Growth media

J Assist Reprod Genet, 1993 Apr, 10(3), 192 - 6
Effects of medium composition on murine and human blastocyst formation and hatching rate; Olar TT et al.; PURPOSE: Murine two-cell embryos (n = 5573) were cultured for 96 hr in human tubal fluid (HTF) medium (n = 2709) or alpha modification of minimum essential medium (MEM; n = 2864) through the hatched blastocyst stage from mid-1990 to mid-1991 . An additional 373 embryos were cultured in MEM or HTF with 0, 1, 5, or 10 ng/ml E . coli endotoxin . A total of 17 patients had supernumerary embryos simultaneously cultured in HTF (n = 48) or MEM (n = 61) . Additionally, pregnancy rates were compared for July to December 1990, when MEM was used as growth medium, and for July to December 1989, when HTF was used . RESULTS: Blastocyst formation was higher (P < 0.001) for murine embryos cultured in MEM (blasts = 95%) compared to HTF (blasts = 70%) . When cultured with endotoxin, blastocyst formation was higher (P < 0.01) for embryos cultured in MEM compared with HTF for controls and at each endotoxin level . No difference in human blastocyst development was observed in HTF and MEM . However, more MEM-cultured blastocysts were cryopreserved (P < 0.05) . There also was a lower spontaneous abortion rate and a higher multiple gestation rate when embryos were cultured in MEM . CONCLUSION: Thus, MEM may result in healthier blastocyst development, especially when culture conditions are substandard, although this is not an acceptable substitution for meticulous technique.

Mol Microbiol, 1993 Apr, 8(2), 205 - 10
Trehalose metabolism in Escherichia coli: stress protection and stress regulation of gene expression; Strom AR et al.; Endogenously synthesized trehalose is a stress protectant in Escherichia coli . Externally supplied trehalose does not serve as a stress protectant, but it can be utilized as the sole source of carbon and energy . Mutants defective in trehalose synthesis display an impaired osmotic tolerance in minimal growth media without glycine betaine, and an impaired stationary-phase-induced heat tolerance . Mechanisms for stress protection by trehalose are discussed . The genes for trehalose-6-phosphate synthase (otsA) and anabolic trehalose-6-phosphate phosphatase (otsB) constitute an operon . Their expression is induced both by osmotic stress and by growth into the stationary phase and depend on the sigma factor encoded by rpoS (katF) . rpoS is amber-mutated in E . coli K-12 and its DNA sequence varies among K-12 strains . For trehalose catabolism under osmotic stress E . coli depends on the osmotically inducible periplasmic trehalase (TreA) . In the absence of osmotic stress, trehalose induces the formation of an enzyme IITre (TreB) of the group translocation system, a catabolic trehalose-6-phosphate phosphatase (TreE), and an amylotrehalase (TreC) which converts trehalose to free glucose and a glucose polymer.

J Neurosci Res, 1993 Apr 1, 34(5), 546 - 61
Manganese induces spreading and process outgrowth in rat pheochromocytoma (PC12) cells; Lin WH et al.; Mn2+ has been shown to promote cell-substrate adhesion and cell spreading in many cell culture systems . In this study, we present data demonstrating that Mn2+ not only promotes spreading, but also induces process outgrowth in rat pheochromocytoma (PC12) cells . In the presence of 1.0 mM MnCl2, cell spreading was apparent by 6 hr, and nearly 50% of the exposed cells extended neurite-like processes . These morphological effects of Mn2+ were both time- and dose-dependent . In the presence of cycloheximide, a protein synthesis inhibitor, both Mn(2+)-induced spreading and neurite outgrowth were prevented, indicating that de novo protein synthesis is required for the effects of Mn2+ to take place . Of the other divalent cations tested, Mg2+, Cd2+, Cu2+, Ni2+, and Zn2+ were ineffective, and only Co2+ partially mimicked the effects of Mn2+ . Although Mn(2+)-induced cell adhesion and spreading have been extensively studied, this is the first report that this divalent cation can cause neurite outgrowth . The neurite outgrowth-promoting effects of Mn2+ were distinct from those of nerve growth factor in that the response to Mn2+ was considerably more rapid, but apparently lacked the ability to sustain continuous outgrowth and networking of neurites . Mn2+ also induced the levels of GAP-43 and peripherin, two proteins associated with neuronal differentiation of PC-12 cells . In cells grown in serum-free defined medium, Mn2+ was capable of promoting neurite outgrowth when the cells were plated on surfaces pretreated with normal growth medium, vitronectin, or fibronectin, while it failed to cause these morphological changes in cells plated on untreated or poly-D-lysine-coated substrata . Similarly, Mn2+ also promoted neurite outgrowth from rat sympathetic neurons attached to laminin-treated substrate, but had no effect on neurons maintained on substrate with polylysine only . The pentapeptide Gly-Arg-Gly-Asp-Ser nearly completely prevented the morphological effects of Mn2+ on PC12 cells . These findings are consistent with a hypothesis that Mn(2+)-mediated alteration of an RGD-dependent extracellular matrix-integrin interaction is responsible for the neuritogenic effects.

Appl Microbiol Biotechnol, 1993 Apr, 39(1), 42 - 7
Induction of ajmalicine formation and related enzyme activities in Catharanthus roseus cells: effect of inoculum density; Moreno PR et al.; In Catharanthus roseus cell cultures the time courses of four enzyme activities, tryptophan decarboxylase (TDC), strictosidine synthase (SSS), geraniol-10-hydroxylase (G10H) and anthranilate synthase (AS), and alkaloid accumulation were compared under two different culture conditions (low-inoculum density and high-inoculum density on induction medium) and a control on growth medium . In growth medium a transient increase in TDC activity was first observed after which G10H reached its maximum activity; only tryptamine accumulated, no ajmalicine could be detected . Apparently, a concerted induction of enzyme activities is required for ajmalicine formation . Cells inoculated in induction medium showed such a concerted induction of AS, TDC and G10H activities . After 30 days the low-density culture had accumulated six times more ajmalicine (in mumoles/g) than the high-density culture . Thus, increase in biomass concentration (high-density cultures) did not enhance the total alkaloid production . The major differences observed in enzyme levels between high- and low-density cultures were in the AS and TDC activities, which were two to three times higher in the low-density culture, indicating that there is a positive correlation between ajmalicine formation and AS and TDC activities.

Placenta, 1993 Mar-Apr, 14(2), 187 - 201
Colchicine inhibits human trophoblast differentiation in vitro; Douglas GC et al.; When cultured in keratinocyte growth medium, mononuclear cytotrophoblast cells aggregate into multicellular colonies which then fuse to form multinucleated syncytiotrophoblast . In an attempt to characterize better the mechanism of human trophoblast differentiation and to obtain information about the role of the cytoskeleton, experiments were performed using cytoskeletal-disrupting drugs and primary cultures of cytotrophoblast cells from term placentae . Addition of colchicine 6 h after plating permitted aggregation but the cells did not form syncytiotrophoblast, as revealed by staining for desmosomes and nuclei . Staining with an anti-tubulin antibody showed that microtubules were present in untreated control cells but absent in colchicine-treated cultures . If colchicine was added 24 h after plating, the cells also failed to differentiate . When cells were exposed to colchicine for the first 24 h after plating and then cultured in the absence of the drug, differentiation proceeded normally . Cells exposed to colchicine for 48 h and then incubated in the absence of the drug failed to form syncytiotrophoblast . The results suggest that a decision to differentiate is made between 24 and 48 h after plating . The effects of colchicine were observed between 2.5 and 250 microM . Beta-lumicolchicine blocked differentiation at 250 microM but was ineffective at lower concentrations . Colchicine also inhibited HCG secretion in a dose-dependent manner; beta-lumicolchicine only caused inhibition at 250 microM . Staining with antitubulin antibody revealed that lumicolchicine-treated cells had intact microtubules . These results suggest a role for microtubules in trophoblast differentiation.

Indian J Exp Biol, 1993 Mar, 31(3), 224 - 30
Modification of radiation damage in transformed mammalian cells by 5-bromo-2-deoxy-uridine and 2-deoxy-D-glucose; Kalia VK et al.; The effects of 2-deoxy-D-glucose (2-DG) and 5-bromo-2-deoxy-uridine (BrdU) on gamma ray (60Co) induced damage were studied in monolayer cultures of transformed mammalian (BHK-21) cells . Micronuclei formation and changes in DNA content dispersion were used as indices of cytogenetic damage . Exposure of cells to BrdU (0.8 microM) for nearly two cell cycles before irradiation significantly increased micronuclei formation in exponentially growing cells . Incubation of irradiated cells under suboptimal growth conditions (in HBSS) for 4 hr, instead of growth medium, decreased the manifestation of damage . However, post-irradiation presence of 2-DG (5 mM, equimolar with glucose; 4 hr) in growth medium or HBSS significantly increased radiation damage . The effects of 2-DG treatment following irradiation in plateau phase were quantitatively less . These results suggest that: (i) radiation induced DNA lesions leading to micronuclei formation in BrdU incorporated cells are partly repairable; (ii) 2-DG could increase radiation induced cytogenetic damage in transformed mammalian cells, possibly by inhibiting the cellular repair processes; and (iii) combination of 2-DG treatment may decrease the BrdU doses required for radiosensitization of proliferating tumour cell populations.

