J Neurosurg, 1983 Sep, 59(3), 461 - 6 Production of collagenase and inhibitor (TIMP) by intracranial tumors and dura in vitro; Halaka AN et al.; The production of collagenase and collagenase inhibitor (TIMP) by various intracranial tumors (25 meningiomas, eight gliomas, seven metastases, four pituitary adenomas, and five others) was studied in short-term organ culture . While meningiomas produced negligible amounts of collagenase, two metastatic carcinomas of bronchial and breast origin produced significant amounts of the enzyme . Cultures of dura from an invasive meningioma and of bone invaded by a meningioma also produced collagenase . In varying amounts, TIMP was detected in culture media from most of the tumors studied; invasive tumors tended to produce less TIMP than noninvasive tumors . The results are discussed in relation to current views on tissue degradation and mechanisms of tumor invasion. Boll Soc Ital Biol Sper, 1983 Aug 30, 59(8), 1190 - 6 {Inflammatory reaction to polyester material in the guinea pig . IV . Production and liberation of beta glucuronidase and lactate dehydrogenase by peritoneal macrophages in culture}; Ventrelli I et al.; Peritoneal macrophages obtained from guinea pigs intraperitoneally injected with minced polyester threads with saline, and from control animals were cultivated and the activities of the lysosomal enzyme beta-glucuronidase and of the cytoplasmic enzyme lactate dehydrogenase were determined in the cells and in the culture media . The production and particularly the release into the medium of beta-glucuronidase increased in the cultures from the treated animals; the LDH production was also markedly increased, and the enzyme was partly lost into the medium, suggesting that the injection of Mersilene into the guinea pigs markedly induces these enzymes in the phagocytes and at the same time probably increases the leakiness of their cell membrane. J Immunol Methods, 1983 Aug 12, 62(1), 59 - 67 Improved fusion technique . I . Human umbilical cord serum, a new and potent growth promoter, compared with other B cell and hybridoma activators; Westerwoudt RJ et al.; Accelerated proliferation of hybridoma cells was observed in the presence of human umbilical cord serum (HUCS) . This had very strong growth-promoting activity, even at a concentration of 2% . A comparison was made between HUCS and other B cell growth promoters, such as lipopolysaccharide (LPS) and dextran sulfate (DxS), macrophage supernatant, and human endothelial culture supernatant (HECS) . The growth-promoting effect of HUCS was superior . Using a microcytotoxicity assay, we found no significant differences in the number of antibody producing clones with the various culture media, except for fetal calf serum. Cancer Res, 1983 Aug, 43(8), 3754 - 8 Comparable growth regulation of five human tumor cell lines by neonatal human lung fibroblasts in semisolid culture media; Kirk D et al.; Cellular growth interactions were studied between neonatal human lung fibroblasts (NLF-13) and human tumor lines derived from carcinomas of the prostate (PC-3, DU145), bladder (J82), and endometrium (HEC-1A) and from a glioma (Hs 683t) . NLF-13 were interacted with tumor cells in soft agar or agarose media using two experimental protocols . In one system, NLF-13 cells were grown as anchored monolayers proliferating under the tumor cell layer . In the second, NLF-13 were embedded directly (nonanchored) into the agar or agarose layer with the tumor cells . The results from both interaction systems were similar for all five tumor lines . Anchored NLF-13 caused a dose-dependent inhibition of tumor growth, whereas nonanchored cells produced a dose-dependent growth stimulation . A time exposure experiment indicated that tumor stimulation and inhibition were biphasic responses to NLF-13 . It was concluded that low concentrations of a diffusible NLF-13 product(s) accelerated tumor growth, whereas high concentrations were inhibitory . Further, the production of the active NLF-13 substance(s) was positively correlated with NLF-13 growth rate . Tumor cell inhibition was irreversible after a 5-day exposure to proliferating NLF-13 cells . Another line of normal neonatal human lung fibroblasts (NLF-147) showed inhibitory properties similar to those described for NLF-13 . However, preliminary studies with fibroblasts from the skin of a Down's syndrome neonate (DS-172) and from a human kidney tumor (KTF-130) have shown both these fibroblast types to have a reduced ability to inhibit tumor cell cultures (J82) compared to the neonatal lung fibroblasts (NLF-13 and NLF-147). Radioisotopes, 1983 Aug, 32(8), 353 - 8 Influences of stable iodine upon the concentration of radioactive iodine by marine organisms; Hirano S et al.; Uptake and loss of radioactive iodine by marine organisms were studied in the artificial seawater in which the concentration as well as chemical forms of both stable and radioactive iodine were controlled . The concentration factors of radioactive iodine by these organisms were clearly dependent upon the concentration of stable iodide ion in the culture media while the concentration of stable iodate ion did not affect the uptake and loss of radioactive iodine . It was observed that the higher the concentration of iodide ion was in the culture media the shorter the biological half-life of radioactive iodine became, and thus the lower the concentration factor resulted in. Gann, 1983 Aug, 74(4), 534 - 41 Erythropoietin production in human renal carcinoma cells passaged in nude mice and in tissue culture; Katsuoka Y et al.; Renal cell carcinoma tissues from two patients, one with and one without erythrocytosis, were successfully transplanted into athymic nude mice . Transplantations of the erythrocytic tumor through six successive generations of nude mice produced a significant (P less than 0.001) elevation in mean hematocrit from 36.5 +/- 2.1% (range 32-42%) to 53.7 +/- 5.1% (range 40-63%), in comparison with a non-erythrocytic tumor which showed a progressive fall in hematocrit from 46.5 +/- 2.0% (range 41-50%) to 36.8 +/- 1.6% (range 33-40%) . Non-grafted control nude mice maintained stable hematocrit levels from an initial level of 45 +/- 0.5% to 46.5 +/- 1.2% when studied over the same time interval . Similarly red cell mass values in the mice transplanted with the erythrocytic tumor (5.04 +/- 1.85 ml/100 g) were considerably higher than in both the non-grafted nude mice (3.39 +/- 0.81 ml/100 g) and the non-erythrocytic tumor-grafted mice (3.8 +/- 0.3 ml/100 g) after 6 generations of transplants . Plasma erythropoietin levels in the erythrocytic tumor-grafted mice (169.4 +/- 83.1 mU/ml) were significantly (P less than 0.02) higher than in the non-grafted controls (22.2 +/- 9.5 mU/ml), and furthermore the erythropoietin levels in the tumor extracts were significantly (P less than 0.02) higher in the tumors from erythrocytic mice (range 54.7 to 234.6 mU/g tumor) than in the tumors from non-erythrocytic mice (range 0.3 to 1.9 mU/g tumor) . In vitro monolayer cultures of these tumors confirmed the higher erythropoietin levels in the erythrocytic renal carcinoma (138 mU/ml) as compared with culture media of non-erythrocytic tumors (15-91 mU/ml) using the fetal mouse liver assay (59Fe incorp . into heme) . The present studies indicate autonomous erythropoietin production by human renal cell carcinomas both in vivo in nude mice and in vitro in tissue cultures. J Cell Biol, 1983 Aug, 97(2), 351 - 8 Induction of chondroitin sulfate proteoglycan synthesis and secretion in lymphocytes and monocytes; Levitt D et al.; The ability of mononuclear leukocytes to synthesize and secrete proteoglycans was evaluated . Using radiolabeling with H2 35SO4, it is shown that peripheral blood mononuclear cells (PBMC) and their major subpopulations (B cells, T cells, and monocytes), as well as mouse spleen cells, all secreted easily detectable proteoglycan . After 24-h labeling periods, 90% of macromolecular 35S could be detected in culture media . This material was primarily (greater than 95%) chondroitin-4-sulfate proteoglycan (CSPG) . Production and secretion of CSPG could be stimulated more than 200% in PBMC and 300% in T cell populations by high concentrations of concanavalin A and phorbol 12-myristate-13-acetate; lipopolysaccharide induced a small (twofold) but reproducible increase in CSPG secretion by adherent mononuclear leukocytes . The CSPG secreted by PBMC was relatively small in size compared to chondrocyte CSPG (130,000 daltons vs . 2-4 million daltons) but possessed similar sizes of glycosaminoglycan chains and greater solubility in low ionic strength solutions . This sulfated polyanion, which was produced endogenously by leukocytes and was actively secreted, might function as a co-mediator or "second messenger" in certain immune responses. Aust J Exp Biol Med Sci, 1983 Aug, 61 (Pt 4), 439 - 49 Growth and gliotic response of astrocytes in vitro; Lolait SJ et al.; We investigated the development of astrocytes in mechanically dissociated primary cultures of foetal and neonatal rat brain grown in different culture media using immunofluorescence and antiserum to glial fibrillary acidic protein (GFAP) . GFAP-positive cells developed at a time corresponding to the 16th day of embryonic development and initially grew more slowly in Dulbecco's modified Eagle's Medium (DMEM) than in Medium 199 (M199) or Modified Eagle's Medium (MEM), but as the cultures matured a greater proportion of GFAP-positive cells was obtained in DMEM, resulting in relatively pure populations of GFAP-positive cells after 1-2 months in vitro . GFAP-positive cells comprised process-bearing and fibroblastoid cell types and cells with intermediate morphology . Cultures in DMEM were also characterized by the appearance after 8-18 days in vitro of thick long bands of glial processes surrounding cyst-like spaces . The morphological change may represent a gliotic reaction to necrosis in vitro. Gastroenterology, 1983 Aug, 85(2), 376 - 84 Collagenase from the culture medium of rabbit colon wall with special reference to the latent type and substrate specificity; Oyamada I et al.; Latent and active forms of collagenase were detected in culture media of the normal rabbit colon . During culture, the collagenase appeared to be produced by surviving and growing mucosas on the degenerated or necrotic colon wall . Type III collagen was most readily degraded by the collagenase, followed by type I and II collagens . The collagenase did not attack type IV or V collagens . The latent collagenase was activated by trypsin and chaotropic agents such as 3 M NaSCN or NaI, and autoactivated gradually during storage . Activated latent collagenase showed the properties of metalloproteinase as in the active collagenase . The apparent molecular weights, determined by calibrated Sephadex G-75, were 39,000 and 31,000 for the latent and active enzymes, respectively . After 12 h of tissue culture, the latent collagenase appeared in the culture media 10-20 h earlier than the active collagenase . The collagenase in the culture media of the early period was mainly the latent form, while the media of the late period contained a large amount of the active form. J Clin Microbiol, 1983 Aug, 18(2), 384 - 8 Comparison of a radiometric method (BACTEC) and conventional culture media for recovery of mycobacteria from smear-negative specimens; Morgan MA et al.; The BACTEC system and three conventional media (Middlebrook 7H10, selective Middlebrook 7H11 {S7H11}, and Lowenstein-Jensen {LJ} were compared for their mean recovery times and recovery rates of mycobacteria from acid-fast, smear-negative clinical specimens . Of the 71 smear-negative, culture-positive specimens recovered from 2,165 submitted smear-negative cultures, the BACTEC system detected 71.8%, compared with 88.7% for the conventional three-medium system . When media were individually compared, BACTEC medium (Middlebrook 7H12) was more successful in recovering mycobacteria (71.8%) than was LJ (62%), Middlebrook medium 7H10 (55.9%), or Middlebrook S7H11 medium (52.1%) . Middlebrook 7H11 medium containing sodium selenate was also evaluated and did not increase the recovery rate or decrease the recovery time of mycobacterial species when compared with LJ, Middlebrook 7H10, S7H11, and 7H12 media . The mean detection time for the BACTEC system was less than that by conventional methods for the seven species of mycobacteria recovered . Detection times for Mycobacterium tuberculosis on the BACTEC system and conventional cultural systems were 13.7 and 26.3 days, respectively. Thromb Res, 1983 Jul 15, 31(2), 219 - 31 Angiotensin II does not elicit any specific prostaglandin secretion in piglet cultured endothelial cells; Ody C et al.; PGE2 and PGF2 alpha were measured in culture media from piglet aortic endothelial cells by radioimmunological analysis . Prostacyclin secretion was evaluated by radioimmunological analysis of its stable metabolite, 6-keto-PGF1 alpha after reverse-phase high pressure liquid chromatography separation . No stimulation of either prostaglandin was detectable in culture media after treatment with angiotensin II (10(-9) to 10(-6) M) for 15 to 120 min at 37 degrees C . Under the same conditions angiotensin II (10(-7) M) elicited a 2 to 3 fold increase in PGE2 and PGF2 alpha secretion when incubated with cultured piglet aortic smooth muscle cells . In addition, we failed to detect specific angiotensin receptors at the surface of intact cultured endothelial cells . Since there was a very rapid increase in prostaglandin secretion after washing or medium changes we suggested that the effects of Angiotensin II on prostacyclin production, demonstrated in perfused organs, could be due to the mechanical stimulation elicited by the contraction of the underlying smooth muscle cells. J Clin Invest, 1983 Jul, 72(1), 371 - 8 Effect of fatty acids on lipid and apoprotein secretion and association in hepatocyte cultures; Patsch W et al.; Increasing availability of free fatty acids (FFA) to