Thirty-seven episodes (73%) were positive on solid media, and 42 episodes (82%) were positive by both nutrient broth and Bactec methods . The value of Gram-stained smears and WBC counts on dialysates is discussed.

Drugs Exp Clin Res, 1988, 14(1), 19 - 23
Itraconazole: increased activity by chlorhexidine; Simonetti N et al.; Chlorhexidine increases the activity of itraconazole against Candida isolates; itraconazole-chlorhexidine combinations show synergistic activity in culture media . The activity of itraconazole is discussed.

J Orthop Res, 1988, 6(4), 525 - 30
The synovial production of collagenase and chondrocyte activating factors in response to cobalt; Ferguson GM et al.; Addition of CoCl2 solutions to the culture media of confluent monolayers of lapine or human synoviocytes stimulated their production of the neutral proteinases collagenase, gelatinase, and caseinase . With lapine cells, maximum stimulation occurred at 10(-7) M CoCl2, while human cells required 10(-4)-10(-5) M CoCl2 to achieve a maximum stimulation . Production of prostaglandin E2 by lapine cells was enhanced some 30-40% by concentrations of CoCl2 that maximally stimulated synthesis of the neutral proteinases, whereas all concentrations of CoCl2 slightly depressed the production of prostaglandin E2 by human cells . Lapine synovial cells that had been stimulated by CoCl2 also produced a substance, or substances, that provoked the synthesis of collagenase, gelatinase, caseinase, and prostaglandin E2 by monolayers of articular chondrocytes . Chondrocytes themselves, however, resisted activation by CoCl2 . These findings may be relevant to the aseptic loosening of joint prostheses.

Drugs, 1988, 35 Suppl 1, 33 - 41
Effects of interleukin-1 and anti-inflammatory drugs on the degradation of human articular cartilage; Shinmei M et al.; It has been suggested that metalloproteases produced by chondrocytes play an important role in cartilage breakdown in joint diseases . The aim of this study was to investigate changes in enzyme activities in human rheumatoid and osteoarthritic articular cartilage . Cartilage fragments were incubated with various drugs for 48 hours . The concentrated culture media were used as enzyme solutions . Collagenase was assayed using FITC-collagen as the substrate . Proteoglycanase (PGase) was measured either by the release of 35S-labelled proteoglycans from cartilage into the medium, or by enzyme assay using proteoglycan monomer bound to fluorescein-conjugated hyaluronic acid as the substrate . Collagenase and proteoglycanase were found only in trace amounts in the concentrated media of healthy cartilage . Interleukin-1 (IL-1) enhanced the enzyme activities significantly . Marked increases of enzyme activities were observed in the concentrated media of rheumatoid (RA) and osteoarthritic (OA) cartilage . The sensitivity to interleukin-1 was also higher in OA and RA cartilage compared with healthy cartilage . Dexamethasone (10(-6) mol/L) markedly depressed enzyme activity . Tiaprofenic acid (4 x 10(-5) mol/L) also decreased enzyme activity, whereas indomethacin (4 x 10(-6) mol/L) and naproxen (3 x 10(-4) mol/L) had no effect.

Transfusion, 1988 Jan-Feb, 28(1), 81 - 3
Photodynamic therapy of viral contaminants with potential for blood banking applications; Matthews JL et al.; A photodynamic method has been evaluated as a means of eradicating viral contaminants with the potential for rendering blood safe for transfusion . Herpes simplex virus type 1 (HSV-1) was tested under flowing conditions in culture media or in blood supplemented with the virus . Hematoporphyrin derivative was used as the sensitizer and was photoactivated with visible light at 630 nm and 5 J/cm2 . HSV-1 in suspension both in culture medium as well as in blood was shown to be killed . The human immunodeficiency virus was also found to be photoinactivated in flowing cell culture medium and, thus, potentially may be inactivated in blood . These findings extend our previous studies which demonstrated that enveloped viruses can be photoinactivated with hematoporphyrin derivative in a static fluid system . Analysis of blood cell number, red cell lysis, plasma proteins, and other standard hematological tests showed no significant change . The possibility that transfusion-associated acquired immunodeficiency syndrome (AIDS) may result from a blood unit infected with human immunodeficiency virus that tested negative makes it imperative that a safe and effective means of viral killing be developed . The system reported here offers promise as an effective approach to this problem.

Biochem Biophys Res Commun, 1987 Dec 31, 149(3), 1149 - 55
Production of human thyroid-stimulating hormone in Chinese hamster ovary cells; Watanabe S et al.; Human thyroid-stimulating hormone (hTSH) has been produced in Chinese hamster ovary (CHO) cells co-transformed with two plasmids: one carrying the alpha subunit cDNA with mouse dihydrofolate reductase gene and the other carrying hTSH beta subunit cDNA . Each cDNA was driven to expression under the control of SV40 early promoter . hTSH and its alpha subunit were secreted into culture media, and their secretion increased with exposure of the cells to increasing concentrations of methotrexate . Gel filtration analysis revealed that the molecular size of the hTSH was the same as that of natural hTSH . Furthermore, the CHO cell-produced hTSH elevated the cyclic AMP level in the rat thyroid cell line FRTL-5 in the same manner as natural hTSH does.

Eur J Biochem, 1987 Dec 30, 170(1-2), 475 - 83
Metabolism of the vitamin D3 derivative (24R)-hydroxycalcidiol by human promyelocytic leukemia cells (HL-60) . Isolation and identification of (5Z) and (5E)-(24R)-19-nor-10-oxo-24-hydroxycalcidiol; Ishizuka S et al.; Human promyelocytic leukemia cells (HL-60 cells) incubated with (24R)-hydroxy{26,27-methyl-3H}calcidiol (0.2 microCi) or non-radioactive (24R)-hydroxycalcidiol (370 micrograms) produced significant quantities of two new vitamin D3 (calciol) metabolites . The metabolites were isolated from HL-60 cell culture media by methanol/chloroform extraction and a series of chromatographic procedures . The two new metabolites were identified as (5Z)- and (5E)-(24R)-19-nor-10-oxo-24-hydroxycalcidiol by HPLC analysis, ultraviolet absorption spectrophotometry, mass spectrometry and Fourier-transform infrared spectrophotometry . According to the isolation and purification procedures, the total amounts of 3.04 micrograms (5Z)-(24R)-19-nor-10-oxo-24-hydroxycalcidiol (lambda max = 310 nm, epsilon = 17070 M-1 cm-1) and 8.89 micrograms (5E)-(24R)-19-nor-10-oxo-24-hydroxycalcidiol (lambda max = 312 nm, e = 24,500 M-1 cm-1) were calculated, assuming an Mr of 418 . The activity of 19-nor-10-oxo-(24R)-hydroxycalcidiol to promote HL-60 cell differentiation was higher than the activity of the precursor (24R)-hydroxycalcidiol suggesting a possible biological action of this metabolite in HL-60 cells.

Eur J Biochem, 1987 Dec 30, 170(1-2), 133 - 42
Heterogeneity of insulin-like growth factor binding proteins between structure and affinity . 2 . Forms released by human and rat liver in culture; Hossenlopp P et al.; Since the liver is considered to be the major source both of circulating insulin-like growth factors (IGFs) and of their specific binding proteins (BPs), human and rat liver explants were cultured in serum-free medium with a view to characterizing the binding proteins released into the medium and to comparing them with serum binding proteins . In the culture media, as in the serum, IGFs are associated with their binding proteins in the form of complexes . In gel filtration experiments the liver IGF-BP complexes eluted as a single, homogeneous peak with a relative molecular mass of about 40,000, which is similar to that of the 'small' complex of serum . Their sedimentation coefficient, estimated from sucrose gradient centrifugation, was 2.9 S . Polyacrylamide gel electrophoresis (SDS-PAGE) of human liver culture media, in which the binding proteins were cross-linked to 125I-labelled IGF I revealed molecular heterogeneity . Three specific bands corresponding to Mr 46,000, 40,000 and 37,000 were observed, which resemble those of the serum small complex, but none of the higher-Mr bands seen in serum . SDS-PAGE followed by transfer onto nitrocellulose and incubation with 125I-labelled IGF I (western blot) led to the identification in human liver culture media of five molecular forms of binding protein with Mr of 41,500, 38,500, 34,000, 30,000 and 24,000, identical to those seen in serum . The relative concentrations of the 41,500 and 38,500-Mr forms varied from one medium to another, but the 34,000 and 30,000-Mr forms were regularly more abundant in the liver culture media than in normal serum . The binding proteins produced by the liver therefore represent the native forms in the circulation (although this does not exclude other sources) . The absence of high-Mr IGF-BP complexes in the liver culture media, and yet the presence of the 41,500 and 38,500-Mr forms, which are the only binding units of the serum 'large' complex (150,000 Mr), indicates that these two binding proteins are capable of binding IGFs to form 'monomeric' IGF-BP complexes . Western-blot analysis of rat binding proteins revealed a certain analogy with the human proteins, three forms having their Mr between 43,000 and 39,000 and three between 32,000 and 24,000 . Liver binding proteins in human adults and foetuses were found to be identical, whereas in the case of serum the 41,500 and 38,500-Mr forms were more abundant in the adult and the 34,000 and 30,000-Mr forms more abundant in the foetus.(ABSTRACT TRUNCATED AT 400 WORDS)

J Biol Chem, 1987 Dec 25, 262(36), 17605 - 12
Papilin: a Drosophila proteoglycan-like sulfated glycoprotein from basement membranes; Campbell AG et al.; A sulfated glycoprotein was isolated from the culture media of Drosophila Kc cells and named papilin . Affinity purified antibodies against this protein localized it primarily to the basement membranes of embryos . The antibodies cross-reacted with another material which was not sulfated and appeared to be the core protein of papilin, which is proteoglycan-like . After reduction, papilin electrophoresed in sodium dodecyl sulfate-polyacrylamide gel electrophoresis as a broad band of about 900,000 apparent molecular weight and the core protein as a narrow band of approximately 400,000 . The core protein was formed by some cell lines and by other cells on incubation with 1 mM 4-methylumbelliferyl xyloside, which inhibited formation of the proteoglycan-like form . The buoyant density of papilin in CsCl/4 M guanidine hydrochloride is 1.4 g/ml, that of the core protein is much less . Papilin forms oligomers linked by disulfide bridges, as shown by sodium dodecyl sulfate-agarose gel electrophoresis and electron microscopy . The protomer is a 225 +/- 15-nm thread which is disulfide-linked into a loop with fine, protruding thread ends . Oligomers form clover-leaf-like structures . The protein contains 22% combined serine and threonine residues and 25% combined aspartic and glutamic residues . 10 g of polypeptide has attached 6.4 g of glucosamine, 3.1 g of galactosamine, 6.1 g of uronic acid, and 2.7 g of neutral sugars . There are about 80 O-linked carbohydrate chains/core protein molecule . Sulfate is attached to these chains . The O-linkage is through an unidentified neutral sugar . Papilin is largely resistant to common glycosidases and several proteases . The degree of sulfation varies with the sulfate concentration of the incubation medium . This proteoglycan-like glycoprotein differs substantially from corresponding proteoglycans found in vertebrate basement membranes, in contrast to Drosophila basement membrane laminin and collagen IV which have been conserved evolutionarily.

J Immunol Methods, 1987 Dec 24, 105(2), 183 - 92
Studies of serum-free culture medium in the generation of lymphokine activated killer cells; Muul LM et al.; Lymphokine activated killer (LAK) cells administered in conjunction with recombinant interleukin-2 can mediate the regression of metastatic tumor in some patients with advanced cancer . In these trials LAK cells were activated in medium containing 2% human type A or AB serum . We have found three commercially available, serum-free culture media which allow development of in vitro LAK activity by human peripheral blood lymphocytes . They are AIMV (Gibco), MASF-3 (Whitaker-MA Bioproducts) and HB-104 (Dupont) . If 2-mercaptoethanol was added to these culture media they were also capable of generating murine LAK cells which were effective in reducing pulmonary metastases in the murine MCA-106 model . Although LAK cells generated in these media have not been tested in humans yet, potentially they could provide a safe, unlimited and less expensive source of culture fluid for generating the large numbers of LAK cells needed for human clinical trials.

Proc R Soc Lond B Biol Sci, 1987 Dec 22, 232(1268), 273 - 87
Effects of 2,3-iminosqualene on cultured cells; Popjak G et al.; 2,3-Iminosqualene (ISq) is a powerful inhibitor of squalene oxide:lanosterol cyclase (EC 5.4.99.7) . When added to lipid-depleted culture media (LDM) of rat hepatoma (H-4-II-E-C3) or Chinese hamster ovary (CHO) cells at a concentration of 10 micrograms ml-1, it causes the cells to float off the substratum in a few days . Lipoproteins in the culture medium completely counteract this effect . Cells in lipoprotein-containing media (FGM) grow normally in the presence of ISq . Irrespective of the culture medium, ISq at 10 micrograms ml-1 causes an almost complete and apparently irreversible inactivation of the squalene oxide cyclase in CHO and H4 cells and the accumulation in the cells of squalene, of squalene 2,3-oxide (mostly), and of squalene 2,3-22,23-dioxide when {14C}acetate or {14C}mevalonate is fed to the cells . Chronic treatment of H4 cells with ISq failed to elicit induction of the cyclase, but increased the conversion of mevalonate into squalene and squalene dioxide, and depressed the conversion of squalene oxide to the dioxide . Cells loaded with squalene and the squalene oxides from mevalonate in the presence of ISq get rid of these substances by rapidly secreting them into the media and by some unidentified metabolic processes.

Biochem Biophys Res Commun, 1987 Dec 16, 149(2), 642 - 8
Secretion of a growth inhibitory factor by ZR-75-1 human breast cancer cells; Blumenthal R et al.; Cultures of the human mammary carcinoma line ZR-75-1 secrete a growth inhibitory factor (GIF) that, when diluted, slows the growth of MDA-MB-231 and MCF-7 cells . Undiluted "conditioned" media prevents cell division from occurring in both human breast cancer lines . ZR-75-1 cells are unaffected by this factor . The amount of GIF in the culture media is related to the confluency of the ZR-75-1 cells . The activity of this GIF is not altered by DNAse or RNAse but is destroyed by heating or trypsin . Growth inhibition is 85-90% reversible if conditioned media is replaced with fresh media.

J Biol Chem, 1987 Dec 15, 262(35), 17206 - 11
Biosynthesis, glycosylation, and intracellular transport of intestinal lactase-phlorizin hydrolase in rat; Buller HA et al.; The biosynthesis of rat intestinal lactase-phlorizin hydrolase was studied by pulse-labeling of jejunal explants from 5-day-old suckling rats in organ culture . Explants were either continuously labeled with {35S} methionine for 15, 30, and 60 min or pulse-labeled for 30 min and chased for various periods of time up to 6 h in the presence or absence of protease inhibitors (PI), leupeptin, phenylmethylsulfonyl fluoride, and soybean trypsin inhibitor . Lactase-phlorizin hydrolase was immunoprecipitated from microvillus membrane (MVM) and ER-Golgi fractions with monoclonal antibodies . After pulse-labeling, lactase-phlorizin hydrolase from the ER-Golgi fraction appeared on SDS-PAGE as one band of approximately 220 kDa, regardless of the presence or absence of PI in the culture media . The 220-kDa protein band could also be labeled after incubation with {2-3H}mannose . In the absence of PI, the 220-kDa band appeared in the MVM by 30 min chase, simultaneously with a 180-kDa band, and by 60 min of chase an additional band of 130 kDa was seen . With increasing time of chase, the relative intensity of the 130-kDa band increased, whereas that of the 220-kDa band decreased, suggesting a precursor-product relationship . When PI were added to the medium, the formation of the 180-kDa band was not affected, but the conversion of the 180-kDa protein to the 130-kDa protein was virtually blocked . These findings suggest that lactase-phlorizin hydrolase is initially synthesized as a glycosylated precursor of 220 kDa, which is transported to the MVM . There it undergoes the following two cleavages: first, to the 180-kDa form, which is not prevented by PI used in these experiments, and second, to the 130-kDa form inhibited by PI.

J Surg Res, 1987 Dec, 43(6), 513 - 20
Modulation of fibroblast proliferation by postsurgical macrophages; Fukasawa M et al.; Macrophages and fibroblasts are major components of postsurgical peritoneal repair . In order to understand the interaction between these two cell types, we studied the effects of spent macrophage culture media on fibroblast proliferation . Rabbits underwent resection and reanastomosis of their small intestine . Peritoneal exudative cells (PEC) were then collected from these animals on postoperative Days 4, 7, and 28 and from nonsurgical controls . PEC (5 X 10(5) cells/ml) were cultured in M-199 with 3% fetal calf serum . After 48 hr the spent media from the cultured PEC were harvested, centrifuged (200g for 10 min), and stored (medium: M-D0, D4, D7, D28) . A second group of rabbits underwent peritoneal wall abrasion followed by collection of fibroblasts directly from the site of injury on postoperative Days 1, 4, and 7 . After Day 7 of culture, fibroblasts were resuspended and seeded into dishes (1 X 10(5) cells in 1 ml medium), to which was added 1 ml of spent PEC culture medium . After 24 hr of incubation, 1 muCi {3H}thymidine was added for an additional 18 hr . Fibroblasts were then collected and the amount of {3H}thymidine incorporated into trichloroacetic acid-precipitable material was quantitated . In one protocol, fresh M-199 with 3% fetal calf serum was used, and in another protocol, "U-medium" (which was previously incubated for 48 hr with fibroblasts) was used . The cytology of the PEC was determined by Wright's staining, nonspecific esterase activity, and phagocytosis . At least 80% of the peritoneal exudative cells were identified as macrophages . Postsurgical Day 7 fibroblasts demonstrated greater {3H}thymidine incorporation compared to fibroblasts from postoperative Days 1 and 4.(ABSTRACT TRUNCATED AT 250 WORDS)

J Lab Clin Med, 1987 Dec, 110(6), 719 - 25
Removal of transferrin from fetal bovine serum; Huebers E et al.; An antiserum against purified fetal bovine serum (FBS) transferrin was produced in rabbits . The isolation of anti-bovine transferrin IgG fraction was achieved by ammonium sulfate precipitation of rabbit hyperimmune plasma followed by ion exchange chromatography on diethylaminoethanol (DEAE) cellulose, at both an acidic and basic pH . The various antibody fractions were analyzed by fast protein liquid chromatography (FPLC) on a high-resolution mono-Q column . The specificity and efficiency of the antibody fractions obtained were tested by titration of a constant amount of fetal bovine serum with increasing amounts of antibody . The completeness of removal of the fetal bovine serum transferrin resulting from the formation of the highly stable antibody antigen complex was monitored by immunologic and radioisotope methods . The removal of FBS transferrin by this antibody precipitation technique did not interfere with the ability of added iron 59-tagged human transferrin to deliver iron to rat reticulocytes, as shown in an in vivo incubation model . The method proved to be effective for the fast and complete removal of bovine transferrin from fetal bovine serum in vitro and will be a prerequisite for more detailed studies of the interaction of transferrin of different species with tissue receptors on cell lines in culture without resorting to completely defined culture media.

Diabetes, 1987 Dec, 36(12), 1372 - 8
Development of rat embryos cultured in glucose-deficient media; Ellington SK; Embryo-culture techniques were used to study the effects of exposure to glucose-deficient culture media on rat embryos during organogenesis . Embryos and their extraembryonic membranes (explants) were cultured either in control media or in media with low or very low glucose concentrations . At the start of the culture the glucose concentrations were 8.4 +/- 0.57, 4.5 +/- 0.10, and 3.5 +/- 0.07 mM . During the cultures, the explants progressively depleted the glucose in the medium until, after 48 h, the glucose concentrations were reduced to 1.1 +/- 0.18, 0.4 +/- 0.13, and 0.3 +/- 0.02 mM, respectively . At glucose concentrations less than 2.5 mM, the rate of glucose uptake by the explants progressively decreased, and embryonic growth and differentiation became increasingly retarded . After 48 h in glucose-deficient culture media, embryos had smaller crown-rump lengths, lower protein contents, and fewer somites than embryos from the control media (P less than .001) . Most of the embryos exposed to the lower glucose concentrations also had severe dysmorphic lesions, especially of the head and branchial arches . The deleterious effects of exposure to low concentrations of glucose on embryonic development appeared not to be reversible . Embryos cultured for 24 h in media with a low concentration of glucose followed by 24 h in control medium grew and developed just as poorly as those cultured for the entire 48 h in the glucose-deficient media.

J Anim Sci, 1987 Dec, 65(6), 1775 - 81
Comparison of Ham's F10 with CO2 or Hepes buffer for storage of equine embryos at 5 C for 24 H; Carnevale EM et al.; Forty equine embryos collected 7 d post-ovulation were stored at 5 C for 24 h in one of two culture media (n = 20/group): 1) Ham's F10 + 10% heat-treated fetal calf serum (FCS) buffered by gassing with 5% CO2, 5% O2 and 90% N2 and 2) Ham's F10 + 10% FCS with Hepes buffer (25 mM) . Embryos cultured in Ham's F10 + CO2 maintained a better quality score and had a larger average increase in diameter (+34.8 micron) than embryos stored in Hepes buffered Ham's F10 (-10.2 micron) . Embryos were transferred surgically into recipient mares that ovulated -3 to +1 d in relation to the donor mare . Twenty embryos cultured in Dulbecco's phosphate buffered saline + 10% FCS and transferred less than 1 h after collection were used as controls . Pregnancy rates were higher (P less than .05) for embryos stored in Ham's F10 + CO2 (70%, 55%) than for embryos stored in Ham's F10 + Hepes (20%, 15%) at 14 and 35 d, respectively . At 14 d, pregnancy rates for control embryos (90%) were similar (P greater than .05) to pregnancy rates for embryos cultured in Ham's F10 + CO2 (70%); however, by 35 d, pregnancy rates were higher (P less than .05) for controls (80%) than for embryos stored in Ham's F10 + CO2 (55%) . It was concluded that Ham's F10 + CO2 was superior to Ham's F10 + Hepes for short-term storage of equine embryos at 5 C, and that satisfactory pregnancy rates could be obtained from transfer of embryos stored in Ham's F10 + CO2 at 5 C for 24 h.

Gastroenterol Jpn, 1987 Dec, 22(6), 709 - 15
Molecular forms of IgA produced by various lymphoid tissues--analysis using high speed liquid chromatography; Arashi M et al.; Molecular forms of immunoglobulin A (IgA) produced by cultured cells from various human lymphoid tissues were analyzed using high speed liquid chromatography (HLC) . IgA secreted into culture media was easily separated into polymeric and monomeric forms by HLC . HLC has the advantages of high resolution, reproducibility, rapidity and technical simplicity in the separation of polymeric and monomeric IgA . Peripheral blood lymphocytes and cells from gut-associated lymphoid tissues, such as mesenteric lymph nodes or large bowel mucosa, secreted predominantly polymeric IgA, whereas lymphoid cells from bone marrow produced mainly monomeric IgA . Spleen cells and tonsillar cells produced nearly equal proportions of polymeric and monomeric IgA . These results suggest that with regard to IgA in serum, the polymer may originate from the gut-associated lymphoid tissues and the monomer may mostly derive from the bone marrow.

Biol Reprod, 1987 Dec, 37(5), 1307 - 16
The effect of ovine trophoblast protein-one on endometrial protein secretion and cyclic nucleotides; Vallet JL et al.; The effect of ovine trophoblast protein-one (oTP-1) on endometrial protein secretion was examined by using a dual radioisotope technique in which 3H- and 35S-methionine were employed to measure relative rates of protein release into the medium by endometrial explant cultures (Exp . I) . Endometrium (200 mg) from Day (D) 12 of the cycle was cultured with either 5 micrograms/ml oTP-1, 5 micrograms/ml bovine serum albumin (BSA) or 1 mM dibutyryl cyclic adenosine 3',5'-monophosphate (DbcAMP) . Culture media from control BSA and treated explant cultures were mixed . Proteins were separated by two-dimensional polyacrylamide gel electrophoresis (2D-PAGE) and detected by fluorography . Individual protein spots were punched from gels, extracted, and their radioactive content measured . Ratios of 3H:35S were used to determine treatment effects . In Experiment II, 3H- and 14C-leucine were used for the dual radiolabel, and the DbcAMP treatment was omitted . In both experiments, a protein having a molecular weight (Mr) of about 70,000 and a pI approximately equal to 4 was increased (p less than 0.01) 200-400% by oTP-1 . Secretion of several other endometrial proteins was also amplified in the presence of oTP-1 . The polypeptides that increased in response to oTP-1 were inhibited by DbcAMP, and vica versa . In Experiment III, endometrial explants from D12 cyclic ewes were cultured for 4 h with either 5 micrograms/ml oTP-1 or 5 micrograms/ml BSA to determine whether oTP-1 influenced concentrations of 3',5'-cyclic adenosine monophosphate (cAMP) and 3',5'-cyclic guanosine monophosphate (cGMP) . Concentrations of cAMP in oTP-1-treated endometrium were lower (p less than 0.1) than in BSA-treated endometrium (0.29 vs . 0.41 pmoles/mg tissue, respectively) . Levels of cGMP were unaffected by oTP-1 . In Experiment IV, endometrium from D14 of the cycle was incubated in medium alone or in medium containing either 2 micrograms/ml oTP-1, 1 microgram/ml oxytocin (OXY), or oTP-1-plus-OXY . None of the treatments significantly affected cAMP levels . In Experiment V, D16 endometrium was collected from pregnant and nonpregnant ewes that had received either 0 or 10 IU OXY i.v . cAMP was higher (p less than 0.01) in endometrium from pregnant ewes compared to nonpregnant ewes (27.9 vs . 13.0 pmoles/mg tissue, respectively), but OXY had no detectable effect on endometrial content of cAMP in either nonpregnant or pregnant ewes.(ABSTRACT TRUNCATED AT 400 WORDS)

Rev Fr Gynecol Obstet, 1987 Dec, 82(12), 745 - 9
{Fertilization in vitro . Perspectives for improving the technics}; Menezo Y; Techniques of embryo cultures at an early stage, in spite of all the progress in that field, still present a major problem from the viability standpoint . The egg interrupts its segmentation, or, if it develops with an apparently normal chronology, the viability after transfer in vivo is very poor . Since a few years, trials in co-culture have been attempted . Culture on fibroblastic sheets gave disappointing results . As far as we are concerned, we have followed two different approaches: culture within the oviduct explanted in vitro and co-culture in the presence of embryonic tissue . The two types of co-culture give promising results, but it seems that the future lies in cultures on tubal epithelial cells . It will be necessary to obtain cellular lineage of these epithelia and to define culture media corresponding to the needs of the embryo as well as of these cells, since the embryo is an autonomous system, extremely sensitive to media alterations.

Atherosclerosis, 1987 Dec, 68(3), 255 - 61
Cholestan-3 beta,5 alpha,6 beta-triol decreases barrier function of cultured endothelial cell monolayers; Hennig B et al.; Cholesterol oxidation products (oxysterols) found in foods may be atherogenic, possibly by altering the barrier function of the vascular endothelium . To investigate this hypothesis, endothelial cells were cultured on micropore filters and the effect of cholesterol and the oxysterol cholestan-3 beta,5 alpha,6 beta-triol (Triol) on albumin transfer across cultured vascular endothelial monolayers (ECM) was studied . Exposure to Triol significantly increased albumin transfer across ECM . The effect of Triol on endothelial cell barrier function was time and concentration dependent, with maximum albumin transfer being reached at 20 microM Triol and after a 24-h exposure . Pure cholesterol, on the other hand, did not affect albumin transfer at concentrations as high as 130 microM . Although an increase in albumin transfer across ECM was observed after a 2-h incubation with Triol-enriched media, a 24-h incubation period was necessary to cause a significant release of cellular lactate dehydrogenase (LDH) into the culture media . Morphological perturbations of the cell monolayers were observed at approx . 14-18 h after cell exposure to Triol-enriched media . Enrichment with cholesterol or vitamin E did not prevent the Triol-induced increase in albumin transfer across ECM . These results suggest that exposure to oxidized cholesterol, but not cholesterol, itself, reduces the ability of the endothelium to act as a selectively permeable barrier to plasma components, and that these events may not be prevented by cholesterol or vitamin E.

