J Bacteriol, 1985 Sep, 163(3), 906 - 12 Productive phage infection in Escherichia coli with reduced internal levels of the major cations; Kuhn A et al.; Bacteriophage-induced changes in the intracellular levels of the major cations of Escherichia coli were studied to investigate the role of ion concentrations for bacteriophage assembly in vivo . Infection of E . coli by phage T4, P1, or lambda caused a transient reduction of intracellular levels of potassium, magnesium, and polyamines . Phages T3 and T7, however, had no detectable effect on the cation concentrations within the cell . In all cases, any reduction in the ion concentrations was restored later in infection . When the intracellular potassium concentration was lowered from 325 to 150 mM with a different osmotic growth medium, the number of phage progeny was only slightly reduced (by a factor of two) . On additional reduction of the intracellular magnesium concentration from 100 to 50 mM by adding the antibiotic polymyxin B to the infected cells, T4 infections, but not T3 or T7, were markedly affected . These studies show that T3, T4, and T7 phage assembly can efficiently occur in vivo over a broad spectrum of ion concentrations. J Clin Microbiol, 1985 Sep, 22(3), 329 - 32 Resistance of leishmanial phosphatases to inactivation by oxygen metabolites; Saha AK et al.; Leishmania donovani promastigotes produce large quantities of two distinct acid phosphatases; a tartrate-resistant enzyme is localized to the external surface of the plasma membrane, and a tartrate-sensitive enzyme is secreted into the growth medium . It was shown previously that preincubation of human neutrophils and macrophages with the tartrate-resistant phosphatase markedly reduced the ability of these host cells to produce superoxide anions in response to stimulation with the activator formyl-methionyl-leucyl-phenylalanine . The possibility that the cell surface acid phosphatase or the phosphatase that is secreted into the extracellular fluid might compromise other host cell functions, especially intracellular ones, depends on the ability of the enzyme to resist exposure to toxic oxygen metabolites (e.g., superoxide anion, hydrogen peroxide, hypochlorite) generated by phagocytic cells . In the present report, we show that both leishmanial acid phosphatases were relatively resistant to inactivation by oxygen metabolites . At pH 5.5, the activity of the tartrate-resistant phosphatase was reduced 50% by incubation for 1 h with each of the following: 30 mM O2-, 500 mM hydrogen peroxide, and 6 mM hypochlorite ion . These concentrations are many fold greater than the concentrations of these substances that are generated by stimulated polymorphonuclear phagocytes . The tartrate-sensitive acid phosphatase differed markedly from the tartrate-resistant phosphatase in that the former was essentially insensitive to even very high concentrations of superoxide anion and hydrogen peroxide . Furthermore, 50% inactivation of the tartrate-sensitive leishmanial phosphatase required exposure to 35 mM hypochlorite for 30 min . These results indicate that the catalytic potential of these two leishmanial acid phosphatases probably survives exposure to toxic oxygen metabolites generated by neutrophils and macrophages. Mutat Res, 1985 Sep, 146(2), 169 - 76 A minor pathway of postreplication repair in Escherichia coli is independent of the recB, recC and recF genes; Sharma RC et al.; After ultraviolet (UV) irradiation, an Escherichia coli K12 uvrB5 recB21 recF143 strain (SR1203) was able to perform a limited amount of postreplication repair when incubated in minimal growth medium (MM), but not if incubated in a rich growth medium . Similarly, this strain showed a higher survival after UV irradiation if plated on MM versus rich growth medium (i.e., it showed minimal medium recovery (MMR} . In fact, its survival after UV irradiation on rich growth medium was similar to that of a uvrB5 recA56 strain, which does not show MMR or postreplication repair . The results obtained with a uvrB5 recF332::Tn3 delta recBC strain and a uvrB5 recF332::Tn3 recB21 recC22 strain were similar to those obtained for strain SR1203, suggesting that the recB21 and recF143 alleles are not leaky in strain SR1203 . The treatment of UV-irradiated uvrB5 recB21 recF143 and uvrB5 recF332::Tn3 delta recBC cells with rifampicin for 2 h had no effect on survival or the repair of DNA daughter-strand gaps . Therefore, a pathway of postreplication repair has been demonstrated that is constitutive in nature, is inhibited by postirradiation incubation in rich growth medium, and does not require the recB, recC and recF gene products, which control the major pathways of postreplication repair. J Cell Biol, 1985 Sep, 101(3), 1071 - 7 Isolation of an adhesion-mediating protein from chick neural retina adherons; Schubert D et al.; Adherons are high molecular weight glycoprotein complexes which are released into the growth medium of cultured cells . They mediate the adhesive interactions of many cell types, including those of embryonic chick neural retina . The cell surface receptor for chick neural retina adherons has been purified, and shown to be a heparan sulfate proteoglycan (Schubert, D., and M . LaCorbiere, 1985, J . Cell Biol., 100:56-63) . This paper describes the isolation and characterization of a protein in neural retina adherons which interacts specifically with the cell surface receptor . The 20,000-mol-wt protein, called retinal purpurin (RP), stimulates neural retina cell-substratum adhesion and prolongs the survival of neural retina cells in culture . The RP protein interacts with heparin and heparan sulfate, but not with other glycosaminoglycans . Monovalent antibodies against RP inhibit RP-cell adhesion as well as adheron-cell interactions . The RP protein is found in neural retina, but not in other tissues such as brain and muscle . These data suggest that RP plays a role in both the survival and adhesive interactions of neural retina cells. J Bacteriol, 1985 Sep, 163(3), 1265 - 6 Regulation of CDP-diacylglycerol synthase activity in Saccharomyces cerevisiae; Homann MJ et al.; The addition of ethanolamine or choline to inositol-containing growth medium resulted in a reduction of CTP:phosphatidate cytidylyltransferase (CDP-diacylglycerol synthase; EC 2.7.7.41) activity in Saccharomyces cerevisiae . The reduction of activity did not occur in the absence of inositol . CDP-diacylglycerol synthase activity was not regulated in a S . cerevisiae mutant strain (opi1; an inositol biosynthesis regulatory mutant) by the addition of phospholipid precursors to the growth medium. Carcinogenesis, 1985 Sep, 6(9), 1359 - 66 Inhibition of metabolic cooperation by phorbol esters in a cell culture system based on adenosine kinase deficient mutants of V79 cells; Gupta RS et al.; In Chinese hamster V79 cells, stable mutants which are greater than 1000-fold resistant to the adenosine analog, tubercidin (Tubr mutants), and which exhibit high degree of cross-resistance to various other adenosine analogs, viz . toyocamycin, formycin A, 6-methylmercaptopurine riboside and 8-azaadenosine, have been isolated . The inability of the mutant cells to phosphorylate {3H}tubercidin and lack of adenosine kinase activity (AK- phenotype) in their cell extracts provide evidence that the mutant cells are unable to convert adenosine analogs into their toxic phosphorylated derivatives . When AK- cells are co-cultured with increasing numbers of parental V79 (AK+) cells in medium containing tubercidin, then due to metabolic cooperation between AK+ and AK- cells, a cell density-dependent decline in recovery of the resistant cells is observed . However, diphtheria toxin resistant (Dipr) mutants of V79 cells, which are altered in elongation factor-2, showed no similar cell density effect . Addition of 0.01 microgram/ml of phorbol ester, 12-O-tetradecanoyl-phorbol-13-acetate (TPA) to growth medium in these experiments markedly enhanced the recovery of the Tubr mutants, but it had no effect on the recovery of Dipr mutants, which suggests that TPA was enhancing recovery of AK- mutants by inhibiting metabolic cooperation between AK- and AK+ cells . Maximum effect of TPA on the recovery of AK- mutants (3- to 5-fold enhancement) was observed at a density of 6 X 10(5) V79 cells/60 mm diameter dish and it was independent of the particular adenosine analog that was used as selective agent . Studies with a number of different phorbol derivatives show that only those phorbol esters which show tumor promoting activity in the mouse skin system inhibited metabolic cooperation between AK+/AK- cells in a dose-dependent manner . An excellent correlation was observed in these studies between the relative tumor promoting activity of various phorbol esters, their relative binding affinities to the cell surface receptors, and the concentrations at which they inhibited metabolic cooperation in the AK-/AK+ cell system . The AK-/AK+ cell system thus provides a new system for examining the effect of tumor promoters on metabolic cooperation between cells. J Natl Cancer Inst, 1985 Sep, 75(3), 527 - 44 Mouse monoclonal antibody to embryonic antigen: development, cross-reactivity with rodent and human tumors, and preliminary polypeptide characterization; Payne WJ Jr et al.; Hybridomas producing IgM and IgG monoclonal antibodies (MoAb) to embryonic or fetal antigens (EA) were obtained in a completely syngeneic system . Lethally irradiated, 13-day-gestation, C57BL/6N mouse fetal cells or KCI extracts of these fetal cells obtained from primaparous donors were used as immunogens in several regimens to induce splenocytes in C57BL/6N mice that were utilized to form the hybridomas following fusion with a mouse myeloma line . Successful growth and cloning of the IgM-producing hybridomas required supplementation with factor(s) produced in the growth medium of the macrophage cell line RAW 264.7 . An enzyme-linked immunosorbent assay (ELISA) was employed to screen the primary fusion hybridomas for antibody directed against fetal cell or adult cell determinants with the use of freshly explanted tissues . Glutaraldehyde-fixed fetal cells as well as crude fetal cell membranes were used as EA+ target cells (i.e., cell lines known to activate T-lymphocyte-mediated tumor resistance) in a solid-phase ELISA to perform quantitative ELISA adsorption tests of the MoAb . The anti-EA monoclonal IgM and the IgG detected common, embryo-specific antigen(s) on mouse, hamster, and human fetuses . Term fetal cells and adult normal tissues of the mouse, hamster, and human did not express cross-reactive determinants for the MoAb by absorption analysis and/or by direct binding in ELISA . EA expression as oncofetal antigens could also be detected with the monoclones on several rodent tumor cell lines tested as well as on a variety of human carcinomas but not on a spectrum of normal human tissues with the use of indirect ELISA absorption and affinity gel and sodium dodecyl sulfate-polyacrylamide gel electrophoresis analyses . Fluorescence analysis with the monoclones demonstrated specific reactivity with the surface of EA+ tumor cells in the FACS IV flow cytometer . The responsible antigen was carried on a 44- and a 200-kilodalton polypeptide. Brain Res, 1985 Aug 12, 340(2), 333 - 40 Differences in cation transport properties of primary astrocyte cultures from mouse and rat brain; Walz W et al.; 42K and 22Na contents and unidirectional fluxes, as well as net accumulation of 42K in response to elevated extracellular K+, were investigated in primary cultures of astrocytes prepared from neonatal rat and mouse brain . The major difference between both species affected the unidirectional K+ influx which was up to 75 times higher in mouse as compared to rat cultures . The flux rates in mouse astrocytes were doubled by measuring uptake in salt solution instead of growth medium, while 42K influx in rat astrocytes was unaffected by such treatment . 22Na transport was very similar in astrocytes from both species . The length of culture period and treatment with DBcAMP (2',3'-dibutyryl cyclic adenyl monophosphate) modified K+ transport but not Na+ transport . Both types of cultures showed the same accumulation of 42K in response to raised medium K+ . Amiloride inhibited 42K influx by 41% and 13% in mouse and rat cultures, respectively . In contrast, furosemide inhibited 42K uptake in rat astrocytes cultures by 50% but had no effect on mouse astrocyte cultures . 50 microM barium chloride markedly inhibited 42K uptake in mouse cultures by 96% (or 1491 nmol X mg-1 X min-1), but inhibited 42K uptake in rat cultures by only 23% (or 9 nmol X mg-1 X min-1) . Ouabain was similarly effective in both types of astrocyte cultures . We conclude that Na+ transport as well as net K+ accumulation and Cl- transport (based on previous studies) properties are reasonably stable and reproduced in primary cultures from both mouse and rat brain.