Growth media

J Protozool, 1987 Feb, 34(1), 68 - 74
Partial purification and characterization of Naegleria fowleri beta-glucosidase; Das S et al.; Naegleria fowleri cells, grown axenically, contain high levels of beta-D-glucosidase which catalyzes the hydrolysis of 4-methylumbelliferyl-beta-D-glucopyranoside (4MUGlc) (Km, 0.9 mM), octyl-beta-D-glucoside (Km, 0.17 mM), and p-nitrophenyl-beta-D-glucopyranoside at relative rates of 1.00, 2.88, and 1.16, respectively (substrate concentration, 3.0 mM) . When the amebae are subjected to freeze-thawing, sonication, and centrifugation (100,000 g, 1 h), 85% of the beta-glucosidase activity appears in the supernatant fraction . The beta-glucosidase was purified 40-fold (34% yield) using a combination of chromatographic steps involving DE-52 cellulose, concanavalin A-Sepharose, and hydroxylapatite followed by isoelectric focusing . The predominant soluble beta-D-galactosidase activity in the Naegleria extract copurifies with the beta-D-glucosidase; the two activities have the same isoelectric point (pI, 6.9), similar heat stabilities, are both inhibited by lactobionic acid (Ki, 0.40 mM), and exhibit optima at pH 4.5, indicating that they are probably the same enzyme . The Naegleria beta-D-glucosidase has an apparent molecular weight of 66,000, a Stokes radius of 25 A, and a sedimentation coefficient of 4.2S . The beta-glucosidase is not inhibited by conduritol beta-epoxide or galactosylsphingosine but is completely inhibited by 1.25 mM bromo conduritol beta-epoxide . The latter compound, when present in the growth medium, inhibits the growth of the organism and profoundly alters its ultrastructure, the main effect being the apparent inhibition of cytokinesis and the generation of multinucleate cells . The issue of the role of the beta-glucosidase in the metabolism of the ameba and its possible role in pathogenic mechanisms are discussed.

Mol Cell Biol, 1987 Feb, 7(2), 622 - 31
Induction of urokinase-type plasminogen activator by UV light in human fetal fibroblasts is mediated through a UV-induced secreted protein; Rotem N et al.; Plasminogen activator was previously shown to be induced by UV light in human cells with low capacity to repair UV-induced DNA lesions . We now show that in human fetal fibroblasts UV light enhanced the two mRNA species coding for the urokinase-type plasminogen activator (uPA) and the tissue-type plasminogen activator, but immunological analysis revealed exclusively uPA activity . Several independent and complementary experiments indicated that induction of uPA was mediated, apparently entirely, through a UV-induced, secreted protein (UVIS) in the growth medium of irradiated cells . First, elevation of uPA mRNA after irradiation was severely blocked by cycloheximide . Second, replacement of conditioned medium in irradiated cells while the rate of plasminogen activator induction was maximal rapidly and completely stopped any further increase in uPA activity . Third, addition of the same removed conditioned medium to nonirradiated cells mimicked UV light in enhancing the level of uPA activity as well as that of uPA mRNA . Fourth, UVIS activity was completely lost by treating the conditioned medium with trypsin but not with nucleases . Kinetic measurements indicated that the accumulation of UVIS rather than the induction of uPA by UVIS conferred the rate-limiting step in the overall process of uPA induction . Both UV light and UVIS acted synergistically with inhibitors of DNA repair for uPA induction . Based on these results, a model is proposed implicating relaxation of DNA torsional stress of an as yet undefined DNA sequence(s) in the induction of UVIS, which is then responsible for activation of the uPA gene.

J Nucl Med, 1987 Feb, 28(2), 209 - 17
Radiometric assay of bacterial growth: analysis of factors determining system performance and optimization of assay technique; Boonkitticharoen V et al.; A quantitative technique for the measurement of 14CO2 released from a bacterial culture was evaluated . The technique uses liquid scintillation counting to record 14CO2 accumulation on a fluor-impregnated filter paper within a double-chambered scintillation vial that also houses the bacterial growth medium . We have successfully identified and corrected the major causes for a variably low detection efficiency, and also established the optimum mixture of reagents for the detection system . Incorporation of Triton X-100 into the scintillation fluid used for the detector reduced the variability between identical assays in a single batch from 50% to 5%, and, in conjunction with an increase in the scintillator concentration, raised the counting efficiency from 30% to 70-88% . The response of the improved detector is linear over a wide range of count-rates . Another significant modification was the interchange of growth and detector chambers . Overall, a 40-fold increase in count-rate during the exponential phase of bacterial growth was obtained by improving 14CO2 detection efficiency, increasing the rate of 14CO2 transfer from liquid to gas phases and enlarging the growth supporting capacity of the detector system . The minimum detection time for bacterial growth was shortened and the exponential phase of bacterial proliferation was lengthened by at least 2 hr . High counting efficiency, precision, and linearity make the improved detector a sensitive and reliable tool for radiometry of bacterial growth and metabolism.

Am J Physiol, 1987 Feb, 252(2 Pt 1), C232 - 8
Isolation, growth, and characterization of a gluconeogenic strain of renal cells; Gstraunthaler G et al.; LLC-PK1 cells, derived from pig kidney, retain several properties of the proximal tubule, but are incapable of gluconeogenesis, due to the lack of fructose-1,6-bisphosphatase (FBPase) {Am . J . Physiol . 248 (Cell Physiol . 17): C181-185, 1985} . Cells incapable of gluconeogenesis require a hexose, pentose, or nucleoside to provide ribose-5-phosphate for RNA biosynthesis . To induce or select cells that express FBPase activity, we cultured LLC-PK1 cells in glucose-free medium . We obtained cells (designated LLC-PK1-FBPase+) that express FBPase activity and are capable of growing in the complete absence of sugars or nucleosides . The cells have apical membrane enzyme activities that differ from those of wildtype cells . Tests of metabolic flow through the gluconeogenic pathway, using 3-mercaptopicolinic acid, a specific inhibitor of phosphoenolpyruvate carboxykinase, confirmed that the cells are gluconeogenic . LLC-PK1-FBPase+ cells grown in medium containing 5 mM glucose for five weekly passages continued to express FBPase activity and apical membrane enzyme activities characteristic of the FBPase+ strain . When switched back to glucose-free medium, they proliferated well . The strain appears to be stable . It should provide a model for studying the relationship between gluconeogenesis and other proximal tubule functions . An incidental finding is that in both strains, the activity of lactate dehydrogenase varied directly with the concentration of glucose in the growth medium, indicating that the expression of lactate dehydrogenase may be regulated by glucose or a metabolite of glucose.

Proc Natl Acad Sci U S A, 1987 Feb, 84(4), 1005 - 9
Production of hepatitis B virus particles in Hep G2 cells transfected with cloned hepatitis B virus DNA; Sells MA et al.; The hepatoblastoma cell line Hep G2 was transfected with a plasmid carrying the gene that confers resistance to G418 and four 5'-3' tandem copies of the hepatitis B virus (HBV) genome positioned such that two dimers of the genomic DNA are 3'-3' with respect to one another . Cells of one clone that grew in the presence of G418 produce high levels of hepatitis B e antigen and of hepatitis B surface antigen . HBV DNA is carried by these cells as chromosomally integrated sequences and episomally as relaxed circular, covalently closed, and incomplete copies of the HBV genome . Viral DNA was detected also in conditioned growth medium at the buoyant densities characteristic for infectious Dane and immature core particles . Finally, HBV-specific components morphologically identical to the 22-nm spherical and filamentous hepatitis B surface antigen particles as well as 42-nm Dane particles were visualized by immunoelectron microscopic analysis . Therefore, we have demonstrated that the Hep G2 cell line can support the assembly and secretion not only of several of the replicative intermediates of HBV DNA but also of Dane-like particles . This in vitro system can now be used to study the life cycle of HBV and the reaction of immunocompetent cells with cells carrying HBV.

Eur J Cell Biol, 1987 Feb, 43(1), 35 - 47
Keratin filament disruption in interphase and mitotic cells--how is it induced?
Tolle HG, Weber K, Osborn M.
We have studied the lability of keratin intermediate filaments in epithelial cell lines to try to understand the molecular mechanism that cause the ultrastructural transition from 10 nm filaments to the ball-like aggregates containing 2 to 3 nm filaments . Our results suggest that different growth conditions used in different laboratories may explain some but not all of the discrepancies in the literature on mitotic keratin filament disruption . Such disruption is not only cell type, but also subclone dependent and can be manipulated in one instance by altering the NaHCO3 concentration of the growth medium . An apparently similar filament to aggregate transition can be induced in interphase cells of some epithelial cell lines by incubation in a cold hypotonic buffer, or when cells are pretreated with phorbol ester and then incubated in cold physiological saline . A putative dialyzable and heat-stable factor present in medium conditioned by the growth of particular epithelial cell types may be required for disruption . Keratin polypeptide phosphorylation may play a role in filament labilization.

Biochim Biophys Acta, 1987 Jan 20, 923(1), 29 - 34
The inhibitory effect of dithiothreitol on the assembly of the filamentous phage fd; Vaccaro M et al.; Assembly of the filamentous phage fd is preceded by the formation of a complex between the viral single-stranded (ss) DNA and the virally coded gene 5 protein (gene 5 protein-ssDNA complex) . The presence of 5 mM dithiothreitol in the growth medium prevents phage production; however, phage infection, phage DNA replication and phage genome expression are still observed . In contrast, the gene 5 protein-ssDNA complex is not formed in the presence of dithiothreitol in vivo, although the complex is not affected by the disulfide reducing agent in vitro . Furthermore, host lipid composition is altered by growth in the presence of dithiothreitol . The zwitterionic lipid, phosphatidylethanolamine, increases while the cationic phospholipid content, cardiolipin and phosphatidylglycerol, decreases . This suggests a role for lipids or membranous structures in the process of gene 5 protein-ssDNA complex formation.

Eur J Biochem, 1987 Jan 15, 162(2), 305 - 9
DNA synthesis in vivo and in vitro in Escherichia coli irradiated with ultraviolet light; Banfalvi G et al.; DNA synthesis was followed in vivo and in permeable Escherichia coli after ultraviolet light irradiation, irradiation and incubation in a growth medium containing chloramphenicol and in unirradiated cells . In vitro, replicative type DNA synthesis was partially restored after incubation of cells in medium containing chloramphenicol, but not in vivo . The DNA was pulse-labeled in permeable cells in the presence of deoxyribonucleoside triphosphates and ribonucleoside triphosphates . dCTP was replaced by 5-Hg-dCTP as a substrate for DNA synthesis . Hg-DNA was separated from cellular nucleic acids on thiol-agarose affinity columns . The 5' termini of newly synthesized DNA were analyzed after treatment with alkaline phosphatase and rephosphorylation with polynucleotide kinase and {gamma-32P}ATP . DNA synthesis in unirradiated permeable E . coli represents a replicative process dependent on ATP and inhibited by novobiocin . About 70% of the nascent DNA carried terminally labeled RNA moiety at its 5' end . In vitro DNA synthesis in irradiated cells was suppressed and hardly influenced by the presence of ATP or novobiocin . The 5'-RNA content of this cell population was less than 5%.

Differentiation, 1987, 34(2), 79 - 87
Density-dependent induction of discoidin-I synthesis in exponentially growing cells of Dictyostelium discoideum; Clarke M et al.; The synthesis of the lectin, discoidin I, by vegetative cells of Dictyostelium discoideum (strain NC4) was monitored using immunoblot analysis and indirect immunofluorescence . Suspension cultures were used, so that the D . discoideum cell density and the concentration of bacteria could be controlled . Discoidin-I production was found to be a function of the relative densities of D . discoideum cells and food bacteria . Synthesis was initiated in exponentially growing D . discoideum cells approximately three generations before depletion of the food supply . In the growth medium of cells producing discoidin I, a soluble activity was detected that caused low-density cells to begin discoidin-I synthesis . This activity was not dialyzable and was destroyed by heat . A similar activity was produced by AX3 cells during axenic growth . Density-dependent induction of other 'early developmental' proteins was also detected in wild-type cells . These findings suggest that the expression of several 'early developmental' genes is regulated by a mechanism that measures cell density relative to food supply, not by starvation per se.

Dev Biol Stand, 1987, 66, 87 - 90
Cross flow diafiltration of serum with basal medium suitable for growth of hybridomas, sterilization and protein reduction; Jungbauer A et al.; Fetal Calf Serum (FCS) was extensively extracted by cross flow diafiltration (Pellicon, Millipore) and sterilized using the basal growth medium (DMEM) for the extraction . Ultrafiltration membranes of 10(5) and 3 X 10(5) Dalton cut off were used respectively . The diafiltrates were used for hybridoma cultivation and the results of growth and mAb-production were compared with standard medium (DMEM + 5% FCS) . Slightly reduced mAb-titers were achieved . These were, however, compensated by decreased concentration of contaminating protein and higher specific mAb/protein ratio as examined by SDS-PAGE and enzyme linked immuno electro transfer blot (EITB).

Int Arch Allergy Appl Immunol, 1987, 82(3-4), 444 - 6
Physicochemical characterization of a major protein allergen, Der p I, from the house dust mite, Dermatophagoides pteronyssinus . Amino acid analysis and circular dichroism studies; Stewart GA et al.; A major house dust mite allergen, Der p I, was isolated from spent growth medium and physicochemically characterized . These studies show that the allergen is monomeric, contains approximately 216 residues and 4 intra-chain disulphide bonds . The N-terminal amino acid is threonine . Circular dichroism studies show that the allergen contains 10% alpha-helical, 50% beta-pleated sheet and 40% random structures.

Prostate, 1987, 10(2), 153 - 61
Primary culture of epithelial cells derived from the rat ventral prostate: formation of three-dimensional acinus-like structure in collagen gel; Kawamura H et al.; Rat ventral prostate epithelial cells were cultured in collagen gel after collagenase digestion . The primary cultures were mainly composed of single and spherical cells . After 10 days incubation in growth medium containing insulin, transferrin, and cholera toxin, there was a 3.8-fold increase in cell numbers, aggregates of which formed three-dimensional acinus-like structures . These structures consisted of one layer of cells surrounding the lumen . The cells were joined together with a junctional complex and had microvilli on the luminal surface and secretory vacuoles in the cytoplasm facing the lumen . The ultrastructural features of the cells were not altered by growth medium containing steroids . This culture system may prove to be very useful in elucidating proliferation, organization, and differentiation of prostatic epithelial cells in relation to the extracellular matrix and stromal cells.

Brain Res, 1987 Jan, 428(1), 133 - 5
Phorbol ester stimulates proliferation of astrocytes in primary culture; Murphy S et al.; Near-confluent primary cultures of astrocytes from the neonatal rat cerebral cortex were transferred to low serum (0.1%) growth medium for 24 h before a single addition of phorbol-12-myristate-13-acetate (0.01-100 ng X ml-1), a phorbol ester which mimics diacylglycerol activation of protein kinase C . After 48 h the cultures were pulsed with {methyl-3H}thymidine . Cultures exposed to phorbol ester exhibited dose-dependent increases in thymidine incorporation which were reversed by amiloride.

Int J Radiat Biol Relat Stud Phys Chem Med, 1987 Jan, 51(1), 29 - 38
The effect of thioredoxin on the radiosensitivity of bacteria; Lunn CA et al.; The ability of Escherichia coli thioredoxin to protect cells from lethal amounts of gamma radiation was tested using bacterial strains engineered to contain different amounts of thioredoxin per cell . Cells grown to late stationary phase demonstrated a decreasing sensitivity to gamma-radiation with increasing amounts of thioredoxin per cell . Exponentially growing cells were equally sensitive to the gamma-radiation regardless of the intracellular concentration of thioredoxin . Cells exhibiting the radiation-resistant phenotype in the stationary phase reverted to the radiation-sensitive phenotype when diluted into fresh growth medium . These results suggest that thioredoxin can protect cells from gamma-radiation under certain metabolic conditions.

Toxicol Pathol, 1987, 15(2), 238 - 40
Effect of 4-hydroxynonenal on c-myc expression; Barrera G et al.; The 4-hydroxynonenal aldehyde (HNE), a product of lipid peroxidation with high biological activity, inhibits cancerous growths in vivo and in vitro . The mechanism by which this aldehyde acts is not yet understood . The c-myc oncogene seems to be involved in the regulation of cellular multiplication and transformation . We evaluated the c-myc expression and the RNA, DNA and protein synthesis in K562 cells . These cells were incubated for 1 hour in presence of several aldehyde concentrations (range 5.10(-7) to 10(-4)), then washed and kept for 20 hours in a growth medium until used . HNE inhibited both the nucleic acids and protein synthesis in a dose dependent manner, and c-myc expression was evaluated in the K562 cells after incubation with 10(-4) M or 10(-6) M HNE . HNE inhibited c-myc expression only at the highest dose . These preliminary results may suggest that the inhibition of c-myc expression is related to nucleic acid synthesis inhibition following HNE exposure.

Eye, 1987, 1 ( Pt 6), 722 - 7
The effects of steroids on the human lens epithelium; Jacob TJ et al.; A study of anterior capsules from cataractous lenses revealed that there are marked abnormalities in epithelial structure associated with cataract . In certain cases a distinctive reticulated pattern was observed in whole mounts of the anterior capsule and of these a higher number than expected were from patients receiving steroid medication . In tissue culture experiments it was found that the presence of steroids in the growth medium (10 microM prednisolone) adversely affected the growth of human lens epithelial cells . These observations are consistent with the hypothesis that steroid-induced cataracts are the result of effects on anterior lens epithelial cell function.

Acta Physiol Hung, 1987, 70(1), 105 - 10
Presence (hPL, prostaglandin) and absence (triiodothyronine, thyroxine) of hormones in Tetrahymena: experimental facts and open questions; Csaba G et al.; The RIA technique detected prostaglandin (PGF2) and human placetal lactogen (hPL) in Tetrahymena cultures grown in bacto tryptone + yeast extract medium which, however, itself contained these hormones . About one to two per cent of the total hormone content of the medium was demonstrated intracellularly . Treatment with diiodotyrosine (T2), which is known to stimulate the growth of Tetrahymena, was followed by a decrease in the intracellular prostaglandin level . Triiodothyronine and thyroxine were not detected in Tetrahymena or in the medium, and did not appear in it on induction with TSH either . In the light of these observations it might well be doubted that prostaglandin was native in Tetrahymena: the use of synthetic media, and/or a reliable demonstration of the hormone content of the growth medium is recommended for evidence of hormone biosynthesis by unicellular organisms.

Membr Biochem, 1987-88, 7(2), 115 - 30
Na+-linked active transport of ascorbate into cultured bovine retinal pigment epithelial cells: heterologous inhibition by glucose; Khatami M; The transport of ascorbate into cultured bovine retinal pigment epithelial (RPE) cells is reported . Primary or subcultured RPE cells were incubated in the presence of 10-500 microM L-{carboxyl-14C}-ascorbate for various periods of time . Accumulation of ascorbate into RPE cells followed a saturable active transport with a Km of 125 microM and a Vmax of 28 pmole/micrograms DNA/min . RPE intracellular water was calculated to be 0.8 pL/cell, and the transported cellular ascorbate concentration was 7.5 +/- 0.8 mM . Replacement of 150 mM NaCl in the incubation media with choline-Cl strongly inhibited (80 +/- 8%) ascorbate uptake into cultured RPE cells . Although the depletion of cellular ATP by 2,4-dinitrophenol and the inhibition of Na+-K+-ATPase by ouabain reduced ascorbate transport into RPE significantly, active transport of ascorbate was not entirely inhibited by these metabolic inhibitors . The ascorbate analogue, D-isoascorbate, competitively inhibited ascorbate transport into cultured RPE with a Ki of 12.5 mM . Cells grown in the presence of 5 to 50 mM alpha-D-glucose in the growth media did not differ in their ability to transport ascorbate . In contrast, the presence of alpha-D-glucose or its nonmetabolizable analogues, 3-0-methyl-glucose, alpha-methyl-glucose, and 2-deoxy-glucose, but not L-glucose or beta-D-fructose, in the incubation media inhibited ascorbate transport . myo-Inositol (10 or 20 mM) also inhibited ascorbate transport into RPE cells . The active uptake of ascorbate into cultured RPE cells was primarily coupled to the movement of sodium ion down its electrochemical gradient . A bifunctional, cotransport carrier possessing an ascorbate-binding site and a sodium-binding site may be involved in the ascorbate uptake system . The inhibition of ascorbate uptake by sugars appeared to be heterologous in nature, occurring between two distinct carrier systems, both of which were dependent on the sodium ions.

Curr Genet, 1987, 11(5), 399 - 406
Saccharomyces cerevisiae strains sensitive to inorganic mercury . III . Tyrosine uptake; Ono B et al.; In Saccharomyces cerevisiae, the HGS2-1 allele confers sensitivities to inorganis mercury (Ono and Sakamoto 1985) and to excess fermentable sugars such as glucose (Sakamoto et al . 1985); exogenous tyrosine antagonizes both inorganic mercury and excess glucose . In this study, the inorganic mercury sensitive strain has been shown to have about twice more glucose-1,6-bisphosphate and slightly less pyruvate than the normal strains, suggesting that the inorganic mercury sensitive strain has the reduced aldolase activity . It has been also shown that the growth retarded cells accumulate trehalose, by which the lower level of glucose-6-phosphate in the inorganic mercury sensitive strain is accounted for, and that inorganic mercury, presumably excess glucose also, causes growth inhibition via depletion of cellular tyrosine . The mechanism how cellular tyrosine is depleted by inorganic mercury or excess glucose is accounted for by the facts that (1) the tyrosine uptake activity is decreased with increase of glucose concentration in growth medium, (2) HGS2-1 enhances the effect of glucose on the tyrosine uptake activity, and (3) inorganic mercury inhibits the tyrosine uptake system by binding to its SH-group(s) . Thus, it is concluded that the role of tyrosine is not to detoxify inorganic mercury nor excess fermentable sugars but simply to counteract depletion of cellular tyrosine induced by them.

Radiat Environ Biophys, 1987, 26(4), 301 - 12
Effects of low X-ray doses in Saccharomyces cerevisiae; Jordan A et al.; Three strains of Saccharomyces cerevisiae with different capacities for repair of radiation damage (RAD, rad18, and rad52) have been tested for their colony forming ability (CFA) and growth rates after application of small X-ray doses from 3.8 mGy to 40 Gy . There was no reproducible increase in CFA observable after application of doses between 3.8 mGy and 4.7 Gy . X-ray doses of 40 Gy causing an inactivation of CFA from 90% to 50%, depending on the repair capacity of the strains used, caused a reduced increase in optical density during 2 h buffer treatment in comparison to unirradiated cells . This reduction however, is reversible as soon as the cells are transferred into nutrient medium . One hour after transfer into growth medium the portions of cells with large buds (G2 and M phase) and cells with small buds (S phase) are drastically different in irradiated cells from those obtained in unirradiated cells . The time necessary for separation of mother and daughter cells is prolonged by X-ray irradiation and the formation of new buds is retarded.

J Neural Transm Suppl, 1987, 24, 247 - 59
Phosphatidylcholine as a precursor of choline for acetylcholine synthesis; Blusztajn JK et al.; It has been hypothesized that the selective vulnerability of certain brain cholinergic neurons in Alzheimer's disease may reflect the unique way that choline is utilized by these neurons, i.e . not only as a component of major membrane phospholipids, e.g . phosphatidylcholine (PC), but also as a precursor of their neurotransmitter, acetylcholine (ACh) . A prolonged utilization of choline liberated from PC, for ACh production, without adequate resynthesis of this lipid, might result in a net loss of the phosphatide followed by an impairment of membrane function and loss of cellular viability . Studies described in this paper, performed on electrically stimulated striatal slices and on cholinergic cell lines, test this hypothesis . 1) Electrically-stimulated striatal slices continue to release ACh, and sustain their free choline and ACh levels, even when perfused with a choline-free medium . Striatal levels of PC decline under these circumstances, and this decline can be blocked by adding tetrodotoxin (which blocks neuronal depolarization) or choline to the medium . The other major membrane phospholipids, phosphatidylserine and phosphatidylethanolamine, also decline proportionately to PC when slices are stimulated in the absence of choline . 2) In a population of purely cholinergic cells (human neuroblastoma, LA-N-2), ACh can be synthesized from choline derived from degradation of endogenous PC formed de novo by methylation of phosphatidylethanolamine . 3) PC content of cells in culture (neuroblastoma X glioma hybrid, NG 108-15) can be altered by adding various amounts of choline to the growth media . The proportion of PC in the cells apparently affects cellular survival and rate of growth . Taken together these data demonstrate that cholinergic neurons utilize the choline stored in PC to synthesize ACh; that this process may lead to a depletion in membrane phospholipids (when choline supply is inadequate); and that the resulting changes in neuronal membrane composition might adversely affect cellular viability.

Nahrung, 1987, 31(4), 285 - 90
Proteolysis by toxigenic Aspergillus nidulans from Nigerian palm produce; Ogundero VW; The submerged cultures of Aspergillus nidulans had optimal growth and protease production at 37 degrees C and within 6 days of incubation . A rapid drop in pH of the growth medium from 6.9 to 4.8 and a subsequent gradual rise was recorded with the period of incubation . The acid-protease produced was purified by a combination of ethanolic precipitation, ultrafiltration and fractionation on DEAE-cellulose and Sephadex G-200 . A single peak showing protease activity was subsequently obtained with a 16-fold increase in specific activity and a recovery value of 36% . The purified enzyme had optimal activity on casein and gelatin at pH 5.4 and a temperature of 40 degrees C.

Microbios, 1987, 50(202), 43 - 59
Effects of lemongrass oil on the morphological characteristics and peptidoglycan synthesis of Escherichia coli cells; Ogunlana EO et al.; The antibacterial effect of lemongrass oil, obtained from the aerial part of Cymbopogon citratus, on cells of Escherichia coli was investigated by electron microscopy and by measuring cell wall formation . Two strains of E . coli K-12 were used, one of which required diaminopimelic acid in the growth medium for its murein formation . Lemongrass oil was found to elicit morphological changes like filamentation, inhibition of septum formation, spheroplast formation, production of 'blisters', 'bulges' or mesosomes, as well as lysis and development of abnormally shaped cells . The incorporation of radioactively labelled diaminopimelic acid into the cell wall murein of strain W7, was inhibited by lemongrass oil in a dose dependent way . The sequence of changes induced by lemongrass oil on bacterial cell morphology and also its interference with murein synthesis in E . coli cells were interpreted to involve the penicillin binding proteins PBP 2 and PBP 3.

Curr Genet, 1987, 11(5), 393 - 8
Biosynthetic and regulatory role of lys9 mutants of Saccharomyces cerevisiae; Winston MK et al.; Derepression of lysine biosynthetic enzymes of Saccharomyces cerevisiae was investigated in lys9 auxotrophs which lack saccharopine reductase activity . Five enzymes (homocitrate synthase, homoisocitrate dehydrogenase, alpha-aminoadipate aminotransferase, alpha-aminoadipate reductase and saccharopine dehydrogenase) were constitutively derepressed in all lys9 mutants with up to eight-fold higher enzyme levels than in isogenic wild-type cells . Levels of these enzymes in lys2, lys14, and lys15 mutants were the same or lower than those in wild-type cells . The regulatory property of lys9 mutants exhibited recessiveness to the wild-type gene in heterozygous diploids . Unlike the mating type effect, homozygous diploids resulting from crosses between lys9 auxotrophs exhibited even higher levels of derepressed enzymes than the haploid mutants . Addition of a higher concentration of lysine to the growth medium resulted in reduction of enzyme levels although they were still derepressed . These results suggest that lys9 mutants represent a lesion for the saccharopine reductase and may represent a repressor mutation which in the wild-type cells simultaneously represses unlinked structural genes that encode for five of the lysine biosynthetic enzymes.

Gene, 1987, 54(1), 147 - 53
Induction of v-mos and activated Ha-ras oncogene expression in quiescent NIH 3T3 cells causes intracellular alkalinisation and cell-cycle progression; Doppler W et al.; We have tested the effect of the expression of the v-mos oncogene, and the activated human Ha-ras oncogene and its proto-oncogene form on the intracellular pH and on cell cycle progression . The Ha-ras proto-oncogene and the oncogenes under the transcriptional control of the MMTV-LTR promoter were introduced into NIH 3T3 cells . The cells were synchronized at G0/G1 in low-serum medium and the transfected genes were induced by glucocorticoid hormone addition to the growth medium . Expression of the v-mos and the activated form of Ha-ras oncogenes mimics the effect of growth factors and activates the Na+/H+ antiporter . The oncogenes increase the internal pH and provoke initiation of S-phase . The proto-oncogene form of Ha-ras does not induce intracellular alkalinisation and has only a weak mitogenic effect . Our results suggest the oncogenic proteins p21ras and p37mos influence the mitogenic signal transduction pathways at a point prior to the activation of the Na+/H+ antiporter.

J Cancer Res Clin Oncol, 1987, 113(1), 31 - 40
Establishment, growth properties, and morphological characteristics of permanent human small cell lung cancer cell lines; Bepler G et al.; Cell lines from SCLC were established with a success rate of 43% from different metastatic sites of treated and untreated patients . All 6 SCLC cell lines grew as floating cell aggregates without substrate adherence . The degree of aggregation ranged from very tight spheroids to very loose sheets and chains . This gross morphological property showed a striking correlation to the PDT, with short PDTs in loose growing cell lines and long PDTs in tight growing cell lines . Cell size and nuclear features, i.e., chromatin pattern and nucleolar prominence, also seemed to correlate with the PDT and gross morphology . All SCLC cell lines had dense core granules by electron microscopical examination . Several different serum-free and serum-supplemented growth media were tested for their feasibility in establishing and permanently growing SCLC . Serum-free SIT medium and SIT2.5 medium provided the best results in liquid culture . For semisolid SCLC cultivation, R 10 medium was superior to all other media tested . These cell lines are currently under intensive biochemical, molecular biological, and cytogenetical investigation in different laboratories and thus provide a tool for studying the biology of lung cancer.

Oncology, 1987, 44(5), 327 - 30
Differential synthesis and replication of DNA in the Neurospora crassa slime mutant versus normal cells: role of carcinogens; Beljanski M et al.; Small quantities of carcinogens, dl-ethionine, thiotepa, actinomycin D, and 1-(2-chloroethyl-3-cyclohexyl)-1-nitrosourea (CCNU) stimulated in vitro deoxyribonucleic acid (DNA) synthesis of the slime mutant of Neurospora crassa, while there was practically no effect on the DNA from the normal wild type 74A strain . All of these compounds caused increased strand separation in the mutant DNA of N . crassa, but no separation of normal DNA strands . The growth (in vivo tests) of the N . crassa slime mutant, but not its wild type, was markedly increased when nontoxic concentrations of one of the carcinogens (dl-ethionine) tested were present in the growth medium . These observations suggest that, unlike the wild type N . crassa, the slime mutant allows an excessive and unscheduled replication, indicating destabilized nature of its DNA.

Experientia Suppl, 1987, 52, 477 - 80
Abnormal copper metabolism and regulation of metallothionein gene expression in Menkes' disease; Leone A et al.; Menkes' kinky hair disease, a lethal X-linked recessive trait, is characterized by abnormal copper accumulation in several non-hepatic tissues . The level of many copper enzymes is severely reduced, leading to damage of the connective and nervous tissues of the patients . Cultured skin fibroblasts from Menkes' patients retain more copper then normal controls, and the excess metal is bound to metallothionein . Low doses of copper in the media induce MT gene transcription in Menkes' but not in normal cells . Transfection experiments using a plasmid containing the mouse MT-I promoter fused to the enzyme chloramphenicol acetyl transferase show that the activation of the mMTI promoter is in trans . Two other effects are observed in Menkes' cells: (a) two heat-shock like proteins are synthesized in response to low doses of copper in the growth medium, and (b) Menkes' cells are more sensitive then normal fibroblasts to copper toxicity . Our interpretation of these results supports a model for a defect in one or more steps in copper metabolism or transport.

C R Seances Soc Biol Fil, 1987, 181(3), 287 - 93
{Giardia intestinalis: influence of the composition of the osmolarity of the medium on in vitro excystation}; Chochillon C et al.; The in vitro excystation of Giardia intestinalis was studied to make the osmolarity (from 50 to 500 mosmol/l) and the components of growth medium (MCI saline solution, MCII glucose solution, MCIII nutritive solution) varying . The percentage of excystation, the viability and the generation time were determined . Excystation was observed in the saline solution between 100 to 450 mosmol/l after cyst acid pepsin incubation . The trophozoite viability was increased by glucose addition (60 min in MCI; 300 min in MCII) . Only a rich medium (MCIII) permitted a generation time from 225 to 425 mosmol/l.

J Bacteriol, 1987 Jan, 169(1), 157 - 63
Immunoelectron microscopic analysis of elongation of type 1 fimbriae in Escherichia coli; Lowe MA et al.; Using 10- and 20-nm-diameter gold particles conjugated to an antifimbrial monoclonal antibody, we analyzed the location of assembly of newly formed subunits on growing type 1 fimbriae of Escherichia coli . Fimbriae were removed from an E . coli K-12-derived strain, CSH50, by blending . Blended cells were allowed to regenerate their fimbriae in growth medium for approximately 25 min, after which they were labeled with a 20-nm-gold-monoclonal antibody probe . Continued outgrowth of these labeled fimbriae was allowed for additional time intervals, after which they were labeled with a 10-nm-gold-monoclonal antibody probe . The resulting fimbriae, double labeled with 10- and 20-nm-diameter gold particles, were examined in an electron microscope . The pattern of labeling on individual fimbrial organelles indicated morphologically that newly synthesized subunits are added to a growing organelle at its base.

Endocr Res, 1987, 13(3), 243 - 50
The effects of monensin on inhibition of steroidogenesis and disruption of the Golgi complex in adrenal cells are both reversible!
Cheng B, Horst IA, Kowal J.
Following 15-30 min exposure to monensin, adrenocorticotropic hormone (ACTH)-stimulated steroidogenesis in cultured adrenal cells is inhibited by 37-48% . Electron microscopic studies reveal that, in monensin-treated cells, the Golgi complexes are disrupted into large vacuolar structures with loss of its organized structure indicating that the action of monensin on the organelles is comparably rapid . The inhibition is fully reversed after removal of the monensin-containing medium and exposure to fresh growth medium for a subsequent 4-24 h prior to stimulation . Concomitant with the restoration of full steroidogenic activity, the disrupted organelles are extensively reorganized in the cells after exposure to fresh growth medium for 4-24 h . These findings, which demonstrate, for the first time, a correlation between the morphology of the Golgi complex and steroidogenic activity, strengthen the possibility that the organelle may be involved in the regulation of steroidogenesis.

Folia Microbiol (Praha), 1987, 32(5), 402 - 10
Subcellular localization of enzymes in Streptomyces aureofaciens and its alteration by benzyl thiocyanate . I . Phosphatases and ATP-glucokinase; Trilisenko LV et al.; Mycelia of a low- and a high-production strain of Streptomyces aureofaciens were converted into protoplasts and divided into five subcellular fractions in order to localize exopolyphosphatases (EC 3.6.1.11), triphosphatase (EC 3.6.1.25), inorganic diphosphatase (EC 3.6.1.1), apyrase (EC 3.6.1.5) and glucokinase (EC 2.7.1.2) . The highest specific activity of enzymes hydrolyzing polyphosphates was found in cytoplasmic vesicles and membranes . Triphosphatase was detected in the periplasmic fraction . Periplasmic vesicles and cytoplasm exhibited a high activity of diphosphatase . Apyrase was found mainly in the fractions of membranes and cytoplasmic vesicles . Glucokinase was a cytoplasmic enzyme . The enzymes were released from membrane structures into cytoplasm or periplasmic space if benzyl thiocyanate (10 microM) was present in the growth medium.

Microbios, 1987, 51(208-209), 151 - 7
Exposure of herpes simplex virus type 1 and its host cells to cigarette smoke in vitro; Bardell D; A 1.0 ml suspension of herpes simplex virus type 1 in tissue culture growth medium was exposed at 37 degrees C to eight puffs of smoke from one cigarette . Each puff consisted of 25.0 ml of mainstream smoke, and was delivered by a mechanical smoking apparatus . At 0, 15 and 60 min after exposure the virus was assayed for infectivity using HEp-2 cells as the host . Neither filter nor non-filter cigarette smoke affected the infectivity of the virus, or the viability of HEp-2 cells treated with smoke in a manner similar to the virus . The filter cigarette contained 19.0 mg of tar and 1.2 mg of nicotine, and the non-filter cigarette had 23.0 mg of tar and 1.4 mg of nicotine . Smoke from four non-filter cigarettes delivered over a 4 h period caused a 4-log10 drop in virus infectivity titre, and smoke from four filter cigarettes caused more than a 1-log10 drop in titre . Smoke from four non-filter or filter cigarettes was highly cytocidal for HEp-2 cells.

Curr Genet, 1987, 12(4), 283 - 9
Particular RNA primer from growth medium differentially stimulates in vitro DNA synthesis and in vivo cell growth of Neurospora crassa and its slime mutant; Dutta SK et al.; Purine rich small "RNA-primer" molecules (about 10-12 nucleotides), secreted into the growth medium of 3-h germinated conidia of N . crassa, strongly stimulated a concentration-dependent in vitro DNA synthesis of N . crassa slime mutant as well as DNAs from the human cancer cells but did not affect that from normal cells . These "RNA-primer" molecules stimulated also in vivo cell growth of N . crassa slime mutant, but not of the N . crassa wild type . Our studies suggest that DNAs from the slime mutant of N . crassa as well as DNAs from human cancerous cells provide increased sites for enhanced in vitro and in vivo replication of DNAs . "RNA-primer" molecules can be hydrolyzed by T1 RNase but not by pancreatic RNase.

Exp Pathol, 1987, 31(4), 231 - 41
Serum modulation of insulin action: effects on rat gingival fibroblast metabolism; Zebrowski EJ et al.; Serum is reported to reduce the sensitivity of cells in culture to insulin . The effect of serum concentration in the growth medium on the responsiveness of control (C) and streptozotocin diabetic (D) rat gingival fibroblasts to insulin was measured by monitoring cellular DNA, RNA, total protein and medium hydroxyproline (collagen) levels, as well as the cellular uptake of C14-alpha-NH2-isobutyrate (alpha-AIB) and H3-2-deoxyglucose (2DG) . The cells were grown in alpha-MEM at 5, 10, 15 or 20% FCS with 0, 10(-12), 10(-10), 10(-8) and 10(-6) M insulin used at each serum level . Insulin effects in the absence of serum were not assessed . For both the C and D rat cells, the DNA increased proportionately with increasing serum and insulin levels . In contrast, RNA and total cell protein increased with increase in insulin and decrease in serum, the magnitude of the effect being greater in C than in D cells . The insulin stimulation of both 2DG and alpha-AIB uptake and of collagen secretion varied inversely with serum concentrations . The magnitude of the insulin-serum interaction on metabolite uptake was greater for the D rat cells . These data indicate that serum significantly reduced the cell response to insulin stimulated metabolite uptake and collagen secretion, but was without apparent effect on the intracellular insulin responsive parameters . They suggest that serum factor(s) interfere with the availability of insulin to the cell and that the D rat cells are most affected.

Pediatr Res, 1987 Jan, 21(1), 72 - 8
Tissue culture of normal and cystic fibrosis sweat gland duct cells . I . Alterations in dome formation; Hazen-Martin DJ et al.; The elucidation of the underlying defect in fluid secretion by cystic fibrosis (CF) sweat glands is hindered by the unavailability of an experimental model for investigating this disease . As a potential model system, a serum-free growth medium was developed that supports the explant growth of epithelial cells from fragments of human skin . Immunohistochemical analysis demonstrated that these epithelial cell outgrowths originated from the duct of the sweat gland . By electron microscopy, the cells were demonstrated to possess keratinocyte-like morphology as noted by the presence of a multilayered outgrowth of cells containing well-defined keratin bundles . Identical outgrowths from skin biopsies of CF patients were compared to normal outgrowths and alterations were noted to occur in dome formation and in the number of intercellular spaces between cells . Doming alterations were also noted to occur in the CF heterozygous state . No differences in cell fine structure or in growth factor requirements for cell proliferation were noted between normal and CF cells . The potential use of this system as a model for CF research is discussed.

