J Comp Pathol, 1985 Apr, 95(2), 209 - 16 The effect of mycoplasmas and acholeplasmas of equine origin on organ cultures of chicken-embryo trachea; Moorthy AR et al.; Chicken-embryo tracheal organ cultures were inoculated with equine strains of Mycoplasma arginini, M . equigenitalium, 2 strains of M . subdolum, Acholeplasma laidlawii and 3 strains of A . oculi . All strains established and multiplied in the explant cultures, but only M . subdolum and A . oculi produced a cytopathic effect on ciliated epithelial cells, causing sloughing of cells and cilia after 6 days . There was a correlation between ciliostasis and increase in titre of both M . subdolum and A . oculi and this relationship was not observed with M . equigenitalium and A . laidlawii . All the strains of acholeplasma multiplied to some extent in organ culture media, but reached higher titres in the presence of explants . Cells infected with the M . subdolum strain showed sloughing of cilia, vacuolization, and increase in size of mitochondria, followed by disorganization of epithelium and marked destruction of subcellular organelles . Mycoplasmas were closely attached to the epithelial surface of the tracheal explant 8 days after infection. In Vitro Cell Dev Biol, 1985 Apr, 21(4), 229 - 36 Free radical damage to cultured porcine aortic endothelial cells and lung fibroblasts: modulation by culture conditions; Bishop CT et al.; Culture conditions modulating cell damage from xanthine plus xanthine oxidase-derived partially reduced oxygen species were studied . Porcine thoracic aorta endothelial cells and porcine lung fibroblasts were maintained in monolayer culture . Cells were prelabeled with 51Cr before xanthine plus xanthine oxidase exposure . Endothelial cells showed 30 to 100% more lysis than fibroblasts and thus seemed more sensitive to this oxidant stress . The effect of cell culture age, as indicated by population doubling level (PDL), was examined . Response of low PDL endothelial cells and fibroblasts subjected to oxidant stress was compared with the response of PDL 15 cells . Both low PDL endothelial cells and fibroblasts responded differently to the lytic effect of xanthine oxidase-derived free radicals than did higher PDL cells . Specific activities of the antioxidant enzymes catalase, manganese superoxide dismutase, copper-zinc superoxide dismutase, glutathione peroxidase, and glucose-6-phosphate dehydrogenase were measured in both low and high PDL fibroblasts and endothelial cells . Antioxidant enzyme specific activities could only partially explain the differences in response to oxidant stress between fibroblasts and endothelial cells and between low and high PDL cells . Cell culture medium composition modulated the rate of production, and relative proportions of xanthine plus xanthine oxidase-derived partially reduced species of oxygen, i.e . superoxide, hydrogen peroxide, and hydroxyl radical . Serum content of medium was important in modulating free radical generation; superoxide production rates decreased 32%, H2O2 became undetectable, and hydroxyl radical generation decreased 54% in the presence of 10% serum . The medium protein and iron content also modulated free radical generation . The data suggest that cell culture media constituents, cell type, and cell culture age greatly affect in vitro response of cells subjected to oxidant stress. J Cell Physiol, 1985 Apr, 123(1), 126 - 31 A comparison of protein synthetic patterns of MDCK cells grown in serum and hormonally defined serum-free medium; Jefferson DM et al.; The development of hormonally defined serum-free media (HDM) for the culture of mammalian cells has allowed the study of specific cellular functions in a totally defined environment . Recently, several reports have indicated that there are differences in the basic cellular physiology of cells cultured in HDM when compared to cells cultured by the more traditional method of using serum-supplemented culture media (SM) . We report here that there are significant changes in the protein synthetic pattern in MDCK cells grown in HDM . There were no changes exhibited during the first passage in HDM, but following 10 passages in HDM there was an increased isotope ratio of 1) plasma membrane proteins with molecular weights of 12,000, 36,000 and 68,000 and 2) endoplasmic reticulum proteins with molecular weights of 12,000 and 37,000 . Additionally, the incorporation of methionine and uridine were significantly increased in cells cultured in HDM for 10 passages . At present, we believe that these changes in the protein synthetic patterns are due at least partially to increased protein synthesis as indicated by monosome/polysome ratios . Therefore, though the use of HDM offers a completely defined system for studying cellular function, results obtained using HDM must be interpreted with caution when comparing them to previous studies that have used SM. Boll Soc Ital Biol Sper, 1985 Mar 30, 61(3), 387 - 94 {Activity of uremic toxins and polyamines on organ culture of 7-day-old chick embryo}; Stabellini G et al.; We studied possible effects of uremic toxins and polyamines (PAs) on organ cultures of chick embryo . We added on culture media the lyophilized of total dialysate and its chromatographic peak II obtained with chromatography with Sephadex G 15 . The total dialysate and peak II showed toxicity with degeneration of the culture, whereas the free PAs, we added, did not effects. Eur J Cancer Clin Oncol, 1985 Mar, 21(3), 317 - 24 Release of an Mr 140,000 glycoprotein in the culture media of certain human sarcoma and melanoma cell lines; Bizik J et al.; A 140 K glycoprotein was detected in the culture media of human sarcoma and melanoma cell lines by labeling with several radioactive amino acid and sugar precursors, followed by sodium dodecylsulfate-polyacrylamide gel electrophoresis and fluorography . In contrast to this, in the culture media of metabolically labeled embryonic and skin fibroblasts this glycoprotein was not found . Likewise, a protein with an identical molecular weight of 140 K was also found in culture media after cell surface labeling of the neoplastic cells but not in the culture media from control cells . The {35S}methionine-labeled 140 K was not split by collagenase and did not appear to be a fragment of fibronectin . We discuss the possibility that secretion of the 140 K glycoprotein is a transformation-related phenomenon. Cell Struct Funct, 1985 Mar, 10(1), 89 - 93 Mouse lymphoma L1210 cells can be arrested in the G1 phase by adjusting cellular cysteine and glutathione; Ishii T et al.; Mouse lymphoma L1210 cells (NCI line) that have low ability to take up cystine became deficient in cellular cysteine and glutathione in normal culture media . The cells entered the resting state during culture when they were seeded at high cell densities . They remained viable and were mostly present in the G1 or G0 phase . In the growth-arrested state, the cellular glutathione content was one order of magnitude lower than in the exponentially growing phase in the presence of 2-mercaptoethanol . In the arrested state, DNA synthesis was almost inhibited, and RNA and protein synthesis decreased markedly . Transfer of the cells to medium containing 2-mercaptoethanol, which improves the utilization of cystine by these cells, produced the rapid recovery of RNA and protein synthesis . DNA synthesis slowly increased, reaching a maximum after a lag period. J Reprod Fertil, 1985 Mar, 73(2), 567 - 77 Examination of the causes of early pregnancy-associated thrombocytopenia in mice; O'Neill C; In mice, neither the bleeding time nor the clotting time of whole blood was different on Day 2 of pregnancy compared with pseudopregnancy . Standardization of the platelet concentration to 10(6)/microliters plasma resulted in a significant reduction in the clotting time of plasma from pregnant animals . This reduction was not due to an increase in the intrinsic or extrinsic pathways of the coagulation cascade but to enhanced platelet factor III activity, indicating increased platelet activation and consumption . Increased activation was not due to immunological recognition of the embryo because thrombocytopenia occurred after syngeneic and allogeneic matings of inbred strains of mice and also after parthenogenetic activation of ova in situ . Injection of embryo culture medium into splenectomized mice induced a significant dose-dependent thrombocytopenia . It occurred within 10 min after injection and persisted for up to 2 h . There was no reduction in platelet count when animals were injected with culture media in which unfertilized ova had been incubated . Early pregnancy-associated thrombocytopenia was caused by the production of platelet-activating factors by the fertilized eggs . The induction of thrombocytopenia by embryo culture media displayed a dose-response curve that was parallel to that of the platelet-activating factor, 1-0-alkyl-2-acetyl-sn-glycero(3)phosphocholine. Am J Pathol, 1985 Mar, 118(3), 398 - 407 Effects of the aminonucleoside of puromycin on glomerular epithelial cells in vitro; Fishman JA et al.; Glomerular epithelial cells (GECs) in vitro provide a useful model for the study of the mechanism(s) underlying the nephrotic syndrome of rats induced by the aminonucleoside of puromycin (PAN) . Some of the toxicities of PAN are nonspecific, in that the constituent molecules of PAN (adenosine and puromycin) cause similar effects in vitro . These include GEC blebbing and rounding, reduced uptake of precursors of protein (leucine) and glycoprotein (glucosamine) synthesis, and increased permeability of the GEC membrane to adenosine . Some of the effects of PAN are not reproduced by adenosine or puromycin and are inhibited by the simultaneous presence of N6-monomethyl adenosine (MMA), a PAN analog and an in vivo blocker of nephrosis due to PAN . These processes may be related to the nephrotic syndrome and include the loss of adhesion to plastic; a reduction in the incorporation of 14C-glucosamine and 35S-sulfate both into molecules removable from the GEC surface by neuraminidase and into those moieties precipitated from the culture media by TCA; a marked reduction in the "ordering" of the lipids of the rigid GEC membrane, which is possibly dependent upon cell-surface proteins . These morphologic alterations in GECs and in the distribution of negatively charged molecules, which are either secreted or on the cell surface, correlate with observations made in PAN-induced nephrosis in rats in vivo . These include changes in the turnover and the array of sialic acid and heparan sulfate glycoprotein on the GECs and the glomerular basement membrane . The in vitro sensitivity of GECs to PAN and the effects of MMA suggest a role for these cells in in vivo aminonucleoside nephrotoxicity, where alterations in both the morphology and the anionic topology of GECs participate in the development of proteinuria. Blood, 1985 Mar, 65(3), 605 - 14 Reversible oxidant-induced increases in albumin transfer across cultured endothelium: alterations in cell shape and calcium homeostasis; Shasby DM et al.; To determine whether reactive oxygen molecules could directly and reversibly increase the transfer of albumin across an endothelial barrier, we measured albumin transfer across monolayers of endothelium cultured on micropore filters before and after exposure to xanthine and xanthine oxidase . Xanthine and xanthine oxidase increased endothelial albumin transfer in a dose-dependent fashion . Parallel phase contrast and fluorescence microscopy demonstrated retraction of adjacent cells from one another and disruption of the actin filaments . The oxidant-induced increases in albumin transfer and changes in cell shape were reversed by removing xanthine oxidase and then incubating the monolayers for 3 1/2 hours in tissue culture media enriched with fetal bovine serum . However, incubation in tissue culture media without serum resulted in progressive injury and cell death . Hence, the brief exposure to oxidants initiated a progressive injury process that was reversed by incubation in serum . Because intracellular and extracellular calcium are important determinants of cell shape, and because some oxidized membrane lipids act as calcium ionophores, we asked whether oxidants altered endothelial calcium homeostasis . Xanthine-xanthine oxidase increased release of 45Ca++ from preloaded cells . The calcium antagonist lanthanum chloride prevented xanthine-xanthine oxidase increases in endothelial albumin transfer and prevented the changes in cell shape; chelation of extracellular calcium inhibited lysis of endothelium by xanthine-xanthine oxidase; and the calcium ionophore A23187 increased endothelial albumin transfer and mimicked the oxidant-induced changes in cell shape . Lanthanum chloride inhibited these effects of A23187 . These data suggest that oxygen radicals can reversibly increase endothelial permeability to macromolecules, that this is associated with reversible changes in endothelial cell shape and actin filaments, and that the changes in cell shape are related to oxidant-induced changes in endothelial calcium homeostasis. Fertil Steril, 1985 Mar, 43(3), 481 - 4 The use of a mobile laboratory cart in a successful university-based human in vitro fertilization program; Gerrity M et al.; The use of a mobile laboratory unit for a successful university-based IVF program is described . The construction of the cart is described in detail . The laboratory cart can be pushed to any site where ovum aspiration is carried out (either operating theater or ultrasound facilities) . Aspirated oocytes can be transferred immediately to culture media and transported back to the gamete handling laboratory for embryo culture . Developing embryos can also be transported to any location in the hospital for transfer . This system is inexpensive and has eliminated the need to duplicate specialized equipment for a gamete handling laboratory adjacent to the operating room . New construction