Prikl Biokhim Mikrobiol, 1993 Mar-Apr, 29(2), 280 - 5
{The action of exogenous RNAse on the cells of lower eukaryotes}; Il'inskaia ON et al.; We studied the mechanism of growth-stimulating effect of various compounds using an exogenous RNase-micromycete model and found that the treatment with microdoses of RNase intensified the reproduction of the yeast Candida valida and growth of Aspergillus fumigatus . At the same time the respiration activity of yeast cells and fungal mitochondria as well as succinate dehydrogenase activity increased . The secretion of total protein, cellulase and cellobiase changed in the presence of RNase in the growth medium . Electrophoretic mobility of Candida valida cells enhanced by 20% . The RNase stimulating effect on microbial cells seems to result from changes in characteristics of the cell surface.

Eur J Haematol, 1993 Mar, 50(3), 127 - 32
Misincorporation of uracil into the DNA of folate- and B12-deficient HL60 cells; Wickramasinghe SN et al.; HL60 cells were cultured for 10 days under various experimental conditions . They were then incubated with 1 mumol/l {5-3H} uridine for 2 hours and their DNA extracted . The DNA was hydrolysed to deoxyribonucleosides with phosphodiesterase and alkaline phosphatase and the hydrolysate subjected to Aminex A6 chromatography . The elution profiles showed that, when compared with control cells, DNA from cells grown in medium deficient in folate, B12 or both folate and B12 contained increased amounts of deoxyuridine (dU) and increased radioactivity in the dU peak . The data demonstrate that misincorporation of uracil into DNA occurs in a myeloid cell line cultured in growth medium deficient in folate, B12 or both folate and B12.

J Bacteriol, 1993 Mar, 175(6), 1823 - 30
Bioenergetics of sulfur reduction in the hyperthermophilic archaeon Pyrococcus furiosus; Schicho RN et al.; The bioenergetic role of the reduction of elemental sulfur (S0) in the hyperthermophilic archaeon (formerly archaebacterium) Pyrococcus furiosus was investigated with chemostat cultures with maltose as the limiting carbon source . The maximal yield coefficient was 99.8 g (dry weight) of cells (cdw) per mol of maltose in the presence of S0 but only 51.3 g (cdw) per mol of maltose if S0 was omitted . However, the corresponding maintenance coefficients were not found to be significantly different . The primary fermentation products detected were H2, CO2, and acetate, together with H2S, when S0 was also added to the growth medium . If H2S was summed with H2 to represent total reducing equivalents released during fermentation, the presence of S0 had no significant effect on the pattern of fermentation products . In addition, the presence of S0 did not significantly affect the specific activities in cell extracts of hydrogenase, sulfur reductase, alpha-glucosidase, or protease . These results suggest either that S0 reduction is an energy-conserving reaction, i.e., S0 respiration, or that S0 has a stimulatory effect on or helps overcome a process that is yield limiting . A modification of the Entner-Doudoroff glycolytic pathway has been proposed as the primary route of glucose catabolism in P . furiosus (S . Mukund and M . W . W . Adams, J . Biol . Chem . 266:14208-14216, 1991) . Operation of this pathway should yield 4 mol of ATP per mol of maltose oxidized, from which one can calculate a value of 12.9 g (cdw) per mol of ATP for non-S0 growth . Comparison of this value to the yield data for growth in the presence of S0 reduction is equivalent to an ATP yield of 0.5 mol of ATP per mol of S0 reduced . Possible mechanism to account for this apparent energy conservation are discussed.

Arterioscler Thromb, 1993 Mar, 13(3), 360 - 6
A new anti-inflammatory leucine derivative, NPC-15669, inhibits growth of cultured human aortic smooth muscle cells; Bennett RL et al.; We have observed that NPC-15669, a leucine derivative with anti-inflammatory activity, reduced the proliferation of human aortic smooth muscle cells (HASMCs) in culture . We used a colorimetric assay and tritiated thymidine to measure the cell density and proliferation of HASMC cultures treated with this agent . We also studied the effect of NPC-15669 on the proliferation and migration of human aortic endothelial cells (HAECs) . Subconfluent HASMC cultures were growth arrested for 2 days . On the third day, growth was stimulated with either growth media (medium M199 containing 10% fetal bovine serum {FBS}), human platelet-derived growth factor (hPDGF), or fibroblast growth factor (FGF) in the absence or presence of NPC-15669 (0.1-50 microM) . Regardless of the stimulating agent for HASMCs (FBS, hPDGF, or FGF), NPC-15669 at concentrations of 10-25 microM caused a significant reduction in thymidine incorporation (36.7% and 77.2% in 10 microM and 25 microM, respectively; p < 0.005) and cell density (25-87%, p < 0.001) compared with control . NPC-15669 did not, however, have an effect on the rate of proliferation or migration of HAECs, even at concentrations up to 50 microM . Two other anti-inflammatory agents, aspirin and dexamethasone, caused substantially and significantly less inhibition, even at high concentrations (50 and 25 microM, respectively) . This study demonstrates that in vitro, NPC-15669 significantly inhibits HASMC proliferation but has no effect on proliferation or migration of HAECs.

FEBS Lett, 1993 Mar 1, 318(2), 186 - 8
Reduction in the amount of 8-hydroxy-2'-deoxyguanosine in the DNA of SV40-transformed human fibroblasts as compared with normal cells in culture; Barciszewski J et al.; DNA damage due to oxidative free radicals is considered to be a major cause of ageing and age-related diseases including cancer . Of more than 20 modifications formed in DNA by the action of hydroxyl radicals, 8-hydroxy-2'-deoxyguanosine (oh8dG) is potentially highly mutagenic and is known to occur most frequently . Using HPLC combined with electrochemical (HPLC/EC) detection of oh8dG, fivefold higher levels of oh8dG are detected in the DNA of cultured normal human skin fibroblasts as compared with SV40-transformed human fibroblasts MRC-5V2 . In comparison, the levels of oh8dG were similar in the growth medium of both types of cells . Applications of this method range from studies on the genomic stability and instability of normal and cancerous cells to the clinical and laboratory testing of toxic substances and drugs.

FEBS Lett, 1993 Mar 1, 318(2), 162 - 6
SC-0051, a 2-benzoyl-cyclohexane-1,3-dione bleaching herbicide, is a potent inhibitor of the enzyme p-hydroxyphenylpyruvate dioxygenase; Schulz A et al.; Growth inhibition of Lemna gibba plantlets by the bleaching herbicide, SC-0051 (2-(2-chloro-4-methanesulfonylbenzoyl)-1,3-cyclohexanedione)) was alleviated by the addition of homogentisic acid to the growth medium . Homogentisic acid is a key intermediate in the biosynthesis of tyrosine-derived plant quinones as well as in tyrosine metabolism . The herbicide prevented the incorporation of radioactivity from {14C}tyrosine into lipophilic plant metabolites and, in rat liver extracts, the herbicide inhibited the conversion of tyrosine to homogentisic acid . The enzyme p-hydroxyphenylpyruvate dioxygenase (EC 1.13.11.27) from both Zea mays seedlings and liver tissues, was found to be subject to strong inhibition by SC-0051 . Inhibition of plant quinone biosynthesis is a new mode of herbicidal action . One of the consequences of quinone depletion in plants by SC-0051 . Inhibition of plant quinone biosynthesis is a new mode of herbicidal action . One of the consequences of quinone depletion in plants in vivo is apparently an indirect inhibition of phytoene desaturation . The enzyme phytoene desaturase itself, however, is not afflicted by the herbicide.

J Immunoassay, 1993 Mar-Jun, 14(1-2), 1 - 19
A capture enzyme-linked immunosorbent assay for virus infectivity titrations as exemplified in an adenovirus system; Everitt E et al.; An enzyme-linked immunosorbent assay (ELISA), employing a capturing antihexon monoclonal antibody specifically recognizing free hexons, was developed for quantitative infectivity titration of adenovirus in a microscale titration assay . The method is based on the quantitative assessment of the total excess production of the major structural protein late in infection in samples consisting of 10(5) virus-infected HeLa cells maintained as stationary suspension cultures . Results are obtained with a coefficient of variation of 10% within 50 hours after virus infection . The method was designed for monitoring substances interfering with viral replication, e.g., neutralizing antibodies or antiviral drugs . Since it measured the total antigen content associated with cells as well as antigens possibly released into the growth medium the general approach should be applicable to any viral system where a structural protein is synthesized in excess.

Int J Artif Organs, 1993 Mar, 16(3), 164 - 71
Biomaterials in medicine--a bioengineering perspective; Courtney JM et al.; Biomaterials are considered with an emphasis on those used in artificial organs . Attention is drawn to the importance of the polymeric biomaterials and factors which affect their properties . Functions of membranes, sorbents, blood tubing, ventricular diaphragms and cell culture substrates are examined in order to obtain a summary of fundamental properties . Observations are made on the importance of blood compatibility assessment and its association with a biomaterial structure-property relationship . Blood-biomaterial interactions are discussed in terms of an overall relationship between the three components--blood, biomaterial and antithrombotic agent, with examples given of factors influencing each component . Cell-biomaterial interactions are examined in the areas of toxicity evaluation and the promotion of cell attachment and growth, where an overall relationship is described for the cell, growth medium and growth factors, and the biomaterial acting as a substrate.