Biochim Biophys Acta, 1986 Dec 19, 889(3), 287 - 300
Effects of cholesterol and lipoproteins on endocytosis by a monocyte-like cell line; Esfahani M et al.; The human monocyte/macrophage-like cell line U937 is a cholesterol auxotroph . Incubation of these cells in the growth medium in which delipidated fetal calf serum has been substituted for fetal calf serum depletes cellular cholesterol and inhibits growth . The cholesterol requirement of these cells for growth can be satisfied by human low-density lipoprotein (LDL), and very-low-density lipoprotein (VLDL), but not by high-density lipoprotein (HDL) . U937 cells can bind and degrade LDL via a high-affinity site and this recognition is altered by acetylation of LDL . This indicates that these cells express relatively high LDL receptor activity and low levels of the acetyl-LDL receptor . The cells were used to study the role of cholesterol in lectin-mediated and fluid-phase endocytosis . Growth of the cells in the medium containing delipidated fetal calf serum results in impairment of both concanavalin A-mediated endocytosis of horseradish peroxidase and concanavalin A-independent endocytosis of Lucifer Yellow . Supplementation of the medium with cholesterol prevents cellular cholesterol depletion, supports growth and stimulates Lucifer Yellow endocytosis but fails to restore horseradish peroxidase endocytosis . However, if the cells are incubated in the presence of no less than 40 micrograms LDL protein/ml to maintain normal cell cholesterol levels, concanavalin A-mediated endocytosis of horseradish peroxidase is activated . The effect of LDL is specific since neither VLDL nor HDL3 at the same protein concentration activates horseradish peroxidase uptake by the cells . Furthermore, the activation of endocytosis by LDL is not inhibited by the inclusion of heparin or acetylation of the LDL indicating that binding of LDL to the LDL receptor is not required for these effects . The mediation of activation of horseradish peroxidase endocytosis by the lectin is presumed to involve binding of LDL to concanavalin A associated with the cell surface which in turn stimulates horseradish peroxidase binding and uptake by adsorptive endocytosis . The rate of fluid endocytosis and endosome formation seems to depend on cellular cholesterol content presumably because cholesterol is involved in maintaining the appropriate plasma membrane structure and fluidity.

Eur J Biochem, 1986 Dec 15, 161(3), 757 - 61
Fructose 2,6-bisphosphate in Dictyostelium discoideum . Independence of cyclic AMP production and inhibition of fructose-1,6-bisphosphatase; Aragon JJ et al.; The occurrence of fructose 2,6-bisphosphate was detected in Dictyostelium discoideum . The levels of this compound were compared with those of cyclic AMP and several glycolytic intermediates during the early stages of development . Removal of the growth medium and resuspension of the organism in the differentiation medium decreased the content of fructose 2,6-bisphosphate to about 20% within 1 h, remaining low when starvation-induced development was followed for 8 h . The content of cyclic AMP exhibited a transient increase that did not correlate with the change in fructose 2,6-bisphosphate . If after 1 h of development 2% glucose was added to the differentiation medium, fructose 2,6-bisphosphate rapidly rose to similar levels to those found in the vegetative state, while the increase in cyclic AMP was prevented . The contents of hexose 6-phosphates, fructose 1,6-bisphosphate and triose phosphates changed in a way that was parallel to that of fructose 2,6-bisphosphate, and addition of sugar resulted in a large increase in the levels of these metabolites . The content of fructose 2,6-bisphosphate was not significantly modified by the addition of the 8-bromo or dibutyryl derivatives of cyclic AMP to the differentiation medium . These results provide evidence that the changes in fructose 2,6-bisphosphate levels in D . discoideum development are not related to a cyclic-AMP-dependent mechanism but to the availability of substrate . Fructose 2,6-bisphosphate was found to inhibit fructose-1,6-bisphosphatase activity of this organism at nanomolar concentrations, while it does not affect the activity of phosphofructokinase in the micromolar range . The possible physiological implications of these phenomena are discussed.

Int J Cancer, 1986 Dec 15, 38(6), 889 - 99
Characterization of the growth inhibited substate induced in murine hepatic tumor cells during in vitro exposure to dimethylsulfoxide; Higgins PJ; Kinetic events associated with the dimethylsulfoxide (DMSO)-induced inhibition of hepatic tumor cell proliferation were studied using established lines of murine liver tumor cells (BW77-2 and Hepa-1/A1) and conditions of polar solvent treatment (1-3% final concentration in the culture medium for a period of 4 days) previously shown to increase the expression of differentiated functions in BW77-2 cells . Cell-cycle substrates of exponentially growing and DMSO-treated liver tumor cell populations were compared by flow cytometric techniques employing recently developed cytochemical criteria to identify hepatocyte cell cycle compartments based on individual cellular RNA and DNA contents (Higgins, 1985) . Suppression of hepatic tumor cell proliferation by DMSO (in non-cytotoxic concentrations) persisted only for the duration of the exposure period . Resumption of cell division was readily observed following removal of the polar solvent from the culture medium . During DMSO treatment, BW77-2 and Hepa-1/A1 cells accumulated in the G1 phase of the cell division cycle (low-population-density 3% DMSO-treated cultures were composed of 88% G1 cells compared to only 48% G1 DNA content cells in control cultures of similar population density) and exhibited a substantial shift to lower mean cellular RNA content . The relatively few S- and G2 + M-phase cells in DMSO cultures also possessed lower RNA contents compared to the corresponding cell cycle compartments in exponentially growing cultures . The mean RNA contents for the G1, S, and G2 + M compartments of DMSO-treated cells approximated 63.8, 78.6, and 74.4%, respectively, of the amounts observed in control cultures . Low-RNA G1 cells in DMSO cultures expressed a continuum of RNA distributions similar in range variation to (but at lower mean cellular RNA content levels than) cycling G1 cells in log-phase growth . Thus, G1 cells in 1% DMSO-treated populations had a mean cellular RNA content of just 25 (arbitrary RNA) units compared to over 40 units for G1 cells in exponential phase growth . Low RNA content, non-replicating, hepatic tumor cells in polar solvent-treated cultures were designated as being in the "Qi" substate (DMSO-induced quiescent-type cells) . Release of BW77-2 cells from Qi, after replacement of the DMSO-containing growth medium by medium without the polar solvent, was characterized by an increase in mean G1 RNA content and recruitment into log-phase growth.(ABSTRACT TRUNCATED AT 400 WORDS)

Neurosci Lett, 1986 Dec 12, 72(2), 215 - 20
1-Methyl-4-phenylpyridinium (MPP+) but not 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) selectively destroys dopaminergic neurons in cultures of dissociated rat mesencephalic neurons; Sanchez-Ramos J et al.; Dopaminergic neurons were studied in cultures of dissociated cells from the ventral mesencephalon of fetal rat embryos (gestational day E15-16) . After a week of growth, 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) or 1-methyl-4-phenylpyridinium (MPP+) was added to the growth medium for 24 h . Dopaminergic neurons were then visualized with tyrosine hydroxylase (TH) immunocytochemistry or catecholamine (CA) cytofluorescence . Concentrations of MPTP in the range of 10 to 100 microM obliterated CA fluorescence without affecting the number of TH-positive neurons . At concentrations greater than 100 microM, MPTP decreased the number of TH-positive neurons as well as the number of all other cell types . MPP+ (0.1-10.0 microM) produced a decrease in the number of TH-positive neurons without decreasing the total number of all cell types . The findings indicate that MPP+ but not MPTP is able to selectively destroy rat dopaminergic neurons in our cultures . The selective toxicity of MPP+ for dopaminergic neurons was partially prevented by pretreatment and co-incubation with mazindol (a selective inhibitor of dopamine uptake) but not by desipramine or deprenil, in confirmation of the notion that MPP+ enters dopaminergic neurons by the specific uptake mechanism for dopamine.

Biochim Biophys Acta, 1986 Dec 10, 884(3), 482 - 9
Non-competitive inhibition of ornithine decarboxylase by a phosphopeptide and phosphoamino acids; Tsirka SA et al.; In Tetrahymena pyriformis the cytosolic ornithine decarboxylase (L-ornithine carboxy-lyase, EC 4.1.1.17) activity is considerably inhibited by the presence of polyamines in the growth medium, while the nuclear ornithine decarboxylase is only slightly affected . Experimental evidence suggests that the presence of putrescine and/or spermidine elicits the appearance of non-competitive inhibitors of ornithine decarboxylase . One of the inhibitors has a molecular weight of 25,000 and properties of antizyme . In addition, two other low molecular weight inhibitors are extracted, one which is a phosphoserine oligopeptide, and the other which is phosphotyrosine . All inhibit non-competitively the homologous and heterologous (Escherichia coli and rat liver) ornithine decarboxylases . Similarly, non-competitive inhibition was obtained when the commercially available phosphoamino acids were tested against the already mentioned ornithine decarboxylases.

J Theor Biol, 1986 Dec 7, 123(3), 333 - 46
The basis of synchronization by repetitive dilution of a growing culture; Koch AL; Cultures of Escherichia coli have been synchronized by periodic dilution with fresh growth medium in the laboratory of Francois Kepes . When diluted by a large factor into complete test medium, the treated cultures undergo up to 12 synchronous divisions . This long term synchrony must result from an adjustment process during the periodic dilution procedure so that all cells have nearly identical biochemical properties . Robert Pritchard (University of Leicester, personal communication) suggested that this phasing would happen if the uptake of a critical nutrient was limited by the surface area of the cell during a portion of the dilution cycle . If his suggestion is valid, a general method for synchronization of almost any organism that grows exponentially and divides by binary fission into equal sized daughters should be achievable . A computer program was devised to simulate the growth of an initially asynchronous culture under periodic dilution with medium containing a single limiting nutrient . Various models of cell shape and growth were tested along with various models for the growth-limiting substrate uptake.

Z Ernahrungswiss, 1986 Dec, 25(4), 228 - 32
Effect of carbon and nitrogen sources on the chemical composition of lipids of Rhizopus delemar; Tahoun MK et al.; Soy bean extract with glucose or oleic acid in the growth medium of Rhizopus delemar affected the production of higher values of biomass, total lipids and total glycerides than when ammonium nitrate was used as a source of nitrogen . The highest amounts of biomass (12.35 g/l) and total lipids (310 mg/g dry biomass) were attained in glucose-grown mycelia . The high proportions of total glycerides and unsaturated fatty acids in lipids of Rhizopus delemar suggest the utilization of such lipid material for nutritional and industrial purposes.

Gut, 1986 Dec, 27(12), 1457 - 63
Effects of short chain fatty acids on a new human colon carcinoma cell line (LIM1215); Whitehead RH et al.; The effects of short chain fatty acids on a colon carcinoma cell line, LIM1215, have been studied . Of the four short chain fatty acids tested only butyrate at 1 mmol/l and 10 mmol/l and acetate at 10 mmol/l had significant effects on this cell line . The addition of butyrate to growth medium affected the growth rate and the production of alkaline phosphatase, dipeptidyl peptidase IV and carcinoembryonic antigen . Butyrate at a final concentration of 1 mmol/l increased the doubling time of the cells from 26 hours to 72 hours and decreased the cloning efficiency of the cells from 1.1% to 0.054% . Alkaline phosphatase concentrations increased rapidly in cells cultured in 1 mmol/l butyrate reaching peak levels after four days with alkaline phosphatase concentrations increasing more than six-fold . Levels of dipeptidyl peptidase IV and carcinoembryonic antigen were also increased after culture in butyrate containing medium . The number of alkaline phosphatase containing and dipeptidyl peptidase IV containing cells increased markedly in butyrate containing cultures . In contrast the number of mucus containing cells decreased in cultures grown in medium containing butyrate . This differentiating effect of butyrate on colon carcinoma cells may be relevant to the presence of butyrate in the colonic contents and the relationship between short chain fatty acids and fibre intake.

Cancer Lett, 1986 Dec, 33(3), 325 - 32
Secretion of a 97 kilodalton glycoprotein by primary cultures of human ovarian epithelial tumor cells; Subba Rao G et al.; The epithelial cells from both control and neoplastic ovaries were grown as primary cultures . The outgrowth of epithelial cells occurred within 3-5 days and the cells formed essentially a monolayer culture covering more than 80% of the surface of the flask within 5 weeks . The incorporation of {3H}fucose into the glycoproteins secreted into the medium was measured in the cells grown for 4 weeks . The secretion of glycoproteins in the tumor cells was twice that of the control . Analysis of the glycoproteins in the medium showed that the ovarian epithelial tumor cells secreted predominantly a 97 kilodalton glycoprotein . The growth of fibroblasts could be inhibited in these cultures by using Falcon Primaria culture flasks and the growth medium containing D-valine.

Br J Cancer, 1986 Dec, 54(6), 933 - 41
The effect of 2-{(aminopropyl)amino} ethanethiol (WR1065) on radiation-induced DNA damage and repair and cell progression in V79 cells; Grdina DJ et al.; The radioprotector 2-{(aminopropyl)amino} ethanethiol (WR1065) was investigated with respect to its ability to affect radiation-induced DNA damage and repair in V79 cells . Studies were performed to evaluate the protector under conditions in which it is known to be effective in reducing the cytotoxic and mutagenic effects of gamma-irradiation . At a concentration of 4 mM, WR1065 protected against the formation of single strand breaks (SSB), as determined by the method of alkaline elution, when it was present during irradiation . The protector appeared, however, to inhibit the subsequent postirradiation repair or rejoining of SSB . While repair was complete within 24 h, the protector reduced the rate of repair by a factor of 3 . This inhibitory effect on the rate of repair did not correlate with either measured differences in cell survival or mutagenesis . The radioprotector was also investigated with respect to its ability to affect cell cycle progression . WR1065 present in the growth medium inhibited the progression of cells through S-phase, and cell-doubling time following a 3 h exposure to the protector was increased from 11 to 18 h . These data are consistent with the well characterized property of thiols to inhibit DNA polymerase activity . It was concluded that, while the presence of WR1065 during irradiation reduced SSB-DNA damage, its effect on the subsequent rejoining of these breaks could not be correlated with its observed effect on protecting against radiation-induced mutagenesis . It may be that the inhibition of cell-cycle progression by the protector allowed more time to enhance the fidelity of repair as measured by the protector's ability to protect against radiation-induced mutagenesis.

Am J Pathol, 1986 Dec, 125(3), 493 - 500
Glomerular endothelial cells secrete a heparinlike inhibitor and a peptide stimulator of mesangial cell proliferation; Castellot JJ Jr et al.; The regulation of cell growth in the kidney glomerulus plays a key role in many physiologic and pathologic processes . In this communication the authors have examined the possible role of glomerular endothelial cells as potential regulators of mesangial cell proliferation . Conditioned medium was collected from confluent cultures of glomerular endothelial cells and tested for its effects on glomerular mesangial cell and vascular smooth muscle cell growth . When glomerular endothelial cell-conditioned medium was mixed 1:1 with normal growth medium, the growth of these two closely related cell types was inhibited by 60-70% . If the conditioned medium was diluted to 1:9, a stimulation of mesangial and smooth muscle cells growth was seen . Approximately 70% of the antiproliferative activity was destroyed by a highly purified heparinase; the other 30% was sensitive to trypsin . Approximately 90% of the mitogenic activity was protease-sensitive . These results suggest that glomerular endothelial cells may participate in part in mesangial cell growth regulation via a heparin-mediated mechanism.

Radiat Res, 1986 Dec, 108(3), 238 - 50
The role of glutathione in the aerobic radioresponse . I . Sensitization and recovery in the absence of intracellular glutathione; Clark EP et al.; The effect of changes in both the intracellular glutathione (GSH) concentration and the concentration of extracellular reducing equivalents on the aerobic radiosensitization was studied in three cell lines: CHO-10B4, V79, and A549 . Intracellular GSH was metabolically depleted after the inhibition of GSH synthesis by buthionine sulfoximine (BSO), while the extracellular environment was controlled through the replacement of growth medium with a thiol-free salt solution and in some experiments by the exogenous addition of either GSH or GSSG . Each of the cell lines examined exhibited an enhanced aerobic radioresponse when the intracellular GSH was extensively depleted (GSH less than 1 nmol GSH/10(6) cells after 1.0 mM BSO/24 h treatment) and the complexity of the extracellular milieu decreased . Although the addition of oxidized glutathione (5 mM GSSG/30 min) to cells prior to irradiation was without effect, much or all of the induced radiosensitivity was overcome by the addition of reduced glutathione (5 mM GSH/15 min) . However, the observation that the exogenous GSH addition restores the control radioresponse without increasing the intracellular GSH concentration was entirely unexpected . These results suggest that a number of factors exert an influence on the extent of GSH depletion and determine the extent of aerobic radiosensitization . Furthermore, the interaction of exogenous GSH with--but without penetrating--the cell membrane is sufficient to result in radiorecovery.

Cancer Res, 1986 Dec, 46(12 Pt 1), 6041 - 8
Development and inheritance of osmotic tolerance in a line of spontaneously transformed BALB/3T3 cells; Hennessey TL et al.; An in vitro line of transformed BALB/3T3 mouse fibroblasts was exposed to a 100 mM increase in the NaCl concentration of its growth medium . The rate of growth, as measured by the incorporation of tritiated thymidine, decreased almost 100-fold during the first 24 h of exposure to the hyperosmotic medium and then increased approximately 10-fold during the second 24 h of exposure . Cell counts of cultures passaged in medium with excess NaCl revealed a gradual increase in growth rate over a period of several weeks . The early kinetics by which salt tolerance developed in this cell line indicated a process of physiological adaptation rather than selection of preexisting variants in the control population . Clonally derived populations exposed to excess NaCl all showed a similar response . Cultures which tolerated a 100 mM increase in NaCl also grew well in medium containing 200 mM sucrose, indicating that their tolerance was not specific for NaCl . Although the initial response of cultures exposed to excess NaCl appeared to be one of physiological adaptation, tolerance for salt became hereditary after continued passage in hyperosmotic medium . Cultures that were returned to control conditions after prolonged exposure to excess NaCl inherited a high level of tolerance for salt which persisted for several hundred generations without selection.

Arch Microbiol, 1986 Dec, 146(3), 214 - 20
Incorporation of mannoproteins into the walls of aculeacin A-treated yeast cells; Valentin E et al.; Inhibition of the synthesis of alkali-insoluble glucan by aculeacin A in Saccharomyces cerevisiae cells caused a decrease in the incorporation of a high molecular weight heterogeneous mannoprotein material and of a 33,000 mannoprotein into the wall network . This was concomitant with the excretion of the latter molecule into the growth medium . Regenerating yeast protoplasts liberated considerable amounts of the heterogeneous material to the medium independently of the presence of aculeacin . The protoplast walls did lack this component and contained only minor amounts of the 33,000 molecule, which was also completely absent from walls of aculeacin-treated protoplasts . Considerable levels of the 33,000 species were immunodetected in the supernatants from treated and untreated protoplasts . These results point to the existence of specific interactions between the glucan network of the yeast cell surface and some of the wall mannoproteins . On the other hand, the presence of a population of SDS-solubilizable mannoproteins in the wall was independent of glucan levels.

Can J Microbiol, 1986 Dec, 32(12), 969 - 72
Regulation of phosphoenolpyruvate carboxykinase and pyruvate kinase in Saccharomyces cerevisiae grown in the presence of glycolytic and gluconeogenic carbon sources and the role of mitochondrial function on gluconeogenesis; Wilson AJ et al.; Phosphoenolpyruvate carboxykinase (PEPCKase) and pyruvate kinase (PKase) were measured in Saccharomyces cerevisiae grown in the presence of glycolytic and gluconeogenic carbon sources . The PEPCKase activity was highest in ethanol-grown cells . However, high PEPCKase activity was also observed in cells grown in 1% glucose, especially as compared with the activity of sucrose-, maltose-, or galactose-grown cells . Activity was first detected after 12 h when glucose was exhausted from the growth medium . The PKase activity was very high in glucose-grown cells; considerable activity was also present in ethanol- and pyruvate-grown cells . The absolute requirement of respiration for gluconeogenesis was demonstrated by the absence or significantly low levels of PEPCKase and fructose-1,6-bisphosphatase activities observed in respiratory deficient mutants, as well as in wild-type S . cerevisiae cells grown in the presence of glucose and antimycin A or chloramphenicol . Obligate glycolytic and gluconeogenic enzymes were present simultaneously only in stationary phase cells, but not in exponential phase cells; hence futile cycling could not occur in log phase cells regardless of the presence of carbon source in the growth medium.

Neurochem Pathol, 1986 Dec, 5(3), 309 - 29
Gangliosides in the nervous system during development and regeneration; Yates AJ; Gangliosides are present in nervous tissues of echinoderms and chordates, but the amounts and patterns differ widely . There are changes in the ganglioside contents of nervous tissues during development in most animals studied . To a large extent, regional differences and changes with development and degeneration in ganglioside composition reflect changing and different proportions of cellular types and subcellular organelles within the tissue . GM1 and GM4 are enriched in myelin; GD1a may be a marker for dendritic arborization . During regeneration of fish optic nerve and rat sciatic nerve there is an increased amount of ganglioside proximal to the regenerating axon tips, which may largely be a result of accumulation . This could provide a relatively large reservoir of ganglioside to become incorporated into the sprouting axolemma . Gangliosides added exogenously to growth medium can induce neuritogenesis of several types of neurons . The mechanisms of this action are unknown but may be related to nerve growth factor, microskeletal organization, membrane fluidity, and other factors . Gangliosides injected into young animals affect brain development, but further studies are required to determine these effects more specifically . Ganglioside administration increases the number of sprouts in regenerating peripheral nerves, but does not seem to accelerate axonal elongation . Parenterally administered gangliosides alter the recovery of brain tissue from a variety of types of lesions, and clinical trials are in progress to determine if they are of benefit in human neurological disorders . The biochemical mechanisms of these in vivo ganglioside effects are poorly understood, but may involve modulation of several enzyme systems as well as other properties of neural membranes, such as fluidity . It is possible that gangliosides may play similar roles and operate through some of the same mechanisms in developing and regenerating nervous tissues.

Virology, 1986 Dec, 155(2), 545 - 56
The noncoding region of HPV-6vc contains two distinct transcriptional enhancing elements; Rando RF et al.; HPV-6vc subgenomic fragments were inserted into an enhancer-dependent expression vector for chloramphenicol acetyltransferase (CAT) and assayed for the presence of transcriptional enhancing elements . A transcriptional enhancing element was detected in the noncoding region (NCR) of the HPV-6vc viral genome when the CAT assays were performed in viral transformed human kidney cell lines (293 and 324K), in human cervical carcinoma cell lines (HeLa and Siha), and in bovine papillomavirus type 1 (BPV-1) transformed mouse cells (C127-53) . The NCR region of the HPV-6b genome was only capable of enhancing transcription of the CAT gene in the HeLa cell line at a level one-third that of the HPV-6vc NCR . The HPV-6vc NCR enhancing activity in C127-53 cells was further stimulated by the addition of sodium butyrate to the growth medium . Localization of the DNA sequences in the HPV-6vc NCR responsible for enhancing transcription revealed two distinct enhancer elements . One element (HPV-6vc position 7218-7544) was active in the 293, HeLa, Siha, and C127-53 cells . The second enhancer element (HPV-6vc position 7544-7971) was only capable of stimulating transcription in HeLa, C127-53, and Siha cells . When the HPV NCR-CAT expression vectors were cotransfected with a competitor plasmid (pNCR75) into C127-53 or HeLa cells then transcriptional enhancement decreased, indicating competition of cellular factors which affect both segments of the HPV-6vc NCR.

Cancer Genet Cytogenet, 1986 Dec, 23(4), 305 - 13
Application of long-term collagenase disaggregation for the cytogenetic analysis of human solid tumors; Limon J et al.; A method has been elaborated for obtaining chromosome preparations from different histologic types of human solid tumors and applied to 60 cases . Such tumors were mechanically dispersed, washed by settling and transferred into flasks containing collagenase (200 U/ml) in the growth medium . Tumor pieces were dissociated in enzyme for 16 hours to 6 days, depending on tumor consistency . The majority of the tumors (80%) required 16-24 hours of such treatment . After disaggregation, the suspension of cells was washed by settling and then centrifuged . Cells were cultured for 24 hours to 6 days (85% of tumors), treated with a hypotonic solution in situ and then fixed . Of the 60 tumors, 37 (60%) displayed a sufficient number of metaphases for banding analysis . The success rate depends primarily on the type of tumor: 70% for soft tissue sarcomas, 40% for bladder carcinomas, and 40% for renal adenocarcinomas . This procedure is gentle, requires no special equipment and is applicable for the study of the full spectrum of human solid tumors.

Appl Environ Microbiol, 1986 Dec, 52(6), 1273 - 9
Action of citrinin on bacterial chromosomal and plasmid DNA in vivo and in vitro; Martin W et al.; Citrinin, a mycotoxin of Penicillium citrinum and other species of the genera Penicillium and Aspergillus, caused the following effects at different concentrations in Escherichia coli . In vivo at 100 micrograms/ml single-strand breaks were caused in the chromosomal DNA . In the presence of 100 micrograms/ml, UV (254 nm)-induced DNA damage was repaired in the bacterial cells without need for a complete growth medium . At 300 micrograms/ml lambda ts prophage was induced in a lysogenic E . coli strain . In an E . coli strain carrying a F' lac plasmid, 4.7% of the cells displayed the Lac- phenotype after treatment with 200 micrograms of citrinin per ml, suggesting elimination of the F' factor . In vitro, DNA repair synthesis was observed at 5 micrograms of citrinin per ml in permeabilized cells, and replicative DNA synthesis was inhibited at 200 micrograms/ml . In these systems synthesis of stable RNAs was slightly diminished at 300 micrograms/ml, and protein synthesis was not affected at concentrations up to 450 micrograms/ml . Lambda and ColE1 plasmid DNA were cleaved in vitro when small amounts of copper ions were present . This DNA-attacking activity was prevented by NADPH, catalase, and superoxide dismutase and by higher concentrations of hydroxyl radical scavengers, suggesting the involvement of free radicals in the mechanism of action of citrinin on DNA.

J Biol Chem, 1986 Nov 25, 261(33), 15725 - 33
Sulfate transport-deficient mutants of Chinese hamster ovary cells . Sulfation of glycosaminoglycans dependent on cysteine; Esko JD et al.; We isolated 59 Chinese hamster ovary cell mutants defective in 35SO4 incorporation into glycosaminoglycans . Thirty-five mutants incorporated {6-3H}glucosamine into glycosaminoglycans normally, suggesting that they were specifically impaired in sulfate incorporation . Cell hybridization studies revealed that the 35 mutants defined a unique complementation group . Pulse-labeling one of the mutants with 35SO4 showed that it possessed a defect in a saturable, 4-acetamido-4-isothiocyanostilbene-2,2'-disulfonic acid-sensitive transport system required for sulfate uptake . Despite the dramatic reduction in 35SO4 incorporation, the mutant synthesized sulfated heparan and chondroitin chains . Incubation of the mutant with {35S}cysteine resulted in the formation of 35SO4, which was subsequently incorporated into the glycosaminoglycans . Similar results were obtained when wild-type cells were incubated in sulfate-free growth medium containing {35S}cysteine, and isotope dilution analysis indicated that about 15 microM of sulfate was derived from cysteine catabolism . We also found that the sulfate transport deficiency rendered the mutant resistant to 5 microM sodium chromate, whereas wild-type cells did not divide under these conditions . However, the mutant also did not proliferate in medium containing 5 microM chromate when grown in the presence of wild-type cells, suggesting that chromate was transported through cell-cell contacts . Since co-cultivating sulfate transport-deficient mutants with mutants defective in xylosyltransferase or galactosyltransferase I partially restored 35SO4 incorporation into glycosaminoglycans, intercellular sulfate transport occurred as well . Therefore, the availability of sulfate for glycosaminoglycan synthesis depends on sulfate uptake, turnover of sulfur-containing amino acids, and sulfate transport between cells.

J Biol Chem, 1986 Nov 25, 261(33), 15625 - 31
The interrelationship of the soluble and membrane-associated folate-binding proteins in human KB cells; Kane MA et al.; Human KB cells produce two immunologically cross-reactive folate-binding proteins: a particulate cell-associated protein which is solubilized by Triton X-100, and a soluble protein which is released into their growth medium . This compartmentation of these two folate-binding proteins provides a convenient system for studies of their biochemical relationship . The two folate-binding proteins behave similarly to the purified particulate and soluble folate-binding proteins of human milk in analysis by radioactive folate binding, Sephacryl S-200 gel filtration profiles, polyacrylamide gel electrophoresis in either Triton X-100 or sodium dodecyl sulfate, and in Triton X-100 binding based on sucrose density gradient ultracentrifugation in H2O and D2O . The two folate-binding proteins were endogenously labeled by pulsing methionine-starved KB cells with {35S}methionine, and each protein was purified to apparent homogeneity by affinity chromatography at different times during the chase with nonradioactive methionine . The time course of the changes in specific activity (moles of {35S}methionine per mole of folate-binding protein) revealed a more rapid initial rate of synthesis and an earlier maximum in specific activity for the cell-associated folate-binding protein than for the soluble folate-binding protein released into the growth medium . Differences in the levels and specific activities of the two folate-binding proteins of cells exposed to cycloheximide compared with simultaneous controls after pulsing with {35S}methionine suggest that, whereas the cell-associated folate-binding protein is probably produced by de novo protein synthesis, the soluble folate-binding protein seems to be produced from a cellular pool of an already synthesized protein . These results combined with the immunologic cross-reactivity of the two folate-binding proteins strongly suggest a precursor-product relationship between them.

Eur J Biochem, 1986 Nov 17, 161(1), 103 - 9
A (re)initiation-dependent cell-free protein-synthesis system from mouse erythroleukemia cells; Bader M et al.; Cultured mouse erythroleukemia cells (MEL cells) can be induced in vivo to erythroid differentiation which is marked by the onset of globin mRNA and haemoglobin synthesis . When these cells are briefly exposed to hypertonic growth medium prior to lysis, the resulting post-mitochondrial supernatants show a high in vitro protein-synthesis activity . Amino acid incorporation is linear up to 60 min; more than 80% of this is due to (re)initiation, as shown by the inhibition with edeine . Extracts from induced cells reach only a third of overall incorporation as compared to extracts from uninduced cells . This reduction of the protein-synthesizing capacity is also observed in vivo . Polyacrylamide gel electrophoresis shows that extracts from uninduced cells faithfully translate their endogenous mRNA, whereas in extracts from induced cells, non-globin protein synthesis is reduced and globin is preferentially synthesized . Haemin (40 microM) as well as purified eukaryotic initiation factor 2 (eIF-2) from rabbit reticulocytes enhance amino acid incorporation in both kinds of extracts, which suggests that both uninduced and induced MEL cells contain a haemin-controlled eIF-2 alpha kinase . This system should be useful for studying the mechanisms controlling protein synthesis in a nucleated differentiating cell.

J Protozool, 1986 Nov, 33(4), 522 - 5
Encystation of Entamoeba invadens IP-1 is induced by lowering the osmotic pressure and depletion of nutrients from the medium; Avron B et al.; Trophozoites of Entamoeba invadens IP-1 can be induced to encyst in simple solutions composed of semipermeable constituents (buffer, salts, or sugars) provided that their osmotic pressure is in the range of 60-160 mosmol/kg . Optimal yield of mature cysts was obtained when the osmotic pressure of the medium was 110 mosmol/kg . Encystation could be obtained in the absence of serum although higher yields were obtained in its presence . No difference in the yield of mature cysts was found when either dialyzed or full serum was used . High yields of encystation were obtained (greater than 70%) in the presence of 5% serum in solutions of NaCl, KCl, or MgSO4, suggesting that the mechanism of encystation is not induced via sodium or potassium channels . Cysts were obtained in the presence of 72 mM glucose, indicating that depletion of a carbon source is not the only requirement for encystation . A rapid change in the density of the Entamoeba cells was observed upon transfer of trophozoites (density 1.061-1.073 g/ml) from growth medium to the low osmotic pressure encystation solutions . Within the first 2 min their density decreased (to 1.050 g/ml), but it soon increased, reaching within 30 min a density higher than 1.120 g/ml . As the encystation process continued to completion, the density of the cells gradually decreased, the mature cysts reaching a density of 1.049-1.061 g/ml.

Biochem Pharmacol, 1986 Nov 1, 35(21), 3857 - 62
Alterations in growth rate and cell cycle kinetics of rat liver tumor cells cultured in ethanol-containing medium . In vitro model of proliferative restriction in response to ethanol exposure; Higgins PJ et al.; Mechanisms related to the growth suppressive effect of acute ethanol exposure on liver cells were investigated using an established line of ethanol-sensitive rat hepatic tumor cells (32IIIA) and recently developed cytochemical methods for analysis of hepatocyte cell cycle kinetics . Exposure of exponentially growing 32IIIA cells to ethyl alcohol (range 10-100 mM in the growth medium) for a period of 3 days resulted in concentration-dependent decreases (4-25%) in final population density and increases (18-35%) in mean population doubling time compared to untreated cells . Viability was unaffected by ethanol exposure in the concentrations indicated and for the duration period utilized, approximating 94% under all experimental conditions . Multiparametric flow cytometric analysis revealed significant ethanol-associated differences in specific growth parameters and growth state compartments of 32IIIA hepatic tumor cell populations . Most prominent was an ethanol-associated and concentration-dependent (a) increase in the fraction of cells in the G1 phase of the cell cycle, (b) increase in the coefficient of variation in the G1 DNA content measurement, and (c) accumulation (in the G1 phase) of cells with a very low mean RNA content . Increases in each of these cytochemically-defined parameters reflected increasing levels of ethanol in the growth medium . This study indicates that the effects of ethanol on cultured cells of hepatic origin are quite complex . It is concluded that the inhibition of proliferation observed during acute ethanol exposure of liver-derived 32IIIA cells in vitro is due to an accumulation of cells in the G1 compartment.

J Urol, 1986 Nov, 136(5), 1141 - 2
An improved method for the preparation and culture of urothelial cells; James MJ et al.; Urothelial cells have been prepared by a new method involving collagenase treatment of the lumen of a ureter . These cells have been identified as epithelial and successfully subcultured . In addition, we have observed that growth rate is significantly increased by the inclusion of an extract of bovine hypothalamus in the growth medium . This system for cell preparation and culture should greatly facilitate studies involving urothelial cells.

Jpn J Cancer Res, 1986 Nov, 77(11), 1153 - 60
Influence of fetal calf serum on growth-inhibitory activity of human recombinant gamma-interferon (GI-3) in vitro; Hanada M et al.; The growth-inhibitory activity of human recombinant gamma-interferon (rIFN-gamma: GI-3) against 30 human cultured cell lines derived from leukemias and lymphoma (9 T-cell, 13 B-cell, 2 nonTnonB and 6 myeloid cell lines) was measured quantitatively by in vitro regrowth assay . Only three myelomonocytoid cell lines (HL-60, U937 and THP-1-0) were found to be sensitive, and HL-60 was the most sensitive . However, the sensitivity of HL-60 was found to be much influenced by both the batch and the concentration of fetal calf serum (FCS) used in the experiment . In the experiments using a certain FCS, GI-3 had a high antiproliferative activity against HL-60 at the optimal concentration (10(4)-10(5) U/ml), when assayed in medium containing 10% FCS . Both lower and higher concentrations of the interferon than 10(4)-10(5) U/ml resulted in decreased antiproliferative activity . When a high concentration (30%) of the FCS was employed in the assay, the antiproliferative activity of GI-3 was also much reduced . In the experiments using the other FCS, no antiproliferative activity of GI-3 against HL-60 was observed on assay in the medium containing 10% FCS, but significant antiproliferative activity was observed in the medium containing 30% FCS from the same source . Experiments using serum-free culture medium revealed that GI-3 itself had both a slight growth-inhibitory action at the optimal concentrations (at about 10(4) U/ml) and growth-enhancing activity at high (10(6) U/ml) concentration . The antiviral titer of GI-3 was stable during 72 hr of incubation in growth medium with or without cells . These findings suggest that there are cofactors in the serum which positively or negatively influence the growth-regulatory activity of GI-3 against HL-60 cells.

Mutat Res, 1986 Nov, 163(2), 167 - 74
Proliferation-dependent reduction of sister-chromatid exchange frequency induced by mitomycin C in human lymphoblastoid cells and its suppression by inhibitors of DNA replication; Tohda H et al.; A high frequency of sister-chromatid exchange (SCE) induced in cells of a human lymphoblastoid cell line, NL3, by 2-h treatment with 1 microM mitomycin C (MMC) was maintained after holding the treated cells in a nonproliferating state for 48 h before cells were transferred into the BrdUrd-containing medium for SCE assay . The same was observed in cells treated with 4-nitroquinoline 1-oxide (4NQO) or ethyl methanesulfonate (EMS) . In contrast, when MMC-treated cells were transferred into a growth medium and allowed to proliferate for various periods of time before SCE assay, MMC-induced SCE frequency decreased with time and reached near control level after 48 h . The reduction in SCE was also observed in 4NQO-treated cells, though to a lesser extent, but not in EMS-treated cells . When hydroxyurea or 1-beta-D-arabinofuranosylcytosine was given as a post-MMC treatment during this recovery process, such a reduction of SCE frequency was suppressed and the extent of the suppression appears to be roughly parallel to their ability to inhibit DNA replication . Cycloheximide and 5-azacytidine also exerted a similar inhibitory effect on the reduction of SCE . Benzamide and caffeine had no appreciable effect . Our results indicate that the SCE-forming lesions induced by MMC can be eliminated only in proliferating cells, probably during DNA replication.

J Bacteriol, 1986 Nov, 168(2), 668 - 72
Regulation of phosphatidylserine synthase from Saccharomyces cerevisiae by phospholipid precursors; Poole MA et al.; The addition of ethanolamine or choline to inositol-containing growth medium of Saccharomyces cerevisiae wild-type cells resulted in a reduction of membrane-associated phosphatidylserine synthase (CDPdiacylglycerol:L-serine O-phosphatidyltransferase, EC 2.7.8.8) activity in cell extracts . The reduction of activity did not occur when inositol was absent from the growth medium . Under the growth conditions where a reduction of enzyme activity occurred, there was a corresponding qualitative reduction of enzyme subunit as determined by immunoblotting with antiserum raised against purified phosphatidylserine synthase . Water-soluble phospholipid precursors did not effect purified phosphatidylserine synthase activity . Phosphatidylserine synthase (activity and enzyme subunit) was not regulated by the availability of water-soluble phospholipid precursors in S . cerevisiae VAL2C(YEp CHO1) and the opi1 mutant . VAL2C(YEp CHO1) is a plasmid-bearing strain that over produces phosphatidylserine synthase activity, and the opi1 mutant is an inositol biosynthesis regulatory mutant . The results of this study suggest that the regulation of phosphatidylserine synthase by the availability of phospholipid precursors occurs at the level of enzyme formation and not at the enzyme activity level . Furthermore, the regulation of phosphatidylserine synthase is coupled to inositol synthesis.

Endocrinology, 1986 Nov, 119(5), 2193 - 200
Norepinephrine and thyroid-stimulating hormone induce inositol phosphate accumulation in FRTL-5 cells; Bone EA et al.; 3H-Labeled inositol phosphate accumulation is observed when prelabeled FRTL-5 cells (a rat thyroid cell line) are exposed to norepinephrine (NE) or TSH . The presence of inositol trisphosphate among the products implicates a phosphodiesterase-catalyzed hydrolysis of phosphatidylinositol 4,5-bisphosphate . The response to NE is much greater than that to TSH . This may be explained by the ability of cAMP to inhibit inositol phosphate accumulation in these cells . The stimulation by NE is inhibited by alpha 1-adrenergic receptor antagonists and is markedly potentiated in medium of reduced Ca2+ concentration . After chronic withdrawal of TSH from the growth medium, the magnitude of the response to NE is considerably reduced; however, there is no substantial shift in the dose-response curve . This reflects the dependency of alpha 1-adrenergic receptor expression on TSH in the FRTL-5 cell . In contrast, the characteristics of inositol phosphate accumulation induced by acute treatment with TSH are similar in cells maintained in the presence or absence of a low concentration of this hormone, and correlate well with the iodide efflux and iodination of thyroglobulin observed in response to TSH . These results support the hypothesis that TSH may mediate certain of its physiological effects through cAMP-independent mechanisms, such as phospholipid/Ca2+ and C-kinase pathways.