charges have been eliminated; and as new egg retrieval methodologies are developed, the use of the mobile laboratory cart will provide our IVF program with maximum flexibility in changing sites within the hospital . Used in conjunction with an extensive quality control program, this modification has not interfered with oocyte fertilizability or with the overall pregnancy rate in the University of Wisconsin IVF program. J Virol, 1985 Mar, 53(3), 786 - 92 In vitro phenotypic markers of a poliovirus recombinant constructed from infectious cDNA clones of the neurovirulent Mahoney strain and the attenuated Sabin 1 strain; Kohara M et al.; Infectious cDNA corresponding to the entire genome of the attenuated Sabin strain of type 1 poliovirus has been inserted into EcoRI site of bacterial plasmid pBR325 . Two consecutive PstI fragments (nucleotide positions 1814 to 3421) of the infectious cDNA of the Sabin 1 strain were replaced by the corresponding DNA fragments prepared from an infectious DNA clone of the genome of the virulent Mahoney strain of poliovirus type 1 . The exchanged segment encodes capsid protein VP1 and part of capsid protein VP3, a region in which a large number of amino acid differences between the attenuated Sabin and the parental, neurovirulent Mahoney strain cluster . The recombinant virus was obtained by DNA transfection of HeLa S3 cells, and several in vitro phenotypes of the virus were compared with those of the parental viruses . The recombinant virus was recognized by a neutralizing monoclonal antibody specific to the Mahoney strain . Growth of the Sabin strain of poliovirus has been shown to be quite dependent upon the bicarbonate concentration (d marker) . The growth of the recombinant virus, however, was not highly dependent upon the concentration of bicarbonate in cell culture media, and thus resembled that of the Mahoney strain . On the other hand, the temperature-sensitive multiplication (rct marker) and the small-plaque morphology of the recombinant virus corresponded to the phenotype of the Sabin 1 strain . The in vitro recombination of infectious cDNA clones of genomic RNA and subsequent analysis of the growth properties of the recombinant virus have allowed us to correlate specific mutations in the genome of an RNA virus with certain biological characteristics of that virus. J Biol Chem, 1985 Feb 25, 260(4), 2565 - 9 Swainsonine treatment accelerates intracellular transport and secretion of glycoproteins in human hepatoma cells; Yeo TK et al.; We are interested in determining whether carbohydrates are important regulatory determinants in the intracellular transport and secretion of glycoproteins . In the present study, we have used swainsonine, an indolizidine alkaloid, to modify the structure of N-glycosidically linked complex oligosaccharides . By inhibiting Golgi mannosidase II, swainsonine prevents the trimming of GlcNAc(Man)5(GlcNAc)2 to GlcNAc-(Man)3(GlcNAc)2, resulting in the formation of hybrid-type oligosaccharides . We find, from pulse-chase experiments using {35S}methionine and immunoprecipitation of individual proteins from culture media, that swainsonine treatment (1 microgram/ml) accelerated the secretion of glycoproteins (transferrin, ceruloplasmin, alpha 2-macroglobulin, and alpha 1-antitrypsin) by decreasing the lag period by 10-15 min relative to untreated cultures . The enhanced secretion was specific for glycoproteins since the secretion of albumin, a nonglycoprotein, was unaffected . When alpha 1-antitrypsin was immunoprecipitated from the cell lysates, sodium dodecyl sulfate-polyacrylamide gel electrophoresis fluorographic analysis demonstrated that the conversion of the high-mannose precursor to the hybrid form in swainsonine-treated cells occurred more rapidly (by about 10 min) than the conversion to the complex form in control cells . Since both the hybrid and complex forms of alpha 1-antitrypsin are terminally sialylated by sialyltransferase in the trans-Golgi, these results suggest that swainsonine-modified glycoproteins traverse the Golgi more rapidly than their normal counterparts . Therefore, accelerated transport within this organelle may account for the decreased lag period of glycoprotein secretion in the swainsonine-treated cultures. Aust J Exp Biol Med Sci, 1985 Feb, 63 ( Pt 1), 91 - 8 Characterisation of rat tumour cell hybrids: procoagulant and fibrinolytic activities; Badenoch-Jones P et al.; The formation of lung colonies after i.v . injection of highly metastatic rat mammary adenocarcinoma cells (MAT 13762) was greatly reduced by concurrent treatment of rats with heparin . The procoagulant activity (PCA) of these cells, and of a non-metastatic adenocarcinoma (DMBA-8) has therefore been measured . These have been compared with PCA expressed by MAT 13762 cell derivatives including a non-metastatic hybrid clone (MAT 13762 X DMBA-8), its metastatic revertant, and clones selected in vivo from lung metastases . Potent PCA was expressed on intact MAT 13762 cells and in their spent culture media, the latter being sedimentable and associated with shed membrane vesicles . Cell-derived PCA, unlike thromboplastin, was equally effective in factor VII-deficient and normal bovine plasma . There were, however, no major differences in the expression of PCA (either cell-associated or shed) between the metastatic and non-metastatic cell types studied . Plasminogen activator (PA) production by these cells has also been measured . The results are discussed in the context of the possible role of fibrin formation and fibrinolysis in the metastatic process. In Vitro Cell Dev Biol, 1985 Feb, 21(2), 99 - 107 In vitro modulation of differentiation by calcium in organ cultures of human and murine epithelial tissue; Sacks PG et al.; The differentiation of epithelial tissue in organ cultures of murine buccal mucosa, various human oral mucosa, and human newborn foreskin was found to be dependent on the calcium concentration of the culture media . In low calcium medium (less than or equal to 0.07 mM) epithelial differentiation was inhibited . The original stratifying layers separate and can be removed, producing a destratified explant . Histologically such an explant consists of a dorsal epithelial layer of basal keratinocytes resting on an intact basal lamina with subjacent stroma . At 0.01 mM calcium, the epithelial layer was one to two cells thick whereas at 0.07 mM it could be three or more layers in thickness with the most superficial cells being spread over the underlying cells . In addition to differentiation, keratinocyte migration over the sides of the explant (epiboly) and epithelial proliferation as determined by {3H}thymidine autoradiography were reduced by culture in low calcium medium . Redifferentiation occurs upon return to normal calcium levels (1.8 mM); addition of hydrocortisone to low calcium media was found to facilitate this redifferentiation. Radiat Res, 1985 Feb, 101(2), 394 - 401 Acute radiation effects on the content and release of plasminogen activator activity in cultured aortic endothelial cells; Ts'ao C et al.; Confluent monolayers from three lines of bovine aortic endothelial cells were exposed to a single dose of 10 Gy of 60Co gamma rays . Seventy-two hours later, the morphology of the irradiated and sham-irradiated monolayers was examined, and cellular DNA and protein contents were determined . In addition, the release of plasminogen activator (PA) activity into the culture media and PA activity in the cell lysates were assayed . Irradiated monolayers maintained their cobblestone appearance, but individual endothelial cells were enlarged considerably compared to sham-irradiated cells . DNA and protein contents in the irradiated monolayers were reduced to 43-50% and 72-95% of the control levels, respectively . These data indicate that radiation induced cell loss (detachment and/or lysis) from the monolayer, with hypertrophy of surviving (attached) cells to preserve the continuity of the monolayer surface . Total PA activity (lysate plus medium) in the irradiated dishes was reduced to 50-75% of the control level . However, when endothelial PA activity was expressed on the basis of DNA content, the irradiated monolayers from two of the three cell lines contained significantly more PA activity than did sham-irradiated monolayers . Most importantly, the percentage of the total PA activity released into the culture medium by irradiated cells (5-22%) was significantly (P less than 0.001) lower than that released by sham-irradiated cells (23-68%) . These data suggest that fibrinolytic defects observed in irradiated tissues in situ may be attributable at least in part to a radiation-induced inhibition of PA release by vascular endothelial cells. J Cell Physiol, 1985 Feb, 122(2), 205 - 9 Induction of stress proteins in chicken embryo cells by low-level zinc contamination in amino acid-free media; Whelan SA et al.; It has been reported that chicken embryo cells deprived of exogenous amino acids for 4 hours synthesize stress (heat-shock) proteins . Herein, we show that amino acid deprivation is not sufficient to cause induction of stress proteins . Zinc contaminating a component of commercial cell culture medium used to prepare amino acid-free medium was an inducer in our cultures . In the absence of exogenous amino acids, the concentration of zinc ions needed for half-maximal induction of stress proteins was an order of magnitude lower than the dose required for cells in complete medium . Histidine and cystine, which have high affinities for zinc ions, were the amino acids most effective in blocking the induction of stress proteins by zinc . Problems posed by heavy metal ions in culture media and biologic fluids for searches for in vivo inducers of the cellular stress (heat shock) response are discussed. Am J Vet Res, 1985 Feb, 46(2), 421 - 3 Cryopreservation of Babesia bigemina for in vitro cultivation; Vega CA et al.; Babesia bigemina-infected RBC and merozoites were cryopreserved and used to initiate in vitro cultures in normal bovine RBC; the cryoprotectant was a final 10% polyvinylpyrrolidone in Vega y Martinez solution . A cooling rate of 20 C/min until -80 C and then rapid transfer to liquid N2 storage was satisfactory . Samples for culture initiation were rapidly thawed at 37 C, washed in Vega y Martinez solution and resuspended in complete culture media containing 10% normal bovine RBC . The optimum culture conditions to reestablish cultures were a 24-well plate (16 mm ID), 5 mm in depth, and an atmosphere of 2% to 5% O2, 5% CO2, and 93% to 90% N2. J Appl Physiol, 1985 Feb, 58(2), 592 - 7 Prostacyclin synthesis in irradiated endothelial cells cultured from bovine aorta; Rubin DB et al.; Confluent monolayers of bovine aortic endothelial cells were examined 2-72 h after exposure to 0.5-5.0 Gy of 60Co gamma-rays . Accumulation of prostacyclin {PGI2, measured as 6-ketoprostaglandin F1 alpha (6-keto-PGF1 alpha)} in the culture media and PGI2 production stimulated by exogenous arachidonate were correlated with cell detachment and release of lactate dehydrogenase (LDH) activity . Platelet adherence to irradiated and control monolayers also was studied . There were simultaneous time- and dose-dependent increases in cell detachment and in the titers of 6-keto-PGF1 alpha and LDH activity in the culture medium . These changes were evident between 4 and 8 h after 5 Gy or at 24 h after 0.5 Gy . Four hours after 5 Gy, both adherent and detached endothelial cells showed a twofold increase in PGI2 production during a 15-min incubation with arachidonate (10 microM) . However, by 72 h this increase was less significant . The accumulation of 6-keto-PGF1 alpha appeared to be related to cell destruction, but radiation also stimulated PGI2 synthesis independent of cell detachment . There was an increased platelet interaction with irradiated monolayers, as a result of platelet adherence to subendothelial matrix exposed after cell detachment . However, irradiation did not alter the nonadherent property of the endothelial cell surface toward platelets. J Protozool, 1985 Feb, 32(1), 76 - 80 Observation on complete schizogony of Plasmodium vivax in vitro; Brockelman CR et al.; Attempts to grow Plasmodium vivax in vitro were made on 43 isolates in three different culture media . Complete schizogony occurred in the new medium SCMI 612 in which 34 out of 43 isolates produced merozoites . The RPMI 1640 and Waymouth media suitable for the cultivation of P . falciparum were also used with markedly less success . Results of the experiments indicate differences in nutritional requirements between the two species of Plasmodium. J Immunol, 1985 Feb, 134(2), 904 - 9 Interleukin 1 increases collagen type IV production by murine mammary epithelial cells; Matsushima K et al.; The effects of human interleukin 1 (IL 1) on collagen type IV production by normal mouse mammary epithelial cells were examined . Human IL 1 was derived from the culture media of peripheral blood monocytes or placental cells that were stimulated with silica . Although crude culture media of silica-stimulated monocytes or placental cells had no enhancing activity for type IV collagen production, IL 1-containing fractions obtained by Sephacryl S-200 gel chromatography and isoelectrofocusing from such media possessed considerable activity . To confirm the effects of IL 1 on collagen production, human monocyte-derived IL 1 was highly purified by sequential isoelectrofocusing, anion-exchange (AX 300), high-performance liquid chromatography (HPLC), and HPLC gel filtration (TSK 3000) . The same HPLC gel filtration fractions contained both an activity that stimulated collagen synthesis by mammary cells and thymocyte growth-promoting activity . These activities of IL 1 differed from a number of other factors, such as epidermal growth factor and another factor produced by placental cells that stimulated type IV collagen production but not thymocyte proliferation . In fact, IL 1 induced 100-fold less collagen type IV