(ABSTRACT TRUNCATED AT 250 WORDS) J Biol Chem, 1985 Aug 5, 260(16), 9123 - 31 Relationships between membrane lipid composition and biological properties of rat myocytes . Effects of aging and manipulation of lipid composition; Yechiel E et al.; Cultures of newborn rat heart myocytes undergo major age-related alterations as demonstrated by comparing 5-6-day-old cells ("young cells") and 14-15-day-old cells ("old cells") . This includes: changes from spherical to elongated shape; sphingomyelin and cholesterol level/cell increase by 100% and 50%, respectively, while the phosphatidylcholine is reduced by 15-20% with almost no change in content of total phospholipids . There is a 50% increase in total protein content/cell while DNA content remain constant . The specific activity of seven marker enzymes representing most subcellular organelles is increased . Beating rate is reduced from 160 +/- 20 to 20 +/- 20 beats min-1 . All the above age-dependent alterations are affected by modification of cellular polar lipid composition . Small unilamellar vesicles of egg phosphatidylcholine added to the growth medium of old cells serve as donor of egg phosphatidylcholine to the cells and as acceptor of cellular sphingomyelin and cholesterol . Sphingomyelin-phospholipid exchange can be separated from cholesterol depletion either by using vesicles of egg phosphatidylcholine/cholesterol mixtures which serve only in the phospholipid exchange process, or by small unilamellar vesicles of sphingomyelin which act only as efficient cholesterol acceptors . Such experiments indicated that the major response of old cells is to alteration in the phosphatidylcholine to sphingomyelin mole ratio, while changes in the cholesterol level induce smaller effects . Thus, reversal of phosphatidylcholine to sphingomyelin mole ratio to the values shown by young cells reverse cellular functions and features which were altered by cell aging to levels found in young cells . This includes: increase in the beating rate back to 160 +/- 20, reduction in the total protein level and in the specific activity per DNA content of seven marker enzymes and reappearance of spherical cell shape . These results suggest that membrane lipid composition has major influence on cellular properties which as described in the accompanying paper (Yechiel, E., Barenholz, Y., and Henis, Y . I . (1985) J . Biol . Chem . 260, 9132-9136), may be mediated through the organization and dynamics of the cell membranes. J Cell Physiol, 1985 Aug, 124(2), 199 - 206 Production of insulin-like growth factor-II (MSA) by endoderm-like cells derived from embryonal carcinoma cells: possible mediator of embryonic cell growth; Nagarajan L et al.; The present study was carried out to determine if an insulin-like growth factor (IGF) type activity might be produced by embryonal carcinoma-derived cells . The cell line used to condition growth medium for the isolation of secreted growth factors was a newly established Dif 5 cell type . Dif 5 cells are a differentiated endoderm-like cell type derived from F9 embryonal carcinoma cells (which possess properties similar to mouse embryonic stem cells) following extensive exposure to retinoic acid . When growth medium conditioned by Dif 5 cells is chromatographed on Sephadex G-75 in 1 M acetic acid two peaks of activity are observed which compete for specific {125I}iodo multiplication stimulating activity (MSA) binding to PYS cells . MSA is the rat homologue of human IGF-II . The high molecular weight fraction (Mr approximately 60K) apparently corresponds to IGF-binding protein as determined by its ability to bind {125I}iodo-MSA . The low molecular weight fraction (Mr approximately 8K) is biologically active as this fraction stimulates {3H}thymidine incorporation into serum-starved chick embryo fibroblasts . Radioimmunoassay data indicate that the IGF-like activity produced by Dif 5 cells is more closely related to IGF-II than to IGF-I . Undifferentiated embryonal carcinoma stem cell lines (F9, Nulli, and PCC4) produced little of this MSA-like activity, while PYS-2 (parietal endoderm-like) cells produced about 16 ng MSA/10(6) cells/24 hr as determined by radioimmunoassay . Dif 5 and PSA-5E (visceral endoderm-like) cells, are found to secrete significant amounts of MSA into the growth medium (30-50 ng MSA/10(6) cells/24 hr) . These findings offer further support to a proposal that MSA (IGF-II) produced by endoderm cells, particularly visceral endoderm, may serve as an early embryonic growth factor. Am J Reprod Immunol Microbiol, 1985 Aug, 8(4), 130 - 1 In vitro fertilization results for women with sperm antibodies in plasma and follicular fluid; Clarke GN et al.; In vitro fertilization (IVF) and embryo transfer results were analyzed for three women with sperm antibodies in their plasma and follicular fluid . These preliminary results suggest that substitution of the patients' serum by replacement serum in the fertilization and embryo growth media may prove to be an effective means of improving IVF treatment for women with sperm immunity. Br J Dermatol, 1985 Aug, 113(2), 237 - 43 Effect of platelet homogenate on in vitro glycosaminoglycans production by dermal fibroblasts from systemic sclerosis patients and normal controls; Falanga V et al.; Confluent cultures of dermal fibroblasts from the involved skin of systemic sclerosis patients (SF) and a matched skin site of normal controls (NF) were investigated for the synthesis of glycosaminoglycans (GAG) in response to various concentrations of human platelet homogenate (PH) . Experiments were carried out in the presence of 1% and 15% human serum (HS) . In the absence of PH, GAG synthesis was higher in SF than NF . An increase in GAG synthesis was demonstrated in both SF and NF as the concentration of PH was increased to 200 micrograms/ml of growth medium . The PH-stimulated GAG synthesis occurred in 15% HS treated SF and NF, but there was no GAG synthesis increase in 1% HS-treated NF . The absolute count of GAG synthesis was always greater in SF than NF . The addition of PH in concentrations higher than 200 micrograms/ml led to cell death of both SF and NF . These findings are the first to indicate a difference between SF and NF response to PH. J Cell Sci, 1985 Aug, 77, 47 - 56 Changes in the transport of mitochondrial Ca2+ during the culture growth cycle of Tetrahymena pyriformis; Kim YV et al.; The properties of the Ca2+ transport system of mitochondria, isolated in various phases of growth of static cultures of Tetrahymena pyriformis, were studied . A large increase in the endogenous energy-dependent Ca2+ content of mitochondria was observed as cultures of T . pyriformis passed through the exponential and stationary phases of growth (approx . 0.25 and 50 nmol Ca2+ per mg mitochondrial protein, respectively) . Simultaneously, the mitochondria dramatically lost their ability to withstand large concentrations of Ca2+ and ADP . However, in the latter case they were able to phosphorylate a large amount of ADP if the strong Ca2+ chelator, ethylene glycol bis-(beta-aminoethyl ether)-N,N,N',N'-tetraacetic acid, was initially present in the incubation medium . Furthermore, all the changes observed in mitochondria from the stationary phase cells were completely reversed when cell proliferation was re-activated after the lag phase, either by reseeding the stationery cells in fresh growth medium or by oxygenation of the old medium . In aerobic conditions even a small addition of Ca2+ was able to induce rapid release of Ca2+ from mitochondria isolated during the stationary phase of growth . It is suggested that the redistribution of Ca2+ between the mitochondria and the cytoplasm at the onset of the lag phase may serve as the main trigger for the subsequent biochemical and morphological changes observed in T . pyriformis. Can J Microbiol, 1985 Aug, 31(8), 707 - 10 Evidence that the mechanisms of fungitoxicity of 8-quinolinol and its bischelate with copper(II) are different; Gershon H et al.; The copper(II) contents of the growth media, Sabouraud dextrose and Czapek-Dox broths, and of the spore inocula of Aspergillus niger (ATCC 1004), Aspergillus oryzae (ATCC 1011), Trichoderma viride (ATCC 8678), and Myrothecium verrucaria (ATCC 9095) were determined by atomic absorption spectrophotometry in a graphite furnace . The test systems composed of Sabouraud dextrose broth and spore inocula of the four fungi contained only a little over 3% of the copper(II) required to form a minimal inhibitory concentration of bis(8-quinolinolato)copper(II) . The test system of Czapek-Dox broth and A . oryzae contained slightly less than 65% of the copper(II) required to form a minimal inhibitory concentration of the bischelate of 8-quinolinol with copper(II) . When the minimal inhibitory concentrations of 8-quinolinol and bis(8-quinolinolato)copper(II) were added simultaneously to the test system of Czapek-Dox broth and A . oryzae, 10% of the combined mixture of toxicants caused complete inhibition of growth indicating synergism between the toxicants . These results together with the observation that alpha-lipoic acid as well as small aliphatic thiol-containing compounds (cysteine, glutathione, dithioerythritol, and dithiothreitol) reversed the toxicity of 8-quinolinol but not the toxicity of bis(8-quinolinolato)copper(II) led to the conclusion that the mechanisms of fungitoxicity of both toxicants are different. Genetika, 1985 Aug, 21(8), 1261 - 5 {Isolation and mapping of a mutation leading to damage in the cytoplasmic-specific component of the fructose transport system in Escherichia coli K-12 bacteria}; Dobrynina OIu et al.; A mutant delta ptsH of Escherichia coli was used to obtain a mutation which damages a function of cytoplasmic specific component of the fructose phosphotransferase system--FPr protein . This mutation was mapped using a number of genetical methods . In conjugational crosses the mutation was localized at 47 min of the E . coli chromosomal map . The gene responsible for this defect was designated fpr . In P1 mediated transduction and in conjugational three-factorial crosses the order of the markers in this region was established as: gyrA-ompC-fpr-ptsF-...-his . The frequency of cotransduction of the fpr gene was 4.5 and 35% with gyrA and ompC markers, respectively . In fructose containing minimal medium the doubling time of the ptsH+fpr mutant was lower than that of the delta ptsHfpr+ mutant . Also, the doubling time of the fpr mutant depends on concentration of fructose in growth medium but is independent of the amount of this sugar in the case of the delta ptsH mutant. Am Rev Respir Dis, 1985 Aug, 132(2), 305 - 10 In vivo and in vitro studies on the use of the guinea pig as a model for virus-provoked airway hyperreactivity; Buckner CK et al.; The parainfluenza 3 (P-3)-infected guinea pig was examined as a model for virus-provoked airway hyperreactivity by measuring changes in airway overflow pressure in response to intravenously (iv) administered histamine . In vitro responses of lung parenchymal strips to several contractile substances were also measured . All studies were conducted 4 days after nasal insufflation with P-3 or P-3 growth medium (control) . Increases in airway overflow pressure caused by histamine were enhanced by P-3 infection, and the dose required to produce a standard level of response was decreased (i.e., there was an increase in sensitivity to histamine) . Enhancement of in vivo histamine responses caused by P-3 was prevented both by cutting the vagus nerves in the midcervical region and by iv administered hexamethonium, 5 mg/kg . The enhancement was not blocked by 1 mg/kg of atropine given iv, but was blocked by a larger dose 5 mg/kg . The larger dose of atropine significantly antagonized responses to histamine in the P-3-infected state . Increases in airway overflow pressure produced by electrical stimulation of the left vagus and nicotine (1 and 10 mg/kg given iv), both studied after bilateral vagotomy and propranolol, 1 mg/kg given iv, were also enhanced by P-3 infection . Atropine, 1 mg/kg given iv blocked the P-3-induced enhancement of responses to vagus stimulation . Propranolol, 1 mg/kg, and phentolamine, 3 mg/kg, given together iv, produced a doubling of the airway overflow pressure only in P-3-infected animals . Propranolol alone or other receptor antagonists did not produce as marked a change in either group of animals.(ABSTRACT TRUNCATED AT 250 WORDS) Biochem Pharmacol, 1985 Aug 1, 34(15), 2721 - 31 Comparison of aryl hydrocarbon hydroxylase and acetanilide 4-hydroxylase induction by polycyclic aromatic compounds in human and mouse cell lines; Jaiswal AK et al.; The human MCF-7 and the mouse Hepa-1 cell culture lines were compared for aryl hydrocarbon hydroxylase and acetanilide 4-hydroxylase inducibility by 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) and benzo{a}anthracene (BA) and TCDD- and BA-specific binding in the cytosol and nucleus . The effective concentration of BA in the growth medium required to induce either enzyme to 50% of its maximally inducible activity (EC50) was the same (5-11 microM) in both MCF-7 and Hepa-1 cells . On the other hand, the EC50 for TCDD in MCF-7 cells (5-25 nM) was more than 40-fold greater than that in Hepa-1 cells (0.4 to 0.6 nM) . P1-450- and P3-450-specific mouse cDNA probes were used to quantitate mRNA induction in the Hepa-1 cell line . P1-450 mRNA was induced markedly by TCDD and benzo{a} anthracene, whereas P3-450 mRNA was induced negligibly . A P1-450-specific human cDNA probe was used to quantitate P1-450 mRNA induction in the MCF-7 cell line . Aryl hydrocarbon hydroxylase inducibility by TCDD or BA always paralleled P1-450 mRNA inducibility in either the mouse or human line . Although the cytosolic Ah receptor in Hepa-1 cells was easily detected by sucrose density gradient centrifugation, gel permeation chromatography, and anion-exchange high-performance liquid chromatography, the cytosolic receptor cannot be detected in MCF-7 cells . Following in vivo exposure of cultures to radiolabeled TCDD, the intranuclear concentration of inducer-receptor complex was at least fifty times greater in Hepa-1 than MCF-7 cultures . The complete lack of measurable cytosolic receptor and almost totally absent inducer-receptor complex in the nucleus of MCF-7 cells was, therefore, out of proportion to its capacity for aryl hydrocarbon hydroxylase and acetanilide 4-hydroxylase inducibility . This MCF-7 line should provide an interesting model for a better understanding of the mechanisms of drug-metabolizing enzyme induction by polycyclic aromatic compounds, including the Ah receptor-mediated mechanism. J Gen Microbiol, 1985 Aug, 131 ( Pt 8), 1903 - 10 Glutamine synthetase of Streptomyces cattleya: purification and regulation of synthesis; Paress PS et al.; Glutamine synthetase (GS; EC 6.3.1.2) from Streptomyces cattleya was purified using a single affinity-gel chromatography step, and some of its properties were determined . Levels of GS in S . cattleya cells varied by a factor of 8 depending upon the source of nitrogen in the growth medium . Of 24 nitrogen sources examined only