J Cell Biol, 1986 Nov, 103(5), 1817 - 27
Mannose 6-phosphate receptor-mediated endocytosis of acid hydrolases: internalization of beta-glucuronidase is accompanied by a limited dephosphorylation; Gabel CA et al.; Endocytosis of acid hydrolases via the cell surface mannose 6-phosphate (Man 6-P) receptor results in the delivery of the enzymes to lysosomes . To examine the fate of the ligand-associated phosphorylated high mannose oligosaccharides, we have analyzed the asparagine-linked oligosaccharides attached to beta-glucuronidase after uptake and processing by Man 6-P receptor-positive mouse L cells . beta-Glucuronidase, double-labeled with {2-3H}mannose and {35S}methionine, was isolated from the growth medium of mouse P388D1 cells . 80% of the {3H}mannose associated with the secreted enzyme was recovered as high mannose-type oligosaccharides, and 24-37% of these units were phosphorylated . Three species of phosphorylated oligosaccharides were identified; high mannose-type units containing either one or two phosphomonoesters, and hybrid-type units containing one phosphomonoester and one sialic acid residue . After endocytosis by the L cells, the beta-glucuronidase molecules migrated faster on an SDS gel, suggesting that the enzymes had been processed within lysosomes . Examination of the cell-associated beta-glucuronidase molecules indicated that: (a) the percentage of phosphorylated oligosaccharides remained comparable to the input form of the enzyme, even after a 24-h chase period, (b) the presence of a single species of phosphorylated oligosaccharide that contained one phosphomonoester, and (c) the positioning of the phosphate within the intracellular monophosphorylated species was comparable to the positioning of the phosphate within the two phosphomonoester species originally secreted by the P388D1 cells . Therefore, the internalized beta-glucuronidase molecules undergo a limited dephosphorylation; oligosaccharides containing two phosphomonoesters are converted to monophosphorylated species, but the one phosphomonoester forms are conserved . A comparison of the phosphorylated oligosaccharides recovered from ligands internalized by the L cells at 37 degrees and 20 degrees C indicated that: (a) molecules internalized at 20 degrees C retain a higher percentage of phosphorylated structures; and (b) at both temperatures the predominant phosphorylated oligosaccharide contains a single phosphomonoester group . The results indicate that the Man 6-P recognition marker persists after endocytosis and delivery to lysosomes and support the possibility that the limited dephosphorylation of the oligosaccharides may occur en route to these organelles.

Ann Inst Pasteur Microbiol, 1986 Nov-Dec, 137B(3), 231 - 7
Changes in the enzyme activities involved in nitrogen assimilation in Mycobacterium smegmatis under various growth conditions; Ahmad S et al.; Glutamate dehydrogenase (aminating) and glutamine synthetase activities were assayed in Mycobacterium smegmatis following growth on various carbon and nitrogen sources . The activities (expressed as nmoles product formed/min/mg crude extract protein) of these two enzymes were higher in crude extracts from glucose-grown cells than in glycerol- or fructose-grown cells . In the presence of succinate, pyruvate, fumarate or acetate in the growth medium, both these enzyme activities were lower than those in citrate-grown cells . The glutamate dehydrogenase (GDH) activity was the same in asparagine and glutamine-grown cells . Ammonium chloride, alanine or glutamic acid, when used as nitrogen source, resulted in low GDH activity as compared to asparagine-grown cells . Glutamine synthetase activity was considerably lower (2-4 fold) when the cells were grown on alanine, glutamine, glutamic acid or ammonium chloride as the nitrogen source than those in asparagine-grown cells . Glutamate and ammonium chloride, when present in the growth medium, repressed both glutamate dehydrogenase and glutamine synthetase, though the degree of repression was small . The results suggest that only a weak transcriptional control operates for these enzyme activities in M . smegmatis.

J Gen Microbiol, 1986 Nov, 132 ( Pt 11), 3221 - 9
Complementation of mutations in Escherichia coli and Bordetella pertussis by B . pertussis DNA cloned in a broad-host-range cosmid vector; Brownlie RM et al.; A gene library of Bordetella pertussis DNA was constructed in Escherichia coli using the broad-host-range cosmid vector pLAFR1 . The average insert size was 24.9 kb . From 500 members of the gene library, clones were identified which complemented trpE, glnA and Thr- mutations in E . coli but none which complemented trpD, trpC, trpB, trpA, proA or Leu- mutations . Four clones were identified which complemented trpE in E . coli . Anthranilate synthase activity was detected in a trpE strain only when it harboured a plasmid from one of these clones; activity was repressed when tryptophan was included in the growth medium . Two clones were identified which complemented glnA of E . coli . A recombinant plasmid from one of these clones also restored some of the nitrogen acquisition functions of glnG and glnL in E . coli . Expression of several B . pertussis virulence-associated products (haemolysin, heat-labile toxin, adenylate cyclase, filamentous haemagglutinin, and the cell-envelope polypeptide of Mr 30,000) was not detected in 500 independent clones . However, by transferring the recombinant plasmids to a mutant of B . pertussis deficient in haemolysin and adenylate cyclase, a plasmid was identified which restored both these activities.

Cancer Res, 1986 Oct, 46(10), 5248 - 58
N,N-dimethylformamide-induced synthesis of an anti-fibronectin reactive protein in cultured human colon carcinoma cells; Marks ME et al.; The cell surfaces of human colon cancer cells before and after exposure to N,N-dimethylformamide (DMF) were probed using radioiodination and immunofluorescent labeling techniques . Growth of the human colon carcinoma cell line HCT MOSER in DMF-supplemented culture medium resulted in monolayer culture growth and marked cell morphology alterations consisting of cellular flattening and elongation . Accompanying the morphology alterations were distinct changes in the cell surface protein composition as determined by 125I labeling and electrophoresis . The cell surface changes associated with growth of HCT MOSER cells in the presence of DMF were dependent upon time of exposure to DMF and DMF concentration . Furthermore, removal of DMF-treated HCT MOSER cells from DMF-containing growth medium caused reversion of both cell morphology and cell surface composition to a state comparable to that of cells not exposed to DMF . The HCT MOSER cell surface alterations produced by DMF included a reduction of radioiodinated surface proteins with molecular weights of 87,000, 120,000, and 180,000 and an increase of a 125I-labeled surface protein with a molecular weight of 200,000-250,000 . Appearance of a surface protein of approximately 200,000 molecular weight and assumption of a fibroblast-like morphology by DMF-treated HCT MOSER cells suggested that this approximately 200,000 molecular weight material might be fibronectin . Immunofluorescent labeling with anti-human fibronectin showed that HCT MOSER cells grown in DMF did manifest an anti-fibronectin immunoreactive material that was only transiently associated with the cell surface before being released . DMF-treated HCT MOSER cultures continued to express surface carcinoembryonic antigen, indicating that the presence of material immunoreactive with anti-human fibronectin was not secondary to proliferation of a contaminating fibroblast population . The response of HCT MOSER cells to DMF paralleled in many ways that previously reported for methylcholanthrene-transformed AKR-2B (AKR-MCA) fibroblasts . However, unlike AKR-MCA cells, HCT MOSER cells did not exhibit an increase in 125I incorporation per microgram DNA as a function of time of exposure to DMF, which suggests that the surface protein with a molecular weight of approximately 200,000 induced by DMF was not retained on the cell surface.

Zh Mikrobiol Epidemiol Immunobiol, 1986 Oct, (10), 25 - 7
{1 of the causes of a change in viability of Escherichia coli L1 in air}; Koniukhov VF et al.; The possibility of changing the fatty acid composition of lipids in E . coli strain BB 20-14 by the introduction of ready lipid vesicles obtained from other E . coli strains into the growth medium has been established . Using E . coli strain BB 20-14 as an example, the dependence of the viability of bacteria on their fatty acid composition has been demonstrated.

Mol Gen Genet, 1986 Oct, 205(1), 51 - 5
Complementation analysis of the wild-type and mutant ompR genes exhibiting different phenotypes of osmoregulation of the ompF and ompC genes of Escherichia coli; Nara F et al.; Expression of the ompF and ompC genes, which encode the major outer membrane proteins, OmpF and OmpC, respectively, is affected in a reciprocal manner by the osmolarity of the growth medium . This osmoregulation is mediated by the OmpR protein, a positive regulator of both genes, which is encoded by the ompR gene . Structural and functional properties of this regulatory protein were studied through complementation analysis of the wild-type and five mutant ompR genes that exhibited differences in osmoregulation of the expression of the OmpF and OmpC proteins . Complementation was carried out with combinations of a host strain and a plasmid, each of which carried either the wild-type or a mutant ompR gene . In some combinations, negative complementation was observed . For example, ompR1, a deletion mutation with an OmpF- OmpC- phenotype, was dominant to OmpF+ or OmpC+ phenotypes conferred by other ompR genes . Positive complementation of two mutant ompR genes was also observed in other combinations, when the two mutations were distantly located from each other on the OmpR protein . These results, together with other observations, support the view that the OmpR protein has a two-domain structure, each domain exhibiting a different role in the expression of the OmpF and OmpC proteins, and that this protein takes a multimeric structure as a functional unit.

Proc Natl Acad Sci U S A, 1986 Oct, 83(19), 7307 - 10
Regulation of melanoma by the embryonic skin; Gerschenson M et al.; This report focuses on the regulation of murine melanoma by the embryonic skin . A surgical technique was developed to allow injection of B16 melanoma cells into the embryo in utero . A significant decrease in incidence of tumors was noted, which correlated with the time of arrival of normally migrating premelanocytes into the skin . Media were conditioned from skin explanted at the time premelanocytes arrive in it; these media inhibited the growth of melanoma cells in vitro . Under optimal conditions the growth of melanoma cells ceased; the cells had altered morphology and failed to proliferate when placed in fresh growth media.

Ann Rheum Dis, 1986 Oct, 45(10), 858 - 64
Growth of monosodium urate monohydrate crystals: effect of cartilage and synovial fluid components on in vitro growth rates; Burt HM et al.; The effects of cartilage and synovial fluid components such as proteoglycans, chondroitin sulphate, hyaluronic acid, phospholipids, and albumin on the growth kinetics of monosodium urate monohydrate (MSUM) crystals were investigated . MSUM seed crystals were added to supersaturated sodium urate solutions, and the rate of decrease in the concentration of growth medium was used as a measure of the growth rate . A second order dependence of growth rate on supersaturation was found, and growth rate constants were determined with an integrated form of the growth equation . The additives, hyaluronic acid, proteoglycan monomer and aggregate, and phosphatidylserine, had no significant effect on the growth rate constant . Chondroitin sulphate and phosphatidylcholine increased the growth rate constant, possibly by promoting further nucleation in the growth medium . Albumin significantly inhibited MSUM crystallisation . The possible implications of these findings on in vivo MSUM crystallisation are discussed.

EMBO J, 1986 Oct, 5(10), 2609 - 16
The v-mos and H-ras oncogene expression represses glucocorticoid hormone-dependent transcription from the mouse mammary tumor virus LTR; Jaggi R et al.; We have subjected the viral mos oncogene (v-mos), the activated human H-ras oncogene {H-ras (A)} and the normal human H-ras protooncogene {H-ras (N)} to the transcriptional regulation of glucocorticoid hormones by in vitro recombination with the promoter region of the mouse mammary tumor virus long terminal repeat (MMTV LTR) and transfection into NIH 3T3 cells . Cell clones were selected which exhibit a transformed phenotype strictly dependent on the presence of hormone in the growth medium . The expression of the chimeric genes as a function of time after hormone stimulation was studied at the level of transcriptional rate, mRNA and protein accumulation . Oncogene expression was stimulated rapidly to high levels, after hormone addition, but declined in the continuous presence of hormone . Measurements of the transcriptional rates in nuclei from LTR v-mos and LTR H-ras (A) transfected cells showed a repression of LTR v-mos and LTR H-ras (A) transcription after the initial stimulation by hormone . LTR H-ras (N) transcription was not affected . An independently transfected LTR H-2Ld construct in LTR v-mos or LTR H-ras (A) containing cells is also transcriptionally repressed . These experiments demonstrated a transcriptional repression effect of the oncogene products on the glucocorticoid hormone-dependent MMTV LTR transcription.

Clin Exp Metastasis, 1986 Oct-Dec, 4(4), 293 - 309
In vitro modulation of the metastatic phenotype . I . Analysis of differentiation forms of the B16 melanoma expressing Met-72 determinants and metastatic activity; Xiang JH et al.; In vitro cultures of a highly metastatic B16 melanoma clone (BL6-10) were found to undergo dramatic changes in morphology and differentiation upon transfer to another culture medium . Specifically, BL6-10 melanoma cells which had been originally selected and adapted for growth in Eagles' Hanks' amino acid supplemented media with 10 per cent newborn calf serum were amelanotic and epitheliod in shape . When these cells were shifted into Dulbecco's modified Eagles medium with 10 per cent fetal calf serum, they became highly melanotic and of spindle/dendritic morphology within 4 days of culture . These morphological changes as well as other parameters were all characteristic of established criteria of melanoma differentiation . Alterations in the differentiation state of our highly metastatic variant, BL6-10, did not result in any change in tumorigenicity but did have profound effects on metastatic potential . All of the morphological and functional characteristics of the differentiated melanoma were found to be reversible by re-plating the cells in their original growth medium and 4 days of in vitro growth . These studies have allowed us to follow and more firmly establish Met-72 antigen expression as a surface marker for metastatic cells of the B16 melanoma, and have provided direct experimental evidence that the less differentiated, Met-72 positive melanoma form is the dominant cell type capable of metastatic potential.

J Am Acad Dermatol, 1986 Oct, 15(4 Pt 2), 789 - 97
Epidermal effects of retinoids: in vitro studies; Eichner R; Cell culture provides controlled conditions in which to investigate the effects of retinoids on the molecular and cell biology of epidermal differentiation . In general, retinoids enhance proliferation and desquamation of cultured epidermal cells and suppress differentiation . In the presence of 10(-6) mol/L retinoic acid, cultured human epidermal cells stratify, but they do not form the granular layer and anucleate, stratum corneum-like superficial layer typical of normal epidermis . Retinoic acid in the growth medium alters keratin synthesis and inhibits the formation of cross-linked envelopes . Expression of other keratinocyte proteins, including filaggrin and components of desmosomes, may also be affected by retinoids . The molecular mechanisms of retinoid action on epidermal cells are still unclear . Cells cultured from normal and pathologic epidermis appear to differ significantly in their responsiveness to retinoids . Recent data suggest that retinoids may modulate gene transcription, stabilize cell membranes, and alter posttranslational processing of several keratinocyte proteins.

Toxicol Appl Pharmacol, 1986 Sep 30, 85(3), 456 - 63
Perfluorodecanoic acid inactivation of a channel for 2-aminopurine in the L5178Y cell membrane and recovery of the channel; Wigler PW et al.; Treatment of L5178Y mouse lymphoma cells with perfluoro-n-decanoic acid (PFDA) in growth medium for 24 hr at 30 degrees C produces a dose-dependent inactivation of a channel in the cell membrane . Activity of the channel was estimated from the initial rate of efflux of a fluorescent purine, 2-aminopurine (AP) . The L5178Y cells were preloaded with 100 microM AP and excess AP was removed . The preloaded cells were put in a flow system, and AP efflux was estimated continuously at 21 degrees C from the fluorescence emission of AP at 370 nm . The AP channel was markedly inactivated by a treatment with 150 micrograms/ml PFDA for 24 hr at 30 degrees C . There was no significant recovery of AP flux after 3 days at 30 degrees C in fresh growth medium; however, recovery was significant after 6 days . Recovery of activity of the AP channel occurs in 1 day at 37 degrees C . The initial rate of AP efflux for control cells increases with AP concentration; the reaction is not saturated at 1000 microM AP . The efflux of AP was inhibited by the presence of uric acid in the external buffer . An apparent inhibition constant value of 355 microM was determined for urate inhibition of AP efflux . These observations suggest the presence of a urate-sensitive channel for AP in the membrane of L5178Y cells . The channel was inactivated by PFDA under conditions that had no significant effect on cell viability.

J Biol Chem, 1986 Sep 25, 261(27), 12441 - 3
10-Thiastearic acid inhibits both dihydrosterculic acid biosynthesis and growth of the protozoan Crithidia fasciculata; Pascal RA Jr et al.; 10-Thiastearic acid is a specific inhibitor of the biosynthesis of dihydrosterculic acid (9,10-methyleneoctadecanoic acid) in the trypanosomatid protozoan Crithidia fasciculata . A 50% inhibition of the biosynthesis of dihydrosterculate is observed in the presence of 4 microM 10-thiastearate in the protozoan growth medium, but little effect is seen on the distribution of the other fatty acids . In addition, the growth of the protozoa is slowed by the presence of 10-thiastearate, with 50% growth inhibition produced at about 10 microM . A possible mechanism of this inhibition and the implication of this result with regard to the design of antiprotozoal agents are discussed.

J Biol Chem, 1986 Sep 5, 261(25), 11896 - 905
The sequential transfer of internalized, cell surface sialoglycoconjugates through the lysosomes and Golgi complex in HeLa cells; Fishman JB et al.; Surface sialoglycoproteins of HeLa cells were labeled by NaB{3H}4 reduction after oxidation with NaIO4, yielding seven major radioactive bands as visualized by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and autoradiography . When labeled cells are reincubated in growth medium, all of these major classes of glycoproteins are internalized and all but one (105 kDa) are recycled, i.e . subsequently reappear on the surface . The surface-labeling patterns over time remain qualitatively similar, but changes in relative specific activity of the bands suggest some preferential degradation of individual glycoproteins . Analytical fractionation at various time points after labeling suggests that the surface molecules pass through the lysosomal compartment and subsequently accumulate in the Golgi and Golgi-related compartments before returning to the surface . Inhibition of lysosomal function with chloroquine or NH4Cl prevents the accumulation and subsequent recycling . The pathway is confirmed with preparative fractionation into surface membrane, prelysosomal, lysosomal, Golgi, and Golgi-related compartments . Sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis demonstrates a degree of preferential handling of the glycoproteins on this pathway, e.g . the 180-kDa band is relatively reduced at the endocytic/prelysosomal stage and the 105-kDa band appears to be degraded in its first passage through the lysosomes . The other bands recycle 10-20 times before being degraded.

J Biol Chem, 1986 Sep 5, 261(25), 11840 - 8
Resistance to copper toxicity of cultured hepatoma cells . Characterization of resistant cell lines; Freedman JH et al.; A series of four cell lines resistant to the toxic effect of copper were developed from Morris rat hepatoma cells by gradually increasing the concentration of copper in the growth medium . The EC50, that concentration of copper that kills and/or inhibits the growth of 50% of the cells after 72 h, increased 4-fold over that for wild type cells in the most resistant cell line . These cells were also resistant to zinc, cadmium, and mercury toxicity, but not to nickel or cobalt . The amount of copper in the soluble protein pool of the resistant cells increased proportionally with the concentration of copper in the medium in which they were maintained . Associated with copper accumulation was the production of an 18-kDa cysteine-rich protein which complexes a significant amount of the metal . It is suggested that resistance to copper toxicity is due to sequestration of the metal by this protein . When resistant cells were removed from the copper-enriched environment, cellular copper levels rapidly fell to that observed for wild type cells, but no reduction in either the EC50 or the level of the cysteine-rich protein was noted . This suggests that a permanent change responsible for copper resistance had occurred which is maintained in the absence of the metal.

Appl Environ Microbiol, 1986 Sep, 52(3), 580 - 2
Effects of monovalent and divalent salts on the phospholipid and fatty acid compositions of a halotolerant Planococcus sp; Miller KJ; The phospholipid headgroup and fatty acid compositions of a halotolerant Planococcus sp . (strain A4a) were examined when cells were grown in the presence of high concentrations of a variety of salts . The fatty acid composition of Planococcus sp . strain A4a was altered primarily as a function of the osmolality of the growth medium . The phospholipid headgroup composition was influenced by both the osmolality of the growth medium and the nature of the cation species present . An increase in the cardiolipin/phosphatidylglycerol molar ratio was detected when cells were grown in the presence of high concentrations of monovalent cations.

Br J Cancer, 1986 Sep, 54(3), 511 - 3
Cytotoxicity of enantiomers of gossypol; Joseph AE et al.; PIP: The effect of gossypol on cell proliferation was studied using 24-well multiwell dishes . Cells were added to each well and incubated for 24 hours before the addition of different concentration of gossypol, which was dissolved in dimethyl sulfoxide immediately prior to use . After a further 3-day incubation, cell growth was determined by measuring cellular protein content . Standards and controls were included and dose response curves constructed by plotting increase in protein content over the 3-day growth period against gossypol concentration . Gossypol inhibited proliferation in tumor-derived cells as well as normal fibroblasts . BCL-D1 cells, a human diploid strain of fibroblasts, were sensitive to gossypol and further studies were undertaken using these cells . In contrast of most of the tumor-derived cells that have been used, these cells maintained consistent patterns of growth over periods of many months . The enantiomers of gossypol were prepared and analyzed as described in Matlin & Zhou (1984) . The (-) enantiomer of gossypol was more cytotoxic than the (+) form . In the case of all the tumor-derived cells, the concentration of the (-) enantiomer required to produce cytotoxicity was -10% of that required in the case of the (+) enantiomer . At lower concentration, gossypol inhibited cellular proliferation, but at higher concentrations the compound was responsible for complete cell lysis . Even with high doses of (-), gossypol cell lysis did not occur rapidly . When gossypol was added to cultures and then removed with thorough washing at time intervals, it was found that 30 minute exposure was adequate to bring about the cytolytic effect . Thus, the biochemical effects of gossypol occurred rapidly after addition even though cellular changes may not be obvious for some hours . Once gossypol has been added to tissue culture medium, there is a loss of cytotoxic potential . When gossypol was added to culture fluid and incubated at 37 degrees Centigrade before addition to cells, the chemical lost -10%/hour of its cytotoxic potential . The likely explanation is a binding of gossypol to plasma proteins that form the serum supplement to the growth medium . More than 10,000 men have participated in clinical trials relating to the anti-fertility effects of gossypol; a low incidence of side effects has been reported . The question arises as to why gossypol fails to have an effect on bone marrow cell proliferation . Most likely the answer lies in the protection afforded by higher protein concentrations .

J Cell Physiol, 1986 Sep, 128(3), 375 - 82
Density-dependent inhibition of expression of syncytiotrophoblastic markers by cultured human choriocarcinoma (BeWo) cells; Burres NS et al.; In the presence of methotrexate, cultured human choriocarcinoma (BeWo) cells undergo a differentiative response that resembles normal trophoblastic development . In the current study, the effects of cell number and population density on drug-induced conversion of BeWo cells from the cytotrophoblastlike to the syncytiotrophoblastlike phenotype were investigated using as markers of differentiation formation of "giant" cells, a process shown to require exogenous purines, and expression of placental (heat-stable) alkaline phosphatase . Giant cell formation, assessed by determination of cell volumes, was reduced in crowded cultures, and addition of hypoxanthine to growth media partially restored methotrexate-induced cell enlargement . Cellular uptake of methotrexate, assessed by following the loss of methotrexate from cell culture fluids during drug exposures, was two-threefold greater in sparsely populated than in densely populated cultures . Although the concentration of methotrexate in culture fluids of crowded cultures declined during exposures of 48 hr, the amount of extracellular drug remaining at 48 hr was well above the threshold for induction of the differentiative response . When culture population was held constant and population density was manipulated by varying the substratum available to cells, methotrexate-induced cell enlargement was inversely related to population density . Expression of placental alkaline phosphatase, salvage of exogenous hypoxanthine, and synthesis of RNA were also reduced at high population densities . These results indicate that expression of markers of methotrexate-induced differentiation of BeWo cells was inhibited in a density-dependent manner that may have been related to reduced cellular uptake of the inducing agent and of exogenous nutrients (purines) from culture fluids.

J Cell Physiol, 1986 Sep, 128(3), 353 - 61
Isolation and partial characterization of endothelial cell extracellular complexes; Hatcher VB et al.; Human endothelial cells release components into the growth medium that stimulate cell-substratum adhesion . Several macromolecular components were isolated by ultracentrifugation of the endothelial cell conditioned medium . The components were heterogeneous, consisting of several sizes when examined by sedimentation velocity and gel filtration . When the extracellular components were evaluated by electron microscopy, structurally discrete particles were observed . The extracellular components and the complexes mediated cell-substratum adhesion to both human umbilical and arterial endothelial cells . The majority of the extracellular components that promote endothelial cell adhesion were pelleted by ultracentrifugation . Although the complexes contained fibronectin, antibodies to fibronectin did not inhibit cell adhesion to the complexes . Significant inhibition of endothelial cell adhesion was observed in the presence of heparin and heparan sulfate . The supernatant fraction following ultracentrifugation of the growth medium contained a component that suppressed endothelial cell adhesion to culture dishes coated with fibronectin, type I collagen, and endothelial cell complexes . SDS-polyacrylamide gel electrophoresis indicated that the complexes contained several components, and the majority of the large-molecular-weight components were pelleted by ultracentrifugation . The conditioned medium from human endothelial cells contains specific complexes that promote cell-substratum adhesion and components that suppress cell-substratum adhesion.

J Bacteriol, 1986 Sep, 167(3), 818 - 27
Initial reactions of xanthone biodegradation by an Arthrobacter sp; Tomasek PH et al.; This study examined the catabolism of xanthone by an Arthrobacter sp . (strain GFB100) capable of growth on xanthone as its main source of carbon and energy . An early catabolic intermediate was 3,4-dihydroxyxanthone . This compound was isolated from the growth medium of a mutant strain of the Arthrobacter sp . which lacked the xanthone-inducible dihydroxyxanthone ring-fission dioxygenase of the wild-type strain . Cell extracts from wild-type xanthone-grown cells oxidized 3,4-dihydroxyxanthone to a yellow ring-fission metabolite . The same yellow compound accumulated in xanthone-grown cultures of a spontaneous mutant which lacked an active, xanthone-inducible, NADPH-linked ring-fission metabolite reductase . The yellow ring-fission metabolite appears to be 4-hydroxy-3-(2'-oxo-3-trans-butenoate)-coumarin, based on its nuclear magnetic resonance spectrum and mass spectral fragmentation pattern, indicating that ring cleavage of 3,4-dihydroxyxanthone was by an extra-diol (meta-fission) mechanism . Enzymatic analyses indicated that growth on xanthone induced a complete gentisate pathway: dioxygenase-catalyzed cleavage of gentisate to maleylpyruvate, isomerization of maleylpyruvate to fumarylpyruvate, and hydrolysis of fumarylpyruvate to fumarate and pyruvate . 4-Hydroxycoumarin was thought to be a likely pathway intermediate linking the early xanthone catabolic steps to the gentisate pathway, since 2-hydroxyacetophenone, a byproduct of 4-hydroxycoumarin hydrolysis, was formed when wild-type cells were cultured with xanthone . Chlorinated 2-hydroxyacetophenones were also obtained from specific chloro-substituted xanthones.

J Bacteriol, 1986 Sep, 167(3), 1089 - 91
Changes in membrane lipid composition of Mycoplasma capricolum affect the cell volume; Romano N et al.; The cellular water volume of Mycoplasma capricolum was markedly increased by a decrease in the cholesterol-to-phospholipid molar ratio in the membrane . An increase in cell volume was also observed with the increase in the phospholipid cell membrane content obtained by the incorporation of exogenous phosphatidylcholine from the growth medium.

J Invest Dermatol, 1986 Sep, 87(3), 305 - 8
Contact inhibitory factor also restores anchorage and serum dependence to hamster melanoma cells; Lipkin G et al.; Conditioned medium (CM) from confluent cultures of the contact-inhibited hamster melanocytic cell line, FF, contains a biologic activity, contact inhibitory factor (CIF), which reversibly restores density-dependent growth to melanoma cells . When a hydrophobic affinity-concentrated extract of CIF-containing CM was incorporated in agarose at a concentration of 1000 micrograms protein/ml, it restored anchorage-dependent growth to RPMI 1846 hamster melanoma cells . Colony-forming efficiency in CIF-treated wells decreased to 5% from levels of 51.5% in controls prepared with regular growth medium . In addition, CIF-containing CM restored serum-dependent growth to RPMI 1846 cells, markedly restricting proliferation in 1% calf serum-containing medium . Control cultures containing 1% calf serum and either complete growth medium or CM from the non-contact-inhibited hamster melanoma line itself, supported proliferation of RPMI 1846 cells to levels 3.9 X and 3.7 X that of CIF-treated cultures, respectively . CIF is the first factor derived from contact-inhibited mammalian cell cultures that has been shown to restore density-, anchorage-, and serum-dependent growth to malignant melanoma cells.

Mikrobiologiia, 1986 Sep-Oct, 55(5), 796 - 9
{Formation of secreted acid phosphatase during the growth of Saccharomyces cerevisiae yeasts on different sources of carbon and nitrogen nutrition}; Semenova IN et al.; The effect of certain components in the growth medium on the secretion of acid phosphatase was studied with Saccharomyces cerevisiae . The presence of phosphate at a concentration of 10 mM in the medium inhibited the formation of repressible forms of this enzyme . The synthesis of the secreted enzyme depended on the sources of carbon and nitrogen nutrition . The enzyme yield was highest in a medium with sucrose as a carbon source and ammonium chloride as a nitrogen source . The secretion of acid phosphatase is stimulated by an increase in the sugar content and a deficiency of the nitrogen source in the medium.

Int J Radiat Biol Relat Stud Phys Chem Med, 1986 Sep, 50(3), 481 - 90
Radiation chemical and physical mechanisms of radiosensitization of single cell systems by iothalamate; Capuano V et al.; The radiosensitizing effect of iothalamate (ITA) has been investigated in bacterial and mammalian cells in order to obtain a better understanding of the physical and radiation chemical mechanisms of sensitization displayed by the drug . In order to distinguish between the two, Escherichia coli B/r cells were irradiated with 9 MeV electrons, which allow only the radiation chemical mechanism to operate, and V79 cells with 250 KVp X-rays, which instead make possible the occurrence of both mechanisms . It has been shown that: Maximum sensitization already occurs in bacteria with 10(-2) mol dm-3 ITA (enhancement ratio (ER) 11.2 in oxygen, 2.7 in nitrogen), while in mammalian cells a concentration higher by a factor of 10 is required (ER 2.2 both in air and nitrogen) . ITA sensitization is inhibited when bacteria are irradiated in growth medium instead of buffer . Such inhibition does not occur with V79 cells . Cysteine and glycerol completely cancel the sensitizing effect of ITA on bacterial cells in both gas phases . Dimethylsulphoxide (DMSO) does the same in nitrogen, while in oxygen it only reduces ITA sensitization to about 50 per cent of the level observed in control conditions . With mammalian cells, all the three scavengers do not modify significantly the enhancement produced by ITA, either in air or in nitrogen . The experimental results are consistent with both postulated mechanisms of sensitization.

Mikrobiologiia, 1986 Sep-Oct, 55(5), 754 - 9
{Changes in the electrical characteristics of bacterial cells in the disruption of the barrier function of the cytoplasmic membrane}; Fomchenko VM et al.; A change in the electro-orientation spectrum followed by a change in the electric characteristics of bacterial cells had been measured when the barrier function of the cytoplasmic membrane was disordered under the action of damaging physical and chemical factors . Analysis of changes in the spectra at a frequency of 10(4) to 10(7) Hz allows one to estimate the degree of cell damage in quantitative terms . The paper presents the results of comparing three procedures for such an estimation: inoculation onto solid growth media; linear regression analysis of an electro-orientation spectrum; comparison of the electro-orientation effect at a high frequency (ca . 5 X 10(5) Hz) and at a medium frequency (ca . 10(4) Hz).

Mol Cell Biol, 1986 Sep, 6(9), 3034 - 41
Transcriptional control of glucoamylase synthesis in vegetatively growing and sporulating Saccharomyces species; Pretorius IS et al.; Three unlinked, homologous genes, STA1, STA2, and STA3, encode the extracellular glycosylated glucoamylase isozymes I, II, and III, respectively, in Saccharomyces species . S . cerevisiae, which is sta0 (absence of functional STA genes in haploids), does carry a glucoamylase gene, delta sta, expressed only during sporulation (W . J . Colonna and P . T . Magee, J . Bacteriol . 134:844-853, 1978; I . Yamashita and S . Fukui, Mol . Cell . Biol . 5:3069-3073, 1985) . In this study we examined some of the physiological and genetic factors that affect glucoamylase expression . It was found that STA2 strains grown in synthetic medium produce glucoamylase only in the presence of either Maltrin M365 (a mixture of maltooligosaccharides) or starch . Maximal levels of glucoamylase activity were found in cells grown in rich medium supplemented with glycerol plus ethanol, starch, or Maltrin . When various sugars served as carbon sources they all supported glucoamylase synthesis, although at reduced levels . In any given growth medium glucoamylase isozyme II synthesis was modulated by functionality of the mitochondria . Synthesis of glucoamylase is continuous throughout the growth phases, with maximal secretion taking place in the early stationary phase . In the various regimens, the differences in enzyme accumulation are accounted for by differences in the levels of glucoamylase mRNA . Both glucoamylase mRNA and enzyme activity were drastically and coordinately inhibited in MATa/MAT alpha diploids and by the presence of the regulatory gene STA10 . Both effects were partially overcome when the STA2 gene was present on a multicopy plasmid . The STA2 mRNA and glucoamylase were coinduced in sporulating STA2/STA2 diploids . A smaller, coinduced RNA species was also detected by Northern blotting with a STA2 probe . The same mRNA species was detected in sporulating sta0 diploids and is likely to encode the sporulation-specific glucoamylase.

Mikrobiologiia, 1986 Sep-Oct, 55(5), 781 - 6
{Mechanism of growth limitations in a recombinant strain of Escherichia coli}; Golovlev EL et al.; A recombinant Escherichia coli strain with the cloned gene of restriction endonuclease EcoR-V was studied . The diauxic growth of this strain under batch conditions, the composition of excreted products and the hyperbolic shape of the chemostat growth curve has made it possible to formulate the following hypothesis . At a high flow rate, one of the first reactions in the tricarboxylic acid cycle or a reaction directly preceding the cycle is a limiting step in the metabolism of E . coli cells . The saturation of the limiting reaction with a substrate at a flow rate higher than the critical one may be responsible for the hyperbolic profile of chemostat curves . The above hypothesis was confirmed by experiments conducted under extreme conditions, in which the composition of the growth medium was changed and the temperature was raised so that the cloned protein synthesis was depressed . Under such conditions, the enzyme composition of the cell was fixed and determined by the physiological state of the culture at a time when the temperature was elevated . The hypothesis was also supported by the results obtained in growing the strain with the induced protein synthesis in media to which various compounds were added.

Biochim Biophys Acta, 1986 Aug 29, 888(1), 10 - 7
Adriamycin transport and sensitivity in fatty acid-modified leukemia cells; Burns CP et al.; The membrane phospholipids of L1210 murine leukemia cells were modified by supplementing the growth medium with micromolar concentrations of polyunsaturated or monounsaturated fatty acids . This procedure results in enrichment of cellular phospholipids by the supplemented fatty acid . Enrichment with polyunsaturated fatty acids resulted in a marked increase in sensitivity to adriamycin as compared to enrichment with monounsaturated fatty acids . The increased cytotoxicity was directly proportional to the extent of unsaturation of the inserted fatty acid, but there was no difference in cells enriched with n-3 compared with n-6 family fatty acids . To explore the mechanism of this observation, we examined whether augmented uptake of the drug might explain the increased cytotoxicity . The uptake of {14C}adriamycin, which was approximately linear at later time points, was only partially temperature dependent and never reached a steady state . Initial uptake at time points prior to 60 s could not be measured due to high and variable rapid membrane adsorption . Cellular accumulation of drug was greater in the docosahexaenoate 22:6-enriched L1210 cells as compared to oleate 18:1-enriched cells and was about 32% greater after 20 min . When L1210 cells were enriched with six fatty acids of variable degrees of unsaturation, the accumulation of adriamycin was directly correlated with the average number of double bonds in the fatty acids contained in cellular phospholipids . There was no difference in efflux of drug from cells pre-loaded with adriamycin . We conclude that the greater accumulation of adriamycin by the polyunsaturated fatty acid-enriched L1210 cells likely explains the increased sensitivity of these cells to adriamycin compared to 18:1-enriched cells.

Biochim Biophys Acta, 1986 Aug 22, 867(4), 234 - 43
Molecular cloning of mRNAs expressed specifically during spherulation of Physarum polycephalum; Bernier F et al.; A cDNA library was constructed using the poly(A)+ RNA extracted from spherulating Physarum polycephalum microplasmodia . This library (740 clones) was screened by differential hybridization with 32P-labeled poly(A)+ RNA from growing plasmodia and developing spherules . The results showed that at least 30% of the clones corresponded to mRNAs expressed specifically in spherulating plasmodia . The 35 spherulation-specific cDNA clones giving the strongest hybridization signals were analysed . From this group, four different sequences complementary to very abundant mRNAs were identified . They each accounted for 1.5% of 4.5% of all the clones in the library and probably represented the most abundant spherulation-specific mRNAs . In addition, four less abundant mRNAs were identified from stage-specific clones giving weaker hybridization signals . These sequences represented individually between 0.3% and 0.7% of the clones in the library . Northern blots showed that these eight different sequences were absent from plasmodia and were most abundant 24-36 h after the induction of spherulation . Similar results were also obtained when spherulation was induced by the addition of a sublethal concentration of ferrous iron ions to the growth medium . Hybridization of the spherule-specific clones to Southern blots of genomic DNA suggested the presence of one copy for each gene.

Biochim Biophys Acta, 1986 Aug 7, 860(1), 1 - 8
Perturbation of Na+ and K+ gradients in human fibroblasts incubated in unsupplemented saline solutions; Dall'Asta V et al.; Changes in the intracellular concentrations of Na+ and K+ of fetal human fibroblasts have been followed after replacement of serum-containing growth media with unsupplemented and serum-supplemented saline solution (Earle's balanced salt solution) . Incubation in unsupplemented salt solution was followed by a progressive increase of the internal Na+ counterbalanced by a decrease of internal K+, without major alterations of the internal osmolarity . After 3 h incubation the intracellular Na+ and K+ concentrations were 120 mM and 50 mM, respectively . These intracellular ion derangements were not associated with a failure of the (Na+ + K+)-ATPase pump, whose activity actually increased with enhanced intracellular Na+ concentration . Ion changes did not take place when serum (in excess of 0.5%, final concentration) was present in the saline solution and a complete restoration to normal of the Na+ and K+ gradients occurred upon addition of serum to cells previously incubated in plain saline solution . The effects of serum were mimicked by furosemide, thus suggesting that channels sensitive to this diuretic are involved in the movement of Na+ and K+ following fibroblast incubation in unsupplemented saline solution.

Biochem Cell Biol, 1986 Aug, 64(8), 811 - 5
The effects of hypoxanthine on methotrexate-induced differentiation of cultured human choriocarcinoma (BeWo) cells; Burres NS et al.; When cultured human choriocarcinoma (BeWo) cells are exposed to methotrexate, proliferation ceases and cells undergo a complex differentiative response that resembles development of normal trophoblast . Although thymidylate starvation has been shown to be causative in methotrexate-induced expression of syncytiotrophoblastic markers by BeWo cells, the role of purine deprivation is uncertain since previous studies utilized growth media containing exogenous purines . This work investigated the effects of hypoxanthine on methotrexate-induced cell enlargement, expression of placental alkaline phosphatase, and morphological differentiation to the syncytiotrophoblast-like phenotype . When methotrexate exposures (1 microM, 48 h) were conducted in a purine-free basal medium supplemented with dialyzed fetal bovine serum, RNA synthesis was greatly reduced and cell enlargement did not occur . Specific methods for removing purines (charcoal extraction and xanthine oxidase treatment) decreased the ability of serum to support cell enlargement during methotrexate exposures, whereas addition of hypoxanthine to culture fluids restored its ability to support maximal increases in cell mass, confirming that purines were the factors lost during dialysis . In contrast, morphologically differentiation to the syncytiotrophoblast-like phenotype and increased expression of placental alkaline phosphatase were unaffected by the availability of purines during exposure to methotrexate.