production by mammary epithelial cells than was needed to induce thymocyte proliferation . Our data suggest that IL 1-like molecules, which reportedly are produced by many tissue cell types, may therefore play a role in promoting a basement membrane formation at stromal-epithelial boundaries. Am J Hosp Pharm, 1985 Feb, 42(2), 343 - 5 Stability of an injectable disulfiram formulation sterilized by gamma irradiation; Phillips M et al.; Stability of an injectable disulfiram suspension sterilized by gamma(gamma) irradiation was tested . Single doses of disulfiram powder in plastic syringes were subjected to 50,000 rads of gamma radiation . Culture media were inoculated with the irradiated drug to test for growth of bacteria, fungi, and mycobacteria . The irradiated drug and nonirradiated controls were analyzed by high-performance liquid chromatography (HPLC) for disulfiram and its major degradation product, diethyldithiocarbamate (DDC) . Ultraviolet absorption spectra of irradiated and nonirradiated disulfiram were obtained . No organisms grew in any of the culture media . HPLC analysis indicated that disulfiram content of the irradiated specimens was not reduced, and DDC was not detected . There were no important differences between the ultraviolet spectra of the irradiated and nonirradiated samples . Disulfiram can be sterilized by gamma irradiation without chemical degradation. J Med Virol, 1985 Feb, 15(2), 149 - 56 Lytic cytomegalovirus replication and the hormones of human pregnancy; Koment RW; The frequency of human cytomegalovirus (CMV) excretion during pregnancy denoting active infection has been demonstrated to increase as gestation advances and at term involves a significant percentage of women . This increase enhances the risk of congenital infection of the fetus . Thus it appears that some factor(s) unique to the condition of pregnancy favors susceptibility to maternal CMV infection . We designed our studies to investigate the possible association of the continuously rising levels of selected hormones and this increased susceptibility . Progesterone, 17 beta-estradiol, and cortisol were added to tissue culture media in final concentrations to match those occurring in term pregnancy serum . Two strains of human foreskin cells, one neonatal and the other fetal, were treated with either single or paired combinations of hormone-containing media . Lytic CMV replication in neonatal foreskin cells was enhanced by a maximum of 5.7-fold when these cells were treated with cortisol . Such enhancement did not occur in the fetal cells . No synergistic effects were seen when cortisol was used in combination with other hormones nor when neonatal foreskin cells were replicated for at least three generations in either single or paired combinations of hormones prior to use . Differential hormonal enhancement of CMV replication in vitro suggests a possible mechanism for the increased incidence of CMV infection observed during human pregnancy. Brain Res, 1985 Feb, 350(1-2), 37 - 49 Selective culture of neurons from rat cerebral cortex: morphological characterization, glutamate uptake and related enzymes during maturation in various culture media; Borg J et al.; Dissociated cerebral cortex of fetal rat was grown in a serum-free, chemically defined medium (CDM) (containing insulin, progesterone, estradiol, transferrin, putrescine, selenium and 15 mM KCl) and compared with cultures grown in a medium containing 20% fetal calf serum (SCM) . Neurons survived well using either medium, but in the serum-free medium the cellular population was exclusively neuronal (at 96%), while glial cells began to proliferate after one week in the SCM . The various cellular morphologies are described in the present report and the presence of immunological markers characteristic of neurons was investigated . Autoradiographic experiments have been performed after incubation with various putative neurotransmitters and we have shown the presence of a strikingly high proportion of glutamatergic neurons in these cultures . Glutamate high affinity uptake was also greatly increased in neuronal cultures maintained in a CDM compared to a SCM, especially in young cultures . The development of different enzymes involved in the metabolism of glutamate was also studied; the specific activity of glutaminase increased in culture and was found to be higher in a CDM than in a SCM, while the inverse was true for glutamine synthetase . The relative proportion of both enzymes in neurons compared to glial cells was opposite, as neuronal cultures had higher levels of glutaminase and glial cultures were enriched in glutamine synthetase activity . It seems that the proportion of glutamate-neurons increases when cultured in a CDM compared to a SCM and we suggest that this culture procedure may provide a purely neuronal population enriched in mature glutamatergic neurons . It may thus be useful for future in vitro studies on glutamate and GABA metabolism in neurons. J Biol Chem, 1985 Jan 10, 260(1), 139 - 46 The intracellular accumulation of UDP-N-acetylhexosamines is concomitant with the inability of human colon cancer cells to differentiate; Wice BM et al.; The relationship between the intracellular concentration of various nucleotides as measured by high-performance liquid chromatography analysis, and the differentiation of 2 human colon cancer cell lines was studied . HT-29 cells were induced to undergo both structural and functional enterocytic differentiation (as determined by electron microscopy and the presence of brush-border specific enzymes, respectively) by changing the carbon source or adding Na butyrate to standard tissue culture media . This differentiation occurred after the cells reached confluency when they were cultured in galactose, uridine, inosine, or without nucleosides (all in the absence of glucose) and in the presence of glucose plus Na butyrate . Cells cultured in 25 mM fructose or glucose +/- nucleosides did not differentiate . In all culture conditions where HT-29 cells did not differentite, the intracellular concentrations of 2 compounds which co-migrated with UDP-N-acetylglucosamine and UDP-N-acetylgalactosamine rose approximately equal to 10-fold at confluency and remained elevated throughout the stationary phase, whereas their concentrations remained constant and low after confluency in cells that underwent differentiation . This indicated that the accumulation of these compounds is associated with the inability of these cells to differentiate since other nucleotides and nucleotide sugars did not change in a similar fashion . Purification of the presumed UDP-N-acetylhexosamines, followed by the identification of the products from their chemical and enzymatic hydrolysis, confirmed the identity of these two peaks . Nucleotide analysis of Caco-2 cells, which undergo enterocytic differentiation after they reach confluency even when cultured on glucose, revealed the same pattern of UDP-N-acetylhexosamine levels as differentiated HT-29 cells, with its concentration remaining relatively constant and very low, even after the cells were confluent . The significance of the accumulation of UDP-N-acetylhexosamines in cells unable to differentiate is discussed. Artery, 1985, 13(2), 95 - 107 Growth of aortic smooth muscle cells from hypertensive obese and lean rats in homologous and heterologous sera; Falkow LJ et al.; Arterial smooth muscle cells (SMC) were derived from the medial thoracic aortas of three groups of rats: (a) obese, hyperlipidemic, hypertensive koletsky rats (OHT); (b) lean, normolipidemic, hypertensive koletsky rats (LHT) and (c) lean, normotensive Wistar rats (NT) . The growth patterns of these cells were subsequently compared in tissue culture media (DMEM) supplemented with pooled 10% homologous or 10% heterologous sera . Morphologically the SMC from all three rat sources grew in culture in typical SMC hill and valley patterns . When the SMC from the three rat sources were cultured in DMEM supplemented with 10% newborn calf serum (NCS) there were no differences in final cell densities and morphologies between the cell types . However, SMC derived from OHT rat aortas grown in DMEM supplemented with pooled 10% homologous serum achieved lower cell densities than SMC derived from LHT and NT rat aortas in their respective homologous sera . There were no differences in final cell densities when LHT SMC and NT SMC were separately cultured inpooled heterologous sera derived from LHT, OHT or NT rats or when OHT cells were cultured in heterologous sera derived from either LHT or NT rats . The OHT serum preferentially affected only the growth and morphology of SMC derived from the obese, hyperlipidemic, hypertensive koletsky rats. J Toxicol Environ Health, 1985, 16(1), 115 - 26 An in vitro system for exposure of lung cells to gases: effects of ozone on rat macrophages; Valentine R; A test system was developed for the in vitro exposure of lung cells to gases . The exposure system was used to evaluate ozone (O3) injury to pulmonary alveolar macrophages (PAM) obtained from Sprague-Dawley rats by bronchopulmonary lavage . Ozone exposures were conducted within temperature-controlled stainless-steel and Plexiglas chambers that contained glass petri dishes affixed to a revolving, inclined platform . Cell exposures were accomplished by rotation of the platform at 1 rpm to alternately expose PAM monolayers to culture media and O3 . The system provided stable, O3-containing atmospheres and permitted simultaneous in vitro exposures at three O3 concentrations . In vitro exposure of PAM monolayers for 2 h at chamber concentrations ranging from 0.2 to 6.1 ppm O3 was associated with a significant, concentration-related reduction of latex-bead phagocytosis in rotated PAM cultures . In contrast, PAM that were similarly exposed in nonrotated dishes placed horizontally and covered with a stationary layer of media (1.5 mm depth) were not affected . Other parameters of cell function, including PAM viability and adherence, were unchanged compared to unexposed or horizontal, nonrotated controls . The inability to observe adverse effects among the nonrotated cultures is consistent with the impaired diffusion of O3 through the comparatively thick media overlay in stationary cultures . The in vitro system provides a realistic simulation of lung cell exposure to O3 and represents a useful model to study the toxicity of gases on cultured cells. Tissue Cell, 1985, 17(4), 461 - 72 Role of alveolar macrophages in endotoxin-induced neutrophilic alveolitis in rats; Rinaldo JE et al.; Although bacterial endotoxins have potent effects on blood monocytes and tissue macrophages, the role of alveolar macrophages in regulating intrapulmonary neutrophil traffic following endotoxemia has not been studied previously . We have previously reported that a single intraperitoneal injection of endotoxin from Escherichia coli serotype 055B5 causes acute lung inflammation by neutrophils (PMN) in rats . The factors which influence the migration of PMN in the lung in this model are unknown . To determine whether macrophage-derived products could play a role in directing migration, we enumerated neutrophils in histologic sections and employed electron microscopy to document the location of neutrophils in the lung in vivo following endotoxin . We also cultured the alveolar macrophages recovered by lung lavage to measure the effect of their culture supernatants on neutrophil migration in vitro . In the first 6 hr following endotoxin, and also 24 hr later, there was an increase in the number of PMN enumerated in the lung parenchyma by light microscopy . Electron microscopy showed the location of the neutrophils to be exclusively intravascular at 6 hr . By contrast, neutrophils were observed in both interstitial and bronchoalveolar spaces at 24 hr, confirming that transvascular migration was active at that time . The pulmonary macrophages which were recovered by lung lavage from groups of rats sacrificed at 4 and at 15 hr following the administration of endotoxin were assayed for the release into culture media of migration-stimulatory activity for neutrophils . Macrophages from animals sacrificed 4 hr following endotoxin released less migration-stimulating activity into media than macrophages from controls . These macrophages could be stimulated to release migration-stimulating activity into culture media at levels comparable to macrophages from controls by the addition of opsonized Zymosan to the culture media . By contrast, macrophages from animals sacrificed 15 hr after endotoxin spontaneously released more migration-stimulating activity for neutrophils than did macrophages from controls . Thus, in this model, a specific increase in the synthesis or release by alveolar macrophages of factors which stimulate the migration of neutrophils in vitro coincided with a transition from intravascular to extravascular alveolar inflammation by neutrophils in vivo . These observations are consistent with the hypothesis that pulmonary alveolar macrophages may contribute to the regulation of alveolar inflammation following endotoxemia by releasing factors which influence the migration of neutrophils. Int J Immunopharmacol, 1985, 7(5), 761 - 8 Thymosin alpha 1 exerts protective effect against the 5-FU induced bone marrow toxicity; Ohta Y et al.; Thymosin alpha 1 was shown to prevent the 5-fluorouracil(5-FU)-induced bone marrow toxicity in BDF1 mice, as determined by the cellularity, haemopoietic stem cells (CFU-s) and granulocyte-macrophage colony forming unit (GM-CFU) . Furthermore, thymosin alpha 1 increased the levels of colony stimulating factor (CSF) in sera or in culture media of spleen cells derived from 5-FU-treated mice . The treatment of spleen cells with anti-Thy 1,2 antibody plus complement abolished completely the CSF production . The in vivo treatment of donor mice with anti-Thy 1,2 antibody following 5-FU abolished completely the capability of their bone marrow cells to save lethally irradiated