glutamine or NH4Cl utilization resulted in very low GS activity . Addition of NH4Cl to a culture with high GS levels appeared to stop further synthesis and resulted in a progressive decrease in the specific activity of the enzyme . The GS inhibitor methionine sulphoximine (MSX) inhibited GS activity but had no effect on exponentially growing cells . The presence of MSX either lengthened or shortened the period between spore inoculation and initiation of exponential growth, depending on the source of nitrogen . In glutamine minimal medium MSX produced earlier and more efficient spore germination while in glutamate or nitrate minimal medium germination was delayed by its presence. Biochim Biophys Acta, 1985 Jul 30, 846(1), 101 - 8 Erythroid differentiation of cultured murine erythroleukemia cells by the spermine analogue canavalmine; Fujihara S et al.; Canavalmine, an analogue of spermine, induced erythroid differentiation of murine erythroleukemia cells 745A, as evidenced by benzidine staining and heme content of cultured cells . Benzidine-positive cells synthesizing hemoglobin appeared on day 4 after addition of 250 microM canavalmine . The canavalmine-induced cell differentiation was inhibited by the addition of agents which alter the structure of the cell membrane, such as local anesthetics (procainamide and lidocaine) or Ca2+ antagonists (nifedipine and verapamil) at dosages not toxic for the cell growth . Canavalmine did not significantly affect the levels of conjugated polyamines in the acid-insoluble fraction of the cells . In contrast, the level of free spermidine in the acid-soluble fraction greatly decreased during the 18 h after canavalmine treatment . Putrescine and spermidine, when added externally to the growth medium, showed dose-dependent inhibition of canavalmine-induced cell differentiation . Neither cadaverine nor spermine had any significant effect . These results suggest that not only structural change of cell membrane but alteration of the polyamine metabolism, especially a regulation of the cellular level of free spermidine, might have a key importance in erythroid differentiation of murine erythroleukemia cells induced by canavalmine. Biochim Biophys Acta, 1985 Jul 9, 835(2), 322 - 30 Utilization of exogenous glycerophosphodiesters and glycerol 3-phosphate by inositol-starved yeast, Saccharomyces uvarum; Paltauf F et al.; Inositol-starved Saccharomyces uvarum cells hydrolyse exogenous glycerophosphodiesters to glycerol 3-phosphate and the corresponding alcohol . Glycerophosphodiesterase activity is highest with glycerophosphoinositol as the substrate, followed by glycerophosphoethanolamine and glycerophosphocholine; the artificial substrate for phosphodiesterases, bis-p-nitrophenylphosphate,is hydrolysed at a similar rate as compared with glycerophosphoinositol . Competition experiments suggest that distinct phosphodiesterases are involved in the hydrolysis of the respective substrates . An Mg2+-dependent glycerophosphate phosphohydrolase with a pH-optimum around neutral cleaves glycerol 3-phosphate to glycerol and orthophosphate . The latter is taken up into cells without first entering the pool of orthophosphate present in the growth medium . Accessibility to substrates with whole cells, adhesion of enzymes to spheroplasts, and solubilization of enzymes by treatment of whole cells with Triton X-100 under mild conditions suggest that phosphodiesterases and glycerol-3-phosphate phosphohydrolase are loosely associated with the outer side of the yeast plasma membrane . Enzyme activities are only marginal in inositol-supplemented cells, but are derepressed not only by inositol deficiency, but also by starvation of orthophosphate. Genetika, 1985 Jul, 21(7), 1099 - 104 {Tryptophan operon of methylotrophic facultative Pseudomonas sp . M bacteria . I . Isolation and characterization of auxotrophic Trp-mutants}; Olekhnovich IN et al.; Eighteen auxotropic trp- mutants of the facultative methylotrophic bacteria Pseudomonas sp . M . induced by nitrosoguanidine were characterized . Trp- mutants were tested for a number of biochemical properties: the capacity to grow on tryptophan intermediates, their accumulation in growth medium and activities of key enzymes . The trpE, trpD, trpC, trpF, trpB and trpA mutants were identified . The trpDC121 mutant with a one-point mutation has been obtained . This mutation caused inactivation of two enzymes--anthranilate-5-phosphoribosyl transferase and indole-3-glycerophosphate synthase . Unusual trpA and trpB auxotrophs with TrpAB- phenotype were described . It may be concluded that this type of mutations cause loss of catalytic activity of a subunit of tryptophan synthase as well as its structural modification . As a result, no active tryptophan synthase complex is formed and hence, the activity of the opposite intact subunit is inhibited. Lab Invest, 1985 Jul, 53(1), 30 - 6 Human tumor cells in culture stimulate glycosaminoglycan synthesis by human skin fibroblasts; Merrilees MJ et al.; The human tumor cell lines, MM-96, FME, HCT-8, HT-29, MCF-7 and T-47D, in culture produced a factor or factors able to stimulate glycosaminoglycan (GAG) synthesis in human skin fibroblasts (HF) . Conditioned growth media from the melanoma MM-96 and the colon carcinoma HT-29 produced a 10- and 8-fold stimulation of HF GAG synthesis, respectively, with an even larger stimulation of hyaluronic acid . Conditioned media from the melanoma FME and the breast carcinomas MCF-7 and T-47D stimulated GAG synthesis 2-fold, whereas media from the colon carcinoma HCT-8 gave a variable response often with no effect on GAG levels . Conditioned media from HF cultures had no effect on tumor cell GAG synthesis . Coculture of tumor cells and HF also resulted in increased GAG synthesis, and the degree of stimulation was similar to that with the conditioned media . Tumor cell-conditioned media were also effective in stimulating GAG synthesis by porcine smooth muscle cells and by chick embryo fibroblasts in culture, although the increase in GAG synthesis was much less than with HF cultures . These findings support the concept that the stromal desmoplasia characteristic of many growing and invasive tumors in vivo arises by tumor cell modulation of GAG synthesis by surrounding normal connective tissue cells. Mutat Res, 1985 Jul, 146(1), 63 - 70 Repair of DNA single-strand breaks in X-irradiated yeast . II . Kinetics of repair as measured by the DNA-unwinding method; Ogorek B et al.; The kinetics of disappearance of single-strand breaks (SSB) from the DNA of X-irradiated stationary yeast cells under liquid-holding conditions was found to proceed in a dose-independent manner up to a dose of at least 2400 Gy, and was found to be complete after incubation of cells for 1 h . This was deduced from data for a yeast wild-type (WT) haploid and diploid strain as well as for rad52 haploid cells defective in DNA double-strand break (DSB) repair . In all cases an initial fast repair component assumed to correspond to SSB repair was observed whereby about 80% of the induced 'unwinding points' disappeared from the DNA with a time constant of about 3 min . Following this fast component, a slower component of removal of 'unwinding points' occurred with a time constant estimated to be 20 min . The molecular nature of these two components of repair is not known . We could find no evidence for the induction of secondary (enzymatic) breaks in the DNA during post-irradiation incubation . Incubation of cells in growth medium after irradiation resulted in similar kinetics as those under liquid-holding conditions . In the absence of an energy source in the medium (i.e . when cells were incubated in buffer or distilled water after irradiation) only 60-80% of the SSB were removed from yeast DNA . Residual SSB disappeared from the DNA only when cells were transferred to a medium containing glucose . The relative mass of DNA unwound per induced strand break (i.e . represented by the slope of the dose-effect curve immediately after irradiation) was found to change slowly with the age of the cell culture under liquid-holding conditions . This effect had to be corrected for in the measurements of strand break repair under these conditions. Antimicrob Agents Chemother, 1985 Jul, 28(1), 15 - 20 Susceptibility of Legionella pneumophila to ofloxacin in vitro and in experimental Legionella pneumonia in guinea pigs; Saito A et al.; The antimicrobial activity of ofloxacin was tested against 15 standard strains and 37 clinical and environmental strains of Legionella pneumophila by agar dilution susceptibility studies with a new growth medium . The ofloxacin MICs were inoculum dependent and ranged from 0.03 to 0.125 microgram/ml . The antibacterial activities of other agents tested relative to ofloxacin were rifampin greater than ofloxacin greater than josamycin greater than pipemidic acid . Ofloxacin, at concentrations equal to or greater than 0.05 microgram/ml, inhibited the growth of L . pneumophila grown in human monocytes . The therapeutic efficacy of ofloxacin in experimental guinea pig L . pneumophila pneumonia was greater than that observed with erythromycin or josamycin therapy; it was less effective than was rifampin . Ofloxacin was very active against intracellular L . pneumophila in these experiments and should be studied in the therapy of human Legionnaires disease. Int J Hyperthermia, 1985 Jul-Sep, 1(3), 255 - 63 Effect of external K+ on protein and DNA synthesis during and after heat shock in rat hepatoma cells; Boonstra J et al.; The effects of extracellular K+ concentrations on protein and DNA synthesis after non-lethal heat shock were studied in the hepatoma cell lines Reuber H35 and HTC . Elevation of the extracellular K+ concentration by equimolar replacement of Na+ by K+ in growth media of Reuber H35 and HTC cells caused an increase of the intracellular K+ content in both cell lines . This property was subsequently used to study the effect of elevated intracellular K+ concentrations on protein and DNA synthesis after hyperthermic treatment at 42 degrees C for 30 min . In normal K+ medium, protein and DNA synthesis were inhibited rapidly after the start of the hyperthermic treatment in both Reuber H35 and HTC cells . Increasing the external K+ concentration of the medium did not influence the inhibition and subsequent recovery of protein synthesis after heat shock in both cell lines . In contrast, in media with elevated K+ concentrations, DNA synthesis after heat-shock was inhibited less in Reuber H35 cells than in cells incubated in normal K+ medium and, furthermore, showed no inhibition in HTC cells . The protective effect of external K+ on DNA synthesis after heat shock was maximal between 50 and 70 mM in the temperature range 42-44 degrees C. In Vitro Cell Dev Biol, 1985 Jul, 21(7), 391 - 401 Pure gelatin microcarriers: synthesis and use in cell attachment and growth of fibroblast and endothelial cells; Wissemann KW et al.; A new type of microcarrier was described using bead emulsion-polymerization techniques . An aqueous solution of gelatin and glutaraldehyde was dispersed in a hydrophobic phase of mineral oil, using Triton X-114 as an emulsifier, and polymerization was initiated . The resultant spherical beads, composed entirely of gelatin, showed excellent mechanical stability to ethanol drying, sterilization, and long-term use in microcarrier spinner cultures . The solid gelatin microcarriers supported the growth of L-929 fibroblast, swine aorta endothelial, human umbilical endothelial, and HeLa-S3 cultures with no adverse effects on cell morphology or growth . The beads were transparent in growth medium and attached cells were clearly visualized without staining . The beads were also compatible with techniques for scanning electron microscopy . Collagenase could be used to entirely digest the gelatin beads, leaving the cells free from microcarriers and suspended in solution while retaining 98% cell viability . The results further showed that after collagenase treatment the cells would populate fresh gelatin microcarriers and grow to confluence . Cell attachment kinetics revealed that the endothelial cells attached to the gelatin beads at the same rate as to tissue culture plates, whereas the fibroblast cells attached to the beads more slowly . However, once the fibroblast cells were attached to the gelatin microcarriers they spread and grew normally. J Bacteriol, 1985 Jul, 163(1), 82 - 7 Uncoupling of osmoregulation of the Escherichia coli K-12 ompF gene from ompB-dependent transcription; Ramakrishnan G et al.; The expression of the genes for the Escherichia coli K-12 outer membrane proteins, ompF and ompC, is subject to osmoregulation and responds to changes in the osmolarity of the growth medium . The transcription of these genes is dependent on the products of the regulatory locus ompB (comprising the genes ompR and envZ) . The native promoter of ompF was replaced with an inducible lpp promoter to eliminate this transcriptional dependence of ompF expression on ompB . As a result, it was possible for the OmpF protein to be produced in an ompB mutant strain that does not normally express ompF . Surprisingly, the expression of ompF under the lpp promoter was still osmoregulated not only in the ompB+ strain but also in two ompB strains tested . These results indicate the involvement of a factor(s) besides the ompR and envZ gene products in the osmoregulation of OmpF production . This factor may interact with a sequence downstream of the ompF promoter . In addition, we show that the expression of ompF under the lpp promoter has no direct effect on ompC expression. J Biol Chem, 1985 Jun 25, 260(12), 7716 - 20 Drosophila ribosomal RNA stability increases during slow growth conditions; Winkles JA et al.; We have developed density labeling pulse-chase methods which, in contrast to a conventional radiolabeling approach, allow us to determine the effectiveness of our chase and to measure RNA stability in vivo without measuring precursor pool specific activities . We have