J Antimicrob Chemother, 1986 Aug, 18(2), 251 - 60
The antimicrobial activity of ciprofloxacin against Legionella species and the treatment of experimental Legionella pneumonia in guinea pigs; Saito A et al.; The antimicrobial activity of ciprofloxacin was tested against 15 standard reference strains, and 37 clinical and environmental strains of Legionella pneumophila by an agar dilution method, using a new growth medium (B-SYE agar) which we devised . The minimal inhibitory concentrations of ciprofloxacin were found to be inoculum dependent, and ranged from 0.02 to 0.06 mg/l at 10(4) cfu inoculum and 0.02 by 0.125 mg/l at 10(6) cfu inoculum . The most potent antibacterial activity was shown by rifampicin, followed by ofloxacin, ciprofloxacin, enoxacin, norfloxacin, erythromycin and pipemidic acid in that order . The therapeutic efficacy of ciprofloxacin in experimental guinea pig pneumonia due to L . pneumophila was fairly good with a survival rate of 80% . From other data of ours, its effectiveness in experimental pneumonia was equal to or greater than that of erythromycin . Further studies would be appropriate to investigate the possibility of using ciprofloxacin for the treatment of human L . pneumophila infection.

Proc Natl Acad Sci U S A, 1986 Aug, 83(16), 6070 - 4
Molecular analysis of the DNA sequences involved in the transcriptional regulation of the phosphate-repressible acid phosphatase gene (PHO5) of Saccharomyces cerevisiae; Bergman LW et al.; The expression of the PHO5 gene of Saccharomyces cerevisiae is transcriptionally regulated in response to the level of inorganic phosphate present in the growth medium . We have identified, by DNA deletion analysis, the sequences (upstream activator sequences) that mediate this response . The sequence 5' CTGCACAAATG 3' is present in two copies located within a 60-base-pair region . The presence of a single copy of the sequence is sufficient for the phosphate-mediated transcriptional response . In addition, a DNA fragment that contains two copies of this sequence will act to repress transcription of a CYC1-lacZ fusion when placed either upstream or downstream of the CYC1 activator sequence.

Prostaglandins Leukot Med, 1986 Aug, 23(2-3), 173 - 8
Eicosanoid regulation of oosporogenesis by Lagenidium giganteum; Kerwin JL et al.; Cell proliferation is inhibited in many biological systems by lipid peroxides and related products derived from polyunsaturated fatty acids . Using developmentally synchronized cultures of Lagenidium giganteum (Oomycetes: Lagenidiales), a facultative parasite of mosquito larvae, it has been documented that oxidative lipid metabolism is necessary for the induction and subsequent maturation of its sexual stage, the oospore . Addition of lipoxygenase inhibitors to liquid cultures of the fungus results in the stage-specific disruption of antheridial induction, gametangial fusion, induction of meiosis and spore cell wall formation . Oosporogenesis is inhibited by these compounds at concentrations which have no discernible effect on mycelial viability or asexual reproduction . Cyclooxygenase inhibition had comparable effects using ibuprofen and to a lesser extent with indomethacin . Phenylbutazone and the salicylates affected oosporogenesis only at concentrations which decreased asexual reproduction or mycelial viability . The inhibitory effects of NDGA on oosporogenesis could be reversed using partly purified eicosanoid extracts from growth media.

Biochem Biophys Res Commun, 1986 Jul 31, 138(2), 699 - 706
31P NMR spectra of rodent and avian fibroblasts transformed by Rous or Kirsten sarcoma viruses; Dimicoli JL et al.; 31P NMR spectra of normal rodent and avian fibroblasts were compared to those of the same cells transformed either by the Rous sarcoma virus (RSV) or by the Kirsten sarcoma virus (Ki-MSV) . Under physiological conditions, the spectra of living or perchloric acid extracted chicken embryo fibroblasts, rat cell line FR3T3 and mouse cell line C127 did not differ from those of their counterparts transformed by RSV or Ki-MSV . However, in the case of FR3T3 cells, on shifting from 37 degrees C to 20 degrees C, and particularly if PBS replaced serum growth medium, a different, though transitory, response of the transformed cells was detected . They then showed, within few minutes, a more rapid ATP depletion with accumulation of fructose 1,6-diphosphate (FDP), as compared to normal control cells.

Virology, 1986 Jul 30, 152(2), 298 - 307
Bovine rotavirus maturation is a calcium-dependent process; Shahrabadi MS et al.; Bovine rotavirus-infected MA-104 cells maintained in the presence and absence of CaCl2 displayed cytopathic effects (cpe) distinct from each other . Lysates of calcium-free cultures were unable to induce cpe in subsequent passages in MA-104 cells, an observation reflected by the demonstration that virus titers of such lysates were drastically reduced . The minimum concentration of CaCl2 in the growth medium required to maintain maximum virus yield was determined to be approximately 0.17 mM . The period of calcium-dependency for infectious virus formation was between 6 and 12 hr postinfection at 37 degrees, a time corresponding to the entire log phase of virus growth . Viruses produced in the absence of calcium were found to be exclusively incomplete single-shelled particles (D particles), as determined by cesium chloride density gradient analysis, electron microscopy, and SDS-polyacrylamide gel electrophoresis . Subsequent examination of virus-specified proteins in infected cells revealed that there was a reduction in the level of the major outer capsid protein (42K) in the absence of calcium . Thus, total inhibition of mature virus production under this condition could be due to the combined effect of reduced production of the 42K protein and incomplete assembly of the virus.

Biochim Biophys Acta, 1986 Jul 10, 859(1), 105 - 9
Cultured heart cell reaggregates: a model for studying relationships between aging and lipid composition; Yechiel E et al.; Cultured heart cells serve as a common model for studying the electronphysiology and pharmacology of intact cells of the myocardium from which they are derived (Sperelakis, N . (1982) in Cardiovascular Toxicology (Van Stel, E.W., ed.), pp . 57-108, Raven Press, New York) . In this study, heart cell reaggregates were used for investigating the relationship between lipid composition and aging of the heart cells . Spherical reaggregates were prepared from newborn, 3- and 18-month-old rats, respectively . They were grown for 6 days in culture and then analyzed for their lipid composition and creatine phosphokinase levels . There was an age-related increase in total phospholipids and cholesterol level per unit of cell protein . Due to a relatively greater increase in the cholesterol, the mole ratio of cholesterol to phospholipids increased with animal age . The phospholipid composition was also affected . Thus, sphingomyelin levels increased, while those of phosphatidylcholine decreased; these alterations became much more pronounced with increasing animal age . All these changes could be affected by adding small unilamellar vesicles composed of egg phosphatidylcholine to the growth medium on the 5th day after seeding . Such treatment resulted in a lesser ratio of cholesterol to phospholipid as well as sphingomyelin to phosphatidylcholine, without reducing the total phospholipid per unit protein; the level of creatine phosphokinase was also reduced . This study demonstrated that cultured heart reaggregates can serve as a model for studying aging of the whole animal . Its main advantage is the ability to employ cells from rats of any desired age . Currently this is not possible for cultured heart monolayers.

J Cell Sci, 1986 Jul, 83, 165 - 79
Intracellular pH in Dictyostelium: a 31P nuclear magnetic resonance study of its regulation and possible role in controlling cell differentiation; Kay RR et al.; Intracellular pH (pHi) has been measured in Dictyostelium discoideum cells by 31P nuclear magnetic resonance . Ax2 cells, newly harvested from growth medium, maintained a pHi of 7.33 +/- 0.04 (17) at an extracellular pH ranging from 3.5 to 6.5 . Below pH 3.5 the cells tend to lyse, whereas at pH values above 6.5 their pHi rises though they remain viable . pHi regulation in acid medium is not dependent on external Na+ or any other inorganic ion and so most probably involves the electrogenic plasma membrane proton pump . No significant change in pHi was detected during development through to the slug stage . Mature stalk cells gave a very acidic phosphate signal (pH less than or equal to 5.5) which was probably vacuolar in origin . Indirect experiments had suggested that pHi might regulate the development of Dictyostelium cells, with low pHi favouring stalk cell and high pHi favouring spore cell differentiation . In particular, two inhibitors of the plasma membrane proton pump, diethylstilbestrol and zearalenone, had been shown to be stalk cell inducers . In the present studies measurements of pHi of cells exposed to these inducers failed to detect the expected drop in pHi . In addition, DIF-1 (a low Mr factor), the natural inducer of stalk cell formation, caused, if anything, a slight alkalinization of the cells . Thus the original theory linking pHi and cell differentiation is not supported by these results and therefore appears to require some modification . Finally, extract experiments revealed the existence of two unidentified abundant phospho-compounds with resonant frequencies close to inorganic phosphate . The existence of these compounds can complicate the interpretation of spectra gained from living Dictyostelium cells.

J Gen Microbiol, 1986 Jul, 132 ( Pt 7), 2013 - 21
A comparative study of the morphology and viability of hyphae of Penicillium expansum and Phytophthora nicotianae during freezing and thawing; Smith D et al.; The changes in morphology of Penicillium expansum Link and Phytophthora nicotianae Van Breda de Haan during freezing and thawing in a growth medium with and without the cryoprotective additive glycerol were examined with a light microscope fitted with a temperature-controlled stage . Viability of 0.5-1.0 mm diameter colonies of both fungi was determined after equivalent rates of cooling to -196 degrees C in the presence or absence of glycerol . In P . expansum shrinkage occurred in all hyphae at rates of cooling of less than 15 degrees C min-1; at faster rates intracellular ice nucleation occurred . The addition of glycerol increased the rate of cooling at which 50% of the hyphae formed intracellular ice from 18 degrees C min-1 to 55 degrees C min-1 . This species was particularly resistant to freezing injury and recovery was greater than 60% at all rates of cooling examined . At rapid rates of cooling recovery occurred in hyphae in which intracellular ice had nucleated . In contrast, during the cooling of Ph . nicotianae in the growth medium, shrinkage occurred and no samples survived on thawing from -196 degrees C . However, on the addition of glycerol, shrinkage during freezing decreased and viable hyphae were recovered upon thawing; at rates of cooling over 10 degrees C min-1 the loss of viability was related to glycerol-induced osmotic shrinkage during cooling rather than to the nucleation of intracellular ice.

Zh Mikrobiol Epidemiol Immunobiol, 1986 Jul, (7), 32 - 7
{Purification and properties of Bordetella pertussis toxin}; Gureeva AA et al.; The modified method for the isolation and purification of B . pertussis toxin has been proposed . Chromatography with the use of hydroxylapatite and lentil lectin--Sepharose 4B has permitted the isolation of the preparation purified 600 times . Its molecular weight is about 90,000 . The preparation has been found to possess leukocytosis-stimulating, histamine-sensitising and hemagglutinating activity . Electrophoretic analysis has revealed that the isolated substance consists of four subunits with molecular weights 28,400, 24,300, 21,800 and 15,200 . This substance has proved to be capable of hydrolyzing NAD+, as well as of suppressing the GTPase activity of transducin, which is indicative of the covalent modification (ADP-ribosylyzing) of GTP-binding protein under the action of B . pertussis toxin . Two methods for the isolation of B . pertussis toxin (from liquid and solid growth media), as well as the isolation of the toxin from different B . pertussis strains, are evaluated.

Radiat Res, 1986 Jul, 107(1), 58 - 72
Quantitation of the involvement of the recA, recB, recC, recF, recJ, recN, lexA, radA, radB, uvrD, and umuC genes in the repair of X-ray-induced DNA double-strand breaks in Escherichia coli; Sargentini NJ et al.; Isogenic Escherichia coli strains carrying single DNA-repair mutations were compared for their capacity for (i) the repair of X-ray-induced DNA double-strand breaks (DSB) as measured using neutral sucrose gradients; (ii) medium-dependent resistance, i.e., a recA-dependent X-ray survival phenomenon that correlates closely with the capacity for repairing DSB; and (iii) the growth medium-dependent, recA-dependent repair of X-ray-induced DNA single-strand breaks (SSB) as measured using alkaline sucrose gradients (about 80% of these SSB are actually parts of DSB) . These three capacities were measured to quantitate more accurately the involvement of the various genes in the repair of DSB over a wide dose range . The mutations tested were grouped into five classes according to their effect on the repair of X-ray-induced DSB: (I) the recA, recB, recC, and lexA mutants were completely deficient; (II) the radB and recN mutants were about 90% deficient; (III) the recF and recJ mutants were about 70% deficient; (IV) the radA and uvrD mutants were about 30% deficient; and (V) the umuC mutant resembled the wild-type strains in its capacity for the repair of DSB.

J Reprod Fertil, 1986 Jul, 77(2), 547 - 58
Culture of epithelial and stromal cells of guinea-pig endometrium and the effect of oestradiol-17 beta on the epithelial cells; Chaminadas G et al.; Epithelial and stromal cells of guinea-pig endometrium were separated by enzymic digestion, isolated by successive centrifugation, and maintained in culture as pure cell types for 5 days on growth medium . On Day 5, ultrastructural studies were performed on the two cell types, demonstrating that epithelial cells can grow as a monolayer composed of cohesive groups of polygonal cells (1.3 X 10(5) cells/cm2), while stromal cells were mostly fibroblastic . The effect of hormones was studied on the epithelial cells in culture . The monolayer was cultured into harvest medium for 3 days to ensure the complete removal of endogenous steroids, then these cells were incubated with 2 X 10(-9) M-oestradiol-17 beta for 3 days . There was a rise in the progesterone receptor level, varying from 1.3 to 10.8 times . The three enzymes known to interfere with oestradiol-17 beta metabolism were present in the epithelial cells grown in our culture conditions . By incubation with oestrone sulphate for 3 days it was demonstrated that, in cultured epithelial cells, oestrone sulphate is converted into oestradiol-17 beta sulphate, and oestrogen sulphates are hydrolysed to active oestrogens.

Mutat Res, 1986 Jul, 166(1), 23 - 8
Repair of DNA double-strand breaks in UV-irradiated Escherichia coli uvrB recF cells is inhibited by rich growth medium; Sharma RC et al.; Ultraviolet (UV)-irradiated uvrB recF and uvrB recB cells of Escherichia coli K-12 showed similar radiation sensitivities when plated on minimal growth medium (MM), however, the uvrB recF cells were much more UV radiation-sensitive than the uvrB recB cells when plated on rich growth medium . Sedimentation analysis of the DNA from UV-irradiated uvrB recF cells suggests that the rich medium killing of uvrB recF cells is due to the inhibition of the repair of UV-radiation-induced DNA double-strand breaks, i.e., the killing is due to the inhibition of the recB-dependent pathway of postreplication repair . Furthermore, we demonstrated that the DNA double-strand breaks that were formed in UV-irradiated uvrB recA200(Ts) cells incubated at 42 degrees C in rich growth medium were not repaired whether the medium during subsequent repair incubation at 30 degrees C was MM or rich growth medium, while DNA double-strand breaks that were formed in MM at 42 degrees C could be repaired in MM or in rich growth medium at 30 degrees C . How the absence of an abrupt slowing of DNA synthesis when UV-irradiated cells are held in rich growth medium (Sharma and Smith, 1985b) may prevent the repair of these DNA double-strand breaks is discussed.

J Bacteriol, 1986 Jul, 167(1), 272 - 8
Intracellular Trp repressor levels in Escherichia coli; Gunsalus RP et al.; A radioimmunoassay for the Trp repressor protein of Escherichia coli was developed with antisera raised against purified Trp repressor protein . This assay was used to directly measure the intracellular Trp repressor content in several E . coli K-12 and B/r strains . Repressor levels varied from 2.5- to 3-fold in response to L-tryptophan concentration in the growth medium (15 to 44 ng of repressor per mg of protein) . Neither cell growth rate nor culture age had a significant effect on repressor concentrations within the cell . Addition of L-tryptophan to the growth medium resulted in lowered intracellular levels of Trp repressor . The absolute amounts of native Trp repressor molecules per cell varied between 120 and 375 dimers in the presence and absence of L-tryptophan in the culture medium, respectively . Assuming an intracellular volume of 7.3 microliters/10(10) E . coli cells, the Trp repressor concentration varied from 270 to 850 nM in response to extracellular tryptophan levels . These findings represent the first direct measurements of Trp repressor levels in E . coli and confirm the autoregulatory nature of the trpR gene.

Int J Radiat Biol Relat Stud Phys Chem Med, 1986 Jul, 50(1), 155 - 65
Reduction of intracellular glutathione content and radiosensitivity; Vos O et al.; The intracellular glutathione (GSH) content of HeLa, CHO and V79 cells was reduced by incubating the cells in growth medium containing buthionine sulphoximine or diethyl maleate (DEM) . Clonogenicity, single-strand DNA breaks (ssb) and double-strand DNA breaks (dsb) were used as criteria for radiation-induced damage after X- or gamma-irradiation . In survival experiments, DEM gave a slightly larger sensitization although it gave a smaller reduction of the intracellular GSH . In general, sensitization was larger for dsb than for ssb, also the reduction of the o.e.r . was generally larger for dsb than for ssb . This may be due to the higher dose rate in case of dsb experiments resulting in a higher rate of radiochemical oxygen consumption . In general, no effect was found on post-irradiation repair of ssb and dsb.

J Bacteriol, 1986 Jul, 167(1), 299 - 304
Incorporation and modification of exogenous phosphatidylcholines by mycoplasmas; Rottem S et al.; The uptake and modification of exogenous phosphatidylcholine (PC) by several Mycoplasma and Spiroplasma species was investigated . While in most Mycoplasma species and in all Spiroplasma species tested the PC appears to be incorporated unchanged from the growth medium, the PC of M . gallisepticum, M . pulmonis, and M . pneumoniae was disaturated PC, apparently formed by modification of 1-saturated-2-unsaturated PC from the growth medium . The modification of the exogenous PC by M . gallisepticum was inhibited by chloramphenicol under conditions that did not affect de novo synthesis of phosphatidylglycerol . A low activity of an endogenous phospholipase A was detected in native M . gallisepticum membranes . The activity was markedly stimulated by treating the membranes with low concentrations of the nonionic detergents . The PC modification was affected by the fatty acid composition of the exogenous PC species . Diunsaturated, 1-saturated-2-unsaturated, and 1-unsaturated-2-saturated PCs were modified to various extents, whereas the disaturated dipalmitoyl PC (DPPC) was not . Both modified and unmodified PCs were incorporated by the cells, but the unmodified DPPC was incorporated at a lower rate and to a lesser extent . The possibility that the incorporation of DPPC into M . gallisepticum cells is associated with the formation of intracytoplasmic membranes is discussed.

J Neurochem, 1986 Jul, 47(1), 223 - 31
Effect of CDP-choline on hypocapnic neurons in culture; Mykita S et al.; Neuronal cultures from chick embryo cerebral hemispheres were protected against a hypocapnic injury by adding to their growth medium 10(-6)M CDP-choline before or after the injury . The protection obtained with CDP-choline was analyzed by a morphometric analysis and showed that pretreatment of neuronal cultures with CDP-choline maintained the number of cell aggregates and of primary neuronal processes at control values after hypocapnic shock . Various experiments showed that the intact molecule was responsible for the protective action, since pretreatment with different concentrations of various nucleosides and nucleotides (up to 10(-5) M), choline, and phosphorylcholine was without protective effect . The addition of CDP-choline after the hypocapnic injury resulted in a protection of the cultures as shown by morphological observation . Incubation of neurons with radioactive choline showed that hypocapnia increased the incorporation of the label into phospholipids whereas the presence of CDP-choline reduced it . The de novo synthesis of choline was affected by neither hypocapnia nor CDP-choline treatment . The results indicate that CDP-choline may have the capacity to protect neurons under conditions of basic pH and that cellular proliferation may be stimulated by the compound.

Mol Cell Biol, 1986 Jul, 6(7), 2298 - 304
A DNA fragment containing the upstream activator sequence determines nucleosome positioning of the transcriptionally repressed PHO5 gene of Saccharomyces cerevisiae; Bergman LW; The functional relationship of nucleosome positioning and gene expression is not known . Using high-copy plasmids, containing the yeast phosphate-repressible acid phosphatase gene (PHO5) and the TRP1/ARS1 vector system, I have determined the nucleosomal structure of the 5' region of the PHO5 gene and demonstrated that the nucleosomal positioning of this region is independent of orientation or position in the various plasmid constructions utilized . However, deletion of a 278-base pair BamHI-ClaI fragment from the 5'-flanking sequences of the PHO5 gene causes the nucleosome positioning to become dependent on orientation or position in the plasmids tested . Use of PHO5-CYC1-lACZ fusions have demonstrated that this DNA fragment contains the sequences responsible for the transcriptional regulation of the PHO5 gene in response to the level of phosphate in the growth media . The nucleosome positioning in the 5' region of PHO5 may be determined by an interaction with the sequences or machinery responsible for transcriptional regulation of the gene.

J Biol Chem, 1986 Jun 15, 261(17), 7871 - 8
Mechanisms of action of chloroalanyl antibacterial peptides . Identification of the intracellular enzymes inactivated on treatment of Escherichia coli JSR-O with the dipeptide beta Cl-LAla-beta Cl-LAla; Boisvert W et al.; The dipeptide beta Cl-LAla-beta Cl-LAla is an antibacterial agent designed to utilize bacterial peptide transport for intracellular delivery of the alanine racemase inactivator beta Cl-LAla . The minimum inhibitory concentrations (MICs) for the peptide against Gram-negative species grown on enriched agar medium range from 1.56 to 12.5 micrograms/ml; MICs are increased to greater than 100 micrograms/ml when D-alanine is included in the medium, indicating that alanine racemase is, in fact, inhibited in sensitive species . When susceptible Gram-negative cells are grown on a minimal medium, D-alanine supplementation alone does not increase the MICs for beta Cl-LAla-beta Cl-LAla, but complete protection is afforded by supplementation with D-alanine, L-valine, L-leucine, and L-isoleucine . In liquid culture, the peptide is: bactericidal and lytic against Escherichia coli JSR-O growing in enriched medium or in minimal medium supplemented with the branched-chain amino acids; only inhibitory against these cells growing in minimal medium supplemented with D-alanine; and ineffective against these cells in minimal medium containing the branched-chain amino acids plus D-alanine . Cells exposed to beta Cl-LAla-beta Cl-LAla (with the protection of the four amino acids) have specific activities of both alanine racemase and transaminase B that are lower than those of cultures not treated with the peptide . Finally, E . coli JSR-O alanine racemase experiences time-dependent loss of activity when exposed to the dipeptide in the presence of aminopeptidases; the dipeptide alone is not an inactivator of the racemase in vitro . These results suggest the following mechanism of action for beta Cl-LAla-beta Cl-LAla: transport of the dipeptide into the cell; intracellular hydrolysis to give accumulation of beta Cl-LAla; and subsequent inactivation of targeted enzymes . Whether inactivation of the racemase or of the transaminase determines the pathophysiologic effects of the peptide depends on the composition of the growth medium.

Biochemistry, 1986 Jun 3, 25(11), 3225 - 30
DNA fragmentation and cytotoxicity from increased cellular deoxyuridylate; Ingraham HA et al.; Previous results from this laboratory have shown that thymidylate deprivation results in dramatic elevation of intracellular dUTP and incorporation of dUMP into DNA . The goal of the present studies was to determine whether the latter changes may play a part in the associated cytotoxicity ("thymineless death"), which is ordinarily assumed to be a direct result of reduced intracellular dTTP . The approach used here was to increase intracellular dUTP without allowing dTTP to diminish and observe the effects on cell viability . dUMP pools were expanded by exposure of cells to deoxyuridine {in cell growth medium containing hypoxanthine, methotrexate, and thymidine (HAT medium)}, resulting in accumulation of dUTP to levels that approached those of dTTP, which were at, or higher than, the levels in untreated cells . In conjunction with this the cells became nonviable, and newly synthesized DNA was fragmented, both of which occur with thymidylate depletion and, we assume, result from the active process of excision repair at the many uracil-containing sites in DNA . The results indicate that, although the relative importance of low dTTP remains unknown, elevated dUTP can account for the cytotoxicity caused by thymidine starvation . Most of the "dTTP" measured by the DNA polymerase assay in cells treated with methotrexate (MTX) (plus purine supplement) was, in fact, dUTP, which may explain some previous observations of only modest depression of dTTP in cells treated with MTX or similarly acting drugs.

In Vitro Cell Dev Biol, 1986 Jun, 22(6), 355 - 9
The growth of WI-38 cells in a serum-free, growth factor-free, medium with elevated calcium concentrations; Praeger FC et al.; The growth of WI-38 cells in serum-free growth medium with and without hormone supplementation in the presence of elevated Ca2+ concentrations was investigated . At 5 mM CaCl2, WI-38 cells seeded at low density without serum or hormone supplementation showed up to a 12-fold increase in cell number at saturation density over that obtained at day 1 . Saturation densities were comparable when either 5 mM CaCl2 or epidermal growth factor (1 mM CaCl2) was used in the presence of insulin, dexamethasone and transferrin . Combining suboptimal doses of epidermal growth factor and CaCl2 resulted in an additive effect on saturation density . Thus, normal human diploid cells are capable of substantial growth in serum-free, hormone-free growth medium . In contrast, confluent cultures refed with the same medium are not responsive to elevated Ca2+ concentrations . In fact, elevated Ca2+ concentrations inhibited the proliferative response of confluent cultures to epidermal growth factor, but enhanced their response to the combined treatment of insulin, transferrin and dexamethasone.

Ecotoxicol Environ Saf, 1986 Jun, 11(3), 277 - 94
On the toxic effects of tetraethyl lead and its derivatives on the chrysophyte Poterioochromonas malhamensis . VII . Protective action of thiol compounds, vitamins, trace elements, and other agents; Roderer G; The influence of 45 different substances on the growth inhibiting effects of triethyl lead chloride (TriEL) in cultures of the unicellular alga Poterioochromonas malhamensis was studied . Ten thiol or disulfide compounds, 9 vitamins, 12 trace elements, 14 miscellaneous agents, and 9 combinations of these agents were tested . The agents were applied to the algal cultures in three different concentrations simultaneously with 10 microM TriEL and incubated for 72 hr . While none of the tested thiol and disulfide compounds remarkably protected the algae from TriEL toxicity, two vitamins (tocopheryl acetate, ascorbic acid), one trace element (zinc), adenosine-5'-triphosphate Na2 salt, cyclic AMP, and concanavalin A as well as combinations of some of these agents were found to suppress markedly the growth inhibiting effects of TriEL . Zinc was the most effective agent; it increased algal growth in TriEL-treated cultures by about 70 times as compared with cultures containing TriEL alone . A combination of 10 essential trace elements was even more protective and almost completely suppressed TriEL toxicity . In contrast to this, some of the other agents potentiated the toxic effects of TriEL (e.g., magnesium, molybdenum, caffeine, deuteriumoxide, chlorpromazine, dimethylsulfoxide) in the tested concentration ranges . The most protective agents Zn, VitC, and VitE did not prevent the inhibitory effects of TriEL on microtubule (MT) assembly in vitro, suggesting that their in vivo protection is based on mechanisms other than direct protecting MTs from the attack of the lead compound . Zn had no direct influence on the stability and half life of TriEL in the growth medium . The lack of protection found by the thiol compounds used suggests that most probably general thiol interaction of TriEL is not its major toxic mechanism of action . It is postulated that the protective action of Zn, VitE, and VitC is directly or indirectly mediated by suppression of TriEL-induced peroxidation processes in the poisoned algae . The protective agents found provide a basis for further screening experiments in order to test their "therapeutic" potency in experimental animals poisoned with organolead.

Mol Pharmacol, 1986 Jun, 29(6), 606 - 13
Characterization of DNA lesions induced by CaCrO4 in synchronous and asynchronous cultured mammalian cells; Sugiyama M et al.; Alkaline elution studies demonstrated CaCrO4-induced DNA single strand breaks and DNA-protein crosslinks . DNA single strand breaks increased following treatment with 10-400 microM CaCrO4 in Chinese hamster ovary cells maintained with a minimal salts/glucose medium . DNA single strand breaks were rapidly repaired when extracellular CaCrO4 was removed even following exposure levels of CaCrO4 (200 microM for 2 hr) which reduced survival to 0.6% . Under these exposure conditions the trypan blue exclusion was greater than 80%, whereas cell growth was inhibited by 46% within 24 hr . The DNA-protein crosslinks induced by 10 microM CaCrO4 were repaired in the absence of metal within 24 hr . In contrast, the amount of DNA-protein crosslinks measured 24 hr after a 2-hr treatment with 50, 100, and 200 microM CaCrO4 remained unchanged at the 50 microM level or increased at the two higher concentrations . Thus, at concentrations of 50 microM or greater there was no repair of the DNA protein crosslinks, and this may have been due to cytotoxicity of the metal . CaCrO4 at 10 or 25 microM exposure for 6 hr also induced DNA-protein crosslinking in Chinese hamster ovary cells maintained in normal tissue culture growth media . The lack of repair of DNA-protein crosslinks at the 25 microM level, which did not substantially reduce cell survival, indicated the persistence of these lesions in a noncytotoxic form . Uptake of CaCrO4 was linear with all of the concentrations tested . Analysis of the cell cycle sensitivity to CaCrO4 revealed that cells in early S phase were the most sensitive to the cytotoxic and strand breaking activity of CaCrO4 . Compared with other phases of the cell cycle, there was also an elevated level of DNA-protein crosslinks when cells were treated in early S phase and incubated 24 hr without CaCrO4 . These results implicate the DNA-protein crosslink as an important lesion that may be responsible for the cytotoxic and carcinogenic properties of chromate.

J Neurochem, 1986 Jun, 46(6), 1859 - 64
Effect of modification of membrane phospholipid composition on phospholipid methylation in aggregating cell culture; Dainous F et al.; The effect of the presence of nitrogenous bases in the growth medium of fetal rat brain aggregating cell cultures was investigated . The presence of either N-methylethanolamine (MME) or N,N-dimethylethanolamine (DME) in the growth medium resulted in significant increase of the corresponding phospholipid, phosphatidyl-N-monomethylethanolamine (PMME) or phosphatidyl-N,N-dimethylethanolamine (PDME) . They represented 28% and 32% of the total phospholipids, respectively . The presence of the new phospholipids was accompanied by a significant decrease of phosphatidylethanolamine (PE) and phosphatidylcholine (PC) . Cells grown in the presence of ethanolamine or choline had only barely detectable amounts of PMME and PDME . Intact cells previously grown with the bases were incubated with {methyl-3H}methionine . Incubation of cells previously grown in presence of the bases MME and DME resulted in a marked increase of radioactivity in the corresponding phospholipids possessing one additional methyl group, PDME and PC respectively . The incorporation of S-adenosyl{methyl-3H}methionine (AdoMet) was examined in cell homogenates incubated in presence or absence of either PMME or PDME acceptors . The addition of these exogenous phospholipids caused a three-or fourfold stimulation of radioactivity incorporated into the total phospholipids of cells grown in the absence of nitrogen bases . The cells grown in presence of either MME or DME in the culture medium did not show an increased incorporation of methyl groups from AdoMet into the total phospholipids after addition of exogenous acceptors . This work suggests that MME and DME incorporated into the corresponding phospholipids function as effective substrates for phospholipid-N-methylation.

J Biochem (Tokyo), 1986 Jun, 99(6), 1735 - 42
Characterization of the fatty acid synthetase system of Curtobacterium pusillum; Kawaguchi A et al.; Curtobacterium pusillum contains 11-cyclohexylundecanoic acid as a major component of cellular fatty acids . A trace amount of 13-cyclohexyltridecanoic acid is also present . Fatty acids other than omega-cyclohexyl fatty acids present are 13-methyltetradecanoic, 12-methyltetradecanoic, n-pentadecanoic, 14-methylpentadecanoic, 13-methylpentadecanoic, n-hexadecanoic, 15-methylhexadecanoic, 14-methylhexadecanoic, and n-heptadecanoic acids . The fatty acid synthetase system of this bacterium was studied . Various 14C-labeled precursors were added to the growth medium and the incorporation of radioactivity into cellular fatty acids was analyzed . Sodium {14C}acetate and {14C}glucose were incorporated into almost all species of cellular fatty acids, the incorporation into 11-cyclohexylundecanoic acid being predominant . {14C}Isoleucine was incorporated into 12-methyltetradecanoic and 14-methylhexadecanoic acids: {14C}leucine into 13-methyltetradecanoic and 15-methylhexadecanoic acids; and {14C}valine into 14-methylpentadecanoic acid . {14C}-Shikimic acid was incorporated almost exclusively into omega-cyclohexyl fatty acids . The fatty acid synthetase activity of the crude enzyme preparation of C . pusillum was reconstituted on the addition of acyl carrier protein . This synthetase system required NADPH and preferentially utilized cyclohexanecarbonyl-CoA as a primer . The system was also able to use branched- and straight-chain acyl-CoAs with 4 to 6 carbon atoms effectively as primers but was unable to use acetyl-CoA . However, if acetyl acyl carrier protein was used as the priming substrate, the system produced straight-chain fatty acids . The results imply that the specificity of the initial acyl-CoA:acyl carrier protein acyltransferase dictates the structure of fatty acids synthesized and that the enzymes catalyzing the subsequent chain-elongation reactions do not have the same specificity restriction.

Mol Biochem Parasitol, 1986 Jun, 19(3), 241 - 9
Polyamine biosynthesis in trichomonads; North MJ et al.; Trichomonas vaginalis, Tritrichomonas foetus and Trichomitus batrachorum grown in modified Diamond's medium all had high concentrations of putrescine and lower concentrations of spermidine and spermine . Ornithine decarboxylase (ODC; EC 4.1.1.17) was detectable in all three species although at significantly different levels . Trichomonas vaginalis had the highest activity (typically around 1.85 nmol min-1 (mg protein)-1), Trichomitus batrachorum the lowest (0.11 nmol min-1 (mg protein)-1) . The Trichomonas vaginalis ODC had an apparent Mr of 230 000 and was severely inhibited by alpha-difluoromethylornithine (DFMO) . S-Adenosyl-methionine decarboxylase (EC 4.1.1.50) could not be detected in T . batrachorum but was present in the other two species . Arginine decarboxylase was apparently absent from all three . All three trichomonad species were able to accumulate spermidine and putrescine from the medium . When T . vaginalis was grown in the presence of DFMO (4 mM), which had little effect on parasite growth, ODC activity was reduced by over 99% and the polyamine content was altered; putrescine concentrations were decreased, those of spermidine and spermine remained the same or were raised . DFMO-treated cells accumulated more exogenous putrescine than untreated control cells . The results suggest that the lack of effect of DFMO on T . vaginalis in culture was due to the parasite being able to accumulate polyamines from the growth medium . It appears, therefore, that testing DFMO and similar compounds in axenic trichomonad cultures may well not give a true indication of their effectiveness in vivo where sources of exogenous polyamines may not be available.

J Cell Physiol, 1986 Jun, 127(3), 403 - 9
Interleukin 3 and cell cycle progression; Kelvin DJ et al.; Interleukin 3 (IL-3) is a regulatory glycoprotein required for the proliferation and differentiation of cells from many if not all hemopoietic lineages . With the emergence of the competence-progression model of cell proliferation, which predicts that growth factors function at specific stages of the cell cycle, we examined the possibility that IL-3 functions at a specific stage of the cell cycle . C-63 cells were developed as a cell line from normal murine bone marrow . They have a mast cell phenotype and require pokeweed-stimulated spleen cell-conditioned medium (CM), a rich source of IL-3, for their continued growth . Exponentially growing cells were transferred from growth medium, which contains CM, to medium lacking CM or IL-3 . After 24 hours, cell viability had decreased 40-50% . The remaining viable cells did not incorporate 3H-thymidine, and displayed a single peak at G1 in a DNA histogram . Restimulation of these cells with CM or IL-3 resulted in a dramatic rise in 3H-thymidine uptake 20-24 hours after restimulation . DNA histograms of restimulated cultures indicated that the cells were progressing in a wave-like fashion throughout the remainder of the cell cycle . The length of time necessary for cells to be in contact with CM or IL-3 before they could progress into the remainder of the cell cycle was also examined . Cells incubated with CM or IL-3 for less than 16 hours could not progress into S phase, whereas cells incubated for 16 hours or longer could progress into S phase and through the remainder of the cell cycle . These data suggest that IL-3 exerts its function at a specific stage of the cell cycle.

Mol Gen Genet, 1986 Jun, 203(3), 533 - 7
Regulation of amino acid synthetic enzymes in Neurospora crassa in the presence of high concentrations of amino acids; Barthelmess IB; Ornithine carbamoyl transferase and leucine aminotransferase of Neurospora crassa represent two of many amino acid synthetic enzymes which are regulated through cross-pathway (or general) amino acid control . In the wild-type strain both enzymes display derepressed activities if the growth medium is supplemented with high (mM range) concentrations of L-amino acids derived from branched pathways, i.e . the aspartate, pyruvate, glycerophosphate and aromatic families of amino acids . A cpc-1 mutant strain, impaired in cross-pathway regulation i.e . lacking the ability to derepress, shows delayed growth under such conditions . In the presence of glycine, homoserine and isoleucine various cpc-1 isolates do not grow at all . Derepression of the wild-type enzymes and the retarded growth of the mutant strain can be reversed if certain amino acids are present in the medium in addition to the inhibitory amino acids.

Anal Biochem, 1986 Jun, 155(2), 379 - 84
A vital staining method for measuring the efficiency of transfection of eucaryotic cells; Domen J et al.; The xylE gene encodes catechol 2,3-dioxygenase, which catalyzes the conversion of catechol to 2-hydroxymuconic semialdehyde . The expression of this gene in eucaryotic cells can be detected simply by addition of catechol to the growth medium of the cells: cells that have a sufficient level of expression of the xylE gene stain yellow because of the accumulation of 2-hydroxymuconic semialdehyde . The number of stained cells is thus dependent upon the transfection efficiency as well as the level of expression of the xylE gene and is a measure of the combined transfection/expression efficiency in a particular cell type . Since the staining procedure does not affect the viability of the culture, the cells can be harvested afterward and analyzed for the expression of other, cotransfected, genes . This system for measuring transfection efficiency is especially useful when only small amounts of tissue are available.

J Bacteriol, 1986 Jun, 166(3), 884 - 91
Osmoregulation of the maltose regulon in Escherichia coli; Bukau B et al.; The maltose regulon consists of four operons that direct the synthesis of proteins required for the transport and metabolism of maltose and maltodextrins . Expression of the mal genes is induced by maltose and maltodextrins and is dependent on a specific positive regulator, the MalT protein, as well as on the cyclic AMP-catabolite gene activator protein complex . In the absence of an exogenous inducer, expression of the mal regulon was greatly reduced when the osmolarity of the growth medium was high; maltose-induced expression was not affected, and malTc-dependent expression was only weakly affected . Mutants lacking MalK, a cytoplasmic membrane protein required for maltose transport, expressed the remaining mal genes at a high level, presumably because an internal inducer of the mal system accumulated; this expression was also strongly repressed at high osmolarity . The repression of mal regulon expression at high osmolarity was not caused by reduced expression of the malT, envZ, or crp gene or by changes in cellular cyclic AMP levels . In strains carrying mutations in genes encoding amylomaltase (malQ), maltodextrin phosphorylase (malP), amylase (malS), or glycogen (glg), malK mutations still led to elevated expression at low osmolarity . The repression at high osmolarity no longer occurred in malQ mutants, however, provided that glycogen was present.

Cell, 1986 May 9, 45(3), 349 - 56
v-erbA cooperates with sarcoma oncogenes in leukemic cell transformation; Kahn P et al.; The v-erbB, v-src, v-fps, v-sea, and v-Ha-ras oncogenes induce avian erythroid progenitor cells to self-renew in an erythropoietin-independent manner . These transformed erythroblasts retain both their capacity to differentiate into erythrocytes and their requirement for complex growth media . However, previous studies showed that erythroblasts transformed by v-erbB plus v-erbA (which by itself is not oncogenic) are blocked in differentiation and grow in standard media . Here we show that the introduction of v-erbA into erythroblasts transformed with v-src, v-fps, v-sea, or v-Ha-ras likewise induces a fully transformed phenotype . It also reduces the capacity of ts sea- and ts erbB-transformed erythroblasts to differentiate terminally in an erythropoietin-dependent manner after a temperature shift . Cooperativity involving v-erbA also occurs in vivo since chicks infected with a retroviral construct encoding v-erbA and v-src develop both acute erythroblastosis and sarcomas.