recipients . Thymosin alpha 1 treatment prevented the damage by such combined treatment . The present study indicates that thymosin alpha 1 exerts its protective effect against the 5-FU-induced bone marrow toxicity, at least partially, through its effect on the maturation of immature T cells to functional T cells which produce various kinds of lymphokines including CSF. Immunol Lett, 1985, 10(3-4), 145 - 9 Murine monoclonal antibodies which recognize adenosine deaminase from calf, mouse, rat and man; Vielh P et al.; Three monoclonal antibodies against calf adenosine deaminase were obtained from mice . All three strongly cross-react with the rat and human forms of adenosine deaminase, and two of them with the mouse enzyme . We show that these reagents can be useful for the preparation of adenosine deaminase-free cell culture media and consider their potential interest for the early immunofluorescence detection of T-cell acute lymphoblastic leukemias. Dev Biol Stand, 1985, 60, 93 - 100 A screening method to develop serum-free culture media for adherent cell lines; Bodeker BG et al.; A screening method was established to develop chemically defined, serum-free culture media for human adherent cell lines . It indicates within 2-6 months whether a certain cell line is suited for routine cultivation under serum-free conditions, and which medium supplements and surface coatings of the culture vessels the cells need for minimal demand . This method was tested with the human cell lines Bowes melanoma and prostate carcinoma 3 (PC 3). Exp Hematol, 1985 Jan, 13(1), 23 - 8 Translation of messenger RNA from a renal tumor into a product with the biological properties of erythropoietin; Saito T et al.; The renal tumor RCC-3-JCK, when transplanted into immunodeficient mice, caused an erythrocytic polycythemia . When grown in culture, the tumor cells secreted a substance into the culture medium that chromatographed by size-exclusion high-performance liquid chromatography similarly to purified human erythropoietin (Ep) and was positive when assayed for Ep by its ability to stimulate erythropoiesis in fetal mouse liver cells (the FMLC assay) . The poly(A) + RNA was extracted from the tumor cells and injected into Xenopus oocytes, inducing the appearance of Ep(FMLC) in the oocyte culture medium . Both the tumor cells and oocyte culture media were fractionated by size-exclusion high-performance liquid chromatography, and two fractions with Ep(FMLC) activity were found in the tumor-cell culture medium . Three active fractions were found in the medium from the mRNA-injected oocytes . The largest component from both culture media had the same elution time as a human standard (Ep) . The poly(A) + RNA was fractionated by sucrose density-gradient centrifugation and the 8S and 10S fractions were found to induce Ep(FMLC) synthesis when they were injected into the oocytes . We conclude that poly(A) + RNA isolated from the Ep-producing tumor RCC-3-JCK included mRNA for Ep and that the Ep was a translational product of Xenopus oocytes injected with this mRNA. Bull Soc Pathol Exot Filiales, 1985, 78(3), 360 - 7 Clinical manifestations of female trichomoniasis and comparison of direct microscopy and culture media in its diagnosis; Imandel K et al.; A group of 125 women attending a gynecologic clinic was studied to assess the value of four culture media, wet smear and Papanicolaou stained smears and also to re-evaluate the clinical picture in the diagnosis of trichomoniasis . There was no significant difference between the results of 4 different culture media but significant difference was obtained from fresh wet vaginal smears and culture media on the one hand and with Papanicolaou stained smears on the other hand . During the survey, cultures were examined daily for five days, and the Oxoid no . 2 medium gave a majority of positive results and diphasic eggs, CM161 Oxoid, 5447 Merck media were in turn having positive results after 3 days incubation at 36 degrees C . R27, egg, CM161 and 5447 culture techniques and Papanicolaou stained smears, identified T . vaginalis in 37 (29.6%), 33 (26.4%), 30 (24%), 28 (22.4%) and 15 (12%) patients respectively, while the wet mount was positive in 22 (17.6%) patients . R27 culture technique identified T . vaginalis in 37 women, and wet mount was positive in 22 patients (P greater than 0.001) . In no case was the vaginal wet mount preparation positive for T . vaginalis and the culture negative . Egg medium detected T . vaginalis in 33 women, in comparison with wet mount positive 22 (P greater than 0.01) . Vaginal discharge was described by 72% of the 125 patients, but only 19 (51.4%) Trichomonas vaginalis were detected . Dysuria was present in 73 (58.4%) cases but only 21 (16.8%) were suffering from trichomoniasis . Physical examination revealed no vaginal discharge in 18 (48.6%) of the women, 12 (22.4%) cases had frothy leukorrhea in women with trichomoniasis. Bioelectromagnetics, 1985, 6(2), 157 - 68 Low-voltage ELF electric field measurements in ionic media; Gundersen R et al.; Low-voltage electric fields were measured in conductive tissue culture media using three techniques: voltage slope, current density-conductivity, and dipole methods . All three methods tested yielded comparable results . However, all three techniques have associated errors . These errors fall into three major categories: those associated with the measurement equipment, those associated with electrodes, and errors in cross-sectional area measurements . Each source of error is discussed so that all can be taken into account during construction and/or testing of exposure equipment. Arch Geschwulstforsch, 1985, 55(2), 93 - 8 Influence of retinoids on growth and melanin content of Harding-Passey-melanoma cells in vitro and B16 transplantable melanoma in vivo; Drewa G et al.; Growth cessation and cell death of exponentially proliferating Harding-Passey melanoma cells (HPM-73 line) in monolayer culture resulted in the presence of 3.3 X 10(-5) M retinal, while retinol and retinoic acid caused growth retardation at 3.3 X 10(-5) M . Also at 1 X 10(-5) M, the growth-inhibitory effect of retinal was more pronounced than that of retinol or retinoic acid . Following serum removal from the culture media, all 3 retinoids at 3.3 X 10(-6) M or 1 X 10(-5) M revealed cytotoxic effects within 3 days as demonstrated by cell loss from the substratum . Thus, the presence of serum has "protective" effects . Addition of retinal, retinoic acid or retinol at 1 X 10(-5) M to cultures in stationary growth phase did not result in cell loss during period of 6 days . C57Bl mice with B16 melanotic melanoma were i.p . injected during 10 days with retinoids (30 or 100 mcg per mouse daily) . All retinoids inhibited B16 tumor growth in vivo . In this respect, retinoic acid was the most effective one . The cellular melanin content of cultured HPM-cells and of B16 melanotic melanoma in vivo was elevated after treatment with retinoids; retinal having the strongest effect. Ann Biol Clin (Paris), 1985, 43(1), 27 - 31 {Culture media for in vitro fertilization}; Menezo Y; The potential of a medium used for IVF-ET must be defined not only by the percentages of successful fertilization and cleavage, but above all by the percentage of term pregnancies . However, they can only be expressed in a well-defined environmental context . The medium has to remain in equilibrium with its gas phase, which can modify the biophysical parameters (pH, pO2, redox potential), and can even be deleterious (presence of NO and CO) . Several factors can also interfere with the results obtained in theoretically identical media . In powder media, a certain lack, of homogeneity and an inferior quality of the water added can be major sources of variability . The definitions of the actual compositions of the media can vary continuously and insidiously if the original datum is not consistently taken as reference . These two points are developed in the paper . A great number of facts have, nevertheless, been established: the optimal biophysical conditions, the mineral composition, the amino acid composition, and the necessity for certain substrates such as pyruvate . Numerous lacunae remain: vitamin requirements, the supply of lipids and macromolecular compounds . The addition of serum to the culture media has not helped to remove these uncertainties . We have recently shown that serum is not indispensable in the IVF-ET medium . This opens the way to an understanding of the mechanisms of in vitro fertilization and early embryonic development. Dermatologica, 1985, 170(3), 136 - 41 Chancroid or chancroidal ulcers; Sehgal VN et al.; 1532 cases with genital ulcers were investigated, of whom 610 presented with features suggestive of chancroid; classic, multiple lesions of chancroid were observed in 312, while its other variants, i.e . dwarf, giant and phagedaenic chancroid were also seen . In addition, 162 cases had a conspicuous morphology characterised by a single ulcer which was well-defined, soft, tender, non-indurated and had weakening edges . These were termed chancroidal ulcers . The latter had a longer incubation period of 8-11 days . Absence of lymphadenopathy was prominent in these cases . The male/female ratio was 27/1 . Persons of low socio-economic status in the sexually vulnerable age-group were predominantly affected . The prepuce, coronal sulcus and glans penis were the common sites of affliction in males, while the labia minora was frequently involved in females . Due to the limited value of gram-stained smears for the detection of H . ducreyi and lack of good culture media, chancroid and chancroidal ulcers should be differentiated clinically. Int Arch Allergy Appl Immunol, 1985, 76(1), 58 - 67 Standardization of Dermatophagoides pteronyssinus: assessment of potency and allergen content in ten coded extracts; Ford AW et al.; As part of the studies to establish an international reference preparation of Dermatophagoides pteronyssinus allergens, ten coded extracts of this mite were assessed in four laboratories . The extracts were compared for total potency using direct RAST, RAST inhibition and quantitative skin tests, and also for composition and major allergen content using crossed immunoelectrophoresis, crossed radioimmunoelectrophoresis, rocket immunoelectrophoresis and radioimmunoassay . In addition, the source materials were examined by light microscopy, and the extracts were examined for the presence of proteins/allergens derived from the culture media . To help with standardization, a new reference pool of sera from patients allergic to D . pteronyssinus was also established (National Institute of Biological Standards and Control, NIBSC, 82/528) . The results showed that techniques are available for measurements of potency and allergen content . In several of the extracts, culture medium derived allergens and antigens were demonstrated . It also became clear that extracts varied not only in their total potency but also in the distribution of the identifiable allergens . In particular, extracts derived from isolated mites contained more AgX and/or Ag 23 relative to their content of antigen P1 (= Ag42) . These studies lead to the choice of an extract for an international reference preparation (NIBSC 82/518) and helped to establish the methods used subsequently in the international collaborative study. J Free Radic Biol Med, 1985, 1(5-6), 443 - 9 Lipid peroxidation products and clastogenic material in culture media of human leukocytes exposed to the tumor promoter phorbol-myristate-acetate; Khan SH et al.; The chromosome-damaging effect of PMA in blood cultures is mediated by secondary products which are formed by the cells in response to the interaction with this tumor promoter . Since this effect could be influenced by antioxidant enzymes and by inhibitors of arachidonic acid metabolism, the present study was undertaken in order to determine whether the formation of these clastogenic substances is concomitant with the formation of AA metabolites and other lipid peroxidation products . Besides the clastogenic effect of ethyl-acetate extracts, the similarities of cytogenetic and biochemical results (conjugated dienes and TBA-reactive material) obtained for the influence of other blood cells than lymphocytes in the culture system, the importance of PHA stimulation and the protective effect of antioxidant enzymes were arguments in favour of a causal relationship between chromosome damage and lipid peroxidation (enzymatic or nonenzymatic) . If AA release from membrane phospholipids was prevented by inhibition of phospholipase A2, neither conjugated dienes nor TBA-reactive material were found, and chromosome damage was reduced considerably . However, the results obtained with inhibitors of the cyclo- and lipoxygenase pathway were not conclusive, and discrepancies were also observed in the time course of appearance of clastogenic material and lipid peroxidation products. Trans R Soc Trop Med Hyg, 1985, 79(5), 613 - 7 Experimental examination of the direct damaging effects of Giardia lamblia on intestinal mucosal scrapings of mice; Anand BS et al.; Giardia lamblia is known to produce functional and structural derangement of the small intestine but the pathogenesis of this defect is not clear . To examine this, mucosal scrapings from the small intestine of mice were incubated with human G . lamblia trophozoites . The integrity of the mucosal cells was assessed by their ability to exclude trypan blue, and by the levels of brush border lactase, sucrase and maltase . As judged by the trypan blue test, more mucosal cells incubated with G . lamblia were found to be damaged than were in the control groups I (mucosal cells alone) and III (mucosal cells plus Giardia culture media) . Similar results were obtained with disaccharidases where again the mucosal cells incubated with G . lamblia showed a statistically significant reduction in the activity of lactase, sucrase and maltase compared to that in the control