Teratology, 1987 Dec, 36(3), 371 - 7
The in vitro embryotoxicity of 5-fluorouracil in rat embryos; Grafton TF et al.; The fluorinated pyrimidine 5-fluorouracil (5-FU) is an effective chemotherapeutic agent that is teratogenic in a number of species . The mechanism for the embryopathic effect of the drug is unknown . We examined the effects of this compound on gestation day 10.5 rat embryos cultured for 48 hours in a rodent whole embryo culture system . Embryos were exposed for 1-4 hours to various doses of 5-FU . Embryolethality was minimal in all treatment groups . The malformation frequency increased with higher doses; within a dose, the malformation frequency increased with longer exposure to the drug . The tail and hindlimb bud were the most commonly affected structures in vitro; tail and leg defects are produced in several species by exposure to the drug in vivo . The embryopathic drug concentration in the culture media (2-8 micrograms/ml) is similar to the plasma level of 2-17 micrograms/ml, which is associated with embryopathy in vivo . Results from this study suggest that the whole embryo culture system is an appropriate model for developmental toxicity studies of 5-FU.

Diabetes, 1987 Dec, 36(12), 1401 - 7
Tissue culture of human fetal pancreas . Effects of human serum on development and endocrine function of isletlike cell clusters; Sandler S et al.; The human fetal pancreas represents a source of insulin-producing beta-cells with a potential for transplantation to diabetic patients . It has previously been shown that such cells can be viably maintained in tissue culture media containing fetal calf serum (FCS) and that these explants continue to synthesize and release insulin . In this study the effects of human serum (HS) on the growth and function of human fetal pancreatic explants have been compared with those of FCS . For this purpose, pancreatic glands, obtained after prostaglandin-induced abortions, were briefly exposed to collagenase, and the digest was cultured in RPMI-1640 medium plus 10% pooled HS or FCS . The outgrowth of isletlike cell clusters (ICCs) was monitored . In 31 of 58 consecutively explanted glands, development of ICCs was observed . In the presence of FCS the outgrowth of ICC took place on top of a fibroblast monocellular cell layer; HS effected less growth of fibroblasts and increased the formation of ICCs about sevenfold compared with explants from the same glands maintained in FCS . However, in the explant cultures with HS, the cell number per ICC, expressed as DNA content, was reduced by 50% . In both FCS and HS the insulin content of the medium showed great variability and progressively declined from day 2 to day 5 . The medium glucagon concentration also decreased but not to the same extent as that of insulin . Immunocytochemical-stained ICCs showed insulin- and glucagon-positive cells scattered among most nonstained, presumably nonendocrine cells.(ABSTRACT TRUNCATED AT 250 WORDS)

Mol Biochem Parasitol, 1987 Dec, 26(3), 257 - 65
Secretory acetylcholinesterases from Brugia malayi adult and microfilarial parasites; Rathaur S et al.; Brugia malayi, a lymphatic filarial parasite, secretes acetylcholinesterase during in vitro cultivation . A significant amount of enzyme activity was detected both in culture media and somatic extracts of adult and microfilarial stages of the parasite . The microfilarial stage produces three times more enzyme than adult parasites as a proportion of total protein . The enzyme has true acetylcholinesterase (AChE) activity as hydrolysis of acetylthiocholine is three times faster than butyrylthiocholine and is inhibited by eserine, a specific inhibitor of AChE . Secretory enzyme from adult female parasite excretory-secretory material (ES) was enriched 23 fold using copper chelating and concanavalin A (Con A) affinity chromatography . The Con A eluate showed a major protein band of 100 kDa and a minor 200 kDa component . The ES enzyme is antigenic and cross reacts with antibodies raised in mice against AChE from electric eel by enzyme-linked immunosorbent assay and immunoprecipitation . Immunoprecipitation of 125I-labelled microfilarial ES and adult ES with anti-electric eel AChE antibodies revealed three proteins of 30, 40 and 200 kDa in microfilariae and two proteins of 100 and 200 kDa in adult female ES . It appears that filarial secretory AChE exists in multiple molecular forms.

Mol Cell Endocrinol, 1987 Dec, 54(2-3), 185 - 95
Regulation of inhibin production by rat granulosa cells; Suzuki T et al.; Inhibin production by cultured granulosa cells from immature diethylstilbestrol (DES)-primed rats was studied in relation to estradiol and progesterone production . The inhibin content in culture media was assayed with a specific radioimmunoassay (RIA) using an antibody to porcine 32 kDa inhibin that recognizes rat inhibin as well . Inhibin production was about 10 ng/ml/2 X 10(4) cells/72 h at the basal levels and was maximally stimulated with 25 ng/ml of follicle stimulating hormone (FSH) to 45 ng/ml which was 4.5 times the basal levels, with an ED50 value of 2.0 ng/ml . A cyclic AMP analog (dibutyryl cyclic AMP) or reagents that promote cAMP production were also effective in inhibin production, indicating that FSH stimulates inhibin production through a cAMP-dependent pathway . Luteinizing hormone (LH) was not effective in producing inhibin from freshly prepared granulosa cells, whereas granulosa cells pre-incubated with FSH for 48 h because responsive to LH regarding inhibin production . Testosterone sensitized the granulosa cells to the FSH stimulation, whereas hydrocortisone (4 ng/ml) decreased the sensitivity of granulosa cells by increasing the ED50 value for inhibin production by FSH about 10 times . A similar effect was observed regarding estradiol production, while progesterone production due to stimulation by FSH was enhanced by the hydrocortisone treatment . Insulin and platelet extract both stimulated inhibin production and enhanced the maximal response of inhibin production due to stimulation by FSH without altering, or even increasing the ED50 values . Epidermal growth factor (EGF), (D-Leu6)Des-Gly10-LHRH N-ethylamide (GnRH agonist) and 12-O-tetradecanoylphorbol-13-acetate (TPA), a potent protein kinase C activator, inhibited both inhibin production and estradiol or progesterone production . Consequently, the regulation of inhibin production was similar to that of estradiol production, but markedly different from that of progesterone . However, inhibin and estradiol production were modulated differently by various growth factors and hormones . These phenomena might account for possible discrete changes in the plasma levels of inhibin and estradiol in vivo.

Mol Cell Endocrinol, 1987 Dec, 54(2-3), 179 - 84
Partial characterization of somatomedin C-like immunoreactivity secreted by breast cancer cells in vitro; Minuto F et al.; The in vitro secretion of immunoreactive somatomedin C/insulin-like growth factor I (IR Sm-C/IGF-I) by two human breast cancer cell lines, MCF-7 and EVSA-T has been studied . IR Sm-C/IGF-I concentration showed a linear increase in serum-free culture media over 72 h of incubation for both cell lines, and a close correlation with cell number (P less than 0.001) . To characterize this immunoreactivity, a pool of conditioned media collected after 72 h of incubation was dialyzed overnight against 1 M acetic acid, lyophilized, and gel filtered on a Sephadex G-50 column . Fractions were determined for Sm-C/IGF-I content and for the presence of a specific carrier for Sm-C/IGF-I . Two peaks of Sm-C/IGF-I-like immunoreactivity were evidenced, the first in the high molecular weight region and the second corresponding to the molecular weight of the free peptide . The first peak evidenced also a specific binding ability for radioiodinated Sm-C/IGF-I, suggesting that the activity found in this region could be interpreted as interference of the specific free binding sites in the immunoassay . Analysis of this peak by polyacrylamide gel electrophoresis demonstrated the presence of a specific binding for Sm-C/IGF-I in a molecular weight range between 35,000 and 45,000 Da, which was not modified in reducing conditions . The binding activity was competitively inhibited by addition of cold Sm-C/IGF-I but not by insulin excess.(ABSTRACT TRUNCATED AT 250 WORDS)

Scand J Immunol, 1987 Dec, 26(6), 611 - 9
Removal of endotoxin from culture media by a polymyxin B sepharose column . The activity of contaminating endotoxin in culture media measured by the interleukin 1 inducing effect on human monocyte cultures and by the Limulus test; Molvig J et al.; The in vitro study of monocytes (Mo) poses several problems . Minor contamination with endotoxin (ET) of media and utensils as well as adherence to glass or plastic surfaces may activate the cells and cause pronounced production of monokines . Many commercially liquid culture media were found to contain ET in concentrations above 25 X 10(-12) g/ml . A simple system for the removal of ET from media and solutions was established by use of a commercially available Polymyxin B Sepharose gel . To measure the lipopolysaccharide (LPS) binding capacity of the gel, known concentrations of LPS were added to culture media, which were passed through a column consisting of the Polymyxin B Sepharose gel . The content of ET and added LPS in media was measured by the Limulus amoebocyte lysate (LAL) test before and after passage of the column . The LPS-binding capacity of the gel was approximately 2.4 X 10(-6) g/10 ml . The biological activity of contaminating ET and added LPS in media, before and after passage of the column, was also characterized by the capacity of the media to induce interleukin 1 (IL-1) secretion in human Mo cultures . The content of IL-1 in Mo culture supernatants was determined by the mouse thymocyte costimulatory (LAF) assay . By comparison of the activity of ET in these different biological systems, it was demonstrated that 15-20 X 10(-12) g/ml of ET stimulate human Mo cultures to IL-1 secretion.

Biol Reprod, 1987 Nov, 37(4), 979 - 88
Synthesis and release of polypeptides by the baboon (Papio anubis) uterine endometrium in culture; Fazleabas AT et al.; This study was designed to identify proteins released in culture by the baboon uterine endometrium . Endometrial tissues from cyclic baboons were minced and cultured in the presence of L-{3H}leucine or L-{35S}methionine for 24 h . The culture media and solubilized tissues were analyzed by one- and two-dimensional gel electrophoresis for secretory products that were uterine-specific . The fluorographs of the one- and two-dimensional gels demonstrated that the proteins released into culture media could be divided into two groups . Group I proteins were present throughout the menstrual cycle and showed minor cyclic variations in intensity, and Group II proteins were those that appeared to be hormonally modulated . Group I was comprised of several high molecular weight proteins (Mr greater than 200,000) and at least five additional proteins ranging in molecular weight from 80,000 to 37,000, with isoelectric points (pIs) of 5.1 to 6.0 . Group II consisted of a protein (Mr 33,000; pI 7.6) that was observed only during the follicular stages of the cycle and two other groups of proteins (Mr 130,000 and 88,000) that were present during the luteal stage . Western blots of tissue culture media incubated with antibodies against human placental proteins (PP) and prolactin demonstrated that PP4 and PP7 were secreted throughout the cycle while PP12, PP16, and prolactin were only present during the luteal stage of the cycle . Thus, it appears that the baboon uterine endometrium, like that of the human, secretes a wide array of proteins in culture . Our results also suggest that a few of these proteins are immunologically similar . Endometrial differentiation during the menstrual cycle altered the secretion of some proteins, whereas the synthesis of others appeared to be dependent on either estrogen or progesterone and were stage-specific.

J Cell Physiol, 1987 Nov, 133(2), 330 - 6
Regulation of glutathione levels in mouse spleen lymphocytes by transport of cysteine; Ishii T et al.; Cysteine and cystine transport activities of resting and activated mouse spleen lymphocytes were characterized in order to examine the contributions of cysteine and cystine to intracellular glutathione contents . Following stimulation with lipopolysaccharide, the lymphocytes markedly increased their capacity to transport cysteine . The uptake of cysteine was mediated mainly by the ASC system (Na+-dependent neutral amino acid transport system especially reactive with alanine, serine, and cysteine) . On the other hand, both the resting and the activated lymphocytes had extremely low cystine transport activities . Because of the instability of cysteine, the culture media usually contained cystine but not cysteine . Therefore, both the resting and the activated lymphocytes rapidly decreased their glutathione contents owing to their poor capacities to take up cystine . The effects of freshly added cysteine on the cellular glutathione contents were examined in the presence of bathocuproinedisulfonate, a nontoxic copper-specific chelator that inhibits autoxidation of cysteine . Cysteine added at 25-400 microM only partially prevented the rapid decrease of the glutathione contents in fresh resting lymphocytes . In the lipopolysaccharide-activated cells, however, cysteine enhanced the cellular glutathione contents in a dose-dependent manner . These results indicate that the enhanced activity of the ASC system increases the level of intracellular glutathione in the presence of cysteine.

Peptides, 1987 Nov-Dec, 8(6), 977 - 82
Comparative studies of hamster calcitonin from pulmonary endocrine cells in vitro; Nylen ES et al.; The lung-associated peptide calcitonin (CT) has been localized by immunocytochemical means to discrete pulmonary endocrine (PE) cells . A long-term cell culture of CT-staining PE cells has been established . The molecular configuration of immunoreactive (iCT) from PE cell extracts was determined by gel chromatography, revealing predominantly large molecular weight forms of iCT . The size distribution characteristics of PE Cell iCT were similar to those of intact hamster lung . In contrast, hamster thyroid extracts contain predominantly 4000 dalton iCT (presumed monomer) and apparent iCT fragments . The culture media of the PE cells were found to contain mainly 4000 dalton iCT . We conclude that although the predominant forms of iCT found within cultured PE cells are distinct from those found within thyroidal C-cells, both iCT producing cells release mainly the monomer into the media . Malignant human bronchial carcinoid cells store predominantly monomeric iCT while secreting large molecular weight forms of iCT . Since the PE cell is the putative precursor cell to neuroendocrine malignancies, the disparity noted in the processing of CT may have significant pathobiological implications.

J Reprod Immunol, 1987 Nov, 12(3), 191 - 200
Human seminal plasma suppresses lymphocyte responses in vitro in serum-free medium; Szymaniec S et al.; The in vitro immunosuppressive properties of human seminal plasma have been re-investigated in serum-free medium in view of recent suggestions that the previously observed effects might be dependent on the presence of exogenous serum co-factors present in the culture media . The present studies reveal that low concentrations of seminal plasma can inhibit the ability of peripheral blood leukocytes to lyse K562 target cells in the absence of fetal calf or new-born calf serum . These inhibitory effects could be achieved by pre-incubating the effector cells in seminal plasma at 37 degrees C prior to use in the natural killer cell assay or by incorporating it into the assay system . Additional studies revealed that human seminal plasma could also inhibit the proliferative response of peripheral blood lymphocytes to phytohaemagglutinin in serum-free HB103 medium . These effects were most marked and consistent if the seminal plasma was present throughout the period of culture . Overall, these studies indicate that the previously reported suppressive effects of human seminal plasma in these systems cannot be entirely attributable to cytotoxic factors generated by exogenous serum components.

J Protozool, 1987 Nov, 34(4), 372 - 7
Studies on the development of metacyclic Trypanosoma brucei sspp . cultivated at 27 degrees C with insect cell lines; Kaminsky R et al.; When transformed procyclic trypanosomes of three stocks of Trypanosoma brucei brucei and one stock of T.b . rhodesiense were grown at 27 degrees C in 25-cm2 flasks containing Anopheles gambiae cells, some of them developed into forms infective for mice . Infectivity titrations on trypanosome suspensions revealed that up to 2.8 X 10(5) metacyclic forms per ml could be produced, and the cultures remained infective for varying periods of up to 72 days when they were terminated . Of the various culture media tested, a mixture of three volumes of trypanosome medium and one volume of Anopheles medium was the most successful . Control cultures of trypanosomes grown in medium without cells were generally not infective, but two of the stocks gave rise to a few sporadic infections . Trypanosome populations could be subpassaged in the Anopheles cell cultures without loss of infectivity . Metacyclic forms separated from infective cultures by DEAE-cellulose columns had a surface coat.

Mol Cell Endocrinol, 1987 Nov, 54(1), 43 - 50
Weak estrogenic activity of phenol red in the culture medium: its role in the study of the regulation of prolactin release in vitro; Hofland LJ et al.; Phenol red, which is commonly used in culture media as a pH indicator, has recently been shown to possess estrogenic properties . In this study we investigated the effects of phenol red on prolactin release and synthesis by cultured female and male rat anterior pituitary cells and on the sensitivity of these cells of dopamine, TRH and somatostatin (SRIF) . It was shown that phenol red stimulated rat prolactin release and cell content in a dose-dependent manner . The effects of 30 microM phenol red, which is the medium concentration in our regular culture medium, and a submaximally active concentration of 17 beta-estradiol (E2) were additive . Male rat pituitary cells were far more responsive to phenol red and also to E2 than female pituitary cells . The antiestrogen tamoxifen (100 nM) significantly inhibited the phenol red-stimulated prolactin release by male rat pituitary cells but caused a 2-fold increase of prolactin release in the absence of phenol red . 30 microM phenol red did not modulate the responsiveness of female and male rat lactotrophs to dopamine, TRH or SRIF . We propose from our results that the estrogenic effect of 30 microM phenol red is too weak in order to alter the responsiveness of rat lactotrophs to dopamine, TRH and SRIF but the presence of phenol red in culture media should be considered when the effects of estrogens and antiestrogens on rat prolactin release and synthesis in vitro are studied.

Cancer Res, 1987 Nov 1, 47(21), 5678 - 83
Tumorigenicity in the nude mouse of cocultures derived from two nontumorigenic cell types, human pituitary adenomas and mouse C3H 10T1/2 fibroblasts; U HS et al.; Human pituitary adenoma tissues were not tumorigenic in the hormonally manipulated nude mouse . Mouse fibroblast cells (C3H 10T1/2) also did not form tumors when inoculated alone into nude mice . When these two tissues were cocultured and coinoculated into nude mice however, the majority of inocula developed progressively enlarging tumors which could be established in tissue culture and passaged through the nude mouse . These tumors were sarcomatous histologically and thus did not resemble any human pituitary adenoma tissue injected . In order to detect any human cells in these tumors, tumor genomic DNA was subjected to Southern analysis using human repetitive Alu and HGH DNA sequence probes . Southern blot analysis of the nude mouse derived tumor genomic DNA revealed no sizeable human DNA in the mouse tumor cell genome indicating the absence of significant numbers of human cells in the tumors or the transfer of human DNA to the mouse cells . The tumors therefore arose from transformed C3H 10T1/2 cells after coculture with the human pituitary adenoma cells . These results implied that the tumorigenic transformation of susceptible C3H 10T1/2 cells in the cocultures occurred as a result of the secretion by the adenoma cells of transforming substances in the culture media or the induction of tumorigenicity through direct cell-cell contact between the two cell types.

J Exp Med, 1987 Nov 1, 166(5), 1484 - 98
Granulocyte/macrophage colony-stimulating factor is essential for the viability and function of cultured murine epidermal Langerhans cells; Witmer-Pack MD et al.; A panning method has been developed to enrich Langerhans cells (LC) from murine epidermis . In standard culture media, the enriched populations progressively lose viability over a 3-d interval . When the cultures are supplemented with keratinocyte-conditioned medium, LC viability is improved and the cells increase in size and number of dendritic processes . Accessory function, as monitored by stimulating activity in the mixed lymphocyte reaction (MLR), increases at least 10-20-fold . The conditioned media of stimulated macrophages and T cells also support the viability and maturation of cultured LC . A panel of purified cytokines has been tested, and only granulocyte/macrophage colony-stimulating factor (GM-CSF) substitutes for bulk-conditioned medium . The recombinant molecule exhibits half-maximal activity at 5 pM . Without activity are: IL-1-4; IFN-alpha/beta/gamma; cachectin/TNF; M- and G-CSF . A rabbit anti-GM-CSF specifically neutralizes the capacity of keratinocyte-conditioned medium to generate active LC . However, GM-CSF is not required for LC function during the MLR itself . We conclude that the development of immunologically active LC in culture is mediated by GM-CSF . The observation that these dendritic cells do not respond to lineage-specific G- and M-CSFs suggests that LC represent a distinct myeloid differentiation pathway . Because GM-CSF can be made by nonimmune cells and can mediate the production of active dendritic cells, this cytokine provides a T-independent mechanism for enhancing the sensitization phase of cell-mediated immunity.

Life Sci, 1987 Oct 19, 41(16), 1953 - 9
Analysis of atrial natriuretic factor biosynthesis and secretion in adult and neonatal rat atrial cardiocytes; Zisfein JB et al.; Atrial natriuretic factor (ANF) is stored in atrial cardiocytes as the 126 amino acid polypeptide, proANF, which is later cleaved to the 24-28 amino acid carboxyterminal peptides, the major circulating forms . Earlier studies have demonstrated that isolated, cultured neonatal rat cardiocytes both store and secrete proANF, which can be cleaved to the smaller circulating form(s) by a serum protease . Since differences may exist between neonatal and adult cardiocytes with respect to ANF synthesis and processing, we compared the forms of ANF stored and secreted by neonatal rat cardiocytes with those of adult cells . Using four to five day cultures of isolated atrial cardiocytes prepared from the hearts of neonatal and adult rats, pulse-chase studies were performed with 35S-cysteine and 35S-methionine . Analysis of ANF stored and secreted by these cells was performed by immunoprecipitation of cell extracts and culture media using antibodies directed to either the carboxyterminus or aminoterminus of proANF followed by SDS-PAGE and autoradiography . Cell extracts from both adult and neonatal cultures were found to contain only a 17-kDa polypeptide, previously identified as proANF . The predominant form found in the culture media was also the 17-kDa peptide, with smaller quantities of its 3-kDa carboxyterminal and 14-kDa aminoterminal cleavage products . We conclude from these studies that proANF is the major form stored and secreted by both adult and neonatal cardiocytes in culture; the activity of the protease that cleaves proANF to the smaller forms found in the circulation is either attenuated or is overwhelmed by high ANF-secretory rates in these cultures . Alternatively, the ANF processing and secretory pathways may be somehow altered in culture such that proANF escapes protease cleavage . Further studies will elucidate the nature and location of this protease.

Rev Argent Microbiol, 1987 Oct-Dec, 19(4), 161 - 4
{Comparison of the efficiency of 2 culture media in the recovery of heterotrophic bacteria damaged with chlorine}; Guerrero JJ; In this study, culture mediums R2A and m-HPC were compared with respect to their efficiency in the recuperation of injured heterotrophic bacteria present in water, which previously was treated with chlorine . The results of total counts obtained by membrane filtration, show that medium R2A was better than m-HPC . These two culture mediums are indicated by the 16th edition of Standard Methods for the Examination of Water and Waste-water . The results obtained may be due to the low concentration of organic matter, or to the presence of yeast extract in the R2A medium.

Anticancer Drug Des, 1987 Oct, 2(2), 117 - 28
A new approach to chemotherapy based on molecular biology and nucleic acid chemistry: Matagen (masking tape for gene expression); Miller PS et al.; The nucleotide sequences of genes contain information which can potentially be used to understand gene function and thus the biological properties of living organisms . This information can also be used to develop innovative new strategies for chemotherapy employing sequence-specific non-ionic oligonucleoside methylphosphonates . These oligonucleotide analogs, termed Matagen (an acronym for masking tape for gene expression), have the following properties: (1) the negatively charged phosphodiester linkage normally found in nucleic acids is replaced with a non-charged methylphosphonate group which confers increased lipophilicity to the oligomer; (2) the oligomers form stable hydrogen-bonded complexes with complementary nucleic acid sequences and retain the fidelity of Watson-Crick base pairing; (3) the lipophilic oligomers cross the cell membrane and also enter various organs of the body; and (4) the methylphosphonate backbone is inherently resistant to nuclease hydrolysis and thus oligomers are taken up intact from cell culture media and remain stable within the cellular environment . Two general strategies are used to block gene expression by Matagens at the mRNA level in mammalian cells . In the first approach, Matagens complementary to specific sites such as the initiation codon region are used to block translation of mRNA . Thus Matagens specifically inhibit translation of rabbit globin mRNA in cell-free systems and rabbit reticulocytes, and vesicular stomatitis virus protein synthesis, but not cellular protein synthesis, in virus-infected cells . In the second approach, Matagens complementary to splice junctions of precursor mRNAs are used to inhibit splicing . For example, a Matagen complementary to the donor splice junction of simian virus 40 (SV40) large T-antigen mRNA inhibits T-antigen synthesis in SV40-infected cells, and a Matagen complementary to the acceptor splice junction of herpes simplex virus (HSV) immediate early pre-mRNA 4 + 5 inhibits HSV replication in virus-infected cells . Two new types of Matagen, one derivatized with the photoactivatable cross-linking group psoralen and the other derivatized with a hydroxyl radical-producing group, EDTA-Fe(II), have been designed to improve the efficacy of Matagen and to overcome some of the problems inherent in physical binding of Matagens to complementary nucleic acids . The Matagen approach provides a new way to design antiviral and chemotherapeutic agents in a rational manner . It combines nucleic acid chemistry and chemotherapy to form a common basis for drug development as well as to provide fundamental knowledge about organisms and humans.

Res Rep Health Eff Inst, 1987 Oct, (13), 3 - 19
Effects of nitrogen dioxide on alveolar epithelial barrier properties; Crandall ED et al.; This study analyzed the effects of nitrogen dioxide (NO2) on alveolar epithelial permeability and transport properties . Primary cultured monolayers of rat Type II pneumocytes, cultured on both nonporous and porous surfaces, were used as models of isolated alveolar epithelium for in vitro exposure to nitrogen dioxide . The effects of nitrogen dioxide exposure for monolayers cultured on nonporous substrata were monitored by observing the changes in the net volume of fluid under the monolayer; for cells cultured on porous substrata, alterations in tissue bioelectric properties were noted . As a first step, primary cultured monolayers of rat Type II pneumocytes plated on nonporous plastic Petri dishes were used to investigate the effects of nitrogen dioxide on alveolar epithelial barrier properties . Such monolayers form fluid filled domes that are thought to result from active solute transport from medium to substratum, with water following passively . We used dome formation as a transport marker . Five-day-old cultures were directly exposed to 30 ppm NO2 in 5 percent CO2 in air at 25 degrees C, by cyclically tilting culture plates from side to side, so that both halves of the monolayer were exposed during each cycle . Exposures consisted of 10 cycles of four minutes each (two minutes per side), for a cell exposure time of 20 minutes . Control plates were simultaneously exposed to 5 percent CO2 in air under identical conditions . One day after the exposure, nitrogen dioxide-exposed monolayers exhibited significant decreases in dome density and individual dome volume, compared to the controls . By 48 hours post-exposure, differences between nitrogen dioxide-exposed and control monolayers were less, but remained significant . These results showed that short-term sublethal exposures to nitrogen dioxide produce a decrease in dome formation in Type II alveolar epithelial cell monolayers . This finding is most likely due to a decrease in the active transepithelial sodium transport rate, or an increase in the permeability of cell membranes or tight junctions, or both . Addition of vitamin E-containing liposomes to the culture media 24 hours pre-exposure did not affect the nitrogen dioxide-induced decrease in dome formation, indicating that under these circumstances no protective effect was provided by the antioxidant.(ABSTRACT TRUNCATED AT 400 WORDS)

Am J Physiol, 1987 Oct, 253(4 Pt 1), G502 - 7
Glycine-extended progastrin processing intermediates: accumulation and cosecretion with gastrin; Sugano K et al.; Glycine-extended intermediates of peptide processing serve as substrates for carboxyl-terminal amidation, hence activation, of many brain-gut peptides . To explore the dynamics of accumulation and secretion of these important intermediates we utilized primary cultures of canine antral mucosal G-cells as a model system . Glycine-extended progastrin processing intermediates (G-Gly) accumulated rapidly in G-cells cultured in ascorbate-deficient media, exhibiting a fourfold increase over a 51-h culture period, while gastrin content fell to less than half of the initial level . In contrast, G-cells cultured in ascorbate-supplemented media accumulated G-Gly at a relatively low rate, while gastrin was preserved at a higher level . Under either condition, G-Gly and gastrin were progressively released into the culture media . The release of both immunoreactivities could be stimulated by bombesin and inhibited by somatostatin in similar fashion . By electron microscopy, the cultured G-cells exhibited no ultrastructural alterations . These data suggest that 1) the cellular homeostasis of G-Gly is regulated by the activity of an ascorbate-dependent amidation enzyme similar to one previously described in pituitary tissues, 2) carboxyl-terminal amidation is not an obligatory step for secretion of gastrin, and 3) the proportions of gastrin and G-Gly cosecreted from G-cells reflect their proportional accumulation within G-cell secretory granules . The physiological relevance of the released G-Gly has yet to be determined.