J Biol Chem, 1986 May 5, 261(13), 5795 - 8
Phosphatidylserine biosynthesis in cultured Chinese hamster ovary cells . III . Genetic evidence for utilization of phosphatidylcholine and phosphatidylethanolamine as precursors; Kuge O et al.; In the preceding paper, we reported that Chinese hamster ovary (CHO) cells contain two different serine-exchange enzymes (I and II) which catalyze the base-exchange reaction of phospholipid(s) with serine and that a phosphatidylserine-requiring mutant (strain PSA-3) of CHO cells is defective in serine-exchange enzyme I and lacks the ability to synthesize phosphatidylserine (Kuge, O., Nishijima, M., and Akamatsu, Y . (1986) J . Biol . Chem . 261, 5790-5794) . In this study, we examined precursor phospholipids for phosphatidylserine biosynthesis in CHO cells . When mutant PSA-3 and parent (CHO-K1) cells were cultured with {32P}phosphatidylcholine, phosphatidylserine in the parent accumulated radioactivity while that in the mutant was not labeled significantly . On the contrary, when cultured with {32P}phosphatidylethanolamine, the mutant incorporated the label into phosphatidylserine more efficiently than the parent . Furthermore, we found that mutant PSA-3 grew normally in growth medium supplemented with 30 microM phosphatidylethanolamine as well as phosphatidylserine and that the biosynthesis of phosphatidylserine in the mutant was biosynthesis of phosphatidylserine in the mutant was normal when cells were cultured in the presence of exogenous phosphatidylethanolamine . The simplest interpretation of these findings is that phosphatidylserine in CHO cells is biosynthesized through the following sequential reactions: phosphatidylcholine----phosphatidylserine----phosphatidylethanolamine--- - phosphatidylserine . The three reactions are catalyzed by serine-exchange enzyme I, phosphatidylserine decarboxylase, and serine-exchange enzyme II, respectively.

Eur J Biochem, 1986 May 2, 156(3), 655 - 9
Deuterium incorporation into Escherichia coli proteins . A neutron-scattering study of DNA-dependent RNA polymerase; Lederer H et al.; Neutron small-angle scattering studies of single protein subunits in a protein-DNA complex require the adjustment of the neutron scattering-length densities of protein and DNA, which is attainable by specific deuteration of the protein . The neutron scattering densities of unlabelled DNA and DNA-dependent RNA polymerase of Escherichia coli match when RNA polymerase is isolated from cells grown in a medium containing 46% D2O and unlabelled glucose as carbon source . Their contrasts vanish simultaneously in a dialysis buffer containing 65% D2O . An expression was evaluated which allows the calculation of the degree of deuteration and match point of any E . coli protein from the D2O content of the growth medium, taking the 2H incorporation into RNA polymerase amino acids to be representative for all amino acids in E . coli proteins . The small-angle scattering results, on which the calculation of the degree of deuteration is based, were confirmed by mass spectrometric measurements.

J Cell Physiol, 1986 May, 127(2), 253 - 60
Expression of phenotypic traits following modulation of colchicine resistance in J774.2 cells; Lothstein L et al.; Development of resistance to colchicine in the mouse macrophage-like cell line J774.2 coincides with the expression of a variety of phenotypic traits . A cloned subline (J7/CLC-20), maintained in 20 microM colchicine, exhibits reduced steady-state association with drug, increased presence of a 140,000-145,000 dalton (140-145 kD) phosphoglycoprotein associated with the plasma membrane, double minute chromosomes and cross-resistance to other drugs . While similar phenotypic traits are observed in J774.2 cells resistant to taxol and vinblastine, differences in the electrophoretic mobilities of the resistance-specific glycoproteins in each of the three sublines suggest that multi-drug resistant sublines exhibit specificity for individual drugs . In an attempt to elucidate the relationships between the phenotypic traits associated with colchicine resistance, the degree of colchicine resistance in J7/CLC-20 cells was modulated and the levels of expression of the phenotypic traits were quantitated . In the absence of colchicine in the growth medium, J7/CLC-20 cells reverted to drug sensitivity within 35 days . A decrease in the level of resistance coincided with coordinate changes in both the quantity of the resistance-specific glycoprotein and the average number of double minute chromosomes . We propose that the emergence and disappearance of the resistance-specific glycoprotein and double minute chromosomes may be closely linked . However, J7/CLC-20 cells which had regained their drug sensitivity after growth in drug-free medium maintained a reduced level of steady-state drug association . The persistence of reduced drug association in cells that have reverted to a drug-sensitive state suggests that this phenomenon, although related to colchicine resistance, need not be the primary or only mechanism of drug resistance.

Cancer Res, 1986 May, 46(5), 2520 - 5
Enhancement in the adhesion of tumor cells to endothelial cells by decreased cholesterol synthesis; Ramachandran CK et al.; Adhesive interactions between tumor cells and the endothelial cells are presumed to be an obligatory step in the metastatic process . Using an in vitro model, we have examined the role of endothelial lipids in the regulation of this interaction . The cholesterol levels of bovine aorta endothelial cell monolayers were inhibited by the addition of compactin, 25-hydroxycholesterol, or 7-ketocholesterol . Metastatic B16 melanoma cells prelabeled with 14C-amino acid mixture were then deposited on this monolayer as a suspension and, at various time intervals, the number of cells adhering to the monolayer was determined . The results indicated that inhibition of cellular cholesterol caused enhancement in cell adhesion . On the other hand, perturbations of the glycosylation in the endothelial cells were without any effect on cell adhesion . The presence of cholesterol dispersion in the growth medium partially reversed the enhancement in cell adhesion caused by the cholesterol inhibitors . Growth in the presence of retinol or dexamethasone (1 microM) also caused enhancement in the adhesiveness of the tumor cells to endothelial cells, possibly because of their effects on cholesterol synthesis . Procaine, a local anesthetic which is known to increase membrane fluidity, also increased the tumor cell-endothelial interaction, suggesting that the membrane fluidity plays an important role in the regulation of cell adhesion.

Acta Pathol Microbiol Immunol Scand {A}, 1986 May, 94(3), 169 - 75
Aggregation of fibronectin by asbestos fibers in the pericellular matrix of cultured human fibroblasts; Vartio T et al.; Asbestos fibers, silica dust and glass fibers induced aggregation of the pericellular matrix fibronectin when added to the growth medium of cultured human fibroblasts as judged by immunofluorescence microscopy . Direct binding experiments and analysis of the bound proteins by (SDS-polyacrylamide gel electrophoresis) revealed an adsorption of purified plasma fibronectin to glass fibers, amosite, crocidolite and silica but not to anthophyllite and chrysotile A and B . The results suggest that aggregation of the pericellular fibronectin by these fibrogenic materials may be involved in the initial stages of the development of fibrosis in vivo.

Radiat Res, 1986 May, 106(2), 166 - 70
Escherichia coli radC is deficient in the recA-dependent repair of X-ray-induced DNA strand breaks; Felzenszwalb I et al.; Escherichia coli K-12 cells incubated in buffer can repair most of their X-ray-induced DNA single-strand breaks, but additional single-strand breaks are repaired when the cells are incubated in growth medium . While the radC102 mutant was proficient at repairing DNA single-strand breaks in buffer (polA-dependent repair), it was partially deficient in repairing the additional single-strand breaks (or alkali-labile lesions) that the wild-type strain can repair in growth medium (recA-dependent repair), and this repair deficiency correlated with the X-ray survival deficiency of the radC strain . In studies using neutral sucrose gradients, the radC strain consistently showed a small deficiency in rejoining X-ray-induced DNA double-strand breaks, and it was deficient in restoring the normal sedimentation characteristics of the repaired DNA.

Mol Gen Genet, 1986 May, 203(2), 346 - 53
Regulation of gene expression by pH of the growth medium in Aspergillus nidulans; Caddick MX et al.; In the fungus Aspergillus nidulans the levels of a number of enzymes whose location is at least in part extracellular (e.g . acid phosphatase, alkaline phosphatase, phosphodiesterase) and of certain permeases (e.g . that for gamma-amino-n-butyrate) are controlled by the pH of the growth medium . For example, at acidic pH, levels of acid phosphatase are high and those of alkaline phosphatase are low whereas at alkaline pH the reverse is true . Mutations in five genes, palA, B, C, E and F, mimic the effects of growth at acid pH whereas mutations in pacC mimic the effects of growth at alkaline pH . palA, B, C, E and F mutations result in an intracellular pH (pHin) which is more alkaline than that of the wild type whereas pacC mutations result in a pHin more acidic than that of the wild type . This indicates that these mutations exert their primary effects on the regulation of gene expression by pH rather than on the pH homeostatic mechanism but that the expression of at least some component(s) of the pH homeostatic mechanism is subject to the pH regulatory system . It is suggested that pacC might be a wide domain regulatory gene whose product acts positively in some cases (e.g . acid phosphatase) and negatively in others (e.g . alkaline phosphatase) . The products of palA, B, C, E and F are proposed to be involved in a metabolic pathway leading to synthesis of an effector molecule able to prevent the (positive and negative) action of the pacC product.(ABSTRACT TRUNCATED AT 250 WORDS)

Cancer Res, 1986 May, 46(5), 2356 - 61
Differential regulation by retinoic acid and calcium of transglutaminases in cultured neoplastic and normal human keratinocytes; Rubin AL et al.; In five lines of cultured human squamous carcinoma cells, transglutaminase activity and envelope competence were highly sensitive to retinoic acid and calcium levels in the growth medium . In cells grown in low calcium medium, these measures of keratinocyte differentiation were reduced . Retinoic acid suppressed envelope competence but total transglutaminase activity was markedly reduced, slightly affected, or greatly stimulated depending upon the cell line and whether the cells were grown in low calcium or 1.8 mM calcium-containing medium . Examination by anion exchange chromatography of the transglutaminase activity in SCC-12B2 cultures showed that expression of the particulate form (type I) of the enzyme was greatly stimulated by calcium . The increase in this activity to high levels that occurs at confluence could be almost completely suppressed by retinoic acid in the medium . The soluble form (type II) in the SCC-12B2 cells was induced in growing or confluent cultures by retinoic acid independent of the calcium concentration in the medium, but the 50% effective concentration (100 nM) for its stimulation was approximately 50-fold higher than the 50% effective concentration for suppression of the type I enzyme (2 nM) . Thus, these enzymes appear to be distinct and independently regulated . This conclusion is supported by the finding that SCC-4 and SCC-9 almost exclusively expressed types II and I forms, respectively . In contrast to the results with neoplastic cells, in cultured normal epidermal cells type I enzyme comprised the overwhelming majority of activity and was only partially (75-90%) suppressible by retinoic acid, while type II enzyme seemed poorly if at all stimulable . Thus, the SCC lines appear appropriate for studying biochemical mechanisms of action of certain physiological agents, the molecular basis for altered regulation of differentiated function in neoplastic cells, and the origin of diversity within tumors.

Mol Biol (Mosk), 1986 May-Jun, 20(3), 639 - 45
{The function of the tet gene in the plasmid pBR322 is not inhibited by expression of an anti-tet gene}; Zabarovskii ER; The possibility of gene suppression by the expression of anti-sense sequences has been tested for tet gene of pBR322 plasmid . Anti-tet gene has been inserted into lac-promoter regulated site of M13mp 10 single-stranded high copy phage vector . To achieve that, HihdIII-BamHI fragment of pBR322 carrying part of the tet gene was inserted into poly-linker of mp 10 . The influence of the anti-tet gene expression on growth parameters of cells with or without tetracycline in the growth media was monitored for JM103 cells . The results indicate that in this system the detectable suppression of the tet gene by anti-tet expression was not manifested.

J Chromatogr, 1986 Apr 25, 377, 155 - 63
Sensitive analysis of asparagine and glutamine in physiological fluids and cells by precolumn derivatization with phenylisothiocyanate and reversed-phase high-performance liquid chromatography; Lavi LE et al.; The analytical methodologies for the determination of free amino acids in plasma, serum, erythrocytes and leukemic cells are described . Deproteinization of the sample by methanol or organic acids is followed by derivatization with phenylisothiocyanate to form stable phenylthiocarbamylamino acid derivatives . The derivatives are separated by reversed-phase high-performance liquid chromatography in 80 min using a 5-microns C18 column (250 X 4 mm I.D.) and monitored by ultraviolet detection at 254 nm . Twenty physiological amino acids are resolved and quantified in plasma and erythrocyte samples . The resolution and sensitivity of the analytical method permitted unequivocal quantification of very low asparagine and glutamine levels in leukemic cells and growth media following treatment with asparaginase and glutaminase enzymes despite the presence of high aspartic and glutamic acid levels.

Experientia, 1986 Apr 15, 42(4), 436 - 7
Influence of medium amino acids on the ouabain sensitive and insensitive 86Rb+-fluxes in HeLa cells; Schaefer A et al.; Components of the 86Rb+-influx in HeLa cells were investigated in Joklik minimal essential medium, or in Earle's balanced salt solution with and without medium amino acids . The presence of amino acids led to the stimulation of the ouabain sensitive 86Rb+-uptake and inhibition of the diuretic-sensitive and residual 86Rb+-fluxes . These results show that the presence of amino acids is an important regulator of the K+/Rb+-fluxes under normal conditions in growth medium.

Biochem Pharmacol, 1986 Apr 15, 35(8), 1337 - 43
Expression of glutathione S-transferase and phenol sulfotransferase, but not of UDP-glucuronosyltransferase, in the human lung tumor cell lines NCI-H322 and NCI-H358; Wiebel FJ et al.; The expression of xenobiotic-metabolizing enzymes was studied in the human lung tumour cell lines NCI-H322 and NCI-H358 . These cells are derived from adenocarcinomas and exhibit features of Clara cells and alveolar type II cells, respectively . Examination of the in vitro activities showed that both cell lines lack UDP-glucuronosyltransferase against the substrates 3-hydroxybenzo{a}pyrene (3-OH-BaP) and 4-hydroxybiphenyl (4-OH-Bph) and that in vitro conjugation of sulfate with 3-OH-BaP was only just detectable . In contrast, both cell lines showed fairly high levels of glutathione-S transferase activity with the substrate 1-chloro-2,4-dinitro-benzene (54.4 and 83.0 nmol/min X mg protein, respectively) and of glutathione (81 and 41 nmole/mg protein, respectively) . The metabolic capacity of intact NCI-H322 and NCI-H358 cells was examined using benzo{a}pyrene (BaP) and 3-OH-BaP as substrates . The cell lines formed sulfate conjugates from 3-OH-BaP (4.5 and 0.4 pmol/min X mg protein, respectively) but did not produce any detectable glucuronides . When cultures of the two cell lines were exposed to BaP, phenolic products accumulated in the growth medium . NCI-H322 cells also formed some sulfate conjugates, whereas such conjugates were barely detectable in the medium of NCI-H358 cells . In contrast A549, a human pulmonary adenocarcinoma cell line known to contain UDP-glucuronosyltransferase activity, efficiently conjugated 3-OH-BaP to glucuronic acid and converted the primary phenolic products formed from BaP to glucuronides . Thus the NCI-H322 and NCI-H358 cells are exceptional in that they possess no or very little glucuronosyltransferase activity but exhibit appreciable monooxygenase activity . The cell lines may therefore be of interest for examining the biological effects of potentially toxic chemicals which are otherwise detoxified by glucuronic acid conjugation . The cells may also be useful as test systems for evaluating the potential cytotoxicity and genotoxicity of chemicals to human lung.

Exp Cell Res, 1986 Apr, 163(2), 337 - 48
Ataxia-telangiectasia cell extracts confer radioresistant DNA synthesis on control cells; Mohamed R et al.; We have investigated in greater detail the radioresistant DNA synthesis universally observed in cells from patients with ataxia-telangiectasia (A-T) . The approach employed in this study was to permeabilize cells with lysolecithin after gamma-irradiation and thus facilitate the introduction of cell extract into these cells . This permeabilization can be reversed by diluting the cells in growth medium . Cells treated in this way show the characteristic inhibition (control cells) or lack of it (A-T cells) after exposure to ionizing radiation . Introduction of A-T cells extracts into control cells prevented the radiation-induced inhibition of DNA synthesis normally observed in these cells . A-T cell extracts did not change the level of radioresistant DNA synthesis in A-T cells . Control cell extracts on the other hand did not influence the pattern of inhibition of DNA synthesis in either cell type . It seems likely that the agent involved is a protein because of its heat lability and sensitivity to trypsin digestion . It has a molecular weight (MW) in the range 20-30 000 D . The development of this assay system for a factor conferring radioresistant DNA synthesis on control cells provides a means of purifying this factor, and ultimately an approach to identifying the gene responsible.

J Neurochem, 1986 Apr, 46(4), 1256 - 62
Gamma-aminobutyric acid affects the developmental expression of neuron-associated proteins in cerebellar granule cell cultures; Meier E et al.; The effect of gamma-aminobutyric acid (GABA) on the expression of the neuron-associated D2 and neuron-specific enolase (NSE) was studied during development in culture of cerebellar granule cells . It was found that the presence of GABA during culture development increased the overall protein content . D2 content was also increased but not above the general increase in protein whereas NSE increased above the general level of protein . The presence of GABA in the growth medium also appeared to accelerate the changes in molecular forms of D2 and NSE seen during neuronal development . This suggests that GABA promotes or accelerates the general maturation of neurons, as these two neuron-associated proteins otherwise differ from each other with respect to their subcellular localization and their physiological and biochemical properties.

J Otolaryngol, 1986 Apr, 15(2), 85 - 8
An investigation of the squamous cell epithelium of the human larynx by the method of tissue culture; Boxall JD et al.; Laryngeal squamous cell epithelium has been grown by the method of tissue culture . Explant outgrowths were examined initially . Subsequently colonies of epithelium were grown from single cells upon a feeder layer of irradiated 3T3 mouse fibroblasts in a suitable growth medium . Human prepuce, which is widely used in skin culture studies, served as a control . There were colonial differences between colonies of the squamous epithelium of the larynx which presented a loose lattice structure and those of the prepuce control which gave rise to compacted growth . However, no obvious differences were noted between squamous epithelial colonies derived from fetal and adult vocal cords epithelium.

Proc Soc Exp Biol Med, 1986 Apr, 181(4), 523 - 8
Conversion to adipocytes of a clonal bone marrow preadipocyte line (H-1/A) and fatty acid composition of the resultant adipocytes; Harigaya K et al.; Conversion to adipocytes and fatty acid composition were investigated in a clonal bone marrow preadipocyte line (H-1/A) . The growing cells exhibited a fibroblastic appearance . After the cessation of growth, triacylglyceride (TG) synthesis in the cells increased as they incorporated precursor from the growth medium and became adipocytes . Hydrocortisone and insulin accelerated the TG synthesis in H-1/A cells in a dose-dependent manner when they were cultured in the growth medium containing 10% horse serum . The rate of conversion to adipocytes was reduced as the concentration of horse serum was decreased, and this reduction was not influenced by the addition of insulin and/or hydrocortisone . These results suggest that conversion to adipocytes of H-1/A cells is primarily dependent on some component(s) of the serum . Conversion to adipocytes of the cells may involve a process of differentiation since the conversion was completely inhibited when the cells were cultured in the presence of bromodeoxyuridine . Fatty acid composition was significantly different between adipose H-1/A cells and adipocytes derived from other marrow preadipocyte line MC3T3-G2/PA6 cells . Unsaturated fatty acids accounted for 76% of the fatty acid composition of adipose H-1/A cells; in contrast, saturated fatty acids constituted 65% of the fatty acid composition of the adipose MC3T3-G2/PA6 cells . These results suggest that there is a heterogeneity of preadipocytes in bone marrow . These two preadipocyte lines thus provide a useful tool for the study of marrow adipocytes and can also be used to analyze the hematopoietic microenvironment through studies of the effect of these cells on hematopoietic cell proliferation.

J Clin Microbiol, 1986 Apr, 23(4), 672 - 8
Growth and cytopathogenicity of Trichomonas vaginalis in tissue cultures; Pindak FF et al.; The primary purpose of this study was to identify the mammalian tissue cultures which were most suitable for investigations of the cytopathogenicity of Trichomonas vaginalis . A recently isolated strain of the organism was inoculated into 15 different tissue cultures which were maintained in an appropriately modified growth medium . Proliferation of the protozoon was accompanied by the progressive disintegration of cell culture monolayers . Initial focal lesions consisting of detached cells and an accumulation of trichomonads gradually enlarged until the entire monolayer was disrupted . When judged by the size of the inoculum required to obtain this effect, differences among the tissue cultures were noted . An inoculum of approximately 10(3) viable trichomonads was sufficient to completely disrupt monolayers of HeLa 229, HeLa, McCoy, HEp-2, and RK-13 cells . To obtain a comparable effect with other cells, 10- to 100-fold higher levels of inoculum were required . Polyethylene glycol concentrates from culture filtrates contained a cell-detaching factor (CDF) which caused detachment and clumping of susceptible cells . Freshly seeded cells in growth medium containing CDF failed to form a monolayer . Aggregates of cells maintained for up to 1 week in the presence of CDF remained viable and formed a monolayer after being washed and suspended in normal growth medium . The activity of the CDF was not lost during 1 week of contact with the cells . The CDF may contribute to the pathogenicity mechanisms of T . vaginalis.

Scand J Dent Res, 1986 Apr, 94(2), 95 - 101
Intraoral bacterial growth on tetracycline-impregnated dentin; Bjorvatn K et al.; Previous studies have shown that tetracyclines react with enamel and dentin in vitro to form slightly soluble compounds with a pronounced and long-lasting antimicrobial capacity . The purpose of the present project was to study the effect of doxycycline-impregnation of dentin on the growth of oral microorganisms in vivo . Slabs of native human dentin were immersed in aqueous solutions of doxycycline HCl, 10 mg/ml, for 10 min, and were subsequently ligated to orthodontic brackets placed bucally on maxillary molars in seven individuals . Untreated native dentin specimens were similarly placed on contralateral teeth . Test and control specimens were removed from the oral cavity after 2 h, 24 or 120 h, respectively . The individual specimens were then placed in 0.5-ml saline and agitated mechanically for 30 s . The resulting bacterial suspensions were plated onto blood agar and various selective growth media . Based on CFU-counts obtained after incubation of the plates, doxycycline-treated dentin specimens showed a significantly lower number of viable microorganisms after 2 h and 24 h in the oral cavity than did the control specimens . The bacterial growth was also less pronounced in the test specimens after 120 h . The effect of doxycycline was similar for all the cultivable bacteria . No significant difference in yeast growth was seen between test and control specimens.

Exp Parasitol, 1986 Apr, 61(2), 229 - 35
Plasmodium falciparum: synchronization of cultures with DL-alpha-difluoromethylornithine, an inhibitor of polyamine biosynthesis; Assaraf YG et al.; DL-alpha-Difluoromethylornithine, an inhibitor of polyamine biosynthesis, was tested for its ability to synchronize Plasmodium falciparum . Asynchronous cultures were pretreated with sorbitol and incubated for 28-30 hr . Then, when cultures consisted of mainly schizont stage parasites, DL-alpha-difluoromethylornithine was added to the growth medium for another 38-47 hr of incubation . Putrescine was added to parasites arrested at the early trophozoite stage . This resulted in a synchronous resumption of growth . After 19 hr, 83% of parasites were at the schizont stage . After 30 hr, more than 98% of the parasites were in the ring form stage . Furthermore, the transformation of early trophozoites to schizonts occurred within 3 hr, with a slight reduction in parasitemia . Synchrony was maintained for 4-5 biological cycles as confirmed also by flow fluorimetry . It appears that this new approach to synchronize P . falciparum cultures is simple, reproducible, and effective.

Appl Environ Microbiol, 1986 Apr, 51(4), 790 - 812
Optimization of the BGM cell line culture and viral assay procedures for monitoring viruses in the environment; Dahling DR et al.; An in-depth study of the continuous cell line designated BGM is described herein, and recommendations are made for standardizing cell culture and viral assay procedures . Based on data gathered from a survey of 58 laboratories using this cell line, a research plan was developed that included the study of growth media, sera, NaHCO3 levels, culture bottles, cell concentration, overlay media, agar, virus infection conditions, and cell-dissociating agents . Additionally, a comparative virus isolation study with BGM cells and nine other cell types was conducted with 37 sewage samples collected from nine different geographic areas . The results of the study indicated that the BGM cell line is superior for virus isolation when compared with the other cell types and that certain media and additives tend to increase BGM cell sensitivity to a specific group of viruses . A standardized procedure for cultivation of BGM cells is described which provides a more effective enterovirus assay system.

J Bacteriol, 1986 Apr, 166(1), 15 - 9
Physical and genetic analysis of the ColD plasmid; Frey J et al.; The plasmid ColD-CA23, a high-copy-number plasmid of 5.12 kilobases, encodes colicin D, a protein of approximately 87,000 daltons which inhibits bacterial protein synthesis . Colicin D production is under the control of the Escherichia coli SOS regulatory system and is released to the growth medium via the action of the lysis gene product(s) . A detailed map of the ColD plasmid was established for 10 restriction enzymes . Using in vitro insertional omega mutagenesis and in vivo insertional Tn5 mutagenesis, we localized the regions of the plasmid responsible for colicin D activity (cda), for mitomycin C-induced lysis (cdl), and for colicin D immunity (cdi) . These genes were all located contiguously on a 2,400-base-pair fragment similar to a large number of other Col plasmids (A, E1, E2, E3, E8, N, and CloDF) . The ColD plasmid was mobilizable by conjugative transfer by helper plasmids of the IncFII incompatibility group, but not by plasmids belonging to the groups IncI-alpha or IncP . The location of the mobilization functions was determined by deletion analysis . The plasmid needs a segment of 400 base pairs, which is located between the mob genes and the gene for autolysis, for its replication.

J Interferon Res, 1986 Apr, 6(2), 137 - 42
The antiproliferative activity of interferons assayed by measuring growth medium color changes related to cell number; Dawson KM et al.; A simple colorimetric assay for estimating cell numbers has been developed based on the observation that the indicator color in cell culture medium is proportional to viable cell number . The assay is performed in multiwell plates to take advantage of the rapid color measurement and computerized data handling capabilities of multiwell scanning spectrophotometers . Since no centrifugation or washing steps are involved, the technique is particularly useful for cells that grow in suspension, although it is equally applicable to monolayer cultures . The assay was developed to measure the antiproliferative activity of interferons on Daudi lymphoblastoid cells but could equally well be applied to other cell growth inhibition or stimulation assays.

Exp Cell Res, 1986 Apr, 163(2), 434 - 44
A procedure to introduce protein molecules into living mammalian cells; Nomura S et al.; Although several methods are now available by which to introduce macromolecules into cultured living mammalian cells, each has limitations on its adoption as a general means, for a variety of purposes . We describe here a simple procedure to introduce protein molecules into various living mammalian cells . This procedure is based upon the finding that mammalian cells, after exposure to a low concentration of a phospholipid (L-alpha-lysophosphatidylcholine) in the presence of high (hypertonic) concentrations of glycerol became permeable to protein molecules and that a significant portion of the exposed cells regain their viability following incubation in the appropriate growth medium . We have demonstrated that diphtheria toxin (A fragment), horseradish peroxidase and antibodies against SV40 T-antigens are incorporated into living mouse erythroleukemia (Friend) cells, baby hamster kidney (BHK) cells and mouse fibroblasts (C3H), respectively . The volume introduced into a single cell (mouse Friend cells) is approx . 3 X 10(-15) liter, which is comparable to those with other systems . Parameters affecting permeability to protein molecules and viability of the treated cells were also investigated with these and other cell lines.

Eur J Biochem, 1986 Mar 17, 155(3), 595 - 602
NADPH/NADH-dependent cold-labile glutamate dehydrogenase in Azospirillum brasilense . Purification and properties; Maulik P et al.; A cold-labile glutamate dehydrogenase (GDH, EC 1.4.1.3) has been purified to homogeneity from the crude extracts of Azospirillum brasilense . The purified enzyme shows a dual coenzyme specificity, and both the NADPH and NADH-dependent activities are equally cold-sensitive . The enzyme is highly specific for the substrates 2-oxoglutarate and glutamate . Kinetic studies with GDH indicate that the enzyme is primarily designed to catalyse the reductive amination of 2-oxoglutarate . The NADP+-linked activity of GDH showed Km values 2.5 X 10(-4) M and 1.0 X 10(-2) M for 2-oxoglutarate and glutamate respectively . NAD+-linked activity of GDH could be demonstrated only for the amination of 2-oxoglutarate but not for the deamination of glutamate . The Lineweaver-Burk plot with ammonia as substrate for NADPH-dependent activity shows a biphasic curve, indicating two apparent Km values (0.38 mM and 100 mM) for ammonia; the same plot for NADH-dependent activity shows only one apparent Km value (66 mM) for ammonia . The NADPH-dependent activity shows an optimum pH from 8.5 to 8.6 in Tris/HCl buffer, whereas in potassium phosphate buffer the activity shows a plateau from pH 8.4 to 10.0 . At high pH (greater than 9.5) amino acids in general strongly inhibit the reductive amination reaction by their competition with 2-oxoglutarate for the binding site on GDH . The native enzyme has a Mr = 285000 +/- 20000 and appears to be composed of six identical subunits of Mr = 48000 +/- 2000 . The GDH level in A . brasilense is strongly regulated by the nitrogen source in the growth medium.

Biochem J, 1986 Mar 15, 234(3), 611 - 5
Quinohaemoprotein alcohol dehydrogenase apoenzyme from Pseudomonas testosteroni; Groen BW et al.; Cell-free extracts of Pseudomonas testosteroni, grown on alcohols, contain quinoprotein alcohol dehydrogenase apoenzyme, as was demonstrated by the detection of dye-linked alcohol dehydrogenase activity after the addition of PQQ (pyrroloquinoline quinone) . The apoenzyme was purified to homogeneity, and the holoenzyme was characterized . Primary alcohols (except methanol), secondary alcohols and aldehydes were substrates, and a broad range of dyes functioned as artificial electron acceptor . Optimal activity was observed at pH 7.7, and the presence of Ca2+ in the assay appeared to be essential for activity . The apoenzyme was found to be a monomer (Mr 67,000 +/- 5000), with an absorption spectrum similar to that of oxidized cytochrome c . After reconstitution to the holoenzyme by the addition of PQQ, addition of substrate changed the absorption spectrum to that of reduced cytochrome c, indicating that the haem c group participated in the enzymic mechanism . The enzyme contained one haem c group, and full reconstitution was achieved with 1 mol of PQQ/mol . In view of the aberrant properties, it is proposed to distinguish the enzyme from the common quinoprotein alcohol dehydrogenases by using the name 'quinohaemoprotein alcohol dehydrogenase' . Incorporation of PQQ into the growth medium resulted in a significant shortening of lag time and increase in growth rate . Therefore PQQ appears to be a vitamin for this organism during growth on alcohols, reconstituting the apoenzyme to a functional holoenzyme.

Exp Cell Res, 1986 Mar, 163(1), 35 - 46
Some structural, biochemical and biophysical characteristics of L-929 cells growing in the presence of hyperosmotic sorbitol concentrations; Clegg JS et al.; Mouse L-929 cells (a fibroblast-like line) were transferred from normal growth medium to one supplemented with 0.3 M sorbitol, doubling the normal external osmotic pressure . After a short lag phase and minimal cell death, the cells began to grow, and the growth rate reached that of controls after about one week . These chronically grown cells (S) have been compared to those of control cultures (C) with regard to general morphology, ability to reverse when returned to normal condition, water content, volume and selected metabolic parameters . S-cell cultures exhibited considerable heterogeneity but most contained vesicle-like cytoplasmic structures, sometimes in abundance . These structures do not appear to be completely bounded by membranes, but that is uncertain . S cells become larger and contain more water than C cells; however, the ratio of total water to total dry mass is indistinguishable from controls suggesting regulation at that level . S and C cells were found to be remarkably similar, on a per cell basis, with regard to their rate of respiration and the incorporation of glucose into metabolites and macromolecules . These results are interpreted in terms of current views on the composition and organization of the aqueous compartments of animal cells.

Radiat Res, 1986 Mar, 105(3), 351 - 69
Glutathione depletion by DL-buthionine-SR-sulfoximine (BSO) potentiates X-ray-induced chromosome lesions after liquid holding recovery; Bertsche U et al.; The impact of intracellular glutathione depletion on chromosome damage induced by X irradiation under aerobic conditions was investigated in two different cell lines, Ehrlich ascites tumor cells (EATC) and Chinese hamster ovary cells (CHO-K1) . Thiol-depleted cell cultures in plateau phase were obtained by prolonged incubation in growth medium containing DL-buthionine-SR-sulfoximine (BSO), a specific inhibitor of gamma-glutamyl-cysteine synthetase . Cells were then assayed using the procedures of G . L . Ellmann (Arch . Biochem . Biophys . 82, 70-77 (1959)), F . Tietze (Anal . Biochem . 27, 502-522 (1969)), and J . Sedlack and R.H . Lindsay (Anal . Biochem . 25, 192-205 (1968)) for non-protein bound SH (NPSH), glutathione (GSH), and total SH (TSH) . In both cell lines GSH was reduced to less than 10% of controls at higher BSO concentrations around 1 mM, whereas TSH and NPSH were affected to only 40-60% . In EATC pretreated with up to 1 mM BSO for 72 h, increased levels of spontaneously occurring micronuclei were found . At BSO concentrations above 200 microM, both cell lines showed a potentiation of chromosome lesions scored as micronuclei and induced under aerobic X irradiation when liquid holding recovery in the original nutrient-depleted medium was performed; the extent of chromosome damage eventually reached that which could be obtained by application of beta-arabinofuranosyladenine (beta-araA), known to inhibit DNA repair processes by blocking DNA polymerases . It is therefore suggested that GSH depletion causes impairment of repair of lesions leading to chromosome deletions and subsequently to micronuclei . In contrast to CHO cell cultures, EATC showed a reversion of the potentiation effect as indicated by a decrease in the micronucleus content during prolonged incubation in the presence of BSO in the millimolar range . This effect could not be correlated to the remaining GSH content of less than 10% but may be due to some accumulation of unknown NPSH components . Since addition of L-cysteine to EATC cultures pretreated with BSO decreased the micronucleus content, cysteine/cystine or a related thiol within the NPSH fraction may be involved in the reestablishment of repair . Thus at least in one cell line, a rather complex response to BSO administration indicated that not only GSH but also other thiols may determine the level of chromosome damage after liquid holding recovery.

Mikrobiologiia, 1986 Mar-Apr, 55(2), 337 - 8
{Aniline oxidation by microorganisms in chemostat cultivation}; Orshanskaia FB et al.; Under the conditions of chemostat cultivation, a mixed culture of microorganisms oxidized aniline at a gradually increasing concentration (up to 2.5 g/litre) as a sole source of carbon and nitrogen . The specific rate of aniline oxidation was as high as 160 mg per 1 g of dry biomass when aniline concentration in the growth medium was 2.5 g/litre . As aniline concentration in the growth medium was gradually raised, selection of the microorganisms took place and the number of the strains decreased from ten to two . The growth rate of the microorganisms fell down abruptly when phosphorus concentration in the medium was below 35 mg per 1 g of aniline.

Mycopathologia, 1986 Mar, 93(3), 173 - 84
Exophiala pisciphila . A study of its development; Gaskins JE et al.; Exophiala pisciphila is a dematiaceous fungus that belongs to a group of fungi known as the 'black yeasts' . It was isolated from the skin lesions of a smooth dogfish, Mustelus canis Mitchill, that had been born in the shark exhibit tank of the New York Aquarium . The different stages of development of this fungus were studied by light microscopy and scanning electron microscopy to illustrate the morphology and surface structures of conidia and mycelium . The list of marine and fresh water fish, which have been infected by Exophiala spp . and Exophiala-like fungi has been up-dated . Potato Dextrose Agar and Malt Agar proved to be the best growth media, while Corn Meal Agar proved to be the best medium for studying the morphological features of the conidia and mycelial development of E . pisciphila, which exhibited polymorphic conidiogenesis.

Proc Natl Acad Sci U S A, 1986 Mar, 83(6), 1752 - 6
Oligosaccharide modification by swainsonine treatment inhibits pulmonary colonization by B16-F10 murine melanoma cells; Humphries MJ et al.; Oligosaccharide moieties of cell-surface glycoconjugates are thought to be involved in recognition events associated with tumor metastasis and invasion . Using swainsonine (SW), an inhibitor of Golgi alpha-mannosidase II that results in the formation of hybrid-type oligosaccharides on N-linked glycoproteins, we have tested the hypothesis that specific glycan structures are required for pulmonary colonization by tumor cells . B16-F10 murine melanoma cells were treated with SW in growth medium and then injected intravenously into syngeneic C57BL/6 mice . This treatment resulted in dramatic inhibition of colonization, but it had no effect on B16-F10 viability or on cellular tumorigenicity after subcutaneous implantation . SW-treated radiolabeled B16-F10 cells were cleared from the lungs at a greater rate than control cells, suggesting that one effect of treatment is to alter tumor cell retention in the target organ . Our results implicate specific glycan structures in pulmonary colonization and offer a potential approach for identification of specific macromolecules involved in tumor cell-organ recognition during metastasis.

J Virol, 1986 Mar, 57(3), 968 - 75
The soluble glycoprotein of vesicular stomatitis virus is formed during or shortly after the translation process; Graeve L et al.; Gs protein is a shorter, soluble form of the viral G protein of vesicular stomatitis virus (VSV) lacking the membrane-anchoring domain . Production of Gs protein appears to be a general property of VSV because infection of BHK-21 cells by five different isolates of the VSV serotype Indiana led in all cases to the synthesis of Gs protein . Moreover, it is formed in a variety of eucaryotic cell lines after VSV infection . In pulse-chase experiments, we observed a time-dependent change in the ratio of G to Gs protein released into the growth medium, suggesting that Gs is formed intracellularly rather than on the cell surface . Further experiments revealed that Gs protein can be synthesized in vitro in the reticulocyte lysate system after addition of a viral mRNA fraction and in a coupled transcription-translation system with VSV core particles . In the presence of microsomal membranes both G and Gs protein were glycosylated in the reticulocyte lysate, confirming that the authentic Gs protein is synthesized in vitro . The addition of various protease inhibitors to the cell-free system and variation of the incubation conditions did not alter the ratio of G to Gs formation . Taken together, these experiments suggest strongly that Gs protein is not a product of a membrane-associated proteolytic activity but is formed during or shortly after the translation process . Our attempts to detect a specific, shorter mRNA coding for the Gs protein by molecular hybridization procedures did not reveal the existence of such a mRNA species.

Exp Hematol, 1986 Mar, 14(3), 234 - 40
Mature T cells are part of adherent cells in human long-term bone marrow cultures; Shibata T et al.; We determined the proportion of lymphocytes in nonadherent and adherent fractions of human bone marrow cells cultured by a Dexter-type continuous marrow culture method . T-lymphocytes, B-lymphocytes, and common alloantigen (CALLA)-positive cells (cells with receptors for CALLA) were sequentially enumerated using commercially available appropriate monoclonal antibodies . Nonadherent cells from weeks 2-5 of culture contained relatively fixed proportions of OKT3-positive (4%-10.4%) and CALLA-positive (5%-6.6%) cells . The adherent cells during the culture period between weeks 6 and 18 contained an average of 34% OKT3-positive cells with a range from 6% to 68.5%, despite a high hydrocortisone concentration of 5 X 10(-5) M added to the growth medium . The T cells retrieved from the adherent layers and resuspended in steroid-free medium responded to PHA stimulation and to mixed leukocyte culture in the same manner as did freshly drawn peripheral blood leukocytes . These results indicate that adherent cell populations include mature T-lymphocytes in human long-term bone marrow cultures . In view of well-documented interactions of nonlymphoid hematopoietic progenitors with T-lymphocytes in the clonogenic culture system, it can be speculated that the adherent T cells also may play a role in proliferation and differentiation of granulocyte-macrophage and erythroid progenitors in this long-term culture system.