used these methods to determine the stability of the embryonic ribosomal RNA inherited by either normally or slowly growing Drosophila melanogaster larvae . If larvae are raised in a rich growth medium, embryonic rRNA decays with a half-life of 48 h . However, if larvae are raised in a poor growth medium, which slows larval growth and prolongs development, the half-life of rRNA increases to 115 h . This is the only example, of which we are aware, directly showing that rRNA half-life increases during slow growth conditions . We propose that the increased stability of rRNA that we find may enable slowly growing larvae to maintain the ribosome levels necessary to continue growth and development under conditions of nutrient deprivation. Eur J Biochem, 1985 Jun 3, 149(2), 437 - 44 Amino acid biosynthesis and sodium-dependent transport in Methanococcus voltae, as revealed by 13C NMR; Ekiel I et al.; Of several methanogenic bacteria examined only Methanococcus voltae readily incorporated exogenous amino acids into cell protein . This was easily shown, since growth in the presence of exogenous amino acids resulted in a loss of signal intensities from those carbon atoms normally labelled by {13C}acetate during biosynthesis . From 80% to 95% of the Ser, Lys, Pro or Val incorporated into protein could be supplied directly from the growth medium . In contrast, Asp and Glu, if supplied to the medium, accounted for only a small percentage of the total acidic amino acid used in protein synthesis . Constitutive transport systems took up a wide range of amino acids at rates of 0.1-4.1 nmol min-1 mg-1 . The transport systems required Na+, with the possible exception of the basic amino acid lysine, and were inhibited by N-ethylmaleimide or 3,3',4',5-tetrachlorosalicylanilide . No interconversion of Ile to other amino acids was detected when cells were given {13C}Ile during growth, whereas the expected labelling of the Asp and Glu families of amino acids resulted when {13C}Asp was provided to the culture . Mc . voltae synthesized its amino acids from acetate via routes fully consistent with those found in Methanospirillum hungatei {Ekiel, I., Smith, I.C.P . & Sprott, G.D . (1983) J . Bacteriol . 156, 316-326} . Propionate could substitute for an auxotrophic requirement for Ile, resulting in the synthesis of Ile with the beta-carbon originating from the carboxyl of acetate and the alpha-carbon from the carboxyl of propionate . No labelling of Ile from {13C}acetate could occur without the fatty acid . These results provide strong evidence for the carboxylation of propionate to form 2-oxobutyrate as intermediate in Ile biosynthesis, and show that the metabolic defect in Ile biosynthesis occurs prior to 2-oxobutyrate synthesis . The presence of constitutive amino acid transport systems and multiple routes for ile biosynthesis make Methanococcus voltae an attractive methanogen for genetic studies. Cancer Res, 1985 Jun, 45(6), 2632 - 41 Relationship between the organization and synthesis of vimentin and the metastatic capability of B16 melanoma cells; Ben-Ze'ev A et al.; The organization and synthesis of the vimentin-containing cytoskeletal network as well as the metastatic capability of B16-F1 melanoma cells were investigated in cells treated with cycloheximide (CH) . The addition of CH to cells for 4-8 h resulted in a marked reversible alteration in the organization of the vimentin-containing network in B16-F1 melanoma cells as well as in a variety of epithelial and fibroblast cell lines . Treatment of cells with CH led to a reduction in the synthesis of vimentin, tubulin, and actin followed by a decrease in the concentrations of mRNAs coding for these proteins . However, out of these three cytoskeletal elements, only the organization of the intermediate filaments was disrupted by CH . Cells treated previously with CH and injected i.v . into syngeneic mice had diminished capacity to form lung metastases as compared to control untreated cells . This effect was reversible, and the metastatic capability recovered to the control level after the drug was removed from the growth medium for 16 h . The possibility that the organization and the synthesis of the cytoskeletal components are related and that the metastatic capability of B16 melanoma is influenced by the organization of the cytoskeletal networks are discussed. J Invest Dermatol, 1985 Jun, 84(6), 513 - 5 The in vitro response of fibroblasts to the fluid that accumulates under a vapor-permeable membrane; Alper JC et al.; We decided to examine whether the mechanism for production of granulation tissue during moist wound healing under a vapor-permeable membrane (VPM) is related to a fibroblast growth-promoting substance in the wound fluid beneath the VPM . The experimental design utilized growth curves performed on synchronized fibroblast cultures derived from 2 normal infants . Cell counts were performed at days 1, 4, 7, and 11 (saturation density) . VPM fluid (MWF, moist wound-healing fluid) from 7 different patients was used to supplement growth medium (GM) in the test growth curves . Both 2% MWF alone and 2% MWF plus 2% human serum (2 + 2) were evaluated for each patient . Control curves were conducted using GM supplemented with 2%, 4%, and 10% human serum (HS) . When 2% MWF alone was added to culture medium, all cells lifted off the surface of the flask within 4-7 days . If (2 + 2) was used to supplement the medium, detachment did not occur . At days 4, 7, and 11, (2 + 2) flasks had significantly greater cell densities than did flasks supplemented with either 2% or 4% HS alone (p less than 0.001) . At days 4 and 7, (2 + 2) cell counts were the same as 10% HS cell counts (p = 0.99) . By day 11, (2 + 2) cell counts exceeded those of 10% HS (p less than 0.01) . We conclude that the fluid that collects under the specific VPM used in this study when added to HS causes synergistic stimulation of fibroblast cell division and an altered pattern of fibroblast growth. Dev Biol, 1985 Jun, 109(2), 288 - 98 Comparative analysis of casein synthesis during mammary cell differentiation in collagen and mammary gland development in vivo; Durban EM et al.; Substrata upon which epithelial cells are cultured modulate their morphology,growth, and ability to differentiate . Mouse mammary epithelial cells cannot be induced to synthesize caseins, a marker of cell differentiation, when grown on a plastic surface . An analysis was made of the effect of time within a collagen matrix on the ability of normal mammary epithelial cells to be induced to synthesize caseins and that response was compared to mammary gland development in vivo . Primary cultures of mammary cells from unprimed virgin BALB/c mice were embedded in rat-tail collagen gel mixtures and maintained in growth medium . Induction medium containing lactogenic hormones was added at various times . The cells were monitored every 3-7 days over a period of 8 weeks for cell growth, casein synthesis, and ability to grow in vivo in cleared mammary fat pads . Casein accumulation was assayed quantitatively by an ELISA competition assay and qualitatively by the immunoblot procedure using specific antisera prepared against purified mouse caseins . No marked differences in cell numbers and transplantability potential were observed among cells cultured for various times in collagen . Mammary cells grown in collagen for up to 8 weeks retained the capacity to grow in vivo as normal ductal outgrowths . The duration of culture within collagen prior to hormonal stimulation did influence the kinetics of casein synthesis . Cells cultured for 1 week in growth medium did not accumulate detectable levels of casein until after 3 weeks of induction, whereas cells cultured for 2 or 4 weeks responded by accumulating caseins after 2 weeks and 3 days of induction, respectively . While the levels of total caseins that accumulated under optimal conditions of induction in culture approached levels found during lactation in vivo, the relative proportion of specific casein polypeptides synthesized in culture was altered from alpha casein (43K) in favor of the beta casein (30K) species . These results suggest that a period of culture within collagen is required to permit mammary epithelial cells to become responsive for hormone-induced differentiation . It is possible that during growth within the collagen the cells synthesize and deposit extracellular matrix components important in modulating gene expression. J Neurochem, 1985 Jun, 44(6), 1816 - 21 Concentration of free amino acids in primary cultures of neurones and astrocytes; Patel AJ et al.; The cellular distribution of free amino acids was estimated in primary cultures (14 days in vitro) composed principally of cerebellar interneurones or cerebellar and forebrain astrocytes . In cultured neural cells, the overall concentration of amino acids resembled that found in brain at the corresponding age in vivo . In the two neural cell types, there were marked differences in the distribution of amino acids, in particular, those associated with the metabolic compartmentation of glutamate . In neuronal cell cultures, the concentrations of glutamate, aspartate, and gamma-aminobutyric acid were, respectively, about three, four, and seven times greater than in astrocytes . By contrast, the amount of glutamine was approximately 65% greater in astroglial cell cultures than in interneurone cultures . An unexpected finding was a very high concentration of glycine in astrocytes derived from 8-day-old cerebellum, but the concentrations of both serine and glycine were greater in nerve cell cultures than in forebrain astrocytes . The essential amino acids threonine, valine, isoleucine, leucine, tyrosine, phenylalanine, histidine, lysine, and arginine were all present in the growth medium, and small cellular changes in the contents of some of these amino acids may relate to differences in their influx and efflux during culturing and washing procedures . The present results, together with our previous findings, provide further support for the model assigning the "small" compartment of glutamate to glial cells and the "large" compartment to neurones, and also underline the metabolic interaction between these two cell types in the brain. Appl Environ Microbiol, 1985 Jun, 49(6), 1392 - 5 Biosynthetic relationship among aflatoxins B1, B2, M1, and M2; Dutton MF et al.; Aflatoxins are a family of toxic, acetate-derived decaketides that arise biosynthetically through polyhydroxyanthraquinone intermediates . Most studies have assumed that aflatoxin B1 is the biosynthetic precursor of the other aflatoxins . We used a strain of Aspergillus flavus which accumulates aflatoxin B2 to investigate the later stages of aflatoxin biosynthesis . This strain produced aflatoxins B2 and M2 but no detectable aflatoxin B1 when grown over 12 days in a low-salt, defined growth medium containing asparagine . Addition of dichlorvos to this growth medium inhibited aflatoxin production with concomitant accumulation of versiconal hemiacetal acetate . When mycelial pellets were grown for 24, 48, and 72 h in growth medium and then transferred to a replacement medium, only aflatoxin B2 and M2 were recovered after 96 h of incubation . Addition of sterigmatocystin to the replacement medium led to the recovery of higher levels of aflatoxins B2 and M2 than were detected in control cultures, as well as to the formation of aflatoxins B1 and M1 and O-methylsterigmatocystin . These results support the hypothesis that aflatoxins B1 and B2 can arise independently via a branched pathway. In Vitro Cell Dev Biol, 1985 Jun, 21(6), 340 - 6 Reversible change in the fibroblast lysosomal enzyme dipeptidyl aminopeptidase-1 (cathepsin C) related to the commercial source of fetal bovine serum in the culture medium; Doughty MJ et al.; The commercial source of fetal bovine serum used to supplement the growth medium of human skin fibroblasts alters the activity of the lysosomal enzyme dipeptidyl aminopeptidase-1 (DAP-1) . Cells grown with one serum were found to have a threefold higher level of DAP-1 than those grown with serum from another source (P less than 0.001) . The effect on DAP-1 activity was specific inasmuch as no differences were found in the activities of a variety of other lysosomal and nonlysosomal hydrolases: DAP-II, DAP-III, DAP-IV, beta-glucosidase, beta-glucuronidase, and N-acetyl-beta-galactosaminidase . The effect is reversible and is observed over a wide range of cell population doublings . Cell growth kinetics were not significantly different with the different sera. J Bacteriol, 1985 Jun, 162(3), 1151 - 5 Dimethyl sulfoxide reductase activity by anaerobically grown Escherichia coli HB101; Bilous PT et al.; Escherichia coli grew anaerobically on a minimal medium with glycerol as the carbon and energy source and dimethyl sulfoxide (DMSO) as the terminal electron acceptor . DMSO reductase activity, measured with an artificial electron donor (reduced benzyl viologen), was preferentially associated with the membrane fraction (77 +/- 10% total cellular activity) . A Km for DMSO reduction of 170 +/- 60 microM was determined for the membrane-bound activity . Methyl viologen, reduced flavin mononucleotide, and reduced flavin adenine dinucleotide also served as electron donors for DMSO reduction . Methionine sulfoxide, a DMSO analog, could substitute for DMSO in both the growth medium and in the benzyl viologen assay . DMSO reductase activity was present in cells grown anaerobically on DMSO but was repressed by the presence of nitrate or by aerobic growth . Anaerobic growth on DMSO coinduced nitrate, fumarate, and and trimethylamine-N-oxide reductase activities . The requirement of a molybdenum cofactor for DMSO reduction was suggested by the inhibition of growth and a 60% reduction in DMSO reductase activity in the presence of 10 mM sodium tungstate . Furthermore, chlorate-resistant mutants chlA, chlB, chlE, and chlG were unable to grow anaerobically on DMSO . DMSO reduction appears to be under the control of the fnr gene. J Bacteriol, 