liver results in enhanced rates of secretion of triglycerides in lipoproteins . However, as FFA uptake increases, triglyceride secretory rates reach a plateau and esterified fatty acids accumulate intracellularly, suggesting that something is limiting lipid transport out of the liver . One possibility could be the limited availability of apoproteins . To test this hypothesis, primary rat hepatocytes in culture were incubated with increasing amounts of FFA (0-2.1 mumol/dish) and the amounts of lipids and apoproteins inside the cells and in culture media were measured; the latter by specific radioimmunoassays . Media also were fractionated on Sepharose 2B and 6B columns and the elution profiles of apoproteins were obtained . With exposure to increasing amounts of free fatty acids, hepatocytes took up more fatty acids and intracellular levels of triglycerides rose (from 71 to 146 micrograms/mg cell protein) . Concomitantly, media triglycerides nearly doubled (31 to 51 micrograms/mg) . Incorporation of {3H}glyceride into cellular and media triglyceride also rose . However, levels of apoproteins A-I, B, C-III3, and E in cells and media were unchanged . The increasing amounts of triglycerides in media were present in larger particles, as demonstrated on gel permeation chromatography . The elution profiles of apoproteins B, C-III3, and E were altered in that a greater proportion of the apoproteins eluted with larger particles . Similar results were obtained when hepatocytes were preloaded with increasing amounts of FFA over 12 h and analyses of cells and media were carried out 8 and 22 h after removal of fatty acids from the media . During loading of cells, accumulation of cellular triglycerides was directly related to media FFA concentrations . During unloading, triglyceride secretory rates were related to cellular triglyceride levels . At higher triglyceride secretory rates larger particles were secreted and a greater proportion of apoproteins was associated with the larger particles, but total amounts of apoproteins in the system did not change . These data lead us to suggest that enhanced rates of apoprotein synthesis need not occur in the response to acute changes in hepatic lipid transport, rather, increased secretion of lipid is brought about by augmented intracellular lipid apoprotein association. In Vitro, 1983 Jul, 19(7), 538 - 50 The proliferation of human tumor cell lines in the presence of different agars, agaroses, and methyl cellulose; Pavlik EJ et al.; Human tumor cell lines, derived from cancers of the colon, ovary, and cervix, were grown in liquid tissue culture media and media made semisolid with agar (Bacto + deoxycholate lactose agar), agarose {LE, ME, Sea Plaque and Sea Prep (15/45)}, and methyl cellulose . The effects of each agent on overall cell proliferation and rate of overall cell proliferation were examined . The agents, used to make media semisolid, were observed to inhibit or, in some cases, enhance cell growth in a fashion that was characteristic of individual cell lines . These phenomena may be of consequence to the optimization of nutrient media for primary tumor cell preparations. Clin Exp Immunol, 1983 Jul, 53(1), 239 - 48 In vitro suppression of fibroblast growth inhibitory lymphokine production by asbestos; Lemaire I et al.; Human peripheral blood mononuclear leucocytes (PBML) stimulated with concanavalin A (Con A) or phytohaemagglutinin (PHA) produced a soluble factor which inhibits lung fibroblast DNA synthesis and growth . Lymphocyte enriched preparations produced significant growth inhibitory activity in the presence of PHA whereas media from adherent mononuclear cells incubated in the presence of the mitogen did not contain similar activity . This fibroblast growth inhibitory factor (FGIF) was non-dialysable, heat stable and resistant to pH 5 . FGIF was also resistant to treatment with chymotrypsin and phosphodiesterase but partially sensitive to treatment with trypsin . Interestingly, there was significant suppression of FGIF production by PBML cultured with PHA in the presence of low concentrations of chrysotile asbestos (5-25 micrograms/ml) . In this regard, asbestos (25 micrograms/ml) was not cytotoxic for lymphocytes but had a damaging effect on monocytes as evidenced by the release of lactate dehydrogenase (LDH) a cytoplasmic enzyme, in their culture media . These findings indicate that stimulated lymphocytes have the ability to inhibit fibroblast proliferation by releasing FGIF and that asbestos interfere with this process . Thus, while FGIF may regulate the extent of connective tissue proliferation during normal repair process, suppression of its production by asbestos may contribute to excessive fibroblast accumulation and fibrosis. Am J Physiol, 1983 Jul, 245(1), C78 - 83 Evidence for active Na+ transport by cultured monolayers of pulmonary alveolar epithelial cells; Goodman BE et al.; Primary cultured type II alveolar epithelial cells grown to confluence on nonporous surfaces form many small fluid-filled hemicysts or domes . These domes are generally thought to result from active transport of solutes from the medium above the cell monolayer to the substratum, with water following passively . We have investigated the characteristics of active transport by primary cultured monolayers of type II alveolar epithelial cells from rat lungs . Changes in dome density were measured after exposure to metabolic inhibitors, Na+ or Cl- transport inhibitors, and low-Na+ or low-Cl- culture media . Metabolic and Na+ transport inhibitors, and low-Na+ medium, lead to disappearance of domes, whereas Cl- transport inhibitors and low-Cl- medium seem to have no effect on dome density . These results suggest the presence of a Na+-dependent active transepithelial transport process across the monolayer, which is responsible for the formation of domes . This finding implies that absorption of fluid by mammalian alveolar epithelium in vivo may be important in the maintenance of normal lung fluid balance. J Invest Dermatol, 1983 Jul, 81(1 Suppl), 55s - 8s Modulation of differentiation by retinoids; Kubilus J; At high levels of vitamin A and other retinoids (3 X 10(-6) M) attachment of human keratinocytes to 3T3-coated plastic dishes is mildly inhibited . Retinoids at this concentration in culture media seem to have an antikeratinizing effect in that cells appear to be less differentiated . Retinoid-treated cultures are less stratified, having fewer cell layers, and display larger intercellular spaces and rounder, less flattened cells . Treated cultures also contain higher percentages of saline-soluble proteins and lower percentages of proteins requiring reduction and/or denaturants for solubility . This suggests that in treated cultures, the most keratinized cells are absent . Growth curves show that those most keratinized cells are sloughed from the dish and appear in the media . Thus at 3 X 10(-6) M, the major retinoid effect is to promote desquamation . At higher concentrations, retinoids are toxic to the keratinocyte, but at lower concentrations, they may be stimulative. Prostaglandins Leukot Med, 1983 Jul, 11(3), 317 - 23 PGE2 generation and release by the larval stage of the cestode, taenia taeniaeformis; Leid RW et al.; The existence of an arachidonate cascade in invertebrate species has been little explored . We therefore sought to determine if two important prostanoid compounds, PGE2 and prostacyclin, PGI2 were generated and released by the larval stage of the cat tapeworm, Taenia taeniaeformis . PGE2 and PGI2 were identified by specific radioimmunoassay after thin layer chromatography of chloroform/methanol extracts of the worms or in vitro saline culture media . We detected a release of 176-182 pg of PGE2 from 2 worms after a 60' incubation at 37 degrees C with 4.5 mM exogenous sodium arachidonate . No release of PGI2 was detected immunochemically although 41 pg of immunoreactive material was identified in chloroform/methanol extracts of the worm alone . We therefore suggest that modulation of host immune responses could occur by the generation and release of prostanoid compounds such as PGE2, lipids which markedly suppress host cellular reactivity to the parasite . Secondly, the lack of any clot formation around the living organism may well reflect PGI2 presence at the surface of the parasite membrane . Although these findings are at present limited to larval cestodes we would propose that they may be more general means of evading host responses than heretofore suspected. JAMA, 1983 Jun 17, 249(23), 3184 - 8 Nosocomial Legionnaires' disease uncovered in a prospective pneumonia study; Muder RR et al.; Most hospitals have yet to record a case of nosocomial legionnaires' disease; the importance of isolation of Legionella pneumophila in the water system of such an institution is unclear . We undertook a prospective pneumonia study in tandem at a veterans hospital where legionnaires' disease was known to be endemic and a community teaching hospital where legionnaires' disease had never been documented . Legionella serological tests were performed on all patients with pneumonia; selective culture media and direct fluorescent antibody testing for Legionella were made readily available . Simultaneous environmental surveys for Legionella were performed . At the community hospital, we discovered that 64% of sites in the water distribution system yielded L pneumophila and that 14.3% of nosocomial pneumonias were legionnaires' disease . The epidemiologic implications of these findings are discussed . Options concerning case detection and eradication measures in the face of hospital water contamination with L pneumophila are presented. Life Sci, 1983 Jun 6, 32(23), 2641 - 7 Teratogenicity of acetaldehyde in vitro: relevance to the fetal alcohol syndrome; Campbell MA et al.; Day 10 rat embryos grown in vitro showed significant retardation in growth and development when culture media contained acetaldehyde . A concentration-response range for acetaldehyde-induced embryotoxicity was defined, from no effect at 5 microM to complete lethality at 100 microM . The relative teratogenicity of ethanol and acetaldehyde, and the potential roles of these compounds in producing the Fetal Alcohol Syndrome are discussed . Despite intensive investigation into alcohol teratogenicity, the mechanism that produces the Fetal Alcohol Syndrome (FAS) remains unknown . Observed anomalies may result from direct embryonic exposure to ethanol or one of its metabolites, or from some indirect effect such as altered placental function or maternal nutritional status . Use of in vitro techniques allows study of direct embryonic exposures in the absence of indirect influences . Under such conditions, ethanol has been found to exert direct embryotoxicity (1) . Rat embryos, grown as cultured explants and subjected to ethanol concentrations of 32.5 or 65 mM, were retarded in growth and development when compared to untreated controls . In this paper, we report direct embrytoxic effects of acetaldehyde, the primary metabolite of ethanol, at concentrations as low as 25 microM . Acetaldehyde teratogenicity has not been extensively studied . Veghelyi et al . (2) and Lambert, Papp and Nishiura (3) employed a combination of ethanol and disulfiram (an inhibitor of acetaldehyde-oxidizing enzymes) . Teratogenic effects exceeded expectations based upon assumption of an additive interaction between these two compounds, and were attributed to elevated maternal blood acetaldehyde . O'Shea and Kauffman (4,5) and Dreosti et al . (6) administered acetaldehyde to pregnant animals by injection . Treatment resulted in retarded growth and development, decreased DNA synthesis, and increased frequencies of malformation and resorption . While these studies imply a role for acetaldehyde in alcohol-induced teratogenesis, indirect effects through altered maternal or placental factors cannot be eliminated . We present here the first concentration-response data for direct embryonic exposure to acetaldehyde. J R Army Med Corps, 1983 Jun, 129(2), 111 - 4 Leptospirosis--an occupational disease of soldiers; Johnston JH et al.; Two cases of leptospirosis are described in soldiers who fell into a river together . One developed Weil's disease due to icterohaemorrhagiae serogroup and the other "Mud-Fever" due to grippotyphosa serogroup . The organisms were initially cultured in standard blood culture media . There were six cases of leptospirosis in BAOR in the 10 years to 1980 giving an incidence 10 times greater than in the civil population of UK and West Germany . Leptospirosis is an occupational disease of soldiers in BAOR and the health risks of fresh water immersion should be publicised. J Gen Microbiol, 1983 Jun, 129 (Pt 6), 1721 - 3 Stimulation of aflatoxin biosynthesis by lipophilic epoxides; Fanelli C et al.; Epoxy fatty acids added to the culture media either with the inoculum or at the end of exponential growth phase stimulated aflatoxin production by toxigenic strains of Aspergillus flavus and Aspergillus parasiticus . This effect did not appear when the unsaturated fatty acids used for the synthesis of the epoxides and the polyhydroxyacids (which can be considered to be derived from the opening of the oxirane ring) replaced the epoxides in the culture media . No significant differences were detected in the lipid fractions (diglycerides, sterols, triglycerides, free fatty acids, sterol esters) extracted from the mycelia grown in the presence of any of the fatty acid derivates. Eur J Obstet Gynecol Reprod Biol, 1983 Jun, 15(2), 63 - 70 The Vienna program of in vitro fertilization and embryo-transfer--a successful clinical treatment; Feichtinger W et al.; A clinical program for in vitro fertilization (IVF) and embryo transfer (ET) is described . First successes were obtained using media and techniques according to Lopata et al . (1980, Fertil . Steril., 33, 117-120) . The 'Vienna IVF and ET-program' was developed after several modifications and led to a pregnancy rate of 34%/ET . Studies on different culture media and their supplements were performed . B2-Menezo-medium supplemented by 15% human serum seemed best suited for human IVF because of its simplicity and readiness for use . However, there was no significant difference in comparison of the fertilization rate between B2 and modified Ham's F10 medium, or statistical difference between the usual 5% O2-5% CO2 and 90% N2 gas mixture for culture and 5% CO2 in air . So the latter can be used as a simpler system . pH measurements established follicular fluid to be very stable at 37 degrees C for several hours both on air and on 5% CO2 . It is recommended as an appropriate medium for preincubating oocytes before insemination; eggs can also be transported in this fluid . Based on clinical experience a system of culturing eggs at one specialized IVF center for different hospitals and gynecologists can be discussed. J Appl Bacteriol, 1983 Jun, 54(3), 329 - 34 Rapid method for selecting appropriate solid media for the enumeration of aerobic micro-organisms; Richard J et al.; A quick and cheap method for selecting appropriate solid culture media has been devised . It consists in the rapid picking of fragments of test colonies with the aid of a rubber strip in which pins are fixed in parallel, dispensing up to 8 colonies simultaneously in the wells of a Microtiter plate and streaking 4 strains at the same time on square Petri dishes containing the media under comparison . The approximate diameters of well-isolated colonies are measured with the aid of a series of calibrated spots . The results corresponded with those given by the spiral plate method used as a reference for colony count and diameter measure. Brain Res, 1983 Jun, 284(2-3), 215 - 21 Gangliosides enhance neurite outgrowth in PC12 cells; Ferrari G et al.; Gangliosides are surface membrane components that have been suggested to play a significant role in the regulation of many cellular events including neuronal differentiation, growth and regeneration . We have chosen PC12 cell as a model system to study the influence of exogenously added glycosphyngolipids on in vitro differentiation and regeneration . A mixture of bovine brain gangliosides, (GM1 21%, GD1a 39.7%, GD1b 16% and GT 19%) or purified GM1 and GT respectively were added to culture media containing NGF on plating day and their effect was monitored on alternate days starting on day 5 . The degree and rate of fiber outgrowth was significantly enhanced by media containing gangliosides at a concentration of 10(-6), 10(-7) M when serum was left out and 10(-3), 10(-4) M when serum was added to the culture medium . The stimulating ganglioside action required the presence of NGF to induce neurite outgrowth . However, binding studies indicated that exogenous gangliosides do not affect NGF binding to PC12 cells, therefore their stimulatory action may be separated from the interaction between NGF and its receptors . Subculturing of NGF-treated cells for 10 days demonstrated that gangliosides treatment also enhanced the NGF stimulated regeneration of neurites . Gangliosides may be incorporated at the level of cell surface, thereby affecting and facilitating membrane phenomena involved in neurite outgrowth. J Immunol Methods, 1983 May 27, 60(1-2), 33 - 45 Use of protein A to remove immunoglobulins from serum in hybridoma culture media; Underwood PA et al.; The levels of protein A-reactive immunoglobulin (PA-Ig) in foetal bovine serum were measured in commercial batches . For tissue culture media incorporating 10% foetal bovine serum, the levels of bovine PA-Ig were of a similar order to those of mouse monoclonal antibodies produced by hybridomas grown in such media . The equilibrium constants were calculated for the binding to protein A-Sepharose of a number of mouse monoclonal antibodies, and of PA-Ig in foetal bovine serum and normal mouse serum . The average affinity of the mouse PA-Ig was 10 times higher than that of the bovine PA-Ig, suggesting that the two could be separated by affinity chromatography on protein A-Sepharose . The mouse monoclonal antibodies, however, displayed a range of affinity 1.5-100 times that of the bovine PA-Ig, indicating that such separation could not be generally applied . The optimal technique involved removing PA-Ig from bovine serum before its inclusion in the culture medium and then purifying the monoclonal antibody on a second protein A-Sepharose column. J Biol Chem, 1983 May 10, 258(9), 5869 - 77 Procollagen IV . Association to tetramers; Duncan KG et al.; Procollagen IV was isolated from culture media of the mouse endodermal cell line PF-HR9 . Some of the triple helical procollagen IV molecules were associated at their NH2 ends to tetramers which were identified by electron microscopy, velocity sedimentation, and electrophoresis . The formation of these tetramers in cell cultures and from isolated procollagen IV molecules was investigated . After an initial noncovalent association, which is reversible, disulfide bonds form between molecules . Even alkylated molecules form disulfide-linked tetramers when exposed to a mixture of reduced and oxidized glutathione . This reaction requires an adequate concentration of procollagen and is not facilitated by added laminin, Ca2+, or Mg2+ ions . Cystine, as a normal constituent of cell culture media, interferes in tetramer assembly, presumably by forming mixed disulfides . Tetramers formed normally and under the influence of glutathione are similar, but probably not identical, and resemble those isolated from fragmented basement membranes . We conclude that the NH2 ends of procollagen IV molecules can associate into tetramers without the help of other molecules and that disulfide bridges subsequently stabilize the association in various ways. Nature, 1983 May 5-11, 303(5912), 64 - 5 Bathocuproine sulphonate: a tissue culture-compatible indicator of copper-mediated toxicity; Mohindru A et al.; Metal-binding chelators may interact with biological systems by either of two mechanisms: they may combine with an essential metal, which can be either freely dissociated or part of an enzyme prosthetic group, or they may react with a metal ion to form a biologically reactive metal-chelate complex . As trace metals are always present as contaminants in serum-supplemented culture media used to study chelating agents, it is frequently difficult to distinguish between the two possibilities . Here we describe the use of a nontoxic, copper-specific chelating agent, bathocuproine sulphonate (Fig . 1) which, by combining with available endogenous copper in a tissue culture preparation, abolished the toxicity of three structurally unrelated chelating agents . These three agents may therefore be considered to be biologically active by the second mechanism. Biochim Biophys Acta, 1983 May 4, 757(1), 47 - 58 Breakdown of cartilage proteoglycan in a tissue culture model of rheumatoid arthritis; Steinberg JJ et al.; Proteoglycan breakdown was studied in a coculture model which mimics the confrontation between synovium and cartilage that occurs in rheumatoid arthritis . Bovine nasal-septum cartilage discs radioactively labeled (35SO2-4 with or without {3H}glucosamine) and 'chased' in non-radioactive medium were cultured in contact with minced rheumatoid synovial membranes for intervals up to 8 days . Synovium-stimulated (2-3-fold) cartilage breakdown was unaffected by ascorbate supplementation . Labeled products (small molecules plus proteoglycan complexes) in culture media were characterized by chromatographic, sedimentation and enzymic digestion methods . Breakdown was dominated by the release of a range of proteoglycan products, fully disaggregated and incapable of reaggregation with added hyaluronate . Because constituent glycosaminoglycans were of uniform size, proteoglycan polydispersity was attributed to differences in core protein length . Hydrocortisone inhibited degradation and partially prevented the shift of proteoglycans to lower average molecular weight . An additional breakdown pattern occasionally noted during the initial 48 h of coculture was characterized by release of a subpopulation of low charge-density proteoglycan bearing shortened glycosaminoglycan chains, consistent with glycosidase action . We conclude that rheumatoid synovia exhibit two distinct cartilage degradative potencies in vitro that may be important in vivo: (a) A variable hyaluronidase-like activity at early culture times, and (b) a dominant proteolytic activity generating an array of disaggregated proteoglycan products that differ largely on the basis of their core lengths . The response to hydrocortisone is consistent with inhibition of proteolysis through the stabilization of cellular membranes. Nippon Sanka Fujinka Gakkai Zasshi, 1983 May, 35(5), 674 - 82 {Virological studies on the feto-maternal tissues infected with rubella virus}; Sugiura K; In studies on the congenital rubella syndrome, trans-placental rubella virus (RV) infection was investigated in vitro with human chorionic, decidual and fetal tissues obtained by artificial abortion from RV-infected pregnant women showing high hemagglutination-inhibiting and complement-fixing RV antibodies (1:512 and 1:16) . RV was isolated from both chorionic (CR) and the fetal cells (FR) derived from RV-infected pregnant woman and the neutralization test disclosed that their antigenicity and biological properties were similar to that of the standard RV strain, M-33 . These CR and FR cells showed a constant release of RV ranging from 2 to 4 log10 FFU (focus forming unit)/0.1ml into the culture media . Moreover, positive staining by immunofluorescent technique (IF) over 70 days seems to indicate RV persistent infection in these cells . However, decidual cells derived from RV-infected pregnant woman gave negative results in the RV release and IF staining . The above evidence strongly indicates that the chorionic cells are easily infected and converted to the RV-carrier . One possible mode of trans-placental RV infection is via an initial infection of the chorionic cells followed by the establishment of persistent RV infection. J Nutr, 1983 May, 113(5), 1032 - 8 Purine synthesis and reutilization in folate-deficient rat hepatocytes; Walzem RL et al.; Although folic acid is known to be involved in the pathways of purine metabolism, the precise changes brought about in purine synthesis, reutilization, pool sizes, and ratios by experimental folate deficiency are not clear . Consequently, these aspects of purine metabolism were measured in hepatocytes from control and folate-deficient rats fed an amino acid diet with and without folic acid, respectively . Purine synthesis and reutilization were measured as the rates of incorporation of {U-14C}glycine and {G-3H}hypoxanthine, respectively, into the adenine and guanine pools of freshly isolated hepatocytes after a 3-hour incubation in folate-free, as well as folate- and/or thymidine-supplemented culture media . Hepatocytes from folate-deficient rats had the same rates of purine synthesis as those from control rats . Purine reutilization, purine pool sizes, and the adenine:guanine ratios were lower in hepatocytes from deficient compared with control rats . Purine synthesis was increased when folic acid or thymidine was added to the culture medium . Although hepatocytes from folate-deficient rats had a lower rate of purine reutilization compared with those from control rats, the reutilization rates did not respond to the addition of folic acid or thymidine to the culture medium . The data suggest that purine synthesis was not impaired but purine reutilization was diminished in folate deficiency . Thymidine was as effective as folic acid in stimulating purine synthesis in both control and folate-deficient hepatocytes. Cytometry, 1983 May, 3(6), 453 - 5 Human T lymphocyte differentiation antigens: effects of blood sample storage on Leu antibody binding; Hensleigh PA et al.; Current studies of human T lymphocytes and their subsets often use quantitative immunofluorescence analysis with monoclonal antibodies against cell surface antigens . With storage of whole blood or separated mononuclear cells for more than a few hours we have found marked changes in lymphocyte analysis using a fluorescence activated cell sorter (FACS) . Experiments were done to determine if these lymphocyte changes were influenced by storage temperature and if lymphocytes could be made more stable by addition of culture media RPMI 1640 to whole blood . Optimal conditions found for blood storage were with with addition of 50% RPMI 1640 and with samples held at room temperature (22 degrees C) . With these storage conditions, delay on FACS analysis up to 24 hours did not result in spurious results . When blood samples are collected in places remote from the laboratory or when batch analysis of serially collected samples is desirable, excessive storage times should be avoided. Brain Res Bull, 1983 May, 10(5), 667 - 74 Serum-free medium for cultures of the postnatal mouse cerebellum: only insulin is essential; Huck S; Serum-free culture conditions for dissociated postnatal mouse cerebellar cells were investigated . This study demonstrates that among various supposed growth-promoting factors only insulin is required as an additive to the basic medium . If viewed by phase contrast microscopy, cultures kept in insulin-supplemented basic medium looked identical to those maintained in the presence of a mixture of growth-promoting factors (insulin, putrescine, transferrin, progesterone, triiodothyronine, selenium) . In addition, quantitative evidence is provided indicating that cellular survival is supported to the same extent by insulin as by this admixture . Insulin concentrations required ranged between 0.3-20 micrograms/ml . By contrast to serum-supplemented culture conditions, no significant proliferation of non-neuronal cells was observed in serum-free culture media . It is expected that by these findings attention will be focused again to the exact role insulin plays