Bone Miner, 1987 Oct, 3(1), 13 - 26
Levamisole and inorganic pyrophosphate inhibit beta-glycerophosphate induced mineralization of bone formed in vitro; Tenenbaum HC; The addition of the organic phosphate, beta-glycerophosphate, to the culture media of osteoid forming cells or tissues will induce formation of mineralized bone . The mechanisms behind this phenomenon are not clearly understood . In order to gain new understanding of organic phosphate induced mineralization of bone it was decided to attempt to inhibit this process in two fundamentally different ways . Firstly, the reversible inhibitor of alkaline phosphatase, Levamisole, was used to help define the role of alkaline phosphatase in mineralization in vitro . Secondly, inorganic pyrophosphate, a known inhibitor of hydroxyapatite (HA) formation was also used . It was hypothesized that inorganic pyrophosphate, in addition to its ability to block HA formation, might also interfere with organic phosphate access to alkaline phosphatase and thereby prevent mineralization . The data show that mineralization is blocked when alkaline phosphatase activity is inhibited by Levamisole prior to but not after osteoid maturation . Inorganic pyrophosphate blocks organic phosphate induced mineralization whether added before or after osteoid formation . Organic phosphate effects on alkaline phosphatase activity are reversed by the addition of inorganic pyrophosphate either before or after osteoid formation . These findings suggest a role for alkaline phosphatase in organic phosphate induced mineralization . The data show further that inorganic pyrophosphate may effect mineralization of bone not only by blocking apatite formation but possibly by modulating organic phosphate metabolism.

Biol Reprod, 1987 Oct, 37(3), 595 - 605
Development of a sensitive enzyme-linked immunosorbent assay for cattle, sheep, rat, and mouse luteinizing hormone; Spearow JL et al.; This manuscript reports the development of a rapid, sensitive, and specific-enzyme linked immunosorbent assay (ELISA) suitable for measuring luteinizing hormone (LH) in cattle, sheep, rats, and mice . The LH ELISA used #15 anti-LH serum-coated 96-well plates and peroxidase-labeled bovine luteinizing hormone (bLH) . Bovine LH labeled with peroxidase by the periodate method was stored at -15 degrees C for over 20 mo without appreciable loss of activity . With bLH-B5 used as the standard diluted in assay buffer, the LH ELISA had a sensitivity of 79.6 +/- 31 pg/ml, and a 50% displacement point of 359 +/- 69 pg/ml . With rat LH-RP-2 (rLH) used as the standard diluted in assay buffer, the LH ELISA had a sensitivity of 102 pg/ml and a 50% displacement point of 531 pg/ml . The LH ELISA was highly specific for LH from several mammalian species . This LH ELISA was seven times more sensitive for bLH than a radioimmunoassay (RIA) with comparable sample volumes . The LH ELISA was validated for measurement of LH in buffer, tissue culture media, and sera . Depending on the sensitivity desired, the LH ELISA can be conducted in 3 to 48 h, produces no hazardous waste, and can easily be automated . Use of this LH ELISA offers improvements in speed, convenience, economy, sensitivity, and safety over comparable RIA procedures . The LH ELISA was also conducted with other monoclonal and polyclonal antibodies to LH . The LH ELISA can be performed with other LH antisera, provided the antisera has a high affinity and specificity for LH and can be uniformly coated on 96-well plates.

J Bacteriol, 1987 Oct, 169(10), 4790 - 5
Characterization of Pi-repressible enzymes secreted in culture media by Neurospora crassa wild-type cells and null-type mutants; Furukawa K et al.; In wild-type mycelial cultures of Neurospora crassa under Pi-limited conditions, alkaline phosphatase, cyclic phosphodiesterases I, II, III, and IV, 5'-nucleotidase, acid and alkaline nucleases, RNase N1, and a newly detected endonuclease were secreted into the culture media . These enzymes were either not produced or were produced in very reduced levels in mutants nuc-1, -2, -3, -4, -5, -6, and -7 and cpd-4 . The proteins were examined by polyacrylamide gel electrophoresis in a manner which allowed the identification of each of them.

J Immunol Methods, 1987 Sep 24, 102(2), 173 - 82
A one-step purification method for monoclonal antibodies based on salt-promoted adsorption chromatography on a 'thiophilic' adsorbent; Belew M et al.; A convenient and fast method for the purification of mouse monoclonal antibodies from the culture media of cloned cells or from ascites fluids by means of salt-promoted chromatography on a 'thiophilic' adsorbent is described . The adsorbent has a capacity to adsorb about 20 mg/ml of immunoglobulins and a broad specificity towards immunoglobulins derived from various animal species irrespective of the type or subclass to which they belong . The recovery of the purified IgG is better than 90% while that for IgM is considerably less, probably due to dissociation occurring during the adsorption-desorption process . This one-step procedure also leads to a considerable concentration of dilute solutions of immunoglobulins . Moreover, the purified Igs are eluted by an essentially salt-free buffer at near neutral pH thus obviating the need for post-treatment of the sample before storage or subsequent conjugation to enzymes for use in immunoassays . This purification method is also well suited for large-scale operations since sample preparation requires only the addition of 0.5 M K2SO4 to the ascited fluid or cell culture medium . The degree of purification obtained is, in certain instances, comparable to that obtained by biospecific affinity chromatography based on antigen-antibody interactions . In contrast to immunosorption and desorption methods, however, there is no risk of contamination of the immunoglobulins purified on the 'thiophilic' adsorbent by foreign proteins.

Proc R Soc Lond B Biol Sci, 1987 Sep 22, 231(1265), 391 - 414
Regulation of 3-hydroxy-3-methylglutaryl coenzyme A reductase: search for the enzyme's repressor derived from mevalonate; Popjak G et al.; Three inhibitors of squalene 2,3-oxide-lanosterol cyclase (AMO 1618, 4,4,10 beta-trimethyl-trans-decal-3 beta-ol (TMD) and 2,3-iminosqualene (ISq} were used to study effects on 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase, and on sterol and polyprenyl synthesis from {14C}acetate and {14C}mevalonate in cultured rat hepatoma (H4) cells . After a 4 h exposure of cultures to AMO 1618 or TMD, followed by removal of the inhibitors, the utilization of {14C}acetate for synthesis of digitonin-precipitable sterols increased about twofold, an increase parallelled by the rise in HMG-CoA reductase . Mevalonate at 2.3 mM counteracted the effects of these inhibitors on the reductase . When (R)-{2-14C}mevalonate at 2.3 mM was included with the two inhibitors in the culture media, the cells were still able to synthesize cholesterol although in lesser amounts than the controls . In the presence of TMD the H4 cells also accumulated {14C}squalene 2,3-oxide and {14C}squalene 2,3-22,23-dioxide . ISq added to cells kept in full-growth medium (10 micrograms ml-1) caused an almost complete and irreversible inactivation of the squalene oxide-lanosterol cyclase but did not inhibit polyprenyl synthesis, as the amount of {14C}mevalonate converted into squalene, squalene 2,3-oxide, squalene 2,3-22,23-dioxide plus a little cholesterol was equal to the amount converted by control cells into cholesterol plus squalene . After a 24 h exposure of cells kept in full-growth medium to ISq (10 micrograms ml-1), the levels of HMG-CoA reductase rose about twofold . ISq completely abolished the suppressive effect of 2.3 mM (R)-mevalonate on the reductase . Chromatin isolated from cell nuclei contains cholesterol, which is renewed biosynthetically . It is argued that the suppressor of HMG-CoA reductase, derived from mevalonate, is a sterol and not a non-steroidal product of mevalonate metabolism.

J Immunol, 1987 Sep 15, 139(6), 1911 - 7
Interleukin 1 induces interleukin 1 . II . Recombinant human interleukin 1 induces interleukin 1 production by adult human vascular endothelial cells; Warner SJ et al.; Interleukin 1 (IL-1) alters several potentially pathogenic endothelial cell (EC) functions . The authors report here that recombinant human IL-1 (rIL-1) alpha (0.1 to 10 ng/ml) or IL-1-beta (1 to 100 ng/ml) induce concentration- and time-dependent increases in IL-1-beta mRNA levels in EC derived from adult human saphenous vein . rIL-1 induced IL-1-alpha mRNA only in EC treated concomitantly with cycloheximide (2 micrograms/ml) . IL-1-beta mRNA production began within 1 hr of exposure to rIL-1, peaked after 24 hr, and declined thereafter . Actinomycin D prevented the appearance of IL-1 mRNA in rIL-1-treated EC . rIL-1 also induced the release of biologically active IL-1 from EC, which was inhibited by cycloheximide (1 microgram/ml) . When compared on the basis of their activity in the thymocyte costimulation assay, rIL-1-alpha and rIL-1-beta were equipotent as inducers of IL-1 production by EC . EC stimulated with rIL-1 produced prostaglandin E2, which inhibits IL-1 production by other cell types and also decreases the responsiveness of thymocytes to IL-1 . When EC were exposed to rIL-1 in the presence of indomethacin (1 microgram/ml), which blocked prostaglandin E2 production, greater amounts of rIL-1-induced IL-1 release were detected, although the inhibitor did not affect IL-1-beta mRNA levels . IL-1-induced IL-1 production was unlikely to be caused by endotoxin contamination of tissue culture media or IL-1 preparations, because the lipopolysaccharide (LPS) antagonist polymyxin B (10 micrograms/ml) blocked LPS-induced IL-1 production by EC but did not affect IL-1 release in response to rIL-1-beta (100 ng/ml) . The IL-1-inducing property of rIL-1-beta was heat-labile, whereas heated LPS stimulated EC IL-1 production . The source of IL-1 in our cultures was not monocyte/macrophages, as treatment of EC with monoclonal antibody to the monocyte antigen Mo2 under conditions that lysed adherent peripheral blood monocytes did not affect production of IL-1 by EC in response to LPS (1 microgram/ml) or rIL-1-beta (100 ng/ml) . IL-1 elicits a coordinated program of altered endothelial function that increases adhesiveness for leukocytes and coagulability . IL-1-induced IL-1 gene expression in human adult EC could thus provide a positive feedback mechanism in the pathogenesis of vascular disease including atherosclerosis, vasculitis, and allograft rejection.

Exp Eye Res, 1987 Sep, 45(3), 443 - 51
Relationship of cholesterolgenesis to DNA synthesis and proliferation by lens epithelial cells in culture; el-Sayed GN et al.; Cholesterolgenesis could be important for both cell growth and DNA synthesis in many cell types . Since the ocular lens seems at least partially dependent upon biosynthesis to supply its required cholesterol, cholesterolgenesis could have a special role in control of cell proliferation and DNA synthesis in the lens . We thus examined the effects of inhibiting sterol synthesis with mevinolin upon cell proliferation, upon accumulation of DNA, sterol and protein mass and upon DNA synthesis by bovine lens epithelial cells cultured in lipoprotein-deficient media . All DNA synthesis in the ocular lens occurs in the monolayer of epithelial cells which covers the anterior surface of this organ . Concentrations of mevinolin which largely prevented synthesis and accumulation of sterol by the cultured lens epithelial cells and stopped proliferation of these cells had no effect on the cell's DNA synthesis or accumulation of DNA mass . The inhibition of proliferation by mevinolin could be completely reversed by cholesterol added to the culture media in the form of low density lipoprotein . These findings indicate that an adequate supply of cholesterol is required by lens epithelial cells to proceed through the cell cycle . Inhibition of cholesterolgenesis by the lens in vivo could have profound effects upon lens growth and development.

Cell Immunol, 1987 Sep, 108(2), 425 - 37
Cytostatic peptide isolation from culture media of mouse peritoneal exudate cells; Csuka I et al.; It was found that the supernatant of mouse PEC culture medium (MCM) (both resident and casein-elicited cells) has an inhibitory effect in vitro on the incorporation of {3H}TdR into DNA of mouse spleen cells . The inhibitory effect in the MCM appears in the first 24 hr and also reaches its maximum value within this time . The inhibitory effect of this factor could not be demonstrated in the extract of freshly harvested M phi cells . The factors responsible for inhibition proved to be heat stable at 80 degrees C for longer than 30 min . Following heat treatment, the crude extract was separated into four fractions absorbing uv light at 280 nm using Sephadex G-25 column chromatography, and the most potent biologically active inhibitory factor was eluted in the last fraction . This fraction could also be obtained with a more effective permeation volume using Trysacryl GF 05 gel chromatography, and the active B fraction from this chromatography could be separated into four subfractions by isotachophoresis (ITP) . The active fraction, which was obtained by Trysacryl GF 05 gel chromatography and further separated by ITP, was found to be highly inhibitory . It contained a peptide-like substance with a molecular mass of approximately 2.0 kDa and had an anionic character at pH 4.0 . The inhibitory effect of MCM cannot be influenced either by inhibitory compounds of protein synthesis or by proteolysis blocking agents . Furthermore, the inhibitory effect is shown to be reversible and is more pronounced on B cells than on T lymphocytes.

J Nucl Med, 1987 Sep, 28(9), 1435 - 40
Cellular sources of thymidine nucleotides: studies for PET; Shields AF et al.; The relative utilization of endogenously synthesized thymidine nucleotides and exogenously supplied thymidine analog was compared in a number of mammalian cell lines, tissues, and tumors . To measure the relative utilization, cells were incubated in tissue culture media containing the thymidine analog {3H}-5-bromo-2'-deoxyuridine (BUDR) . After extraction of the DNA, the degree of substitution of the thymidine by BUDR was determined by density gradient centrifugation . All the cell lines and tissues tested utilized both exogenous BUDR and endogenous thymidine sources to a similar extent . The relative utilization of the exogenous pathway could be manipulated by varying the exogenous concentration of BUDR . Our results demonstrate that one can predict the relative utilization of these two pathways and can calculate the effective specific activity of the intracellular thymidine nucleotide pool . Such information is needed in interpreting 11C-labeled thymidine uptake as measured by positron emission tomography.

Sci Sin {B}, 1987 Sep, 30(9), 941 - 5
In vitro pollen plant induction and embryoid clone establishment of Panax ginseng; Du LG et al.; In anther culture of Panax ginseng, its callus formation showed a wide adaptability to culture media . Large numbers of calli were induced on media exhibiting better effects of induction . Supplements of 5 mg 2,4-D/1 and 1 mg KT/1 to the media proved to be much effective . Regeneration of the whole plantlets from anther culture of Panax ginseng is usually quite difficult . During the past three years, however, sixteen of the 100 medium formulae tested were proved to be suitable . The formulae of MS + 0.5 mg BA/1 + 2 mg GA/1 + 1000 mg LH/1 + 3% sucrose were considered good and effective . A visibly differentiated body, which was light-milky white and later turned into a light green spot, was formed 40 days after the callus was transferred to the differentiation media . This body differentiated subsequently into buds, roots and, eventually, seedlings . The embryoid clones have been established in order to maintain its ability of continual differentiation into plantlets through successive culturings of many generations . The test-tube ginseng thus formed were transferred to regular flowerpots and grew well . Based upon chromosome examination of the callus cells and the root tips, we tentatively affirmed that the majority of these regenerated from anther of Panax ginseng were originated from pollen cells.

Andrologia, 1987 Sep-Oct, 19(5), 579 - 84
Sperm wash in three culture media: maximization of motile sperm recovery during swim-up incubation; Vijayakumar R et al.; Three different culture media commonly used during in vitro gamete manipulations were studied for their efficacy in sperm wash procedure . Highest numbers of motile sperm were recovered at 6 hours following incubation in WT-6 and Ham's F-10 media . However, WT-6 yielded higher motile sperm numbers than Ham's F-10 . Swim-up sperm number reached a peak at 3 hours following incubation in BWW . A period of 2 to 6 hours of incubation of sperm pellets overlayed with sperm wash media resulted in highly enriched motile sperm fractions free of dead spermatozoa and seminal debri.

J Anim Sci, 1987 Sep, 65(3), 841 - 60
Description and validation of an in situ autoperfusion method to determine nutrient absorption and metabolism in bovine small intestine; Murray RA et al.; Holstein bull calves, 8 to 12 wk of age, were anesthetized with halothane gas . An approximate 20-cm section of small intestine, 60 to 90 cm proximal to the ileocecal junction was clamped to isolate blood circulation to a single set of arcuate vessels and to form an intestinal segment fitted for infusion and drainage . The vein was catheterized to allow total venous collection . Donor blood was transfused via jugular vein to replace venous drainage . This technique was evaluated in four calves by exposing the lumen to eight replications (12 or 20 min incubation, 30-min wash with 39 C saline) of 16 mM L-Met (14C-labeled) . Time course appearance of Met in venous blood indicated similar rates and patterns of absorption for individual calves . There were no clinically significant alterations in jugular blood chemistry profiles across replications . Four calves were used to evaluate the effect of three isotonic perfusion media (saline, Krebs-Ringer bicarbonate and M-199 tissue culture media) on Lys and Met absorption . Venous flow rates and absorption of Lys were faster with Krebs buffer than with other media . Perfusate medium did not influence venous flow rates or absorption of Met . Effect of restricting venous flow on absorption of Lys and Met was evaluated in two calves . Flow was alternately controlled (6.5 ml/min) or allowed to flow freely (mean = 12.2 ml/min) . Restricting flow decreased steady-state absorption . Light and scanning microscopy indicated maintenance of mucosal tissue integrity throughout 8 h of anesthesia . Results demonstrate validity of the in situ technique to study nutrient absorption in the young bovine.

Endocrinology, 1987 Sep, 121(3), 1089 - 98
An improved in vitro bioassay for follicle-stimulating hormone (FSH): suitable for measurement of FSH in unextracted human serum; Padmanabhan V et al.; FSH bioactivity was measured by means of FSH-dependent aromatase activity (conversion of androgen substrate to estradiol) . Assay sensitivity was optimized by the use of immature (7-10 days old) rats as Sertoli cell donors, serum-free medium for incubation, phosphodiesterase inhibitor (methylisobutylxanthinine), serial dilution of FSH in medium containing 1% BSA, delayed addition of FSH for 72 h after cell plating, and 19-hydroxyandrostenedione (2.5 X 10(-6) M) as the aromatizable androgen substrate . The method consisted of subjecting the decapsulated immature rat testes to a 2-step collagenase dispersion, plating the cells in medium {Dulbecco's Modified Eagle's Medium-Ham's F-10 (1:1)} containing growth factors and methylisobutylxanthinine for 72 h, adding increasing doses of FSH to the standard curve or small volumes of serum to the test vials as well as 19-hydroxyandrostenedione for 24 h, and measuring estradiol by RIA in dilutions of the medium . Using NIAMDD human (h) FSH-2 as the bioassay standard, the useful range of the assay was 0.01-5.0 ng/ml . Specificity was determined by the addition of graded doses of hLH, hTSH, ACTH, hGH, hPRL, and hCG . The minor degree of FSH bioactivity observed in a few hormone preparations was accounted for by the degree of FSH contamination in them . Mean intra- and interassay coefficients of variation were 9% and 11%, and the index of precision was 0.049 . This bioassay was used to determine the bioactive FSH content of pituitary extracts, tissue culture media, elutions from columns, and isoelectrically focused samples . More importantly, small quantities of human sera gave responses parallel to the standard curve in a minimum of two dilutions . The bio- to immunoreactive ratios, expressed as the mean +/- SEM (NIAMDD-hFSH-2), were 0.66 +/- 0.2 in boys (n = 6), 0.78 +/- 0.2 in pubertal girls (n = 6), 1.18 +/- 0.2 in men (n = 13), 1.24 +/- 0.1 in postmenopausal women (n = 30), 1.94 +/- 0.3 in the follicular phase (n = 19), 6.2 +/- 1.4 in the ovulatory phase (n = 19), and 1.6 +/- 0.4 in the luteal phase (n = 19) of the normal menstrual cycle . These results indicate that the bio- to immunoreactive hFSH ratio in the circulation, is dependent upon the hormonal milieu of the subject.

J Biol Chem, 1987 Aug 25, 262(24), 11779 - 84
Characterization of a recombinant fusion protein of the finger domain of tissue-type plasminogen activator with a truncated single chain urokinase-type plasminogen activator; Gheysen D et al.; Human recombinant single chain urokinase-type plasminogen activator (recombinant scu-PA) and a hybrid between human tissue-type plasminogen activator (t-PA) and scu-PA, obtained by ligation of cDNA fragments encoding the NH2-terminal region (amino acids 1-67) of t-PA and the COOH-terminal region (amino acids 136-411) of scu-PA, were expressed in a mammalian cell system . The proteins were purified from conditioned culture media containing 2% fetal calf serum by chromatography on zinc chelate-Sepharose, immunoadsorption chromatography on an insolubilized murine monoclonal antibody directed against urokinase, benzamidine-Sepharose chromatography, and Ultrogel AcA 44 gel filtration . Between 180 and 230 micrograms of the purified proteins were obtained per liter of conditioned medium, with a yield of approximately 18% and a purification factor of 720-1900 . On sodium dodecyl sulfate gel electrophoresis under reducing conditions, the proteins migrated as single bands with approximate Mr 50,000 for recombinant scu-PA and Mr 43,000 for the t-PA/scu-PA hybrid . Following conversion to urokinase with plasmin, the proteins had a specific amidolytic activity comparable to that of natural scu-PA . Both proteins activated plasminogen directly with Km = 0.53 and 1.4 microM and k2 = 0.0034 and 0.0027 s-1, respectively . Both proteins did not bind specifically to fibrin and had a comparable degree of fibrin selectivity as measured in a system composed of a whole human 125I-fibrin-labeled plasma clot suspended in human plasma . It is concluded that this chimeric protein, consisting of the NH2-terminal "finger-like" domain of t-PA and the COOH-terminal region of scu-PA, has very similar enzymatic properties as compared to scu-PA, but has not acquired the fibrin affinity of t-PA.

Biochim Biophys Acta, 1987 Aug 15, 920(3), 195 - 204
Effects of ursodeoxycholic acid, analogues of ursodeoxycholic acid and combination of bile acids on bile acid synthesis in cultured rat hepatocytes; Kubaska WM et al.; The effect of individual 7 beta-hydroxy bile acids (ursodeoxycholic and ursocholic acid), bile acid analogues of ursodeoxycholic acid, combination of bile acids (taurochenodeoxycholate and taurocholate), and mixtures of bile acids, phospholipids and cholesterol in proportions found in rat bile, on bile acids synthesis was studied in cultured rat hepatocytes . Individual steroids tested included ursodeoxycholate (UDCA), ursocholate (UCA), glycoursodeoxycholate (GUDCA) and tauroursodeoxycholate (TUDCA) . Analogues of UDCA (7-methylursodeoxycholate, sarcosylursodeoxycholate and ursooxazoline) and allochenodeoxycholate, a representative of 5 alpha-cholanoic bile acid were also tested in order to determine the specificity of the bile acid biofeedback . Each individual steroid was added to the culture media at concentrations ranging from 10 to 200 microM . Mixtures of taurochenodeoxycholate (TDCA) and taurocholate in concentrations ranging from 150 to 600 microM alone and in combination with phosphatidylcholine (10-125 microM) and cholesterol (3-13 microM) were also tested for their effects on bile acid synthesis . Rates of bile acid synthesis were determined as the conversion of added lipoprotein {4-14C}cholesterol or {2-14C}mevalonate into 14C-labeled bile acids and by GLC quantitation of bile acids secreted into the culture media . Individual bile acids, bile acid analogues, combination of bile acids and mixture of bile acids with phosphatidylcholine and cholesterol failed to inhibit bile acid synthesis in cultured hepatocytes . The addition of UDCA or UCA to the culture medium resulted in a marked increase in the intracellular level of both bile acids, and in the case of UDCA there was a 4-fold increase in beta-muricholate . These results demonstrate effective uptake and metabolism of these bile acids by the rat hepatocytes . UDCA, UCA, TUDCA and GUDCA also failed to inhibit cholesterol-7 alpha-hydroxylase activity in microsomes prepared from cholestyramine-fed rats . The current data confirm and extend our previous observations that, under conditions employed, neither single bile acid nor a mixture of bile acids with or without phosphatidylcholine and cholesterol inhibits bile acid synthesis in primary rat hepatocyte cultures . We postulate that mechanisms other than a direct effect of bile acids on cholesterol-7 alpha-hydroxylase might play a role in the regulation of bile acid synthesis.

J Immunol, 1987 Aug 15, 139(4), 1191 - 8
The murine lymphocyte receptor for IgE . IV . The mechanism of ligand-specific receptor upregulation on B cells; Lee WT et al.; Rodent B cells respond to culture with IgE by increasing their IgE-specific Fc receptors (Fc epsilon R) . The mechanism of this upregulation was characterized on Fc epsilon R+ murine B cell hybridoma lines . Measurements of {35S}methionine incorporated into the Fc epsilon R over time indicated that IgE did not appreciably increase the rate of Fc epsilon R synthesis . In contrast analysis of Fc epsilon R decay from surface radioiodinated B hybridoma cells demonstrated that IgE acted to slow the rate of Fc epsilon R degradation . Very little endocytosis of monomeric IgE was seen; this, combined with the observation that lysomotropic agents failed to inhibit Fc epsilon R degradation suggested that decay occurs at the cell surface . A soluble receptor immunoassay was developed, using monoclonal anti-Fc epsilon R, and this assay demonstrated that cell-bound IgE inhibited the release into the culture media of soluble immunoreactive Fc epsilon R . Examination of the soluble Fc epsilon R by SDS-PAGE after isolation with monoclonal anti-Fc epsilon R demonstrated that it was 10,000 m.w . smaller than the cell-associated Fc epsilon R . IgE affinity columns failed to bind the Fc epsilon R fragment, indicating that the ligand binding activity was largely lost . Thus this study demonstrated that IgE-dependent Fc epsilon R induction on B cells occurs because IgE upon binding to the B cell surface, inhibits the proteolytic cleavage and release of the Fc epsilon R into the surrounding medium, and it is this inhibition of degradation that causes the higher Fc epsilon R levels.

Cancer Res, 1987 Aug 15, 47(16), 4243 - 7
Influence of gangliosides on primary and metastatic neoplastic growth in human and murine cells; Alessandri G et al.; The influence of gangliosides on tumor growth and frequency of metastasis in vivo as well as on growth and motility of neoplastic cells in vitro was tested utilizing human and rodent cell populations . In mice receiving injections of a ganglioside mixture twice daily the tumor volume, the number of spontaneous metastases per animal, and the number of mice with metastasis was approximately double that of controls . Preincubation of neoplastic cells with the ganglioside mixture doubled the number of metastatic foci in the lungs of mice receiving the cells by i.v . injection . Addition of a ganglioside mixture to the culture medium enhanced motility of neoplastic cells about 3-fold . This finding was similar to that observed for capillary endothelium . The presence of gangliosides in the culture media for a 48-h incubation period about doubled the number of neoplastic cells as compared to controls; the same was observed for capillary endothelium . The data are interpreted to indicate that gangliosides improve growth and mobilization of capillary endothelium and neoplastic cells . Both events may concur in enhancing tumor growth in vivo, the first by improving angiogenesis, the second by direct action on the neoplastic cell population.

Nature, 1987 Aug 13-19, 328(6131), 621 - 4
Alternative RNA splicing affects function of encoded platelet-derived growth factor A chain; Collins T et al.; Platelet-derived growth factor (PDGF) is a basic protein of relative molecular mass 30,000 (Mr 30K) composed of two polypeptide chains, designated PDGF A and PDGF B . The B-chain is encoded by the c-sis gene, the cellular counterpart of the simian sarcoma virus transforming gene v-sis . The PDGF A-chain cDNA clones recently isolated and sequenced from a transformed human clonal glioma cell line represent at least two alternatively spliced transcript species differing by 69 base pairs at the C-terminus . Here we demonstrate that the normal human umbilical vein endothelial cell (EC) A chain precursor lacks the 15 carboxy-terminal, highly basic amino acids encoded by the larger tumour cell cDNA . Surprisingly, culture media from monkey kidney cells (COS) transfected with the endothelial cDNA clone contained much less mitogenic activity than media from cells transfected with the longer tumour cell-derived A-chain cDNA . This functional difference appeared to be due to inefficient assembly or secretion of the recombinant endothelial-type growth factor . This suggests that some transformed cells may use alternative RNA splicing to modify normal growth factors and by so doing increase the efficiency of mitogen assembly or secretion.