groups . These findings suggest that G . lamblia causes direct damage to the small intestinal epithelial cells and that this effect is not mediated through factors such as bacterial proliferation, bile salt deconjugation and immunological reactions. Pharmacology, 1985, 30(6), 339 - 47 Role of carbonic anhydrase in bone resorption induced by prostaglandin E2 in vitro; Hall GE et al.; The possible role of carbonic anhydrase in bone resorption induced by prostaglandin E2 (PGE2) was studied using an in vitro neonatal mouse calvarial culture system . PGE2 (10(-6) M) was effective in stimulating resorption, as assessed by calcium release into culture media . This enhanced resorption was accompanied by significant increases in calvarial carbonic anhydrase activity over control values at 48 and 96 h . At 48 h, bones treated with PGE2 had 20% more carbonic anhydrase activity than controls . By 96 h, treated bones contained 79% more carbonic anhydrase activity than controls . PGE2-induced bone resorption was inhibited by the carbonic anhydrase inhibitor acetazolamide in a dose-dependent fashion from 10(-5) to 10(-4) M, with 77% inhibition observed at 10(-4) M . The acetazolamide analogue CL 13,850 (N-t-butylacetazolamide), which does not inhibit carbonic anhydrase, failed to inhibit PGE2-induced resorption . These results are consistent with the hypothesis that carbonic anhydrase is a necessary component of the osteoclastic bone resorptive mechanism. Fertil Steril, 1985 Jan, 43(1), 129 - 34 Effects of human serum and plasma on development of mouse embryos in culture media; Shirley B et al.; Mouse embryos were cultured in Ham's F-10 medium (unsupplemented) and Ham's F-10 medium supplemented with either 15% fetal cord serum, 15% fetal cord plasma, 15% maternal serum, or 15% maternal plasma . None of the blood supplements significantly increased the numbers of embryos that developed to blastocysts, and the last three of the blood supplements inhibited embryo development . The cause of the inhibition was not identified . Estradiol concentrations of blood samples used as media supplements were found not to correlate with inhibition of embryo. Clin Exp Hypertens A, 1985, 7(10), 1395 - 407 Production and release of inactive renin by human vascular smooth muscle cells; Ohashi H et al.; Human arterial smooth muscle cells were obtained from surgically excised tissues and cultured by the explant method . The cultured cells had both active and inactive forms of an angiotensin I forming enzyme . About a five-fold increase in the activity was obtained by trypsin treatment . This renin-like enzyme was also found in abundance in the culture media, mostly in an inactive form . Most of the enzyme activity, either before or after the activation, was suppressed by an antibody specific to human renin . The inactive enzyme was activated to some extent also by acidification and by cold exposure . The molecular weight of the inactive enzyme was estimated to be approximately 49,000 by gel filtration . These results suggest that human vascular smooth muscle cells can produce renin and release an inactive form of renin, and can be a potential source of plasma inactive renin under certain conditions such as the anephric state. J Submicrosc Cytol, 1985 Jan, 17(1), 41 - 7 Frog skin epithelium interactions with a bovine purified type I collagen gel in culture; Denefle JP et al.; Isolated epithelium of the ventral frog skin was superimposed to purified type I collagen gels and then cultured . The epithelial anchoring process was dependent on the collagenase type used for epithelial isolation . With type I collagenase, lamina densa remained attached to the basal epithelial pole and epithelial anchorage to the gel was effective . But this anchorage could only take place if gels were precoated with culture media supplemented with fetal calf serum . In these fibrillar gels, 2 types of collagen striated fibers assemblies occurred . In the upper zone, striated fibers assembly was cell-dependent . The basal pole of Malpighian cells generated numerous filopodia inserted into the gel . These filopodia polarized the collagenous assembly around and along them . In the inner zone, aggregates with cholesteric texture were assembled without superimposed epithelium . Their shape and width seemed to be related to the acidosoluble collagen concentration. J Cell Biol, 1985 Jan, 100(1), 245 - 50 Giant axonal neuropathy: a conditional mutation affecting cytoskeletal organization; Klymkowsky MW et al.; Giant axonal neuropathy (GAN) results from autosomal recessive mutations (gan-) that affect cytoskeletal organization; specifically, intermediate filaments (IFs) are found collapsed into massive bundles in a variety of different cell types . We studied the gan- fibroblast lines WG321 and WG139 derived from different GAN patients . Although previous studies implied that the gan- IF phenotype was constitutive, we find that it is conditional . That is, when cells were grown under the permissive condition of medium containing over 2% fetal calf serum, most cells had normal IF organization . IF bundles formed when gan- cells were transferred to the nonpermissive condition of low (0.1%) serum . Microtubule organization appeared normal in the presence or absence of serum . The effect of serum starvation was largely blocked or reversed by the addition of BSA to the culture media . We found no evidence that the gan- phenotype depends upon progress through the cell cycle . We discuss the possible role of serum effects in the etiology of GAN and speculate as to the molecular nature of the gan- defect. Ann N Y Acad Sci, 1985, 442, 195 - 204 Culture factors affecting the success rate of in vitro fertilization and embryo transfer; Quinn P et al.; The development of one-cell mouse zygotes to the blastocyst stage in vitro has been used as a quality control for the media and handling procedures employed for human in vitro fertilization and embryo transfer (IVF/ET) . One-cell mouse zygotes were placed in culture in medium containing bovine serum albumin . Aliquots of the same batch of medium containing female patients' homologous serum were used for the fertilization and culture of human oocytes . The following procedures were associated with high rates of mouse embryo development and human pregnancies following IVF/ET: adequate gassing and equilibration of the medium, double-rinsing of pipets and catheters used to handle embryos, use of a HEPES-buffered medium for manipulating embryos in the absence of an atmosphere containing 5% CO2, control of excessive temperature in the vicinity of the embryos, and ET using medium containing 50% patient's serum . The institution of these procedures gave more consistent pregnancy rates . However, there was no obvious association between fertilization and cleavage of human oocytes and the quality of the medium ascertained by the mouse embryo development test . In a continuing trial, we are comparing two culture media (modified Tyrode's and a medium formulated on the composition of human fallopian tube fluid {HTF}) and two culture techniques (culture in medium under oil in petri dishes and in loosely capped tubes) . Significantly more mouse zygotes developed in HTF medium compared to Tyrode's medium . In a randomized 2 X 2 factorial trial with human IVF/ET, the highest pregnancy rate occurred when fertilization and culture were carried out in HTF medium under oil, but numbers are not yet sufficient to show any statistical difference between treatments. J Natl Cancer Inst, 1985 Jan, 74(1), 191 - 5 Prostaglandin E2 production by EL 4 leukemia cells from C57BL/6 mice: mechanism for tumor dissemination; Mahan M et al.; Murine EL 4 leukemia cells contained and secreted prostaglandin E2 (PGE2) . PGE2 was secreted into the culture media during in vitro growth, as well as into plasma during growth in the peritoneum of inbred C57BL/6 mice . For the study of the role of PGE2 in tumor dissemination, migration of EL 4 cells out of glass capillary tubes was used as an in vitro model for tumor spread in a host . PGE2 enhanced the in vitro migration of EL 4 cells while indomethacin, a prostaglandin synthesis inhibitor, reduced the extent of migration . When EL 4 cells were allowed to migrate into medium containing both indomethacin and PGE2, the cells exhibited an enhanced migration ability suggesting an extrinsic effect on cells by PGE2 . The participation of tumor-derived PGE2 in promoting tumor spread in a host was supported by demonstration that EL 4 cells grown in indomethacin-treated mice secreted less PGE2 and had reduced in vivo dissemination ability . These results indicated that tumor spread was promoted by tumor-derived PGE2 . The extent of migration and dissemination can be reduced by prostaglandin synthesis inhibitors such as indomethacin. Aust J Biol Sci, 1985, 38(4), 411 - 20 Influence of environmental factors on the metabolism of glucose by preimplantation mouse embryos in vitro; Edirisinghe WR et al.; The metabolism of glucose by late preimplantation mouse embryos was studied in a variety of media whose composition had been changed to reflect the environmental conditions in the uterus more closely than do standard culture media . The effects of combinations of energy substrates, the presence or absence of amino acids and the level of potassium in the medium were investigated . The use of energy substrates for in vitro culture at levels present in the uterine environment resulted in rates of synthesis and degradation of glycogen pools similar to those obtained using standard in vitro culture conditions but elevated incorporation into non-glycogen macromolecules . Amino acids influenced the metabolism of glucose by limiting the entry of glucose carbon into the non-glycogen macromolecular pool and directing more glucose into the synthesis of acid-soluble glycogen . Increasing the K+ concentration to 60 mM in the culture medium caused a small but significant increase in the number of eight-cell embryos degenerating during culture for 24 h but the metabolism of glucose was unaffected over this time . At the time of morula transformation to the blastocyst this level of potassium ions suppressed glycogen synthesis by 50% over 5 h but did not affect its turnover during chase culture . It is concluded that factors other than those studied here contribute to the maintenance of the low glycogen levels found in uterine embryos. J Free Radic Biol Med, 1985, 1(1), 51 - 7 Treatment of lymphocyte cultures with a hypoxanthine-xanthine oxidase system induces the formation of transferable clastogenic material; Emerit I et al.; Culture medium of lymphocyte cultures that have been exposed to the superoxide generating system hypoxanthine plus xanthine oxidase (X-XO) contains substances with chromosome damaging properties . This is demonstrated by the ability of ultrafiltrates of such culture media to induce chromosomal aberrations and sister chromatid exchanges in the lymphocytes of blood test cultures . Culture medium becomes active about 15 hours after the addition of X-XO and stimulation by phytohemagglutinin . Concomitant with the accumulation of clastogenic material, assays for conjugated dienes and thiobarbituric acid-reactive material which measure lipid-peroxidation become positive in the culture media . When cells are pretreated with superoxide dismutase or glutathione peroxidase before the addition of X-XO neither clastogenic substances nor lipid peroxidation products are detected . Catalase is a less efficient protector. Clin Exp Hypertens A, 1985, 7(11), 1563 - 82 Effects of alterations in cell phenotype and hypokalemia on sodium-potassium pump activity in rabbit vascular smooth muscle; Little PJ et al.; We investigated in cell culture, how alterations in phenotype accompanying proliferation of rabbit aortic smooth muscle and chronic hypokalemia could affect the Na,K pump . Total rubidium-86 uptake as well as ouabain and frusemide-sensitive uptake into cells was measured in physiological salts solution (PSS), PSS containing 5% foetal calf serum and PSS containing foetal calf serum plus 15 microM monensin . In physiological salts solution 90% of the rubidium-86 uptake into contractile or synthetic state cells was frusemide-sensitive and less than 8% ouabain-sensitive . Total and frusemide-sensitive rubidium-86 uptakes, measured in PSS or PSS containing foetal calf serum were similar in cells cultured and maintained in the contractile phenotype, cells in the synthetic phenotype and those which had recently reverted from the synthetic to contractile phenotype . When cells were sodium loaded in the presence of monensin and foetal calf serum, ouabain-sensitive rubidium-86 uptake was 50% higher in cells which were maintained in culture in the contractile phenotype . Frusemide-sensitive rubidium-86 uptake was similar in each cell phenotype . To examine how cell culture in hypokalemic media would affect the Na,K pump, we determined ouabain-sensitive rubidium-86 uptake in the presence of monensin plus foetal calf serum in cells incubated for 24 hours in low and normal potassium containing culture media . Ouabain-sensitive uptake was 20% higher in cells cultured in a 0.76 mM potassium medium than in those cultured in 5.4 mM potassium medium . Frusemide-sensitive rubidium-86 uptake was unaffected . These results demonstrate that 'maximal' Na,K pump activity in sodium-loaded cells is reduced when cells change from the contractile to synthetic phenotype . This reduction appears only very slowly reversible when cells revert from the synthetic to contractile phenotype . Prolonged hypokalemia increases 'maximal' activity of the Na,K pump. Fed Proc, 1985 Jan, 44(1 Pt 1), 19 - 24 Arachidonic acid metabolites and endothelial injury: studies with cultures of human endothelial cells; Johnson AR et al.; Human endothelial cells in culture can metabolize arachidonic acid to prostaglandin (PG) I2 (prostacyclin), PGE2, and PGF2 alpha, and to various nonpolar products . Similar metabolites are formed by cultured pulmonary arterial and venous cells and umbilical venous cells . In studies with umbilical venous endothelial cells, PG formation was stimulated by mechanical agitation or by treatment with histamine or melittin . Histamine stimulated prostacyclin release into cell culture media without damaging the cells, but melittin injured the cell membrane as indicated by the loss of 51Cr from labeled cells . Media collected from mechanically agitated cells inhibited the release of chromium by melittin . Heating to 56 C or acidification of the media did not reverse this protective effect . Prostacyclin added to labeled cells before challenge with melittin partially inhibited 51Cr release, but PGE2 or leukotrienes B4, C4, and D4 did not . In experiments with pulmonary endothelial cells, histamine augmented the release of hydroxyeicosatetraenoic acids (HETEs) and other nonpolar metabolites . The major nonpolar product comigrated on high-pressure liquid chromatography with di-HETEs . 