in the central nervous system. In Vitro, 1983 May, 19(5), 373 - 5 Zinc levels in zinc-stabilized insulin are inhibitory to the growth of cells in vitro; Chaproniere-Rickenberg DM et al.; Adult human prostatic epithelium was cultured in a defined medium consisting of RPMI 1640 supplemented with transferrin, insulin, epidermal growth factor, dexamethasone, and vitamin A . In the presence of insulin, stabilized with zinc, maximum epithelial multiplication was obtained at an insulin concentration of 0.03 to 0.1 U/ml, corresponding to a zinc concentration of 1.4 X 10(-7) M . At higher insulin concentrations, growth stimulation declined . Zinc-free insulin, on the other hand, stimulated cell multiplication with an optimum concentration of 0.3 to 1.0 U/ml . At this concentration, the maximum growth was twice that obtained with zinc-stabilized insulin . Results demonstrate that growth inhibition caused by zinc limits the concentration of zinc-stabilized insulin, which can be used in serum-free, defined culture media. J Clin Invest, 1983 May, 71(5), 1161 - 74 Role of insulin in lipoprotein secretion by cultured rat hepatocytes; Patsch W et al.; To study the effect of insulin on lipoprotein synthesis and secretion by the liver, apoprotein and lipid levels were measured in primary rat liver cell cultures grown on fibronectin-coated dishes . Triglycerides, phospholipids, apoprotein (apo) B, apo-E, and apo-C-III3 all accumulated in culture media linearly for periods up to 20 h . During incubations, cellular triglyceride contents increased slightly, while cellular apoprotein and phospholipid contents remained constant . In the absence of insulin, rates of accumulation in media of triglycerides, apo-B, apo-C-III3, and apo-E were 2.5 +/- 0.3 micrograms/mg and 33 +/- 5, 24 +/- 3, and 162 +/- 32 ng/mg cell protein per h, respectively . On gel permeation chromatography and density gradient ultracentrifugation, the majority of apoproteins in media were found to be associated with very low density lipoproteins (VLDL) and very little eluted or sedimented with albumin . Incubations in the presence of 50-800 microU/ml of insulin resulted in dose-dependent decreases of triglyceride, phospholipid, apo-B, and apo-E accumulation in the media, paralleled by increases in the cellular contents of these lipoprotein components . The inhibitory effects of insulin on secretion were reversible . Levels of apo-C-III3 and albumin were not affected by insulin . In addition to decreasing secretory rates, the proportion of apo-B, apo-E, and apo-C-III3 associated with VLDL also decreased after the addition of insulin . Concomitantly, the proportion of apo-B eluting with LDL and apo-C-III3, and apo-E eluting near albumin increased . Control experiments, in which exogenous 125I-VLDL or endogenously labeled {14C}VLDL were added to cultures, revealed that the insulin-induced differences in VLDL accumulation and the lipid association of media apoproteins were not due to differences in the processing of VLDL by cells cultured in the presence or absence of insulin . Therefore, it appears that insulin may inhibit the secretion of VLDL perhaps by reducing the intracellular association of lipids and apoproteins. J Cell Physiol, 1983 May, 115(2), 217 - 23 Culture of quiescent arterial smooth muscle cells in a defined serum-free medium; Libby P et al.; An ideal medium for metabolic studies would maintain cultured vascular smooth muscle cells in a quiescent, viable state, as they are in normal arteries in vivo, and would be chemically defined so that the concentrations of hormones and nutrients could be manipulated precisely . In unsupplemented serum-free media these cultures lose protein and DNA, indicating impaired viability . Addition of maximally effective concentrations of insulin (10(-6) M) and transferrin (5 micrograms/ml) prevents loss of DNA and produces near neutral protein balance . Further addition of ascorbic acid (10(-4) M) actually promotes net gain of protein with little or no increase in DNA . Ascorbate consistently increased noncollagen protein synthesis by cultured aortic smooth muscle cells . This novel action of the vitamin did not require insulin but was additive to the effect of this hormone, and was produced by isoascorbate, but not by a variety of other reducing agents . Thus, vascular smooth muscle cells can be maintained in a quiescent but noncatabolic state in simple chemically defined culture media . This finding should facilitate studies of the effects of nutrients and hormones on the metabolism of these cells under conditions that resemble those in the normal artery in vivo . Such an approach may also prove valuable for culture of other differentiated cell types that do not usually divide in the intact organism. J Bacteriol, 1983 May, 154(2), 669 - 75 Regulation of outer membrane porin protein synthesis in Escherichia coli K-12: ompF regulates the expression of ompC; Ozawa Y et al.; The relative amounts of the OmpF and OmpC proteins in the outer membrane of Escherichia coli K-12 are affected differentially by high concentrations of substances like sucrose in culture media in such a manner that a decrease in the amount of the OmpF protein appears to be compensated for by a reciprocal increase in the OmpC protein . When an ompF mutation was introduced, OmpC synthesis became almost independent of sucrose and occurred at the fully induced level even in the absence of sucrose . On the other hand, introduction of an ompC mutation did not affect the sucrose-dependent profile of OmpF synthesis . The effect of the ompF mutation was also examined with the ompC-lac fusion strain, in which expression of beta-galactosidase is under the control of the ompC promoter . The expression of beta-galactosidase coded for by the ompC-lac fusion in the ompF+ and ompF- strains was essentially the same as that of the OmpC protein, being sucrose dependent in the ompF+ strain and sucrose-independent in the ompF mutant . From these results we conclude that sucrose in the medium primarily regulates ompF gene expression, which in turn regulates ompC gene expression at the transcriptional level . This sequential regulatory mechanism is discussed in relation to the function of the ompB locus. Z Naturforsch {C}, 1983 May-Jun, 38(5-6), 468 - 70 Electromagnetic modulation of biological processes: ATPase function and DNA production by Raji cancer cells in vitro; Colacicco G et al.; Addition of either ATP or ouabain to the culture medium markedly depressed thymidine incorporation into DNA in Raji cells . The electromagnetic field, pulsating at very low frequency, did not affect DNA synthesis in normal culture media nor did it alter its ouabain-inhibition, but it partially reversed the ATP-inhibition . In spite of the presence of ATP, ouabain prevented stimulation of ATPase and DNA synthesis by the field . Although no mechanism is known for the action of either ATP or the field, the results may be interpreted in light of existing speculations . In the absence of the field, external ATP may go into an ATP pool that either blocks ATPase or feeds adenyl cyclase, which hinders DNA synthesis . In contrast, the electromagnetic field may either turn off adenyl cyclase or simply stimulate the ATP-depressed ATPase. Muscle Nerve, 1983 May, 6(4), 283 - 90 Acetylcholine receptor turnover in clonal muscle cells: role of plasmin and effects of protease inhibitors; Romstedt K et al.; Characteristics of acetylcholine receptors were evaluated in G8-1, a continuous skeletal muscle line . Peak binding of 125I-alpha-bungarotoxin was in 10-day-old contractile myotubes at 4-8 nm . Turnover was studied using two different methods; both indicated half-times as little as half as long as previously reported for primary cultures . The effects of a variety of protease inhibitors on receptor turnover were assessed to determine if G8-1 receptors were less stable or turned over faster because of increased neutral protease activity . Leupeptin, antipain, and chloroquine markedly slowed receptor degradation . Inhibitors of plasmin or plasminogen activator had definite but less dramatic effects on receptor turnover . Results from studies in which plasmin was increased in the tissue culture media indicated that a small but definite acceleration of receptor turnover occurred . In clonal G8-1 cells, total number of acetylcholine receptors is controlled by negative feedback and although the major pathway for receptor degradation is lysosomal, plasmin may play a role in initiating receptor internalization. Virology, 1983 Apr 30, 126(2), 548 - 62 Rapid and selective shutoff of plasma protein production in herpes simplex virus type 2-infected hepatoma cells; Isom HC et al.; The effect of herpes simplex virus type 2 (HSV-2) infection of hepatoma McA-RH7777 cells on the production of alpha-fetoprotein (AFP) and several other secreted plasma proteins was examined . Cells infected with HSV-2 were labeled with {35S}methionine for various times postinfection (p.i.) . Culture media and intracellular extracts were immunoprecipitated with plasma protein antibodies and examined by polyacrylamide gel electrophoresis . The relative levels of AFP and several other plasma proteins decreased substantially in infected cells compared with mock-treated controls; however, the level of at least one secreted plasma protein was unchanged after infection and the level of another increased after infection . Hybridization analyses with AFP cDNA showed that AFP RNA decreased to 43 and 30% of controls at 3.5 and 6.5 hr p.i., respectively . These observations were supported by the results of cell-free translation of isolated poly(A)-containing RNA . HSV-2 proteins were not detectable at times p.i . when AFP synthesis, secretion, and mRNA levels were significantly diminished . No decrease in AFP levels was observed in cells infected with ultraviolet-irradiated virus . We conclude that HSV-2 infection of McA-RH7777 cells leads to a rapid decrease in synthesis and secretion of specific plasma proteins which is apparent, at least for AFP, at the level of mRNA . This shutoff is not common to all McA-RH7777 cell proteins and, although seen early after infection, most likely requires the expression of a virus gene(s). J Parasitol, 1983 Apr, 69(2), 319 - 34 Schistosoma mansoni: morphologic changes induced by maintenance in vitro; Carlisle S et al.; Adults of Schistosoma mansoni were incubated in several culture media at various time intervals ranging from 2 to 96 hr . The morphologic changes induced by the incubation were documented using both scanning and transmission electron microscopy . These included changes in the tegument, esophageal cells, and cecum . Variability was noted among worms within experimental groups and the surface changes on single worms were frequently observed to have patchy distribution . Based on morphologic changes observed, culture media were ranked as adequate, mediocre, or inadequate . RPMI-1640 + 50% fetal calf serum, Eagle's MEM with Earle's salts (no serum), and McCoy's 5A Medium + serum were judged adequate . Basal Eagle's Medium, Triple Eagle Medium, NCTC-135, and Earle's Balanced Salt Solution (all with or without serum) were judged mediocre . Hanks' Basal Salt Solution (with or without serum) was judged as severely inadequate . All media tested gave better results in the presence of serum . These factors point to the necessity for the use of carefully selected culture media, as well as adequate controls and sampling techniques in the interpretation of in vitro experiments with S . mansoni. Endocrinology, 1983 Apr, 112(4), 1490 - 8 Uteroglobin production by cultured rabbit uterine epithelial cells; Rajkumar K et al.; Uteroglobin (UG) is a secretory protein produced by the rabbit endometrium and its production is increased during cell differentiation which occurs during early pregnancy or pseudopregnancy . In the present study, the optimal conditions for UG production by rabbit endometrial epithelial cells in culture were examined . Metabolic labeling studies showed the incorporation of {35S}methionine into UG molecules by the endometrial epithelial cells in culture . Accumulation of UG in culture media was linear for at least a period of 24 h . These cells do not catabolize exogenously added radiolabeled UG . Endometrial cells obtained from virgin female rabbits at different times after the administration of human CG (hCG) and put in culture were found to make different amounts of UG . The maximal UG production was found in cells taken from pseudopregnant rabbits 4 days after hCG administration . Cycloheximide (28 micrograms/ml) inhibited the production of UG by the cells in culture whereas actinomycin-D (5 micrograms/ml) and cordycipin (50 micrograms/ml) increased its production . Inhibition of DNA synthesis by hydroxyurea (10(-3) M) did not affect the UG production . The production of UG was significantly less when cells were cultured on attached or floating collagen gels as compared to cells grown on plastic Petri dishes . The amino acid content of Ham's F-12 medium was shown to be adequate for maximal UG production; lowering this amino acid concentration decreased the amount of UG accumulated in the medium over a 24-h period . Increasint the number of cultured cells per dish resulted in an increased UG production per cell. Cancer Res, 1983 Apr, 43(4), 1748 - 60 Newly established uterine cervical cancer cell line (SKG-III) with Regan isoenzyme, human chorionic gonadotropin beta-subunit, and pregnancy-specific beta 1-glycoprotein phenotypes; Nozawa S et al.; The production of Regan isoenzyme (heat-stable, L-phenylalanine-sensitive term-placental alkaline phosphatase), human chorionic gonadotropin beta-subunit, and pregnancy-specific beta 1-glycoprotein by newly characterized human uterine cervical cancer cell lines, SKG-IIIa and SKG-IIIb, is reported . These cell lines were derived from a moderately differentiated epidermoid cancer partially mixed with epidermoid clear-cell components . At the end of the first 4 months in culture 2 