Plant Physiol, 1993 Mar, 101(3), 773 - 9
An osmotic stress protein of cyanobacteria is immunologically related to plant dehydrins; Close TJ et al.; Dehydrins are a family of desiccation proteins that were identified originally in plants (T.J . Close, A.A . Kortt, P.M . Chandler {1989} Plant Mol Biol 13: 95-108; G . Galau, T.J . Close {1992} Plant Physiol 98: 1523-1525) . Dehydrins are characterized by the consensus amino acid sequence domain EKKGIMDKIKEKLPG found at or near the carboxy terminus; the core of this domain (KIKEKLPG) may be repeated from one to many times within the complete polypeptide . Dehydrins generally accumulate in plants in response to dehydration stress, regardless of whether the stimulus is evaporation, chilling, or a decrease in external osmotic potential . Polyclonal antibodies highly specific to the consensus carboxy terminus of plant dehydrins were used to search for dehydrins in cyanobacteria, many of which are known to survive desiccation . A 40-kD osmotic-stress-induced protein was identified in Anabaena sp . strain PCC 7120 . The 40-kD protein was usually not detected in logarithmic cultures and was induced by shifting the growth medium to higher solute concentrations . Several solutes have inductive effects, including sucrose, sorbitol, and polyethylene glycol (PEG) . Measurements of osmotic potential suggest that a shift of -0.5 MPa (sucrose and PEG) or -1.2 MPa (sorbitol) is sufficient to induce synthesis of the 40-kD protein . Glycerol, which is highly permeable, was not an inducer at -1.2 MPa (0.5 M), nor was the plant hormone abscisic acid . Induction appears to be evoked by a shift in osmotic potential approximately equal in absolute magnitude to the expected turgor pressure of bacterial cells in logarithmic phase growth . A dehydrin-like polypeptide was also identified among osmotically induced proteins from two other filamentous, heterocyst-forming cyano-bacteria . A 40-kD protein was observed in Calothrix sp . strain PCC 7601, and in Nostoc sp . strain Mac-R2, an osmotic-induced doublet at 39 and 40 kD was observed . From these data, it appears that cyanobacteria produce a dehydrin-like protein under osmotic stress.

J Neuroimmunol, 1993 Mar, 43(1-2), 69 - 78
Interleukin-3 and encephalitogenic activity of SJL/J myelin basic protein-specific T cell lines; Zhao ML et al.; Myelin basic protein (MBP)-specific SJL/J T cells were cultured in normal growth medium or growth medium supplemented with 10% culture supernatant from WEHI-3 cells, a source of interleukin-3 (IL-3), or with recombinant IL-3 . T cell lines cultured with IL-3 supplementation were more encephalitogenic compared to parallel lines cultured without this supplement . There was little difference in antigen-specific proliferative response or expression of cell surface markers CD3, CD4, CD8, IL-2R, or alpha/beta TCR in the parallel lines . Supernatant fluids from antigen-stimulated T cells from each cycle were tested for the presence of IL-2, IL-3, IL-4, granulocyte macrophage colony-stimulating factor (GM-CSF), tumor necrosis factor-alpha (TNF alpha/beta) and transforming growth factor-beta (TGF beta) . No significant difference in IL-2, IL-4, GM-CSF, TNF alpha/beta, or TGF beta levels were seen when supplemented and unsupplemented cultures were compared . Supernatant culture fluids contained an activity that was highly stimulatory for the IL-3-dependent mouse mast cell line, MC/9 . This activity was attributable to a combination of at least three factors that varied in relative concentrations throughout the course of the experiments . Based on neutralization by monoclonal antibodies, MC/9 stimulating activity in early passage lines was attributable entirely to IL-3 and GM-CSF . The fraction of the MC/9 stimulatory activity that could be neutralized by monoclonal antibody to IL-3 decreased with increasing stimulation cycle while the fraction neutralized by anti-GM-CSF antibodies remained relatively constant . At the time that the lines lost encephalitogenicity, the activity neutralizable by anti-IL-3 had dropped to low levels in the culture supernatants; however, MC/9 stimulatory activity remained present in the supernatants . This was due to GM-CSF and a third unidentified factor.

Mutat Res, 1993 Mar, 299(1), 9 - 18
Cytotoxic and genotoxic properties of tert.-butyl-p-quinone (TBQ) in an in vitro assay system with Chinese hamster V79 cells and in strain D7 of Saccharomyces cerevisiae; Rogers CG et al.; tert.-Butyl-p-quinone (TBQ), a major metabolite of the phenolic antioxidant tert.-butyl-4-hydroxyanisole (BHA), was examined for cytotoxic and genotoxic properties in an in vitro assay system with Chinese hamster V79 cells and in diploid strain D7 of Saccharomyces cerevisiae . TBQ was prepared from BHA by oxidative demethylation with sodium nitrite at low pH . Spectroscopic analyses identified the crystalline reaction product as TBQ with a purity close to 100% . Cytotoxicity of TBQ was determined by cloning efficiency in the absence of hepatocyte activation . TBQ reduced colony size of V79 cells at 0.4 micrograms/ml, prevented growth of 50% of the cells at 0.6 micrograms/ml, and was lethal to 100% of the cells at concentrations above 1.0 micrograms/ml . TBQ was 6-7 times more cytotoxic to V79 cells than TBHQ, a related BHA metabolite, and 100 times more cytotoxic than BHA . At dose levels of 0.2, 0.4 and 0.6 micrograms/ml of medium, TBQ did not increase significantly the frequency of sister-chromatid exchanges (SCE) in V79 cells and did not consistently increase the frequency of mutation to thioguanine resistance (TGR) at the hgprt gene locus either alone or with activation by rat hepatocytes . Incubation with TBQ for 4 h at pH 3.6 without activation resulted in only small increases in the frequency of gene conversion and reverse mutation in strain D7 of Saccharomyces cerevisiae . However, exposure to TBQ alone in growth medium for 24 h, produced inconsistent results . From these studies it was concluded that TBQ was cytotoxic but not genotoxic to V79 cells; and may be weakly genotoxic to strain D7 of S . cerevisiae.

Biochim Biophys Acta, 1993 Feb 20, 1172(1-2), 175 - 80
Molecular cloning and expression of rabbit pancreatic cholesterol esterase; Colwell NS et al.; Rabbit pancreatic cholesterol esterase (CEase, carboxyl ester lipase, EC 3.1.1.3) has been cloned from a lambda gt11 library of adult rabbit pancreatic cDNA . The open reading frame consists of 1788 nucleotides which encodes 576 amino acids of the functional protein and a 20 amino acid leader peptide . When compared to other species, the greatest homology is observed between residues 82-248 with little or no homology at the C-terminal end where proline-glutamate-serine-threonine (PEST) segments are a characteristic feature of the human CEase . Rabbit CEase (RCEase) retains the active-site serine (gxsxg), the active-site histidine and the tentative heparin binding site (KKRCLQ) at similar positions in comparison to pancreatic CEases of other species . When rabbit CEase cDNA is expressed in monkey kidney (COS-7) cells, enzymatic hydrolytic activity is detected in the growth medium as is a 67 kDa protein by Western blotting with polyclonal anti-CEase antibody . Northern blot analysis shows two mRNA (2.2 and 3.2 kb) species.

Biochim Biophys Acta, 1993 Feb 17, 1175(3), 277 - 82
Increased accumulation of drugs in a multidrug resistant cell line by alteration of membrane biophysical properties; Callaghan R et al.; Growth of CHRC5 multidrug resistant cells in media enriched in a saturated C-17 fatty acid, heptadecanoic acid, resulted in these cells accumulating vinblastine at a rate and to an extent comparable to that of the parental cell line AB1 . The fatty acid-enriched growth media had no effect on the ability of AB1 cells to take up vinblastine . The action of amphiphiles on the uptake of rhodamine dyes by CHRC5 cells was compared with the increased dye accumulation affected by verapamil . Membrane rigidifying agents, such as the saturated fatty acid stearic acid, or the cholesterol derivatives, cholesteryl hemisuccinate and cholesteryl phosphorylcholine, as well as a membrane fluidizing unsaturated fatty acid, linoleic acid, could significantly increase dye uptake, although not as well as verapamil . These results taken in conjunction with other reports in the literature, demonstrate that multidrug resistance is sensitive to alterations of membrane properties . They suggest that perturbation of the membrane to either increased or to decreased membrane fluidity can lower the level of resistance.