1985 Jun, 162(3), 1111 - 9 Regulation of capsular polysaccharide synthesis in Escherichia coli K-12: characterization of three regulatory genes; Gottesman S et al.; The synthesis of the Escherichia coli capsular polysaccharide varies with growth medium, temperature of growth, and genetic background . lac fusions to genes necessary for capsule synthesis (cps) demonstrated that these genes are regulated negatively in vivo by the lon gene product . We have now isolated, characterized, and mapped mutations in three new regulatory genes (rcs, for regulator of capsule synthesis) that control expression of these same fusions . rcsA and rcsB are positive regulators of capsule synthesis . rcsA is located at min 43 on the E . coli map, whereas rcsB lies at 47 min . rcsC, a negative regulator of capsule synthesis, is located at min 47, close to rcsB . All three regulatory mutations are unlinked to either the structural genes cpsA-F or lon . Mutations in all three rcs genes are recessive to the wild type . We postulate that lon may regulate capsule synthesis indirectly, by regulating the availability of one of the positive regulators. Virus Res, 1985 Jun, 2(4), 345 - 58 Nonpermissive infection of lymphoblastoid cells by vesicular stomatitis virus . II . Effect on viral morphogenesis; Wethers JA et al.; The human B-lymphoblastoid cell line Raji is nonpermissive for infection by vesicular stomatitis virus (VSV) . The VSV particles released from Raji cells display a more heterogeneous distribution in equilibrium sucrose density gradients than particles released from BHK cells . The particles released from Raji cells contain approximately one-half to one-third as much viral matrix protein, relative to the nucleocapsid protein, as is normal . They also contain a higher proportion of the unglycosylated form of the G protein . The particles released from Raji cells are unstable and many disintegrate in the growth medium . Most of them deform when subjected to ultracentrifugation prior to fixation . The ratio of plaque-forming units to physical particles is much lower for the virions released from Raji cells. J Bacteriol, 1985 Jun, 162(3), 888 - 96 Transport of vitamin B12 in Escherichia coli: cloning of the btuCD region; DeVeaux LC et al.; The transport of vitamin B12 in Escherichia coli requires a specific vitamin B12 receptor protein in the outer membrane and the tonB gene product . In addition, the btuC gene, located at min 38 on the genetic map, has been found to influence vitamin B12 uptake or utilization . The btuC function is required for the growth response to vitamin B12 when the outer membrane transport process (btuB or tonB function) is defective . However, even in a wild-type strain, btuC is required for proper transport of vitamin B12 . Additional mutations in the vicinity of btuC were isolated as lac fusions that produced a phenotype similar to that of a btuC mutant . The btuC region was cloned by selection for complementation of a btuC mutation . Complementation testing with plasmids carrying various deletions or transposon Tn1000 insertions demonstrated that the new mutations defined a separate, independently expressed locus, termed btuD . The coding regions for both genes were identified on a 3.4-kilobase HindIII-HincII fragment and were 800 to 1,000 base pairs in length . They were separated by a 600- to 800-base-pair region . The gene order in this portion of the chromosome map was found to be pps-zdh-3::Tn10-btuD-btuC-pheS . Expression of beta-galactosidase in the btuD-lac fusion-bearing strains, whether proficient or defective in vitamin B12 transport, was not regulated by the presence of vitamin B12 in the growth medium. J Bacteriol, 1985 Jun, 162(3), 925 - 32 Effects of light, oxygen, and substrates on steady-state levels of mRNA coding for ribulose-1,5-bisphosphate carboxylase and light-harvesting and reaction center polypeptides in Rhodopseudomonas sphaeroides; Zhu YS et al.; The mRNA levels specific for ribulose-1,5-bisphosphate carboxylase, light-harvesting I polypeptides alpha and beta, and reaction center polypeptides L and M were assayed by use of a series of DNA probes specific for each cognate mRNA . Both the steady-state amounts and sizes of the specific mRNAs were measured as a function of the light intensity incident to the culture, the presence or absence of oxygen, and the type of substrate present in the growth medium . Northern hybridization revealed at least two and possibly three transcripts for ribulose-1,5-bisphosphate carboxylase . The cellular level of mRNA specific for ribulose-1,5-bisphosphate carboxylase increased in consort with enzyme activity as a function of both light intensity and reducing state of the substrate . Neither mRNA nor enzyme activity was detectable in aerobically grown cells . For the light-harvesting I and reaction center polypeptides there exist two transcripts, the larger of which appears to be a polycistronic mRNA possessing information for all four polypeptides and a smaller transcript specific for only the alpha and beta polypeptides of the light-harvesting I complex . The regulation of each of these mRNAs was affected by light and oxygen, but was not significantly affected by the oxidation-reduction state of the substrate. Biochem Biophys Res Commun, 1985 May 31, 129(1), 262 - 7 Isolation and purification of canadaphore, a siderophore produced by Helminthosporium carbonum; Letendre ED et al.; A new siderophore was isolated and purified from the spent growth medium of the fungus Helminthosporium carbonum by solvent extraction and reverse phase high pressure liquid chromatography . This new molecule has been assigned the name Canadaphore . Canadaphore was detected in culture filtrates after 15 days of growth and production was maximal after growth of H . carbonum to maximal stationary phase in modified Fries basal medium . Production of Canadaphore was completely suppressed when the organism was grown in medium supplemented with iron . Mass spectral analysis yielded a molecular mass of 680 Daltons for the iron-Canadaphore complex and 627 Daltons for the iron-free molecule . Spectroscopic analysis indicates that Canadaphore is a siderophore of the hydroxamate type. Biochim Biophys Acta, 1985 May 14, 815(2), 233 - 44 Biochemical and immunological characterization of the cell surface of the fish pathogenic bacterium Aeromonas salmonicida; Evenberg D et al.; The identification of lipopolysaccharide as periodic acid-Schiff positive material, present in the membrane fraction of the fish pathogenic Gram-negative bacterium Aeromonas salmonicida, analyzed by SDS-polyacrylamide gel electrophoresis, is shown . Such analysis has revealed several periodic acid-Schiff positive bands and many membrane proteins among which a pathogenicity-related Mr 54000 protein as a constituent of an additional surface layer outside the outer membrane (Evenberg et al., (1982) Biochim . Biophys . Acta 684, 241-248) . The latter protein, designated as additional cell envelope protein or ACE protein, has been purified and characterized in our laboratory (Evenberg and Lugtenberg, (1982) Biochim . Biophys . Acta 684, 249-254) . Most strains produce both high and low molecular weight lipopolysaccharide species, presumably corresponding with the presence and (virtual) absence, respectively, of an O-antigenic chain . The property to produce high molecular weight lipopolysaccharide can be lost upon subculturing in laboratory growth media and such is greatly enhanced by the prior loss of the ability to produce ACE protein . Lipopolysaccharide and ACE protein were identified as the major antigens . A new polysaccharide-like antigen, designated as PS-antigen, was detected . Moreover, immunological indications for the presence of a lipoprotein in A . salmonicida are described . The surface localization of the antigens was determined by testing whether preadsorption of antisera by intact cells decreased the binding of IgG to these antigens, or decreased the ability of the sera to agglutinate cells . According to these criteria lipopolysaccharide, ACE protein and PS-antigen are the major surface-located antigens . Material cross-reactive with lipopolysaccharide, ACE protein and PS-antigen has been found in a large number of strains . Several lines of evidence indicate the presence of interactions between ACE protein and lipopolysaccharide . Based on these results a molecular model of the cell envelope of virulent A . salmonicida is presented. Biochim Biophys Acta, 1985 May 14, 815(2), 170 - 4 Effects of cytochalasin B on Na+ content and cell volume of Entamoeba histolytica; Bakker-Grunwald T et al.; Cells of Entamoeba histolytica accumulated K+ and extruded Na+ compared to the concentrations of those ions present in the growth medium . Pinocytic activity, measured by the uptake of horseradish peroxidase of 125I-polyvinylpyrrolidone, was high (up to 0.3 ml/ml cells per h) . Upon addition of cytochalasin B, at a concentration (20 microM) that completely blocked pinocytosis, cells lost up to 40% of their Na+ content within 90 min; K+ content was not affected or increased slightly compared to control cells without the inhibitor . Cation loss was associated with cell shrinkage . The dose-response curves for the effects of cytochalasin B on pinocytosis and Na+ content were identical . These data provide direct evidence that pinocytosis is an important component of the homeostatic system for Na+. Anesthesiology, 1985 May, 62(5), 634 - 6 Sterility of anesthetic multiple-dose vials after opening; Schubert A et al.; Despite the widespread use of multiple dose vials (MDV) for anesthetic medications, there is a paucity of data concerning the sterility of in-use MDV . The purpose of this study was to analyze the frequency of bacterial contamination of MDV used in current anesthesia practice . The authors collected weekly samples from 351 in-use MDV for seven consecutive weeks and cultured them using appropriate bacterial growth media . The vials contained drugs including neuromuscular blockers, anticholinergics, and an induction agent . They were sampled from locations designated for elective as well as emergency surgery . Six vial subgroups were studied with multiple samplings for 6-48 days . One-half of all opened vials remained in use after 4-9 days, while less than 5% remained after 6 weeks . No vial yielded bacteria . The authors conclude that the incidence of MDV contamination with live bacteria is low for the anesthetic medications studied . This appeared to be true even for vials with increasing duration of use and for vials from locations where emergency surgery commonly was performed. J Infect Dis, 1985 May, 151(5), 890 - 4 In vitro effect of phagocyte cationic peptides on Coccidioides immitis; Segal GP et al.; Several cationic peptides that were isolated from rabbit granulocytes exerted fungicidal activity against arthroconidia of Coccidioides immitis in vitro . The fungicidal effect of the cationic peptides required at least 4-8 hr of contact between peptide and fungal cells and appeared to be dependent upon active fungal metabolism . The fungicidal activity was inhibited by increases in the tonicity of the growth medium but was not inhibited by changes in pH . These findings provide a potential mechanism whereby phagocytic cells may limit the spread of infection due to C . immitis. Cancer Res, 1985 May, 45(5), 2320 - 5 Induction of chromosomal damage in Chinese hamster ovary cells by soluble and particulate nickel compounds: preferential fragmentation of the heterochromatic long arm of the X-chromosome by carcinogenic crystalline NiS particles; Sen P et al.; Treatment of intact Chinese hamster ovary cells with crystalline NiS and NiCl2 resulted in the induction of chromosomal aberrations which included gaps, breaks, and exchanges . The incidence of these aberrations increased in a time- and concentration-dependent fashion . NiCl2 was more potent in inducing chromosomal aberrations in cells that were maintained with a salts/glucose medium during metal treatment than when cells were treated in culture growth medium . Chromosomal aberrations induced by NiCl2 occurred randomly among the autosomal arms; however, the heterochromatic centromeric regions of the chromosomes were preferentially damaged . In addition to inducing the same type of aberrations found with NiCl2, crystalline NiS particles also caused the selective fragmentation of the heterochromatic long arms of the X-chromosomes . This fragmentation was attributed to the difference in the mechanism of delivery of nickel ions from phagocytized crystalline NiS particles which aggregate around the nuclear membrane and release large amounts of nickel ions from a dissolving phagocytized particle . Previous studies have demonstrated that treatment of intact cells with crystalline NiS particles produces a considerably higher level of nickel in the nucleus compared with similar exposure to water-soluble NiCl2 . Since heterochromatin is known to form the inside lining of the interface nucleus, nickel ions, as they are solubilized from a phagocytized particle and enter the nucleus, are likely to encounter heterochromatin before they interact with euchromatin . In contrast, nickel ions derived from NiCl2 do not preferentially accumulate in the cell, and those ions that enter the cell are taken up by a nonphagocytic mechanism . It is proposed that when cells are treated with high levels of NiCl2 in an attempt to achieve the cellular levels of nickel produced by NiS phagocytosis, this overloading results in cytotoxic responses rather than the preferential fragmentation of heterochromatin observed with particles . Since liposome-mediated delivery of NiCl2 also results in fragmentation of the long arm of the X-chromosome, the selective breakage of heterochromatin by NiS particles may be due solely to the mechanism of Ni2+ delivery in cells. J Bacteriol, 1985 May, 162(2), 565 - 70 H2, N2, and O2 metabolism by isolated heterocysts from Anabaena sp . strain CA; Smith RL et al.; Metabolically active heterocysts