J Biol Chem, 1987 Aug 5, 262(22), 10855 - 62
Characterization of a fusion protein consisting of amino acids 1 to 263 of tissue-type plasminogen activator and amino acids 144 to 411 of urokinase-type plasminogen activator; Nelles L et al.; A hybrid human cDNA was constructed by splicing of a cDNA fragment of tissue-type plasminogen activator (t-PA), encoding 5'-untranslated, the pre-pro region and amino acids Ser1-Thr263, with a cDNA fragment of urokinase-type plasminogen activator (u-PA), encoding amino acids Leu144-Leu411 . The cDNA fragments were obtained from full length t-PA cDNA, cloned from Bowes melanoma poly(A)+ mRNA, and from full length u-PA cDNA, cloned from CALU-3 lung adenocarcinoma poly(A)+ mRNA . The hybrid (t-PA/u-PA) cDNA was expressed in Chinese hamster ovary cells and the translation product purified from the conditioned cell culture media . On SDS-gel electrophoresis under reducing conditions, the protein migrated as a single band with approximate Mr 70,000 . On immunoblotting, it reacted both with rabbit antisera raised against human t-PA and against human u-PA . The urokinase-like amidolytic activity of the protein was only 320 IU/mg but increased to 43,000 IU/mg after treatment with plasmin, which resulted in conversion of the single-chain molecule (t-PA/scu-PA) to a two-chain molecule (t-PA/tcu-PA) . The specific activity of the protein on fibrin plates was 57,000 IU/mg by comparison with the International Reference Preparation for Urokinase . Both the single-chain hybrid (t-PA/scu-PA) and the two-chain plasmin derivative (t-PA/tcu-PA) bound specifically to fibrin, albeit more weakly than t-PA . The t-PA/tcu-PA hybrid had a higher selectivity for fibrin than tcu-PA, measured in a system composed of a whole human 125I-fibrin-labeled plasma clot immersed in human plasma . Both hybrid proteins activated plasminogen directly with Km = 1.5 microM and k2 = 0.0058 s-1 for t-PA/scu-PA and with Km = 80 microM and k2 = 5.6 s-1 for t-PA/tcu-PA . CNBr-digested fibrinogen stimulated the activation of plasminogen with t-PA/tcu-PA (Km = 0.20 microM and k2 = 1.2 s-1) . It is concluded that these t-PA/u-PA hybrid proteins combine, at least to some extent, the fibrin-affinity of t-PA with the enzymatic properties of u-PA (either scu-PA or tcu-PA), which in some assays result in improved fibrin-mediated plasminogen activation.

Biol Reprod, 1987 Aug, 37(1), 117 - 26
Detection of a progesterone-dependent secretory protein synthesized by cat endometrium; Boomsma RA et al.; Uterine flushings and culture media from endometrial explants incubated in the presence of radiolabeled amino acids were analyzed using one-(1-D) and two-dimensional (2-D) gel electrophoresis to identify proteins synthesized by the endometrium and subsequently released into the uterine lumen . 1-D and 2-D analyses of uterine flushings and culture media of endometrial explants obtained from 7- to 11-day pregnant cats (pre-implantation) showed a Mr 30,000 protein that appeared on 2-D gels as a family of macromolecules with isoelectric points between 6.5 and 7.0 . This family of macromolecules was also present in the culture media of implantation-site tissue obtained from 12- to 16-day pregnant cats and of nonimplantation-site endometrium obtained form 12- to 28-day pregnant cats . The Mr 30,000 protein was absent in uterine flushings and culture media from estrous and 3- to 5-day-pregnant cats . In ovariectomized, steroid-treated animals, the Mr 30,000 protein was only detected in flushings and media from those animals treated with progesterone, regardless of the presence or absence of estradiol-priming and/or simultaneous estradiol treatment . In daily flushings obtained from ovariectomized, steroid-treated cats equipped with an indwelling uterine catheter, the Mr 30,000 protein was absent during the 14 days of estradiol treatment and was first detected 3-4 days after the onset of estradiol plus progesterone treatment . This protein was not detected in serum from estrous, 9-day pregnant, ovariectomized, and ovariectomized, steroid-treated animals . This study shows that 1) a progesterone-dependent protein, with an approximate molecular weight of 30,000 and an isoelectric point of 6.5-7.0, first appears within the uterine lumen soon after the arrival of the blastocyst and continues to be present during implantation; 2) the synthesis and release of the Mr 30,000 protein is dependent on progesterone regardless of the presence or absence of estradiol; and 3) the onset of secretion of the Mr 30,000 protein requires 3-4 days of continuous progesterone treatment in the estradiol-primed cat.

J Steroid Biochem, 1987 Aug, 28(2), 227 - 31
Studies on the mechanism of the antiandrogenic effect of a putative 5 alpha-reductase inhibitor; Vazquez MH et al.; The mechanism of the antiandrogenic effect of 5,10-seco-19-norpregnane-4,5-diene-3,10,20-trione (secosteroid), reputedly an irreversible inhibitor of 5 alpha-reductase, was investigated . Its addition (10 microM) to culture media effectively suppressed the synthesis of rat epididymal proteins specifically induced by 0.1 microM testosterone (T) or dihydrotestosterone (DHT) . Under the same conditions, secosteroid did not change the rate at which labeled T was metabolized to 5 alpha-reduced compounds . In a comparative study, secosteroid inhibited 5 alpha-reductase in an isolated microsomal fraction while not affecting the enzyme activity in minced tissue . Secosteroid was shown to be a competitor of the binding of {3H}T and {3H}DHT (both at 4 nM) to the epididymal cytosol androgen receptor, with ID50 of 1 microM for the former and 4 microM for the latter, thus explaining the mechanism involved in its antiandrogenic properties.

J Clin Invest, 1987 Aug, 80(2), 300 - 7
Biosynthesis and secretion of human colonic mucin glycoproteins; Smith AC et al.; Synthesis and secretion of colonic mucin glycoprotein species were assessed during in vitro culture of colonic mucosal explants . DEAE-cellulose chromatography of endogenously labeled mucin glycoproteins from explant tissue demonstrated the presence of six mucin species (I-VI) similar to those identified earlier in surgical specimens of human colonic tissue . The relative proportions of mucin species I-VI in tissue explants remained constant throughout a 30-h culture period . However, the proportional representation of the various mucin species in media was significantly different from that found in tissue, which suggests that some mucin species (I, II, and III) are differentially secreted, whereas others (IV and V) are retained within intracellular pools . Radiolabeled precursors were incorporated into mucin species I, II, and III at a 2.0-2.6-fold greater rate than their concentration in tissue, supporting the concept that these glycoproteins were both synthesized and secreted at a greater rate than species IV and V . Colonic mucosal explants from patients with ulcerative colitis showed greater than 90% reduction of species IV . However, the amount of species IV recovered from culture media of ulcerative colitis explants was comparable to normal controls . It appears that mucin species IV is differentially secreted rather than retained within intracellular pools in mucosa of patients with ulcerative colitis.

Fertil Steril, 1987 Aug, 48(2), 258 - 64
Use of individual human follicles to compare oocyte in vitro fertilization to granulosa cell in vitro luteinization; Hill GA et al.; Granulosa-lutein (G-L) cells from individual follicles aspirated during cycles of in vitro fertilization-embryo transfer were examined after 3 and 6 days in culture . G-L cells from follicles that contained an oocyte that fertilized in vitro were compared with G-L cells from follicles that contained an oocyte that did not fertilize in vitro . Spent culture media was assayed for progesterone at days 3 and 6 of culture and luteinizing hormone/human chorionic gonadotropin (LH/hCG) receptor content of G-L cells was determined at day 6 . G-L cell cultures from follicles that contained an oocyte that fertilized in vitro produced significantly more progesterone over 3 and 6 days of culture than those obtained from follicles in which the oocyte did not fertilize . Furthermore, LH/hCG receptor content after 6 days was significantly higher in G-L cells obtained from follicles with fertilized oocytes compared with follicles with unfertilized oocytes . Increased progesterone output and LH/hCG receptor acquisition demonstrate more maturation or "luteinization" by G-L cells aspirated from individual follicles that contain oocytes that fertilized in vitro.

Agents Actions, 1987 Aug, 21(3-4), 341 - 4
Catabolin/interleukin-1 regulation of cartilage and chondrocyte metabolism; O'Byrne EM et al.; Catabolin/interleukin-1 effects on metabolism were studied in bovine nasal cartilage organ culture and articular chondrocyte cell culture . Keratan sulfate (KS) and hyaluronic acid (HA) were determined by an ELISA; prostaglandin E2 by RIA, sulfated glycosaminoglycan using dimethylmethylene blue and proliferation by incorporation of tritiated thymidine . Gel filtration of untreated 4-day organ culture media indicated that large sulfated and KS-containing proteoglycans were released and eluted in the void volume . Catabolin/interleukin-1 increased release of sulfated glycosaminoglycans and these were of lower molecular weight with an altered distribution of KS . Catabolin/interleukin-1 treatment of chondrocytes caused a decrease in KS production and proliferation but an increase in HA and in prostaglanding E2 production . Alterations of the chondrocyte metabolism by catabolin/interleukin-1 causing proteoglycan matrix degradation and modulation of chondrocyte glycosaminoglycan biosynthesis and proliferation may play a role in cartilage erosion and failure to repair in arthritic diseases.

Dig Dis Sci, 1987 Aug, 32(8), 878 - 82
Arachidonic acid stimulation of mucus production by rat gastric cultured cells; Terano A et al.; The effect of arachidonic acid (AA) on mucus production (synthesis and secretion) by rat gastric monolayer-cultured cells was investigated . For the study of mucus synthesis, the rate of incorporation of {3H}glucosamine into the cultured cells was measured . The rate of release of glycoprotein into the culture media from the cells, which were incubated in the medium containing {3H}glucosamine for 24 hr in advance, was determined for the study of mucus secretion . Prostaglandins in the medium were measured by radioimmunoassay . AA (10(-4) M) significantly increased mucus synthesis and secretion by the cultured cells (P less than 0.01) . PG (E2 and I2) synthesis by the cultured cells was significantly enhanced by AA (10(-4) M) (P less than 0.05) . An addition of indomethacin to the culture medium abolished this effect of AA . These results suggested that gastric mucus production was enhanced by AA in vitro and that this effect may be mediated by endogenous PG production.

J Neurochem, 1987 Aug, 49(2), 487 - 94
Monoclonal antibodies against glutaraldehyde-conjugated dopamine; Chagnaud JL et al.; Four mice were immunized with dopamine (DA)-glutaraldehyde (G)--protein conjugates over a period of 8-10 weeks . Polyclonal antisera, obtained at various intervals, were tested using an enzyme-linked immunosorbent assay (ELISA) . All had anti-conjugated DA antibodies . As soon as good antibody affinity was detected between 10(-10) and 10(-6) M, the mouse yielding the highest apparent affinity was killed, and the spleen was dissected out . Hybridomas were obtained from spleen cells fused with SP2/O/Ag myeloma cells . Supernatant culture media of hybridomas were tested for the presence of anti-conjugated DA antibodies with the ELISA method . Selected hybridomas giving good antibody affinity and specificity were then cloned by the limiting dilution technique . The resulting supernatant culture media were again tested by ELISA . Clones that gave a high antibody affinity (10(-10)-10(-8)M) for G-conjugated DA were used for histochemical localization of DA in rat brain . G-fixed rat brains were sectioned from the telencephalon to the mesencephalon, reduced with sodium borohydride, and prepared for peroxidase-antiperoxidase immunocytochemistry using supernatant (diluted 1:100) or ascites fluid (diluted 1:50,000) . Dense networks of very fine fibers were observed in the striatum, septum, and cortex . Numerous immunoreactive cell bodies were found in the ventral tegmental area, the substantia nigra, the hypothalamus, and the dorsal raphe . The ELISA tests and adsorption controls suggested that the monoclonal antibody allowed highly specific detection of DA in tissues.

J Biol Chem, 1987 Jul 25, 262(21), 9956 - 61
Proatrial natriuretic factor is phosphorylated by rat cardiocytes in culture; Bloch KD et al.; Proatrial natriuretic factor (proANF) is phosphorylated in primary cultures of neonatal rat cardiocytes . Rittenhouse et al . (Rittenhouse, J., Moberly, L., O'Donnell, M . E., Owen, N . E., and Marcus, F . (1986) J . Biol . Chem . 261, 7607-7610) observed that cyclic AMP-dependent protein kinase phosphorylated synthetic peptides related to atrial natriuretic factor (ANF) and that phosphorylated ANF peptides were more effective in stimulating Na/K/Cl cotransport in smooth muscle cells than nonphosphorylated forms . In our studies, rat cardiocytes in culture were incubated with {32P}orthophosphoric acid, and ANF-related peptides in cell extracts and culture media were isolated using antisera to ANF . Both atrial and ventricular cardiocytes contained and secreted phosphorylated proANF, a 126-amino acid precursor of ANF . Phosphorylated and nonphosphorylated isoforms of proANF were resolved by isoelectric focusing; approximately 35% of the proANF secreted by cardiocytes was phosphorylated . proANF is phosphorylated on a serine residue localized to a 42-amino acid tryptic fragment (proANF residues 26-67) . We conclude that proANF is phosphorylated by rat cardiocytes but not within the portion of the molecule destined to become ANF (proANF residues 99-126) . Phosphorylation may have a role in the cellular mechanisms of proANF storage and secretion or in the modulation of potential biological activities of the circulating amino-terminal portion of proANF.

Life Sci, 1987 Jul 20, 41(3), 273 - 80
Human macrophages degrade tryptophan upon induction by interferon-gamma; Werner ER et al.; Human peripheral blood mononuclear cells, monocytes-macrophages and T-cells were stimulated with human recombinant interferon-gamma, interferon-alpha and phytohemagglutinin . The culture supernatants were analyzed for tryptophan, kynurenine, 3-hydroxyanthranilic acid, anthranilic acid and neopterin by high performance liquid chromatography . Tryptophan was decreased and the four other compounds were increased in supernatants of peripheral blood mononuclear cells activated by interferon-gamma (250 U/ml), interferon-alpha (10.000 U/ml) and phytohemagglutinin (1 microgram/ml) . After splitting of peripheral blood mononuclear cells by adherence, the monocytes and macrophages but not the T-cells degraded tryptophan upon stimulation by interferon-gamma in a dose dependent manner . Supernatants of phytohemagglutinin stimulated but not of resting T-cells were found to induce tryptophan degradation by macrophages, the active principle being neutralized by an antiserum for interferon-gamma . Thus phytohemagglutinin acts by activating T-cells to release interferon-gamma which in turn induces macrophages to degrade tryptophan . In all experiments the appearance of neopterin in the culture media was correlated to the observed tryptophan degradation.

FEBS Lett, 1987 Jul 13, 219(1), 56 - 64
Dexamethasone-dependent expression of beta 1-24 corticotropin stimulated adenylate cyclase during adipose conversion of 3T3-F442A cells; Feve B et al.; When 3T3-F442A preadipocytes were grown in culture media supplemented with corticosteroid poor fetal calf serum and insulin they differentiated into adipocytes . Glycerophosphate dehydrogenase, a marker of terminal differentiation, developed a 600-fold increase of activity whereas the adenylate cyclase system remained unresponsive to the synthetic ACTH(1-24) analog . In contrast, 3T3-F442A adipocytes, differentiated in the presence of dexamethasone, exhibited an adenylate cyclase activity which was stimulated 4-fold by ACTH(1-24) . The stimulation of the adenylate cyclase activity by GTP gamma S remained unchanged (about 20-25-fold) suggesting that the G regulatory coupling protein was not functionally modified by dexamethasone . Binding studies with 125I-ACTH revealed that specific cellular binding could be evidenced in dexamethasone-treated cells while control adipocytes did not exhibit any specific binding of 125I-ACTH . These findings lend support to the hypothesis that the setting off of this ACTH responsiveness in 3T3-F442A cells is regulated by dexamethasone after cells are committed to adipose differentiation.

Nucleic Acids Res, 1987 Jul 10, 15(13), 5461 - 75
Butyrate selectively activates the metallothionein gene in teratocarcinoma cells and induces hypersensitivity to metal induction; Andrews GK et al.; The expression of metallothionein genes (MT-I and MT-II) was shown to be enhanced within 2 h of addition of 2.5-5 mM sodium butyrate to cultures of teratocarcinoma cells . Both undifferentiated stem cells (F9 and OC15) and differentiated cells (PSA5E and OC15 END) reacted similarly to butyrate by increased accumulation of MT mRNAs . As expected, all of the teratocarcinoma cells that were tested also responded to Zn2+ and Cd2+ by 5- to 10-fold increases in MT mRNA accumulation within 2-24 h of metal addition to the culture media . Surprisingly, MT genes in cells pretreated with butyrate were hypersensitive to metal induction, and this was demonstrated by accumulated transcript levels and by synthesis of MT protein . The maximal metal response was obtained by exposure of cells to butyrate for around 5-8 h together with 10 microM heavy metals . Metal additions to culture media over a range of concentrations and times only induced half the levels of MT mRNA that were achieved by butyrate plus metals . Butyrate enhanced the rate of accumulation of MT mRNA in response to metals, increased the sensitivity of the MT gene to metals, and protected cells from toxic effects of high concentrations of metals . The butyrate and metal ion responses were selective in that no accumulation of c-myc, c-fms, HSP-70, or AFP mRNA was detected . However, c-fos mRNA accumulated in cells exposed to toxic concentrations of metals (50 microM and higher) and this was also potentiated by butyrate treatment . These results suggest that butyrate alters the chromatin conformation of both the MT-I and MT-II genes leading to an accentuated transcriptional response to metals.

Tsitologiia, 1987 Jul, 29(7), 818 - 24
{Growth-stimulating effect of UF-irradiated blood . I . The radiation dose dependence of the initial growth potencies of blood and of the functional state of the target cells}; Firulina II et al.; UV irradiation (UVI) of donor blood in the apparatus used in hospitals of the USSR with the therapeutic aim of autotransfusion of UV-irradiated blood (AUVIB), results in an increase of connective tissue cell growth potency: being added into culture media the supernatants of irradiated blood stimulate DNA-synthetic and proliferative activity of cultured human embryonic cells . The high activity of cells persists for about 2 days . The effect is great with low initial levels of cell proliferative activity . In this case the effect is maximum (about 125% of the control) . It is suggested that the above effect may be involved in the mechanism of stimulation of regeneration processes in the organism after AUVIB.

J Reprod Fertil, 1987 Jul, 80(2), 569 - 76
In-vitro synthesis of a low molecular weight lipid-soluble luteotrophic factor by conceptuses of cows at day 13-18 of pregnancy; Hickey GJ et al.; Two culture systems for maintenance of Day 13-18 conceptus tissue were developed . Harvested culture media were assayed for luteotrophic activity by determining their ability to stimulate progesterone synthesis by dispersed bovine luteal cells . Significant luteotrophic activity was found in 80% of the 31 tissue culture media studied . A series of experiments carried out to determine the nature of the luteotrophic activity indicate that it is a small (Mr less than 10,000), heat-labile, lipid-soluble substance that is adsorbed by dextran-coated charcoal . The nature and activity of this factor, together with its synthesis by the early bovine conceptus, suggest that it may have a significant role in stimulating progesterone synthesis by the corpus luteum during early pregnancy.

Development, 1987 Jul, 100(3), 431 - 9
In vitro analysis of glucose metabolism and embryonic growth in postimplantation rat embryos; Ellington SK; The glucose metabolism and embryonic development of rat embryos during organogenesis was studied using embryo culture . Glucose uptake and embryonic growth and differentiation of 10.5-day explants (embryos + membranes) were limited by the decreasing glucose concentration, but not the increasing concentration of metabolites, in the culture media during the second 24 h of a 48 h culture . No such limitations were found on the embryonic development of 9.5-day explants during a 48 h culture although glucose uptake was slightly reduced at very low concentrations of glucose . From the head-fold stage to the 25-somite stage of development, glucose uptake was characteristic of the stage of development of the embryo and not the time it had been in culture . Embryonic growth of 9.5-day explants was similar to that previously observed in vivo . Glucose uptake by 9.5-day explants was dependent on the surface area of the yolk sac and was independent of the glucose concentration in the culture media (within the range of 9.4 to 2.5 mM) . The proportion of glucose converted to lactate was 100% during the first 42h of culture then fell to about 50% during the final 6h . The protein contents of both the extraembryonic membranes and the embryo were dependent on the glucose uptake.

J Androl, 1987 Jul-Aug, 8(4), 259 - 66
Quantification of bovine sperm separation by a swim-up method . Relationship to sperm motility, integrity of acrosomes, sperm migration in polyacrylamide gel and fertility; Parrish JJ et al.; The number of bovine spermatozoa separated in a swim-up procedure was quantified using an electronic cell counter . In an initial test of the swim-up procedure, non-frozen sperm samples with different ratios of live to dead cells were prepared and tested for the number of spermatozoa counted by the swim-up procedure . In ejaculates from six bulls, the number of spermatozoa swimming up was related to the number of live cells present (R2 = 0.97) . Next, sperm quality of frozen-thawed semen immediately after thawing was measured at 37 C by swim-up sperm count, sperm motility, spermatozoa with an intact acrosome and migration in polyacrylamide gel and then compared with the fertility of the semen used for artificial insemination . Twenty-nine ejaculates of frozen-thawed semen from 11 bulls were evaluated . Correlations with fertility were highest on an ejaculate basis for motility (r = 0.41, P = 0.05) and for swim-up sperm count (r = 0.35, P = 0.06) . On a bull basis, swim-up sperm count had the highest correlation with fertility (r = 0.59, P = 0.06) . In a multiple regression model to predict male fertility that included all described measures of semen quality, a R2 value of 0.69 was obtained . This is the first report showing that the ability of spermatozoa to swim out of a more dense medium (whole milk-glycerol extender) into culture media is quantitatively related to in vivo fertility.

J Clin Microbiol, 1987 Jul, 25(7), 1216 - 20
Disposable reversed-phase chromatography columns for improved detection of carboxylic acids in body fluids by electron-capture gas-liquid chromatography; Daneshvar MI et al.; Disposable reversed-phase chromatography columns were tested for their effectiveness in removing unreacted trichloroethanol (TCE) from derivatized samples for gas-liquid chromatography analysis . Derivatized acidic chloroform extracts of saponified whole cells of Mycobacterium species, spent culture media, and derivatized acidic chloroform extracts of serum and cerebrospinal fluids from patients with tuberculous meningitis were tested . Samples were added to preconditioned reversed-phase chromatography columns, and various solvents and solvent mixtures were tested to determine maximum recovery of the TCE derivatives . With this procedure, we were able to quickly remove the TCE reagent and efficiently recover TCE-derivatized carboxylic acids . Use of these columns improved the reagent cleanup procedure, simplified the derivatization step, permitted increased detection of trace components, such as tuberculostearic acid, in body fluids, and improved the selectivity of the procedure for detection of carboxylic acids.

Eur J Obstet Gynecol Reprod Biol, 1987 Jul, 25(3), 203 - 8
Randomized trial of two media used for in vitro fertilization; Parinaud J et al.; Culture media are important components of IVF . Their selection must meet the need for efficiency, and also economic and practical requirements . From these standpoints, we compared two widely used media: Ham's F10 supplemented with cord serum and Menezo B2 used without serum . The test followed a randomized protocol using two series of 159 and 162 oocytes . Since no difference was seen in efficiency assessed from cleavage rate, the discussion focuses on cost and ease of use.

Mem Inst Oswaldo Cruz, 1987 Jul-Sep, 82(3), 379 - 84
Role of divalent cations, pH, cytoskeleton components and surface charge on the adhesion of Trichomonas vaginalis to a polystyrene substrate; Silva Filho FC et al.; The process of adhesion of three different strains of Trichomonas vaginalis to a polystyrene substrate was analysed . The process of adhesion was dependent on the time of incubation and the pH of the phosphate-buffered solution (PBS) in which the parasites were suspended . The highest indices of adhesion were observed after an incubation time of 60 min at pH 6.6 . The adhesion index increased when the parasites were incubated in the presence of culture media or when Ca++ or Mg++ was added to the PBS solution, whereas cytochalasin B, trypsin or neuraminidase reduced adhesion . Incubation of the parasites in the presence of poly-L-lysine facilitated the process of adhesion . Incubation of the parasites or polystyrene beads in the presence of poly-L-lysine led to important changes in their surface charge.

Gen Comp Endocrinol, 1987 Jul, 67(1), 126 - 41
Fundulus heteroclitus gonadotropin(s) . I . Homologous bioassay using oocyte maturation and steroid production by isolated ovarian follicles; Lin YW et al.; Isolated ovarian follicles from several species were cultured to develop an in vitro bioassay system for Fundulus heteroclitus gonadotropin . An extract of F . heteroclitus pituitaries, when tested in heterologous systems using follicles from Rana pipiens . Xenopus laevis, and Carassius auratus, was ineffective in provoking either germinal vesicle breakdown or steroid production . In a homologous system using F . heteroclitus follicles, F . heteroclitus pituitary extract was capable of inducing both germinal vesicle breakdown and steroid production in a dose-dependent fashion . Testosterone, estradiol-17 beta, and 17 alpha-hydroxy,20 beta-dihydroprogesterone were detected in both the culture media and the follicle extracts after F . heteroclitus pituitary extract stimulation . The steroidogenic responses resulting from the pituitary extract stimulation were dependent on the size and stage of follicular development . Only large vitellogenic follicles (1.2-1.4 mm diameter) were able to produce 17 alpha-hydroxy,20 beta-dihydroprogesterone and testosterone . Small vitellogenic follicles (less than 1.2 mm) were unresponsive to stimulation by F . heteroclitus pituitary extracts as scored by either germinal vesicle breakdown or production of 17 alpha-hydroxy, 20 beta-dihydroprogesterone and testosterone . However, estradiol-17 beta production was detected in follicles of a much wider size range: Follicles as small as 0.8 mm diameter were responsive to F . heteroclitus pituitary extract stimulation and produced a large quantity of estradiol-17 beta . There was a marked seasonal sensitivity of F . heteroclitus follicles to pituitary extract stimulation in vitro . Follicles obtained from fish outside of the breeding season (January) were less responsive to stimulation by pituitary extract or steroid . The same preparation of pituitary extract was capable of provoking germinal vesicle breakdown in follicles obtained in May . Pituitary extracts prepared during October through January were also less potent than those prepared during the breeding season (February through September) . We conclude that F . heteroclitus gonadotropin(s) shows a noticeable species specificity and that F . heteroclitus follicles exhibit both a season- and a size-dependent responsiveness to gonadotropin(s) . Hence, with a judicious use of the appropriate types of F . heteroclitus ovarian follicles, we have been able to demonstrate that in vitro oocyte maturation and steroid production are sensitive, homologous bioassays for F . heteroclitus gonadotropin(s).

Pathology, 1987 Jul, 19(3), 277 - 80
Isolation of genital mycoplasmas from the blood of neonates and women with pelvic infection using conventional SPS-free blood culture media; Kelly VN et al.; Standard blood culture media used in our laboratory were tested for their ability to support the growth of Mycoplasma hominis and Ureaplasma urealyticum . Small inocula (approximately 10 colony forming units per ml) of both organisms grew in diphasic tryptone soya medium but not in any of several media containing sodium polyanetholesulphonate (SPS) including a modified Schaedler broth (RWH anaerobic medium) and two BACTEC media (6B and 7D) . Both organisms were inhibited even by very low concentrations of SPS but grew well in the Royal Women's Hospital (RWH) anaerobic medium when SPS was omitted . During a 22-month period, routine "blind" plating of the aerobic blood cultures on to mycoplasma agar resulted in isolation of M . hominis or U . urealyticum from 12 women with postpartum or postoperative pelvic infection, and from 3 neonates . Genital mycoplasmas represented 35% of significant isolates from adult blood cultures.

J Reprod Fertil, 1987 Jul, 80(2), 463 - 72
Partial purification and characterization of chorionic gonadotrophin in plasma and in culture medium of trophoblast cells from the marmoset monkey (Callithrix jacchus); Saunders PT et al.; A biologically active gonadotrophin has been purified from the media of long-term cultures of trophoblast cells of the common marmoset monkey by a combination of precipitation and chromatography . Marmoset chorionic gonadotrophin (CG) is a glycoprotein which binds Concanavalin A and wheat germ agglutinin . The protein purified from culture media exists as several isoelectric species with pI in the range pH 3.5-4.5 . On gel filtration it eluted with an apparent molecular weight of 68-72,000 but on PAGE migrated as if it was 58-65,000 . A glycoprotein with similar characteristics has been recovered from plasma samples of pregnant marmosets . Biological activity of partly purified CG from media, as determined by a mouse testicular cell bioassay, was 1-3 i.u./mg protein.