11-HETE and 15-HETE were identified in media from histamine-stimulated cells by comigration with standards . Neither of these compounds nor 12-HETE protected endothelial cells from melittin-induced injury . It is not yet known if any arachidonic acid metabolites can modulate endothelial cell injury. Trans Assoc Am Physicians, 1985, 98, 116 - 22 On the circulating form of atrial natriuretic factor; Cantin M et al.; Isolation and sequencing of IR-ANF from plasma reveal that the circulating form of ANF is a 28-amino acid peptide (Ser 99-Tyr 126) which inhibits basal aldosterone secretion from suspensions of rat zona glomerulosa cells to the same extent as synthetic Arg 101-Tyr 126 . The same form (minus two N-terminal amino acids) (Arg 101-Tyr 126) has been isolated from culture media of atrial cardiocytes and sequenced . Structure activity studies of shorter, N- or C-terminally truncated versions of synthetic Arg 101-Tyr 126 show that the latter is the most potent one. Allerg Immunol (Leipz), 1985, 31(3), 189 - 93 {Isolation of monocytes by adherence to gelatin-layered surfaces}; Gotze D et al.; The method for purification of monocytes using adherence to gelatin coated glass surfaces described by Chien et al . was optimized by drastic shortening the incubation time and modifying the culture media . After one adherence step we obtained monocytes with a purity of 73-78% and a recovery of 53% . Thiol-protein-disulfide-oxidoreductase (TPO), a new enzyme marker of monocytes, was found to be also valid for monocytes obtained by adherence methods . Comparing the number of TPO-containing monocytes with other markers (alpha-naphthylacetate esterase, peroxidase, phagocytosis of latex particles, acridine orange fluorescence, antigens detected by the monoclonal BL-M/G antibody) almost identical values were found. Comp Biochem Physiol A, 1985, 80(2), 247 - 56 Structural effects of external media on isolated myelinated axons; Orson NV et al.; Myelinated axons were isolated from the sciatic nerve of Xenopus laevis by desheathing and teasing, and were mounted for short-term light microscope observation in different media . Fibres mounted in a conventional physiological saline showed a tendency to gross structural changes, including collapse of axons and separation of the axon from the myelin sheath . With isotonic media based on 120mM potassium aspartate, or 120mM potassium glutamate, structural stability and axoplasmic function (assessed by persistence of particle transport) were well maintained . The speeds of retrograde particle transport in fibres immersed in potassium aspartate or potassium glutamate medium ranged from 0.05 to 3.15 micron/sec with mean speeds of 1.05 and 1.2 micron/sec . Transport persisted for up to 3 hr . Isotonic media based on amino acids but lacking K+ or Na+ were unsatisfactory short-term culture media and destabilized myelin structure . Inclusion of 60 mM KCl in the medium reduced structural disruption and allowed particle transport to persist. Curr Genet, 1985, 10(3), 197 - 203 Characterization of cpd-1 and cpd-2 mutants which affect the activity of orthophosphate regulated cyclic phosphodiesterase in Neurospora; Hasunuma K et al.; Enzymatic and genetic characterization of cpd-1 and cpd-2, which exhibit rhythmic conidiation in liquid media and on solid media, were described with band (bd) strain as a reference . Cpd-1 and cpd-2 showed reduced growth in orthophosphate-free cyclic 3',5'-AMP media, while bd showed wild-type level of growth in the media . In low-phosphate media, cpd-1 and cpd-2 produced 19.2% and 9.8% of orthophosphate-regulated cyclic phosphodiesterase (cPDase) in culture media, while bd produced 123% . The intracellular levels of cPDase with Km of 1 x 10(-5) M in high-phosphate media in cpd-1, cpd-2 and bd were about 20%, 15%, and 10% of that in wild-type, respectively . In low-phosphate media roughly equal levels of cPDase with Km of 1 x 10(-5) M were produced in all strains, whereas the production of cPDase with Km of 2 x 10(-3) M was reduced in cpd-2, and that of cPDase with Km of 1 x 10(-2) was reduced in cpd-1 and cpd-2 . The levels of intracellular cyclic 3',5'-AMP in cpd-1, cpd-2, and bd in high-phosphate media were 13.1%, 10.1%, and 69.6% of that in wild-type . Adenylate cyclase activity in cpd-1, cpd-2, bd, and cr-1 was 69.3%, 34.0%, 63.2%, and 20.3% of that of wild-type (74A) . The levels of Mg++-stimulated cyclic phosphodiesterase in cpd-1, cpd-2, bd, and cr-1 at 0.2 microM cyclic 3',5'-AMP were 199%, 137%, 329%, and 293% of that of wild-type.(ABSTRACT TRUNCATED AT 250 WORDS) Tumour Biol, 1985, 6(3), 233 - 42 The influence of pergonal on in vitro production of placental protein 5 (PP5) by ovarian tumour cells; Sinosich MJ et al.; A radioimmunoassay for the measurement of placental protein 5 (PP5) has been developed using a second antibody-polyethyleneglycol method to separate free from bound ligand . PP5 immunoreactivity was detected in culture media from a cell line derived from a small cell carcinoma of the ovary (SCCWm2) . The culture-derived PP5 shares immunological identity with pregnancy and ovarian follicular PP5 . Affinity interactions between PP5 and matrices such as heparin or thrombin-Sepharose were similar and independent of the origin of PP5 . PP5 was heterogeneous with two forms, a monomer (18k) and dimer (36k) being detected in all biological fluids examined . The dimer was predominant in ovarian follicles and the SCCWm2 cell line, while the monomer predominated in pregnancy serum . The basal rate of PP5 secretion by the SCCWm2 cell line was 0.61AU/24h/10(5) cells . Incubation of cells in the presence of 150mIU Pergonal stimulated PP5 production 20.6 fold and following removal of Pergonal the production rate was maintained at 18.0 times the basal rate . No choriconic gonadotrophin, pregnancy-specific beta 1-glycoprotein, pregnancy associated plasma protein-A or alpha fetoprotein was detected in the culture medium of the SCCWm2 cell line. Acta Derm Venereol, 1985, 65(4), 277 - 81 Collagen synthesis and degradation by epidermolysis bullosa fibroblasts; Oakley CA et al.; Collagen synthesis was measured in fibroblasts cultured from the skin of six patients with epidermolysis bullosa simplex, from six patients with dystrophic epidermolysis bullosa and six age-matched controls without skin disease . Both groups of patients' fibroblasts synthesized approximately twice as much collagen (dpm/cell) as the controls . Synthesis of other proteins showed a smaller increase . Collagenase activity in culture media from four fibroblast lines per group was measured, using a 3H-collagen substrate, both before and after trypsin treatment to activate procollagenase . As expected, the dystrophic group had the highest activity (30% more than controls) and the result was little affected by trypsin: the enzyme appeared to be in active form . Enzyme activity in the simplex group was increased from 67% to 114% of control values by trypsin treatment . The excessive collagen synthesis in both dystrophic and simplex fibroblasts may be a consequence of their greater collagenase activity and suggests an unsuspected dermal involvement in epidermolysis bullosa simplex . Our data confirm an excessive secretion of collagenase by dystrophic fibroblasts but suggest that the enzyme's state of activation may be the important aetiological feature of the dystrophic disease. J Biol Chem, 1984 Dec 25, 259(24), 15123 - 30 A kinetic comparison of the processing and secretion of the alpha beta dimer and the uncombined alpha and beta subunits of chorionic gonadotropin synthesized by human choriocarcinoma cells; Peters BP et al.; Human choriocarcinoma cells (JAR) synthesize the alpha and beta subunits of the glycoprotein hormone chorionic gonadotropin (hCG) (R.W . Ruddon, C.A . Hanson, A . H . Bryan, G.J . Putterman, E.L . White, F . Perini, K . S . Meade, and P.H . Aldenderfer (1980) J . Biol . Chem . 255, 1000-1007) . In addition to the hCG dimer (alpha beta), JAR cells secrete uncombined alpha and beta subunits into the culture medium (L.A . Cole, R.J . Hartle, J.A . Laferla, and R.W . Ruddon (1983) Endocrinology 113, 1176-1178) . Pulse-chase studies with {35S}methionine or {3H}mannose were carried out in order to compare free alpha, free beta, and the alpha beta dimer with regard to the kinetics of synthesis, N-linked oligosaccharide processing, and secretion and to determine the kinetics of alpha-beta subunit combination . A panel of three antisera was used to immunoprecipitate directly the free subunits and the alpha beta dimer sequentially from the same cell lysates and culture media . The alpha subunit of hCG was synthesized in a slight molar excess (1.2-1.5-fold) over the beta subunit, and alpha beta dimer was rapidly formed by combination of the intracellular alpha and beta precursors . Dimer formation was already apparent in JAR cells following a 10-min biosynthetic labeling incubation with {35S}methionine . The combination of subunits ceased by 30 min of chase even though 51% of alpha and 44% of beta remained free within the cells . Combination of the alpha and beta precursors had occurred before their N-linked oligosaccharides were processed beyond the Man8GlcNAc2 structure . The initial trimming of glucosyl and mannosyl units from the high-mannose oligosaccharides of the hCG precursors occurred more rapidly for free alpha and CG-alpha than for free beta and CG-beta . JAR cells accumulated alpha precursors bearing mostly Man8GlcNAc2 units and beta precursors bearing Man8GlcNAc2 units that represent the substrates of the rate-limiting step in the secretory pathway . In spite of the fact that their N-linked oligosaccharides were trimmed at different rates, free alpha, free beta, and alpha beta dimer were all secreted into the medium at the same rate, with a half-time of 35 min . The secreted hCG forms were stable in the chase medium between 4 and 8h, indicating that extracellular degradation, combination of free subunits to form dimer, or dissociation of dimer to form free subunits did not occur.(ABSTRACT TRUNCATED AT 400 WORDS) Thromb Res, 1984 Dec 15, 36(6), 591 - 7 Ethinylestradiol and d-norgestrel regulation of plasminogen activator in a human melanoma cell line; Kjaeldgaard A et al.; The influence of ethinylestradiol (EE2) and d-norgestrel (d-Ng) was studied in a melanoma cell line producing a tissue-type plasminogen activator (t-PA) very similar to or identical with the t-PA isolated in extracts from human uterus . The cell cultures were exposed to the two contraceptive steroids by addition of EE2 or d-Ng dissolved in a week alcoholic solution to the culture media, in which the released t-PA was assayed by an immunoradiometric method . A strongly stimulating effect of ethanol (0.76% w/v) on the t-PA production was demonstrated . Whereas, EE2 in the concentration of 1.7 X 10(-7)M and d-Ng in the concentration of 8.6 X 10(-7)M both caused a significantly reduced secretion of t-PA, and this effect was independent of whether the cell cultures were grown to confluency in the presence of the two synthetic steroids or not . It was concluded, that the two contraceptive steroids had an inhibitory effect on the production of t-PA in melanoma cell culture. Biochem Pharmacol, 1984 Dec 15, 33(24), 3951 - 6 Inhibition of the estradiol-induced growth of cultured human breast cancer cells by the anti-estrogens tamoxifen, desmethyl-tamoxifen, 4-hydroxy-tamoxifen and enclomiphene; Marth C et al.; The growth effects of tamoxifen (T), desmethyl-tamoxifen (dMeT), 4-hydroxy-tamoxifen (OHT) and enclomiphene (Clo) on cultured human breast cancer cell lines have been related to published binding affinities for the estrogen receptor . Only in cells which were stimulated by estrogens did these anti-estrogens markedly inhibit growth . In both estrogen sensitive cell lines tested, 734 B and ZR 75.1, the anti-estrogen activity showed the identical rank: OHT much greater than Clo approximately equal to T = dMeT; this anti-proliferative potency agrees with reported affinities of these compounds for the estrogen receptor . In culture media containing defined amounts of estradiol we observed that a 10,000-fold molar excess of OHT was required to inhibit the estradiol-induced growth, but the estradiol-independent proliferation was not affected. J Biol Chem, 1984 Dec 10, 259(23), 14973 - 8 Selective intracellular degradation of fibrinogen and its reversal in cultured hepatocytes; Grieninger G et al.; Primary monolayer cultures of chick embryo hepatocytes were employed in pulse-chase experiments to examine plasma protein synthesis and secretion . The fates of {35S}methionine-labeled fibrinogen and transferrin were monitored in cell extracts and in spent culture media . It was found that hepatocytes, which were maintained in the absence of added hormones or serum, released into the medium virtually all of the label of transferrin but only 30% of the label in fibrinogen . The remainder of the labeled fibrinogen was retained by the