isolated from wild-type Anabaena sp . strain CA showed high rates of light-dependent acetylene reduction and hydrogen evolution . These rates were similar to those previously reported in heterocysts isolated from the mutant Anabaena sp . strain CA-V possessing fragile vegetative cell walls . Hydrogen production was observed with isolated heterocysts . The ratio of C2H4 to H2 produced ranged from 0.9 to 1.2, and H2 production exhibited unique biphasic kinetics consisting of a 1 to 2-min burst of hydrogen evolution followed by a lower, steady-state rate of hydrogen production . This burst was found to be dependent upon the length of the dark period immediately preceding illumination and may be related to dark-to-light ATP transients . The presence of 100 nM NiCl2 in the growth medium exerted an effect on both acetylene reduction and hydrogen evolution in the isolated heterocysts from strain CA . H2-stimulated acetylene reduction was increased from 2.0 to 3.2 mumol of C2H4 per mg (dry weight) per h, and net hydrogen production was abolished . A phenotypic Hup- mutant (N9AR) of Anabaena sp . strain CA was isolated which did not respond to nickel . In isolated heterocysts from N9AR, ethylene production rates were the same under both 10% C2H2-90% Ar and 10% C2H2-90% H2 with or without added nickel, and net hydrogen evolution was not affected by the presence of 100 nM Ni2+ . Isolated heterocysts from strain CA were shown to have a persistent oxygen uptake of 0.7 mumol of O2 per mg (dry weight) per h, 35% of the rate of whole filaments, at air saturating O2 levels, indicating that O2 impermeability is not a requirement for active heterocysts. Prostaglandins, 1985 May, 29(5), 819 - 30 Prostaglandins or prostaglandin like substances are implicated in normal growth and development in oomycetes; Herman RP et al.; The prostaglandin synthesis inhibitors aspirin and indomethacin inhibit the growth of Achlya caroliniana, A . ambisexualis and Saprolegnia parasitica in a dose-related manner . In addition, the inhibitors cause the formation of a characteristic asterisk-shaped colony . This abnormal colony morphology does not appear to be dependent on medium composition, since three different nitrogen and five differentcarbon sources all support the abnormal growth in the presence of 0.1 mM indomethacin . The abnormal colony morphology is the result of abnormal branching . Inhibitor grown colonies are more densely branched than controls, with shorter distances between branches . Inhibited colonies allowed to grow for greater than ten days escape the inhibition and assume a normal gross colony morphology and size, however, they do not reproduce sexually . The addition of 2 micrograms/ml PGF1 alpha to the growth medium partially overcomes the growth inhibition caused by indomethacin . The data suggest a role for prostaglandin or prostaglandin-like compounds in oomycete development. Proc Natl Acad Sci U S A, 1985 May, 82(9), 2761 - 3 Mutations in the phosphoglucose isomerase gene can lead to marked alterations in cellular ATP levels in cultured fibroblasts exposed to simple nutrient shifts; Plesner P et al.; The generation of ATP in a hamster fibroblast mutant devoid of the enzyme phosphoglucose isomerase (PGI) has been studied and compared with that in the parental line, which is PGI+ . Both cell lines could be maintained for 24 hr in hexose media devoid of L-glutamine . Under these conditions in mannose medium, both the parental line and the PGI mutant maintained high intracellular ATP levels . With glucose under the same conditions, the parental line was able to keep the ATP level high . In contrast, the mutant line lost most of its ATP pool after incubation with glucose; the ATP/ADP ratio fell about 80% after incubation in glucose medium . Addition of pyruvate, with or without glucose, preserved the ATP pool at high levels even in the mutant, as did the presence of L-glutamine . When the PGI mutant was maintained for 3-4 days in growth medium, containing 4 mM L-glutamine and 10% dialyzed calf serum, in which glucose was replaced by mannose, the UDP-glucose pool dwindled and mediated control of the hexose uptake system did not ensue, in contrast to results of the same exposure to glucose-containing medium. Cancer Res, 1985 May, 45(5), 2065 - 9 Switch in differentiative response to maturation inducers of human promyelocytic leukemia cells by prior exposure to alkaline conditions; Fischkoff SA et al.; The myeloid lineage to which HL-60 promyelocytic leukemia cells will differentiate in response to a chemical differentiation inducer can be switched by altering the pH of the growth medium . Cells passaged previously at pH 7.2 become neutrophiles, and those passaged previously at pH 7.6 become eosinophiles after 5 to 7 days of culture in the presence of 0.5 mM butyric acid . Butyric acid and its analogues are unique in that all other chemical maturation inducers tested, such as dimethyl sulfoxide and retinoic acid, promote neutrophilic differentiation regardless of the prior culture history of the cells . This suggests that lineage commitment and maturational commitment are mechanistically separate processes in this multipotential cell line and can be independently manipulated experimentally. Am J Physiol, 1985 May, 248(5 Pt 1), C425 - 35 Regulation of hormonal responsiveness in LLC-PK1L cells grown in defined medium; Roy C; LLC-PK1L cells, a kidney-derived cell line, were able to grow in a chemically defined medium . Growth of the cells in the presence of retinol, ergocalciferol, d-alpha-tocopherol, 3,3',5-triiodothyronine, hydrocortisone, l-carnitine, d-l-methionine-S-methylsulfonium chloride, insulin, transferrin, cholesterol, and sodium linoleate increased the number of vasopressin receptors by 20- to 40-fold . All the newly detectable vasopressin receptors were coupled to the adenylate cyclase activity with similar efficiency . The same growth conditions did not alter the basal adenylate cyclase activity or the responses to calcitonin, parathyroid hormone, prostaglandin, adenosine, and GTP . In contrast, the increased responsiveness of the adenylate cyclase to vasopressin was associated with a reduced response to isoproterenol . Such an inverse correlation was also found when the time course of vasopressin receptor induction was studied . The supplemented medium permitted the growth of cells for several weeks . The effects of the enriched medium were fully reversible when we returned to the original cell growth medium . Thus such a cellular system appears as a useful tool for further work in cellular and kidney endocrinology and for detailing the molecular mechanisms of receptor-adenylate cyclase regulations. Mutat Res, 1985 May, 145(3), 129 - 36 A common pathway for protection of bacteria against damage by solar UVA (334 nm, 365 nm) and an oxidising agent (H2O2); Tyrrell RM; Pre-exposure of growing bacterial populations to low concentrations of hydrogen peroxide (H2O2) protects a repair-proficient strain of Escherichia coli (AB1157) very strongly and a rec A strain (AB2463) to a lesser extent from the lethal action of subsequent exposure to 5 mM H2O2 in buffer . The conditioning procedure also protects AB1157 and AB2463 from the toxic effects of UVA (334 nm, 365 nm) radiation but not UVB (313 nm) or UVC (254 nm) radiations . Pretreatment of growing AB1157 with low fluences of UVA (365 nm) radiation leads to the induction of resistance to H2O2, an effect which apparently requires protein synthesis . As in a previous report, the treatment of growing populations with low concentrations of H2O2 enhanced the resistance of such populations to H2O2 challenge in the growth medium . However, when H2O2 (+ Cu2+)-treated bacteriophage were subsequently infected into AB1157 under optimal inducing conditions, their resistance was not enhanced relative to infection into untreated bacteria . We conclude that the primary mechanism for the inducible effects observed could be the induction of H2O2 scavenging activity by low concentrations of H2O2 either introduced into the growth medium directly or produced by low fluences of UVA irradiation. Cancer Res, 1985 May, 45(5), 2123 - 7 Inhibitors of poly(adenosine diphosphoribose) synthetase, examination of metabolic perturbations, and enhancement of radiation response in Chinese hamster cells; Ben-Hur E et al.; 3-Aminobenzamide, a specific inhibitor of poly(adenosine diphosphoribose) synthesis, has been shown to enhance the response of mammalian cells to ionizing radiation and alkylating agents . Observations such as these usually have been taken to be an indication of the involvement of poly(adenosine diphosphoribose) in the repair of DNA damage . It has been reported that some inhibitors of adenosine diphosphoribosyl transferase (ADPRT) affect cell viability, glucose metabolism, and DNA synthesis when present at low concentrations in the growth medium for extended periods (e.g., lymphoid cells exposed to a few millimolar for 24 h {Milam, K . M., and Cleaver, J . E . Science (Wash . DC), 223: 589, 1984}) . The latter report questioned previous interpretations of radiation results based on the use of ADPRT inhibitors which enhance cell killing . We have studied the enhanced radiation lethality of Chinese hamster cells using higher concentrations of these inhibitors, but for shorter periods, in an effort to determine if metabolic perturbations are produced and if they are relatable to enhanced cell killing . The compounds used were 2-aminobenzamide, 3-aminobenzamide, 4-aminobenzamide, benzamide, and nicotinamide, compounds which show a large variation in their potency as inhibitors of ADPRT . It was found that none of the compounds was toxic at the highest doses used (20 mM for 2 h) and that, during a 2-h period, the potent inhibitor 3-aminobenzamide had little or no effect on DNA synthesis . Two h is long enough to yield a near-maximum radiosensitization with 3-aminobenzamide . Although glucose metabolism was affected to varying degrees (up to a 50% inhibition by 4-aminobenzamide in 2 h), there was no correlation between effectiveness in inhibiting ADPRT and effectiveness in inhibiting glucose metabolism . A correlation was observed, however, between the inhibitory potential of ADPRT and the enhancement of radiation response . When used for sufficiently short times, we conclude that the effects at even high concentrations of a potent inhibitor of ADPRT (e.g., 3-aminobenzamide) are consistent with an involvement of poly(adenosine diphosphoribose) synthesis in the expression of a radiation-induced end point like cell killing. Lab Invest, 1985 May, 52(5), 559 - 67 Explant culture of human submandibular gland epithelial cells: evidence for ductal origin; Sens DA et al.; As an approach to investigating the disease cystic fibrosis, attempts were undertaken to culture from human submandibular glands epithelial cells with a potential for manifesting the cystic fibrosis genetic defect . To initiate the culture of submandibular gland epithelial cells, tissue fragments from glands were explanted as a function of both the composition of the serum-free growth medium and of the matrix utilized to coat the culture vessel growth surface . A morphologically homogeneous growth of submandibular gland epithelial cells, uncontaminated by fibroblasts, was obtained, once optimum culture conditions were defined . Light microscopic examination of these explant cultures in a transverse plane of section demonstrated variation in the outgrowth according to distance from the explant . At its outer margin, the outgrowth consisted of one or two layers of viable low cuboidal cells, and more centrally, it was multilayered . Mitotic figures were observed in the periphery of the outgrowth . In the region, a few cells removed from the periphery where the outgrowth consisted of about three to six cell layers, dilated intercellular spaces, indicative of secretion of fluid and ions into the spaces, separated the basal cuboidal cells . Overlying cells were increasingly flattened toward the culture surface and devoid of nuclei . Centrally, near the explant, the multilayer appeared completely involuted throughout . Ultrastructural examination in a transverse plane of the multilayered region with viable basal cells confirmed these observations showing wide spaces separating the cuboidal basal cells, keratinization of midstratum cells, and complete involution of the upper layer of ghost-like cells . These cells cultured from the submandibular gland reacted positively to immunochemical staining for keratin. J Biol Chem, 1985 Apr 25, 260(8), 4637 - 47 Biosynthesis of the beta-lactam antibiotic, thienamycin, by Streptomyces cattleya; Williamson JM et al.; Radioactive- and stable isotope-containing substrates were used to identify the biosynthetic precursors of the beta-lactam antibiotic, thienamycin, in Streptomyces cattleya . Acetate is utilized by the organism to form C(6) and C(7) of the beta-lactam ring . The two carbons of the hydroxyethyl group attached to C(6) are both derived from the methyl of methionine . The cysteaminyl side chain attached to C(2) is derived from cysteine . Selective inhibition of thienamycin and cephamycin C biosynthesis has been achieved either through the addition of metabolic inhibitors or through manipulation of the growth medium . These results suggest that the two beta-lactam antibiotics, thienamycin and cephamycin C, are formed by different biosynthetic pathways. J Biol Chem, 1985 Apr 25, 260(8), 4799 - 806 Characterization of an Escherichia coli mdoB mutant strain unable to transfer sn-1-phosphoglycerol to membrane-derived oligosaccharides; Fiedler W et al.; A procedure for the isolation of mutants affected in components containing glycerol derived from phospholipids yielded two mutant strains that contain membrane-derived oligosaccharides (MDO) devoid of glycerol (Rotering, H., Fiedler, W., Rollinger, W., and Braun, V . (1984) FEMS Microbiol . Lett . 