J Appl Bacteriol, 1987 Jul, 63(1), 47 - 52
The effect of a surfactant in media for the enumeration, growth and identification of airborne fungi; Madelin TM; Air sampling on to culture media for viable fungi is complicated by the presence of fast-growing species . A non-ionic surfactant (Triton N-101) incorporated into the medium effectively reduced the spread of such species and thus facilitated the enumeration and identification of fungal colonies collected by an Andersen sampler . The concentration of surfactant from 250 to 8000 ppm was not critical . Sixteen species of common moulds inoculated on surfactant media showed restricted radial growth but never total inhibition . Surfactant did not prevent sporulation and did not greatly affect morphological characteristics . Rose bengal media performed poorly in comparative tests . These observations indicate the practical value of this surfactant in aeromycological studies.

J Biol Chem, 1987 Jun 5, 262(16), 7651 - 7
The in situ kinetics of dopamine beta-hydroxylase in bovine adrenomedullary chromaffin cells . Intravesicular compartmentation reduces apparent affinity for the cofactor ascorbate; Menniti FS et al.; The Km of dopamine beta-hydroxylase for its cofactor, ascorbic acid, was determined in situ in primary cultures of bovine adrenomedullary chromaffin cells and in isolated chromaffin vesicles . A range of intravesicular ascorbate concentrations in chromaffin cell cultures (1.1-31.2 mM) was achieved by varying the number and concentration of ascorbate additions to the culture media . The rate of octopamine synthesis from tyramine displayed a Michaelis-Menten relationship with respect to ascorbate concentration and an apparent Km of dopamine beta-hydroxylase for ascorbate of 15.0 +/- 2.0 mM was determined . In isolated chromaffin vesicles, with an initial intravesicular ascorbate concentration of approximately 10 mM, ascorbate consumption during beta-hydroxylation occurred as a first order process . This indicated that dopamine beta-hydroxylase was not saturated at this initial ascorbate concentration . When isolated chromaffin vesicles were prepared with different intravesicular ascorbate concentrations, the rate of octopamine synthesis displayed a Michaelis-Menten relationship with respect to ascorbate with an apparent Km of 17.0 +/- 5.0 mM . Ascorbate consumption also occurred as a first order process in ascorbate-loaded chromaffin-vesicle ghosts which had initial ascorbate concentrations of approximately 30 mM but which were depleted of other small molecules such as catecholamines . These results indicate that the in situ Km of dopamine beta-hydroxylase for ascorbate (approximately 15 mM) is 25-fold higher than it is for the purified or partially purified enzyme assayed under optimal conditions in vitro (0.6 mM) . The factor(s) which decreases the enzyme affinity for ascorbate, relative to in vitro, resides in the chromaffin vesicle interior and is also retained in chromaffin-vesicle ghosts . The mechanism of this effect remains to be determined . The Km value determined in these experiments is close to the estimated intravesicular ascorbate concentration of bovine chromaffin granules in vivo (4), suggesting that the availability of ascorbate could become a factor in regulating the rate of dopamine beta-hydroxylation.

Cancer Lett, 1987 Jun, 35(3), 321 - 6
Thyroid hormone affects the expression of neoplastic transformation induced by DNA-transfection; Leuthauser SW et al.; Thyroid hormone can dramatically modulate oncogenic transformation of cells in culture . To further investigate this we have used DNA-mediated gene transfer (transfection) to transform cells grown in the presence (+T3) or absence (-T3) of thyroid hormones . Removal of thyroid hormones from the culture media greatly reduced the appearance of transformed foci subsequent to transfection . However, +T3 or -T3 media had no effect on the appearance of ouabain-resistant (ouar) colonies following transfection of ouabain-sensitive (ouas) cells with DNA isolated from ouar cells and selection in 3 mM ouabain . These results suggest that thyroid hormone does not effect the uptake or integration of exogenous DNA, but instead may modify the expression of transformation.

Invest Ophthalmol Vis Sci, 1987 Jun, 28(6), 945 - 53
Human trabecular meshwork organ culture . A new method; Johnson DH et al.; A new method has been developed for organ culture of human trabecular meshwork . Human eyebank eyes are sectioned at the equator and lens and vitreous are removed . The anterior segments are placed in a modified culture dish, cornea side up, and then sealed in place . Culture media (Dulbecco's modified Eagle media) are perfused through a cannula built into the bottom of the dish . The sealed system forces media to flow through the trabecular meshwork, Schlemm's canal, and exit through the normal limbal pathways . Intraocular pressure can be maintained at normal (or elevated) levels throughout the culture period . Corneal clarity is maintained . Trabecular cells remain in normal position on trabecular beams and maintain many of their usual morphologic characteristics . Cultures appear to be viable for up to 4 weeks . This technique should allow study of intact human trabecular meshwork in a controlled experimental fashion.

Eur J Cancer Clin Oncol, 1987 Jun, 23(6), 689 - 95
Tachykinin production by carcinoid tumours in culture; Norheim I et al.; Tissue specimens from 5 patients with metastatic midgut carcinoid tumours were kept in organ culture for up to 6 months . The tumour cells were confined to the suspension in the form of condensed cell clusters and appeared to retain their endocrine characteristics . Radioimmunoassay for tachykinin immunoreactivity showed high concentrations in 4 out of 5 culture media . The concentrations were highest in the beginning of the experiment, but subsequently decreased . The 4 patients from which these tumours were taken had all elevated tachykinin concentrations in extracted plasma . The fifth culture medium had low tachykinin concentration, and the concentration in extracted plasma from this patient was within the normal range . Reversed-phase high-performance liquid chromatography of the culture media with elevated tachykinin concentrations revealed immunoreactive components with the characteristics of synthetic neuropeptide K, neurokinin A and eledoisin, components also found in plasma and tumour tissues of carcinoid patients . Our findings indicate that carcinoid tumour cells produce tachykinins . These peptides are biologically very active, resulting in flush and hypotension when infused intravenously into normals, and might contribute to the clinical symptoms of the carcinoid syndrome.

Agents Actions, 1987 Jun, 21(1-2), 149 - 59
Effects of anti-inflammatory drugs on cartilage recovery from catabolin-induced degradation; Strathy GM et al.; Effects of aspirin (200 micrograms/ml), hydrocortisone (10 micrograms/ml), sodium aurothiomalate (100 micrograms/ml), and indomethacin (10 micrograms/ml) on recovery of cartilage from interleukin 1 or catabolin-induced degradation were examined in this initial in vitro study . The experimental protocol involved a "degradative phase" of eight days during which cartilage plugs were incubated in the presence or absence of spent human rheumatoid synovial culture media . A "recovery" period of six days followed during which the effects of the aforementioned drugs on treated cartilage were analyzed . Incorporation of {35S}sulfate and {3H}proline precursors, and total contents of hydroxyproline and glycosaminoglycan in cartilage were determined two, four, and six days after insult . Aspirin treatment caused a rise in total proteoglycan content over degraded controls (p less than 0.002), however, this increase was not associated with increased {35S}sulfate incorporation into glycosaminoglycans . Hydrocortisone resulted in a delayed rise in proteoglycan content concommitant with increased {35S}sulfate uptake, whereas sodium aurothiomalate treatment was without effect on proteoglycans . Indomethacin treatment was associated with an increased release of newly synthesized macromolecules by cartilage into the media (p less than 0.01) . These results suggest that common anti-inflammatory drugs may exhibit distinctly different effects on the in vitro synthesis and retention of proteoglycans by cartilage explants previously exposed to a degradative phase . Further work is necessary to assess the influence of drug concentration in this experimental system.

J Cell Biochem, 1987 Jun, 34(2), 101 - 12
Types I and IV collagenolytic and plasminogen activator activities in preovulatory ovarian follicles; Palotie A et al.; During ovulation, enzymatic degradation of the extracellular matrix occurs within and around the graafian follicles . In this study, the activities of several different proteolytic enzymes were measured in the culture media of follicles taken from pregnant mare serum gonadotropin (PMSG)-primed immature rats . At 52 h after PMSG, the follicles were cultured for 2 to 15 h in media with or without human chorionic gonadotropin (hCG) . Type I collagenase activity in hCG-stimulated follicles gradually increased within 6 h to 3.3-fold above that of the controls . Relatively pure populations of granulosa cells produced type I collagenase to a similar extent . Likewise, type IV collagenase increased 3.8-fold by 6 h after exposure of the follicles to hCG . In contrast, plasminogen activator activity increased by 3.9-fold at 2 h after hCG, but was negligible at 4, 6, and 15 h after incubation . These results suggest that plasminogen activator may activate both type I and type IV collagenase in hCG-stimulated ovulatory follicles.

Mol Cell Endocrinol, 1987 Jun, 51(3), 273 - 6
Phorbol ester stimulates human granulosa-luteal cell cyclic adenosine 3', 5'-monophosphate and progesterone production; Jalkanen J et al.; Human granulosa-luteal cell production of cyclic adenosine 3', 5'-monophosphate (cAMP) and progesterone (P) were studied in response to 12-O-tetradecanoyl-phorbol-13-acetate (TPA) . TPA specifically increased cAMP synthesis in a dose-dependent manner . A 7-fold increase occurred at a TPA concentration of 1 ng/ml . Time-course studies indicated that the increase in accumulation of cAMP into culture media became detectable at 4 h and continued up to 72 h . TPA also enhanced P synthesis, but the increase was statistically significant only at 72 h . Indomethacin prevented TPA-stimulated cAMP and P production . The results suggest that TPA stimulates granulosa-luteal cell cAMP and P production, and that the action of TPA is mediated by the increase in prostaglandin synthesis.

Biol Reprod, 1987 Jun, 36(5), 1289 - 95
Activation of plasminogen by the early bovine embryo; Menino AR Jr et al.; Activation of the plasma zymogen plasminogen to the enzyme plasmin by the early bovine embryo was evaluated . Sixteen-cell embryos to early morulae were collected at death from handmated synchronized and superovulated crossbred beef cows . Embryos were cultured in Ham's F-12 medium supplemented with 15 mg/ml bovine serum albumin containing 0, 15, 30, 60 or 120 micrograms/ml plasminogen in a humidified atmosphere of 5% CO2 in air at 37 degrees C . Cultures were observed every day, and stage of development was recorded . Medium was collected at 24-h intervals, starting at initiation and continuing through 288 h of culture . Plasminogen activator and plasmin levels in the culture media were determined, using a caseinolytic assay . The percentages of embryos developing to the initiating hatching blastocyst, hatched blastocyst, attached blastocyst, and attached blastocyst with trophoblastic outgrowth stages were not significantly different between the five levels of plasminogen . Initiation and completion of hatching, however, accelerated as plasminogen concentration increased in the culture media . Plasminogen activator production, expressed as milliunits X ml-1 X h-1 X viable embryo-1, was low for the first 48 h of culture, increased between 48-120 h, and tended to plateau thereafter . Plasminogen activation, measured indirectly as the plasmin concentration in a microdrop of medium and expressed as microgram plasmin X ml-1 X h-1 X viable embryo-1, followed plasminogen activator production, and was consistently low for the first 48-72 h of culture . Embryonic activation of plasminogen increased sharply thereafter, and also plateaued after 120 h.

Int J Gynaecol Obstet, 1987 Jun, 25(3), 241 - 8
In vitro studies on the control of human myometrial gap junctions; Garfield RE et al.; In this study human myometrial tissues were examined for the presence of gap junctions by quantitative electron microscopy before and after incubation in tissue culture media with and without indomethacin . The area of gap junctions was very low in tissues from pregnant women at term but not labor, before incubation . After 24 and 48 h incubation without any treatment, segments of some of the same tissues developed many gap junctions and other tissues contained few junctions . Prostaglandin E (PGE), prostaglandin F (PGF) and prostaglandin F metabolite (PGF metabolite) levels in the media at various times were measured by radioimmunoassay . The prostaglandins increased progressively during the incubation period . Treatment of tissues with indomethacin decreased prostaglandin levels in the media and increased the numbers of gap junctions in those control tissues that developed few junctions over the same incubation interval . We conclude that the capacity of human myometrial tissues to develop gap junctions in vitro may depend upon a maturational stage in preparation for labor . Furthermore, our results suggest that products of the cyclo-oxygenase or lipoxygenase pathways may control the presence of gap junctions in the human myometrium and that changes in synthesis in these patterns may occur as part of the maturational process.

Dev Biol, 1987 Jun, 121(2), 548 - 58
Comparison of the effects of elevated K+ ions and muscle-conditioned medium on the neurotransmitter phenotype of cultured sympathetic neurons; Raynaud B et al.; Neuronal depolarization and culture media conditioned by certain nonneuronal cells (CM) are known to exert opposite effects on the expression of cholinergic and noradrenergic traits in cultured rat sympathetic neurons . We have compared their effects on the developments of choline acetyltransferase (CAT), tyrosine hydroxylase (TOH), dopa decarboxylase (AADC) and acetylcholinesterase (AcChE) in these cultures . A macromolecular factor which was partially purified from CM increased CAT development in a dose-dependent manner and depressed the development of TOH and AADC by 5- to 10-fold . In the presence of intermediate concentrations of this partially purified factor, both CAT and catecholamine synthesizing enzymes developed to high levels, whereas high concentrations caused a long-lasting, but not total, impairment of TOH development . The effects of CM on both CAT and AADC activities resulted from variations in the number of immunotitratable enzyme molecules . Conversely, K+ ions (30-40 mM) depressed the development of CAT by 90% and stimulated TOH development 2.5-fold . Cultures grown with CM in high K+ medium had similar CAT and TOH activities as compared to those cultures grown without CM in low K+ medium suggesting that CM and K+ ions had antagonistic effects on the expression of these enzymes . However, K+ ions did not affect the development of AADC in these cultures . CM suppressed in a reversible manner the development of the 16 S form of AcChE . In the presence of 40 mM K+, the rate of development of AcChE was reduced . In particular, the development of 16 S AcChE was strikingly impaired, although not totally suppressed . The effect of elevated K+ ions on the percentage of 16 S AcChE was rapidly reversible . It is concluded that CM and elevated K+ ions have antagonistic effects on CAT and TOH, but not on AADC development; AcChE, in particular its asymmetric 16 S form, is regulated independently of the cholinergic/noradrenergic status of sympathetic neurons.

Brain Res, 1987 May 12, 411(1), 102 - 7
Characterization of PGE2 inhibition of corticotropin releasing factor-mediated ACTH release; Sobel DO; The role of prostaglandin E2 (PGE2) on the mechanism of corticotropin releasing factor (CRF) induced adrenocorticotropin (ACTH) release was studied in primary rat pituitary cell culture . The continuous incubation of pituitary cells with PGE2 inhibited CRF-stimulated ACTH with an ED50 of 1.2 X 10(-9) M PGE2 . PGE2, however, did not alter the spontaneous release of ACTH . PGE (10(-8) M) significantly decreased 10(-10) M, 10(-9) M, and 10(-8) M CRF-mediated ACTH release by 42%, 47%, and 31% of total CRF stimulated ACTH release . Time course experiments demonstrated a PGE2-induced inhibition by 20 min of CRF incubation which continued for 3 h . After a 2-h incubation with PGE2, the wash-out of PGE2 from the culture medium just prior to the addition of CRF eliminated the inhibitory activity of PGE2 . PGE2 decreased the amount of CRF-stimulated ACTH from cells incubated with cycloheximide (P less than 0.01) . The inhibitory activity of PGE2 (10(-8) M) on CRF-stimulated ACTH was unaltered by the addition of 3 mM or 7 mM CaCl2 to the standard culture media (1.6 mM CaCl2) . The inhibition of CRF-induced ACTH release by maximal inhibitory concentrations of PGE2 (10(-7) M) and cortisol (5 X 10(-7) M) were not additive . In conclusion, PGE2 may play an important role in modulating pituitary ACTH release . Its inhibitory activity occurs by 20 min of CRF incubation, is in part independent of protein synthesis, requires the continued presence of PGE2, is not reversed with CaCl2, and is not additive with the inhibitory activity of cortisol.

Anticancer Res, 1987 May-Jun, 7(3 Pt B), 381 - 3
Primary human tumor cells and continuous cell lines differ in clonal growth in soft agar; Katoh A et al.; This study is a sequel in our continuing efforts to improve the colony forming efficiency of the Human Tumor Colony Assay . We have previously reported that the use of culture media supplemented with rat red blood cells and a low oxygen environment resulted in notable improvements in the clonal growth of three different tumor cell lines (colon, breast and melanoma) . We now report, however, that the application of these methods to cells from 18 different samples of primary human tumors resulted in no improvements in growth . Our results suggest that caution must be exercised in extrapolating results obtained from the use of homogeneous cell lines to that of cells from human solid tumors.

In Vitro Cell Dev Biol, 1987 May, 23(5), 361 - 6
Extraction of an erythrotropin-like factor from bovine serum albumin (Cohn fraction V); Congote LF; Different batches of commercially available bovine serum albumin (Cohn fraction V) were tested in a serum-free medium for their ability to stimulate thymidine incorporation in erythroid cells of fetal bovine liver . All preparations stimulated thymidine incorporation . Crystallized, charcoal-treated, or fatty acid-free albumin had substantially lower thymidine incorporation-stimulating activities than the crude preparations . The albumin preparations also had a synergistic effect with respect to erythropoietin on erythroid cells from rat liver, a typical property of erythrotropins . One gram of one of the batches of Cohn fraction V was fractionated by reversed-phase high performance liquid chromatography (HPLC) . The fraction with thymidine incorporation-stimulating activity had a similar elution position as erythrotropin isolated from fetal bovine serum . Further purification using reversed-phase HPLC in the presence of trifluoroacetic acid and heptafluorobutyric acid and gel permeation HPLC resulted in the isolation of a factor that is very similar to fetal bovine serum erythrotropin . It has practically the same specific activity as the purified fetal peptide in the rat liver bioassay . These results suggest that many of the beneficial effects of the albumin preparations added as supplement of serum-free tissue culture media may be due to the presence of erythrotropin-like factors.

Carcinogenesis, 1987 May, 8(5), 689 - 97
Enhanced morphological transformation of early passage Syrian hamster embryo cells cultured in medium with a reduced bicarbonate concentration and pH; LeBoeuf RA et al.; Recent studies from our laboratory have demonstrated that clonal cell proliferation of early passage Syrian hamster embryo (SHE) cells is optimal at a bicarbonate concentration in the culture medium of 8.9 mM (pH 6.65-6.75) under the experimental conditions reported . The purpose of the studies reported here was to examine whether morphological transformation induced by benzo{a}pyrene (BP) was enhanced under optimal culture conditions for SHE cell proliferation . Culture media of pH 6.70, 7.11 and 7.34 under incubator conditions of 10% CO2 in air were obtained by the addition of 0.75 (8.9 mM), 2.25 (26.8 mM) and 3.75 g/l (44.6 mM) of NaHCO3 respectively to a modified formulation of Dulbecco's modified Eagles medium . The frequency of morphological transformation of SHE cells was increased at 8.9 mM bicarbonate (pH 6.70) relative to media containing 26.8 or 44.6 mM bicarbonate (pH 7.11 and 7.34 respectively) . Additionally, the isolate of embryo cells and lot of fetal bovine serum used supported transformation induced by BP at 8.9 mM bicarbonate (pH 6.70), but did not with media of higher bicarbonate concentration and pH . The duration of cell culture and the no . of colonies per plate influenced the amount of increase of morphological transformation observed at 8.9 mM bicarbonate relative to media of higher bicarbonate concentration . Initial studies have shown that a fraction of morphologically transformed colonies generated at reduced bicarbonate concentration were tumorigenic in newborn hamsters . These results are discussed in terms of the potential utility of low bicarbonate concentration cultured SHE cells for transformation studies.

J Periodontol, 1987 May, 58(5), 349 - 51
In vitro evaluation of extracts of mineralized tissues for their application in attachment of fibrous tissue; Somerman MJ et al.; A primary objective in the treatment of periodontal diseases is attachment of fibrous tissue to root surfaces . An in vitro method was used to evaluate agents for their potential in enhancing this attachment process . Guanidine EDTA protein extracts of alveolar bone, cementum, and dentin in culture media were added to petri dishes and incubated at 37 degrees C for 1 hour . Human gingival fibroblasts were added to the dishes and incubated for an additional 90 minutes . Following incubation, cells attaching to the dish were quantified electronically, using a Coulter Counter . Extracts of alveolar bone and cementum enhanced cell attachment while dentin extract had no effect . These results indicate that both cementum and bone contain attachment proteins which could prove beneficial for attachment of fibrous tissue to root surfaces.

EMBO J, 1987 May, 6(5), 1281 - 6
Transforming growth factor-beta is a strong and fast acting positive regulator of the level of type-1 plasminogen activator inhibitor mRNA in WI-38 human lung fibroblasts; Lund LR et al.; We have studied the mechanism of a transforming growth factor-beta (TGF-beta)-stimulated production of type-1 plasminogen activator inhibitor (PAI-1) in WI-38 human lung fibroblasts . TGF-beta causes an early increase in the PAI-1 mRNA level which reaches a maximal 50-fold enhancement after 8 h . Blocking of protein synthesis with cycloheximide causes an equally strong increase in the level of PAI-1 mRNA . Quantitative studies of the effect of TGF-beta on PAI-1 protein levels in cell extracts and culture media by using monoclonal antibodies are consistent with the effect on PAI-1 mRNA . The results suggest a primary effect of TGF-beta on PAI-1 gene transcription, and also suggest the possibility that the transcription of this gene in non-induced cells may be suppressed by a short-lived negatively regulating protein . Urokinase-type (u-PA) and tissue-type (t-PA) plasminogen activators are decreased in the culture media of TGF-beta-treated cells concomitantly with the increase in PAI-1 accumulation . These findings show that a primary and important biological effect of TGF-beta may be an overall decreased extracellular proteolytic activity, and give an insight into the molecular mechanisms underlying TGF-beta action at the genetic level.

Cancer Res, 1987 May 1, 47(9), 2380 - 4
Nutritional requirements of human malignant (leukemic) cell lines: implications for adjuvant therapy; Beebe DP et al.; Metabolic requirements of malignant cell lines derived from patients with chronic myelogenous and acute lymphoblastic leukemias were compared to those of proliferating normal cells (mitogen-stimulated human lymphocytes) and circulating blasts from acute myeloblastic and acute lymphoblastic leukemias . Requirements were judged by degree of amino acid (AA) utilization in short-term cultures and assessed by the effect of selective AA deprivation on cell growth . Cell growth was measured by DNA synthesis and growth rate analysis . Six AAs (serine, threonine, methionine, valine, phenylalanine, and lysine) were appreciably utilized (52-87%) by IM-9, CEM, MOLT-4, and K-562 cells, but little or no utilization of these or any other AAs were noted in HSB cells, in leukemic blasts, or in mitogen-stimulated normal lymphocytes in short-term culture . Omission of lysine from culture media greatly inhibited cell growth (DNA synthesis by 91%), and cell density (by 83%) of IM-9 cells . However, omission of lysine, valine, serine, threonine, methionine, or phenylalanine had less of an effect on CEM and MOLT-4 cell lines . These observations demonstrate that under the conditions used the IM-9 cell line is uniquely dependent on extracellular lysine levels in contrast to the other cell lines studied . This suggests that human malignancies other than acute lymphoblastic leukemia which exhibits an obligate dependence on extracellular asparagine might be manageable by enzymatic degradation in vivo or by dietary restriction of indispensable AAs.

J Cell Physiol, 1987 May, 131(2), 276 - 83
Characterization of the receptor for protease nexin-I:protease complexes on human fibroblasts; Howard EW et al.; Fibroblasts as well as several other cell types, secrete a number of protease inhibitors into their culture media . Among these inhibitors are the protease nexins, a class of proteins which covalently bind serine proteases, thereby inactivating their specific targets . Protease nexin-I, first discovered in human foreskin fibroblasts, binds thrombin, plasmin, and urokinase with high affinity, forming covalently linked complexes . Human fibroblasts bind complexes of protease nexin-I and its target protease via a cell-surface, high-affinity receptor . We have analyzed a number of characteristics of this receptor, and found them to be typical of class II receptors in general . At 4 degrees C binding of PN-I:protease complexes was competed by heparin . In addition, binding was independent of the particular protease bound to the PN-I; purified complexes of PN-I with thrombin or urokinase competed equipotently for {125}I-thrombin:PN-I binding . As the pH of the binding buffer was lowered, binding to cells increased . A twofold increase in binding was attained by lowering the pH from 7.5 to 4.5 . This phenomenon was not due to irreversible, pH-induced changes to either the cell surface or the labeled complexes . At 37 degrees C, the removal of labeled complexes from culture medium was rapid; approximately 80% was removed by 4 hours under given conditions . The internalization of complexes was also very rapid, with an estimated ke (endocytic rate constant) of 1.0 min-1 . At neutral pH, fibroblasts bind complexes in a saturable manner . Scatchard analysis yields a receptor number of 250,000 per cell and a Kd of 1 nM.

Placenta, 1987 May-Jun, 8(3), 257 - 64
Response of human chorionic gonadotrophin to luteinizing hormone-releasing hormone stimulation in the culture media of normal human placenta, choriocarcinoma cell lines, and in the serum of patients with gestational trophoblastic disease; Kim SJ et al.; In vitro and in vivo responses to luteinizing hormone-releasing hormone (LHRH) stimulation of human chorionic gonadotrophin (hCG) production were evaluated . We cultured placental tissues of ten weeks and term pregnancy, choriocarcinoma tissues, and monolayers of the BeWo cell line, and added serial dilutions of LHRH (1, 5 and 10 micrograms) to the media for five to seven days . In in vivo experiments, 100 micrograms LHRH was intravenously administered to 20 normally cycling women (control group), 27 women who were 'possible remission', and 21 women with 'minimal resistance' to gestational trophoblastic disease . After injection of LHRH, blood samples were collected at 0, 30, 60, 90 and 120 minutes . The concentrations of immunoreactive beta-hCG in in vitro culture media, and in sera from patients, were measured before and after LHRH stimulation by double-antibody radioimmunoassay . These in vitro and in vivo experiments revealed that normal and malignant trophoblastic cells responded to the LHRH stimulation by producing immunoreactive beta-hCG . Therefore, LHRH stimulation may be useful in detecting residual choriocarcinoma cells in gestational trophoblastic disease patients during their periremission periods.

Biochemistry, 1987 Apr 7, 26(7), 2067 - 70
Identification and subcellular localization of a 21-kilodalton molecule using affinity-purified antibodies against alpha-transforming growth factor; Hazarika P et al.; Monospecific antibodies were generated against each of six different peptide sequences derived from rat and human alpha-transforming growth factor (alpha-TGF) . The affinity-purified antibody to the 17 amino acid carboxyl-terminal portion of the molecule proved most useful in detecting alpha-TGF . When used in a peptide-based radioimmunoassay, it was possible to measure nanogram quantities of native alpha-TGF in conditioned cell culture media . When used to analyze cell lysate, these antibodies specifically recognized a 21-kilodalton protein species . Indirect immunofluorescence localization procedures revealed a high concentration of alpha-TGF in a perinuclear ring with a diffuse cytoplasmic distribution . These results suggest that a precursor form of alpha-TGF has a cellular role beyond that of an autocrine growth factor.

Lipids, 1987 Apr, 22(4), 241 - 9
Influence of environmental medium on fatty acid composition of human cells: leukocytes and fibroblasts; Delplanque B et al.; Fibroblasts in culture and leukocytes have been widely used to study fatty acid and lipoprotein cellular metabolism . The present investigations were designed to study the role of nutritional and environmental factors on lipid metabolism in these two types of cells . Leukocytes freshly isolated from human blood and fibroblasts cultured in media enriched in human serum (HS) have relatively similar fatty acid distributions . However, more important differences are observed in fibroblasts cultured in media enriched with HS or with fetal bovine serum (FBS) . It is obvious that the quantity and quality of fatty acids are very different in FBS and HS, but intracellular regulation ensures relative homogeneity of saturated (SFA) and monounsaturated fatty acids (MUFA) in the cells, particularly in phospholipids . The first modifications induced by different media (FBS or HS) are detected on cellular growth; the differences seem to be due more to the fatty acid (FA) quantitative supply than to the FA quality of each culture medium . The major modifications in FA composition induced by different culture media concern the polyunsaturated fatty acids (PUFA) of phospholipids, especially the n-6 family . The intracellular linoleic acid level depends on the level in the medium, but intracellular n-6 metabolite levels depend both on the level in the medium and on the growth state of the cells . The n-3 family seems to be less affected by the quality of the medium in our experiment, and the cells maintain a stable docosahexaenoic acid (22:6n-3) level . A higher content of the n-3 family in the medium induces a higher level of eicosa- or docosapentaenoic acid, rather than docosahexaenoic acid itself.(ABSTRACT TRUNCATED AT 250 WORDS)

J Biol Stand, 1987 Apr, 15(2), 117 - 26
Sterility testing of immunological products: comparative efficacy of media and effect of preservatives; Thornton DH et al.; A comparison was made of the abilities of various culture media to support the growth of a range of micro-organisms commonly recommended as control strains in tests for the sterility of immunological products . The effects of phenol, cresol, formaldehyde and thiomersal on the growth of these organisms were studied . Attention is drawn to some limitations of the current pharmacopoeial test methods.