sublines with different morphologies were identified . In nude mice, SKG-IIIa produce clear-cell epidermoid cancer with much glycogen, while SKG-IIIb grew as a moderately differentiated epidermoid cancer rich in tonofilaments . The presence of Regan isoenzyme was established by biochemistry, enzyme cytochemistry, immunocytochemistry, and immunoelectrophoresis . However, the copresence of small amounts of early placental alkaline phosphatase was also demonstrated . The alkaline phosphatase specific activities of SKG-IIIa cells and SKG-IIIb cells were 3.7 and 1.4 nmol per mg protein per min, respectively . The existence was proven by radioimmunoassay of human chorionic gonadotropin beta-subunit (SKG-IIIa, 5.0 mlU/mg protein; SKG-IIIb, 4.4 mlU/mg protein), pregnancy-specific beta 1-glycoprotein (SKG-IIIa, 0.7 ng/mg protein) in the culture media as a tumor cell product . The described cell lines may serve as a more representative model system for studies of regulation of oncodevelopmental genes in gynecological tumors in general and in epidermoid cervical cancer in particular. Biochimie, 1983 Apr-May, 65(4-5), 283 - 9 Evidence for the involvement of activated oxygen in fungal degradation of lignocellulose; Bes B et al.; Oxygen has been shown to be necessary as a cosubstrate for the fungal degradation of lignins . In this work, the active forms of oxygen were tentatively identified in three ways: --effect of chemically generated active radicals and molecular species on lignocellulosic complexes, --use of activated oxygen scavengers in culture media of ligninolytic fungi, --characterization of active forms of oxygen by specific reactions . The data obtained strongly suggest that two main oxygen species are involved, namely OH radical and singlet oxygen (1O2) . Chemical or enzymic scavengers inhibit the degradation of lignocelluloses by Phanerochaete chrysosporium . The fungus has been demonstrated to synthesize OH. J Clin Microbiol, 1983 Apr, 17(4), 601 - 4 Value of routine aerobic subculturing of unvented blood culture bottles; Pfaller MA et al.; The value of performing routine aerobic subcultures of both vented and unvented blood culture bottles has not been evaluated critically . We studied 4,954 pairs of blood culture bottles consisting of one vented biphasic tryptic soy broth bottle (Roche Diagnostics) and one unvented Thiol broth bottle (Difco Laboratories) . A total of 736 isolates were detected, of which 124 (17%) were in the Thiol broth bottle only . Some 15 isolates were detected only by subculturing the Thiol broth, and 13 of these isolates either were contaminants or were detected in previous positive cultures . Similar results were obtained when the unvented Thiol broth bottle was paired with a vented Difco tryptic soy broth bottle . Analysis of these pairs revealed a total of 360 isolates detected in 2,669 pairs of bottles, of which 83 isolates (23%) were in the Thiol broth bottle only . There were 11 isolates seen only in subcultures of the Thiol broth bottle, and 8 of these were probable contaminants . Thus, routine subculturing of unvented Thiol broth bottles had limited value . These results may differ with the use of other culture media or subculturing procedures . We recommend that each laboratory evaluate critically its experience with aerobic subcultures from unvented bottles. Environ Res, 1983 Apr, 30(2), 281 - 90 Asbestos-induced alterations of human lymphoid cell mitogenic responses; Bozelka BE et al.; Using mitogenic assays, we have investigated the short term effects of two asbestos (amosite and chrysotile) fibers on lymphocyte functions in vitro . These oppositely charged fibers produced different alterations in mitogenesis . The blastogenic responses of concanavalin-A (Con-A) and pokeweed mitogen stimulated human peripheral blood mononuclear cells (PBMN) were significantly increased by the inclusion of 6 micrograms of chrysotile to the culture media . Amosite fibers proved to be inhibitory in all tests . When PBMN were depleted of monocytes, asbestos-related alterations of Con-A responsiveness were unchanged among the remaining cells . However, the addition of chrysotile to phytohemagglutinin (PHA) cultures resulted in a significant increase of the mitogenic response . When PBMN were enriched for T lymphocytes, and again cultured with the mitogens and fibers, the Con-A response still displayed impressive enhancement with chrysotile . In contrast to an intact PBMN population, PHA-induced blastogenesis among these T-enriched lymphocytes was significantly elevated . These experiments demonstrate that asbestos can induce significant changes in the functional integrity of PBMN following a relatively short exposure time in culture. J Biol Chem, 1983 Mar 25, 258(6), 4037 - 44 Proteolytic processing of human preproapolipoprotein A-I . A proposed defect in the conversion of pro A-I to A-I in Tangier's disease; Gordon JI et al.; The primary translation product of human intestinal apolipoprotein A-I mRNA was isolated from wheat germ and ascites cell-free translation systems . Comparison of its NH2-terminal sequence with that of plasma high density lipoprotein-associated A-I showed that it is initially synthesized as a preproprotein . Like rat preproapolipoprotein A-I, it contains an 18-amino acid prepeptide and a 6-amino acid propeptide . The highly unusual COOH-terminal Gln-Gln dipeptide present in the rat pro-segment is also represented at the same position in the human sequence . The functional division of the 24-amino acid NH2-terminal extention into pro- and presegments was verified by finding that the stable intracellular form of A-I in a human hepatoma cell line was the proprotein . Edman degradation of radiolabeled intracellular and extracellular A-I indicated that this apolipoprotein was secreted without proteolytic cleavage of its hexapeptide prosegment . Therefore, it appears that apolipoprotein A-I undergoes an additional proteolytic processing step before it is fully integrated into plasma high density lipoprotein . Two-dimensional gel electrophoresis of purified proapolipoprotein A-I isolated from the hepatocyte cell culture media indicated that it corresponds to isoforms 2 and 3, the basic A-I isoproteins which are the precursors of plasma A-I and the predominant plasma A-I isoforms found in patients with Tangier's disease (Zannis, V . I., Lees, A . M., Lees, R . S., and Breslow, J . L . (1982) J . Biol . Chem., 257, 4978-4986) . Therefore this pathologic state probably arises from a defect in the conversion of proapolipoprotein A-I to apolipoprotein A-I. Biochem Biophys Res Commun, 1983 Mar 16, 111(2), 353 - 9 Effect of thyrotropin on glycosaminoglycans synthesized by primocultured thyroid cells; Giraud A et al.; The synthesis of glycosaminoglycans (GAGs) was investigated in porcine thyroid cells under the influence or not of thyrotropin . After labelling with {3H} glucosamine and {35S} SO4(2-), enriched GAG-fractions prepared from culture media, cells, and eventually substrate adhering materials, were analyzed by cellulose acetate electrophoresis combined with specific degradations . They comprised heparan sulfate and hyaluronic acid together with an unknown sulfated component labile to endo-beta-galactosidase . Whereas global labellings of newly made GAGs were not significantly modified by thyrotropin, we reproducibly observed with the hormone a substantial increase in the proportion of hyaluronic acid {3H} label and, when cells organized into follicles, of the proportion of cell-associated {3H} GAGs . This system thus offers an interesting model to study how the responsiveness to an hormone and the reorganization that follows might implicate specific glycoconjugates. Biochemistry, 1983 Mar 1, 22(5), 1289 - 97 Physical and chemical properties of human type III procollagen; Gerard S et al.; Type III procollagen was isolated from the serum-free culture media of human foreskin fibroblasts by adsorption to controlled-pore glass beads and chromatography of the eluted procollagen pool on diethylaminoethylcellulose {Gerard, S., & Mitchell, W . M . (1979) Anal . Biochem . 96, 433-447} . Sodium dodecyl sulfate (NaDodSO4) electrophoresis in 1% agarose-2% acrylamide gels with or without prior sample reduction revealed the predominance of a band with retarded mobility as compared to human procollagen I {hupro(I)} . Digestion of hupro(III) with pepsin yielded a product whose electrophoretic mobility was retarded for both the intact trimer and its reduced monomeric subunit as compared to that for the respective bands of rat skin (type I) collagen . NaDodSO4-polyacrylamide gel electrophoresis of bacterial collagenase-digested hupro(III) demonstrated disulfide-bonded propeptides which upon reduction were replaced by two distinct monomeric propeptide bands . The amino acid composition of hupro(III) was similar to that of hupro(I) but contained increased amounts of HO-Pro and Cys and less Thr, Ala, Val, and Arg . Sedimentation equilibrium analysis in 1 M CaCl2 yielded at extrapolated zero concentration a Mr of 505 +/- 25K . A {hupro(III) - collagen(III)} circular dichroic difference spectrum suggests approximately 10% alpha helix . The zero-order mutarotation rate of hupro(III) (vo = 55.0 X 10(-5) s-1) was twice that of hucol(III) (vo = 25.4 X 10(-5) s-1) at 20 degrees C, which may reflect the influence of the interchain disulfide-bonded carboxyl propeptides on the process of collagen fold formation. Am J Trop Med Hyg, 1983 Mar, 32(2), 296 - 9 Comparison of microscopy and culture in the detection of Leishmania donovani from splenic aspirates; Lightner LK et al.; Three culture media were compared with Giemsa-stained smears for the detection of Leishmania in splenic aspirates from Kenyan patients with visceral leishmaniasis . Ninety-nine splenic aspirates obtained from 26 patients at various times before, during, and after treatment were cultured in Schneider's Drosophila medium and RPMI medium 1640 (each supplemented with 20% fetal bovine serum) and McConnell's modification of Senekje's medium overlayed with 0.9% saline . From 13 splenic aspirates obtained before treatment, amastigotes were identified microscopically in all and promastigotes were cultured in 12 . During and after treatment, Schneider's medium was the most sensitive method for detecting parasites, followed by microscopic examination of stained smears which was more sensitive than either of the other two media tested . Results indicate that, for initial diagnosis, both culture and direct microscopy of aspirates should be employed. Am J Pathol, 1983 Mar, 110(3), 247 - 53 In vitro suppression of myelopoiesis by adherent murine splenocytes in experimental disseminated histoplasmosis; Caldwell CW et al.; Supernatant culture media obtained from adherent spleen cell preparations of mice experimentally infected with Histoplasma capsulatum yeast cells suppressed in vitro growth of human myeloid cells . This suppression was significantly greater than that obtained when splenocytes from normal mice or those inoculated with killed yeast cells were used . The use of indomethacin partially blocked this suppressive effect . A direct relationship was observed between the levels of prostaglandin E (PGE) per 10(6) adherent cells in the splenocyte preparations and the degree of in vitro myelosuppression . These findings suggest a possible mechanism for the leukopenia frequently observed in disseminated histoplasmosis. Dev Biol, 1983 Mar, 96(1), 46 - 62 Analysis of cartilage differentiation from skeletal muscle grown on bone matrix . III . Environmental regulation of glycosaminoglycan and proteoglycan synthesis; Nathanson MA; The ability of numerous nutritional and topographic factors to influence differentiation of embryonic mesenchyme has given rise to several theories which attempt to explain the development of muscle and cartilage from these similar-appearing cells . Some theories are challenged by the observation that a substratum of demineralized bone is capable of supporting the transformation of skeletal muscle into cartilage in vitro and that the potential to form cartilage still resides within cloned myoblasts and fibroblasts of skeletal muscle . In the present study, culture media CMRL-1066, minimal essential medium (MEM), and F-12 provide varied nutritional environments and are tested for their ability to support the morphological and biochemical transformation of skeletal muscle into cartilage . Morphologically, CMRL-1066 reproducibly supports hyaline cartilage formation, whereas MEM does so in only one out of three explants onto demineralized bone, and F-12 is incapable of supporting formation of a hyaline matrix . Biochemically, each medium is sufficient to elicit synthesis of cartilage-like patterns of sulfated glycosaminoglycans and proteoglycan monomer . Synthesis of hyaluronic acid (HA) initially increases in explants grown in CMRL-1066, but decreases prior to chondrogenesis . MEM elicits a similar increase in HA synthesis, but the subsequent decrease is not as rapid . In F-12, synthesis remains depressed throughout the experiment . The data show that increases in HA synthesis occur concurrent with the appearance of fibroblast-like cells, which normally precede chondroblasts . Decreases in HA synthesis correlate well with the onset of chondrogenesis . Explants grown in CMRL-1066 reproducibly from cartilage and synthesize the greatest amounts of proteoglycan aggregate . Those grown in MEM form cartilage infrequently, synthesize reduced amounts of proteoglycan aggregate-like material, and contain greater amounts of HA, of low molecular weight . The data demonstrate that chondrogenesis can be subtly regulated by environmental factors, and such factors regulate both the morphological and biochemical expression of the phenotype through HA synthesis. Am J Hosp Pharm, 1983 Mar, 40(3), 400 - 3 Evaluating aseptic technique of pharmacy personnel; Brier KL; A procedure for validating the effectiveness of a training program in aseptic technique for admixture personnel