Biochim Biophys Acta, 1993 Feb 13, 1161(2-3), 249 - 56
The chloroperoxidase from the fungus Curvularia inaequalis; a novel vanadium enzyme; van Schijndel JW et al.; The presence of vanadium-containing bromoperoxidases in various types of seaweed is well-documented . We now report that the terrestrial fungus Curvularia inaequalis excretes a novel chloroperoxidase which also contains vanadium as a prosthetic group . The chloroperoxidase is excreted in the medium as the only protein and is, therefore, almost purely obtained . Atomic absorption spectroscopy measurements showed that the chloroperoxidase contained vanadium, which was essential for enzymatic activity, in a stoichiometry of 1 mol vanadium per mol of enzyme . When the fungus was grown in media containing low concentrations of vanadate (VO4(3-)) or when vanadate was absent, the enzyme was excreted in an apoform . Addition of vanadate to the apoenzyme purified from the medium, dialyzed holo-enzyme or growth medium led to incorporation of the metal and to a subsequent increase in specific activity from 0.7 to about 7.5 units/mg . The reduced enzyme showed an axially symmetric EPR spectrum (g(o) = 1.971, Ao = 91.7 x 10(-4) cm-1) with 16 hyperfine lines that is essentially the same as the EPR spectrum of the vanadium-containing bromoperoxidase of the seaweed Ascophyllum nodosum . This demonstrates that the active sites in the two enzymes are very similar . The chlorinating and brominating activities of the chloroperoxidase from C . inaequalis were also studied and compared to those of the vanadium bromoperoxidase from A . nodosum . The chlorinating reaction catalyzed by the chloroperoxidase had a pH optimum around 5.5 and the Km for Cl- was small (0.25 mM at pH 4.5), but the logarithm of its value increased linearly with increasing pH . At high bromide concentrations, the pH optima of chloroperoxidase and bromoperoxidase in the brominating reaction were about the same (5.5) . However, at low bromide concentrations the pH optimum of the chloroperoxidase was at higher pH values than that of the bromoperoxidase.

Cell, 1993 Feb 12, 72(3), 365 - 78
ZIP1 is a synaptonemal complex protein required for meiotic chromosome synapsis; Sym M et al.; ZIP1 is a novel meiosis-specific gene required for chromosome synapsis and cell cycle progression in S . cerevisiae . zip1 strains undergo homologous chromosome pairing, but are defective in synaptonemal complex (SC) formation . The zip1 mutation confers a uniform arrest in meiosis prior to the first division . zip1 strains display nearly wild-type levels of commitment to meiotic recombination; however, mature reciprocal recombinants are not formed until cells are released from meiotic arrest by return to growth medium . DNA sequence analysis of ZIP1 reveals structural homology to a number of proteins containing coiled coils . Immunofluorescence experiments using anti-ZIP1 antibodies demonstrate that the ZIP1 protein localizes to synapsed meiotic chromosomes but not to unsynapsed axial elements . Taken together, these data suggest that ZIP1 is a component of the central region of the SC . We propose a model in which ZIP1 acts as a molecular zipper to bring homologous chromosomes in close apposition.

J Surg Res, 1993 Feb, 54(2), 157 - 62
Comparison of techniques for harvesting enterocytes for transplantation; Nguyen BL et al.; Selective enterocyte transplantation is a potential alternative to small intestinal transplantation for treatment of the short bowel syndrome . Our aim was to compare chelation and enzymatic methods of isolating enterocytes with respect to initial cell yield and characteristics and in vitro growth . Enterocytes were harvested from adjacent ileal segments in 35 rabbits using chelation with EDTA and warm trypsinization . Determinations were made of initial viability by trypan blue exclusion, cell yield, and proportion of intact crypts . Cells (5 x 10(6)) were seeded in growth media into culture vessels . Cell attachment was estimated by measuring cells liberated by Dispase at 24 hr . In vitro growth was assessed at 14 and 28 days . Although total cell yield (15.0 +/- 9.9 vs 12.5 +/- 8.3 x 10(6) cells/cm) and intact crypts were similar, the trypsin technique resulted in cells with higher initial viability (86 +/- 7 vs 71 +/- 18%, P < 0.05) . Cell attachment (7 +/- 8 vs 8 +/- 4%) and enterocyte disaccharidase activity were similar using both techniques . While the number of epithelial cell growth foci was not significantly different at 14 days, there was significantly greater surface coverage on both plain (44 +/- 20% vs 1 +/- 0%, P < 0.05) and Matrigel-coated (80 +/- 14 vs 16 +/- 25%, P < .05) vessels at 28 days with trypsin-isolated cells . Trypsinization resulted in a cell population which had a higher percentage of viable cells but a similar proportion of intact crypts and differentiated cells compared to those resulting from the chelation technique . Trypsin-liberated cells had greater capacity for in vitro growth particularly on Matrigel-coated surfaces.(ABSTRACT TRUNCATED AT 250 WORDS)

J Dairy Sci, 1993 Feb, 76(2), 600 - 5
Effects of beta-carotene and alpha-tocopherol on rumen bacteria in the utilization of long-chain fatty acids and cellulose; Hino T et al.; Addition of safflower oil to a growth medium depressed the growth of mixed rumen bacteria above 200 mg/L and did not significantly increase bacteria, even at lower concentrations . However, when 10 mg/L of beta-carotene were added to 50 to 100 mg/L of safflower oil, bacterial growth was significantly increased . When more than 200 mg/L of safflower oil were present, beta-carotene markedly restored the growth capacity . alpha-Tocopherol was more effective than beta-carotene, although it inhibited growth at high concentrations . The combination of beta-carotene and alpha-tocopherol (each 5 mg/L) exerted partially additive effects . beta-Carotene plus alpha-tocopherol enhanced bacterial cell yield in the presence of safflower oil, caprate, stearate, or linoleate, suggesting that beta-carotene and alpha-tocopherol increase the utilization of fatty acids . beta-Carotene plus alpha-tocopherol also stimulated cellulose digestion in the presence of 100 mg/L of safflower oil, evidently through the increased growth of cellulolytic bacteria.

J Cell Biochem, 1993 Feb, 51(2), 165 - 74
4-Quinolones cause a selective loss of mitochondrial DNA from mouse L1210 leukemia cells; Lawrence JW et al.; The 4-quinolone antibiotics nalidixic acid and ciprofloxacin are potent inhibitors of the bacterial type II topoisomerase DNA gyrase . Treatment of mouse L1210 leukemia cells with these drugs resulted in a delayed inhibition of cell proliferation . Prior to inhibition of cell proliferation, there was a time-dependent decrease in the cellular content of mitochondrial DNA (mtDNA) . The decrease in mtDNA was associated with a decrease in the rate of mitochondrial respiration and an increase in the concentration of lactate in the growth medium . Inhibition of cell proliferation by 4-quinolones was reversible upon drug washout . However, there was a 2- to 4-day lag before the growth rate returned to normal levels . This was preceded by an increase in mtDNA content and mitochondrial respiration . These studies suggest that inhibition of mammalian cell proliferation by 4-quinolone drugs is related to the selective depletion of mtDNA.

Int J Cancer, 1993 Feb 1, 53(3), 499 - 505
Persistence of plasmin-mediated pro-urokinase activation on the surface of human monocytoid leukemia cells in vitro; Tapiovaara H et al.; Human leukemia cell lines, unlike those from adherent tumors, have been shown to continuously activate the pro-urokinase (pro-u-PA) they produce . In the present study we found that, in normal cell-culture conditions in 10% FCS the plasminogen activation cascade works continuously on monocytoid leukemia cells, which expressed plasmin activity and active u-PA on their cell surface . This plasmin catalyzed the conversion of the produced pro-u-PA to active 2-chain urokinase (tcu-PA), and was derived from bovine serum plasminogen by the activity of cell-bound tcu-PA . Plasmin generation was abolished and pro-u-PA accumulated in cell cultures that were grown for several days, either in the presence of serum thoroughly depleted of plasminogen, or in the presence of 1 mM tranexamic acid . Plasmin generated on the cell surface was found to be present in 2 enzymatically active fragments, of M(r) 85,000 and M(r) 50,000, which were slowly released into the growth medium . These fragments could activate pro-u-PA in serum-free growth medium . Most of the bound plasmin could be washed off cells with 10 mM tranexamic acid, but complete removal of plasmin from the cell surface required washing of the cells with acid-glycine pH 3.0.

Am J Clin Nutr, 1993 Feb, 57(2 Suppl), 313S - 314S
Selenium-dependent regulation of type I 5'-deiodinase expression; Oertel M et al.; Selenite concentration regulates activity and expression of the p27 substrate-binding subunit of type I 5'deiodinase (5'-D) and of a protein labeled with bromoacetylthyroxin (BrAcT4), or p30, with yet unknown function in a porcine-kidney epithelial cell line (LLC-PK1) cultured in serum-free medium . p27 is metabolically labeled by 75-selenite and affinity labeled by BrAc{125I}T4 . Compared with glutathione peroxidase, expression of the p27 5'D subunit (5'-DI) is observed at 10-fold-lower concentrations of selenium in the growth medium, suggesting an intracellular hierarchy of selenite utilization . Selenium deficiency retards cell growth and prevents 5'-DI expression and may thus impair thyroid hormone action in vivo.