J Invest Dermatol, 1987 Apr, 88(4), 434 - 8
In vitro growth characteristics of melanocytes obtained from adult normal and vitiligo subjects; Puri N et al.; The in vitro growth characteristics of melanocytes obtained from uninvolved and perilesional skin of vitiligo vulgaris subjects have been investigated in comparison to those from healthy adult donors . Normal human melanocytes have been found to grow exponentially in the presence of 10(-11) M cholera toxin and 10 ng/ml of 12-O-tetradecanoylphorbol-13-acetate in routine tissue culture media . They could be trypsinized up to 3-4 passages . Melanocytes of the uninvolved skin of vitiligo subjects manifested a lag of 8-11 days for the onset of growth and they could not be passaged . Melanocytes obtained from both hypo- and hyper-pigmented perilesional skin failed to grow under these conditions . Only in a few cases where the perilesional skin was normally pigmented did the melanocytes manifest some growth after a lag of 15 days . The initial seeding capacity of the melanocytes from uninvolved and perilesional skin of vitiligo patients were, respectively, 50% and 25% of the normal individuals . Vitiligo lesions themselves gave rise to unidentified dendritic cells that survived for 10-15 days without manifesting any growth . Our results suggest that melanocytes of individuals with vitiligo are defective . This fact has to be taken into account in any theory on the etiology of vitiligo.

Biochem Pharmacol, 1987 Apr 1, 36(7), 995 - 1002
Proteoglycan degradation by a chondrocyte metalloprotease . Effects of synthetic protease inhibitors; Caputo CB et al.; Synthetic inhibitors of a chondrocyte metalloprotease (CMP) were assessed for potency . Proteoglycan core protein was used as substrate . The IC50 values were between 2 X 10(-6) and 7 X 10(-6) M for two types of inhibitors, thiol tripeptides and N-carboxyalkyl peptides . Hydroxamic acid peptides were more potent, with IC50 values of 3.2 X 10(-8) to 6.0 X 10(-8) M . These results confirm inhibitory concentrations reported using a proteoglycan-polyacrylamide bead assay . The slopes of the dose-response curves for the thiol compounds were steeper than the slopes for the other two types of compounds . All of the culture media tested inhibited CMP to some extent . Some media also interfered with inhibitor activity . In Ham's F10 nutrient medium, minimum CMP inhibition occurred, and all four hydroxamic acid peptides retained their activity for 1-2 days at 37 degrees . One thiol peptide compound assayed lost activity in 1 hr in thiocyanate-treated serum . All four hydroxamic acid peptides assayed retained activity in thiocyanate-treated serum after 3 days at 37 degrees . The hydroxamic acid peptides may provide a way to block endogenous CMP activity in vivo and to assess the role of CMP in normal and experimentally altered cartilage . They are more potent than other known CMP inhibitors . They retain activity in culture media and serum conditions used for in vivo and in vitro tests of CMP activity and toxicity.

J Cell Sci, 1987 Apr, 87 ( Pt 3), 375 - 81
Regulation of epitectin production in a malignant cell line; Bader SA et al.; RT112 cells, a line derived from a human bladder carcinoma, produce epitectin at very low levels in standard culture media; but production and secretion of this mucin are greatly increased when the cells are exposed to hyperosmotic conditions . It appears that hyperosmolarity, by inducing an increase in intracellular sodium, entrains an increase in intracellular free calcium . Evidence is presented for the view that it is the increase in intracellular free calcium that provides the more direct stimulus for the enhanced production of epitectin.

Zentralbl Bakteriol Mikrobiol Hyg {A}, 1987 Apr, 264(1-2), 33 - 40
Detection of Shiga-like (SL) toxins of enteropathogenic Escherichia coli (EPEC) of human, porcine, calf, and lamb origin on Vero and HeLa S3 cells: a comparative study; Baloda SB et al.; One hundred and forty eight strains of human, porcine, calf and lamb origin belonging to different enteropathogenic O:H serotypes isolated in 13 countries in four continents were tested for production of Shiga, Shiga-like (SL) and other cytotoxins on Vero cells and HeLa (S3 subline) cells in tissue cultures . Altogether, 45% human strains and 89% porcine strains were defined as strong toxin producers (toxin titre greater than or equal to 1:100) on Vero or HeLa S3 cells while 31% of human and 9% porcine strains were regarded as moderate to weak toxin producers (toxin titre less than 1:100) . Twenty three percent of human and 1.5% of porcine strains were negative for Shiga or SL-toxin . Polymyxin release of Shiga or SL toxins from bacterial colonies of blood agar grown cultures is recommended as it is simple and effective method facilitating the detection of even low levels of toxins in EPEC or non-EPEC strains . Of the ten strains from calves and lambs, only four were strong toxin producers when cell-free culture supernatants were tested while a polymyxin release method showed that 8 strains were strong toxin producers . One strain was negative by both methods . The high proportion of Shiga/SL toxin negative strains in all O:H serotypes of human origin (but not of porcine origin, especially O 139 serogroup) suggests that systematic studies should continue to look for new toxins in freshly isolated strains grown under in vivo like conditions, e.g . in iron depleted culture media.

Zentralbl Bakteriol Mikrobiol Hyg {A}, 1987 Apr, 264(1-2), 120 - 30
Microcalorimetric assessment of liquid culture media; Allerberger FJ et al.; Microcalorimetry constitutes an analytic tool to register the heat effects produced by the metabolic processes taking place in a bacterial culture . Since these depend on the nutrients supplied in the media, microcalorimetry allows conclusions to be drawn about the nature and quality of the culture medium when using a standard germ . Taking Columbia Broth as an example, we showed that faulty weighing of the dry substrate, incorrect pH or overheating during the dissolving or autoclaving procedures could be detected by the use of the microcalorimetry . A microcalorimetric assessment to compare media of the same name produced by different manufacturers was carried out and significant differences were observed . We consider this microcalorimetric technique to be a valuable tool in the assessment of liquid culture media.

Endocrinology, 1987 Apr, 120(4), 1633 - 8
Follicle-stimulating hormone and somatomedin-C stimulate inhibin production by rat granulosa cells in vitro; Zhang ZW et al.; The direct effect of somatomedin-C (Sm-C) and FSH on inhibin production by rat granulosa cells in vitro has been examined . FSH stimulated accumulation of inhibin in culture media in a dose-dependent manner with maximal stimulation (6-fold) being observed at a dose of 300 ng FSH/ml . Addition of Sm-C (30 ng/ml) either alone or in the presence of FSH (3-300 ng/ml) increased inhibin production (up to 5-fold) . Sm-C alone was effective over the physiological dose range of 3-100 ng/ml . Concomitant addition of FSH (100 ng/ml) and Sm-C (3-100 ng/ml) resulted in a significant increase in inhibin production at all doses of Sm-C . The dose-dependent effects of FSH and Sm-C were also time dependent with a synergistic effect apparent after 48 h of culture . The Sm-C induced FSH inhibitory activity of granulosa cell culture media was confirmed as authentic inhibin by the demonstration of a dose-dependent neutralization of this activity by a monoclonal antibody raised against purified bovine inhibin . The data indicate a direct role for both FSH and Sm-C in ovarian inhibin production and provide additional evidence for an autocrine-paracrine role for Sm-C in granulosa cell differentiation.

Arthritis Rheum, 1987 Apr, 30(4), 424 - 30
Human recombinant interleukin-1 beta stimulates glycosaminoglycan production in human synovial fibroblast cultures; Yaron I et al.; Human recombinant interleukin-1 beta (rIL-1 beta) stimulated glycosaminoglycan (GAG) production in human synovial fibroblast cultures . A dose-dependent increase in GAG production was found, to a maximum of 500% . Increase was detected at doses as low as 1 pg/ml of rIL-1 beta, reached a maximum at 10-100 pg/ml, and was apparent 10 hours after addition of rIL-1 beta . Stimulation of GAG was always accompanied by increased accumulation of prostaglandin E (PGE) in culture media and by increased collagenase production in approximately one-half the experiments . Indomethacin (5 micrograms/ml) completely inhibited PGE stimulation by rIL-1 beta, but only partially inhibited that of GAG overproduction and had no effect on collagenase production . Hydrocortisone (2 micrograms/ml) inhibited stimulation of all 3 parameters . Stimulation of hyaluronate in synovial cultures prevailed over that of sulfated GAG, which occurred to a lesser extent . Our results support earlier suggestions that interleukin-1 is a major active mononuclear cell factor that is capable of inducing profound changes in connective tissue cell function.

Cell Immunol, 1987 Apr 1, 105(2), 411 - 22
Regulation of human peripheral blood monocyte collagenase by prostaglandins and anti-inflammatory drugs; Wahl LM et al.; Macrophages, which produce the collagenolytic enzyme collagenase, are commonly found at sites of connective tissue destruction in chronic inflammatory lesions . Since tissue macrophages are derived from circulating peripheral blood monocytes, we used these less-differentiated, more readily available cells to examine the production and regulation of collagenase . Human monocytes, isolated in large quantities by counterflow centrifugal elutriation, were shown to produce substantial amounts of collagenase when stimulated by concanavalin A (Con A) and to a lesser extent with lipopolysaccharide, while unstimulated monocyte cultures produced negligible collagenase . Collagenase was detected in the culture media within the first 24 hr of culture after activation with peak production at 48 hr . Analysis of the intracellular regulation of collagenase revealed that synthesis of this enzyme required a prostaglandin (PGE2)-dependent step since indomethacin-inhibited enzyme synthesis was reversed by PGE2 . Additionally, dibutyryladenosine cyclic monophosphate (dBcAMP) restored collagenase synthesis in indomethacin-blocked cultures, indicating a PGE2-dependent generation of cAMP requirement for collagenase production similar to that demonstrated in experimental animals systems . In additional studies, anti-inflammatory drugs which are known to modulate connective tissue destruction were analyzed for their influence on monocyte-derived collagenase . Dexamethasone, colchicine or retinoic acid all inhibited collagenase synthesis by monocytes in a dose-dependent manner although the effect of these drugs on monocyte PGE2 synthesis differed . Dexamethasone inhibited PGE2 synthesis, which resulted in the suppression of collagenase . However, PGE2 production was unaffected by colchicine whereas retinoic acid caused a significant increase in PGE2 levels . Inhibition of collagenase synthesis by dexamethasone, but not colchicine or retinoic acid, could be reversed by PGE2 or phospholipase A2 . These findings provide insight into the intracellular events regulating monocyte collagenase synthesis and also implicate monocytes as a target of anti-inflammatory agents which ameliorate connective tissue degradation associated with chronic inflammatory lesions.

J Neurol Sci, 1987 Apr, 78(2), 139 - 50
Tissue culture evidence for a circulating neurotoxin in Huntington's chorea; Perry TL et al.; We explored with tissue culture techniques the possibility that a circulating neurotoxin might cause the premature loss of certain populations of neurons that characterizes Huntington's chorea (HC) . Explants of striatum from newborn rats were grown in culture media containing 30% by volume of serum from drug-free HC patients or from healthy control subjects . Glutamic acid decarboxylase (GAD), the enzyme which synthesizes gamma-aminobutyric acid (GABA), was later assayed in these explants as an indicator of the health of GABAergic striatal neurons . The sera of 7 of 8 HC patients decreased GAD activity markedly when present as 30% of the tissue culture medium . When present in lower concentration (15%), HC sera either decreased or increased GAD activity in explants . Deproteinization of sera with perchloric acid did not abolish these effects on GAD activity . A depressant effect on GAD activity was detected in the cerebrospinal fluid of 1 of 4 HC patients tested . These experiments suggest the presence of a circulating neurotoxin, possibly excitotoxic to GABAergic striatal interneurons, and probably a small molecule . Identification of this substance could lead to an effective preventive treatment for persons genetically at risk for HC.

J Immunol, 1987 Mar 15, 138(6), 1999 - 2007
Activated macrophages and antibodies against the plant lectin, GSI-B4, recognize the same tumor-associated structure (TAS); Takacs B et al.; Activated macrophages that were stabilized with either formalin or glutaraldehyde absorbed two polypeptides (Mr 100,000 and 60,000) from detergent extracts of all of the tumor cell lines tested, but not from detergent extracts of normal human peripheral blood lymphocytes . A major polypeptide (Mr 95,000) was retained from spent culture media of tumor cell lines . Polypeptides with molecular sizes of 100,000 and 60,000 daltons were also adsorbed by activated macrophages from detergent extracts of chicken embryo cell membranes, suggesting an oncofetal nature for these proteins . The 100,000 dalton polypeptide, but not the 60,000 dalton component, was found to be available to lactoperoxidase-catalyzed cell surface iodination . Polypeptides with identical molecular sizes could be adsorbed to immobilized galactopyranoside, indicating that they are vertebrate lectins . Activated macrophages and affinity adsorbents prepared by the covalent coupling of galactopyranoside to agarose also bind the plant lectin isolectin B4 prepared from the seeds of Griffonia simplicifolia . On the basis of these findings, we put forth the hypothesis that macromolecules of the same specificity, that is affinity to galactopyranosyl residues, must show homologies in their binding sites . We have predicted therefore that antisera prepared against this plant lectin should cross-react with galactopyranosyl-binding vertebrate lectins present on the surface of tumor cells . In this communication, we also report the generation of hybridomas that produce antibodies reactive with both the plant and vertebrate lectins . Inhibition experiments that make use of various mono- and disaccharides suggest that the specificities of these antibodies are for determinants intimately associated with the galactosyl binding site on the lectin molecule . Two of the antibodies were found to have moderate selectivity for tumor cells when tested in an immunohistochemical procedure that made use of fresh-frozen or paraffin-embedded sections of human biopsy material . These two antibodies on immunoblots of tumor cell membrane extracts reacted with a polypeptide with an apparent molecular size of 100,000 daltons.

J Immunol, 1987 Mar 15, 138(6), 1845 - 51
Characterization of a monoclonal antibody directed against the murine B lymphocyte receptor for IgE; Rao M et al.; A rat hybridoma producing a high-affinity IgG2a monoclonal antibody (B3B4) directed against against the murine lymphocyte IgE receptor (Fc epsilon R) was established by using purified Fc epsilon R from Fc epsilon R+ murine hybridoma B cells as immunogen . The monoclonal and polyclonal anti-Fc epsilon R inhibited the binding of IgE to the murine lymphocyte Fc epsilon R and were also used to isolate the Fc epsilon R . B3B4 specifically recognized only the 49-Kd Fc epsilon R on murine B lymphocyte as determined by immunoprecipitation and SDS-PAGE analysis . In addition to its reaction with intact Fc epsilon R, B3B4 also recognized Fc epsilon R fragments that were present in the culture media of Fc epsilon R+ hybridoma cells . The predominant fragments isolated were 38 Kd and 28 Kd by SDS-PAGE analysis . When tested for reactivity with other cell types, B3B4 was highly specific for murine B lineage cells in that it did not significantly react with Fc epsilon R on macrophages and T cells and, in addition, did not react with the high affinity mast cell Fc epsilon R . B3B4 completely blocked IgE rosetting, and a reciprocal inhibition of binding was seen in a dose-dependent fashion between IgE and B3B4, indicating a close proximity of the IgE and B3B4 binding sites . Saturation binding analysis indicated that the Fab' fragment of B3B4 bound to twice as many sites/cell as IgE, suggesting that there are two identical B3B4 determinants per 49-Kd Fc epsilon R or that the IgE binding site is formed by the association of at least two 49-Kd Fc epsilon R . However, unlike IgE, neither B3B4 nor F(ab')2-B3B4 nor Fab'-B3B4 were very effective in causing Fc epsilon R upregulation on murine hybridoma B cells; in fact, B3B4 prevented this upregulation when added in combination with IgE . These results suggest that a site-specific interaction provided only by IgE may be essential for ligand-specific upregulation . Both polyclonal and monoclonal antibodies will be useful in further studies concerning the functional relationship between the membrane Fc epsilon R and the soluble Fc epsilon R fragments.

Ann Thorac Surg, 1987 Mar, 43(3), 341 - 7
Legionnaires' disease: an emerging surgical problem; Korvick JA et al.; Legionnaires' disease is an important, although often overlooked, complication in the patient postoperatively . Up to 50% of all nosocomial legionellosis in the hospitals reviewed was found in surgical patients . Patients undergoing a transplant procedure are at highest risk, but occurrence is common in the surgical patient undergoing general anesthesia, endotracheal intubation, or both . Aerosolization, aspiration, and direct instillation of contaminated water during manipulation of the respiratory tract are likely mechanisms of transmission . The usual clinical presentation is that of a nonspecific pneumonia . Specialized laboratory techniques including selective culture media, direct fluorescent antibody stains, and serological detection of antibodies are necessary for accurate diagnosis . If these tests are not routinely available, Legionnaires' disease may remain undiagnosed . Environmental surveillance of the hospital water distribution system is advisable for hospitals with a large surgical case load . If transplantation is performed, such surveillance is mandatory.

Gan To Kagaku Ryoho, 1987 Mar, 14(3 Pt 1), 749 - 53
{Primary cultures of various differentiated human cells and their transfer (3) . Apparatus and instrument for primary culture}; Yamane I; Clean bench, water distilled, low speed centrifuge, CO2-incubator and invert microscope were described and discussed as those necessary for the primary culture of differentiated human cells . Also the instruments including knives, filter sterilization equipment, the reagents including culture media were described and discussed as ones necessary for those primary cultures.

Vet Immunol Immunopathol, 1987 Mar, 14(3), 233 - 44
In vitro effects of prostaglandin E1, prostaglandin E2, indomethacin, histamine, and tuftsin on chemiluminescence response of bovine polymorphonuclear leukocytes; Phillips TR et al.; The in vitro effects of prostaglandin E1 (PGE1), prostaglandin E2 (PGE2), indomethacin, histamine, and tuftsin on the chemiluminescence response of bovine polymorphonuclear cells (PMN) were determined . Addition of PGE1, PGE2, indomethacin, and histamine in vitro significantly suppressed the chemiluminescence response of bovine PMN's, whereas tuftsin had no effect . Suppression was dependent upon the continued presence of PGE1, PGE2, and histamine in the culture media . However, indomethacin's suppressive effect remained even after it was removed from the culture media . Hydrogen peroxide generated chemiluminescence was suppressed by high concentrations of indomethacin and histamine . Results of this study suggest possible pharmacologic or regulatory mechanisms for certain of these immune modulators in the control of the oxidative burst reaction of bovine PMN's.

Acta Pathol Jpn, 1987 Mar, 37(3), 413 - 23
Fibronectin in human hepatocellular carcinoma (HCC) and HCC cell lines; Tsumagari J; The localization of fibronectin (FN) in human hepatocellular carcinoma (HCC) was studied in thirty-six HCC tumors (19 autopsy and 17 surgical specimens), two xenografted tumors of HCC to BALB/c mice and three HCC cell lines . The synthesis of FN was also examined in three HCC cell lines . FN was demonstrated on the endothelial surface of the blood spaces of cancerous tissue . Cytoplasmic and intercellular localization of FN was also observed . But there was no correlation between the localization pattern of FN and metastasis . In the two xenografted HCC tumors, FN was found in association with the blood vessels of the tumor tissue and between the HCC tumor cells . In all 3 HCC cell lines, FN was localized on the surface and in the cytoplasm of some HCC cells . FN was detected in the serum-free culture media of three HCC cell lines by immunoelectroblotting . The electrophoretic pattern of FN synthesized by these cell lines was different from that of plasma-FN and resembled that of cellular-FN synthesized by normal liver fibroblasts.

J Gen Virol, 1987 Mar, 68 ( Pt 3), 793 - 803
Beta 2 microglobulin enhances the infectivity of cytomegalovirus and when bound to the virus enables class I HLA molecules to be used as a virus receptor; Grundy JE et al.; We have previously demonstrated that human cytomegalovirus (CMV) binds the host protein beta 2 microglobulin (beta 2m) from body fluids or from cell culture media . In this report we have examined the effect of the beta 2m on viral infectivity . We have shown that the addition of human purified beta 2m, or a fraction of foetal calf serum corresponding to bovine beta 2m, to culture medium increased the amount of infectious extracellular CMV, compared to that from cells grown in serum-free medium . Metabolic labelling experiments demonstrated that this effect was not due to an increase in the amount of extracellular virus but to an increase in the infectivity of the virus present in extracellular fluids . We concluded that the binding of beta 2m by CMV increased its infectivity . We have shown that CMV and beta 2m compete for binding sites on fibroblasts . As the main binding site on cells for beta 2m is the class I HLA heavy chain we compared the binding of CMV to the Raji and Daudi cell lines which express or lack expression of class I HLA molecules . The binding of radiolabelled beta 2m-coated CMV was significantly higher to Raji cells than to Daudi cells . Furthermore, CMV could compete with beta 2m for binding to Raji cells, although the reverse was not true . These results demonstrate that CMV can use class I HLA molecules as a virus receptor . We propose that when coated with beta 2m, CMV has the capacity to displace beta 2m from the class I HLA heavy chain-beta 2m dimer on the cell surface and bind to cells . The fact that beta 2m enhances infectivity suggests that such binding leads to productive infection of cells.

Am J Physiol, 1987 Mar, 252(3 Pt 1), E304 - 12
Transport epithelial characteristics of cultured bovine pituitary follicular cells; Ferrara N et al.; Confluent monolayers of polygonal epithelioid cells were obtained from enzymatically and mechanically dispersed bovine anterior pituitaries (AP) and pars tuberali (PT) . The ultrastructure of the cells composing the monolayer was consistent with the follicular or folliculostellate cells (FC) of the pituitary, i.e., lack of secretory granules; formation of follicles in culture; interdigitations with neighboring cells with numerous tight junctions; presence of extensive microfilaments; and sparse rough endoplasmic reticulum and Golgi apparatus . Culture media from monolayers of first passage cultures contained little if any of the AP hormones' luteinizing hormone, prolactin, and ACTH . Shortly after reaching confluency, regions of the monolayer bulge away from the surface of the culture dish to form domes . Dome formation has been described only with cultures of cells that function as transport epithelia in vivo . FC cultured on polycarbonate filters were placed in Ussing chambers . A transepithelial potential difference of approximately 1.1 mV and a resistance greater than 300 omega cm2 were detectable 4-5 days after plating . The short-circuit current (Isc) was decreased 70% by amiloride applied to the mucosal surface and further decreased by the addition of ouabain at the serosal surface . The beta-adrenergic agonist isoproterenol increased the Isc and this action was prevented by a beta-antagonist . These observations indicate that pituitary FC in culture behave as a transport epithelium . Considering the organization of FC in the AP and PT, they suggest a regulatory role for FC in the maintenance of the ionic composition of the interstitial fluid of the pituitary gland.

Cancer Res, 1987 Feb 15, 47(4), 1161 - 9
Glycoproteins distinguishing non-small cell from small cell human lung carcinoma recognized by monoclonal antibody 43-9F; Pettijohn DE et al.; Cell lines derived from human squamous lung carcinoma release large amounts of a soluble glycoprotein into the culture media, having very high molecular weight (greater than 2 X 10(6} and mucin-like properties . A monoclonal antibody called 43-9F has been generated that recognizes a carbohydrate epitope on the glycoconjugate . The epitope is also present on a diverse set of smaller glycoproteins (Mr 50,000-200,000) distributed primarily on the surface of the squamous lung carcinoma cells . A sensitive assay using the 43-9F antibody in a dot blot procedure has been devised that is able to detect an amount of antigen less than that possessed by a single squamous lung carcinoma cell . This assay, and also conventional immunofluorescence and immunohistochemical assay procedures, have been used to screen different normal cells, normal tissues, cancer cells, and tumor biopsy specimens for the antigen . In the normal lung the 43-9F antigen is found only on cells of some of the seromucous glands . In the normal digestive system it is associated in certain organs only with a limited population of mucosal epithelial cells . Other organ systems lack any reactive cells . The cells of most human non-small cell lung carcinomas and their released glycoconjugates have large amounts of the 43-9F epitope, while small cell lung carcinomas and the glycoconjugates released by small cell lung cancer cells lack the epitope . The oligosaccharide recognized by the 43-9F antibody may therefore provide a useful marker to distinguish the different lung carcinomas and for investigating the different cells of origin of these tumors.

J Immunol Methods, 1987 Feb 11, 96(2), 157 - 64
Third component of rat complement . Purification from plasma and radioimmunoassay in culture media from cell lines; Guiguet M et al.; A double antibody radioimmunoassay for rat C3 has been developed . The assay required the preparation of C3 from plasma . A new purification procedure using ion exchange high performance liquid chromatography is described . The final product was homogeneous on SDS-PAGE analysis . Rat C3 has an apparent molecular weight of 187,000 and is composed of two polypeptide chains with molecular weights of 125,000 and 73,000, respectively . The purified C3 antigen with high hemolytic reactivity, as assessed by its specific functional activity, was used in preparing anti-C3 sera to perform a specific radioimmunoassay for quantifying C3 in the presence of heterologous sera contained in the cell culture media . All the validating criteria, such as precision, recovery and dilution studies, were investigated . The high sensitivity of the method allowed replicate determination of C3 in small aliquots of the cell culture medium.

Am J Physiol, 1987 Feb, 252(2 Pt 2), R209 - 15
Changes in pituitary growth hormone cells prepared from rats flown on Spacelab 3; Grindeland R et al.; Anterior pituitaries from "small" (250 g) and "large" (400 g) rats flown on the 7-day Spacelab 3 mission were pooled and trypsinized into two single-cell suspensions . Compared with ground-based controls, flight cells appeared to contain more intracellular growth hormone (GH) but release less GH over a 6-day culture period . After implantation into hypophysectomized rats, both sets of flight cells released only 50% of the GH compared with the control cells . Glands from large flight rats contained 44% somatotrophs compared with 37% for controls; small animals showed no difference . There were no striking differences in somatotroph ultrastructure between cells in the four groups . Western blot analysis indicated that there were no major differences in immunoactive GH variants . High-performance liquid chromatography fractionation of culture media indicated that small flight cells released much less of a high-molecular weight variant rich in GH bioactivity . The results suggest that GH cells from rats exposed to microgravity may experience secretory dysfunction . The possibility that this occurs directly at the pituitary cell level is discussed.

Surgery, 1987 Feb, 101(2), 228 - 33
Effects of tumor bearing and removal on blood levels of lipids, lipolytic activity, and glycerol and on carcass weight in the rat; Devereux DF et al.; In this animal model we investigated the effects of sarcoma bearing and complete or incomplete tumor excision on blood levels of lipids, lipolytic activity, and glycerol and on carcass weight . We also sought causes that could account for the apparent increase in lipid mobilization . Tumor-bearing (TB) rats had depleted retroperitoneal fat stores, elevated blood triglycerides and cholesterol, and normal thyroid, hepatic, and renal function during tumor bearing . Catecholamine, growth hormone, and glucagon levels were not different between TB and nontumor-bearing (NTB) groups . Serum lipolytic activity and glycerol levels, which were elevated during tumor bearing, returned to NTB levels after complete tumor excision, along with restoration of retroperitoneal fat depots and carcass weight . The elevated serum lipolytic activity of animals that had tumors only incompletely excised rose significantly higher after the tumor regrew; this was accompanied by a failure to regain carcass weight . Previous work has shown that elevated lipolytic activity can also be demonstrated in culture media conditioned by growth of certain animal or human tumor cell lines in cell culture, free of host influences . This suggests that the actual tumor cell and not the host may be at least partly responsible for the lipid derangements seen in this model.