and for monitoring the aseptic technique of those trained is described . Double-strength soybean casein digest broth was prepared as the culture media . Technicians were instructed to prepare 50 sample i.v . admixtures after the structured one-week orientation and training period . The sample admixtures were cultured for sterility by a total-culturing method . An ongoing random-sampling plan was implemented to monitor the technique of the trained technicians . Six technicians completed the orientation program, and five compounded the validation admixtures aseptically . The ongoing monitoring revealed that the technicians who were validated have continued to perform at acceptable levels . This program validated that the personnel had adequate training in aseptic technique and the necessary skills for performing the aseptic manipulations required in i.v . admixture compounding. Acta Virol, 1983 Mar, 27(2), 130 - 7 Regulation of interferon production . Superinduction by dihydrorifampicin in human and chick embryo fibroblasts and mouse L929 cells; Kara J et al.; Dihydrorifampicin (DHR), a new reversible inhibitor of RNA polymerases I and II in eukaryotic cells, exhibited a very high enhancing effect on the production of interferon (IFN) in cultures of human and chick embryo fibroblasts and mouse L929 cells induced by poly(I) . poly(C) . The titres of interferon produced in human fibroblast cultures superinduced with poly(I) . poly(C), cycloheximide and DHR were 128 times higher as compared with cultures treated with poly(I) . poly(C) only . A similar superinduction of interferon by DHR was observed in mouse and chicken cell cultures, IFN titres in culture media were 40-60 times higher in comparison with cultures treated only with the inducer . In comparison with actinomycin D in the superinduction experiments, DHR was not toxic and allowed much higher yields of IFN . The use of DHR may be especially advantageous for the superinduction and production of human fibroblast IFN in cultures of human diploid fibroblasts. J Immunol Methods, 1983 Feb 25, 57(1-3), 111 - 9 An enzyme-linked immunoabsorbent assay for measuring antibodies against muscle acetylcholine receptor; Dwyer DS et al.; An enzyme-linked immunoabsorbent assay has been developed for measuring anti-acetylcholine receptor antibodies from sera of patients with myasthenia gravis or tissue culture supernatants from hybridomas . Acetylcholine receptor from a detergent extract of muscle tissue was bound indirectly to microtiter plates via a monoclonal anti-receptor antibody already coupled to the polyvinyl plates . Myasthenic sera or antibodies in tissue culture media were then tested for binding to the acetylcholine receptor attached to the monoclonal antibody . Anti-receptor antibodies were detected in the serum of 80% of myasthenic patients when assayed by this method and the levels of antibody corresponded fairly well with antibody titers determined by an immunoprecipitation assay . Occasional myasthenic patients had serum antibodies which reacted specifically with the monoclonal antibody attached to the microtiter plate . The assay described here was far less time-consuming than immunoprecipitation assays, required only small quantities of receptor, and did not require the use of radioisotopes such as 125I-alpha-bungarotoxin. J Biol Chem, 1983 Feb 25, 258(4), 2065 - 7 Biosynthesis of von Willebrand protein by human endothelial cells . Identification of a large precursor polypeptide chain; Wagner DD et al.; von Willebrand protein subunit is first synthesized by human endothelial cells as a larger precursor of 260-kDa that is slowly cleaved to its 220-kDa form . Both the precursor and the finished product are glycosylated . Small amounts of uncleaved precursor are secreted into the culture media . Disulfide bonded polymers typical for von Willebrand protein are also formed inside the cells . In comparison to fibronectin the biosynthetic processing of von Willebrand protein is longer as a delay in secretion is observed . The presence of a higher cellular pool of von Willebrand protein as compared to fibronectin indicates a storage compartment for von Willebrand protein in the endothelial cells. Am J Obstet Gynecol, 1983 Feb 15, 145(4), 397 - 401 Factors affecting human sperm penetration of zona-free hamster ova; Berger T et al.; The effects of in vivo and in vitro aging of hamster ova, protein supplementation of culture media, sperm concentration, and sperm motility on penetration of zona-free hamster ova by human sperm were investigated . Penetrability of ova was significantly lowered by 2 to 4 additional hours of in vivo aging or by an additional 3 hours of in vitro aging . A comparison was made of the effects of the addition to the media of human preovulatory serum and human serum albumin, and the penetrating ability of human sperm was increased with the addition of 10% human preovulatory serum . Sperm motility was also better maintained in the presence of 10% serum . Maximum penetration occurred after 3 hours of sperm-egg interaction following a 3-hour preincubation period with a sperm concentration of 5 x 10(6) motile sperm per milliliter . When motile sperm concentration was maintained at 1 X 10(7) motile sperm per milliliter, there was no correlation between penetrating ability and percentage of motility . These factors should be controlled to allow reproducible results with the hamster test. J Immunol, 1983 Feb, 130(2), 874 - 7 C3 cleavage products stimulate release of prostaglandins by human mononuclear phagocytes in vitro; Rutherford B et al.; Human monocytes cultured for up to 48 hr in serum-free, chemically defined culture media released low levels of prostaglandin . C3b, C3bi, and C3c stimulated an indomethacin-sensitive, dose-responsive increase in the amount of monocyte prostaglandin released by 18 hr after treatment . Native C3 and C3d, which do not bind to monocyte receptors, failed to stimulate increased prostaglandin release . Lymphocytes, treated and untreated, produced 10(-2) to 10(-3) as much prostaglandin as the monocytes . These data support the concept that monocytes are a significant source of leukocyte prostaglandin . They also introduce an important new biologic function for the C3 fragments C3b, C3bi, and C3c. Radiat Res, 1983 Feb, 93(2), 340 - 52 Polyamines and polyamine biosynthesis in cells exposed to hyperthermia; Gerner EW et al.; The issue of how polyamines act to sensitize cultured cells to the lethal effects of hyperthermia was investigated using Chinese hamster cells which were induced to express thermotolerance . Intracellular levels of these naturally occurring polycations were manipulated in certain situations by treating whole cells with methylglyoxal bis-(guanylhydrazone), an inhibitor of the S-adenosyl-L-methionine decarboxylases . Exogenous spermine as low as 100 microM in the culture media dramatically sensitized cells expressing thermotolerance to the lethal effects of subsequent 42 degrees C exposures . When thermotolerance was differentially induced in cultures exposed to 42.4 degrees C by varying the rate of heating from 37 to 42.4 degrees C, the most resistant cells had the highest levels of intracellular spermidine and spermine . This finding was explainable in part by the observation that the putrescine-dependent S-adenosyl-L-methionine decarboxylase activity was minimally affected in cells expressing the greatest degree of thermotolerance . When this enzyme activity was inhibited by drug, lowered intracellular polyamine levels did not correspond with subsequent survival responses to heat . Interestingly, cultures treated with methylglyoxal bis-(guanylhydrazone) 24 hr previous to heat exposure showed a reduced capacity to express rate of heating-induced thermotolerance . Together, these results demonstrate that the polyamines, especially spermidine and spermine, enhance hyperthermia-induced cell killing by some mechanism involving the plasma membrane . Further, our data suggest that methylglyoxal bis-(guanylhydrazone) can act to affect thermal responses by a mechanism(s) other than modification of intracellular polyamine levels. Zh Mikrobiol Epidemiol Immunobiol, 1983 Feb, (2), 80 - 4 {Preparation and use of solid culture medium for Leptospira isolation}; Soboleva GL; Recommendations on the preparation and use of a solid culture medium are given; age changes in the colonies of leptospires belonging to different serovars and serogroups have been studied in their dynamics; the absence of relationship between the form of the colonies, the serovar and serogroup of the cultures, their virulence, as well as the region, time and source of their isolation has been established, which makes it impossible to use these parameters for the differentiation of Leptospira strains belonging to different serovars on solid culture media. Br J Vener Dis, 1983 Feb, 59(1), 21 - 9 Factors affecting the attachment of Treponema pallidum to mammalian cells in vitro; Wong GH et al.; Attachment of Treponema pallidum (Nichols) to mammalian cells is probably the first step in the pathogenesis of syphilis . It may also be important for the multiplication of T pallidum in vitro . When factors affecting the attachment of T pallidum to mammalian cells in vitro were studied significantly greater numbers of treponemes were found to attach to baby rabbit genital organ (BRGO) cells than to five other mammalian cell lines . When attached to BRGO cells T pallidum survived longer in vitro than unattached treponemes . Eagle's minimal essential medium was superior to three other culture media in increasing attachment and maintaining the survival of treponemes . Dithiothreitol (0.25-1.0 mmol/l) had no effect on the attachment of T pallidum to BRGO cells . Anaerobic conditions were superior to microaerophilic conditions, and the latter were superior to aerobic conditions for the attachment and survival of T pallidum to BRGO cells . Within the range of concentrations tested the number of treponemes attached to the BRGO cells was directly dependent on the concentrations of viable treponemes in the inoculum . Greater numbers of treponemes attached to actively metabolising BRGO cells than to quiescent or slowly growing cells. J Histochem Cytochem, 1983 Feb, 31(2), 307 - 17 Effect of cisplatin on the plasma membrane phosphatase activities in ascites sarcoma-180 cells: a cytochemical study; Aggarwal SK et al.; To study the effects of cisplatin {cis-dichlorodiammine-platinum (II)} on tumor cells in the presence or absence of the immune system, animals with ascites sarcoma-180 tumor burden were treated with therapeutic dose levels (9 mg/kg) . Similarly, ascites sarcoma-180 cells were maintained in tissue culture media containing the same levels of the drug . Cell samples were taken from the animals at 12-hr intervals for 3 days, whereas samples were drawn from the tissue cultures at 15-, 30-, 45-, and 60-min and at 2-, 3-, 4-, and 5-hr intervals . Treated and untreated cells from in vitro and in vivo experiments, when checked for alkaline phosphatase, 5'-nucleotidase, Ca2+-ATPase, and Na+-K+-ATPase, show a gradual decrease in activity on the plasma membrane . It takes about 60 min for inactivation of any enzyme in vitro, whereas it takes 2 days in in vivo experiments . Quantitative analysis show alkaline phosphatase activity drops from 9.7 to 4.9 nmol in just 15 min, and drops further to 0.79 nmol after 2 hr . Inactivation of various plasma membrane enzymes, resulting in permeability changes, is probably responsible for cell death. J Antimicrob Chemother, 1983 Feb, 11(2), 135 - 49 In-vitro studies of a new oral azole antimycotic, BAY N 7133; Yamaguchi H et al.; The in-vitro antifungal activities of BAY N 7133, a new orally absorbed synthetic azole derivative against 364 strains of fungal pathogens were studied in direct comparison with another orally active azole compound ketoconazole, using an agar dilution procedure . BAY N 7133 was found to have a broad spectrum of antifungal activity and to inhibit most pathogenic yeasts and fungi at concentrations ranging from less than or equal to 0.04 to 10 mg/1, with the exception only of some strains of zygomycetic pathogens . It was equal to or even greater than ketoconazole in activity against most of the fungal pathogens tested . Composition and pH of culture media and inoculum size altered the effect of BAY N 7133 . No in-vitro development of drug resistance was demonstrated . BAY N 7133 can be considered to have a possible usefulness in the treatment of human mycoses. J Biol Chem, 1983 Jan 10, 258(1), 547 - 54 Abnormal glycosylation of human fibronectin secreted in the presence of monensin; Ledger PW et al.; Detailed studies of the effects of the ionophore monensin upon the glycosylation of secreted fibronectin have been carried out . Human fibroblasts in culture were incubated in 1 microM monensin for several hours, following which radiolabeled glucosamine or mannose was added to the cultures . Parallel incubation and labeling of control cultures were done . Labeled fibronectin was isolated from the culture media by gelatin-Sepharose chromatography, from cell surfaces by urea extraction, and from intracellular locations by cell lysis followed by immunoprecipitation . Detailed comparison of the glycopeptides released from fibronectin by pronase and of the oligosaccharides liberated by hydrazinolysis was carried out, particularly focusing on the secreted fibronectin, using gel filtration, high performance liquid chromatography, and concanavalin A chromatography, in conjunction with the use of endoglycosidase H and specific exoglycosidases . We demonstrate that fibronectin in the medium of monensin-treated cultures differs in its glycosylation pattern from the control fibronectin . High mannose oligosaccharides are abundant in the monensin-derived fibronectin, whereas the control protein contains primarily complex oligosaccharides . Monensin apparently does not alter the initial glycosylation of fibronectin since the high mannose