Oncogene, 1993 Feb, 8(2), 503 - 7
Alternative mRNA forms and open reading frames of the max gene; Vastrik I et al.; The max gene encodes a heterodimeric partner of Myc . We have recently identified an alternative max mRNA (delta max) that contains an additional internal exon introducing an in-frame translational termination . Here we have studied the expression of human max mRNAs by Northern blotting analysis . In addition to the major 2.3-kb mRNA form, four bands were identified . Our results indicate that these bands represent differentially spliced mRNA forms, which contain altogether three open reading frames . In addition to the previously identified Max and delta Max proteins, sequence analysis of a 3.5-kb mRNA form predicted a protein that resembles delta Max in structure . Like delta Max, this protein enhanced the number of transformed foci in the ras-myc co-transformation assay . Although the 3.5-kb mRNA represents a minor form in actively proliferating cells, a shift from the major 2.3-kb mRNA to the 3.5-kb form was observed in response to high cell density or acidification of the growth medium . Our results indicate the presence of several differentially spliced mRNA forms of the max gene, and suggest a possible mechanism for the production of functionally distinct Max proteins.

Indian J Biochem Biophys, 1993 Feb, 30(1), 71 - 2
Further studies on the influence of aminophylline on lipids of Microsporum gypseum; Bindra A et al.; Aminophylline added to the growth medium of Microsporum gypseum for varying periods exhibited varied effects on lipid synthesis . A decreased incorporation of {14C}acetate into both lipids and phospholipids was initially observed which showed increase after 24 hr of incubation . Short-time exposure of aminophylline also resulted in decreased activity of glycerol-3-phosphate acyltransferase, the key enzyme of lipid synthesis, which, however, got stimulated on longer incubation, supporting the decreased/increased synthesis of lipids during short/long time exposure . Changes in the intracellular levels of cAMP at different time points account for the induced alterations in lipid synthesis, possibly due to formation of metabolites of aminophylline during growth of the organism.

J Cell Sci, 1993 Feb, 104 ( Pt 2), 307 - 15
Non-genotoxic hepatocarcinogenesis in vitro: the FaO hepatoma line responds to peroxisome proliferators and retains the ability to undergo apoptosis; Bayly AC et al.; A range of hepatoma cell lines (RH1, HTC, FaO, 7800C1 and MH1C1), has been studied with the aim of establishing an in vitro model to investigate the molecular mechanisms of hepatocarcinogenicity induced by the peroxisome proliferator class of non-genotoxic carcinogens . In view of speculation that peroxisome proliferators suppress hepatocyte apoptosis in vivo, we have placed particular emphasis on evaluating whether hepatoma cell lines retain the ability to undergo apoptotic cell death . Expression of the liver-specific differentiation marker albumin and the peroxisome proliferator-activated receptor (PPAR) was highest in the Reuber hepatoma cell line, FaO . This cell line also demonstrated the most marked response to the peroxisome proliferator nafenopin with a 2.2-fold induction of the microsomal enzyme cytochrome p450IVA1 . This response was found to display intercellular heterogeneity by immunocytochemistry . Thus, the FaO cell line maintained characteristics of hepatocytes, both in vivo and in vitro, in terms of expression of constitutive and inducible markers . However, none of the cell lines tested mirrored the hyperplastic response of hepatocytes to nafenopin, since no increase in cell growth kinetics was observed on addition of nafenopin to the growth medium . The mode of cell death in confluent FaO cultures was characterised as apoptosis, by fluorescence microscopy and agarose gel electrophoresis of extracted DNA . Cells detaching from confluent FaO cultures exhibited chromatin condensation and DNA fragmentation patterns characteristic of cels undergoing apoptotic death.Interestingly, no apoptosis was seen in monolayer cells, suggesting that apoptosis in vitro is associated with cell shrinkage and detachment similar to that documented for the liver in vivo.(ABSTRACT TRUNCATED AT 250 WORDS)

Arch Biochem Biophys, 1993 Feb 1, 300(2), 734 - 7
The expression of human relaxin in yeast; Yang S et al.; The chemically synthesized DNA-coding sequence for an artificial single chain human relaxin consisting of a B chain, an Arg-Arg-Glu-Phe-Lys-Arg-connecting peptide, followed by the A chain, was used to construct two plasmids which were introduced into Saccharomyces cerevisiae . Expression of the relaxin-coding sequence was under the control of either the yeast TDH3 promoter or the CUP1 promoter . The yeast alpha-factor signal sequence was used to direct the protein into the secretory path, and the appearance of human relaxin in the growth medium was confirmed by radioimmunoassay and immunoblotting . Partially purified human relaxin from yeast was biologically active in the mouse symphysis pubis assay and radioreceptor assay.

Int J Cancer, 1993 Feb 1, 53(3), 478 - 85
Distinct P-glycoprotein expression in two subclones simultaneously selected from a human colon carcinoma cell line by cis-diamminedichloroplatinum (II); Yang LY et al.; Two drug-resistant sublines, CP2.0 and RT, were simultaneously selected by cis-diamminedichloroplatinum (CDDP) from the human colon carcinoma cell line LoVo by the conventional method of continuous drug exposure . The 2 sublines differed in morphology, growth kinetics and pattern of gene expression . Genetic signature analysis indicated that the lines were independent subclones but that both arose from LoVo . These sublines were maintained in a growth medium containing 2.0 micrograms/ml CDDP . However, CP2.0 cells were 3 times more resistant to CDDP than were RT cells . Although both were cross-resistant to mustargen and 5-fluorouracil, only CP2.0 was resistant to Adriamycin and vincristine . Western-blot analysis, immunocytochemical staining and in vitro phosphorylation experiments indicated that the level of P-glycoprotein was significantly elevated in CP2.0 but not in RT . Despite the differences between these sublines, they possess similar CDDP-resistance mechanisms, including decreased intracellular CDDP accumulation, elevated levels of glutathione and metallothionein-like proteins, increased glutathione transferase-pi mRNA, and enhanced susceptibility to CDDP cytotoxicity after treatment with DL-buthionine-{S,R}-sulfoximine . Nevertheless, our results suggest that, in certain tumor types, P-glycoprotein-mediated multi-drug resistance and CDDP-resistance phenotypes can coexist in cells with primary resistance to CDDP.

In Vitro Cell Dev Biol, 1993 Feb, 29A(2), 127 - 34
A cobblestone cell isolated from the human omentum: the mesothelial cell; isolation, identification, and growth characteristics; Pronk A et al.; Normal human mesothelial cells (NHMC) were isolated from pieces of human omentum . The cell yield was approximately one million cells per square centimeter omentum . The mesothelial cells were identified by their positive staining with monoclonal antibodies against cytokeratins 6 and 18 . Transmission electronmicroscopy of cultured NHMC revealed many microvilli on the apical surface and many mitochondria and pinocytotic vesicles in the cytoplasm, indicating active transmembrane transport . Growth of NHMC was directly related to the concentration of human serum or of fetal bovine serum in the growth medium . Addition of epidermal growth factor with or without hydrocortisone resulted in a significant increase of NHMC growth; when endothelial cell growth factor, insulin, or hydrocortisone were added no such increase was observed . Seeding NHMC at densities less than 3000/cm2 did not result in monolayer formation . The mesothelial cells were serially passed in growth medium M199 with added 10% fetal bovine serum up to 7 passages . However, after Passage 4 the cells changed into giant cells with an irregular pattern, and a lack of intracellular cytokeratin expression was observed for most of the cells.

Carcinogenesis, 1993 Feb, 14(2), 285 - 9
Inactivation of a tumor suppressor function in immortal Syrian hamster cells by N-methyl-N'-nitro-N-nitrosoguanidine and by 5-aza-2'-deoxycytidine; West RW et al.; Clonal lines of immortal Syrian hamster cells were previously isolated that either suppressed (supB+) tumorigenicity in hybrids with a malignant hamster cell line (BP6T) or had lost this suppression ability (supB-) . Neither line was tumorigenic or showed anchorage-independent growth in normal growth medium . SupB- cells, but not supB+ cells, grew in agar supplemented with the growth factors EGF, PDGF and insulin (EPI), providing a selective assay for the supB- phenotype . After treatment of supB+ cells with either N-methyl-N'-nitro-N-nitrosoguanidine (10-300 ng/ml) or 5-aza-2'-deoxycytidine (25-250 ng/ml), and an expression period of 4-8 weeks, a dose-dependent increase in altered cells that grew in agar supplemented with EPI was observed . Cell lines derived from colonies in agar showed persistent EPI-stimulated growth in agar, and decreased suppression of growth in agar for hybrids with BP6T cells . Thus, carcinogen-induced loss of the tumor suppressor phenotype has been demonstrated.

Brain Res, 1993 Jan 29, 602(1), 41 - 4
Effect of ascorbate on Na(+)-independent and Na(+)-dependent uptake of {3H}norepinephrine by rat primary astrocyte cultures from neonatal rat cerebral cortex; Kimelberg HK et al.; We have previously reported that primary astrocyte cultures prepared from neonatal rat brains show Na(+)-dependent, tricyclic antidepressant-sensitive, high-affinity uptake of {3H}norepinephrine ({3H}NE) . Other workers, however, using primary astrocyte cultures from neonatal mice, have failed to find such uptake . This prompted us to examine possible reasons for the variability of the uptake in primary astrocyte cultures such as growth conditions and the effect of ascorbic acid . The presence of ascorbic acid increased the Na(+)-dependent uptake of NE by inhibiting the Na(+)-independent component . Na(+)-dependent uptake in rat cultures occurs when either fetal bovine or horse serum are present in the growth media, but not in a serum-free growth medium . Other workers have shown a species difference such that, even under optimal uptake conditions where rat astrocyte cultures exhibit Na(+)-dependent {3H}NE uptake, mouse astrocyte cultures do not.