22, 61-68) . MDO are found in the periplasmic space of Escherichia coli and other Gram-negative bacteria, and they may comprise up to 7% of the cells dry weight . The biosynthesis of MDO is osmoregulated (Kennedy, E . P . (1982) Proc . Natl . Acad . Sci . U . S . A . 79, 1092-1095) and linked to the metabolism of phospholipids (van Golde, L . M . G., Schulman, H., and Kennedy, E . P . (1973) Proc . Natl . Acad . Sci . U . S . A . 70, 1368-1372) . This leads to substitution of MDO with sn-1-phosphoglycerol and phosphoethanolamine (Kennedy, E . P., Rumley, M . K., Schulman, P., and van Golde, L . M . G . (1976) J . Biol . Chem . 251, 4208-4213) . MDO also contain succinate in O-ester linkage . We now report that one mutant strain lacks phosphoglycerol transferase I activity and thus is unable to transfer sn-1-phosphoglycerol residues from phosphatidylglycerol to MDO . The mdoB gene affected in this mutant has been located at 99.2 min on the E . coli chromosome . The ethanolamine content of MDO isolated from the mutant strain is elevated, whereas the number of succinate residues is not affected . The only phenotype of mdoB mutants we found is a dramatic reduction of the diglyceride content observed in dgk mdoB double mutants when the beta-glucoside arbutin is present in the growth medium. Thromb Haemost, 1985 Apr 22, 53(2), 165 - 9 Vascular smooth muscle cells inhibit the plasminogen activators secreted by endothelial cells; Laug WE; Pure cultures of bovine endothelial cells (EC) produce and secrete large amounts of plasminogen activators (PA) . Cocultivation of EC with vascular smooth muscle cells (SMC) resulted in a significant decrease of PA activities secreted by the EC, whereas the cellular PA activities remained unaffected . Secreted PA activities were absent in the growth medium as long as the SMC to EC ratio was 2:1 or higher . The PA inhibitory activity of the SMC was rapid and cell-to-cell contact was not necessary . The PA inhibitory activity was present in homogenates of SMC as well as in the medium conditioned by them but not in the extracellular matrix elaborated by these cells . Serum free medium conditioned by SMC neutralized both tissue type (t-PA) and urokinase like (u-PA) plasminogen activators . Gel electrophoretic analysis of SMC conditioned medium followed by reverse fibrin autography demonstrated PA inhibitory activities in the molecular weight (Mr) range of 50,000 to 52,000 similar to those present in media conditioned by bovine endothelial cells or fibroblasts . Regular fibrin zymography of SMC conditioned medium incubated with u-PA or t-PA revealed the presence of a component with a calculated approximate Mr of 45,000 to 50,000 which formed SDS resistant complexes with both types of PA . These data demonstrate that vascular SMC produce and secrete (a) inhibitor(s) of PAs which may influence the fibrinolytic potential of EC. Biochim Biophys Acta, 1985 Apr 17, 839(2), 181 - 90 Identification in various chlorate-resistant mutants of a protein involved in the activation of nitrate reductase in the soluble fraction of a chlA mutant of Escherichia coli K-12; Giordano G et al.; We report some properties of Protein PA which has been isolated from the soluble fraction of a chlB mutant after anaerobic growth in the presence of KNO3 . This protein has been identified by its capacity to reactivate nitrate reductase present in the soluble fraction of a chlA mutant by the complementation process . The presence of active Protein PA in the chlB mutant is independent of the presence of oxygen or of nitrate during growth . In contrast, the addition of sodium tungstate to the growth medium leads to the formation of inactive Protein PA which is not able to activate nitrate reductase in the chlA-soluble extract by complementation . Inactive Protein PA has been quantitated immunologically . The partial purification of Protein PA has been achieved from various chlorate-resistant mutants (chlA-chlG) . The establishment of particular complementation systems comprising the soluble extracts of chlA or chlB mutants and partially purified Protein PA from soluble fractions of different chlorate-resistant mutants, has allowed the quantitative estimation of this protein . The analysis by 'rocket immunoelectrophoresis' using an antiserum specific for Protein PA has shown that inactive Protein PA is present in approximately equivalent amounts in the chlA, chlE, chlG and chlD mutants. J Bacteriol, 1985 Apr, 162(1), 276 - 9 Transformation of Azotobacter vinelandii with plasmid DNA; Glick BR et al.; Azotobacter vinelandii cells can be transformed at high frequencies with the broad-host-range plasmids pRK2501, RSF1010, and pGSS15, using a modification of the procedure developed by Page and von Tigerstrom (J . Bacteriol . 139:1058-1061, 1979) for chromosomal DNA-mediated transformation . The frequency of transformation per microgram of plasmid DNA per viable cell with pRK2501 and pGSS15 was about 5 X 10(-2) and 2 X 10(-2), respectively . With RSF1010, transformation frequencies ranged from 3 X 10(-4) to 4 X 10(-2) . With each plasmid, the frequency of transformation was independent of the phase of the growth cycle . When concentrations of pRK2501 ranging from 0.1 to 51 micrograms of DNA were tested, the frequency of transformation was directly proportional to the amount of DNA . This linear response indicated that, although the uptake of plasmid DNA with this procedure may be inefficient, there is a high probability that once inside a cell the plasmid will be stably maintained . Cells that have been transformed with pRK2501 did not grow well on transforming medium which lacks iron and contains fixed nitrogen . However, on growth medium which contains iron and lacks fixed nitrogen, transformants produced distinctive colonies larger than those of nontransformed cells . Resistance to kanamycin due to transformation by pRK2501 was stably maintained for at least 10 successive generations in the absence of selective pressure . The present protocol should facilitate the molecular cloning of genes in Azotobacter spp. J Bacteriol, 1985 Apr, 162(1), 263 - 70 Effects of temperature and sodium chloride concentration on the phospholipid and fatty acid compositions of a halotolerant Planococcus sp; Miller KJ; The phospholipid headgroup composition and fatty acid composition of a gram-positive halotolerant Planococcus sp . (strain A4a) were examined as a function of growth temperature (5 to 35 degrees C) and NaCl content (0 to 1.5 M) of the growth medium . When the growth temperature was decreased, the relative amount of mono-unsaturated branched-chain fatty acids increased . When Planococcus sp . strain A4a was grown in media containing high NaCl concentrations, the relative amount of the major fatty acid, Ca15:0, increased . The relative amount of anionic phospholipid also increased when the NaCl concentration of the growth medium was increased . The increase in anionic phospholipid content resulted from a decrease in the relative mole percent content of phosphatidylethanolamine and an increase in the relative mole percent content of cardiolipin. J Biol Stand, 1985 Apr, 13(2), 129 - 34 Stainer and Scholte's pertussis medium with an alternative buffer; Lothe RA et al.; A comparative study of the Stainer and Scholte chemically defined growth medium (SS) for Bordetella pertussis, and a modification of it (MSS), was made . The Tris buffer which is reported to have adverse effects was replaced in MSS with sodium glycerophosphate . By using generation time as a measure of the growth promoting properties of the media, we found that the MSS was superior to SS for B . pertussis strain CN5612/67, which was used as a vaccine strain in Norway . Additionally by employing the 'dilution to extinction' method we showed that MSS supported B . pertussis growth from smaller inocula than the original SS medium . No significant differences in the quality of vaccines made from organisms grown in the two alternative media were observed in animal tests for potency and safety . The difference in buffers in SS and MSS had no influence on the formation of the agglutinogens, as tested by agglutination with specific factor sera . It was confirmed that liquid media gave a better total expression of the agglutinogens than solid media. J Biol Chem, 1985 Mar 10, 260(5), 2777 - 81 Solid-state 13C and 15N nuclear magnetic resonance studies of alanine metabolism in Aerococcus viridans (Gaffkya homari); Jacob GS et al.; Transport and metabolism of D- and L-alanine by Aerococcus viridans (Gaffkya homari) have been studied using cross-polarization magic-angle spinning 13C and 15N NMR spectroscopy of lyophilized whole cells, isolated cell walls, and crude extracts . For equimolar concentrations in the growth medium, about 10 times more D-alanine than L-alanine is transported into the cells . Examination of cells labeled with D-{13C}alanine and L-{epsilon-15N}lysine by double cross-polarization magic-angle spinning 15N NMR indicates that only about 20% of the D-alanine present in cell-wall peptidoglycan comes directly from the growth medium . The rest is produced by de novo synthesis . Most of the labeled D-alanine is found within peptidoglycan precursors or inverted to L-alanyl residues of soluble proteins. Arch Inst Pasteur Tunis, 1985 Mar-Jun, 62(1-2), 69 - 89 {Study of the lipolytic system of Penicillium verrucosum var cyclopium (Westling) . II . Conditions for the production of the lipase system}; Glenza A et al.; The specie Penicillium verrucosum var cyclopium (Westling) has showed that Media containing peptones, especially, trypticase, as a source of nitrogen and maltose or glucose as a source of carbon are most efficient for a good stimulation of the lipolytic activity . Salt, especially Mg++ ion and some oligo- elements exhibit a marked effect on enzyme production . On the other hand, addition of lipids to the growth medium inhibited the lipase production . Shaking of the medium decreases the amount of lipase production but allows an early growth of P . verrucosum var cyclopium (Westling). Mikrobiologiia, 1985 Mar-Apr, 54(2), 191 - 6 {Biotin transport in the yeast Trichosporon cutaneum}; Almukhova EA et al.; Biotin transport was studied in the yeast Trichosporon cutaneum and was shown to be the active transport with Km = 1.5 X 10(-7) M . The process depended on the pH and temperature whose optimal values were pH 6.8-7.2 and 35-40 degrees C . T . cutaneum biotin permease was highly specific for biotin and was inhibited by biotin analogs . Biotin uptake by the cells was inhibited by glucose . Excess biotin in the growth medium caused partial repression of its transport system in the yeast . Synthesis of biotin permease by T . cutaneum depended on the nature of a catabolite present in the growth medium . The rate of biotin transport was highest in the cells grown in the presence of ramnose or arabinose and lowest in the cells utilizing galactose, lactose or xylose. Mikrobiologiia, 1985 Mar-Apr, 54(2), 274 - 9 {Simultaneous cultivation of the fungi Trichoderma longibrachiatum and Endomycopsis fibuligera}; Gavristov AV et al.; Combined cultivation of the following microorganisms was studied: the fungus Trichoderma longibrachiatum producing cellulases and the yeast Endomycopsis fibuligera producing glucoamylase . A growth medium was found to maintain the activity of these enzymes at a high level in the both microbial monocultures . The effect of the yeast inoculation time on the enzyme activity was studied during combined cultivation of the two organisms . When the yeast was inoculated during the first two days of the fungal growth, the enzyme activities were 40-70% of those during the growth of the monocultures . The yeast did not grow when it was inoculated by the 4th day of the fungal growth . When the yeast was added to the fungus earlier, the activity of cellulases fell down . Possible reasons for these phenomena are discussed. EMBO J, 1985 Mar, 4(3), 761 - 7 Cloning and expression of the chromosomal immune interferon gene of the rat; Dijkema R et al.; The chromosomal immune interferon gene of the rat (IFN-gamma) was identified by screening a recombinant rat lambda phage library with a human IFN-gamma cDNA probe . In contrast to the genes of other rat IFNs, this rat IFN-gamma chromosomal gene contains introns and its structural organization closely resembles that of the human and murine IFN-gamma genes . The rat IFN-gamma gene encodes a signal sequence of 19 amino acids followed by the mature IFN-gamma protein of 137 amino acids . The gene was expressed under control of the simian virus 40 (SV40) early promoter in Chinese hamster ovary (CHO) cells deficient in dihydrofolate reductase (DHFR) after co-transformation with a plasmid containing the mouse DHFR gene . Initial transformants with a DHFR+ phenotype produced IFN-gamma titres ranging from 20 to 1600 units/ml . After stepwise increases in the concentration of methotrexate (MTX) in the growth medium of transformed CHO cells, MTX-resistant clones producing 80 000-100 000 units per ml were isolated . Protein analysis of supernatants of these MTX-resistant cells by polyacrylamide gel electrophoresis revealed a product with an apparent mol . wt . of 18 000 daltons which was not detectable in the growth medium of DHFR+ transformants that did not produce IFN . The product was identified as rat IFN-gamma and constituted approximately 5% of the proteins excreted from these cells. Can J Microbiol, 1985 Mar, 31(3), 305 - 9 Ultrastructural changes observed in Rickettsia rickettsii incubated at different temperatures in cell-free medium; Pontefract RD et al.; When cells of Rickettsia rickettsii were suspended at different temperatures in growth medium free of host cells, ultrastructural changes were observed in some of these organisms . Depending upon the temperature and length of incubation, loosely organized cells developed into compact, intensely stained, rod-shaped organisms . These compact cells closely resembled the morphology of the original culture of Rickettsia used for inoculation . Morphological changes were primarily noted in cells maintained at 21 degrees C . The viability