oligosaccharides are present on both control and monensin-treated intracellular fibronectin . We suggest, therefore, that monensin, by impairing intracellular translocation through the Golgi region, allows incompletely processed forms of fibronectin to reach the cell surface and to be released into the culture medium. Nature, 1983 Jan 6, 301(5895), 89 - 92 Hypomethylation distinguishes genes of some human cancers from their normal counterparts; Feinberg AP et al.; It has been suggested that cancer represents an alteration in DNA, heritable by progeny cells, that leads to abnormally regulated expression of normal cellular genes; DNA alterations such as mutations, rearrangements and changes in methylation have been proposed to have such a role . Because of increasing evidence that DNA methylation is important in gene expression (for review see refs 7, 9-11), several investigators have studied DNA methylation in animal tumours, transformed cells and leukaemia cells in culture . The results of these studies have varied; depending on the techniques and systems used, an increase, decrease, or no change in the degree of methylation has been reported . To our knowledge, however, primary human tumour tissues have not been used in such studies . We have now examined DNA methylation in human cancer with three considerations in mind: (1) the methylation pattern of specific genes, rather than total levels of methylation, was determined; (2) human cancers and adjacent analogous normal tissues, unconditioned by culture media, were analysed; and (3) the cancers were taken from patients who had received neither radiation nor chemotherapy . In four of five patients studied, representing two histological types of cancer, substantial hypomethylation was found in genes of cancer cells compared with their normal counterparts . This hypomethylation was progressive in a metastasis from one of the patients. Klin Wochenschr, 1983 Jan 3, 61(1), 1 - 16 {Nerve cell clonal lines in culture--models for studying the molecular basis of neuropharmacological actions}; Herken H; Nerve cell lines with stable properties are isolated from neuroblastomas, glioblastomas or pheochromocytomas by periodic cloning using defined culture media . After the action of different drugs, these cells show all morphological and biochemical signs of differentiation and maturation . Depending on the origin of the clone, the cell lines synthesise typical neurotransmitters, which are stored in vesicles . It is demonstrated on cell lines which synthesise catecholamines that noradrenergic and dopaminergic clones are particularly suitable test objects for basic research in neuropharmacology. Microbiol Immunol, 1983, 27(4), 377 - 87 Inhibition of macrophage DNA synthesis by immunomodulators . I . Suppression of {3H}thymidine incorporation into macrophages by MDP and LPS; Nagao S et al.; Oil-induced guinea pig peritoneal exudate macrophages were found to incorporate actively {3H}thymidine without any tissue fluids such as conditioned medium, lymphokines or inflammatory tissue exudates . The {3H}thymidine incorporation was markedly suppressed by macrophage stimulants such as muramyl dipeptide (MDP) or bacterial lipopolysaccharide (LPS), while glucosamine incorporation was simultaneously increased by these stimulants . The degree of suppression of thymidine incorporation depended on the cell density, the concentrations of the stimulants, and sera or culture media used . The exposure of macrophages to MDP for 30 min was sufficient to cause significant suppression. J Pediatr Gastroenterol Nutr, 1983, 2(1), 62 - 70 Quantification of immunoglobulins after organ culture of human duodenal mucosa; Fluge G et al.; Duodenal biopsies from 33 celiac and 16 nonceliac patients were kept in organ culture for 24 h . Quantities of immunoglobulins were measured by rocket immunoelectrophoresis in mucus removed from the biopsy surface after culture and in culture media . Increased amounts of immunoglobulins were recorded after culture of biopsies from celiac disease patients in the exacerbation state; but only in 11 of 33 celiac mucosae could an increment be detected after culture in the presence of gluten compared to culture on gluten-free medium . The amount of IgA showed a significant correlation with radioactivity of mucus and culture medium after {14C}leucine incorporation during culture . In such experiments autoradiograms of immunoprecipitates disclosed in vitro synthesis of IgA, whereas 47% of the IgG precipitates were radionegative . Amounts of IgA corresponded significantly to serum concentration of this immunoglobulin, whereas for IgM and IgG no such correlation existed . Quantification of immunoglobulins seems to be unsuitable as a method of evaluating in vitro gluten toxicity in celiac disease. Cancer Chemother Pharmacol, 1983, 11(1), 8 - 15 Properties of anticancer agents relevant to in vitro determinations of human tumor cell sensitivity; Pavlik EJ et al.; The physical properties of 59 anticancer agents have been examined with respect to solubility in tissue culture media, binding to ultrafiltration materials, and molecular absorbance and fluorescence behavior . Methods for dissolving these agents, which are compatible with in vitro sensitivity testing of human tumor cells to anticancer agents, are reported in this paper . The potential for anticancer agent binding to cellulose nitrate/cellulose acetate and teflon membrane ultrafilters was documented, and quantitation of these anticancer agents based upon absorbance and fluorescence spectroscopy was performed . Post-filtration quantitation of anticancer agents was found to be a reliable method for determining the actual drug concentrations available in tumor cell sensitivity testing in vitro . The properties documented herein are pharmacologically relevant parameters related to in vitro determinations of human tumor cell sensitivity to anticancer agents. Acta Vitaminol Enzymol, 1983, 5(2), 119 - 24 Effect of retinol on macromolecular synthesis in cultured cell lines; Ferrari N et al.; The effects of retinol on SK Mel 28 and HeLa cell lines were studied with regard to DNA and protein synthesis . It was found that vitamin A added to the culture media at a 50 microM concentration causes a 60% inhibition of DNA synthesis, that the inhibition is reversible and that this treatment does not select retinol-resistant clones . The synthesis of cytoplasmic, acid-soluble nuclear proteins and that of non-histone chromosomal proteins is inhibited as soon as the vitamin reaches the cell. Med Interne, 1983 Jan-Mar, 21(1), 13 - 7 Investigations upon the incidence of atmospheric fungi in the town of Craiova; Popescu IG et al.; The present investigation was suggested by the fact that bronchial asthma (of all types) presents in Craiova and the district of Dolj a relatively increased incidence which may be correlated with certain findings concerning the incidence of atmospheric fungi in the houses and working places of asthmatic subjects . Petri dishes with Czapek medium were exposed monthly in the open air, in 17 houses of asthmatic subjects and in a large bakery unit . The prevalence of various types of fungi was observed on the culture media and a correlation could be made with the seasons . In the houses of asthmatics Penicillium was found to predominate and in certain months of the year Aspergillus as well, which may have a certain etiopathogenic role in bronchial asthma . In certain months Cladosporium and Alternaria also appeared . In the open air the number of colonies was smaller but the incidence was identical . In the bakery unit Penicillium was predominant in 3 out of 5 months, Aspergillus prevailling in all the other months of the year . Cladosporium and Alternaria were also rather frequent . Other fungi sometimes found in relatively large proportions (Mucor, Rhizopus, Verticillium) were not in themselves etiopathogenically involved in bronchial asthma. Environ Mutagen, 1983, 5(1), 49 - 57 Synergism in the transformation of hamster embryo cells treated with formaldehyde and adenovirus; Hatch GG et al.; Formaldehyde is a large production volume chemical widely distributed in research laboratories, industrial workplaces, and home and personal environments . Inhalation studies with formaldehyde have documented its ability to produce squamous cell carcinomas in rats . When primary hamster embryo cells were treated by gaseous exposure to formaldehyde or by incorporation into the medium, a dose-related increase in the frequency of SA7 virus transformation was produced . The length of chemical treatment and the time interval before subsequent addition of transforming virus was critical, with two-hr treatment times as the most efficient . Treatment by gaseous exposure permitted utilization of lower treatment concentrations . Determination of formaldehyde concentrations in culture media of bioassay dishes treated by this method documented that 2.2 micrograms/ml produced significantly enhanced viral transformation . Exposure of hamster embryo cells to formaldehyde by these methods produces reproducible and quantitative genotoxic effects. Invest Ophthalmol Vis Sci, 1983 Jan, 24(1), 113 - 8 The effect of inhibition of glutathione reductase on the detoxification of H2O2 by rabbit lens; Giblin FJ et al.; Mechanisms by which the lens protects against H2O2 are believed to include the metabolism of glutathione (GSH) . In the present study, rabbit lenses were exposed to constant concentrations of H2O2 (0.01 to 0.1 mM) that were maintained in culture media with the use of a peristaltic pump . The rate at which H2O2 entered the lens was proportional to its concentration in the medium and reached 2.6 mumol H2O2/lens/3 hr at 0.1 mM H2O2 . Up to 0.06 mM H2O2, a concentration that approximates that present in normal rabbit aqueous humor, the activity of the hexose monophosphate shunt (HMPS) increased linearly with no significant decrease in the concentration of lens GSH . However, at 0.1 mM H2O2, there was indication of oxidative damage to the lens as shown by a sharp decrease in HMPS activity and a coincident drop in the concentration of GSH . Pretreatment of lenses with 1,3-bis(2-chloroethyl)-1-nitrosourea (BCNU), an inhibitor of glutathione reductase (GR), blocked the normal threefold stimulation of HMPS activity occurring in the presence of 0.06 mM H2O2 and resulted in accumulation of oxidized GSH . This result demonstrated the inability of H2O2 to react directly with NADPH in the lens . BCNU was shown not to affect the potential of the HMPS to respond to compounds other than H2O2 since it did not alter methylene blue-stimulation of HMPS activity . The study supports the hypothesis that detoxification of H2O2 in the aqueous humor is linked to the metabolism of GSH in the lens and demonstrates that lenses with impaired GR activity are more susceptible to oxidative damage by peroxide. Dev Biol, 1983 Jan, 95(1), 219 - 26 Studies on the development of melanophores in in vitro cultured amphibian neural plates; Matsuda M; Various parts of neural plates of Japanese newt (Cynops pyrrhogaster) neurula embryos were cultured alone in drops of culture media (Niu-Twitty's balanced salt solution or modified Leibovitz L-15 medium) with or without fetal calf serum (FCS) . Although none of the parts gave rise to melanophores in a medium without FCS, some produced melanophores in a medium with FCS . The localization of melanophore-producing areas in the neural plates corresponded to that of Tada's (1944) findings . The assumption that FCS affects survival and development of melanophores is excluded, because neural fold cells do not require FCS to develop into melanophores . Therefore, there may be in FCS some factor which acts on the specialization of neural plate cells into melanophores . The results of this experiment suggest that this factor may be heat labile . The findings also indicate that FCS does not induce melanophores in gastrula ectoderm, but only affects neurula neural plate cells so as to give rise to melanophores. Dev Biol Stand, 1983, 55, 77 - 8 Production of a feline parvovirusvaccine using monolayer cell systems in roller flasks and microcarriers; Bergman R et al.; A parvovirus-strain originating from a case of spontaneous panleukopenia has been adapted and propagated in a feline lung fibroblastic cell line . Cultivation of parvovirus infected monolayer cells was carried out in 11 glass flasks containing 50 ml media and on microcarriers in 0.5 - 101 stirring flasks with 0.5-61 media . Harvest of propagated virus from flask monolayer and microcarrier cultures was performed daily for 5 days during one week . The yields of virus antigen per/ml tissue culture media were equal in the two systems, as monitored by hemagglutination and ELISA assays . The seeding and harvesting procedures of virus were found to be simpler with the microcarrier system than with roller flasks . The scaling up is in progress and comparable results have been obtained in 101 scale. Connect Tissue Res, 1983, 12(1), 17 - 31 Biochemical characterization of collagens and of a non-collagenous protein synthesized by guinea pig lung fibroblasts in culture; Bashey RI et al.; Employing various radioactive amino acids, protein biosynthesis by guinea pig lung fibroblasts has been studied in monolayer culture . The cells were shown to synthesize and secrete several collagenous and noncollagenous proteins . The biosynthesized macromolecules have been characterized employing molecular sieve and ion exchange chromatography (DEAE-cellulose column), SDS-polyacrylamide gel electrophoresis, and enzymatic digestion . It was found that the guinea pig lung fibroblasts synthesized mainly type I procollagen molecules which appeared in the media in various stages of cleavage . The intact procollagen molecules and the various processed products were identified by their electrophoretic migration in SDS-polyacrylamide slab gels and further characterized by 1) elution position on SDS-agarose columns under reducing conditions; 2) hydroxyproline