FEBS Lett, 1993 Jan 25, 316(2), 181 - 5
Molecular weight-determination of biosynthetically modified monomeric and oligomeric muropeptides from Escherichia coli by plasma desorption-mass spectrometry; Caparros M et al.; The presence of certain D-amino acids in the growth media of Escherichia coli results in the accumulation of 2 major and 3-5 minor new muropeptides in the murein sacculus . Preliminary data suggested that the major muropeptides correspond to a monomer and a cross-linked dimer with one residue of D-amino acid per molecule . We have analyzed several D-amino acid-modified muropeptides by plasma desorption-mass spectrometry . Our results confirmed that the general structures of the major modified muropeptides are: GlucNAc-MurNAc-L-Ala-D-Glu-m-A2pm-D-X, and GlucNAc-MurNAc-L-Ala-D-Glu-m-A2pm-D-Ala; GlucNAc-MurNAc-L-Ala-D-Glu-m-A2pm-D-X, being X a residue of the D-amino acid . These results corroborate the usefulness of this technique for the structural analysis of muropeptides.

Gene, 1993 Jan 15, 123(1), 51 - 8
Analysis of hemolysin operons in Actinobacillus pleuropneumoniae; Frey J et al.; Among the twelve different serotypes of Actinobacillus pleuropneumoniae, the causative agent of swine pleuropneumonia, a strongly active hemolysin I (HlyI) is produced by serotypes which are particularly virulent, and less active hemolysin II (HlyII) is produced by all serotypes except type 10 . In the serotypes 1, 5a, 5b, 9, 10 and 11, which produce HlyI, the hemolysin (hly) operon consists of a structural hlyIA gene, encoding pre-HlyI, an activator gene, hlyIC, necessary for the activation of pre-Hly to active Hly, and two genes, hlyIB and hlyID, involved in Hly secretion . These genes are clustered in the order, hlyICABD . This is characteristic to RTX toxin (repeats in the structural toxin) operons . The HlyII operons in all serotypes producing HlyII consist only of the pre-HlyII-encoding gene, appA, and its activator gene, appC . The serotypes, which produce HlyII, but not HlyI, contain a truncated HlyI operon, with the promoter, hlyIB and hlyID, and a small segment of the C terminus of hlyIA . This partial HlyI operon might have been formed by deletion of hlyIC and most of hlyIA . In serotype 3, which produces HlyII, but no HlyI, and which releases only minute amounts of this Hly into the growth medium, none of the hlyI genes and consequently no Hly secretion genes were found . The above results postulate that HlyII is secreted via the products of hlyIB and hlyID, and explain the low amount of HlyII secreted by serotype 3 . Cloning and analysis of the structural genes encoding pre-HlyI and pre-HlyII among the different serotypes revealed differences in the hlyIA genes which are highly similar in the serologically related serotypes 1, 9 and 11, and differ from the serotypes, 5a, 5b and 10 . The hlyIIA genes, in contrast, seem to be conserved in all serotypes.

Cancer Res, 1993 Jan 15, 53(2), 393 - 400
8-Chloroadenosine mediates 8-chloro-cyclic AMP-induced down-regulation of cyclic AMP-dependent protein kinase in normal and neoplastic mouse lung epithelial cells by a cyclic AMP-independent mechanism; Lange-Carter CA et al.; The 8-chloro analogue of the regulatory molecule, cyclic AMP (cAMP), modulates the intracellular concentrations of cAMP-dependent protein kinases (PKA) and inhibits both in vitro and in vivo growth of several neoplastic cell types . Because 8-chloro-cyclic AMP (8-Cl-cAMP) can be converted to 8-chloroadenosine (8-Cl-adenosine) by serum enzymes contained in cell growth media, we tested whether 8-Cl-cAMP effects were mediated by its adenosine metabolite in normal and neoplastic cell lines of mouse lung epithelial origin . 8-Cl-adenosine, directly added to cells or derived from exogenously applied 8-Cl-cAMP, specifically decreased the intracellular concentration of the type I isozyme of cAMP-dependent protein kinase (PKA I) . 8-Cl-adenosine and 8-Cl-cAMP were equipotent at inhibiting cell growth, and elicited similar changes in the proportion of cells in the G1, S, and G2-M phases of the cell cycle . The presence of adenosine deaminase, which converts 8-Cl-adenosine to 8-chloroinosine, completely prevented growth inhibition by 8-Cl-cAMP and the concomitant diminution of PKA I . 8-Cl-cAMP had no discernible effect on cells when its conversion into 8-Cl-adenosine was prevented by 3-isobutyl-1-methyl-xanthene, an inhibitor of phosphodiesterase . 6-(p-Nitrobenzyl)-thioinosine, an inhibitor of adenosine uptake, protected cells from cytostasis, indicating that 8-Cl-adenosine acts intracellularly . 8-Cl-adenosine greatly decreased RI (regulatory subunit of PKA I) and PKA catalytic (C) subunit protein concentrations without affecting RII (regulatory subunit of the PKA type II isozyme) or intracellular cAMP levels . Northern blot analysis of PKA subunit mRNAs following treatment of each cell line with 8-Cl-adenosine demonstrated decreased C alpha mRNA expression, increased RII alpha mRNA, and no change in RI alpha mRNA abundance . Our results indicate that 8-Cl-adenosine inhibits lung cell growth and induces PKA I down-regulation via a cAMP-independent mechanism.

Science, 1993 Jan 8, 259(5092), 241 - 4
Tyrosine phosphorylation of actin in Dictyostelium associated with cell-shape changes; Howard PK et al.; When Dictyostelium cells that have initiated their developmental program upon starvation are returned to growth medium, there is a rapid and transient de novo tyrosine phosphorylation of a 43-kilodalton protein . This protein was found to be actin . Most of the phosphorylation occurred in a single, minor acidic isoform of actin . Developing cells that had been returned to growth medium lost their pseudopod extensions, became round, and had reduced adhesion to the substratum . These effects occurred with kinetics that matched the increase in tyrosine phosphorylation of actin . In mutant cell lines in which the gene for the phosphotyrosine phosphatase PTP1 had been disrupted, tyrosine phosphorylation of actin was rapid and more prolonged . These cells responded with proportionally accelerated kinetics of cell rounding . Cell lines overexpressing PTP1 had diminished amplitude and duration of actin tyrosine phosphorylation and exhibited diminished cell-shape change and an accelerated return to the extended cell-shape morphology seen in starved cells.

Arch Tierernahr, 1993, 43(1), 45 - 51
Heat-induced formation of soluble Maillard reaction products and its influence on utilization of glucose by rumen bacteria; Marounek M et al.; In a series of experiments with pure strains of rumen bacteria the effect of heat-induced formation of soluble Maillard reaction products on the utilization of glucose was examined . Maillard reaction products were prepared from glucose and amino acids, which were dissolved in a growth medium and autoclaved at 100 degrees C and 120 degrees C . Glucose was utilized almost completely in all cultures, no matter whether it was bound in Maillard products or not . The complexing of glucose with amino acids lowered the growth rate and growth yields in most rumen bacteria studied . In some strains, however, the effect of the Maillard reaction was small . It was concluded that the heat damage of carbohydrates in feedstuffs could be attributed to final stages of the Maillard reaction (formation of insoluble polymers), rather than to initial ones.

Agents Actions, 1993 Jan, 38(1-2), 44 - 7
Release of polyamines in cultures of rat parotid and liver cells; Nilsson BO et al.; Rat parotid gland and liver cells were cultured for 6 and 24 h . The cells as well as their growth medium were analyzed on their content of the polyamines putrescine, spermidine and spermine . In control medium the content of polyamines was very low but already after 6 h substantial amounts of all three polyamines under study had been released into the medium from parotid as well as from liver cells . The release was much more pronounced from parotid compared to liver cells . Putrescine was accumulated in parotid cells as well as in the medium indicating that a new synthesis of this amine occurred in these cells.