Cancer, 1986 Mar 1, 57(5), 1011 - 8
Effect of dibutyryl cyclic AMP on morphologic features and biologic markers of a human salivary gland adenocarcinoma cell line in culture; Yoshida H et al.; Dibutyryl cyclic AMP (dB-cAMP) has marked effects on the growth, morphologic features, and biologic markers of a human salivary gland adenocarcinoma cell line in culture . A cell line with ultrastructure and biologic markers specific to the intercalated duct cells of human salivary glands was cultivated in the presence of dB-cAMP . Major alterations, such as process formation and expression of myofilaments and oxytocin receptor in addition to myosin and S-100 protein, were observed in those cells with a phenotype similar to myoepithelial cells . Both the anchorage-independent and anchorage-dependent growths were markedly suppressed in the presence of dB-cAMP . After the removal of dB-cAMP from the culture, the treated cells returned rapidly to the phenotype of the untreated cells . These findings indicate that reversible differentiation into the myoepithelial cells of a human salivary gland adenocarcinoma cell line occurs in growth medium containing dB-cAMP.

Zh Mikrobiol Epidemiol Immunobiol, 1986 Mar, (3), 18 - 21
{Enhancing the effectiveness of microbiological nutrient media through balancing their content by an experimental analytical method}; Ivanova LG et al.; The possibility of successful use of the analitico-experimental method for the development and improvement of balanced microbiological growth media, economical in composition, has been shown . By this method a new variant of the semisynthetic medium for growing Bordetella pertussis vaccine strain has been obtained, thus making it possible to achieve a considerable increase in the yield of the biomass without decreasing the immunogenicity of the culture . The use of the balanced medium in the production of vaccines may alone give the overall economic effect totalling 11000 roubles per 10000 liters.

Arch Surg, 1986 Mar, 121(3), 285 - 8
Effects of gastrin, glutamine, and somatostatin on the in vitro growth of normal and malignant human gastric mucosal cells; Moyer MP et al.; This study evaluated the dose-related trophic effects of glutamine, gastrin, and somatostatin on the in vitro growth of human gastric cancer cells and normal human gastric mucosal cells . Quadruplicate cell cultures were seeded into growth medium with or without glutamine, gastrin, or somatostatin . After 72 hours' incubation, cells were counted and their numbers compared with those of controls . Glutamine and gastrin stimulated the growth of both normal and malignant gastric mucosal cells . Compared with normal cells, the malignant cells responded to these growth factors at lower concentrations . Somatostatin enhanced growth of gastric cancer cells at all concentrations and inhibited growth of normal cells at high concentrations . Further studies on the responsiveness of gastric adenocarcinoma to gastrointestinal tract hormones may elucidate mechanisms of oncogenesis and suggest new therapeutic avenues for patients with gastric cancer.

Genetika, 1986 Feb, 22(2), 194 - 9
{The tryptophan operon of the facultative methylotrophic bacteria Pseudomonas sp . M . III . Characteristics of regulatory 5-MTR mutants}; Maksimova NP et al.; Regulatory 5-DL-methyltryptophan (5-MT)-resistant mutants of facultative methylotrophic Pseudomonas sp . M . were obtained . They are able to excrete tryptophan into the growth medium (60 to 300 g/ml) . 5-MTR regulatory mutants are characterized by depression of trpE, trpD and trpC genes, which causes the production of intermediates of tryptophan biosynthesis and results in trpA and trpB genes induction as well as in two-fold activation of N-5-phosphoribosyl anthranilateisomerase (trpF gene product) . Besides, all mutants demonstrate reduction of synthase feed-back inhibition about 4-11-fold . Together with tryptophan excretion, 5-MTR regulatory mutants are able to excrete tyrosine and unable to utilize this amino acid as the sole carbon source, which points to multiple nature of the selective effect of 5-MT.

Am J Physiol, 1986 Feb, 250(2 Pt 1), C314 - 8
Role of cell replication in regulation of Na-coupled hexose transport in LLC-PK1 epithelial cells; Moran A et al.; The glucose concentration in growth medium has been shown to regulate the number of sodium-coupled glucose transporters in LLC-PK1 epithelial cells . Epithelia grown in high concentrations of glucose express fewer transporters than epithelia grown in low concentrations of glucose . In the present work, the effect of a dose of ionizing radiation sufficient to block the incorporation of thymidine was examined in order to gauge the importance of cell replication in the hexose transport regulatory process . The low rate of thymidine incorporation in the plateau phase was completely eliminated by ionizing radiation . Under conditions of irradiation that completely blocked thymidine incorporation, down-regulation, namely the loss of alpha-methylglucoside-concentrating capacity, brought about by switching the epithelium from low to high glucose-containing medium, is independent of the irradiation and therefore most likely is also independent of cell replication . In contrast, the up-regulatory phenomenon is strongly impaired by radiation . This impairment may be due to specific radiation impairment of gene expression necessary for the up-regulatory process . It is apparent from the dose-response data that up-regulation is not inhibited by irradiation in a simple manner and is not inhibited at the same radiation dose as cell replication.

J Parasitol, 1986 Feb, 72(1), 170 - 4
Carbohydrate utilization by Trichomonas gallinae: effects on growth and metabolic activity under nongrowth conditions; Matthews HM; Trichomonas gallinae used 13 of 29 carbohydrates for growth . Quantitative relationships between final populations, acid production, and cellular glycogen contents varied depending on the substrate . The effect of growth on different carbohydrates on the subsequent utilization of carbohydrates by cells under nongrowth conditions was studied by measuring carbohydrate uptake, changes in cellular glycogen content, and gas production . Two major utilization patterns were found . Cells grown on maltose or starch used these substrates well, but cells grown on other sugars did not . All cells used glucose, fructose, galactose, and mannose, but cells grown on maltose or starch did not use them as well as cells grown on other sugars . All cells used ribose slightly but not xylose or arabinose . Turanose, a disaccharide yielding high populations in growth medium, was not used under nongrowth conditions.

Biophys J, 1986 Feb, 49(2), 485 - 91
Repair of near-ultraviolet (365 nm)-induced strand breaks in Escherichia coli DNA . The role of the polA and recA gene products; Miguel AG et al.; The action of near-ultraviolet (UV-365 nm) radiation in cellular inactivation (biological measurements) and induction and repair of DNA strand breaks (physical measurements) were studied in a repair-proficient strain and in polA-, recA-, uvrA-, and polA uvrA-deficient strains of Escherichia coli K-12 . The induction of breaks in the polA and polA uvrA strains was linear with dose (4.0 and 3.7 X 10(-5) breaks/2.5 X 10(9) daltons/Jm-2, respectively) . However, in the recA-, uvrA-, and repair-proficient strains, there was an initial lag in break induction at low doses and then a linear induction of breaks at higher doses with rates of 4.6, 2.8, and 3.2 X 10(-5) breaks/2.5 X 10(9) daltons/Jm-2, respectively . We interpret these strain differences as indicating simultaneous induction and repair of breaks in polymerase 1 (polA)-proficient strains under the 0 degrees C, M9 buffer irradiation conditions that, for maximum efficiency, require both the polA and recA gene products . Strand-break rejoining also occurred at 30 degrees C in complete growth medium . We propose that at least three (and possibly four) distinct types of pathways can act to reduce the levels of 365-nm radiation-induced strand breaks . A quantitative comparison of the number of breaks remaining with the number of lethal events remaining after repair in complete medium at 30 degrees C showed that between one and three breaks remain per lethal event in the wild-type and recA strains, whereas in the polA strain one order of magnitude more breaks were induced.

Radiat Res, 1986 Feb, 105(2), 180 - 6
Characterization and quantitation of DNA strand breaks requiring recA-dependent repair in X-irradiated Escherichia coli; Sargentini NJ et al.; The repair of X-ray-induced DNA single-strand breaks was studied after the completion of growth-medium-independent repair in Escherichia coli K-12 . A comparison of the sedimentation of DNA from bacteriophages T2 and T7 was used to test the accuracy of our alkaline and neutral sucrose gradient procedures for determining the molecular weight of bacterial DNA . The repair of DNA single-strand breaks by cells incubated in buffer occurred by two processes . About 85% of the repairable breaks were resealed rapidly (t1/2 = less than 6 min), while the remainder were resealed slowly (t1/2 = approximately 20 min) . After the completion of the repair of DNA single-strand breaks in buffer, about 80% of the single-strand breaks that remained were found to be associated with DNA double-strand breaks . The subsequent resuspension of cells in growth medium allowed the repair of both DNA single- and double-strand breaks in wild-type but not in recA cells . Thus the recA-dependent, growth-medium-dependent repair of DNA single-strand breaks is essentially the repair of DNA double-strand breaks.

J Exp Zool, 1986 Feb, 237(2), 221 - 30
Hormonal regulation of growth and life span of bullfrog tadpole tail epidermal cells cultured in vitro; Nishikawa A et al.; Epidermal cells were dissociated from tails of the bullfrog tadpole, Rana catesbeiana, and cultured to investigate their response to steroid and thyroid hormones . Charcoal-treated serum (CTS) was used in the growth medium when cells were to be grown in the absence of steroid and thyroid hormones . The cells could be maintained for 2 weeks with a small increase in cell number in medium that contained CTS (CTS medium) . Addition of cortisol to CTS medium increased both cellular attachment to the culture dishes and the proliferation of the attached cells with an optimum concentration of 5 X 10(-7) M . The cells remained viable and attached for at least a week . Cortisol stimulated the rate of protein synthesis 1.8-fold but did not alter the rate of DNA synthesis . The cells did not proliferate in the medium containing triiodothyronine (T3) and detached themselves from the dish within 5 days, which occurred in a dose-dependent manner with a maximum effect at 10(-8) M . It drastically decreased the rate of DNA synthesis but did not influence the rate of protein synthesis . These responses of cells to cortisol and T3 may reflect growth and death of tail epidermal cells in vivo at metamorphosis.

EMBO J, 1986 Feb, 5(2), 311 - 6
Evidence that expression of c-fos protein in amnion cells is regulated by external signals; Muller R et al.; While c-fos expression is normally very low in certain cell types but transiently inducible by growth factors (e.g . in fibroblasts), other cells (e.g . amnion cells) exhibit an apparently constitutive expression in vivo . Here we show that in primary amnion cells c-fos protein expression rapidly drops to undetectable levels following plating in normal growth medium . However, c-fos expression is inducible by, and maintained at high levels in the presence of, dialyzed placenta- or embryo-conditioned medium . These observations suggest that c-fos expression in primary amnion cells is regulated by placenta- and embryo-derived factor(s), providing further evidence for the hypothesis that transcription of the c-fos gene may generally be controlled by external signals . We also show that proliferation of primary amnion cells is not dependent on a high c-fos expression, suggesting that the function of c-fos is more likely to be associated with other cellular functions in the differentiated amnion cell.

Mol Gen Genet, 1986 Feb, 202(2), 194 - 9
Molecular analysis of mutant ompR genes exhibiting different phenotypes as to osmoregulation of the ompF and ompC genes of Escherichia coli; Nara F et al.; Expression of the ompF and ompC genes coding for major outer membrane proteins is osmoregulated by solutes, such as sucrose and NaCl, in the growth medium . The OmpR protein, a positive regulator of these genes, is involved in the osmoregulation (Dairi et al . 1985; Nara et al . 1984) . In the present work, five mutant ompR genes exhibiting different phenotypes of osmoregulation were cloned and sequenced . Three of them, ompR1, ompR2 and ompR20, were previously isolated mutants . The others, ompR3 and ompR4, were isolated in the present work . The ompR1 mutation resulted in the deletion of 19 amino acids near the C-terminus of the OmpR protein . The ompR3 and ompR4 mutations resulted in Arg15 to Cys and Arg71 to Thr conversions, respectively, at the N-terminal portion, whereas the ompR20 and ompR2 mutations resulted in Arg150 to Cys and Val207 to Met conversions, respectively, at the C-terminal portion . Based on these results, the structure and function of the OmpR protein are discussed in relation to the mechanism of osmoregulation.

Environ Res, 1986 Feb, 39(1), 205 - 31
On the toxic effects of tetraethyl lead and its derivatives on the chrysophyte Poterioochromonas malhamensis . VI . Effects on lorica formation, mitosis, and cytokinesis; Roderer G; Effects of tetraethyl lead (TEL) and derivatives triethyl lead (TriEL), diethyl lead (DiEL), and inorganic lead (Pb) on lorica formation of the unicellular alga Poterioochromonas malhamensis were investigated by light and electron microscopy . Lorica formation is microtubule (MT)--mediated and disturbed by agents interfering with MTs . TEL, largely ineffective as such, inhibited lorica formation of P . malhamensis when the lead compound was illuminated during or before the experiment . TriEL inhibited most; DiEL produced qualitatively effects similar to those of TriEL at about 10 times higher concentrations . Inorganic lead was even less toxic and did not selectively inhibit lorica formation of the algae . Low concentrations of TriEL (5 to 7.5 microM) selectively disturbed lorica formation, causing formation of numerous stalk-less loricae which exhibited gross and ultrastructural alterations like those induced by the antimitotic drug colchicine . The effects of TriEL on mitosis and cytokinesis of P . malhamensis were also investigated . The most sensitive mitotic phase was metaphase, which, however, accumulated only up to 5% after treatment of the cells with toxic concentrations (10 microM) of TriEL for 24 hr (control, 2%) . On the other hand, up to 15% telophases (including binucleated cells) and even multinucleated cells with up to eight nuclei per cell were found, indicating that cytokinesis was considerably more effectively disturbed by TriEL than mitosis . In giant multinucleated algae, mitoses normally proceeded synchronously; some asynchronous mitoses were found . Beside normal-looking mitotic spindles in giant algae, multipolar spindles, disoriented spindles, and metaphase-like chromosome arrays completely lacking MTs were observed by electron microscopy . The effects of TriEL on cytokinesis of the algae were largely reversible . Giant cells spontaneously recovered and underwent cytokinesis after transferred into TriEL-free growth medium . Colchicine acted qualitatively identical to TriEL (accumulation of metaphases and telophases: 5 and 19%, respectively), but TriEL was about 600 times more toxic than colchicine . Unlike colchicine, its derivative, colchicine, which is known not to interfere with MTs, remained without any selective inhibitory influence on mitosis and cytokinesis of the algae, although much more toxic than the parent compound . From the inhibitory effects of TriEL and the close qualitative similarities to the effects of colchicine, it is concluded that TriEL selectively interferes with cytoplasmic and mitotic MTs of the algae, thereby causing the observed inhibitory effects on lorica formation, mitosis, and cytokinesis.

Cancer Res, 1986 Feb, 46(2), 770 - 7
Emergence of differentiated subclones from a human salivary adenocarcinoma cell clone in culture after treatment with sodium butyrate; Azuma M et al.; A human salivary gland adenocarcinoma cell line, which has ultrastructure and biological markers specific to the intercalated duct cells of human salivary glands, was cultured in 5 mM sodium butyrate for 12 days; then the cells were trypsinized, subcultured for an additional 16 days, and then transferred to growth medium without sodium butyrate . Morphological changes appeared about 1 wk after return to growth medium without sodium butyrate; cells being spindle or stellate in shape appeared in the treated cells, whereas the untreated cells were polygonal in shape . This morphologically altered phenotype persists after more than 14 mo of culture in growth medium without sodium butyrate . Of 40 subclones isolated, 2 clonal cell lines were established from the subculture and characterized . The other 38 subclones were accompanied by cell death during the subcultures . The clonal lines exhibited a phenotype similar to myoepithelial cells such as myosin, beta-chain of S-100 protein, myofilaments, and oxytocin receptor in addition to decreased tumorigenicity and anchorage-independent growth . These findings indicate that commitment to differentiation into myoepithelial cells and conversion from malignant to normal phenotype occur in a human salivary gland adenocarcinoma cell line following the sodium butyrate treatment.

J Biol Chem, 1986 Jan 25, 261(3), 1293 - 8
Regulation of polyamine biosynthesis in tobacco . Effects of inhibitors and exogenous polyamines on arginine decarboxylase, ornithine decarboxylase, and S-adenosylmethionine decarboxylase; Hiatt AC et al.; Treatment of tobacco liquid suspension cultures with methylglyoxal bis(guanylhydrazone) (MGBG) an inhibitor of S-adenosylmethionine decarboxylase, resulted in a dramatic overproduction of a 35-kDa peptide on sodium dodecyl sulfate-polyacrylamide gel electrophoresis (Malmberg, R.L., and McIndoo, J . (1983) Nature 305, 623-625) . MGBG treatment also resulted in a 20-fold increase in the activity of S-adenosylmethionine decarboxylase . Purification of S-adenosylmethionine decarboxylase from MGBG-treated cultures revealed that the overproduced 35-kDa peptide and S-adenosylmethionine decarboxylase are identical . Precursor incorporation experiments using {3H} methionine and {35S}methionine revealed that MGBG does not induce any increased synthesis of S-adenosylmethionine decarboxylase but rather stabilizes the protein to proteolytic degradation . The half-life of the enzyme activity was increased when MGBG was present in the growth medium . In addition to stabilizing S-adenosylmethionine decarboxylase, MGBG also resulted in the rapid and specific loss of arginine decarboxylase activity with little effect ornithine decarboxylase . The kinetics of this effect suggest that arginine decarboxylase synthesis was rapidly inhibited by MGBG . Exogenously added polyamines had little effect on ornithine decarboxylase, whereas S-adenosylmethionine and arginine decarboxylase activities rapidly diminished with added spermidine or spermine . Finally, inhibition of ornithine decarboxylase was lethal to the cultures, whereas inhibition of arginine decarboxylase was only lethal during initiation of growth in suspension culture.

Biochemistry, 1986 Jan 14, 25(1), 203 - 11
Studies of anaerobic and aerobic glycolysis in Saccharomyces cerevisiae; den Hollander JA et al.; Glucose metabolism was followed in suspensions of Saccharomyces cerevisiae by using 13C NMR and 14C radioactive labeling techniques and by Warburg manometer experiments . These experiments were performed for cells grown with various carbon sources in the growth medium, so as to evaluate the effect of catabolite repression . The rate of glucose utilization was most conveniently determined by the 13C NMR experiments, which measured the concentration of {1-13C}glucose, whereas the distribution of end products was determined from the 13C and the 14C experiments . By combining these measurements the flows into the various pathways that contribute to glucose catabolism were estimated, and the effect of oxygen upon glucose catabolism was evaluated . From these measurements, the Pasteur quotient (PQ) for glucose catabolism was calculated to be 2.95 for acetate-grown cells and 1.89 for cells grown on glucose into saturation . The Warburg experiments provided an independent estimate of glucose catabolism . The PQ estimated from Warburg experiments was 2.9 for acetate-grown cells in excellent agreement with the labeled carbon experiments and 4.6 for cells grown into saturation, which did not agree . Possible explanations of these differences are discussed . From these data an estimate is obtained of the net flow through the Embden-Meyerhof-Parnas pathway . The backward flow through fructose-1,6-bisphosphatase (Fru-1,6-P2-ase) was calculated from the "scrambling" of the 13C label of {1-13C}glucose into the C1 and C6 positions of trehalose . Combining these data allowed us to calculate the net flux through phosphofructokinase (PFK) . For acetate-grown cells we found that the relative flow through PFK is a factor of 1.7 faster anaerobically than aerobically.(ABSTRACT TRUNCATED AT 250 WORDS)

Anal Biochem, 1986 Jan, 152(1), 136 - 40
Determination of the number of endothelial cells in culture using an acid phosphatase assay; Connolly DT et al.; A simple assay is described in which small numbers of endothelial cells in culture can be determined by measuring acid phosphatase activity . After removal of the growth medium from cells grown in 96-well culture plates, the cells are lysed in buffer containing the detergent Triton X-100 and the phosphatase substrate p-nitrophenyl phosphate . After 2 h at 37 degrees C, the reaction is stopped with sodium hydroxide, and color development is determined using a rapid multiwell plate reader . The assay detects 100 to 10,000 cells per well . The assay has been used to determine growth curves for endothelial cells in the presence and absence of endothelial cell growth factor from bovine hypothalamus and to monitor fractions during purification of the growth factor . Minor modifications in the assay allow it to be fully automated.

J Toxicol Environ Health, 1986, 17(1), 101 - 17
Biological availability of nickel arsenides: cellular response to soluble Ni5As2; Gurley LR et al.; Particulate Ni5As2 has been shown to be highly cytotoxic and carcinogenic . By measuring the solubility of Ni5As2 particles in a variety of aqueous solutions, we have determined that particulate Ni5As2 that might be produced during oil-shale retorting could be mobilized to the environment and made available to the cells of living organisms, including humans . Ni5As2 was five times more soluble in ground water taken from aquifers surrounding a major oil-shale source in Colorado, U.S.A., than in distilled water . It was also two times more soluble in oil-shale product water from an above-ground retort than in distilled water . Thus, it is possible that Ni5As2 could be solubilized and mobilized to the environment by the flooding of abandoned in situ retorts with ground water or by the disposal of oil-shale product water by spraying it on spent shale beds . Particulate Ni5As2 was found to be 12 times more soluble in culture growth medium than in distilled water, and much more soluble in solutions of amino acids, inorganic salts, organic constituents of culture medium, and 15% calf serum . These observations suggest Ni5As2 particles in airborne dust would be dissolved when they came in contact with the biological fluids of the lung and gastrointestinal tract . The availability to cells of the soluble products of Ni5As2 was demonstrated by measuring its effects on cell proliferation . As little as 1 ppm soluble Ni5As2 retarded Chinese hamster (CHO) cell proliferation in culture, and 4 ppm resulted in cell death . Flow cytometry measurements indicated there was a preferential cytotoxic effect on S-phase cells . Despite this, many cells survived to form colonies, causing concern that Ni5As2 might cause genetic damage that could be passed on to future cell generations . This did not appear to be the case, however, for no mutations could be detected at the hypoxanthine-guanine phosphoribosyl transferase (HGPRT) locus in cells that survived the cytotoxic effects . This suggests that Ni5As2 carcinogenesis might be caused by epigenetic rather than mutagenic mechanisms.

J Cell Biol, 1986 Jan, 102(1), 289 - 97
On the mechanism of rapid plasma membrane and chloroplast envelope expansion in Dunaliella salina exposed to hypoosmotic shock; Maeda M et al.; Dunaliella salina cells rapidly diluted from their normal 1.71 M NaCl-containing growth medium into medium containing 0.86 M NaCl swelled within 2--4 min to an average volume 1.76 X larger and a surface area 1.53 X larger than found in control cells . Morphometric analysis of thin section electron micrographs revealed that certain organelles, including the chloroplast, nucleus, and some types of vacuoles, also expanded in surface area as much or more than did the entire cell . It is likely that glycerol, the most important osmotically active intracellular solute, was present in high concentration within these organelles as well as in the cytoplasm itself . Thin section and freeze-fracture electron microscopy were utilized to trace the origin of membrane material whose addition permitted the large increase in plasma membrane surface area and the equally large growth of the chloroplast outer envelope . The findings indicated that the plasma membrane's expansion resulted from its selective fusion with numerous small (less than or equal to 0.25 micron diam) vesicles prevalent throughout the cytoplasm . In contrast, new membrane added to the chloroplast outer envelope was drawn from an entirely different source, namely, elements of the endoplasmic reticulum.

Antonie Van Leeuwenhoek, 1986, 52(6), 491 - 506
Mathematical modelling of lipid production by oleaginous yeasts in continuous cultures; Ykema A et al.; A mathematical model was constructed to describe the influence of the carbon to nitrogen ratio (C/N-ratio) of the growth medium on lipid production by oleaginous yeasts . To test this model and to determine some relevant model parameters, the oleaginous yeast Apiotrichum curvatum ATCC 20509 was grown in continuous cultures at various C/N-ratios and dilution rates . It appeared that when nitrogen is limiting for the formation of biomass, the remaining glucose can be converted to storage carbohydrate and storage lipid . No clear dependence of carbohydrate yield on the C/N-ratio could be demonstrated, but lipid yield increased gradually with increasing C/N-ratios . The maximal dilution rate for lipid producing yeast cells appeared to be optimal at relatively low C/N-ratios . It can be concluded that the experimental results fitted well with the mathematical model . By using this model, lipid yield and lipid production rate can be calculated at any C/N-ratio of the growth medium and optimum operation conditions can be predicted for the production of microbial lipids.

Comp Biochem Physiol B, 1986, 85(2), 369 - 73
Fatty acid composition of whole bodies, specific tissues and cell lines of two lepidopteran insects; Stanley-Samuelson DW et al.; We detail the fatty acid compositions of last larval instars of two lepidopterans, Spodoptera frugiperda and Trichoplusia ni, two tissues from T . ni, a cell line derived from each species and the respective larval and cell culture media . Larval whole-body and specific tissue fatty acid profiles exhibited the major features commonly found in previous lepidopteran analyses, whereas the cell-line fatty acid compositions were substantially different from the compositions of both their growth media and larvae of their respective species . It appears that these cell-line patterns result from increased monoene biosynthesis in response to low levels of exogenous polyunsaturated fatty acids, a commonly observed essential fatty acid deficiency symptom in whole animals.

Radiat Environ Biophys, 1986, 25(2), 141 - 9
Induction of ELF transmembrane potentials in relation to power-frequency electric field bioeffects in a plant root model system . I . Relationship between applied field strength and cucurbitaceous root growth rates; Brayman AA et al.; Seminal roots of Cucumis sativus and Cucurbita maxima were exposed to 60 Hz electric fields of 100-500 V X m-1 in a conducting aqueous inorganic growth medium . Root growth rates were measured to produce a dose-response relationship for each species . The species were selected for study because of their familial relationship, reported sensitivity to 60 Hz, 360 V X m-1 electric fields, and differing average root cell sizes . The latter characteristic influences the magnitude of ELF membrane potentials induced by constant-strength applied electric fields, but does not affect the magnitude of the electric field strength tangent to the cell surface . The difference in average root cell size between C . sativus (smaller cells) and C . maxima (larger cells) was used to evaluate two alternate hypotheses that the observed effect on root growth is stimulated by the electric field tangent to the cell surface, or a field-induced perturbation in the normal transmembrane potential of the cells . The results of the dose-response relationship studies are qualitatively consistent with the hypothesis that the effect is elicited by induced transmembrane potentials . The smaller-celled roots showed a substantially higher response threshold {C . sativus; E0TH approximately 330 V X m-1} than did the larger-celled species {C . maxima; E0TH approximately 200 V X m-1} . At field strengths above the response thresholds in both species, the growth rate of C . sativus roots was less affected than that of C . maxima roots exposed to the same field strength.

Digestion, 1986, 34(3), 161 - 8
Incubation of rat hepatic tumor cells with ethanol and acetaldehyde in vitro: effects on growth rate, albumin secretion and cellular protein content; Higgins PJ et al.; The in vitro response (defined as changes in growth rate, cellular protein content, and albumin secretion) of liver epithelial cells to the putative hepatotoxins ethanol and acetaldehyde were evaluated using the well-characterized 32IIIA 6/2d rat liver tumor clonal cell line . Exposure of hepatic tumor cells to ethanol (50-100 mM) for a period of 3 days reduced final population density (apparently due to a drug-induced increase in mean cell cycle transit time), reduced secretion of albumin, and increased the mean cellular protein content . Since these ethanol-associated effects were also observed in cells cultured in growth medium containing acetaldehyde (0.1 mM), and were inhibited by simultaneous addition of pyrazole, the changes in the parameters measured in this study appear to be induced by products of ethanol metabolism . These data complement recent in vivo studies implicating acetaldehyde as an inhibitor of hepatocyte secretory function . The 32IIIA 6/2d liver cell system, thus, responds to certain hepatotoxic compounds in a manner analogous to the in vivo organ and may facilitate future analysis of molecular mechanisms underlying alcohol-induced liver disease under defined culture conditions.

Int J Biochem, 1986, 18(9), 813 - 20
Preparation and purification of the proteinase inhibitor, leupeptin, from culture filtrates of Streptomyces lavendulae; Ning MC et al.; Leupeptin (N-acetyl-L-leucyl-L-leucyl-L-argininal) was produced as an extracellular metabolite by Streptomyces lavendulae . Addition of lysine to the growth medium stimulated leupeptin production from 200 to 1400 micrograms/ml . Incubation of late exponential phase mycelium in a synthetic medium was used to prepare single labelled (3H) and double labelled (3H/14C) inhibitor for metabolic studies . An improved purification scheme that generates leupeptin of high purity with good recoveries is also reported.

Basic Appl Histochem, 1986, 30(1), 85 - 92
The effect of extracellular matrix modifications on UDP-glucose dehydrogenase activity in cultured human skin fibroblasts; Rizzotti M et al.; The effect of modifications of the extracellular matrix on the biosynthesis of glycosaminoglycans was investigated in human skin fibroblast cultures by studying UDPGDH activity in order to evaluate: a), the histoenzymological and biochemical modifications induced by chondroitinase ABC treatment (new experimental conditions were developed in order to obtain minimum cell damage); b), the reversibility of these modifications; c), the effect of growing the cells in the presence of chondroitinsulfate; d), the specificity of the modifications induced . The results demonstrated that our experimental conditions specifically affected intracellular UDPGDH activity . Chondroitinase ABC treatment induced a reversible increase of UDP-glucuronic acid synthesis . On the contrary, the presence of chondroitinsulfate in the growth medium completely inhibited UDPGDH activity.

Mol Cell Biol, 1986 Jan, 6(1), 38 - 46
Structure of the transcriptionally repressed phosphate-repressible acid phosphatase gene (PHO5) of Saccharomyces cerevisiae; Bergman LW et al.; We developed a high-copy-number plasmid system containing the entire structural and regulatory sequences of the phosphate-repressible acid phosphatase (PHO5) gene and the TRP1/ARS1 replicator sequences of the yeast Saccharomyces cerevisiae to investigate the mechanism of repression-derepression of transcription . The resulting plasmid was used to transform either wild-type cells or a number of strains which contain mutations in various trans-acting regulatory loci for the production of acid phosphatase . Results of analysis of mRNA levels isolated from the transformed strains grown under repressed or derepressed conditions suggested that normal transcriptional regulation of the gene persisted, although gene copy number was significantly increased . Analysis of changes in linking number (i.e., the number of negative supercoils) of the plasmid isolated under repressed and derepressed growth conditions revealed that the transcriptionally inactive plasmid contained approximately three more negative supercoils than the transcriptionally active plasmid . This difference in topological state was similarly seen in a plasmid containing a sequence-related acid phosphatase gene (PHO11) under the same regulatory control system, but it was not seen in plasmids isolated from some strains containing mutations which caused either fully constitutive or nonderepressible production of acid phosphatase . Finally, analysis of the nucleosome positioning along the inactive gene sequence revealed that an abnormally broad internucleosomal spacer is present in a region presumed to function in the regulation of transcription by the level of Pi in the growth media.

Exp Lung Res, 1986, 11(4), 263 - 75
Characterization of rat alveolar type II cells in vitro by immunological, biochemical, and morphological criteria; McMahon JB et al.; Using an anti-rat surfactant apoprotein antiserum which specifically reacts with cytoplasmic structures in alveolar type II cells on histopathology sections of rat lung, we have examined the immunoreactivity of pulmonary type II cells in vitro . Single cell suspensions of lung tissue were prepared from male Fischer 344 rats by intratracheal elastase digestion according to standard published methods . Cytocentrifuged preparations of the resulting cell suspensions revealed that approximately 40% of the cells stained positive for surfactant apoprotein using an immunoperoxidase staining technique . Without further cell fractionation steps, the cell suspensions were plated at colonial densities in growth medium . The cells that attached after 24 hours of incubation and at daily intervals were analyzed for surfactant apoprotein immunoreactivity as well as for proliferation, morphology, and phospholipid biosynthesis . The percentage of immunopositive cells increased with time from 75% at day 1 to 94% at 4 days after plating . This increase was paralleled by a linear increase in the number of immunopositive cells, which expanded into cell colonies . During the initial 5 days in vitro, the immunopositive cells retained their epithelial morphology and contained cytoplasmic osmiophilic bodies . Phospholipid biosynthesis by the isolated lung cells was analyzed and the data revealed that the rate of incorporation of 14C-choline into phosphatidylcholine increased with time in culture . These studies indicated that the anti-rat surfactant apoprotein antisera can be used to identify and quantitate functional alveolar type II cells in vitro . Thus the specific antisera may facilitate studies of type II cells undergoing various environmental alterations both in vivo and in vitro.

Eur J Cell Biol, 1986 Jan, 39(2), 475 - 80
Vacuolar pH is one factor that regulates hydrolase secretion; Hohman TC et al.; Lysosomal hydrolases are continually secreted by Acanthamoeba as a consequence of membrane cycling between the vacuolar compartment and the cell surface . In pinocytosing amoebae acid hydrolases can be separated into two groups on the basis of their secretion kinetics . We have previously shown that in Acanthamoeba acid hydrolases are almost exclusively restricted to a single compartment, digestive vacuoles, and that pH-dependent differential binding of hydrolases to vacuolar membrane can account for the different rates of hydrolase secretion from this compartment . In this report we show that the hydrolase secretion pattern changes and that all of the hydrolases are released with the same kinetics after phagocytosis of yeast or in growth media supplemented with ammonium acetate or chloroquine, but not after phagocytosis of polystyrene beads . The changes in the pattern of hydrolase secretion correlate with changes in vacuolar pH . The vacuolar pH of pinocytosing amoebae and amoebae saturated with beads is about 4.8 . This value is increased to 6.8 by accumulation of weak bases and to about 6.1 when digestive vacuoles are saturated with yeast . These results indicate that vacuolar pH modulates hydrolase transport and secretion.

Comp Biochem Physiol B, 1986, 83(1), 57 - 61
Heme synthesis in Trypanosoma cruzi: influence of the strain and culture medium; Salzman TA et al.; The activity of the following enzymes involved in the biosynthesis of porphyrins was determined in two strains of Trypanosoma cruzi (Y and CL) grown in two culture media (LIT and Warren): succinyl coenzyme A synthetase (Suc.CoA-S), 5-aminolevulinate synthetase (ALA-S), 4,5-dioxovaleric acid transaminase (DOVA-T), 5-aminolevulinate dehydratase (ALA-D), porphobilinogenase (PBGase), deaminase and heme synthetase (Heme-S) . The amount of 5-aminolevulinic acid (ALA) and porphobilinogen, porphyrins and heme was also determined . ALA and PGB were detected in both strains of T . cruzi . However, ALA was not detected in epimastigotes of the Y strain grown in the LIT medium . The content of ALA and PBG varied according to the strain and the growth medium . No free porphyrins and heme were detected in both strains of T . cruzi . The activity of Suc.CoA-S and DOVA-T was markedly influenced by the strains of the parasite and the growth medium . No significant DOVA-T activity was detected in epimastigotes of the CL strain grown in the Warren's medium . No significant activity of ALA-D, PBGase and deaminase was detected in T . cruzi . Activity of Heme-S was detected in both strains of T . cruzi when mesoporphyrin, protoporphyrin or deuteroporphyrin was used as substrate . The enzyme activity was influenced by the strain of the parasite, the growth medium and the substrate used.

Arch Virol, 1986, 87(1-2), 49 - 59
Studies on Autographa californica nuclear polyhedrosis virus replication in Spodoptera littoralis cells including virus-induced protein synthesis; Roberts PL; The replication of the Autographa californica nuclear polyhedrosis virus in Spodoptera littoralis cells has been investigated . Various cytopathic changes were detected by light and electron microscopy and progeny enveloped virus particles, some occluded within polyhedra, were later seen in the nucleus of infected cells . Infectious virus was released into the growth medium and increased exponentially from ca . 10 to 24 hours post infection and then slowly increased over the next 4 days . In comparison, virus was released ca . 3.5 hours earlier from Spodoptera frugiperda cells . Total rates of DNA and protein synthesis were drastically reduced during the late stages of virus replication when cell death was occurring . By ca . 18 hours post infection, a clear switch from host to virus induced protein synthesis had occurred and a total of 39 virus-induced polypeptides of M.W . 12 to 120 X 10(3) were detected . These included polyhedrin of M.W . 33 X 10(3), which was particularly prominent during the late stages of virus replication, and a major virus structural protein of M.W . 42 X 10(3).

Endocrinology, 1986 Jan, 118(1), 52 - 7
The stimulation of sugar transport in heart cells grown in a serum-free medium by picomolar concentrations of thyroid hormones: the effects of insulin and hydrocortisone; Gordon A et al.; Chick embryo heart cells were propagated in a defined serum-free medium . They formed a confluent, synchronously contracting monolayer that is not different from myocytes grown in serum containing media . The uptake of 2-deoxy-D-{1-3H}glucose in these cells was stimulated by exposure to physiological concentrations of T3 (1 pM) and T4 (10 pM) . Actinomycin-D and puromycin did not block the stimulation of 2-deoxy-D-{1-3H}glucose uptake when given with T3 throughout a 6-h incubation period . Cells grown in the absence of both insulin and hydrocortisone were unresponsive to T3 . Insulin at 200 nM restored the sensitivity of the cells to 0.1 pM T3 . Addition of 10 nM hydrocortisone to the growth medium enhanced the effects of T3 synergistically . The T3-stimulated sugar uptake was completely blocked by 5 X 10(-6) M cytochalasin B, suggesting that T3 acts, like insulin, by the translocation of glucose transporters to the plasma membrane.

Endocrinology, 1986 Jan, 118(1), 393 - 407
A complex noncoordinate regulation of alpha-lactalbumin and 25 K beta-casein by corticosterone, prolactin, and insulin in long term cultures of normal rat mammary cells; Ray DB et al.; The concentrations of PRL, corticosterone, and insulin required by long term cultures of normal rat mammary cells to produce alpha-lactalbumin (alpha LA) and the 25,000 mol wt beta-casein were evaluated with a variety of hormone ratios and concentrations . For these studies a double antibody RIA for beta-casein capable of measuring 0.5 ng beta-casein/100 microliter growth media was developed and used along with our previously reported RIA for alpha LA . PRL was active at physiological levels (0.05-0.15 micrograms/ml) and quantitatively stimulated beta-casein more than alpha LA, whereas physiological levels of corticosterone (0.05-0.15 micrograms/ml) quantitatively stimulated alpha LA more than beta-casein . The concentration of corticosterone greatly altered the magnitude of the cells' response to insulin and PRL for alpha LA output by cells from either virginal or midpregnant rats . Insulin also enhanced production of these milk proteins, but very little effect was measured in the physiological range . alpha LA was increased more by insulin than by PRL, and beta-casein was enhanced more by insulin than by corticosterone . Cells from midpregnant rats required less insulin to stimulate beta-casein production than to stimulate alpha LA . Cells from virginal rats required a supraphysiological insulin level to stimulate both beta-casein and alpha LA under these conditions . These cells generally require 5-6 weeks to achieve a steady-state rate of milk protein output . The complexities of our observations help explain some of the conflicting reports in the literature concerning which hormone is of prime importance for quantitatively increasing the synthesis of a particular milk protein, particularly since high hormone levels are often employed and time in culture varies considerably among reports . We conclude that lower levels of all these hormones can and should be used in vitro . Our messenger RNA (mRNA) studies using cloned complementary DNA probes for two rat casein mRNAs show that cells grown for 2 months with hormones contain significant amounts of both alpha- and beta-casein mRNAs . Simultaneous quantification of beta-casein mRNA levels and rates of beta-casein protein production in these long term cell cultures indicated that a substantial portion of their beta-casein protein production is regulated by the amount of its mRNA . This could be controlled by mRNA synthesis and/or mRNA degradation.(ABSTRACT TRUNCATED AT 400 WORDS)

J Cell Physiol, 1986 Jan, 126(1), 10 - 20
Growth requirements and characterization of rat cervical epithelial cells in culture; Wright TC Jr et al.; The extended culture of rat cervical epithelial cells can be achieved in the absence of a fibroblast feeder layer by utilizing collagen gels and a complex growth medium . The medium contains a 1:1 mixture of RPMI-1640 and Ham's F12 supplemented with 7.5% porcine serum and epidermal growth factor, cholera toxin, transferrin, insulin, and hydrocortisone . Under these culture conditions the cells show rapid log-phase growth and high saturation densities while retaining the ultrastructural characteristics of immature squamous metaplastic cells of the rat uterine cervix even after extended passage . In a manner similar to epithelial cells from a variety of sources, rat cervical epithelial cells form hemicysts at confluence in vitro when cultured on impermeable substrates . The development of these methods for culturing cervical epithelial cells provides an experimental system for the study of factors important in regulating the growth and differentiation of metaplastic squamous epithelial cells.