Exp Mol Pathol, 1987 Feb, 46(1), 64 - 77
Isolation and maintenance of monolayer hepatocytes from the livers of toxin-treated rats; MacDonald JR et al.; Hepatocytes were isolated from male Sprague-Dawley rats 30 min after oral challenge with carbon tetrachloride (CCl4), or 1 hr after ip injection of D(+)-galactosamine (GAL) . Cell preparations of comparable yield and initial viability were obtained from toxin-treated and respective control animals with equivalent 1-hr attachment efficiencies to tissue culture plastic . Cells from toxin-treated rats exhibited significant degeneration over 48 hr in culture . This degeneration included loss of viable cell density on the monolayer and increased lactate dehydrogenase activity in the culture media compared to controls . Toxicity expressed in culture was dose dependent for both CCl4 (0.1-2.5 ml/kg) and GAL (100-400 mg/kg) . Loss of cell viability in vitro and ultrastructural degeneration followed a time course consistent with hepatocellular necrosis produced by these agents in vivo . As a model for studying late occurring events in the progression from initiation of toxic cell injury to cell death, this methodology offers several potential advantages over strict in vivo or in vitro models . The analytical advantages of an in vitro cell model are incorporated with initiation of injury in vivo by physiologically relevant doses of hepatotoxins . Limited bioactivating potential of monolayer hepatocytes, and time course limitations of suspension hepatocytes for toxicity studies, are also circumvented in this model.

Parasitology, 1987 Feb, 94 ( Pt 1), 39 - 48
Isoenzyme characterization of trypanosomes of the subgenus Herpetosoma; Mohamed HA et al.; Isoenzyme analysis was used to characterize 6 species of trypanosomes of the subgenus Herpetosoma using 13 different enzyme systems . The species studied were Trypanosoma lewisi, T . musculi, T . grosi, T . microti, T . evotomys and T . nabiasi which cannot be distinguished on morphological grounds . Extracts for thin-layer starch-gel electrophoresis were prepared from cultures of insect forms in either Schneider's Drosophila or Grace's insect tissue culture media with foetal calf serum or a nutrient agar medium . Extracts of T . lewisi and T . musculi bloodstream forms were also run for comparison . All parasites gave distinct patterns which enabled them to be differentiated on one or more enzyme systems . Two types of computer analysis were used to group the parasites; using these techniques the murine parasites T . lewisi, T . musculi and T . grosi fell into one broad group, and T . microti and T . evotomys of microtine rodents formed another . These findings are in accord with earlier observations on the behavioural characteristics of these parasites in their mammalian host and their vector (fleas) . The clear differences observed provide the basis for the application of other biochemical and immunological techniques for differentiation within this subgenus of trypanosomes.

J Invest Dermatol, 1987 Feb, 88(2), 207 - 11
Human keratinocytes synthesize and secrete the extracellular matrix protein, thrombospondin; Wikner NE et al.; Thrombospondin (TSP) a glycoprotein originally identified as the endogenous lectin of platelets, is also synthesized by fibroblasts, endothelial cells, pneumocytes, smooth muscle cells, and macrophages . Thrombospondin is subdivided into functional domains which bind specifically to heparin, fibronectin, collagen, and to specific cellular receptors . It is found within the basement membranes of kidney, lung, smooth muscle, and skin . Thus TSP may serve as an important link between cells and matrices . Thrombospondin also has been reported at the epidermal-dermal junction . We wished to determine whether human keratinocytes synthesize and secrete TSP . Pure human keratinocytes were grown in defined medium without fibroblast feeder layers . Immunofluorescent staining with either rabbit polyclonal or mouse monoclonal antibodies to human platelet TSP yielded specific granular staining within the cytoplasm of keratinocytes . Culture media and cellular lysates were harvested from cultures metabolically labeled with {35S}methionine . Trichloroacetic acid precipitation, sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), and autoradiography revealed a major labeled band comigrating with purified platelet TSP in both the media and the cellular lysates . Immunoprecipitation with either the polyclonal or the monoclonal anti-TSP antibodies followed by SDS-PAGE and autoradiography identified this band as TSP . Thus keratinocytes in culture synthesize and secrete TSP . Thrombospondin may play an important role in epidermal interactions with extracellular matrix.

Zh Mikrobiol Epidemiol Immunobiol, 1987 Feb, (2), 3 - 7
{Cultivation of the gonococcus in nutrient broths}; Osmanova MM et al.; The dynamics of the multiplication of gonococci and the parameters of their growth have been studied in the process of batch cultivation in liquid culture media with different content of bovine blood serum . The protective and stimulating action of the serum on the growth of gonococci has been shown . The optimization of the process of cultivation has been carried out; as a result, the growth of test strains in a culture medium containing no serum has been achieved . Phasic changes in the ultrastructure of gonococci in the process of their cultivation in liquid media have been followed . 9- to 12-hour cultures of gonococci grown in liquid culture media have been found the most valuable and physiologically active.

J Rheumatol, 1987 Feb, 14(1), 28 - 32
Tetracyclines inhibit human synovial collagenase in vivo and in vitro; Greenwald RA et al.; To determine if tetracyclines can inhibit human synovial collagenase from rheumatoid tissue, paired synovial tissue (or synovial fluid) was collected from 7 patients before and after oral administration of minocycline (100 mg BID) for 10 days . With each patient serving as his own control, the postminocycline collagenase activities fell an average of 67% from pretreatment values . Qualitative SDS-PAGE revealed decreased loss of alpha collagen components and reduced formation of alpha A digestion fragments . Addition of minocycline or a chemically modified tetracycline to synovial culture media in vitro profoundly inhibited collagenase activity . Further study of this action of tetracyclines could serve as a probe of the role of collagenase in rheumatoid arthritis and lead to development of agents capable of modifying the tissue destructive actions of collagenase.

Curr Eye Res, 1987 Feb, 6(2), 381 - 9
Corneal deturgescence during organ culture; Walkenbach RJ et al.; A simple method is described to monitor rabbit and human corneal deturgescence while the tissue is suspended in tissue culture media . The composition of the media is such that the cornea slowly thickens at 4 degrees but exhibits the classic "temperature reversal" thinning phenomenon when incubated at 23 degrees-37 degrees . Each cornea is cultured in a closed eye bank corneal viewing chamber during swelling and deswelling phases of an experiment, minimizing direct handling of the tissue . Optimal conditions for observing corneal deturgescence are described . This protocol has several advantages over corneal perfusion techniques and could be further developed for routine use in eye banks as a functional test of donor suitability for keratoplasty.

J Virol Methods, 1987 Feb, 15(3), 167 - 75
Competitive and blocking enzyme-linked immunoassay for detection of fetal bovine serum antibodies to bovine viral diarrhea virus; Katz JB et al.; A competitive blocking enzyme-linked immunoassay (CELIA) was developed to detect bovine viral diarrhea virus (BVDV) antibodies in undiluted fetal bovine serum (FBS) . The CELIA was based on competition of serum BVDV antibodies with biotin-labelled anti-BVDV immunoglobulins (Ig) for a limited quantity of solid-phase BVDV antigen . Antigen preparation was simple, FBS could be tested undiluted, and detergent-containing washes were unnecessary . A series of dilutions of postnatal bovine BVDV antiserum prepared in FBS and a set of 147 undiluted abbatoir FBS samples were tested by both CELIA and serum neutralization tests (SNT) . CELIA results on both sets of specimens correlated positively with SNT titers (r = 0.99 and r = 0.85) . Relative to the SNT, CELIA sensitivity was 100%; specificity was 76% . CELIA detected a level of BVDV antibody below the 1:2-titer threshold detectable with the SNT . Advantages, limitations, and theoretical differences between the CELIA and SNT are discussed . A similar comparison of CELIA with non-competitive enzyme-linked immunoassay approaches to BVDV serodiagnosis is made . It is concluded that the CELIA is valuable in selecting only BVDV-seronegative FBS for use in virologic cell culture media.

Horm Metab Res, 1987 Feb, 19(2), 62 - 4
Spent media from immature seminiferous tubules and Sertoli cells inhibit adult rat Leydig cell aromatase activity; Papadopoulos V et al.; Adult rat Leydig cell aromatase activity is stimulated 2.5 fold by LH or dbcAMP . Spent media prepared from seminiferous tubules or Sertoli cells of immature rats depress both the basal and the LH stimulated estradiol syntheses (25 and 20% decreases, respectively) . These inhibitory effects are further enhanced when FSH is added to the culture medium of seminiferous tubules or Sertoli cells . Rat serum as well as culture media from other cell lines are ineffective while seminiferous tubule media from other immature animals (mouse, guinea-pig, calf) inhibit the aromatase activity . This Sertoli cell factor is a heat stable protein (molecular weight greater than 10 kDa), different from the LHRH-like Sertoli cell compound, which acts on the aromatase activity at a step beyond the adenylate cyclase.

Jpn J Cancer Res, 1987 Feb, 78(2), 144 - 52
Retrovirus produced by a lymphoid cell line from an infant with acute lymphoblastic leukemia; Miki T et al.; A lymphoid cell line CK-a was established from peripheral blood of an infant with acute lymphoblastic leukemia of non-T, non-B cell type with mediastinal tumor . The CK-a cells were positive for surface immunoglobulins, Epstein-Barr virus-specific nuclear antigen, HLA-DR and Leu 12 antigens, and negative for sheep erythrocyte-rosette-receptor, and Leu 1, 2, 3 and 4 antigens . Budding particles were detected in electron micrographs of ultrathin sections of the CK-a cells . In the culture media of CK-a cells, particles with a buoyant density of 1.16 g/ml and labeled with {3H}uridine and {35S}methionine but not with {3H}thymidine were found to carry reverse transcriptase activity which preferred Mg2+ to Mn2+ . Enveloped particles of 80 to 120 nm in diameter were detected in the fractions at 1.16 g/ml by electron microscopy . Thus, the particles had properties compatible with a definition of Retroviridae, and were tentatively named CK virus (CKV) . The genome size of CKV RNA determined by agarose-acrylamide composite gel electrophoresis was 6.1 +/- 0.2 kb . Immune electroblotting assay detected antibody reactive with a CKV protein with a molecular weight of 67,000 in the serum of the patient, but not in sera of an adult T cell leukemia patient and healthy controls . No syncytia were formed by mixed cultures of CK-a and XC cells.

Hokkaido Igaku Zasshi, 1987 Jan, 62(1), 122 - 31
{Kinetics of hemopoietic stem cells in hypoxic culture}; Ishikawa Y; In an effort to develop more nearly optimal conditions for the clonal growth of hemopoietic stem cells (CFC, BFU-E, CFU-mix and Mast-CFC) iv vitro, I examined the influence of low oxygen tension (7% O2) on plating efficiency . The numbers of colonies derived from human bone marrow CFC increased by 1.7 fold in 7% O2 than under a gas phase containing air (19% O2) . Bursts obtained from bone marrow BFU-E and mixed colonies from CFU-mix increased by about 2.5 fold in 7% O2 . Total cell count of mixed colonies showed that the average cell per colony gassed with 7% O2 were 900 compared with 511 in 19% O2 . However the subpopulation of CFC and composed cell type of mixed colonies were not different in the two gas phase . With mouse spleen cell in 7% O2 a dramatic increase in Mast-CFC numbers without 2-ME and decreased enhancement by 2-ME were seen . Blood gas analysis of human bone marrow showed a Po2 of 51.8 +/- 14.5 mmHg, which was close to O2 tension in culture media of gas phase contained 7% O2 . These data showed that physiological O2 tension enhances hemopoietic stem cell proliferation in vitro, and that part of the enhancing effect by 2-ME is due to a prevention of O2 toxicity at 19% O2.

Am J Hum Genet, 1987 Jan, 40(1), 15 - 31
Gaucher disease: genetic heterogeneity within and among the subtypes detected by immunoblotting; Fabbro D et al.; The genetic heterogeneity of Gaucher disease subtypes and variants was investigated by immunoblotting of fibroblast extracts . For these studies polyclonal and monoclonal antibodies were raised to acid beta-glucosidase preparations containing a single N-terminal amino acid sequence that was colinear with that encoded by the beta-Glc cDNAs . Three forms (Mr approximately equal to 67,000, 64,000-61,000, and 58,000) of cross-reacting immunologic material (CRIM) were observed in control individuals . Decreased amounts of the same CRIM forms were detected in most type 1 Gaucher disease patients, but single CRIM forms of variable molecular weight were observed in several non-Jewish type 1 variants . One or two CRIM forms of variable molecular weight were found in neuronopathic (type 2 and type 3) patients . The amount of CRIM was severely decreased in the majority of the type 2 and type 3 patients; one American black type 2 patient was CRIM negative . With this one exception, one CRIM form was detected in the cell-free culture media from all normal or Gaucher disease fibroblasts that had an Mr approximately 2,000 greater than the highest respective intracellular molecular-weight form . All intra- or extracellular CRIM forms were reduced to a single form after deglycosylation with N-Glycanase . In addition, the radioactivity from {3H}Br-conduritol B epoxide, a specific covalent inhibitor of beta-Glc, localized to the CRIM forms of beta-Glc on immunoblots . These results indicate that all subtypes and variants of Gaucher disease result from mutations that alter the stability and/or processing of beta-Glc . Furthermore, the heterogeneity of the CRIM patterns within and among the variants of Gaucher disease cause the diagnostic usefulness of immunoblotting to be restricted to those families in which the phenotype has been well established.

Gen Comp Endocrinol, 1987 Jan, 65(1), 9 - 11
17 Beta-estradiol secretion by the ovary of the hypophysectomized chick embryo; Weniger JP et al.; Ovaries of intact and hypophysectomized 16-day-old chick embryos were cultured in medium 199 for 24 hr, and 17 beta-estradiol released into the culture media was measured by radioimmunoassay . The difference of the means of the two series of measurements was not significant . It is concluded that 17 beta-estradiol secretion by the chick embryo ovary is not under pituitary control, at least not before the stage of 16 days of incubation.

Gan To Kagaku Ryoho, 1987 Jan, 14(1), 211 - 9
{Primary cultures of various differentiated human cells and their transfer}; Yamane I; From our long experience with primary cultures of human differentiated cells, we have been able to come to the following conclusions . The culture media routinely employed have been developed using established cell lines but not primary cells as the growth marker . Therefore the culture media for primary cultures should be modified from those routinely employed . In addition the concentrated supplementation of serum to basal media dose not contribute to the growth of primary differentiated cells, and in fact on the contrary, is advantageous for the growth of non-target fibroblasts which would hamper the growth of the target cells . A hypoxic culture environment is more favorable for primary cultures especially when smaller cell numbers are used as the inoculum . The primary culture cells are more fragile in comparison with established cell lines . Therefore, the low-temperature cell diopersion procedure is recommended with the use of diluted crystalline trypsin saline . On the basis of the above findings, we have successfully carried out primary and transfer cultures of human esophageal and gall bladder epithelial cells, skin keratinocytes and endothelial cells . Their epithelial mature was proved by the presence of keratin in their cytoplasm, and the phenotype of endothelial cells was evident from the presence of Factor VIII-related antigen in their cytoplasm . Culture of endothelial cells requires the supplementation of a specific growth factor which we have utilized and isolated from conditioned medium of human diploid fibroblasts . Tiny amounts of the factor were shown to, induce the vascularization of rabbit eye cornea.

J Steroid Biochem, 1987, 27(1-3), 185 - 92
Are estrogen receptors cytoplasmic or nuclear? Some immunocytochemical and biochemical studies; Parikh I et al.; The subcellular localization of estradiol receptor (ER) has been examined using various experimental approaches . Immunocytochemical studies using the monoclonal antibody JS 34/32, raised against calf uterine cytosolic ER, yielded only equivocal results . In general, cells and tissues pretreated with estradiol showed positive immunostaining in the nuclei whereas those not exposed to the steroid did not show any staining . Nuclear translocation of ER was examined in intact MCF-7 cells using compounds which are known to influence receptor activation . When MCF-7 cells were exposed to molybdate (20 mM), nuclear translocation was completely inhibited while dithiothreitol (20 mM), dibutyryl cAMP (1 microM) and dibutyryl cGMP (1 microM) increased the translocation 2-3-fold . Phenol red, at the range of concentrations generally used in tissue culture media, also increased translocation . The physiological validity of such translocation was examined using cellular progesterone receptor (PR) synthesis as a specific parameter . When MCF-7 cells were grown in media containing phenol red for 48 h, the PR synthesis increased significantly . We further examined whether cytoskeletal proteins are involved in the translocation of ER . Colchicine, an inhibitor of microtubule assembly, inhibited translocation of ER in MCF-7 cells at 1-10 microM . PR synthesis was also inhibited by colchicine in a dose-dependent manner . It may be concluded from these and other published data that ER may not be located at all times in a single subcellular compartment but may rather exist in a dynamic equilibrium between the plasma membrane, cytoplasm and nucleus.

J Hirnforsch, 1987, 28(5), 479 - 84
Scanning electron microscopical observations on retina cells in culture; Grosse G et al.; In cell cultures of the retina from 10 days old chicken embryo photoreceptors, bipolar neurons, synaptic contacts, and supporting cells of Muller were demonstrated by scanning electron microscopy . It was found that the longer the cells survive in culture media they form constantly growing cell aggregates . After 12 days in vitro large multilayered cell complexes covered by an epithelioid cell layer had developed . Merely at the border of such aggregates typical retinal cells could be observed . The occurrence of a dense epithelioid cell layer covering the aggregate of neuronal cells is interpreted as a general adaptational reaction of the cells to the long lasting survival under in vitro conditions.

Drug Chem Toxicol, 1987, 10(1-2), 133 - 56
Asbestos in peripheral lung culture a species comparison of pulmonary tissue response; Placke ME et al.; We have recently developed culture techniques to allow the long-term maintenance of adult peripheral lung tissue in vitro from a variety of mammalian species including hamster, rat, bovine, and human . The technique involves perfusion of the major airways with agarose gel and culture media followed by thin sectioning and culture on porous surgical foam . Cross sections of lung lobes 1-2 mm thick have been cultured for periods beyond four weeks with maintenance of structural and biochemical integrity of the lung . In vitro exposure of lung explants to crocidolite asbestos through the airways produced fibrotic and hyperplastic lesions similar to those reported after in vivo exposure . The incidence and severity of interstitial fibrous was concentration-dependent, including the human specimens, and the morphologic appearance of the lesions was similar in explants derived from each species . The lung explant model is well suited for further mechanistic evaluations of asbestos-induced lung lesions . It is notable that the pulmonary lesions were produced without the possibility for recruitment of hematogenous inflammatory cell populations.

Exp Gerontol, 1987, 22(4), 263 - 9
Comparison of calcium effect on in vitro calcitonin and parathyroid hormone release by young and aged thyroparathyroid glands; Wongsurawat N et al.; Serum immunoreactive parathyroid hormone (iPTH) and calcitonin (iCT) levels are higher in young than aged rats . However, serum calcium concentration does not change with age suggesting that the calcium regulation of PTH and CT secretion may be affected by aging . We compared iPTH and iCT secretion in vitro at low and high calcium concentrations using thyroparathyroid glands removed from young (2-3 months), adult (12-13 months), and old (24-27 months) F-344 male rats fed regular rat chow . Glands from each animal were incubated for 3 h in serum-free culture media containing 1.0 mM calcium and then transferred to media containing 2.5 mM calcium for another 3 h . Immunoreactive PTH and iCT concentrations of the media after each incubation period were determined by radioimmunoassay . Immunoreactive PTH and iCT secretion per pair of glands was significantly higher in glands from older animals regardless of calcium concentration . The decrease in iPTH, and increment in iCT, secretion in response to 2.5 mM calcium by glands from old rats was smaller than that observed for glands from young animals . These age-related changes in the regulation of secretion by calcium may contribute to the increased iPTH and iCT secretion and serum levels seen in older animals.

Ann Biol Clin (Paris), 1987, 45(3), 368 - 72
Quality control of culture media for in vitro fertilisation; Naaktgeboren N; The organisation of a control programme to evaluate the quality of culture media for human in vitro fertilisation is described . The development of mouse embryos to the blastocyst stage in vitro was used as control system . It is shown that it was possible to screen for embryo toxic chemicals in the media so that these can be avoided in the human culture . The influence of temporary pH shifts on the mouse embryo development and in vitro fertilisation in the mouse are mentioned . Exposure to low pH before fertilisation caused a drastic reduction of the development of mouse embryos . No clear association is found between pregnancy rate using in vitro fertilised human oocytes and mouse embryo development . The control system cannot be used to improve media for human in vitro fertilisation.

Neoplasma, 1987, 34(2), 129 - 38
Retrovirus like particles produced by human embryonal cells and cell lines derived from human malignancies . II . Protein structure; Prachar J et al.; Very small amounts of retrovirus-like particles were isolated from tissue culture media of various types of human cell lines derived from malignant as well as normal cells . The aim of the present study is the characterization of protein profiles of these isolates . The comparison of protein profiles of isolated human virus-like particles with the profiles of well characterized animal as well as human exogenous (LAV/HTLV-III) retroviruses revealed a 25k protein (p25) to be a major protein or at least one of the protein components of human retrovirus-like particles.

Nutr Cancer, 1987, 10(3), 137 - 44
In vitro inhibitory effect of onion extract on hamster buccal pouch carcinogenesis; Niukian K et al.; In vitro studies were performed that used varying concentrations of onion extract added to cell cultures of an epidermoid carcinoma cell line derived from hamster buccal pouch carcinoma (HCPC-1) . The studies demonstrated tumor growth inhibition beginning after 24 hours of incubation at an onion extract concentration of 25% and above in culture media . After 4 days and 10 days of incubation, there was a noted decrease in tumor proliferation . The plating efficiency for 24 hours was observed to produce a 54-89% inhibition in plating density . The results indicated here provide in vitro evidence of the inhibitory and cytotoxic activity on an oral carcinoma cell line.

J Pineal Res, 1987, 4(3), 267 - 75
Lower tryptophan:phenylalanine ratios in culture media increase medium: pineal melatonin ratios in early dark but not late light phase; Catala MD et al.; Pineals from male Long-Evans rats (60-65 days old; adapted to a 0700-1900 photoperiod) were cultured for 6 h either in light (1200-1800) or in dark (1800-2400) . The objective was to ascertain the effects of tryptophan (trp) and phenylalanine (phe) levels and ratios in the culture medium on melatonin levels in the pineals and their respective media . Total culture (pineal + medium) melatonin levels, determined by RIA, were similar under all conditions . However, in cultures during the early dark phase (1800-2400) lower trp:phe ratios in the medium led to lower pineal:medium ratios of melatonin content . In cultures during the late light phase (1200-1800) the trp:phe ratio had little impact on the pineal:medium distribution of melatonin . Trp:phe ratio rather than absolute level of either amino acid appeared responsible for this effect . Functionally this means that during early dark phase, but not late light phase, movement of melatonin from cultured pineal to medium is progressively facilitated by lower trp:phe ratios . It remains to be determined to what extent darkness per se and/or endogenous pineal rhythmic mechanisms have a permissive role in the action of trp:phe ratio on pineal melatonin release . A melatonin compartmentalization/release effect of these or other amino acids, or their ratios, has not been reported previously and may possibly contribute to mechanisms for melatonin's transport or release at night.

Comp Biochem Physiol B, 1987, 86(3), 607 - 11
Progesterone fate in rabbit cornea; Navarro-Ruiz A et al.; Excised cornea from adult New Zealand rabbits were incubated with progesterone-4-14C in Eagle's media for 96 hr . Samples were inactivated at intervals of 24 hr incubation periods . The following metabolites of progesterone were isolated: 20 alpha-Hydroxy-4-pregnen-3-one, 20-hydroxy-4-pregnen-3-one, 5 alpha-pregnane-3,20-dione; 5 beta-pregnane-3,20-dione and 6 beta-hydroxy-4-pregnen-3,20-dione . 20 alpha-Hydroxy-pregnen-3-one was the predominant metabolite of progesterone-4-14C . A linear increase was observed throughout 96 hr . The opposite was found for 5 alpha and 5 beta pregnane-3,20-dione . Compounds remaining at the origin of the paper chromatograms contained 6 beta-hydroxy-4-pregnen-3,20-dione and other still unidentified metabolites of progesterone-4-14C . Presence of 20 alpha and 20 beta-reductase; 5 alpha and 5 beta-reductase and 6 beta-hydroxylase enzyme systems are involved in corneal progesterone metabolism . No fungal neither bacterial enzymatic biotransformation occurred in the culture media.

Dev Pharmacol Ther, 1987, 10(1), 47 - 59
Trophic influence of sympathetic neurons on the cardiac alpha-adrenergic response requires close nerve-muscle association; Drugge ED et al.; In tissue culture, sympathetic neurons exert a trophic effect on myocardial cells to alter the alpha-adrenergic response from positive to negative chronotropism . We hypothesized that this neurotrophic effect might be a result of the release of a humoral substance into the bulk phase of the culture medium by the neurons . The negative chronotropic response to alpha stimulation persisted in nerve-muscle cultures pretreated with the muscarinic antagonist atropine, indicating that acetylcholine is not the trophic agent . Further, variations in the concentration of norepinephrine, epinephrine or dopamine in the nerve-muscle culture media do not account for the presence of a negative chronotropic response to alpha stimulation . Finally, muscle cells grown in the same petri dish with innervated muscle cells, to allow conditioning of the muscle cell environment by the neurons, do not acquire a negative chronotropic response to alpha stimulation . The results of this study clearly demonstrate that this trophic effect is not due to the release of a humoral substance into the bulk phase of the culture medium, but requires close nerve-muscle association . This suggests that the trophic effect may involve localized release and transsynaptic transfer of a chemical substance other than a neurotransmitter.

J Neurosci Res, 1987, 17(1), 51 - 9
Development of transferrin-positive oligodendrocytes in the rat central nervous system; Connor JR et al.; Transferrin is the second most abundant plasma protein and functions to transport iron . It is an essential constituent in culture media for virtually all cells . In a recent study, we reported that transferrin (Tf) is specifically located in oligodendrocytes in the rat nervous system . This investigation examines immunohistochemically the development of Tf in the cerebral cortex, corpus striatum, and spinal cord . Tf is first seen in oligodendrocytes in the spinal cord white matter at 5 days of age . The immunoreactivity is confined to the white matter in the periphery of the spinal cord between 5 and 8 days of age . By 10-12 days of age, the number of immunoreactive oligodendrocytes in the spinal cord white matter increases considerably, corresponding to the onset of myelination . Tf-positive oligodendrocytes are first found in the gray matter at 15 days of age . By 30 days of age, the number and distribution of Tf-positive oligodendrocytes in both the brain and spinal cord have reached the adult pattern . The results of this study demonstrate a spatial and temporal association between Tf development and myelinogenesis . This suggests that part of the process of differentiation of oligodendrocytes includes the accumulation of Tf, perhaps in order to support the metabolic demands associated with the production and maintenance of myelin.

Hum Reprod, 1987 Jan, 2(1), 23 - 7
Quantitative aspects of protein synthesis in non-cultured and cultured rabbit blastocysts; Jung T et al.; Day 4 rabbit blastocysts were cultured in Ham's F-10 medium supplemented either with homologous serum or uterine flushings . Development was assessed by leucine and methionine incorporation at 24 h and 48 h, respectively, after initiation of culture and compared with day 4 and day 5 non-cultured controls . After 24 h in culture, incorporation data were similar for both media groups . After 48 h, a significantly higher protein synthesis activity was found in blastocysts which had access to uterine secretions in vitro . However, incorporation levels of day 5 controls were not reached . Modes of incorporation of leucine and methionine revealed to be very similar in all experimental groups but ranged on a different level . Leucine was incorporated on a 5.5-fold higher level than methionine . We conclude that (i) supplementation of culture media with uterine secretions is beneficial for blastocyst development, and (ii) further studies should focus on non-serum components within the uterine fluid and their significance for embryonic development.

Mol Gen Mikrobiol Virusol, 1987 Jan, (1), 19 - 23
{Various characteristics of the structure and synthesis of procollagens produced by cultured skin fibroblasts from patients with Danlos-Ehlers syndrome type I}; Sokolov BP et al.; The electrophoretic mobilities of the collagen and procollagen type I and III chains synthesized by the fibroblasts isolated from patients with type I Ehlers-Danlos syndrome as well as a set of peptides obtained by splitting of pro alpha 1(I) and pro alpha 2(I) type I procollagens by cyanbromide are not different from the normal ones . The fact demonstrates the absence of long insertions or deletions, or the sufficient defects in intracellular chain modifications . The changes were also nor registered for the ratio of type I and III collagens from the digested by pepsin preparations of protein accumulating in the culture media of the cultured skin fibroblasts from patients . The studied strains of cultured fibroblasts from patients suffering the Ehlers-Danlos syndrome have the trend to increased accumulation of partially processed chains of proc alpha 1(I) and proc alpha 2(I) type I procollagen and to the increased ratio of pro alpha 1(I) to pro alpha 2(I).