J Craniofac Genet Dev Biol, 1993 Jan-Mar, 13(1), 6 - 17
Effects of 5-fluorouracil on collagen synthesis during quail secondary palate development; Benkhaial GS et al.; A study was undertaken to examine the involvement of collagen synthesis during palate development in quail where mammalian-type shelf reorientation is absent . Teratological observation showed that 100 micrograms 5-fluorouracil (FU) on day 4 of incubation increased the gap between the palatal shelves . Light microscopic observation indicated that the quail palatal shelves develop as horizontal ridges on day 5 and approximate on day 8 of incubation but never fuse . FU treatment affected the approximation of palatal shelves . In separate experiments, both the control and FU-treated quail embryonic palates, which were dissected between days 5 and 10 of incubation, were incubated in a growth medium supplemented with 14C-proline . The rate of collagen synthesis, total protein, hydroxyproline (HYP) levels, and collagen isotype were determined . The result showed that in control palates the rate of collagen synthesis increased fivefold between days 6 and 8 of incubation but dropped thereafter . In FU-exposed palates, the rate of collagen synthesis was lower than that in controls . It increased threefold between days 7 and 8 of incubation . High performance liquid chromatographic measurement of HYP levels indicated that, in comparison to controls, HYP accumulation in FU-treated palates was reduced by 50% on day 6 and 75% on day 8 of incubation . Total protein content in FU-treated palate were also 50-70% lower than those in their control counterparts between days 5 and 10 of incubation . Gel electrophoresis showed that only type I collagen was synthesized in the developing palate of both the control and FU-treated quail embryo . An analysis of results of the present study, along with the data from literature on mammals, corroborate the proposition that an increased collagen synthesis may contribute to the acquisition of volume of vertebrate's secondary palatal shelf for its continuing morphogenesis.

Boll Chim Farm, 1993 Jan, 132(1), 23 - 8
{Microbiological validation of a film system for monitoring total aerobic bacteria on surfaces and laboratory garments in controlled areas}; Dal Maso G et al.; The microbiological control of the environment is a real issue inside the pharmaceutical production plants . Actually the most used methods are the Contact Plates and the Swabs . The purpose of this study is to evaluate the performance of a new "ready to use" method called Petrifilm SM (produced by 3M Laboratories) . Petrifilm consists of a dry-rehydratable growth medium coated on a flexible film . Petrifilm is tested in comparison with Swabs and Contact Plates to monitor the microbiological pollution on labs surfaces and operator's overalls . The results obtained are comparable with the above mentioned methods, but with important advantages being less bulky, considerably time saving and highly reproducible . Hence, we think that Petrifilm S, can be successfully used instead of the other two more traditional methods.

Arch Virol, 1993, 129(1-4), 251 - 63
Exchange of the cellular growth medium supplement from fetal bovine serum to Ultroser G increases the affinity of adenovirus for HeLa cells; Blixt Y; A comparative investigation was performed on the process of attachment of adenovirus type 2 to HeLa cells cultivated in the presence of 3.5% fetal bovine serum (FBS-cells) or 2% Ultroser G (USG-cells) . The initial rates of virus attachment were markedly higher at temperatures between 10 and 35 degrees C for the virus binding to USG-cells than to FBS-cells . This was not caused by a higher amount of available virus-recognizing cellular receptor sites or cellular receptor units recognizing the viral fiber, but could be explained by a higher affinity of virions for USG-cells as compared to FBS-cells . Studies of virus attachment to cells, pretreated with neuraminidase and/or wheat germ agglutinin, suggested that the cellular receptor sites of FBS-cells were masked to a higher extent by sialic acid than the cellular receptor sites of USG-cells.

Arch Toxicol, 1993, 67(1), 76 - 8
On the toxicity of low doses of tetrasodium-ethylenediamine-tetraacetate (Na-EDTA) in normal rat kidney (NRK) cells in culture; Hugenschmidt S et al.; The toxicity of tetrasodium-ethylenediaminetetraacetate (Na-EDTA) was investigated in normal rat kidney cells (NRK-52E, American Type Culture Collection) in culture . Cell death and reduced colony-forming ability were observed at Na-EDTA concentrations < 100 microM, that is at concentrations lower than that required to chelate all the Ca2+ in the growth medium . No toxicity was observed when cells were exposed to 100 microM Zn- or Fe-EDTA, but the same concentration of Cu-EDTA was as toxic as Na-EDTA . Continuous exposure of the cell cultures to 5 microM Na- or Zn-EDTA for up to 7 weeks yielded no indications of toxicity.

Neuroscience, 1993 Jan, 52(2), 333 - 46
Grafting in acute spinal cord injury: morphological and immunological aspects of transplanted adult rat enteric ganglia; Jaeger CB et al.; We have studied allogeneic transplants of adult rat enteric ganglia in order to evaluate their use as donor tissue for eventual autografts in rodent spinal cord injury models . Female Sprague-Dawley rats of similar weights served either as transplant donors or as recipients . A glass micropipette of 0.8 mm diameter was used to create a local penetrating injury of the lower thoracic spinal cord and the transplant material was pressure injected through the pipette within the neural parenchyma . Ganglia of the myenteric plexus adhering to the stratum longitudinal muscularis were dissected from portions of the jejunum and ileum . Following partial enzymatic digestion and mechanical disruption of the myenteric plexus and muscle tissue (labeled with adherent rhodamine conjugated microbeads), reaggregates of myenteric plexus and muscle were suspended in growth medium and cultured in vitro for one to two days prior to transplantation . Transplants were examined at three, four, six, and eight weeks after surgery . Some of the donor tissue was grown in vitro, in order to determine its cellular composition . These cultured explants were fixed after 10 days, and like myenteric plexus and muscle grafts, were stained histochemically for acetylcholinesterase and observed by fluorescence and light microscopy . At the earlier post-transplantation periods, grafts contained several clusters of enteric ganglion cells that were positive for acetylcholinesterase and exhibited ultrastructural features characteristic of the enteric nervous system . They had well-defined boundaries . Reactive astrocytes and their processes remained located within the host spinal cord adjacent to the boundary region of the grafts . Likewise, macrophages were located in areas abutting the graft . Newly formed vasculature penetrated the graft interior and appeared to be continuous with the host vessels . Grafts grown for at least eight weeks were characterized by interdigitating boundaries . Finger-like protrusions of graft tissue containing fibroblasts and collagen intermixed with adjacent gray and white matter of the host cord . Such transplants also had reactive astrocytes and ED1-positive macrophages . At this later stage, several groups of ganglion cells were identified that were intensely acetylcholinesterase-positive; however, only two of four grafts were recovered, whereas two of the transplants degenerated . We postulate that degeneration of allogeneic grafts may occur as a result of ongoing immune responses of the host which could be prevented by use of autogeneic enteric ganglia . Our studies show that fully differentiated enteric ganglia can survive transplantation to acutely injured spinal cord of adult rats.

Cell Prolif, 1993 Jan, 26(1), 77 - 88
The in vitro response of human tumour cells to desferrioxamine is growth medium dependent; Voest EE et al.; Iron chelating agents have been demonstrated to inhibit tumour cell growth . However, in vitro and in vivo results using desferrioxamine a hexadentate iron chelating agent, for anti-cancer treatment are not always in agreement . Therefore, we have studied the response of three human tumour cell lines (HL-60 promyelocytic leukaemia, MCF-7 breast cancer and HepG2 hepatoma), grown in culture medium supplemented with either human pooled (HPS) or fetal bovine serum (FBS), to desferrioxamine . Desferrioxamine, at micromolar concentrations, induced severe cytotoxicity in all tumour cell lines grown in FBS medium . When grown in HPS medium, comparable desferrioxamine cytotoxicity was observed in the millimolar range . The addition of 50% saturated human transferrin to FBS medium resulted in protection against desferrioxamine cytotoxicity . HL-60 cells were further studied for iron metabolism characteristics . HL-60 cells, grown in medium with FBS, were found to have an 8.4 fold increase in surface transferrin receptor (TfR) expression (P < 0.001) as compared with HL-60 cells grown in medium with HPS . However, iron uptake of HPS cultured HL-60 cells, after incubation with saturated human transferrin, was higher, resulting in a higher concentration of iron in HPS cultured HL-60 cells as compared with FBS cultured cells (1.72 +/- 0.02 mumol/g protein v . 1.32 +/- 0.14 mumol/g protein; P < 0.001) . Using desferrioxamine it was shown that TfR expression is dependent on the biological availability of iron in the cell . Consistent with the lower iron content in FBS cultured cells, we conclude that the cytotoxicity of desferrioxamine is dependent on the ability of cells to replenish cellular iron stores from the culture medium . Cells grown in FBS medium lack this ability and are therefore more susceptible to desferrioxamine.

Mol Microbiol, 1993 Jan, 7(1), 151 - 7
Definition of nitrite and nitrate response elements at the anaerobically inducible Escherichia coli nirB promoter: interactions between FNR and NarL; Tyson KL et al.; Transcription initiation at the Escherichia coli nirB promoter is induced by anaerobic growth and further increased by the presence of nitrite or nitrate in the growth medium . Expression from this promoter is totally dependent on the transcription factor, FNR, which binds between positions -52 and -30 upstream of the transcription startsite . The 20 base pairs from position -79 to -60 contain an inverted repeat of two 10-base sequence elements that are related to sequences at the NarL-binding site at the E . coli narG promoter . Comparison of these, and sequence elements at other promoters regulated by NarL, suggests a consensus NarL-binding sequence . Mutations in the putative NarL-binding site at the nirB promoter decrease FNR-dependent anaerobic induction, suggesting that NarL acts as a helper to FNR during transcription activation . These mutations also suppress induction by nitrite: single mutations at symmetry-related positions have similar effects, whilst double mutations have more severe effects, probably because two NarL subunits bind to the inverted repeat . Disruption of narL decreases nitrite induction of the nirB promoter whilst not suppressing induction by nitrate, suggesting that there may be a second nitrate-responsive factor . Nitrate induction was, however, suppressed by double mutations at symmetry-related positions in the NarL-binding site, suggesting that this putative second factor may bind to sequences similar to those recognized by NarL.