Arch Oral Biol, 1986, 31(11), 717 - 22
Biochemical and morphological studies of human diploid and fluoride-resistant fibroblasts in vitro; Sato T et al.; Fluoride-resistant (FR 30) lines were produced from human diploid fibroblast (NDU-1) cells by progressively increasing the concentration of fluoride (F) in the growth medium up to 1.58 mM . This concentration completely inhibited the growth of the original cells . The resistant cells had decreased incorporation of {14C}-leucine and an increased rate of the cell division . The activities of acid phosphatase, alkaline phosphatase, lactate dehydrogenase, leucine aminopeptidase, glutamic oxaloacetic transaminase, glutamic pyruvic transaminase and alpha-hydroxybutyrate dehydrogenase in the FR 30 cells were lower than in the NDU-1 cells . The FR 30 cells had irregular shapes and sizes; the amount of undeveloped rough endoplasmic reticulum and the number of lysosomes were increased . These biochemical and morphological changes in FR 30 cells suggest that their metabolic activities were depressed by F, although they have some degree of F resistance.

Invest New Drugs, 1986, 4(4), 347 - 57
Cell cycle kinetics responses of human stomach cancer cells to reduction in polyamine levels by treatment with alpha difluoromethylornithine in vitro; Barranco SC et al.; Treatment of human gastric cancer clones in vitro with low doses of DFMO (5 mM) produced elongation of the cell population doubling times and lowering of the saturation densities . By contrast, DFMO treatment of normal human skin fibroblasts altered only the saturation density . The lack of an effect of 5 mM DFMO on the doubling time of normal fibroblasts may be directly related to baseline intracellular putrescine levels, which were about 2.5 times higher than in the cancer cells . The same dose of DFMO caused a rapid decrease in intracellular polyamine levels in the tumor clones . The effects on the doubling time and saturation density were almost totally abolished by the addition of 50 microM putrescine to the growth medium during the first 24 h of treatment with DFMO . Exposure to 5 mM DFMO for 24 h caused the human gastric cancer cells to become blocked in G1 phase only, and this led to a reduction in the fraction of cells in S phase . The G1 block was reversible and this cohort of cells eventually passed through S phase and then through G2 and M . A higher 100 mM dose of DFMO and longer exposure times for both doses produced cell cycle changes and death of more than 90% of the cell population . These data suggest that cell kinetics changes observed under these experimental conditions may reflect polyamine-related alterations in the biochemical events of cell cycle progression kinetics; but may also be the result of DFMO-induced loss of cell viability.

Biochimie, 1986 Jan, 68(1), 167 - 79
Genetic and physiological characterization of new Escherichia coli mutants impaired in hydrogenase activity; Wu LF et al.; The Mu dl (ApR lac) bacteriophage was used to generate mutants of Escherichia coli which were defective in formate hydrogenlyase . Three mutants were chosen for further analysis: they lacked hydrogenase (hydrogen: benzyl viologen oxidoreductase) activity, but produced normal levels of fumarate reductase activity and two- to three-fold reduced levels of benzyl viologen (BV)-dependent formate dehydrogenase activity . Two of them (hydC) were shown to contain about 4-fold reduced amounts of formate hydrogenlyase and fumarate-dependent H2 uptake activities . The third one (hydD) was totally devoid of both activities . Their insertion sites were located at 77 min on the E . coli map . Subdivision of these mutants into two classes was subsequently based on the restoration capacity of hydrogenase activity with high concentration of nickel in the growth media . Addition of 500 microM NiCl2 led to a complete recovery of hydrogenase activity, and to the concomitant restoration of normal BV-linked formate dehydrogenase, formate hydrogenlyase and fumarate-dependent H2 uptake activities in the hydC mutants . The hydD mutant was insensitive to the effect of nickel . Expression of the lac operon in hydC and hydD mutants was induced by anaerobiosis . It was not increased by the addition of formate under anaerobic conditions . The presence of nitrate resulted in slightly reduced beta-galactosidase activities in the hydC mutants, whereas those found in the hydD mutant reached only one third of the level obtained in its absence . Fumarate had no effect on both classes . Moreover, in contrast to the hydD locus, the hydC::Mu dl fusions were found to be dependent upon the positive control exerted by the nirR gene product and were totally repressed by an excess of nickel . In addition, the low levels of overall hydrogenase-dependent activities found in a nirR strain were also relieved by the presence of nickel . Our results strongly suggest that the pleiotropic regulatory gene nirR is essential for the expression of a gene (hydC) involved in either transport or processing of nickel in the cell, whose alteration leads to a loss of hydrogenase activity.

Exp Cell Res, 1986 Jan, 162(1), 127 - 41
Cell configuration and adhesive properties of metastasizing and non-metastasizing BSp73 rat adenocarcinoma cells; Raz A et al.; The pattern of cell substrate interaction, the cell surface composition and the organization of cytoskeletal elements was studied in tumour cell variants of the BSp73 rat adenocarcinoma displaying different metastatic capabilities and cell configuration . The non-metastasizing AS variant cells adhered to the substrate and spread via vinculin-containing focal contacts . These cells also synthesized, secreted and assembled fibronectin at the pericellular area . The metastasizing ASML variant cells adhered to the substrate at a slower rate via thick cytoplasmic protrusions, but were removed from the substrate by trypsin-EDTA slower than the non-metastasizing AS variant cells . The ASML cells also synthesized very low levels of both vinculin and fibronectin, displayed a diffuse pattern of actin and tubulin organization, and were unable to spread on the substrate . Spreading could not be induced in the ASML cells by seeding the cells on an extracellular matrix derived from bovine corneal endothelial cells or on concanavalin A (conA)-coated substrates, or by the addition of db-cAMP to the medium . The metastasizing cells expressed a unique and abundant cell surface glycoprotein of Mr 170 000 which was also shed into the growth medium . The relationships among the adhesive properties, the organization of cell surface components and of the cytoskeleton in the tumour cell variants, and the expression of their metastatic phenotype is discussed.

Miner Electrolyte Metab, 1986, 12(1), 32 - 41
Sodium cotransport processes in renal epithelial cell lines; Rabito CA; Cell growth and synchronization, gene expression and regulation, maintenance of epithelial cell polarization are major functional aspects of the nephron which have been difficult to approach with conventional preparations . Certainly, without the introduction of tissue culture techniques, the opportunity to analyze these issues could not have been afforded . Several renal epithelial cell lines with differentiated characteristics of proximal and distal segments of the nephron are already available . LLC-PK1, derived from a normal Hampshire pig kidney, shows multiple differentiated characteristics of in vivo epithelia . Specifically, this cell line has Na+-dependent sugar, amino acid, and phosphate cotransport systems with similar characteristics as those present in the renal proximal tubule . The expression of the Na+-dependent sugar transport system in LLC-PK1 cells depends on the growing conditions of the cells . For instance, isolated cells obtained by trypsin-EDTA treatment of confluent monolayers or exponentially growing cells do not express the Na+-dependent sugar transport system . Full expression occurs after the monolayer reaches confluency . Expression of the transporter can also be modified by agents that affect the differentiation of other cellular systems, such as the Friend erythroleukemia cells . The expression of the Na-sugar cotransport system is also regulated by the concentration of glucose in the growth medium . Low glucose concentration increases the sugar influx through the Na+-coupled apical membrane transporter by increasing the number rather than the affinity of the transporter . This effect appears to be mediated through the sugar metabolism of the cell . The expression of the Na+-amino acid cotransport system in LLC-PK1 cells and the epithelial cell line MDCK derived from a normal dog kidney also responds to regulatory signals associated with cell growth or amino acid deprivation . LLC-PK1 cells and the cell line OK derived from an opposum kidney, shows a Na+-dependent phosphate cotransport system modulated by parathyroid hormone and cyclic nucleotides that will prove to be an excellent model not only to study the mechanisms of the action of the hormone, but to study the mechanisms involved in the expression and modulation of the Na+-dependent phosphate cotransport system.

Chem Biol Interact, 1985 Dec 31, 56(2-3), 239 - 49
Metabolism and specific benzopyrene metabolite modification of DNA in early S by human lung epithelial and fibroblast cells leading to the expression of an abnormal phenotype; Milo GE; Examination of the high pressure liquid chromatographic profiles of ethyl acetate extractable benzo {a} pyrene (B(a)P)-metabolites from human lung fibroblast and type II epithelial cells after S phase entry indicated that B(a)P-7,8-diol and 9,10-diol species were produced following the oxygenation of B(a)P . These metabolites were detected intracellularly and in the extracellular growth medium . Both cell types appeared to release extracellularly, elevated amounts of the B(a)P-7,8-diol species . It was interesting to note of the 4 pmol of oxygenated metabolites localized intracellularly, in the fibroblast, that we identified two major metabolites, B(a)P-9,10 and -7,8-diol species . Lung epithelial cells metabolize intracellular B(a)P extensively, greater than or equal to 93% of the parent B(a)P . No tetrols were detected intracellularly or extracellularly in the treated fibroblast cells . The treated epithelial cells produced both tetrols and sulfate conjugates . The extent of observed modification of early S phase nuclear DNA of lung epithelial cells was 7.5 +/- 4.9 adducts per 10(6) bases and 4.2 +/- 2.7 adducts per 10(6) bases in lung fibroblasts . The major adduct formed in both cell types was 7 beta-BPDE-I-dG . Under conditions for transformation, both the B(a)P treated lung epithelial cells and lung fibroblasts treated in early S with either B(a)P or BPDE-I yielded populations that exhibited properties of anchorage independent growth and cellular invasiveness . Metabolism and the presence internally of metabolites did not correlate with the extent of modification of DNA in early S.

Neurosci Lett, 1985 Dec 4, 62(2), 283 - 9
Neurite-promoting effects of 12-O-tetradecanoylphorbol-13-acetate on chick embryo neurons; Hsu L; The addition of the phorbol ester 12-O-tetradecanoylphorbol-13-acetate (TPA) to defined growth medium enhanced the survival of ciliary neurons in dissociated cultures and promoted the rapid development of neurites from sympathetic and parasympathetic ganglion explants . Explants of the central nervous system including the cerebral cortex and spinal cord of chick embryos were, however, unresponsive to either 10 or 100 ng/ml of TPA.

Appl Environ Microbiol, 1985 Dec, 50(6), 1556 - 7
Preparation and testing of an autolysate of fish viscera as growth substrate for bacteria; Clausen E et al.; The aqueous soluble phase of acidified and autolyzed fish viscera was used as the nitrogen source in a growth medium for bacteria . The bacteria tested grew faster and produced higher yields of cell mass on this growth medium than on corresponding media with standard tryptone preparations as the nitrogen source.

Immunology, 1985 Dec, 56(4), 615 - 23
Leukaemia x fibroblast hybrid cells augment the antibody response to sheep red blood cells in inbred mice; Cohen EP et al.; ASL-1 x LM(TK-) hybrid cells, an established murine leukaemia x fibroblast hybrid cell line, augment the antibody response to sheep red blood cells in inbred mice, as determined by the plaque assay method . The intraperitoneal injection of viable hybrid cells or of growth medium conditioned by the cells leads to an increase both in the total number as well as the proportion of cells forming antibodies to sheep red blood cells . CSF-1, (M-CSF), is detected by radioimmunoassay in the medium conditioned by the hybrid and LM(TK-) cells, but not ASL-1 parental cells . Prior treatment of the conditioned medium with CSF-1 antiserum reduces its capacity to augment the antibody response, and its proliferative stimulus on cells from the marrow indicating that CSF-1 may be at least partly responsible for the adjuvant effect observed . The intraperitoneal implantation of diffusion chambers containing viable CSF-1 producing hybrid cells, like the cells themselves, also leads to an increase in the number of spleen cells forming antibodies to sheep red blood cells.

J Bacteriol, 1985 Dec, 164(3), 1324 - 31
Differential expression of hydrogenase isoenzymes in Escherichia coli K-12: evidence for a third isoenzyme; Sawers RG et al.; The cellular contents of the nickel-containing, membrane-bound hydrogenase isoenzymes 1 and 2 (hydrogenases 1 and 2) were analyzed by crossed immunoelectrophoresis . Their expression was differentially influenced by nutritional and genetic factors . Hydrogenase 2 content was enhanced after growth with either hydrogen and fumarate or glycerol and fumarate and correlated reasonably with cellular hydrogen uptake capacity . Hydrogenase 1 content was negligible under the above conditions but was enhanced by exogenous formate . Its expression was greatly reduced in a pfl mutant, which is unable to synthesise formate, but was restored to normal levels when the growth medium included formate . A mutation in the anaerobic regulatory gene, fnr, led to low overall hydrogenase activity and greatly reduced levels of both isoenzymes and abolished the formate enhancement of hydrogenase 1 content . Formate hydrogenlyase activity was similarly reduced in the fnr strain but, in contrast, was restored, as was overall hydrogenase activity, to normal levels by growth in the presence of formate . Low H2 uptake activity was found for the fnr strain under all growth conditions examined . Hydrogenase 1 content, therefore, does not correlate with formate hydrogenlyase activity and its role is unclear . A third hydrogenase isoenzyme, immunologically distinct from hydrogenases 1 and 2, whose expression is enhanced by formate, is present and forms part of the formate hydrogenlyase . We suggest that the effect of the fnr gene product on formate hydrogenlyase expression is mediated via internal formate.

Exp Cell Res, 1985 Dec, 161(2), 509 - 16
Effect of cholesterol and growth factors on the proliferation of cultured human skin fibroblasts; Ostlund RE Jr et al.; A cholesterol-deficient growth medium for human skin fibroblasts was prepared by adding to Eagle's Minimum Essential Medium a bovine serum treated with ultracentrifugation to remove bulk lipoproteins followed by silicic acid adsorption to remove residual lipoproteins and cholesterol . Cell growth was slow, but the daily cell doublings could be increased by 76% by including 7.5 micrograms purified cholesterol/ml in the medium . Cell growth in cholesterol-deficient culture medium could be increased to that seen with medium containing 15% untreated fetal bovine serum by the inclusion of the following growth factors: epidermal growth factor (EGF), cortisol, non-essential amino acids, insulin, transferrin and selenium . Cholesterol increased the proliferation of these rapidly-growing cultures by 19% . No effect of cholesterol was observed in transformed L-cell mouse fibroblasts.

Infect Immun, 1985 Dec, 50(3), 701 - 8
Specific nature of Trichomonas vaginalis parasitism of host cell surfaces; Alderete JF et al.; The adherence of Trichomonas vaginalis NYH 286 to host cells was evaluated by using monolayer cultures of HeLa and HEp-2 epithelial cells and human fibroblast cell lines . Saturation of sites on HeLa cells was achieved, yielding a maximal T . vaginalis NYH 286-to-cell ratio of two . The ability of radiolabeled NYH 286 to compete with unlabeled trichomonads for attachment and the time, temperature, and pH-dependent nature of host cell parasitism reinforced the idea of specific parasite-cell associations . Other trichomonal isolates (JH31A, RU375, and JHHR) were also found to adhere to cell monolayers, albeit to different degrees, and all isolates produced maximal contact-dependent HeLa cell cytotoxicity . The avirulent trichomonad, Trichomonas tenax, did not adhere to cell monolayers and did not cause host cell damage . Interestingly, parasite cytadherence was greater with HeLa and HEp-2 epithelial cells than with fibroblast cells . In addition, cytotoxicity with fibroblast cells never exceeded 20% of the level of cell killing observed for epithelial cells . Elucidation of properties of the pathogenic human trichomonads that allowed for host cell surface parasitism was also attempted . Treatment of motile T . vaginalis NYH 286 with trypsin diminished cell parasitism . Incubation of trypsinized organisms in growth medium allowed for regeneration of trichomonal adherence, and cycloheximide inhibited the regeneration of attachment . Organisms poisoned with metronidazole or iodoacetate failed to attach to host cells, and adherent trichomonads exposed to metronidazole or iodoacetate were readily released from parasitized cells . Coincubation experiments with polycationic proteins and sugars and pretreatment of parasites or cells with neuraminidase or periodate had no effect on host cell parasitism . Colchicine and cytochalasin B, however, did produce some inhibition of adherence to HeLa cells . The data suggest that metabolizing T . vaginalis adheres to host cells via parasite surface proteins in a specific receptor-ligand fashion . Furthermore, parasitism of epithelial cells appears to render this cell type more susceptible than fibroblast cell types to contact-dependent cytotoxicity.

J Gen Microbiol, 1985 Dec, 131 ( Pt 12), 3171 - 7
Influence of cysteine deprivation on chlamydial differentiation from reproductive to infective life-cycle forms; Allan I et al.; The effects of omission of individual amino acids from growth medium on the differentiation of Chlamydia trachomatis DK-20 (serotype E) during infection of cycloheximide-treated McCoy cells are described . As judged by inclusion body staining with acridine orange, omission of cysteine from the medium severely retarded differentiation of reproductive reticulate body (RB) to infective elementary body (EB) forms . The effect appeared specific to cysteine in that omission of other amino acids had little or no effect on differentiation once RBs appeared . On restoration of cysteine, culture infectivity increased and inclusions contained organisms which, by cytochemical and morphological criteria, were differentiating to infective forms, indicating that cysteine deprivation did not irreversibly inhibit differentiation . Impairment of RB to EB differentiation in cysteine-less medium was also observed for three strains of Chlamydia psittaci and 10 other strains of C . trachomatis . It is suggested that the effect arises via the biosynthetic requirement for cysteine for provision of three cysteine-rich proteins, whose synthesis and insertion into the outer membrane have previously been shown to accompany RB to EB differentiation of C . psittaci 6BC and C . trachomatis 434 (serotype L2) . Synthesis of cysteine-rich outer membrane proteins during differentiation may thus be common to all chlamydiae.

J Gen Microbiol, 1985 Dec, 131 ( Pt 12), 3199 - 209
Cyclic AMP and the stimulation of trehalase activity in the yeast Saccharomyces cerevisiae by carbon sources, nitrogen sources and inhibitors of protein synthesis; Thevelein JM et al.; Addition of glucose to acetate-grown cells of Saccharomyces cerevisiae caused a rapid transient increase in the cAMP level followed by a 10-fold, transient increase in the activity of trehalase . Ethidium bromide and acridine analogues inhibited both glucose-induced responses in a similar way, confirming the role of the cAMP signal as the second messenger in the sugar-induced activation of trehalase . When nitrogen sources or protein synthesis inhibitors were added after the transient glucose-induced increase in the trehalase activity, a rapid reactivation of trehalase occurred . In this case, however, there was only a very small increase in the cAMP level, which appeared to be insignificant . When the nitrogen source or the protein synthesis inhibitor was added together with glucose, the trehalase activity remained high for a much longer time also without a significant effect on the cAMP level . When a membrane depolarizing agent was added together with the glucose, both the trehalase activity and the cAMP level remained high . Reversibility experiments in which trehalase was activated to different degrees also showed that for high sugar-induced trehalase activation a high cAMP level is needed, while nitrogen sources stimulate trehalase activity without affecting cAMP levels . In cell extracts, both cAMP and cGMP were able to activate trehalase, the latter however only at 10-fold higher concentrations . The cGMP level in vivo was about 10-fold lower than the cAMP level and was not significantly affected by nitrogen sources or protein synthesis inhibitors . Hence, neither cAMP nor cGMP seem to be involved as the second messenger in the stimulating effect of nitrogen sources and protein synthesis inhibitors on trehalase activity in yeast . Since all other evidence obtained here and before strongly points to regulation of trehalase by a 'cAMP-dependent' protein kinase, we suggest that the presence of a nitrogen source in the growth medium of yeast induces the rapid synthesis of an alternative second messenger able to activate this or another protein kinase.

J Bacteriol, 1985 Dec, 164(3), 1376 - 80
Excretion of alkaline phosphatase by Escherichia coli K-12 pho constitutive mutants transformed with plasmids carrying the alkaline phosphatase structural gene; Lazzaroni JC et al.; Escherichia coli alkaline phosphatase constitutive mutants carrying a pst or a phoS mutation and a plasmid-bearing gene phoA+ excreted into the growth medium up to 50% of the total alkaline phosphatase production . This excretion was pH dependent and did not involve drastic modifications of the cell envelope . Alkaline phosphatase accounted for 80% of total released proteins . Amplification of gene phoA+ was a necessary condition for excretion to occur . When the beta-lactamase structural gene bla+ was coamplified with gene phoA+, both enzymes were excreted . pst-transformed excretory strains did not show the pleiotrophic phenotype previously described for lky mutants.

J Pharmacol Exp Ther, 1985 Dec, 235(3), 657 - 64
Selective regulation of beta-1 and beta-2 adrenergic receptors by atypical agonists; Neve KA et al.; The interactions of the atypical agonists pindolol and celiprolol with beta adrenergic receptors were compared with those of the full agonist, isoproterenol . Studies were carried out using intact cells as well as membranes prepared from C6 glioma cells . Computer-assisted analysis of dose-response curves resulting from the inhibition of the binding of {125I}iodopindolol by the beta-1 and beta-2 selective compounds ICI 89,406 and ICI 118,551 revealed that approximately one-third of the beta adrenergic receptors on these cells were beta-1 receptors . Addition of GTP to the binding assay simplified the dose-response curve for inhibition of the binding of {125I}iodopindolol by isoproterenol and diminished the potency of the agonist . GTP had no effect on the binding of pindolol or celiprolol, suggesting that these drugs do not induce the formation of a ternary complex with the receptor and the guanine nucleotide-binding protein for stimulation of adenylate cyclase activity . When added to the growth medium of intact C6 cells, isoproterenol induced a 40-fold increase in cyclic AMP accumulation . Pindolol and celiprolol, however, caused no elevation of enzyme activity . Addition of isoproterenol to the growth medium of intact cells resulted in an 80% decrease in the density of both beta-1 and beta-2 adrenergic receptors within 8 hr . Growing cells in the presence of pindolol or celiprolol induced a 50% decrease in the density of beta-2 receptors, which was inhibited by beta adrenergic antagonists.(ABSTRACT TRUNCATED AT 250 WORDS)

Cell Mol Neurobiol, 1985 Dec, 5(4), 333 - 52
The effects of exposure to exogenous fatty acids and membrane fatty acid modification on the electrical properties of NG108-15 cells; Love JA et al.; The role of membrane lipid composition in determining the electrical properties of neuronal cells was investigated by altering the available fatty acids in the growth medium of cultured neuroblastoma X glioma hybrid cells, clone NG108-15 . Growth of the cells for several days in the presence of polyunsaturated fatty acids (linoleic, linolenic, and arachidonic) caused a pronounced decrease in the Na+ action-potential rate of rise (dV/dt) and smaller decreases in the amplitude, measured by intracellular recording . Oleic acid had no effect on the action potentials generated by the cells . In contrast, a saturated fatty acid (palmitate) and a trans monounsaturated fatty acid (elaidate) caused increases in both the rate of rise and the amplitude . No changes in the resting membrane potentials or Ca2+ action potentials of fatty acid-treated cells were observed . The membrane capacitance and time constant were not altered by exposure to arachidonate, oleate, or elaidate, whereas arachidonate caused a small increase in membrane resistance . Examination of the membrane phospholipid fatty acid composition of cells grown with various fatty acids revealed no consistent alterations which could explain these results . To examine the mechanism for arachidonate-induced decreases in dV/dt, the binding of 3H-saxitoxin (known to interact with voltage-sensitive Na+) channels was measured . Membranes from cells grown with arachidonate contained fewer saxitoxin binding sites, suggesting fewer Na+ channels in these cells . We conclude that conditions which lead to major changes in the membrane fatty acid composition have no effect on the resting membrane potential, membrane capacitance, time constant, or Ca2+ action potentials in NG108-15 cells . Membrane resistance also does not appear to be very sensitive to membrane fatty acid composition . However, changes in the availability of fatty acids and/or changes in the subsequent membrane fatty acid composition lead to altered Na+ action potentials . The primary mechanism for this alteration appears to be through changes in the number of Na+ channels in the cells.

J Exp Med, 1985 Dec 1, 162(6), 2089 - 106
Functional properties of a unique subset of cytotoxic CD3+ T lymphocytes that express Fc receptors for IgG (CD16/Leu-11 antigen); Lanier LL et al.; A subset of peripheral blood T lymphocytes coexpressing CD3 and IgG Fc receptors (FcR) (CD16/Leu-11 antigen) have been identified, isolated, and functionally characterized . The CD3+, CD16+ cells were established in short-term culture using growth medium containing interleukin 2 (IL-2) . Both the freshly isolated cells and the cultured cell line stably expressed the CD3+, CD16+ phenotype . Furthermore, a majority of these T cells lacked either CD4 or CD8 expression . Like in vitro-activated cytotoxic T lymphocytes and natural killer (NK) cells, the CD3+, CD16+ cells showed numerous azurophilic granules . Although these cells failed to mediate significant levels of NK cell-mediated cytotoxicity even after stimulation with IL-2, they efficiently functioned as effectors of antibody-dependent cellular cytotoxicity (ADCC) . The Ig isotype specificity of the ADCC was analyzed using an isotype switch-variant family of a murine anti-HLA monoclonal antibody (mAb) . Similar to the CD3-, CD16+ NK cell population, the CD3+, CD16+ T cells preferentially used the IgG2a antibody to mediate ADCC . The CD3+, CD16+ cells demonstrated a proliferative response when cocultured with either a NK-sensitive tumor cell line, K562, or a NK-insensitive B lymphoblastoid cell line, CCRF-SB . The response against CCRF-SB was significantly inhibited by anti-IL-2 receptor antibody, whereas the response against K562 was only partially diminished . Cytotoxicity was also induced in the CD3+, CD16+ population by the presence of anti-CD3 mAb, indicating that cytotoxicity can be triggered by stimulation via the CD3-T cell antigen receptor complex . By isolating these CD3+, CD16+ cells from the peripheral blood of a normal, healthy individual, it has been possible to extensively study the morphology, antigenic phenotype, and functional behavior of this unique subset of T lymphocytes expressing IgG FcR.

FEBS Lett, 1985 Nov 25, 193(1), 49 - 53
Stimulation of protein accumulation in HeLa cells by inhibitors of DNA replication . Ferritin; Menozzi FD et al.; Incubation of HeLa cells for 24 h with either hydroxyurea (HU), aphidicolin (APHI), thymidine (T) or butyrate (BU), substances used to inhibit replication and accumulate cells at the G1/S interphase, followed by the elimination of the inhibitor and the addition of iron to the growth medium, results in an immediate (HU, APHI, T) or slightly delayed (BU) increased accumulation (18-24-fold higher than the basal level) of ferritin . Under the same experimental circumstances, 5-azacytidine is without effect . As a result of the action of these inhibitors on the structure of DNA, it is proposed that ferritin genes remain accessible to RNA polymerase allowing the accumulation in the cytoplasm of mature ferritin mRNA ready to be mobilized by iron for the production of ferritin molecules.

J Biol Chem, 1985 Nov 25, 260(27), 14838 - 43
Evidence for negative control of cya transcription by cAMP and cAMP receptor protein in intact Escherichia coli cells; Mori K et al.; The transcriptional regulation of cya by cAMP and its receptor protein (CRP) has been studied by S1 nuclease and RNA dot blot assays . The crp- Escherichia coli cells were shown to produce about 5-fold more cya mRNA than do the wild type cells . The effect of cAMP and CRP on the cya transcription was directly examined by introducing a crp plasmid into the cells and/or by adding cAMP exogenously . The levels of cya mRNA in crp+ cells decreased with increasing concentrations of cAMP in the growth medium . The repressive effect of cAMP on cya transcription was strongly enhanced in cells carrying a multicopy crp plasmid . These results indicate that the cya transcription is negatively regulated by cAMP-CRP complex in intact cells.

Anal Biochem, 1985 Nov 15, 151(1), 118 - 24
Counting cells as DNA: estimation of ovine pituitary cells on Sephadex columns; Metcalf MG; A technique is described for estimating the number of sheep pituitary cells remaining on Bio-Gel P2-Sephadex G-25 columns at the conclusion of perifusion experiments . After they were washed to remove growth medium, the cells were lysed by sonication in a hypotonic solution . The resultant DNA was eluted through a 10-micron filter and measured by fluorometric analysis after reaction with the Hoechst fluorochrome, H33258 . DNA recovery increased in parallel with the number of cells added to a column (correlation coefficient for 52 observations on 9000-500,000 cells, 0.993; interassay coefficient of variation, 8.4%) . The relevance of this technique to the general problem of counting cells in the presence of a finely divided solid phase is discussed . The loss of pituitary cells from 42 columns during a 10-h perifusion study ranged from less than or equal to 10% (24 columns) to greater than 10-25% (10 columns), greater than 25-50% (6 columns), and greater than 50% (2 columns) . It is concluded (i) that pituitary cells mixed with Sephadex and Bio-Gel may be counted as DNA and (ii) that the measurement of pituitary cell loss is a necessary prerequisite for the valid interpretation of the results of perifusion experiments.

Biochim Biophys Acta, 1985 Nov 13, 826(2-3), 151 - 3
Acid DNAase activity from Dictyostelium discoideum; Guyer RB et al.; We have isolated and partially characterized an acid endonuclease activity from the cellular slime mold, Dictyostelium discoideum . This activity comprises more than 90% of the nonspecific DNA-endonuclease activity of the vegetative cells . Its molecular weight is about 44 000, and its activity is enhanced 7-fold by Mg2+ . The pH optimum for the nicking activity depends upon NaCl concentrations, being at pH 5.0 in 207 mM NaCl, and at pH 5.8 in 7 mM NaCl . Large quantities of this enzyme activity are released into the growth medium or buffer, with detectable amounts appearing within 15 min of incubation.

Brain Res, 1985 Nov 11, 347(1), 196 - 9
Acquisition of glial fibrillary acidic protein-containing fibres by astroglial cells in primary culture is orchestrated by a communicable factor; Wilson J et al.; In a previous study we discovered that primary cultures initiated from the whole brain of 21-day foetal rats contained astroblasts that concertedly acquired glial fibrillary acidic protein (GFAP) fibres . The mechanism of this burst of cytoskeletal differentiation could not be investigated in these cultures because it occurred too quickly (completed within 2 h) . We report that cultures initiated from the region of the third ventricle display an extended burst of GFAP acquisition whose rate could be markedly reduced by medium changing . Temporary medium deprivation or the addition of cytosine arabinoside to the growth medium had no effect . Our findings suggest that an as yet uncharacterised communicable factor is involved in the orchestration of cytoskeletal differentiation in culture . This factor may be responsible for synchronising the appearance of GFAP-positive cells in the periventricular regions of the foetal brain.

Nature, 1985 Nov 7-13, 318(6041), 73 - 5
Sarcoma viruses carrying ras oncogenes induce differentiation-associated properties in a neuronal cell line; Noda M et al.; The growth-promoting and/or differentiation-blocking activities of Kirsten (Ki-MSV) or Harvey murine sarcoma virus (Ha-MSV) on various types of cells in vitro are well documented . Here we report an unexpected effect of these viruses on a rat phaeochromocytoma cell line, PC12 . PC12 cells, which multiply indefinitely in growth medium, are known to respond to nerve growth factor (NGF) by cessation of cell division and expression of several properties resembling those of differentiated sympathetic neurones . We have found that Ki- and Ha-MSV mimic some, if not all, of the activities of NGF in PC12 cells, and there is evidence that the viral oncogenes, v-Ki-ras and v-Ha-ras, are responsible for this phenomenon . This system may be of value for studying the mechanism of action of the v-ras genes as well as the regulatory mechanism of growth and differentiation in neuronal cells.

Mikrobiologiia, 1985 Nov-Dec, 54(6), 883 - 8
{Effect of the exogenous tryptophan on biosynthesis of ergot alkaloids in Penicillium sizovae}; Kozlovskii AG et al.; Various concentrations of exogenous L and D-tryptophan as well as of their analogue D,L-6-methyltryptophan were added to the growth medium of Penicillium sizovae during its inoculation and after the active growth of the fungus was over . The authors studied the effect of these compounds on the accumulation of exocellular alkaloids and biomass as well as on the synthesis of proteins, the content of free tryptophan in the cells, and the activity of tryptophan synthetase . As was shown in experiments using labeled tryptophan, this amino acid is a direct precursor of alkaloids in the culture.

Allergy, 1985 Nov, 40(8), 586 - 91
Cultivation of fungi in synthetic and semi-synthetic liquid medium . I . Growth characteristics of the fungi and biochemical properties of the isolated antigenic material; van der Heide S et al.; Four allergologically important fungi, viz . Aspergillus fumigatus, Alternaria Penicillium notatum, and Cladosporium herbarum, were cultured in a pure synthetic medium and the patterns of growth as characterized by the pH, protein and carbohydrate concentration of the culture fluid, were studied . A fumigatus and P . notatum showed a similar growth pattern, characterized by a rapid decrease in the pH of the culture medium (pH 7.4----4.0), while proteins were slowly released and saccharose poorly consumed . In contrast, A . alternata and C . herbarum demonstrated a different pattern of growth, in which the pH of the culture hardly changed during incubation . Enrichment of the synthetic medium with yeast extract greatly improved the growth of all four fungi, as was confirmed by the enhanced yield of antigenic material and strongly increased consumption of saccharose . The yeast extract especially changed the growth pattern of A . fumigatus and P . notatum, which now is characterized by three phases . Phase I: fall in pH of the growth medium and excretion of proteins; phase II: increase in pH and fall in protein concentration; phase III: stabilization of pH at alkaline values and renewed excretions of proteins . It is concluded that during cultivation of the fungi, the metabolic state of the culture changes, influencing the antigenic composition of the extracts obtained after different periods of cultivation.

Mycopathologia, 1985 Nov, 92(2), 111 - 3
Nutrient requirements in germination of conidiospores of Aspergillus niger V . Tieghen; Abdel-Rahim AM et al.; Germination of conidiospores of A . niger was stimulated by the addition of some carbohydrates and nitrogenous compounds to the growth medium . Glucose at 0.5 and 1.0 mg/ml concentrations was the best sugar for germination . Sucrose and fructose were the second best . Glutamic acid and valine were the most effective nitrogenous compounds for the spore germination of the fungus . Glucose, sucrose, and glutamic acid initiated germination within the first 6 h of incubation . When cellobiose, galactose and valine were used germination started after 9 h . With leucine, cysteine and arginine, however, not only the percentage of germination reduced, but also the latent period increased, significantly . A period of 15 h was needed before germination had been started.

J Clin Invest, 1985 Nov, 76(5), 1727 - 32
Effects of bile and bile salts on growth and membrane lipid uptake by Giardia lamblia . Possible implications for pathogenesis of intestinal disease; Farthing MJ et al.; We have shown previously that ox and pig bile accelerate in vitro growth of Giardia lamblia . We have now investigated the possible mechanisms by which mammalian biles promote parasite growth . Growth effects of (a) ox, pig, guinea pig, and human biles, (b) pure bile salts, and (c) egg and soybean lecithins were studied in the presence of a lecithin-containing growth medium . Individually, dilute native bile and pure sodium taurocholate (TC), glycocholate (GC), and taurodeoxycholate (TDC) promoted parasite growth; growth was most marked with biles of high phospholipid content, with biles enriched in more hydrophobic bile salts (ox approximately equal to human greater than pig greater than guinea pig) and with micellar concentrations of GC and submicellar concentrations of TC and TDC . By measuring uptake of radiolabeled biliary lipids from bile and bile salt-supplemented growth medium, we showed that the parasite consumed bile lipids, with the rank order lecithin greater than bile salts . Apparent net uptake of cholesterol was considered to be due to exchange, since net loss of cholesterol from the growth medium was not detected . Although bile and bile salt-stimulated parasite growth was associated with enhanced lecithin uptake, reduction in generation time was observed at low bile and bile salt concentrations when lecithin uptake was similar to bile free controls . Thus, bile salts may stimulate Giardia growth initially by a mechanism independent of enhanced membrane phospholipid uptake . However, since Giardia has no capacity to synthesize membrane lipid, biliary lecithin may be a major source of phospholipid for growth of this parasite.

J Bacteriol, 1985 Nov, 164(2), 896 - 903
Isolation, identification, and structural analysis of the mycobactins of Mycobacterium avium, Mycobacterium intracellulare, Mycobacterium scrofulaceum, and Mycobacterium paratuberculosis; Barclay R et al.; Methods were devised to purify the cell-associated, iron-binding compounds known as mycobactins from the closely related species Mycobacterium avium, Mycobacterium intracellulare, and Mycobacterium scrofulaceum (i.e., the MAIS complex of organisms) . The mycobactins from these three species showed a structure that is common to the mycobactins from all the mycobacteria examined to date . However, these mycobactins were unique in that they had more than one alkyl chain . The M . scrofulaceum mycobactins differed from other MAIS mycobactins by a shift in the position of the double bond in the R1 alkyl chain . Traces of other mycobactin types were observed in ethanol extracts of the three species, and examination of the chromatographic properties of these mycobactins showed that each species produced five mycobactin types . Each mycobactin could be subdivided further by the length of its R1 alkyl chain . No differences in the production of these novel mycobactin were observed among species . Mycobactins from three strains of Mycobacterium paratuberculosis and two wood pigeon strains of Mycobacterium avium which had lost their original growth requirements for mycobactin after repeated subculturing in laboratory growth media were examined by thin-layer chromatography and high-pressure liquid chromatography . Each organism produced a mycobactin with similar chromatographic properties to those synthesized by MAIS organisms . M . paratuberculosis NADC 18 produced at least two components in our laboratory, and nuclear magnetic resonance analysis of the major component showed this mycobactin to be identical to that produced by M . intracellulare M12 . However, a sample of mycobactin J isolated by Merkal and McCullough (Curr . Microbiol . 7:333-335, 1982) from M . paratuberculosis NADC 18 was different from our isolates and appeared to correspond to a minor mycobactin component we had seen by thin-layer chromatography . No reason for this difference could be evinced . Our findings indicate that there is a close taxonomic relationship between M . paratuberculosis and the MAIS complex.

Cancer Res, 1985 Nov, 45(11 Pt 1), 5496 - 504
Effects of selenium on cell proliferation in rat liver and mammalian cells as indicated by cytokinetic and biochemical analysis; LeBoeuf RA et al.; Studies were conducted in vivo with regenerating liver and in vitro with mammalian cells to determine the effects of selenium on cell proliferation and the stages of the cell cycle affected by selenium . Six ppm selenium as Na2SeO3 fed to weanling male F344 rats for 6 wk significantly reduced the percentage of 3H-labeled hepatocyte nuclei by one-half compared to 0.1 ppm selenium when {methyl-3H}thymidine was injected at 23 h post-two-thirds hepatectomy . Sampling was done at 30 h post-hepatectomy . A trend towards decreased 3H per DNA per labeled cell was also observed, suggesting that selenium decreased the rate of DNA synthesis as well as delaying the entry of cells into S phase (i.e., increasing the duration of G0-G1) . Studies in vitro with H-4 "minimal deviation" hepatomas and 3T3 mouse fibroblasts demonstrated that selenium decreased the growth of these cells in a dose-dependent manner, and this inhibition was reversible upon removal of selenium from the growth medium . Cytokinetic analysis using fluorescence flow cytometry and microscopic techniques indicated that selenium treatment increased the duration of G1, S, and G2 phases of the cell cycle, while having no effect on mitosis under the conditions of our experiments . Biochemical analyses of H-4 cells demonstrated that selenium treatment caused a significant dose-dependent increase in oxidized and reduced glutathione (GSSG and GSH) as well as in the GSSG:GSH ratio as was previously observed in liver in vivo . In addition, glutathione reductase activity as well as the oxidized nicotinamide adenine dinucleotide phosphate:reduced nicotinamide adenine dinucleotide phosphate ratio was significantly increased with selenium treatment . These results indicate that selenium affects all "synthetic" stages of the cell cycle, and elevated GSSG or the GSSG:GSH ratio may explain the antiproliferative effects of selenium on cells.