cells, gradually disappearing in a manner suggestive of its intracellular degradation . To stimulate fibrinogen production on as many levels as possible, fetal bovine serum was added to the medium of the cultured cells . Serum elicited an increase in the level of fibrinogen mRNA which was accompanied by a 7-fold increase in the rate of fibrinogen synthesis as well as the complete release of fibrinogen label, resulting in an overall 20-fold enhancement in the hepatocellular output of this protein . Thus, both the amount of fibrinogen synthesized as well as the amount ultimately secreted are subject to modulation by the hepatocellular environment. Vet Parasitol, 1984 Dec, 17(1), 65 - 73 The migration of larval Toxocara canis in mice . I . Migration through the intestine in primary infections; Abo-Shehada MN et al.; Toxocara canis second stage larvae (L2) hatched in the stomach of mice and within 2 h reached all parts of the small intestine, the posterior half being the preferred site for larval penetration . Following penetration at the base of crypts of leiberkuhn they followed tortuous routes in the lamina propria, and entered the tunica muscularis obliquely . Larvae only appeared to be arrested in their migration in the submucosa and lamina propria by an inflammatory reaction composed mainly of lymphocytes and eosinophils . Larvae were seen entering and within lymphatic vessels as well as the peritoneal cavity and invasion of the vascular system followed though actual penetration of intestinal blood vessels was not seen . Proteolytic enzyme activity was detected in culture media in which L2 larvae were maintained. Tropenmed Parasitol, 1984 Dec, 35(4), 209 - 11 Development of onchocerca lienalis and O . volvulus from the third to fourth larval stage in vitro; Lok JB et al.; Third-stage larvae of Onchocerca volvulus and O . lienalis were observed to molt to the fourth stage in various cell-free in vitro systems . The percentage of O . lienalis completing the molt was similar in the three culture media and two gas phases tested ranging from 44.8% (1:1 IMDM:NCTC + 5% CO2: 95% N2) to 56.7% (L-15 + 5% CO2: 95% air) . Percent molting in O . volvulus ranged from 0% (F12(K) + 5% CO2: 95% N2) to 33.3% (L-15 + 5% CO2: 95% N2) . All media were supplemented with either 20% FCS or 20% horse serum . Molting by O . lienalis occurred on days 2-5 in culture . Molting by O . volvulus was observed as early as day 5 and as late as day 10 . Incomplete casting of the third-stage cuticle was frequently observed in O . volvulus . Larvae of both species entered a lethargus 24-48 hours prior to the onset of molting . Maximum survival in culture was 42 days for O . lienalis and 25 days for O . volvulus . Significant growth of larval O . lienalis was noted early in the culture period, but neither species continued development to the fifth stage. J Biochem (Tokyo), 1984 Dec, 96(6), 1831 - 9 X-ray diffraction studies on chromatophore membrane from photosynthetic bacteria . III . Basic structure of the photosynthetic unit and its relation to other bacteriochlorophyll forms; Nakamoto S et al.; We have performed X-ray diffraction studies on photosynthetic units of Rhodospirillum rubrum and solubilized *B800 + B890 complex from chromatophores of Chromatium vinosum, to investigate the homology of their molecular structures . The native chromatophores of Chromatium vinosum, which contain other bacteriochlorophyll forms, were examined by an X-ray diffraction technique, in order to assess the interactions between the complexes as well as the molecular structures of the bacteriochlorophyll forms . The subchromatophore particles, solubilized by Triton X-100 from cells of Chromatium vinosum, exhibit a major absorption maximum at 881 nm and a minor one at 804 nm, consisting of bacteriochlorophyll form *B800 + B890 . The near-IR absorption spectrum of the particle is very similar to that of chromatophores of Rhodospirillum rubrum although the major absorption maximum is shifted slightly . The X-ray diffraction pattern of the subchromatophore particles is very similar to that of chromatophores of Rhodospirillum rubrum . Thus, the subchromatophore particles are considered to be the "photoreaction unit" of Rhodospirillum rubrum . Since the bacteriochlorophyll form, *B800 + B890, is common in the purple bacteria, it is strongly suggested that the photoreaction unit is the basic and common structure existing in the photosynthetic units of purple bacteria . Chromatium vinosum cells exhibit different near-IR absorption spectra, depending on the culture media and also on the intensity of the illumination during culture . The chromatophores from these cells give different equatorial X-ray diffraction patterns . These patterns are much broader than that of solubilized subchromatophore particles, though they have common features . Thus, the molecular structures in the photosynthetic units are different, depending on their constituent bacteriochlorophyll forms.(ABSTRACT TRUNCATED AT 250 WORDS) Prostaglandins, 1984 Dec, 28(6), 769 - 81 Stimulation of arachidonic acid metabolism in primary cultures of osteoblast-like cells by hormones and drugs; Feyen JH et al.; The effects of parathyroid hormone (PTH), dihydroxycholecalciferol (1,25-(OH)2 D3), thrombin, epidermal growth factor (EGF) and 12-o-tetradecanoylphorbol-13-acetate (PMA) on the biosynthesis and release of arachidonic acid metabolites were studied in primary cultures of osteoblast-like cells isolated from 18-day-old chick embryo calvaria . Cells were labelled with (14C)-arachidonic acid for 30 h . The radioactive eicosanoids were extracted from the cell culture media after a further 30 h stimulation period and analysed on a PRP-1 column by HPLC . The radioactive products were characterized by co-elution of (3H) standard prostanoids . Osteoblasts showed a basal release of the prostanoids 6-keto-PGF1 alpha, TXB2, PGF2 alpha, PGE2, PGD2 and PGB2, the latter being the most abundant one . Indomethacin (10(-5) M) effectively inhibited the basal release, but not that of an as yet unidentified compound . The release of prostanoids was stimulated by PTH (2 U/ml), thrombin (0.4 NIH/ml), EGF (50 ng/ml) and PMA (25 ng/ml), the latter being by far the most potent one . 1,25-(OH)2D3 was found to slightly inhibit the prostanoid release . These results indicate: (1) primary cultures of osteoblasts synthesize several prostaglandins, thromboxane B2 and one unidentified product . (2) the action on bone of PTH and the various drugs tested may be, at least partly, mediated by an increased prostaglandin production by osteoblasts . Clearly this does not apply to 1,25-(OH)2D3. Nippon Yakurigaku Zasshi, 1984 Dec, 84(6), 499 - 507 {Immunopharmacological actions of neurotropin (4) . Effect of neurotropin on mouse peritoneal macrophages}; Yoshii H et al.; It was reported that neurotropin (NSP), an extract isolated from the inflamed skins of rabbits inoculated with vaccinia virus, activates murine T cell functions participating in cell-mediated immunity . The present study was undertaken to examine the effect of NSP on plastic dish-adherent macrophages (M phi) from ddY mice in vitro . Total activities of beta-glucuronidase and N-acetyl-beta-D-glucosaminidase in resident peritoneal M phi was slightly enhanced when the M phi were cultured with NSP (10-1000 micrograms/ml) for 48 and 96 hr, but no enhancement was noted in 24 hr culture . Intracellular activity of lactate dehydrogenase (LDH) was also strongly enhanced in a dose-dependent manner by culturing with NSP for 48 and 96 hr . The enhanced LDH activity in the M phi cultured with NSP for 96 hr was completely inhibited by cycloheximide, an inhibitor of protein synthesis . In addition, consumption of glucose in the culture media by the M phi was also enhanced by culturing with NSP for 96 hr . Intracellular activity of LDH and glucose consumption of plastic dish-nonadherent cells from normal mouse peritoneal cells, however, was not enhanced by NSP in 96 hr culture . In regard to allogeneic M phi-mediated cytostatic activity to P815-X2 mastocytoma, NSP had no effect on cytostatic activities of the resident and thioglycollate-induced M phi, although NSP by itself dose-dependently inhibited the growth of P815-X2 mastocytoma without affecting cell viability . These results suggest that NSP biochemically activates mouse peritoneal M phi in vitro, but the M phi activated by NSP can not inhibit the growth of P815-X2 mastocytoma. J Neurochem, 1984 Dec, 43(6), 1737 - 44 GABA induces functionally active low-affinity GABA receptors on cultured cerebellar granule cells; Meier E et al.; The effect of gamma-aminobutyric acid (GABA) and its agonists muscimol and 4,5,6,7-tetrahydroisoxazolo{5-4-c}pyridin-3-ol (THIP) on the development of GABA receptors on cerebellar granule cells was studied by cultivation of the cells in media containing these substances . It was found that the presence of 50 microM GABA in the culture media led to the induction of low-affinity GABA receptors (KD 546 +/- 117 nM) in addition to the high-affinity receptors (KD 7 +/- 0.5 nM) which were present regardless of the presence of GABA in the culture media . The functional activity of the GABA receptors was tested by investigating the ability of GABA to modulate evoked glutamate release from the cells . It was found that GABA could inhibit evoked glutamate release (ED50 10 +/- 3 microM) only when the cells had been cultured in the presence of 50 microM GABA, 50 microM muscimol, or 150 microM THIP, i.e., under conditions where low-affinity GABA receptors were present on the cells . This inhibitory effect of GABA could be blocked by 120 microM bicuculline and mimicked by 50 microM muscimol or 150 microM THIP whereas 150 microM (-)-baclofen had no effect . It is concluded that GABA acting extracellularly induces formation of low-affinity receptors on cerebellar granule cells and that these receptors are necessary for mediating an inhibitory effect of GABA on evoked glutamate release . The pharmacological properties of these GABA receptors indicate that they belong to the so-called GABAA receptors. Can J Biochem Cell Biol, 1984 Dec, 62(12), 1335 - 42 Somatomedin-like activity from cultured embryo-derived cells: partial characterization and stimulation of production by epidermal growth factor (urogastrone); Atkison PR et al.; We have measured the appearance in cell culture media of a somatomedin-like activity (SLA) that cross-reacts in a basic-somatomedin radioreceptor assay and radioimmunoassay and that is produced by cultures of normal fetal-derived human (WI-38 fibroblasts) and mouse (embryonic palate) tissues and by cultures of a Simian virus 40 transformed WI-38 fibroblast cell line (WI-38 VA13/2RA subline) . In addition to human growth hormone (hGH), epidermal growth factor (urogastrone) (EGF-URO), human placental lactogen (hPL), and porcine insulin (INS) were able to stimulate the production of SLA in serum-free cultures of the normal WI-38 fibroblasts; human prolactin (hPRL) caused only a marginal stimulation of SLA production in these cultures . In order of the magnitude of the stimulation of SLA production, the effects of the several hormones tested were EGF-URO greater than INS congruent to hPL greater than hGH much greater than hPRL . The half-maximal stimulatory effect of EGF-URO in the WI-38 cultures was observed at a concentration of about 1 nM . In the mouse palate organ cultures, EGF-URO also stimulated SLA production; the effect of EGF-URO was more pronounced in palate organ cultures derived from day 13 embryos compared with tissue obtained from embryos at days 14 and 15 . The SLA produced by the human VA13/2RA subline was characterized further by gel filtration under acid conditions, by Procion dye column chromatography, and by high pressure liquid chromatography (HPLC).(ABSTRACT TRUNCATED AT 250 WORDS) Zh Mikrobiol Epidemiol Immunobiol, 1984 Dec, (12), 48 - 50 {Use of different media for growing the producer of the restriction enzyme XBA I}; Perel'man EV et al.; The possibility of using culture media prepared from local ingredients and intended for growing the producers of restricting enzyme Xba 1 has been demonstrated . The yield of restricting enzyme Xba 1 per g of crude biomass, obtained with the use of peptone-yeast medium prepared from ingredients supplied by Difco Laboratories (USA), has proved to be 4 times greater than that obtained with the use of peptone-yeast medium prepared from local ingredients . At the same time the use of casein-saline medium ensures the yield of the enzyme, similar to that obtained with the use of peptone-yeast medium prepared from ingredients supplied by Difco Laboratories, but with a greater content of nonspecific nucleases. Horm Metab Res, 1984 Dec, 16(12), 663 - 6 In vivo and in vitro effect of liver on somatomedin generation; Minuto F et al.; The serum concentration of circulating somatomedins was measured in the blood of healthy donors and subjects with hepatic cirrhosis, and in culture media from in vitro explants of healthy and cirrhotic human liver . Serum levels of somatomedin bioactivity were significantly lower in cirrhotic subjects (0.42 +/- 0.03 U/ml; M +/- SEM) compared with age matched controls (0.99 +/- 0.03 U/ml) . Radioreceptor assay of somatomedin concentrations confirmed this reduction in cirrhotic patients (0.89 +/- 0.06 U/ml) compared with controls (1.32 +/- 0.05 U/ml) . A parallel reduction in somatomedin circulating binding ability was also observed (99.43 +/- 7.28% in cirrhotic and 123.5% +/- 10.8% in normal subjects) . In vitro explants from normal human liver tissue produced a significant increase (0.57 +/- 0.09 U/ml) in somatomedin bioactivity contained in the medium (0.29 +/- 0.06 U/ml), while a decreased bioactivity (0.12 +/- 0.06 U/ml) was observed with explants of cirrhotic livers . These results support a role of liver in the biosynthesis of both somatomedin and somatomedin binding protein. Gann, 1984 Dec, 75(12), 1089 - 99 Interconversion of