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 pro