Cancer Res, 1985 Nov, 45(11 Pt 1), 5328 - 34
Uptake of hematoporphyrin derivative by normal and malignant cells: effect of serum, pH, temperature, and cell size; Bohmer RM et al.; Normal and malignant cells were incubated with hematoporphyrin derivative (HPD) and their uptake and retention of HPD were analyzed by flow cytometry . In standard growth medium the amount of HPD taken up by cells was proportional to the added HPD concentration and reached a plateau level after 5-6 h of incubation . The uptake occurred in two steps; within seconds a large amount of HPD became loosely bound to the cells, presumably the outer membrane . This was followed by a slower uptake of HPD into the cytoplasm . The loosely bound portion could be washed from the cells by medium containing either fetal calf serum or serum albumin . At low temperatures the uptake into the cytoplasm was strongly reduced . A major determinant of HPD uptake was the concentration of serum in the medium . At any particular concentration of HPD below 200 mg/liter, increasing concentrations of fetal calf serum or bovine serum albumin resulted in a reduction in the amount of HPD taken up by cells . A further factor affecting uptake was the pH of the medium . At low pH (pH 6) the rate of HPD incorporation was much higher than at pH 7.4 . Under identical conditions of incubation, HPD uptake was proportional to cell size as estimated using the low angle light scatter signal in the flow cytometer . Our data suggest that acidic pH, differences in extracellular serum concentrations of malignant tumor tissue, as well as the increased size of tumor cells may play an important role in the selective uptake of HPD by malignant tumors.

Allergy, 1985 Nov, 40(8), 592 - 8
Cultivation of fungi in synthetic and semi-synthetic liquid medium . II . Immunochemical properties of the antigenic and allergenic extracts; van der Heide S et al.; Four allergologically important fungi, viz . Aspergillus fumigatus, Penicillium notatum, Alternaria alternata, and Cladosporium herbarum were cultured in a liquid synthetic medium, with or without addition of 0.1% yeast extract (YE) . After 10 and 28 days of cultivation, immunochemical properties of the fungal extracts, characterised by precipitation pattern and IgG- and IgE-binding capacity, were studied . In pure synthetic medium no large differences were observed in number of precipitates, as measured by DID, among the four antigenic fractions of each fungus (metabolic and mycelial antigens of both early and late phase cultures) . Addition of YE to the growth medium hardly changed the number of precipitates and generally caused a decrease in IgG-binding capacity of the extracts . In contrast to these observations were the findings with the IgE-binding capacities of the fungal extracts as measured by RAST . Most allergenic fractions demonstrated an increase (sometimes strong) in IgE-binding capacity after YE was added to the growth medium . It is concluded that the time of cultivation influences the immunochemical characteristics of fungal extracts and that mycelial as well as metabolic antigens of both early and late phase cultures should be used in order to obtain a wide spectrum of allergens in extracts used for diagnostic purposes.

Cancer Res, 1985 Nov, 45(11 Pt 1), 5263 - 9
Kinetics of protein synthesis inactivation in human T-lymphocytes by selective monoclonal antibody-ricin conjugates; Leonard JE et al.; Immunotoxins synthesized with the pan-T-cell monoclonal antibody T101 and ricin, acetylricin, or ricin A-chain have been compared . Native ricin was acetylated with N-acetylimidazole to block the galactose-binding site of the toxin B (binding)-chain . In the presence of lactose, both whole-ricin-containing immunotoxins were selectively cytotoxic but the ricin A-chain conjugate was less effective in blocking cellular protein synthesis . Immunotoxin-treated cells cultured in fresh growth medium exhibited no growth, declining viabilities, and no protein synthesis activity . Lymphocytes treated with T101:ricin or ricin did not form clusters or colonies when plated in 0.3% Bacto-agar . Ammonium chloride markedly enhanced the efficacy of T101:ricin and T101:ricin A-chain . Our results suggest that: (a) all immunotoxins were selectively cytotoxic; (b) in the presence of ammonium chloride the effectiveness of the T101:ricin A-chain conjugate approached that of T101:ricin; and (c) the toxin B-chain may facilitate conjugate internalization and/or processing.

Cancer Res, 1985 Nov, 45(11 Pt 1), 5480 - 8
Differential cellular retention of vincristine and vinblastine by cultured human promyelocytic leukemia HL-60/Cl cells: the basis of differential toxicity; Ferguson PJ et al.; Differential toxicity of vincristine and vinblastine against cells of a cloned subline of human promyelocytic leukemia (HL-60/Cl) was dependent on exposure conditions . During continuous exposures of 48 h, vincristine and vinblastine were equitoxic with drug concentrations that inhibited proliferation rates by 50% of 7.6 and 8.1 nM, respectively . When cells were subjected to 4-h exposures and transferred to drug-free medium, the drug concentration of vinblastine that inhibited proliferation rates by 50% (1.1 microM) was significantly greater than that of vincristine (41 nM) . Analysis by flow cytometry of the effects of equitoxic drug exposures on cell-cycle progression suggested that vincristine and vinblastine acted by the same mechanism (G2-M phase inhibition) . {3H}Vincristine and {3H}vinblastine were bound to serum proteins in growth medium to the same extent (25%) over a wide range of concentrations, and the amounts of "free" extracellular drug did not decrease during prolonged exposures . Analysis by high-performance liquid chromatography of extracts of cultures incubated with growth-inhibitory concentrations of {3H}vincristine or {3H}vinblastine indicated little, if any, metabolism of either drug by cells or culture fluids; after 24 h, 85-95% of radioactivity was recovered from cells or growth medium as unchanged vincristine or vinblastine . At concentrations from 6 nM to 6 microM, vinblastine entered cells rapidly, reaching maximum levels within 0.5-2 h, and the relationship between maximal cell-associated drug and extracellular free vinblastine was linear . Although uptake of vincristine was slower than that of vinblastine, the cellular content of vincristine reached that of vinblastine during prolonged (12-24 h) exposures, and the amounts of cell-associated drug, relative to extracellular drug concentrations, indicated considerable "concentrative" accumulation (intra: extracellular ratios, greater than 100) . When drug exposures were ended by transfer of cells to drug-free medium, vinblastine was released from cells more rapidly and to a greater extent than vincristine, independent of whether exposures were 4 or 24 h . Rates of uptake and release of vinblastine (50 nM) were unaffected by depletion of cellular adenosine triphosphate, suggesting that rapid release was not mediated by an energy-dependent efflux system.

J Am Intraocul Implant Soc, 1985 Nov, 11(6), 558 - 63
Neodymium:YAG laser interaction with intraocular lenses: an in vitro toxicity assay; Lindstrom RL et al.; Use of the Nd:YAG laser is an effective technique to open an opacified posterior lens capsule . However, in the presence of a posterior chamber intraocular lens (IOL), precise focusing of the laser on the capsule is required to avoid pitting the lens optic . The question has been raised whether toxic products may result from laser damage to the IOL . We addressed this issue in the present study by exposing primary human corneal endothelial cell and human corneal organ cultures to solutions produced by purposefully hitting IOLs immersed in cell growth medium with a Nd:YAG laser . The lenses studied were lathe-cut polymethylmethacrylate (PMMA), injection-molded non-UV PMMA, injection-molded UV PMMA, and cast-molded UV PMMA . Samples of each material were irradiated in a holder containing 1 ml of cell culture medium using the following conditions: 5, 10, and 50 laser bursts at 10 millijoules (mJ), and 50 laser bursts at 5 mJ . The solutions were applied to the endothelial cell cultures (all materials) and to the corneal organ cultures (injection-molded non-UV lenses only) . There was no toxicity in either assay for any of the materials studied.

Am J Pathol, 1985 Nov, 121(2), 298 - 310
The in vitro growth and characterization of the skeletal muscle component of Wilms' tumor; Garvin AJ et al.; Skeletal muscle differentiation within a Wilms' tumor is a well-documented histopathologic entity thought to occur at a relatively low incidence and influence prognosis . A serum-free hormonally defined growth medium has been developed, allowing the long-term growth of the skeletal muscle component of Wilms' tumors . Eight Wilms' tumors have been grown under these conditions . Three cases grew a homogeneous population of cells which ultrastructurally displayed all stages of myogenesis through myotubule formation . They also possessed immunoreactivity for skeletal muscle myosin and myoglobin and synthesized the M and B subunits of creatine kinase . Of interest was the finding that the ability to yield skeletal muscle cultures was limited to those cases which exhibited skeletal muscle fibers in vivo . This technique is also a very sensitive marker for identifying Wilms' tumors possessing a myoid component . A second serum-free hormonally defined medium has also been developed that supports the long-term culture of a unique cell type from Wilms' tumors which contain a myoid component . These cells are spindle-shaped and exhibit all of the characteristics of early myoblasts.

J Mol Biol, 1985 Oct 20, 185(4), 701 - 12
Genetic dissection of stringent control and nutritional shift-up response of the Escherichia coli S10 ribosomal protein operon; Freedman LP et al.; The S10 operon of Escherichia coli is autogenously regulated by L4, one of 11 ribosomal proteins encoded by the operon . We have previously shown that L4 regulates transcription of the operon by modulating the level of read-through at an attenuator in the S10 leader . To determine the physiological roles of both L4-mediated attenuation and the regulation of transcription initiation, we have constructed mutations eliminating their two regulatory targets, the S10 leader and the S10 promoter . Our results indicate that stringent control requires only the S10 promoter and therefore is mediated at the level of initiation . However, growth-medium-dependent control after a nutritional shift-up involves regulation of both initiation of transcription at the promoter and transcription read-through at the attenuator.

Eur J Biochem, 1985 Oct 15, 152(2), 323 - 9
Gelatin-degrading activity secreted by cultured macrophages from human blood; Vartio T; Gelatin-binding proteins (fibronectin and the 95 000-Mr protein {T . Vartio, T . Hovi & A . Vaheri (1982) J . Biol . Chem . 257, 8862-8866} were isolated by gelatin-agarose from the growth medium of cultured human monocyte/macrophages and the 95 000-Mr protein was further separated from fibronectin under nondenaturing conditions by preparative polyacrylamide gel electrophoresis . In the latter the proteins were eluted from the bottom of the tube gel into fractions which were then tested for ability to degrade native or heat-denatured type I collagen (gelatin) . When solutions from fractions containing the 95 000-Mr protein were incubated with gelatin, degradation was revealed by analysis of the reaction mixtures in the sodium dodecyl sulfate/polyacrylamide gel electrophoresis . Native type I collagen as well as native or heat-denatured fibronectin or other plasma proteins were unaffected when tested similarly . The degradation of gelatin was calcium-dependent and was inhibited by serum, sulfhydryl and metal-chelating reagents, but not with serine proteinase inhibitors . Gelatin was degraded optimally at pH 7-9 and at 41 degrees C and 37 degrees C and less effectively at 22 degrees C . Native type I collagen was degraded at 41 degrees C but not at 37 degrees C or 22 degrees C . The results show that cultured human macrophages secrete highly specific gelatin-degrading metal-proteinase activity which is associated with the 95 000-Mr gelatin-binding protein.

Appl Environ Microbiol, 1985 Oct, 50(4), 868 - 71
Charcoal agar, a new growth medium for the fish disease bacterium Renibacterium salmoninarum; Daly JG et al.; Charcoal is an effective replacement for serum in media for the isolation and culture of Renibacterium salmoninarum, the causative agent of bacterial kidney disease in salmonid fish . The medium, KDM-C, contains 10 g of peptone, 0.5 g of yeast extract, 1 g of L-cysteine hydrochloride, 1 g of activated charcoal, and 15 g of agar per liter and is adjusted to pH 6.8 with NaOH before autoclaving . Eight strains of R . salmoninarum grew from dilute inocula as well on KDM-C as on a standard serum-containing medium (KDM-2) . The medium was effective for both primary isolations from fish and repeated transfers and has potential value for antigen preparation and physiological studies.

Eur J Clin Microbiol, 1985 Oct, 4(5), 483 - 7
Gas chromatographic fatty acid profiles for characterisation of mycobacteria: an interlaboratory methodological evaluation; Larsson L et al.; Three species of mycobacteria were cultured and processed for cellular fatty acid analysis by capillary gas chromatography in three laboratories to study interlaboratory variations of the resulting chromatographic profiles . Largely consistent and characteristic fatty acid profiles were obtained, although there were minor quantitative variations in the patterns due to methodological differences (cultivation, hydrolysis, derivatization, gas chromatographic conditions etc.) . The following points were important for achieving informative and reproducible results . A chemically defined growth medium (e.g., Proskauer-Beck) provides more consistent profiles than the lipid-rich Lowenstein-Jensen medium . Harvesting directly into the digesting solution (NaOH or HCl in methanol) followed by heating or autoclaving is a simple and reliable way of releasing fatty acids . Care should be taken to ensure reproducible detection of long-chain alcohols either by using acid methanolysis or including a base-wash step in the procedure following alkaline hydrolysis . The temperature of the gas chromatographic injector should be at least 325 degrees C . A capillary column of a minimum length of 10 m coated with a methyl silicone is adequate . Our results indicate the possibility of recommending a practical and reproducible gas chromatographic procedure for mycobacterial characterisation.

J Appl Toxicol, 1985 Oct, 5(5), 306 - 14
Welding fumes and chromium compounds in cell transformation assays; Hansen K et al.; Fumes generated from mild steel and stainless steel welding were collected on paper filters and tested in the BHK and SHE cell transformation assays . Fumes from the manual metal arc welding of stainless steel (MMA/SS) had a toxic and transforming effect attributable to their Cr(VI) content . The fumes from metal inert gas stainless steel (MIG/SS) welding also had a toxic effect but this was 2-3 times greater than that expected from their soluble Cr(VI) content based on the activity of soluble Cr(VI) from pure chromium compounds . When collected in an impinger, the fumes from MIG/SS were found to contain approximately 10 times the soluble Cr(VI) content of samples collected on filters . This additional Cr(VI), when collected in a water impinger, also exhibited a greater toxicity compared with that found for the additional Cr(VI) collected in an impinger filled with growth medium . This comparison implies the presence of a short-lived biologically active Cr(VI) species usually lost in conventional sampling techniques . It also implies that there is a detoxification step associated with the formation of Cr(VI) organic complexes . Relatively insoluble Cr(VI) compounds showed a higher toxic and transforming effect in the BHK assay than could be ascribed to the soluble Cr(VI) content of the medium, indicating the importance of phagocytosis as a pathway for the uptake of Cr(VI) and other toxic substances from particulates.

In Vitro Cell Dev Biol, 1985 Oct, 21(10), 597 - 602
Culture of sweat gland epithelial cells from normal individuals and patients with cystic fibrosis; Collie G et al.; Recent electrophysiological and pharmacological studies have confirmed previous clinical evidence that the gene defect in cystic fibrosis is strongly expressed in the sweat gland . This has provided a major impetus to efforts to culture the cells of this tissue in order to provide a source of experimental material for molecular studies . Toward this end, eccrine sweat glands were isolated from collagenase treated skin specimens and the secretory coil and the reabsorptive duct separated . Segments of each portion of the gland were transferred to a plastic or collagen substrate and covered with serum-containing or serum-free defined growth media . Epithelial cell outgrowth took place in both media but fibroblast overgrowth occurred in the presence of serum at concentrations as low as 1% . In serum-free medium both secretory and reabsorptive cells formed tightly joined epithelial sheets, first as monolayers and later as multilayers consisting of at least six cell layers . Growth continued for approximately fifteen generations each of about two and a half days . Remarkably large domes or hemicysts with diameters as great as two cm were formed, apparently attesting to the retention of the capacity of the cells to actively transport ions and water . Ultrastructurally, cells which grew out from the secretory coil resembled the fluid secreting clear cells; neither dark cells nor myoepithelial cells were propagated.

J Cell Biol, 1985 Oct, 101(4), 1591 - 8
Biochemical studies on cell fusion . II . Control of fusion response by lipid alteration; Roos DS et al.; The preceding communication (Roos, D.S . and P.W . Choppin, 1985, J . Cell Biol . 101:1578-1590) described the lipid composition of a series of mouse fibroblast cell lines which vary in susceptibility to the fusogenic effects of polyethylene glycol (PEG) . Two alterations in lipid content were found to be directly correlated with resistance to PEG-induced cell fusion: increases in fatty acyl chain saturation, and the elevation of neutral glycerides, including an unusual ether-linked compound . In this study, we have probed the association between lipid composition and cell fusion through the use of fatty acid supplements to the cellular growth medium, and show that the fusibility of cells can be controlled by altering their acyl chain composition . The parental Clone 1D cells contain moderately unsaturated fatty acids with a ratio of saturates to polyunsaturates (S/P) approximately 1 and fuse virtually to completion following a standard PEG treatment . By contrast, the lipids of a highly fusion-resistant mutant cell line, F40, are highly saturated (S/P approximately 4) . When the S/P ratio of Clone 1D cells was increased to approximate that normally found in F40 cells by growth in the presence of high concentrations of saturated fatty acids, they became highly resistant to PEG . Reduction of the S/P ratio of F40 cells by growth in cis-polyunsaturated fatty acids rendered them susceptible to fusion . Cell lines F8, F16, etc., which are normally intermediate between Clone 1D and F40 in both lipid composition and fusion response, can be altered in either direction (towards either increased or decreased susceptibility to fusion) by the addition of appropriate fatty acids to the growth medium . Although trans-unsaturated fatty acids have phase-transition temperatures roughly similar to saturated compounds, and might therefore be expected to affect membrane fluidity in a similar manner, trans-unsaturated fatty acids exerted the same effect as cis-unsaturates on the control of PEG-induced cell fusion . This observation suggests that the control of cell fusion by alteration of fatty acid content is not due to changes in membrane fluidity, and thus that the fatty acids are involved in some other way in the modulation of cell fusion.

Blood, 1985 Oct, 66(4), 1002 - 5
Regulated proliferation of primitive hematopoietic progenitor cells in long-term human marrow cultures; Cashman J et al.; We have examined the cycling status of various classes of erythroid and granulopoietic progenitor populations maintained for many weeks in standard normal long-term human marrow cultures . These were initiated with a single inoculum of marrow aspirate and were routinely fed by weekly removal of half of the nonadherent cells and replacement of half of the growth medium . Progenitors of large erythroid colonies (more than eight erythroblast clusters) present in the nonadherent fraction and progenitors of small granulocyte/macrophage colonies (fewer than 500 cells) present in both the nonadherent and adherent fractions were found to be actively cycling at all times examined (28% to 63% kill following a 20-minute exposure to 20 microCi/mL of high specific activity 3H-thymidine) . In contrast, progenitors of large granulocyte/macrophage colonies (more than 500 cells) and progenitors of large erythroid colonies (more than eight erythroblast clusters), present in the adherent layer, consistently alternated between a quiescent state at the time of each weekly medium change and a proliferating state two to three days later (0% to 13% kill and 21% to 49% kill, respectively) . Additional experiments revealed that the activation of primitive progenitors in the adherent layer was not dependent on the addition of fresh glutamine or hydrocortisone, nor on the physical manipulations involved in changing the growth medium . These studies provide the first direct evidence that normal long-term human marrow cultures support the continued turnover of a variety of early hematopoietic progenitor cell types . Further, they indicate that the proliferative activity of the most primitive of these progenitors is regulated by stage-specific cell-cell interactions that are subject to manipulation.

Endocrinology, 1985 Oct, 117(4), 1409 - 17
Selection and characterization of a breast cancer cell line resistant to the antiestrogen LY 117018; Bronzert DA et al.; We have selected and cloned a stable variant of the MCF-7 human breast cancer cell line (LY 2) that is resistant to LY 117018 (LY), a potent antiestrogen that inhibits cell growth at concentrations as low as 10(-10) M . The cell line was selected by increasing the concentration of LY in the growth medium in a stepwise manner from 10(-8) to 10(-6) M as the cells become resistant . LY2 has been cloned in soft agar and carried for over 50 passages with no change in resistance . Other antiestrogens, such as tamoxifen and 40-hydroxytamoxifen no longer inhibit cell proliferation of LY 2 . The cell line is still responsive to estrogen in a cell proliferation assay, but contains somewhat less estrogen receptors than MCF-7 . The cytosolic estrogen receptor sediments to a 4S position on high salt sucrose density gradient centrifugation and is completely shifted to a denser gradient region when the receptor is incubated with a monoclonal antiestrophilin . The nuclear estrogen receptor when covalently labeled with {3H}tamoxifen aziridine has the same mol wt (62,000) in both MCF-7 and LY2 cells, when determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis . In a competitive binding assay, LY 117018 competes for {3H}estradiol binding to its cytosol receptor with the same Ki in both MCF-7 and LY2 cells . When the induction of estrogen-specific proteins was examined, no detectable progesterone receptor could be detected in either estrogen-induced or control LY2 cells, in contrast to MCF-7 cells . However, both 52,000- and 160,000-dalton proteins were estrogen inducible in the medium of LY2 and MCF-7 cells, as measured by labeling with {35S}methionine . The phenotypic stability of the antiestrogen resistance in LY2 cells coupled with the cross-resistance the antiestrogens of widely different structures make this cell line an ideal model system for the study of hormone resistance in human breast cancer . In addition, while the mechanism of resistance is currently not elucidated, the selective loss of estrogen-inducible functions in this cell line may provide powerful clues for future study.

J Periodontol, 1985 Oct, 56(10), 592 - 6
Inhibitory effect of periodontally diseased root extracts on the growth of human gingival fibroblasts; Olson RH et al.; Cementum shavings obtained from periodontally diseased and nondiseased areas of 100 removed, single-rooted teeth were extracted with either pyrogen-free water (PFW) for 5 minutes, 1 M citric acid for 5 minutes or 45% phenol-PFW for 90 minutes at 65 degrees C . The extracts were membrane-filtered, dialyzed exhaustively versus PFW, lyophilized, weighed and then dissolved in complete growth medium . The phenol-water or citric acid extracts of cementum shavings from periodontally diseased roots were positive for endotoxin by the limulus lysate assay (LLA) . Pyrogen-free water extracts of diseased or phenol-water extracts of nondiseased cementum shavings were negative, or only slightly positive, respectively, for endotoxin by LLA . Media containing the various extracts were added to logarithmically growing cultures of human gingival fibroblasts (HGF) . Separate cultures of HGF were exposed to Escherichia coli endotoxin at concentrations of 50, 100, 250 and 500 micrograms/ml to determine the growth-inhibitory effects of a known endotoxin . Cell growth was analyzed by measuring the incorporation of tritiated thymidine into cells . Suppression of HGF growth from 30 to 49% by E . coli endotoxin was concentration-dependent and linear over the concentration range of endotoxin tested . Pyrogen-free water extracts of diseased (endotoxin negative) or phenol-water extracts of nondiseased cementum shavings (slightly endotoxin positive) did not effect HGF growth . However, citric acid or phenol-water extracts of diseased cementum shavings (highly endotoxin positive) significantly suppressed HGF growth 58% and 61%, respectively . These results indicate that citric acid is effective in removing cytotoxic substances, presumably endotoxin, from cementum shavings and suggest that citric acid treatment is effective clinically in detoxifying periodontally diseased root surfaces.

Radiat Res, 1985 Oct, 104(1), 109 - 15
Growth-medium-dependent repair of DNA single-strand and double-strand breaks in X-irradiated Escherichia coli; Sargentini NJ et al.; The X-ray resistance of logarithmic phase cells of Escherichia coli K-12 is enhanced threefold by growth in rich medium versus minimal medium (N . J . Sargentini, W . P . Diver, and K . C . Smith, Radiat . Res . 93, 364-380, 1983) . In this work, X-ray-induced DNA strand breaks were assayed by sedimentation in alkaline and neutral sucrose gradients to correlate the enhanced survival of rich-medium-grown cells with an enhanced capacity for DNA repair . While rich-medium-grown cells showed no enhanced capacity for repairing DNA single-strand breaks in buffer, i.e., fast, polA-dependent repair, they did show an enhanced capacity to repair both single-strand and double-strand breaks in growth medium, i.e., slow, recA-dependent repair . This enhanced capacity for DNA repair in rich-medium-grown cells was inhibited by rifampicin post-treatment, indicating the requirement for de novo RNA synthesis . Kinetic studies indicated that the repair of DNA double-strand breaks was a complex process . Relative to the sedimentation rate in neutral sucrose gradients of nonirradiated DNA, the sedimentation rate of X-irradiated DNA first changed from slow to very fast . Based on alkaline sucrose gradient sedimentation studies, all the strand breaks had been repaired during the formation of the very fast sedimenting DNA . With continued incubation, the sedimentation rate of the DNA on neutral sucrose gradients decreased to the normal rate.

J Neurosci, 1985 Oct, 5(10), 2662 - 71
Isolation and characterization of rat schwannoma neurite-promoting factor: evidence that the factor contains laminin; Davis GE et al.; Rat RN22 schwannoma cells in vitro release into their growth medium a macromolecular factor that, when bound to polyornithine-coated culture substrata, will stimulate neuritic regeneration from axotomized peripheral and central neurons . During the purification of this factor, the neurite-promoting activity co-purifies with laminin immunoreactivity as measured by an enzyme-linked immunoadsorbent assay . The purified factor has an immunoreactivity per milligram of protein similar to that of purified rat yolk sac tumor laminin . After sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) under reducing conditions, the purified factor exhibits a major band at 200 kilodaltons (kD) and two minor ones at about 130 and 35 kD . The 200-kD band comigrates with the 200-kD band of purified rat laminin . After SDS-PAGE under non-reducing conditions, the rat schwannoma factor and rat laminin both exhibit a band in the 900-kD range with the schwannoma factor band migrating slightly faster than the laminin one . The 200-kD (reducing conditions) and 900-kD (non-reducing conditions) bands of both the schwannoma factor and laminin are stained by immunoblotting with antisera raised against rat and human laminin and against a partially purified preparation of the schwannoma factor . On immunoblots the 400-kD band of laminin (a band not seen in the schwannoma factor preparation) also stains with all three antisera . When the antibodies from each of the three antisera are immobilized on protein A-agarose beads, the beads will completely remove from solution the neurite-promoting activities of both the schwannoma factor and laminin . Antibodies raised against rat laminin fail to block the neurite-promoting activity of the purified schwannoma factor but totally block that of rat laminin . In contrast, antibodies raised against the schwannoma factor will block the neurite-promoting activities of both the schwannoma factor and laminin . By rotary shadowing electron microscopy the schwannoma factor preparation exhibits cross-shaped images similar but not identical to those previously reported for rat and mouse laminin . In addition, the schwannoma factor preparation contains images resembling proteoglycans.

J Bacteriol, 1985 Oct, 164(1), 331 - 7
Kinetics of uptake and incorporation of meso-diaminopimelic acid in different Escherichia coli strains; Wientjes FB et al.; The rate at which the peptidoglycan precursor meso-diaminopimelic acid (DAP) is incorporated into the cell wall of Escherichia coli cells was determined by pulse-label experiments . For different E . coli strains, the incorporation rate was compared with the rate of uptake of DAP into the cell . With E . coli W7, a dap lys mutant generally used in this kind of studies, steady-state incorporation was reached only after about 0.75 of the doubling time . This lag period can be ascribed to the presence of a large internal DAP pool in the cells . An E . coli K-12 lysA strain was constructed which could be grown without DAP in its medium . Consequently, due to the higher specific activity of the added {3H}DAP, faster incorporation and higher levels of radioactivity in the peptidoglycan layer were observed in the K-12 lysA strain than in the W7 strain . In addition, uptake and incorporation were faster in steady state (within about 0.2 of the doubling time), indicating a smaller DAP pool . The lag period could be further diminished and the incorporation rate could be increased by feedback inhibition of the biosynthetic pathway to DAP with threonine and methionine . These results make MC4100 lysA a suitable strain for studies on peptidoglycan synthesis . To explain our observations, we suggest the existence of an expandable pool of DAP in E . coli which varies with the DAP concentration in the growth medium . With 2 microgram of DAP per ml, the size of the pool is severalfold the amount of DAP contained in the cell wall . This pool can be partly washed out of the cells . Grown without DAP, MC4100 lysA still has a small pool caused by endogenous synthesis, which accounts for the fact that steady-state {3H}DAP incorporation in the lysA strain still shows a lag period.

Mol Cell Biol, 1985 Oct, 5(10), 2662 - 8
Induction of adenine salvage in mouse cell lines deficient in adenine phosphoribosyltransferase; Turker MS et al.; Adenine phosphoribosyltransferase (APRT) (EC 2.4.2.7) pseudorevertant cell lines were isolated under selective conditions requiring adenine salvage for survival; yet they were found to be deficient in measurable APRT activity and resistant to the purine analog 2'6'-diaminopurine (DAP) (M.S . Turker, J . A . Tischfield, P . Rabinovitch, P.J . Stambrook, J.J . Trill, A.C . Smith, C.E . Ogburn, and G.M . Martin, manuscript in preparation) . Adenine salvage was examined in two APRT pseudorevertant cell lines, their two APRT homozygous deficient parental cell lines, and a genotypic APRT revertant cell line (i.e., one with measurable APRT activity and DAP sensitivity) . Adenine accumulation was observed in both revertant phenotypes and was demonstrated by high-performance liquid chromatography to be linked with adenine metabolism . The ability to salvage adenine declined substantially in the pseudorevertant cell lines when they were removed from selective media containing inhibitors of de novo 5'-AMP synthesis (alanosine and azaserine); for one pseudorevertant cell line this decline was accelerated by the addition of DAP to the medium . The readdition of alanosine or azaserine to the growth medium of the pseudorevertant lines induced adenine salvage to its previous levels . An APRT-like cross-reacting material was found in the pseudorevertant cell lines, although its relationship to adenine salvage is unknown . A low level of constitutive adenine salvage was found in the parental APRT-deficient lines, and it was also possible to induce adenine salvage in these cell lines . These findings suggest a novel regulatory mechanism for adenine salvage.

J Appl Bacteriol, 1985 Oct, 59(4), 353 - 5
A note on the use of metal species in microbiological tests involving growth media; Bird NP et al.; The feasibility of using traditional growth media for biological testing of metal species, for example as potential microbiocides, was investigated . Significant interactions between both of the representative metal species studied, Cu2+ and FeEDTA, and the test media were found . It is recommended that the use of growth media for tests on metal species should be avoided.

EMBO J, 1985 Oct, 4(10), 2627 - 33
Identification of proteins involved in the regulation of yeast iso- 1-cytochrome C expression by oxygen; Arcangioli B et al.; On the basis of a gel electrophoresis retardation assay, protein(s) which interact specifically with the upstream activating site (UASc) of the yeast iso-1-cytochrome C (CYC1) gene were identified and separated by heparin ultrogel chromatography . DNase I protection experiments indicate that these factors protect a 23-bp sequence overlapping the UASc site previously defined . The specific binding activity is strongly reduced in extracts prepared from a wild-type strain grown anaerobically . It is absent in a mutant strain blocked in the biosynthesis of heme but it is restored upon the addition of the missing precursor, delta amino levulinic acid (dALA) to the growth medium . In contrast, the binding activity does not differ significantly in extracts form a wild-type strain grown in either glucose or glycerol as carbon source . These data strongly argue that the CYC1 UAS binding protein(s) that we have identified mediate the oxygen and heme control of cytochrome C biosynthesis.

Proc Natl Acad Sci U S A, 1985 Oct, 82(20), 6750 - 4
Methionine-sensitive glycolysis in transformed cells; Boerner P et al.; Glycolysis in several tumor cell lines grown in tissue culture was inhibited by methionine . Kirsten murine sarcoma virus-transformed rat kidney cells (K-NRK) were inhibited 60-75% by 10 mM methionine, whereas normal rat kidney (NRK-49F) cells showed little or no inhibition . Inhibition of glycolysis in K-NRK cells was manifest 2-4 hr after exposure to the amino acid . Glycolysis in a chemically transformed cell line of Madin-Darby canine kidney cells was also sensitive to methionine, but maximal inhibition (75%) required 18-24 hr of incubation with the amino acid . Under the same conditions glycolysis in the nontransformed canine cells was less than 20% inhibited by methionine . In Ehrlich ascites tumor cells grown in tissue culture, 10 mM methionine inhibited glycolysis by about 50% . Inhibition of glycolysis, even by 50 mM methionine, was rapidly reversible . Within 2 hr after removal of methionine the rate of glycolytic activity was restored to that observed in control cells . Furthermore, inhibition by methionine required a minimum level (7%) of serum in the growth medium and inhibition was not sensitive to cycloheximide . Only amino acids that are transported by system A (including the nonmetabolized analogue methylaminoisobutyric acid) specifically inhibited glycolysis in tumor cells . The only exception was phenylalanine, which was toxic to both transformed and normal cell lines.

Exp Eye Res, 1985 Oct, 41(4), 487 - 95
Effect of hydrocortisone on corneal endothelial cells in vitro; Samples JR et al.; The effect of hydrocortisone on the growth potential of human and baboon corneal endothelial cells in tissue culture was evaluated . Hydrocortisone-21-sodium succinate was incorporated into growth medium at 10(-3) to 10(-7) M concentrations in a standard tissue culture medium . Inclusion of hydrocortisone into growth medium at higher concentrations (e.g . 10(-4) and 10(-3) M) resulted in a significantly reduced cell growth and a decreased level of DNA synthesis . Higher concentrations of hydrocortisone induced morphological alterations in the corneal endothelial cells in vitro . The incorporation of 5% dextran into the growth medium failed to alter the negative hydrocortisone effects . These experiments suggest the potential adverse effects of hydrocortisone for the corneal endothelium in vitro when used at higher concentrations.

Atherosclerosis, 1985 Sep, 56(3), 345 - 58
Accumulation and mobilization of cholesteryl esters in cultured human fibroblasts exposed to free cholesterol-rich phospholipid vesicles; Lundberg B et al.; The uptake of free cholesterol (FC) into cells by surface transfer and its esterification to cholesteryl esters (CE) has been studied with a system of FC-egg phosphatidylcholine (EPC) vesicles and human lung fibroblasts in serum-free growth medium . The influx of FC was dependent on the molar ratio of FC to EPC in the donor vesicles . The FC incorporated by surface transfer was available for esterification in the cells . Incubations with FC/EPC vesicles with a FC/EPC molar ratio of 0.5: 1 gave a small net decrease in cellular CE, while 1:1 vesicles gave a mild increase . When the cells were incubated with 2:1 FC/EPC vesicles an extensive accumulation of CE was demonstrated, which was accentuated if albumin was present in the medium . The CE accumulated in the form of lipid droplets within the cells . The largest of these droplets exhibited positive birefringence with formee crosses, that is typical for liquid crystals of cholesteryl esters . If cells loaded with CE were incubated with vesicles with low FC/EPC ratios a net efflux of CE was noted . The present study demonstrates that the uptake of FC from lipid vesicles by surface transfer can reproduce typical features of foam cells in early atherosclerosis.

Infection, 1985 Sep-Oct, 13(5), 235 - 9
Relationship between the bactericidal and bacteriolytic activity of cephalosporins and changes in the cell volumes of Escherichia coli cultures; Schulz E et al.; The bactericidal effect of cefoxitin and cefotaxime in relation to concentration and exposure time, as demonstrated by the killing curve diagrams of Escherichia coli cultures, was compared with the degree of bacteriolysis and the cell volume increase measured by the coulter counter-channel analyser system . Human plasma ultrafiltrate was used as the growth medium . Cefoxitin has a higher bactericidal activity than cefotaxime . With increasing concentrations the bactericidal efficacy of cefoxitin increases more rapidly in the lower range of concentrations (2-10 mg/l) than in the higher range (10-40 mg/l) . In contrast, the bactericidal effect of cefotaxime in the range 0.06-1.2 mg/l is virtually constant and can only be increased by high levels (10-40 mg/l) . The morphometric effect of cefoxitin on E . coli cultures, as demonstrated by volume distribution curves, is characterized by intensive and rapidly appearing bacteriolysis 20 min after exposure to the antibiotic without a preceding increase in bacterial cell volume . Higher concentrations result in an earlier onset of bacteriolysis . In contrast, the application of cefotaxime reveals a massive increase in bacterial cell volume (more than five-fold) with a delayed (greater than 2 h) onset of bacteriolysis . High cefotaxime concentrations reduce the extent of bacterial cell volume increase, associated with an earlier and more intensive onset of bacteriolysis . With both cephalosporins, the bacterial cell alterations are particularly dependent on the exposure time . There is evidently a close correlation between bactericidal and bacteriolytic activity . This is valid both for the two cephalosporins and generally for the concentration-activity relationships.

Diabetologia, 1985 Sep, 28(9), 641 - 4
Influence of glucose, insulin and sera from diabetic patients on the prostacyclin synthesis in vitro in cultured human endothelial cells; Aanderud S et al.; The effects of glucose, insulin and sera from Type 1 (insulin-dependent) diabetic patients on the synthesis of prostacyclin in vitro were studied in confluent primary cultures of human endothelial cells . The stable metabolite, 6-keto-prostaglandin F1 alpha, was measured in growth medium after 24 h of incubation with endothelial cells in a buffer incubated with the cells for 10 min on a rocker platform, and in a buffer solution of ruptured cells . Glucose (11, 15, 20 or 25 mmol/l) and glucose (11 mmol/l) plus insulin (10(3), 10(4) or 10(6) mU/l) in growth medium did not have any effects on the prostacyclin synthesis . The prostacyclin synthesis was significantly reduced in cell cultures incubated with medium supplemented with 10% serum from patients with Type 1 diabetes (p less than 0.01) compared with cultures incubated with pooled serum from healthy blood donors . These data suggest that diabetic sera inhibit the prostacyclin synthesis in cultured endothelial cells unrelated to the glucose and insulin levels.

J Clin Pathol, 1985 Sep, 38(9), 1052 - 4
Improved method for isolation and growth of Chlamydia trachomatis in McCoy cells treated with cycloheximide using polyethylene glycol; Mohammed NR et al.; The sensitivity of non-replicating McCoy cells pretreated with polyethylene glycol for the isolation of Chlamydia trachomatis from clinical specimens and for the growth of a laboratory strain was compared with the sensitivity of untreated non-replicating cell cultures . The concentration of polyethylene glycol in different solutions and the time of addition to the cell culture medium were critical . A concentration of 35% polyethylene glycol in barbitone added to the cell culture growth medium either immediately before or immediately after infection with chlamydia increased the number of inclusions detected . The rate of isolation obtained from clinical specimens was also increased when cell cultures treated with polyethylene glycol were used.

Mutat Res, 1985 Sep, 146(2), 177 - 83
A mechanism for rich-medium inhibition of the repair of daughter-strand gaps in the deoxyribonucleic acid of UV-irradiated Escherichia coli K12 uvrA; Sharma RC et al.; Ultraviolet-irradiated Escherichia coli K12 uvrA(B,C) cells show higher survival if plated on minimal growth medium (MM) rather than on rich growth medium (RM) . This phenomenon has been referred to as 'minimal medium recovery' (MMR) . UV-irradiated (4 J/m2) uvrA cells showed a similar rate of protein synthesis, whether incubated in MM or RM, however, they showed a severe depression in DNA synthesis when incubated in MM that lasted for about 30 min, and the normal rate of DNA synthesis was not reestablished until about 60 min after irradiation . When a sample of these same cells was switched to RM immediately after UV-irradiation, there was only a slight slowing of DNA synthesis, and the normal rate of synthesis was reestablished by 60 min . An additional mmrA mutation or growth retardation by valine blocked both this extra DNA synthesis in RM, and the inhibitory effect of RM on survival . These findings suggest that the absence of a marked delay in DNA synthesis observed in RM may be responsible for the inhibitory effect of RM on the survival of UV-irradiated excision-deficient cells . Two hypotheses, which are not mutually exclusive, are proposed and supported by data to explain why a fast rate of DNA synthesis after UV-irradiation partially inhibits postreplication repair and enhances cell lethality.

J Bacteriol, 1985 Sep, 163(3), 1120 - 5
Ferric iron reductase of Rhodopseudomonas sphaeroides; Moody MD et al.; Ferric iron reductase activity was examined in the facultative photosynthetic bacterium Rhodopseudomonas sphaeroides . The specific activities of extracts from cells grown under phototrophic and aerobic conditions were similar and not affected by the concentration of iron in the growth media . The activity was resolved by ion-exchange column chromatography into two fractions, designated iron reductase A and iron reductase B, with molecular weights of 41,000 and 32,000, respectively . Both of these soluble cytoplasmic enzymes required the presence of flavin mononucleotide for activity and utilized NADH to reduce iron supplied as ferric citrate . Iron reductase B was responsible for the majority of activity in crude extracts and was purified 556-fold by conventional protein purification techniques . The apparent Km values of iron reductase B for NADH, Fe3+, and flavin mononucleotide were determined to be 18.2, 8.3, and 3.2 microM, respectively.

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