content; 3) change in elution position on SDS-agarose after pepsin digestion; 4) chromatographic separation on DEAE-cellulose columns and electrophoretic mobility of the various peaks; and 5) susceptibility to collagenase digestion . Slab gel electrophoresis under non-reducing conditions of aliquots of culture media after limited pepsin digestion indicated the presence of disulfide-bonded type III collagen alpha-chains . In addition, the lung fibroblasts synthesized a non-collagenous protein composed of two disulfide-bonded chains . The individual chains appeared to have a molecular weight of approximately 220,000 when examined under reducing conditions . This protein has been identified as fibronectin based on its molecular weight, its resistance to collagenase attack, its susceptibility to protease digestion and its precipitation with specific antisera to fibronectin. Physiol Chem Phys Med NMR, 1983, 15(1), 81 - 7 Polyamine metabolism in McCoy cells: III . Comparative studies of the metabolic fate of exogenous putrescine in human fibroblasts and McCoy cultures; Fan MZ et al.; The fate of exogenously added 14C-putrescine following incubation for 24 hours with McCoy and human skin fibroblast cultures was examined . The nature of the polyamine derivatives found were quite different indicative of a difference in the cellular metabolism of polyamines . Exogenously added putrescine (PUT) was metabolized by both McCoy and human skin fibroblast cultures to form spermidine (SPD), spermine (SPM), gamma-aminobutyric acid (GABA) and some unidentified compounds . Within the experimental period of observation, human cultured fibroblasts metabolized PUT more efficiently than McCoy cells and converted more than 50% of it into SPD, SPM, GABA and unknown compounds . Monoacetyl putrescine (MAP) was formed by human skin fibroblasts . It was mainly identified in the culture medium . No MAP was detectable either intracellularly or extracellularly in McCoy cultures . The percentage of 14C-radioactivity found as PUT in the culture medium was greater in McCoy cells (86.0%) than in human fibroblasts (53.9%) . The reverse was true for the percentage distribution of 14C-radioactivity as PUT inside the cells . No low Mr conjugates of SPD or SPM were found in the medium or intracellularly with either culture type . Some low Mr putrescine conjugates were found in the culture media; these were identified by the liberation of PUT upon acid hydrolysis. Oncodev Biol Med, 1983, 4(4), 281 - 8 Production of placental protein 5 (PP5) by non-malignant human fibroblasts in culture . Comparison with 'pregnancy-specific' glycoprotein (SP1); Nisbet AD et al.; Placental protein 5 (PP5) was detected by radioimmunoassay in the culture media from 11/19 (58%) of cell lines derived from non-malignant human fibroblasts . One line was studied in detail . Its rate of PP5 secretion was polyphasic, with maxima of 3.1 pmol/10(6) cells per 24 h on day 1, 1.7 on day 8 and 2.0 on day 14, and minima of 0.9 on day 4, 1.0 on day 10 and 0.7 on day 15 of culture . A discordance with the secretion of pregnancy-specific glycoprotein (SP1) was demonstrated; the SP1 concentration was constant initially at 2.9 pmol/10(6) cells per 24 h, followed by a steady decrease to 0.5 on day 15 . A small amount of PP5 was retained in the cells, but usually less than 5% of that secreted . When the culture medium was changed daily, more PP5 and SP1 were produced than when the cells were grown in conditioned media . A cell line derived from a human fibrosarcoma produced SP1 at rates exceeding that of the normal fibroblasts, but PP5 production was not detected in the fibrosarcoma. Acta Med Scand Suppl, 1983, 671, 117 - 9 Transplantation of isolated pancreatic islets: current status and prospects; Henriksson C; Complete and permanent reversal of diabetes by transplantation of isolated pancreatic islets has been repeatedly demonstrated experimentally, whereas islet isolation and transplantation in man are associated with largely unsolved problems: the standard isolation procedure including disruption of acini and collagenase digestion is ineffective . Improvement of the islet yield is currently obtained in animal and human trials by using substances selectively acting on exocrine tissue and others protecting the islets at the isolation as well as in tissue culture media . Islet tissue banks and repeated transplantations seem to be realistic also in man . Islets are advantageously implanted intraportally in the liver . They are rejected soon without protection or efficient immunosuppression . Intense research concerns islet-containing diffusion chambers, macrophage suppression, new immunosuppressive agents and induction of immunological tolerance . A successful solution of the technical and immunological problems will aggravate a shortage of donor tissue, already noticeable in the experimental work: this will make the recipient selection crucial. Acta Histochem Suppl, 1983, 27, 253 - 8 {Effect of growth factor preparations on the proliferation of in vitro cultivated aortic endothelial cells}; Halle W et al.; A functionally and structurally well-defined calf aortic endothelial cell line has been used to characterize tissue preparations with mitogenic activity by means of cell proliferation kinetics . The following preparations were tested: An extract from corpora lutea (pig), produced by stepwise ammonium sulfate precipitation (CL-S3), a fraction obtained from CL-S3 extract by ion exchange chromatography (CM-S4), and a chorioallantoic membrane preparation (CAM preparation) . Under optimal cultivation conditions 100 micrograms CL-S3, 5 micrograms CM-S4, or 100 micrograms CAM preparation per ml medium increased the cell number and growth rate of the endothelial cells in approximately like manner after an incubation period of 48 hours . Compared with CL-S3 these results demonstrate a 20 fold enrichment of the mitogenic activity in the CM-S4 fraction . Under suboptimal cultivation conditions (i.e . a low cell number at the seeding and a reduced nutritive quality) 100 micrograms CL-S3 per ml medium stimulated the cell proliferation up to 12 times at the end of a 6-days-cultivation-period . These results are discussed in connection with the use of these preparations in the angiogenesis research and with respect to the application of selected preparations in cell culture media in order to reduce the serum concentrations. C R Seances Acad Sci III, 1983, 296(6), 293 - 6 {Aromatization of testosterone by the rat embryo testis}; Weniger JP et al.; After culturing testes from 19 to 20-day-old Rat embryos in vitro in the presence of 3H-testosterone, 3H-oestradiol could be identified in the culture media by recrystallization to constant specific activity . However, the conversion rate of testosterone to oestradiol was low, approximately 0,03%. Enzyme, 1983, 29(1), 44 - 53 Isoenzyme pattern and immunological properties of arginase in normal and hyperargininemia fibroblasts; Konarska L et al.; Arginase deficiency is an inborn error of the last step in the urea cycle and leads to profound hyperargininemia . The enzyme deficiency has been demonstrated in the liver and red blood cells . In cultured patient fibroblasts, the activity is normal . Arginase exists in multiple molecular forms only one of which is missing in hyperargininemic patients . In fibroblasts, three arginase isoenzymes can be demonstrated by DEAE-cellulose column chromatography, two by electrophoresis and by immunoprecipitation methods . From the present data, it is improbable that part of the A1 isoenzyme in fibroblasts originates from fetal calf serum arginase which supplements the culture media . None of the techniques for the separation and analyses of arginase isoenzyme allows to differentiate between the normal and the arginase-deficient phenotype . A possible explanation would be that the defect in A1 arginase observed in the liver is the result of a regulatory defect. In Vitro, 1983 Jan, 19(1), 31 - 40 Organ and species specificity of epithelial growth; Heckman CA; Previous work in this laboratory demonstrated that rat respiratory airway epithelial cells grown from tissue explants undergo concurrent division and differentiation in culture . In the present studies, this model system has been used to optimize conditions for epithelial growth, with the goal of facilitating the culture of epithelium from other organs and species . Fibroblasts appeared to limit the expansion of epithelial outgrowths; their growth was controlled by using delipidated serum, rather than serum, in the culture media, with addition of adrenergic antagonists . In nonsupplemented media, rat tracheal fibroblasts doubled in number in about 3 d . The doubling time was slowed to more than 9 d by the serum substitution in conjunction with the alpha-antagonist phenoxybenzamine . Rat tracheal epithelial cultures maintained under identical conditions doubled in less than 4 d, so that a growth advantage of threefold was achieved . The combination of conditions also seemed to inhibit growth of fibroblasts from other species . However, even when the problem of fibroblast overgrowth was obviated, the respiratory airway epithelia of the mouse, hamster, and marmoset failed to show self-sustaining growth in culture . The rat epithelium continued to grow for over 6 wk . A number of other organs from the rat, notably esophagus, skin, and salivary gland, showed self-sustaining growth after removal of explants . Although epithelial outgrowths were formed by explants from certain organs of the hamster, only those from the kidney and thyroid continued to grow after removal of the tissue explants . Therefore, growth failure in cultured epithelia, quantitatively defined, is common and frequently unrelated to the problem of fibroblast overgrowth . The rat seems to be the species of choice for studies on the isolated epithelia from most organ sites. J Biol Response Mod, 1983, 2(3), 263 - 71 Fetal calf serum and 2-mercaptoethanol induce anti-trinitrophenyl antibody production: II . Preferential stimulation of B cells in culture; Soderberg LS et al.; Fetal calf serum (FCS) and 2-mercaptoethanol (2-ME) are commonly used in culture media because they enhance lymphocyte responsiveness . We had previously reported that FCS and 2-ME generate high antitrinitrophenyl (TNP) antibody plaque-forming cell responses in unprimed murine spleen cells without added antigen . This was due in part to a component in FCS, which was antigenically cross-reactive with TNP . In the present report, we studied the mitogenic activity of FCS and 2-ME, which also contributed to the high anti-TNP response . Such mitogenic activity could not be replaced by B- or T-cell mitogens . Both FCS and 2-ME were required for the mitogenic activity, which was directed primarily at B cells . Purified T cells and spleen cells from B-cell-depleted mice were unresponsive to FCS and 2-ME stimulation . The mitogenic activity was not due to lipopolysaccharide (LPS) contamination of FCS, since cells of the LPS-unresponsive mouse strain, C3H/HeJ, were stimulated by FCS and 2-ME. Int J Tissue React, 1983, 5(2), 153 - 8 Effect of dexamethasone and combinations of hormones on 7-day chick embryo lung cells; Richmond JE; Seven-day chick embryo lung was dissociated into single cells and the capacity of these cells to incorporate 1-3H-glucosamine and 1-14C-glycine in a rotational-mediated aggregation system upon the addition of hormones was determined . Incorporation of 1-3H-glucosamine was stimulated 50% by 10(-8) M dexamethasone while 1-14C glycine uptake was not markedly influenced . Dexamethasone, 10(-8) M and 10(-6) M insulin gave rise to a 90% enhancement in 1-3H-glucosamine incorporation and a 20% increase in 14C-glycine uptake . When the aggregating cells were incubated with 10(-8) M dexamethasone and 10(-8) M 3,3',5-tri-iodo-thyronine, glucosamine incorporation was augmented by 40% and glycine by 25% . The combination, 10(-8) M 3,3',5-tri-iodo-thyronine, 10(-8) M dexamethasone and 10(-6) M insulin led to a 70% stimulation in glucosamine labelling and a 50% increase in 1-14C-glycine uptake . Very little change in the incorporation of the hexosamine and glycine into the soluble culture media was observed by adding either of the hormones; however, there appeared to be a slight increase in the incorporation of both tracers when the cells were incubated with the combination of dexamethasone and insulin. Cell Tissue Res, 1983, 232(1), 157 - 76 Effect of 17 alpha-methyltestosterone, estradiol-17 beta and synthetic LHRH on production of gonadotropic hormone in pituitaries of rainbow trout (organ culture); Fahraeus-van Ree GE et al.; Pituitary glands from 6-month-old sexually immature female rainbow trout, Salmo gairdneri, were kept in organ culture for 48 or 72 h . Certain groups of pituitaries were cultivated for 48 h on either control medium or medium with 17 alpha-methyltestosterone (MT), or with estradiol-17 beta (E2) in concentrations of 8.5 X 10(-7) M . Other groups of pituitaries were cultivated for 72 h on control medium, or for 48 h on either control medium or MT-medium or E2-medium, and subsequently for 24 h on medium with synthetic LHRH in concentrations of 8.5 X 10(-7) M and 8.5 X 10(-10) M . Gonadotropic (GTH) cells are identified by Alcian Blue-Periodic Acid Schiff-Orange G staining and the double-antibody immunoenzyme-cytochemical technique using anti-carp beta GTH as the first antibody . A quantitative histological procedure was used to study the nuclear size of the GTH cells in response to the different hormones . Secretory activity was estimated by measuring the gonadotropin (GTH) content in extracts of pituitaries, plasma, and the culture media every 24 h by radioimmunoassay . Cultivation on MT- or E2-enriched medium results in an increase of the total amount of GTH in the pituitary and medium, an accumulation of GTH in GTH-cells (approximately 20 percentage points) and an increase in their nuclear size, indicating a stimulation of GTH synthesis . Howeve