biological characteristics of small cell lung cancer depending on culture conditions; Terasaki T et al.; Two distinct cell lines were obtained from a single heterotransplanted tumor which had originated from a primary focus of small cell carcinoma of the lung (SCCL) . They were maintained separately from the beginning in culture media with and without fetal calf serum supplementation . Cells in the serum-free medium grew mostly floating in loose aggregates and showed poor cell cohesiveness, scanty cytoplasm and a few intracytoplasmic small dense-cored granules; all of these features are characteristics of oat cell type SCCL . On the other hand, cells in the serum-supplemented medium grew mostly floating in flatter and more closely associated clumps, were larger, and showed increased cell cohesiveness, occasional tubular structures, better developed organelles including dense-cored granules, and an increased number of cell attachments; these features are characteristics of intermediate cell type SCCL . The modal number of chromosomes differed from each other . Neuron-specific enolase (gamma enolase) and aromatic L-amino acid decarboxylase (ADC) activities in cell pellets were significantly higher in both lines than in control non-small cell lung cancer cell lines . The alpha/gamma type enolase ratio was lower, as was the ADC activity, in serum-free cultures than in serum-supplemented cultures . Interchange of the culture medium induced changes of the growth pattern and cell type from "oat cell type" to "intermediate cell type" and vice versa . The chromosomal number also partially changed . These findings suggest that cultured cells of SCCL alter their growth pattern and cell type depending on the culture conditions and that the selective growth of one cell type might then take place. Coll Relat Res, 1984 Dec, 4(6), 453 - 65 Susceptibility of interstitial rabbit collagens to rabbit articular chondrocyte collagenase; Kresina TF et al.; Conditioned culture media from confluent rabbit articular chondrocytes maintained in serum-free monolayer culture contained metal-dependent neutral pH collagenolytic activity degrading Type I, II and III rabbit {125I}-labeled collagens . This collagenolytic activity degraded Type II collagen more slowly than Type I collagen and Type III collagen at 37 degrees C . By contrast, collagenolysis by chondrocyte cytosolic protein, lysosomal granule protein and residual lysosomal membrane protein was highly specific for Type II collagen . Although collagenolytic activity against all the collagen isotypes tested was predominantly in a latent form after 24 h of culture, increasing levels of constitutive collagenolytic activity was measured with increasing culture time . These results are consistent with a differential degradation of rabbit interstitial collagens by rabbit chondrocyte collagenase . The data suggest a cellular compartmentalization of collagenolytic activity with specificity toward Type II collagen. Arthritis Rheum, 1984 Dec, 27(12), 1397 - 404 The production of collagenase by adherent mononuclear cells cultured from human peripheral blood; Louie JS et al.; Mononuclear cells were isolated from human peripheral blood by Ficoll-Hypaque centrifugation, and the cells adherent to plastic substrata were cultured in serum-free media supplemented with lactalbumin hydrolysate . These cell cultures, which consisted predominantly of monocyte-macrophages as judged by nonspecific esterase staining, accumulated collagenase in the medium . This collagenase resembled other vertebrate collagenases in that it cleaved native triple-helical type I collagen at a locus 3/4-length away from the amino-terminal end of the molecule . The collagenase activity was inhibited by Na2EDTA, dithiothreitol, and fetal calf serum, while the addition of Ca++ or N-ethylmaleimide enhanced the enzyme activity . The accumulation of collagenase in the culture media was markedly enhanced by the incubation of cells with concanavalin A or phorbol myristic acetate . In the presence of cycloheximide, the levels of collagenase activity were markedly reduced, suggesting that active protein synthesis was required to express the enzyme activity . In additional experiments, monocytes were further purified by counterflow centrifugation-elutriation . The collagenase production was markedly increased in cultures enriched in monocyte-macrophages and devoid of polymorphonuclear leukocytes . The accumulation of collagenase in monocyte cultures incubated for 48 hours in the presence of concanavalin A or phorbol myristic acetate was of the same order of magnitude as in parallel cultures containing the same number of polymorphonuclear leukocytes purified by Ficoll-Hypaque centrifugation and Plasmagel sedimentation.(ABSTRACT TRUNCATED AT 250 WORDS) Mech Ageing Dev, 1984 Nov, 28(1), 83 - 97 Structural comparisons of fibronectins isolated from early and late passage cells; Sorrentino JA et al.; Fibronectins isolated from culture media conditioned by the growth of early (young) and late (old) passage human fibroblasts were compared by affinity chromatography and polyacrylamide gel electrophoresis . The results indicated that fibronectins from young and old cells were similar, but not identical . The fibronectin synthesized by old cells migrated more slowly on NaDodSO4 polyacrylamide gels in the presence of reducing agent than did fibronectin from young cells . The apparent molecular weight difference for purified fibronectins compared on gradient gels was estimated to be 5-10 000 daltons . The molecular weight difference was also evident in unpurified fibronectins in whole conditioned media . Both young and old fibronectins formed disulfide bonded dimers in the absence of reducing agents indicating that the molecular weight difference was not generated by proteolytic cleavage at the C-terminus of the molecule . Further, both fibronectins were bound by heparin-Sepharose, thiol-activated-Sepharose, and gelatin-Sepharose resins . Comparison of peptide maps, generated by limited proteolytic digestion revealed several differences . In particular, a polypeptide of molecular weight approx . 160 000 was larger in old cell fibronectin than in young cell fibronectin . This polypeptide had heparin binding activity, but lacked affinity for gelatin. J Clin Invest, 1984 Nov, 74(5), 1890 - 4 Iron and copper promote modification of low density lipoprotein by human arterial smooth muscle cells in culture; Heinecke JW et al.; Modification of low density lipoproteins by human arterial smooth muscle cells was characterized by increased electrophoretic mobility and increased content of malondialdehyde-like oxidation products reactive with thiobarbituric acid . Lipoprotein modification was promoted by micromolar concentrations of iron or copper in the culture medium and was metal ion concentration- and time-dependent . The ability of diverse media to promote smooth muscle cell-mediated low density lipoprotein modification correlated with their iron concentration . Therefore, metal ion concentration of culture media contributes substantially to low density lipoprotein modification in vitro . Human monocyte-derived macrophages took up and esterified the cholesterol from modified low density lipoprotein more extensively than from native low density lipoprotein . Metal ion-mediated modification of low density lipoprotein may be a contributing factor to the pathogenesis of arteriosclerosis. Invest Ophthalmol Vis Sci, 1984 Nov, 25(11), 1254 - 7 In vitro incorporation of proline into keratoconic human corneas; Rehany U et al.; Corneal buttons were obtained from four young adults with advanced keratoconus following perforating keratoplasty . The corneal buttons were incubated in organ culture media containing 3H-proline . Autoradiographs of corneal sections showed that the incorporation of the radioisotope was significantly higher in all layers of the kerotoconic corneas than that found in the normal controls, indicating an increased protein biosynthesis in the former . It is suggested that in spite of the found increased synthetic activity, slow destruction and thinning of the cornea in keratoconus might occur as the result of inadequate compensation for tissue loss due to increased collagenolytic activity in the disease. J Immunol, 1984 Nov, 133(5), 2624 - 8 Estradiol regulation of secretory component in the uterus of the rat: evidence for involvement of RNA synthesis; Wira CR et al.; The present studies were undertaken to characterize the response of uterine secretory component (SC) to estradiol . Administration of estradiol for 3 days to ovariectomized rats before incubation of uterine tissues resulted in a marked accumulation of SC in the incubation media . When uteri from ovariectomized rats treated with progesterone or testosterone were incubated, very little SC accumulated in the media, indicating that the estradiol-stimulated increase is hormone-specific . When uteri from rats that received estradiol for 6 days were compared with uteri from 3-day treated rats, SC release during a 24-hr incubation period was the same . This finding indicates that in the presence of prolonged estradiol exposure, SC production continues . The estradiol-induced accumulation of SC in culture is not due to the release of pre-formed uterine SC . When tissue SC levels were measured after 3 days of estradiol treatment, very little tissue SC was found relative to that released into culture media during 24 hr of incubation . The addition of actinomycin D to the incubation media markedly inhibited SC release by uteri from estradiol-treated rats . The release of SC was also inhibited by alpha-amanitin, a known inhibitor of Type II polymerase . These studies demonstrate that estradiol stimulation of SC is markedly reduced by inhibitors of RNA synthesis, and suggest that estradiol regulation of SC is mediated through uterine mRNA synthesis. Vet Res Commun, 1984 Nov, 8(4), 309 - 16 Attempts to establish the scrapie agent in cell lines; Elleman CJ; Attempts were made to establish a persistent infection with scrapie agent in four murine cell lines . Of the four lines tested, viz . P388D1, P3-NS1-Ag-1 (NS1), L cells and an NS1 spleen cell hybrid, only the NS1 cell line showed any evidence of agent replication . Ten per cent dimethylsulphoxide (DMSO) was included in the culture media of L cells inoculated with scrapie agent . This treatment raised the initial levels of scrapie agent associating with the L cells but did not result in a persistently infected cell line . An effect of DMSO in the inoculum was observed in mice inoculated intraperitoneally, the incubation period of the disease was considerably shortened. Hypertension, 1984 Nov-Dec, 6(6 Pt 2), III27 - 32 Humoral trophic influence on cardiovascular structural changes in hypertension; Yamori Y et al.; Since the early development of structural cardiovascular change in spontaneously hypertensive rats (SHR) and stroke-prone SHR (SHRSP) indicated the involvement of non-pressure-dependent factors in this process in hypertension, smooth muscle cells (SMC) from the aorta of SHR, SHRSP, and normotensive Wistar-Kyoto rats (WKY) were investigated under tissue culture conditions free from blood pressure and humoral factors in vivo . By the observation of such factors as growth rate and DNA or protein synthesis vascular SMC from these rats with genetic hypertension were proved to have intrinsically greater growth activity independently of blood pressure . Although serum from SHR and SHRSP had no specific stimulative effect on SMC growth, circulating epinephrine may accelerate cardiovascular structural changes because isoproterenol added to the culture media enhanced ornithine decarboxylase (ODC) activity . Moreover, SMC from SHR and SHRSP showed greater thymidine incorporation than those from WKY even in response to lower extracellular Na+ concentration . Local nutritional conditions of SMC, which were proved to have a great effect on the morphology and structure of cultured SMC, may be a basic determinant of the development of hypertension-induced structural vascular changes or lesions. Dev Biol, 1984 Nov, 106(1), 89 - 96 Early differentiation of vertebrate spinal neurons in the absence of voltage-dependent Ca2+ and Na+ influx; Bixby JL et al.; The development of the action potential and responses to neurotransmitters have been described for a population of embryonic spinal neurons developing in vivo . A comparable pattern is seen for spinal neurons developing in dissociated cell culture . The impulse appears very early in this developmental sequence, and the action potential involves a large inward Ca2+ current . Since Ca2+ is a ubiquitous intracellular regulator, we questioned whether a large influx of Ca2+ is necessary for the subsequent differentiation of membrane properties . Embryonic Xenopus neurons grown in normal culture medium do not make Ca2+- or Na+-dependent action potentials in their cell bodies in a Ca2+-free saline containing tetrodotoxin (TTX) . To achieve a chronic blockade of impulse activity, neurons were grown in a medium in which Ca2+ was replaced by Mg2+, and to which 1 mM EGTA was added . In some instances TTX was present . Neurons grown in these experimental culture media extend neurites more rapidly than controls . Action potentials cannot be elicited from neurons when examined in experimental medium . However, examination in saline reveals that the change in the ionic dependence of the impulse is indistinguishable from that observed in neurons grown in normal medium . Furthermore, the time of onset of responses to GABA is unaffected by this experimental treatment . Thus the expression of Ca2+- and Na+-dependent action potentials seems not to play a part in the early differentiation of these membrane properties . However, the later development of GABA sensitivity is reduced. Tohoku J Exp Med, 1984 Nov, 144(3), 305 - 13 Effect of estradiol, prostaglandin E2 and prostaglandin F2 alpha on incorporation of {3H}uridine by preimpla