of the cells was also affected by the temperature of incubation. Eur J Immunol, 1985 Mar, 15(3), 216 - 21 Murine intestinal intraepithelial lymphocytes II . Comparison of freshly isolated and cultured intraepithelial lymphocytes; Ernst PB et al.; Highly enriched preparations of intraepithelial lymphocytes (IEL) containing a large subpopulation of granulated cells were isolated from the murine small intestinal mucosa . We cultured IEL in media containing interleukin 2 (growth media conditioned with 20% concanavalin A supernatant; Con A CM) or mast cell growth factor(s) (growth media conditioned with 40% WEHI-3 supernatant; WEHI CM) and compared the physical and functional properties of the cultured cells to freshly isolated IEL . IEL cultured in Con A CM developed enhanced cytotoxicity against YAC-1, compared to freshly isolated IEL, and spontaneous cytotoxicity for P815 targets . Most of these cultured cells were Thy-1+ Lyt-1- Lyt-2+, and contained cytoplasmic granules similar to those seen in electron photomicrographs of other cytotoxic cell populations . IEL cultured in WEHI CM gave rise to cells that morphologically resembled mast cells . Unlike freshly isolated IEL, the cells stained metachromatically, contained 200-450 ng of histamine/10(6) cells and expressed high-affinity receptors for IgE . Our data clearly show that, although IEL do not themselves have physical characteristics of mast cells, they do contain mast cell precursors . In addition, IEL grown in the presence of T cell growth factors give rise to an activated cytotoxic cell population which is mostly granulated and Thy-1+ Lyt-1- Lyt-2+. Mikrobiologiia, 1985 Mar-Apr, 54(2), 280 - 3 {Various characteristics of growth kinetics and exohydrolase synthesis in Aspergillus foetidus and Zygofabospora marxiana}; Astapovich NI et al.; The kinetics of growth and synthesis of exohydrolases (polygalacturonases and proteinases) were studied in Aspergillus foetidus and Zygofabospora marxiana . The processes of growth and synthesis of polygalacturonases were found to be shifted in time . Endopolymethylgalacturonase of the micromycete and endopolygalacturonase of the yeast are not accumulated in the cells, but are mainly secreted into the growth medium . The maximal value of polygalacturonase bound to the cell coincides with the maximal rate of synthesis of the enzyme secreted into the medium and with the maximal level of cell-bound alkaline and acid proteinases . The activity of alkaline proteinase is not found in the growth medium of the cultures, and only traces of acid proteinase were detected . It is possible that proteinase are involved in processing of endopolygalacturonases. J Biol Chem, 1985 Feb 25, 260(4), 2295 - 300 The fate of glucosylceramide (glucocerebroside) in genetically impaired (lysosomal beta-glucosidase deficient) Gaucher disease diploid human fibroblasts; Saito M et al.; Diploid human infant skin fibroblasts cultured from normal infants and Gaucher disease infants, with genetically defective lysosomal glucosylceramide:beta-glucohydrolase activity, had a full range of homologous glycosphingolipids from the simplest (glucosylceramide) to higher neutral derivatives (lactosyl-, trihexosyl- and tetrahexosylceramide) and anionic sialo derivatives (gangliosides) (sialosyllactosyl-, disialosyllactosyl-, sialosylgangliotriaosyl-, and mono- and disialosylgangliotetraosylceramide) . Although excessive storage of glucosylceramide in histiocytes is pathognomonic for Gaucher disease, we found that Gaucher disease fibroblasts contained 1.23 +/- 0.08 nmol of glucosylceramide/mg cell protein; normal infant cells, 1.11 +/- 0.48 . When we aged infantile Gaucher disease fibroblasts for 20 days beyond their confluency state, we found no increased accumulation of glucosylceramide, but a 1.5-2-fold increase in trihexosylceramide, sialosylgangliotetraosylceramide, and disialosyllactosylceramide . Gaucher disease fibroblasts took up and could not degrade but, instead, effectively converted pulse-chase 3-O-{3H}glucosylceramide supplied in the growth medium in liposomes into higher glycosphingolipids, especially the plasma membrane ganglioside, sialosyllactosylceramide . When grown with extracellular particulate {3H}glucosylceramide, infantile Gaucher fibroblasts localized it and higher labeled homologues in the plasma membrane; glucosylceramide did not accumulate in the lysosomes . These findings indicate that fibroblasts that are genetically deficient in lysosomal glucosylceramide:beta-glucosidase avoid pathological lysosomal accumulation by relegating undegradable glucosylceramide to an anabolic compartment where glucosylceramide is converted into more highly glycosylated glycosphingolipids. S Afr Med J, 1985 Feb 16, 67(7), 241 - 2 The effect of surgical glove powder on cleavage of two-cell mouse embryos in an in vitro fertilization programme; Kruger TF et al.; The effect of surgical glove powder on the development of early mouse embryos was studied . Embryos from F1 hybrid mice (C57 B1/6 X CBA) were suspended in Whittingham's T6 growth medium with 10% human serum, using Petri dishes (Falcon 3001) . Contamination was brought about by a sterile, powdered, surgical glove touching the surface of the growth medium for less than a second in group I, and in group II the same procedure was followed but the glove was rinsed beforehand with sterile, four times distilled water and air-dried . In the control group (group III) no contamination with surgical glove powder occurred . In group I only 9 of 137 embryos (7%) reached the blastocyst stage, in contrast with 110 of 196 (56%) in group II and 258 of 287 (90%) in group III . The differences in results between groups I and III, groups I and II, and groups II and III were found to be statistically significant (P less than 0,001) by the chi-square test . It is concluded that surgical gloves are a potent inhibitor of early embryonic growth . In an in vitro fertilization programme including follicle aspiration and embryo transfer, contamination of embryos with these gloves should be avoided at all costs. Can J Biochem Cell Biol, 1985 Feb, 63(2), 85 - 90 Cerulenin inhibition of lipid synthesis and its reversal by exogenous fatty acids in Mycobacterium smegmatis ATCC 607; Mahajan S et al.; Cerulenin inhibited the lipid synthesis of Mycobacterium smegmatis ATCC 607 over the range of 0.5-1.8 microgram/mL with complete inhibition at 1.8 microgram/mL, as monitored by {14C}glycerol incorporation into lipids . Exogenous fatty acids failed to restore the lipid synthesis at 1.8 microgram/mL; however, the addition of palmitic acid to the growth medium partially restored the lipid synthesis when cerulenin concentration was decreased to 1.6 microgram/mL . Fatty acid analysis of cerulenin plus palmitic acid supplemented cultures revealed that exogenously supplied fatty acid was incorporated into cellular phospholipids . Further investigations with 1.6 microgram/mL of cerulenin and {14C}acetate and {32P}orthophosphoric acid showed that cerulenin inhibited the synthesis of saturated plus unsaturated fatty acids and phospholipids . Pulse-chase studies with {14C}acetate revealed decreased synthesis and degradation of each of the phospholipid components. Cancer Res, 1985 Feb, 45(2), 504 - 8 Decreased intracellular glutathione concentration and increased hyperthermic cytotoxicity in an acid environment; Freeman ML et al.; Chinese hamster ovary (CHO) cells were heated at either pH 7.2 to 7.4 or 6.7 to 6.8 in order to determine if conditions which suppress the development of thermotolerance (pH 6.7 to 6.8) reduce intracellular levels of glutathione (GSH) . When the pH of the growth medium was reduced from 7.2 to 6.7, a 25 to 30% reduction in GSH was observed in cells maintained at 37 degrees . Cells heated at 42 degrees in medium adjusted to pH 6.7 had lower levels of GSH compared to cells heated at pH 7.2 . Cells were also heated for 1 hr at 43 degrees and then incubated at 37 degrees for up to 9.5 hr prior to GSH measurement . The GSH levels of cells treated at pH 7.3 increased approximately 20% above control, whereas treatment at pH 6.7 resulted in a 20% reduction compared to control . Chinese hamster ovary cells were exposed to 5 mM buthionine sulfoximine (BSO) prior to and during 42 degrees heat treatment . BSO exposure at either pH 7.3 or 6.8 reduced the GSH concentration to approximately 65% of control and increased thermal cytotoxicity . The thermal sensitivity of cells incubated at 42 degrees and pH 7.3 was compared to that of cells incubated at pH 6.8 . Decreasing the pH from 7.3 to 6.8 increased sensitivity by a factor of 1.87 in the absence of BSO, whereas decreasing the pH in the presence of BSO increased sensitivity by only 1.50 . In summary, these results suggest that the increase in thermal sensitivity observed when Chinese hamster ovary cells are heated in acid medium is due partly to the depletion of GSH. J Protozool, 1985 Feb, 32(1), 25 - 31 Further studies of dopamine metabolism and function in Tetrahymena; Gundersen RE et al.; The large amounts of dopamine accumulated by cells of Tetrahymena pyriformis strain NT-1 and secreted into their growth medium were found to depend primarily upon an extracellular, non-enzymatic conversion of tyrosine to L-dihydroxyphenylalanine (L-DOPA); L-DOPA was then rapidly taken into the cells and transformed into dopamine enzymatically . Efforts to find physiologically significant dopamine binding sites on the cell surface or dopamine-sensitive adenylate cyclase activity were unsuccessful, suggesting that the catecholamine does not function in Tetrahymena as it does in higher animals. Virus Res, 1985 Feb, 2(1), 29 - 33 Increase of virus yields and releases of Borna disease virus from persistently infected cells; Pauli G et al.; Borna disease virus grows to low titres in persistently infected cells with an infectious particle to cell ratio of 0.01 to 0.05 . Inclusion of n-butyrate in the growth medium enhances infectivity yields up to 1 log . This effect is time and concentration dependent . In hypertonic medium with an excess of NaCl, KCl or Na2SO4 up to 50% of the total infectious virus yield is released from the cells . Released supernatant virus (buoyant density in sucrose rho = 1.22 g/cm3) is more heat stabile than cell-bound virus (rho = 1.18 g/cm3) . The access to cell-free (released) virus opens new possibilities for the characterization of this neurotropic agent. Genetika, 1985 Feb, 21(2), 344 - 6 {Maintenance stability of the pBR322 and pBR327 vector plasmids in Escherichia coli cells during multiple passages}; Potapova IA et al.; Stability of pBR322 and pBR327 plasmids was studied . Plasmid-containing Escherichia coli strains were grown in liquid growth medium without selection pressure . Plasmid pBR327 was shown to be more stable in E . coli CSH54 cells than pBR322 . Essential heterogenity of individual plasmid-containing clones was recognized by the maintenance stability of plasmid DNA . The indicated clones with high stability failed to be cured from pBR327 plasmid by means of acridine orange . High stability of plasmid maintenance and the failure to cure cells containing this plasmid are suggested to correlate with and to be essentially determined by the cell functions. DNA, 1985 Feb, 4(1), 33 - 8 Expression of a cloned human fibrinogen cDNA in Escherichia coli: synthesis of an A alpha polypeptide; Lord ST; The construction of a plasmid, p166.9, for the controlled synthesis of the A alpha-chain of human fibrinogen in Escherichia coli is described . The plasmid combines the tac promoter, constructed for controlled high-level peptide expression, with the promoter and signal peptide codons from an E . coli plasmid beta-lactamase gene and a cDNA of the A alpha-chain of human fibrinogen . The tac promoter is repressed in lacIQ strains of E . coli and induced by isopropylthio-beta-D-galactoside (IPTG) . Protein blot analysis of lysates of cells carrying p166.9 demonstrates the IPTG-dependent synthesis of polypeptides which cross react with antisera to the A alpha-chain of human fibrinogen . The largest and predominant species corresponds to an apparent molecular weight of 63,000 . When the cell growth media or cell lysates are treated with thrombin, the enzyme which normally releases fibrinopeptide A (FPA) from the A alpha-chain, FPA-like peptides are detectable by radioimmunoassay with antiserum prepared against human FPA . Thrombin-treated cell growth media prepared 4 hr after IPTG induction contained 340 ng/ml of FPA-like material; using a mass ratio of 40 for FPA to A alpha, this indicates that the A alpha-peptide concentration in the culture media is 13 micrograms/ml. Carcinogenesis, 1985 Feb, 6(2), 181 - 8 Analysis of isoenzymes in normal and carcinogen-treated human endometrial stromal cells in culture; Nelson KG et al.; The isoenzyme patterns of lactate dehydrogenase (LDH), hexokinase, phosphofructokinase, and aldolase were investigated in cultured normal and carcinogen-treated human endometrial stromal cells . Both normal and carcinogen-treated cells had similar phosphofructokinase and aldolase isoenzymes . Distinctive changes in hexokinase and LDH isoenzyme patterns were found in the carcinogen-treated stromal cells . The LDH isoenzyme patterns of the carcinogen-treated stromal cells were shifted toward the muscle LDH forms . This is comparable to the alteration of LDH isoenzyme profiles observed in cell lines established from human uterine sarcomas . The two tissue culture media used affected the LDH isoenzyme patterns of endometrial stromal cells but differences between the LDH isoenzyme patterns of control and carcinogen-treated cells were detected regardless of the growth medium used . Total LDH activity was not significantly different in control and carcinogen-treated stromal cells . The hexokinase isoenzyme patterns expressed by the carcinogen-treated stromal cells were distinctly different from the normal hexokinase patterns . The treated stromal cells contained both hexokinase I and II, whereas the normal cells c