Complement, 1987, 4(2), 87 - 98
Identification of a spontaneously shed fragment of B cell complement receptor type two (CR2) containing the C3d-binding site; Myones BL et al.; CR2 is a 140-kilodalton glycoprotein expressed on B lymphocytes which binds to both C3d and Epstein-Barr virus (EBV) . The present study identified a 72-kilodalton C3d-binding protein (gp72) in the spent culture media of Raji B lymphoblastoid cells as a spontaneously shed fragment of the 140-kilodalton CR2 molecule . Both polyclonal and monoclonal antibodies (AB) were used in several assay systems to detect antigenic determinants shared between the gp72 fragment and CR2 . Rabbit antibodies to either intact CR2 or gp72 blocked C3d receptor activity, and this inhibitory activity was removed by absorption of anti-CR2 with purified gp72.OKB7, a monoclonal anti-CR2 AB that blocks both C3d and EBV binding to CR2, reacted specifically with both CR2 and gp72, whereas both anti-B2 and HB-5 monoclonal anti-CR2 AB, that block neither of these receptor sites, were unreactive with gp72 . The data suggested that the gp72 fragment was not present as such in intact cells, but rather was a product of cells generated by proteolysis of CR2 . Thus, intrinsically labeled gp72 was isolated from Raji cell media by affinity chromatography on OKB7-Sepharose, but only intact CR2 was isolated from the Raji cell fraction solubilized in the presence of protease inhibitors . Several lines of evidence suggested that gp72 was not a second type of C3d receptor that was distinct from CR2 . First, Raji cells expressed nearly identical amounts of OKB7 and HB-5 epitopes when analyzed by flow cytometry or radioimmune assay, excluding the possibility that B cells expressed OKB7 antigens in both CR2 and a distinct HB-5-negative C3d receptor . Second, all Raji cell surface C3d receptor activity was associated with HB-5-reactive CR2 molecules . We conclude that gp72 represents a spontaneously shed proteolytic fragment of CR2 that contains the C3d-binding site and the closely associated OKB7 epitope.

J Natl Cancer Inst, 1987 Jan, 78(1), 95 - 9
Analysis and stability of retinol in plasma; Peng YM et al.; A simple, precise, and specific high-performance liquid chromatography (HPLC) method was developed for the simultaneous measurement of retinol (ROH), 13-cis-retinoic acid (13-cRA), and 4-oxo-13-cRA . The average recovery of ROH from serum or plasma was 95%, and the precision of the assay was less than 5% . With this HPLC method, a series of studies was carried out to evaluate the stability of ROH in various matrices . ROH was stable under our HPLC assay conditions as well as in plasma- and in serum-enriched culture media; however, ROH was not stable in aqueous matrices . Serum or heparinized plasma may be routinely used for measurement of ROH concentrations, providing EDTA, oxalate, and citrate are not used as anticoagulants . Because of ROH stability, blood samples can be kept on ice in the dark for at least 24 hours prior to separation of plasma . In addition, plasma samples containing ROH can be stored for up to 1 year at -20 degrees C without loss of stability.

Adv Exp Med Biol, 1987, 230, 151 - 65
Analysis of proteins secreted by the human endometrium in vivo and in vitro; MacLaughlin DT et al.; Human uterine luminal fluids contain over two dozen proteins distinct from those of serum as detected by two dimensional gel electrophoresis and silver-type protein staining . Eighty-one percent of these uterine fluid proteins can be detected in vitro by radiolabeled methionine incorporation studies and the vast majority of these products are epithelial in origin . The major recognizable menstrual cycle phase-dependent change in the protein pattern in these gels was the appearance of a protein group (number 27) of approximately 25,000 mw and pI of 5.8 - 6.3 . This group of proteins was found in nearly all mid- and all late secretory phase fluids or culture media and in none obtained earlier in the cycle . As yet, however, it has not been possible to induce these proteins in proliferative specimens in vitro by the addition of estrogens and/or progestins, though studies along these lines are continuing . Although we cannot be certain, it appears as though protein group number 27 is distinct from, but similar in several respects to, other proteins of human endometrium reported in the literature.

Exp Cell Biol, 1987, 55(6), 313 - 21
Contribution of the extracellular matrix to growth properties of cells from a preneoplastic outgrowth: possible role of hyaluronic acid; Elstad CA et al.; Hyaluronic acid (HA) accumulates around actively growing normal and tumorigenic mammary epithelial cells and has been implicated as a modulator of cell proliferation . We have tested the role of exogenous HA presented in several different forms in in vitro growth regulation of a cell line (CL-S1) derived from preneoplastic mouse mammary tissue . This cell line grows slowly and synthesizes very little HA . We first assessed growth of CL-S1 cells seeded onto actual matrix generated by CL-S1 cells themselves (which has a low HA content) or by a related tumorigenic cell line, +SA, that generates an HA-rich matrix . Growth on both these HA-containing substrata was significantly enhanced above control values on plastic . Growth on the +SA biomatrix was over 5 times greater than on tissue culture plastic and significantly greater than that seen with all other treatments . Differences in growth responses of CL-S1 cells seeded atop CL-S1- and +SA-derived matrices could be attributable to differences in matrix HA content . As a more direct test of this possibility, growth responses of CL-S1 cells to HA covalently bonded to tissue culture dishes and to HA dissolved in culture media were tested . Growth on the prepared HA substrata was consistently twice that on plastic . In soluble form, HA at a concentration of 100 micrograms HA/ml culture medium, stimulated CL-S1 growth 196 and 125% of control in monolayer cultures, respectively, seeded at low (approximately equal to 10(2) viable cells/cm2) and high (approximately equal to 10(4) viable cells/cm2) densities on plastic . Higher HA concentrations inhibited growth at low seeding densities.(ABSTRACT TRUNCATED AT 250 WORDS)

Dev Neurosci, 1987, 9(3), 174 - 82
Mouse spinal cord neurons in serum-free culture media: suitability for patch clamp studies on chemical and electrical excitability; Salamanca MC et al.; Methods were devised for the serum-free culture of spinal cord neurons derived from 12- to 13-day mouse embryos . Neurons exhibited good attachment if plated for 24 h on poly-d-lysine-coated dishes in the presence of serum . Cultures were subsequently fed with a serum-free medium consisting of minimum essential medium, Earle's salts and the N1 supplement, i.e . insulin, putrescine, transferrin, progesterone and selenium . After 3 weeks in vitro, growth and survival of neurons in this medium were comparable to results obtained using serum-supplemented medium . The presence of putrescine was not essential for the beneficial effects of N1, while insulin was required for long-term survival in serum-free media . Neurons maintained in serum-free media for 3 weeks retained aspects of electrical and chemical excitability characteristic of serum-grown cells.

Ann Biol Clin (Paris), 1987, 45(3), 361 - 7
Problems related to the laboratory part of treatment by in vitro fertilization and embryo transfer; Leroy F et al.; The successive stages leading to fertilization in mammals are reviewed in this article . Methods of human sperm preparation for IVF are described and the "ideal" delay between oocyte pick-up and insemination time is discussed, as well as methods to reduce the incidence of polyspermy . Different culture media and their supplementation are mentioned, as well as a semi-quantitative embryonic scoring system, defined by the IVF team of the Saint-Pierre Hospital in Brussels . Finally the optimal transfer time, and the handling of embryos at replacement are discussed.

Aust J Biol Sci, 1987, 40(1), 105 - 13
A rapid, sensitive and reliable assay for inhibin bioactivity; Lee VW et al.; A rapid 2-day quantitative assay for inhibin bioactivity based on FSH secretion from pituitary cells of immature female rats is described . The bioassay exhibited steeper slopes, improved precision and greater (fourfold) sensitivity compared with a previously established pituitary FSH cell content assay . Whole pituitary glands were used for the preparation of pituitary cells and the method for cell dispersion required a single enzymatic treatment with trypsin . Cells (180,000 viable cells per well) were dispensed into culture media containing inhibin and incubated for 48 h . Media were removed and assayed for FSH by radioimmunoassay . Using a ram rete testis fluid preparation as standard the inhibin dose-response curves of 25 consecutive experiments showed indices of precision of -0.08(mean){range -0.04 to -0.17} and Finney's G values of 0.017{0.003-0.06} . The mean ED40 was 0.17 units of inhibin activity per well with interassay variation of 16.2% at this point of the dose-response curve . The assay had a practical capacity of 400 wells, permitting the measurement of dose-response curves of at least 40 unknowns with three dose points and triplicate wells per dose . The assay is specific for inhibin-containing preparations from several animal species . Overall, the assay is simple, precise, and sensitive, indicative of its applicability to the measurement of inhibin samples with low inhibin bioactivity and to the screening of large numbers of fractions during inhibin purification.

Aust J Biol Sci, 1987, 40(1), 105 - 13
A rapid, sensitive and reliable assay for inhibin bioactivity; Lee VW et al.; A rapid 2-day quantitative assay for inhibin bioactivity based on FSH secretion from pituitary cells of immature female rats is described . The bioassay exhibited steeper slopes, improved precision and greater (fourfold) sensitivity compared with a previously established pituitary FSH cell content assay . Whole pituitary glands were used for the preparation of pituitary cells and the method for cell dispersion required a single enzymatic treatment with trypsin . Cells (180,000 viable cells per well) were dispensed into culture media containing inhibin and incubated for 48 h . Media were removed and assayed for FSH by radioimmunoassay . Using a ram rete testis fluid preparation as standard the inhibin dose-response curves of 25 consecutive experiments showed indices of precision of -0.08(mean){range -0.04 to -0.17} and Finney's G values of 0.017{0.003-0.06} . The mean ED40 was 0.17 units of inhibin activity per well with interassay variation of 16.2% at this point of the dose-response curve . The assay had a practical capacity of 400 wells, permitting the measurement of dose-response curves of at least 40 unknowns with three dose points and triplicate wells per dose . The assay is specific for inhibin-containing preparations from several animal species . Overall, the assay is simple, precise, and sensitive, indicative of its applicability to the measurement of inhibin samples with low inhibin bioactivity and to the screening of large numbers of fractions during inhibin purification.

Oncogene, 1987, 1(3), 297 - 300
Accumulation of c-fos mRNA in slices of mouse submandibular gland incubated in vitro; Barka T et al.; Incubation of slices or isolated lobules of murine submandibular gland at 37 degrees C in physiologic solutions or in tissue culture media, or dissociation of the cells by collagenase-hyaluronidase treatment, increased the steady-state level of c-fos mRNA without any additional stimulus . This activation of c-fos expression required the presence of Na+ and K+ but not extracellular Ca2+ . It was augmented by depolarizing concentrations of K+ and by veratridine, and inhibited by high concentrations of amiloride . Alterations in membrane permeability and in ion fluxes and/or perturbation in membrane phospholipids may play a role in this transitional activation of the c-fos gene expression in incubated tissue slices in which the cells are not viable and undergo a necrobiotic process.

Arch Androl, 1987, 19(2), 149 - 58
Mouse embryo growth in different culture media: selection of a medium for quality control cross-testing of human in vitro fertilization conditions; Vijayakumar R et al.; A total of 2070 two-cell mouse embryos were recovered from 89 superovulated female hybrid mice . Six different culture media were tested . The various media supported mouse embryo development as follows (percentage mean +/- SD, n = 10): Hopp and Pitts medium (H&P) 87 +/- 5 Dulbecco's modified; Eagle's medium supplemented with 10% (volume/volume, v/v) fetal bovine serum (DMEM) 80 +/- 4; Ham's F-10 +/- 15.0% (v/v) human fetal cord serum (hFCS) 79 +/- 3; Whittingham's T-6 medium (WT-6) 60 +/- 4; Ham's F-10 +/- 7.5% (v/v) hFCS 55 +/- 5; Krebs-Ringer low bicarbonate buffer (KRLBB) 42 +/- 6 . In H&P, DMEM, WT-6, and Ham's F-10 medium supplemented with hFCS, the pH was maintained within a narrow range of 7.30-7.45 and adequate level of oxygenation was achieved during 72 h in culture . KRLBB had poor buffering capacity and attained ineffective levels of oxygenation during culture . Superior mouse embryo development from two-cells to morulae and hollow blastocysts occurred in H&P, Ham's F-10 + 15% hFCS, and DMEM . Ham's F-10 medium supplemented with hFCS is routinely checked for its ability to support mouse two-cell embryo development to morulae and blastocysts . This is done in conjunction with H&P medium as the control.

Circ Shock, 1987, 23(4), 295 - 303
Endotoxin-induced production of thromboxane and prostacyclin by equine peritoneal macrophages; Morris DD et al.; Equine peritoneal macrophages were isolated and cultured in vitro to assess their ability to produce thromboxane (TxA2) and prostacyclin (PGI2) in response to endotoxin . Peritoneal macrophages (2.5 x 10(6)/ml) were incubated in tissue culture media, containing 1) no additive (nonstimulated control), 2) endotoxin (0.5 to 100 ng/ml) or 3) the calcium ionophore, A23187 (0.95 microM) for two and six h . Concentrations of the stable metabolites of TxA2 and PGI2 thromboxane B2 (TxB2) and 6-keto-prostaglandin F1 alpha (6-keto-PGF1 alpha), in the incubation media were determined by radioimmunoassay . The concentrations of both metabolites increased from two to six h incubation . Endotoxin increased the production of TxA2 and PGI2 over the nonstimulated control values at both two and six h and endotoxin-induced concentrations of 6-keto-PGF1 alpha were higher at six than at two h . The response of macrophages to A23187 was similar to endotoxin . Mean eicosanoid concentrations did not differ among the range of endotoxin concentrations at either time; however there was significant curvilinear regression between endotoxin concentration and TxB2 at both times, and between endotoxin and 6-keto-PGF1 alpha at two h . The results indicate that equine macrophages may be a significant source of TxA2 and PGI2 during endotoxemia.

C R Acad Sci III, 1987, 305(12), 459 - 64
{Human placental tissue: a source of natural hematopoietic regulators}; Martin-Thouvenin V et al.; In this paper, it has been shown that human placental tissular extracts are a potent source of natural haemopoietic growth factors . The colony-stimulating activities (CSA) recovered by extraction from washed placental pulp were active both on human and murine haemopoietic progenitors, from monocytic and granulocytic lineages . Crude tissular extracts contained CSA titers at least ten fold the titers usually found in placenta culture media . Placenta is the only human tissue easily available for the study of natural tissue-bound haemopoietic regulators . Extraction on an industrial scale, as proposed for the first time in this paper, should also benefit the identification and purification of new minor molecular classes of growth and maturation factors or inhibitors involved in human haemopoiesis.

Eksp Onkol, 1987, 9(3), 28 - 31
{Ratio of polyamine synthesis and oxidation enzymes during cell proliferation and differentiation}; Shliakhovenko VA et al.; Difluoromethylornithine (DFMO) was studied for its effect on the aminoxidase activity in the culture media during proliferation and differentiation of mouse fibroblasts . Single introduction of difluoromethylornithine into the culture medium increases the polyamine oxidase activity during 12 to 24 hours after the action . In the subsequent periods DFMO in 0.1 to 1 mM concentrations inhibits the proliferation and differentiation, but 0.01 mM concentration leads to their acceleration, which correlates with higher or lower aminoxidase activities respectively in the culture media.

Invest New Drugs, 1987, 5(1), 21 - 9
Effect of interferon alpha, interferon beta, and interferon gamma on the in vitro growth of human renal adenocarcinoma cells; Kuebler JP et al.; Interferon-alpha, interferon-beta, and interferon-gamma differ in their antiproliferative effects for several cell lines . Interferons were thus assessed for their activity in inhibiting proliferation of three renal cell carcinoma cell lines . The malignant epithelial phenotype of each of these cell lines was confirmed by electron microscopy, histology, karyotype and tumorigenicity . When compared on an anti-viral unit basis, naturally produced interferon-beta was more effective than natural interferon-alpha for all cell lines and clones . Proliferation of each of the cell lines was inhibited by interferon-gamma . In all cases, removal of interferons from culture media resulted in resumption of the rate of cell growth after a variable delay of 6-10 days . If the antiproliferative effects of interferons predominate in mediating tumor regression, clinical response may depend upon the type of interferon to which the tumor is exposed.

Virchows Arch A Pathol Anat Histopathol, 1987, 410(5), 375 - 81
Ewing's sarcoma lines synthesize laminin and fibronectin; Scarpa S et al.; Immunoelectron microscopy was employed to detect laminin and fibronectin cell surface expression on five Ewing's sarcoma lines plus a normal fibroblast line as control . Monospecific antibodies to both glycoproteins were detected on tumour cell and fibroblast layers with colloidal gold--protein A conjugates . All five tumour lines were positive for fibronectin and/or laminin, whereas the fibroblast line expressed fibronectin only, as expected . Fibronectin displayed a dense granular pattern, typically in the cell-cell and cell-matrix adhesion areas; laminin displayed a punctate pattern . 3H-leucine metabolical labelling was also used to demonstrate laminin and fibronectin synthesis . The labelled proteins released in the culture media were separated by molecular weight on SDS-PAGE and identified by immunoprecipitation with the monospecific antibodies . The results substantiated the immunoelectron microscopy data . These findings indicate that Ewing's sarcoma lines produce a complex extracellular matrix including fibronectin and laminin, in addition to the collagens described by other workers . Histogenetic classification of this tumour in terms of extracellular matrix proteins synthesis is thus more difficult than has been supposed . The same complexity must also be borne in mind when using the matrix components as an aid to Ewing's sarcoma differentiation from other childhood tumours.

Parasitol Res, 1987, 73(1), 9 - 14
In vitro cultivation of Herpetosoma trypanosomes in insect cell tissue culture media; Mohamed HA et al.; The cultivation of Herpetosoma trypanosomes in insect tissue culture media supplemented with foetal calf serum is described . Trypanosoma lewisi and T . musculi, which can be grown in blood agar media, were compared with four other species of Herpetosoma trypanosomes, T . microti, T . evotomys, T . grosi and T . nabiasi, in their growth in Schneider's Drosophila medium, Grace's, Mitsuhashi-Maramorosch, RPMI 1640, TCM 199 and nutrient blood agar media . Schneider's Drosophila and Grace's media supplemented with 20% foetal calf serum proved the most suitable media for growth of all parasites except T . nabiasi from rabbits which was not successfully established . Primary cultures were passaged after approximately 3 weeks and were maintained to continuously produce metacyclic trypomastigotes which produced less virulent infections although they maintained their infectivity to their respective hosts . The growth patterns in culture and morphology of the parasites are described.

Endocr Res, 1987, 13(1), 85 - 95
Response of anterior pituitary cells to culture media; Cronin MJ et al.; We examined the ability of four commonly used culture media to support prolactin (PRL), growth hormone (GH), and adrenocorticotropic hormone (ACTH) release, as well as the inhibitory PRL response to dopamine . After a week of primary culture, rat anterior pituitary cells from both genders were studied over a 4 hour period . Whereas ACTH secretion was similar across the various media, PRL and GH release were lessened with M199 and F10, respectively . Dopamine inhibited PRL release under all media conditions which was inconsistant with the reported lack of a dopamine effect with RPMI-1640 medium . These data confirm the postulate that media culture conditions can determine the degree of expression of constituitive phenotypes in anterior pituitary cells.

Arch Dermatol Res, 1987, 279(5), 341 - 6
Plasmin induces acantholysis in skin organ cultures; Hunziker T et al.; Addition of human plasminogen to three different pemphigus plasma samples showed a synergistic effect on acantholysis in the skin organ culture model . Human plasmin itself, without addition of pemphigus plasma, induced typical acantholytic changes in the skin explants, causing different types of acantholysis in a dose- and time-dependent manner: in the presence of 3 CU plasmin per ml culture medium, focal suprabasilar acantholysis of pemphigus vulgaris type could be detected after 72 h incubation, whereas 15 CU/ml caused extended acantholysis of pemphigus foliaceus type in the upper epidermal layers after 24 h, and extended acantholysis of benign chronic pemphigus (Hailey-Hailey disease) type comprising all layers of the epidermis after 48 h incubation . Plasminogen activator levels (Mr 55,000 urokinase type) in tissue extracts of skin explants and in culture media were reduced after 24 and 48 h incubation with pemphigus IgG as compared to control experiments with normal human IgG; this probably resulted from urokinase inactivation by reaction with inhibitors . These results lend support to the hypothesis proposed by Hashimoto et al . in 1983 that the plasminogen activator-plasmin system could play an essential role in the protease mechanisms of pemphigus acantholysis.

Connect Tissue Res, 1987, 16(1), 41 - 56
Extracellular matrix-induced synthesis of a low molecular weight collagen by fetal calf ligament fibroblasts; Sage H et al.; Fetal calf ligamentum nuchae fibroblasts, cultured from animals of different gestational age, synthesize a unique, low molecular weight collagen termed FCL-1 (Sage, H., Mecham, R., Johnson, C., and Bornstein, P., 1983, J . Cell Biol . 97:1933-1938) . Previous studies on the elastogenic differentiation of these cells in vitro demonstrated that the extracellular matrix (ECM) protein elastin was specifically induced in undifferentiated fibroblasts when they were grown on ligament ECM isolated from animals at later stages of development (Mecham, R.P., Madaras, J.G., and Senior, R.M., 1984 . J . Cell Biol . 98:1804-1812) . To investigate the expression of FCL-1 as a function of developmental age, we grew fetal calf ligament fibroblasts from an 85 d (first trimester) animal (FCL 85d) on three different substrata: ligament from a 120 d (second trimester) animal, ligament from a 270 d (term) animal, and unmodified plastic tissue culture dishes . FCL 270d fibroblasts were grown on plastic substrata and served as a differentiated cellular control . Analysis of metabolically radiolabeled proteins from both the culture media and the cell layers showed that the synthesis of FCL-1 was selectively increased in those cells cultured on ligament ECM . For FCL 85d fibroblasts grown on 120 d and 270 d ligaments, FCL-1 comprised 17% and 22%, respectively, of the culture medium proteins that precipitated at concentrations of ammonium sulfate from 20-50% . FCL 85d and 270d fibroblasts grown on plastic substrata yielded values of 2.5% and 1.0%, respectively . This effect appeared to be specific for this collagen and did not reflect a general increase in the synthesis of connective tissue proteins of the ECM (e.g., types I and III procollagen) . As percent of total newly-synthesized cellular protein, the output of FCL-1 was 10-fold higher by FCL 85d cells grown on 270d ligament ECM (5.8%) as compared to that of the same cellular population grown on a plastic surface (0.56%) . The presence of the ligament ECM also altered the levels and distribution of secreted proteins between the culture medium and the cell layer . These studies provide evidence for differential expression of the novel collagen FCL-1 by FCL fibroblasts during development and suggest that such expression is affected, at least in part, by interaction of the cell with a ligament ECM.

In Vitro Cell Dev Biol, 1987 Jan, 23(1), 2 - 9
Synthesis of barbituric acid derivatives and their effect on survival of functional hepatocytes from adult rats in primary culture; Miyazaki M et al.; Ten barbituric acid (BA) derivatives were synthesized and tested for their potency for supporting survival of functional hepatocytes from adult rats in primary culture . Of the 10 BA derivatives, 7 compounds (C-2, 3, 4, 5, 6, 9, and 10) efficiently supported hepatocyte survival for at least 2 wks in primary culture . Especially C-5, 6, and 9 showed excellent efficiency for such action . The optimum concentrations of the BA derivatives for observing the morphological and biochemical effects differed from each other . The maintenance of hepatocytes was attained only in the continuous presence of the BA derivatives in the medium . The morphologic features of hepatocytes surviving in the presence of the BA derivatives resembled those of hepatocytes 24 h after inoculation . The surviving hepatocytes secreted remarkably large amounts of albumin into the culture media . Tyrosine aminotransferase (TAT) activity was higher in the 1-wk-old cultures treated with C-5, 6, and 9 than in the freshly isolated hepatocytes . The addition of dexamethasone (10 microM) caused a 1.7 to 2.1-fold induction in TAT activity . The basal levels of TAT activity and the induction rates increased in the cultures treated with C-5 and 6 from Week 1 to 2 of primary culture.

AIDS Res Hum Retroviruses, 1987, 3(4), 387 - 400
Purification of 120,000 dalton envelope glycoprotein from culture fluids of human immunodeficiency virus (HIV)-infected H9 cells; Pyle SW et al.; The outer envelope glycoprotein, gp120, has been purified from large volumes (greater than 100 liters) of HTLV-IIIB-infected H9 cell culture fluids using immunoaffinity chromatography resins prepared from immunoglobulins of AIDS patients plasma . By using a single-step immunoaffinity purification, between 7 and 28 micrograms of gp120 was recovered from each liter of culture fluid which represented between 60 and 95% of the total envelope glycoprotein present in the fluid . Envelope glycoprotein in culture media was concentrated more than 10,000 times over the starting material . The water-soluble gp120, containing trace contaminating proteins, was purified to apparent homogeneity by preparative polyacrylamide gel electrophoresis (PAGE) . Envelope glycoprotein purified from culture fluids was immunogenic in laboratory animals in both native and PAGE-purified forms and was reactive with AIDS patient sera in immunoassays.

Acta Derm Venereol, 1987, 67(2), 100 - 5
Dopaquinone addition products in cultured human melanoma cells; Carstam R et al.; The concentrations of dopa, cysteinyldopas, 5-S-glutathionyldopa, gamma-glutamyl-5-S-cysteinyldopa and 5-S-cysteinylglycinedopa, were analysed in homogenates of cultured human melanoma cells and in culture media . Cysteinyldopas were found to be the major catechol in the cells, with a molar concentration more than a hundred times that of dopa . 5-S-Glutathionyldopa was found in the same amount as dopa, while the quantity of 5-S-cysteinylglycinedopa was one order of magnitude less . gamma-Glutamyl-5-S-cysteinyldopa was not present in detectable amounts . In the medium the concentrations of dopa, 5-S-cysteinylglycinedopa and of 5-S-glutathionyldopa were about one half of those in the cells, while the concentration of cysteinyldopas was about 2% . The ratio between 2-S-cysteinyldopa and 5-S-cysteinyldopa when incubating dopa and cysteine with tyrosinase was identical with the ratio between the analogically synthetised isomers of glutathionyldopa . Consequently, from the calculation of these ratios in cells and media one cannot deduce whether cysteinyldopas arise from the direct addition of cysteine to dopaquinone, or from degradation of glutathionyldopa . Oxidation of 5-S-glutathionyldopa gives a red chromophore with maximum absorption at 480 nm which develops into a black pigment.

Gynecol Obstet Invest, 1987, 23(1), 60 - 6
Production of pregnancy-associated plasma protein-A (PAPP-A) by cultured tumour granulosa cells; Sinosich MJ et al.; Ten ovarian and 2 cervical tumour cell lines were analysed for the production of pregnancy-associated proteins . Pregnancy-associated plasma protein-A (PAPP-A) was detected by radioimmunoassay in culture media of 2 out of 4 (50%) tumour granulosa cell lines (mean = 104 microIU/10(5) cells/24 h) but not in any ovarian (n = 6) or cervical (n = 2) tumour cell lines . By contrast, human chorionic gonadotrophin (hCG), pregnancy specific beta 1-glycoprotein and alpha-fetoprotein (AFP) were not detected in any of the PAPP-A positive media . Only two cell lines produced hCG (58.5 and 25.5 mIU/10(5) cells/24 h) . No AFP was produced by any of these 12 cell lines, whereas placental protein 5 was positive in 7 . None of these proteins were detected in the culture media of 4 cell lines . In vitro derived PAPP-A was immunologically indistinguishable from either pregnancy or ovarian follicular PAPP-A . All PAPP-A species interacted reversibly with immobilised heparin and were determined by molecular sieve chromatography to have an apparent molecular weight of 820,000 daltons . Cultured tumour granulosa cells specifically synthesised and secreted a large protein which was immunologically and physicochemically indistinguishable from in vivo (pregnancy and ovarian follicular) derived PAPP-A.

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