J Bacteriol, 1993 Jan, 175(1), 214 - 21
Osmotic repression of anaerobic metabolic systems in Escherichia coli; Gouesbet G et al.; The influence of the osmolarity of the growth medium on anaerobic fermentation and nitrate respiratory pathways was analyzed . The levels of several enzymes, including formate dehydrogenase, hydrogenase, and nitrate reductase, plus a nickel uptake system were examined, as was the expression of the corresponding structural and regulatory genes . While some functions appear to be only moderately affected by an increase in osmolarity, others were found to vary considerably . An increase in the osmolarity of the medium inhibits both fermentation and anaerobic respiratory pathways, though in a more dramatic fashion for the former . fnr expression is affected by osmolarity, but the repression of anaerobic gene expression was shown to be independent of FNR regulatory protein, at least for hyd-17 and fdhF . This repression could be mediated by the intracellular concentration of potassium and is reversed by glycine betaine.

J Virol, 1993 Jan, 67(1), 277 - 87
Release, uptake, and effects of extracellular human immunodeficiency virus type 1 Tat protein on cell growth and viral transactivation; Ensoli B et al.; During acute human immunodeficiency virus type 1 (HIV-1) infection or after transfection of the tat gene, Tat protein is released into the cell culture supernatant . In this extracellular form, Tat stimulates both HIV-1 gene expression and the growth of cells derived from Kaposi's sarcoma (KS) lesions of HIV-1-infected individuals (AIDS-KS cells) . Tat protein and its biological activities appear in the cell supernatants at the peak of Tat expression, when the rate of cell death is low (infection) or cell death is undetectable (transfection) and increased levels of cytoplasmic Tat are present . Tat-containing supernatants stimulate maximal AIDS-KS cell growth but only low to moderate levels of HIV-1 gene expression . This is due to the different concentrations of exogenous Tat required for the two effects . The cell growth-promoting effects of Tat peak at between 0.1 and 1 ng of purified recombinant protein per ml in the cell growth medium and do not increase with concentration . In contrast, both the detection of nuclear-localized Tat taken up by cells and the induction of HIV-1 gene expression or replication require higher Tat concentrations (> or = 100 ng/ml), and all increase linearly with increasing amounts of the exogenous protein . These data suggest that Tat can be released by a mechanism(s) other than cell death and that the cell growth-promoting activity and the virus-transactivating effect of extracellular Tat are mediated by different pathways.

Yeast, 1993 Jan, 9(1), 77 - 84
In Saccharomyces cerevisiae, protein secretion into the growth medium depends on environmental factors; Rossini D et al.; In the budding yeast Saccharomyces cerevisiae the cell wall, mainly composed of mannoproteins and glucans, constitutes a barrier to protein excretion in the growth medium . In this paper we have studied the effects of different environmental parameters on excretion of Escherichia coli beta-galactosidase obtained by exploiting the glucoamylase II signal sequence . Excretion of the unglycosylated beta-galactosidase was detectable only in cells grown in rich medium, was affected by temperature (36 degrees C > 30 degrees C >> 24 degrees C) and slightly stimulated by reducing agents . On the contrary, glycosylated proteins, such as alpha-galactosidase and glucoamylase II, were excreted to a good extent under all tested conditions of medium composition, growth temperature and pH . These data indicate that optimization of environmental parameters may help the excretion of heterologous proteins, offering advantages for protein purification.

J Pharmacol Exp Ther, 1993 Jan, 264(1), 463 - 8
Tetrahydroaminoacridine is neurotrophic and promotes the expression of muscarinic receptor-coupled phosphoinositide turnover in differentiating cerebellar granule cells; Sunaga K et al.; We have investigated whether 9-amino-1,2,3,4-tetrahydroacridine (THA), a drug with potential antidementia activity, has a trophic action on differentiating cerebellar granule cells by using the method of {3H}inositol incorporation into inositol-containing phospholipid . Addition of THA (30-50 microM) prevented the extensive neuronal degeneration which occurred in the growth medium containing "low" K+ (15 mM) . These effects were similar to the neuroprotective action caused by the presence of 100 microM N-methyl-D-aspartate (NMDA) . Neurotrophic effects of THA and NMDA on cells grown in low K+ were also demonstrated by direct microscopic examination of cellular morphology . Measurement of phosphoinositide (PI) response in the rescued cells indicated that NMDA modestly promoted the PI response to carbachol and norepinephrine but markedly stimulated the activity induced by glutamate . In contrast, although THA had little or no influence on the maturation of the norepinephrine- and glutamate-induced PI response, it selectively enhanced the activity stimulated by carbachol . Furthermore, the THA treatment drastically increased the Vmax value of carbachol-induced PI turnover with no significant alteration in the EC50 value . Scatchard analysis of the binding of N-{3H}methylscopolamine to intact granule cells indicated a selective increase in the maximum binding value in cells grown in THA-supplementing medium . These observations suggest that THA seems to selectively up-regulate muscarinic cholinergic receptors.

Reg Immunol, 1993 Jan-Feb, 5(1), 18 - 27
Characterization of an inhibitory factor derived from epithelial cells of the small intestine; Llana T et al.; Rat small intestine epithelial cell (EC) culture conditioned media (ECCM) contains a factor that has inhibitory activity against: a) the response of freshly isolated lymphocytes to Concanavalin A (Con A) or IL-2, b) the proliferative response to antigen of primed lymphocytes, and c) the normal growth of transformed cell lines derived from several species . Inhibition is reversible, and not the result of a cytotoxic effect . The inhibitory activity is enterocyte derived, effective at low concentration (1-2% in growth media), and can be derived from freshly isolated EC . Biochemical analysis indicates the inhibitory activity is associated with a protein with an approximate molecular weight of 32 kd, and an isoelectric point (PI) in the range of 3-5 . The protein is trypsin sensitive, and labile with prolonged heating at 56 degrees C . The described activity differs from previously reported mucosally-derived inhibitory activities on the basis of molecular weight, and its ability to inhibit the growth of several cell lines . We suggest that this factor can provide the immunomodulatory activity necessary to produce the low level of intestinal T cell reactivity that is observed in vivo.

Microbios, 1993, 74(298), 23 - 8
The in vitro lysozyme susceptibility of Candida species cultured in sucrose supplemented media; Samaranayake YH et al.; The in vitro sensitivity to lysozyme of twelve isolates of Candida (three isolates each of C . albicans, C . tropicalis, C . glabrata and C . krusei) pre-incubated in sucrose supplemented media, was determined by a growth inhibition assay . In general, C . albicans and C . tropicalis exhibited increased susceptibility to lysozyme as the sucrose concentration in the growth medium was reduced . Candida krusei demonstrated the reverse effect and C . glabrata isolates showed different trends . Although generally the Candida species examined were susceptible to lysozyme in the decreasing order of C . glabrata, C . albicans, C . tropicalis and C . krusei, differences in susceptibility were noted among different isolates within a given species . These results imply that salivary lysozyme and dietary carbohydrates may exert selective pressure on oral colonization by Candida species.

Arch Dermatol Res, 1993, 285(7), 385 - 92
Growth and differentiation of normal human melanocytes in a TPA-free, cholera toxin-free, low-serum medium and influence of keratinocytes; Donatien P et al.; Melanocyte cultures were obtained from a modification of the keratinocyte culture system MCDB153 . Either promelanocytes or mature melanocytes were selected from epidermal cell primary cultures . Pure subcultures of actively dividing melanocytes of both types were grown in a low-serum medium totally deprived of TPA and cholera toxin called melanocyte growth medium (MGM) . Early passaged cells from MGM primary cocultures were similar to normal adult human melanocytes in vivo, exhibiting numerous melanosomes, strong dopa positivity and a high dendricity . The ability of MGM to support melanocyte growth was mainly a consequence of its basic composition, combined with a low serum concentration . Bovine pituitary extract significantly enhanced melanocyte growth . Using complete MGM, in the absence of mitogens and keratinocytes, cell growth was maintained, but the differentiation of melanocytes decreased . The presence of keratinocytes was found to promote melanocyte growth . The coculture system used strongly suggests the action of soluble keratinocyte-derived factors . Keratinocyte contact was necessary to sustain melanocyte dendricity and melanization . Melanization and dendricity behaved mostly as independent features when keratinocyte influence was withheld . Our results underline the essential role of keratinocytes in the regulation of melanocyte growth and differentiation in a physiological culture system.

C R Acad Sci III, 1993, 316(5), 469 - 73
{A second gene involved in the formation of disulfide bonds in proteins localized in Escherichia coli periplasmic space}; Belin P et al.; A novel mutant of Escherichia coli was isolated whose phenotype is similar to those of dsbA strains . For instance, it is unable to express pH 2.5 acid phosphatase, glucose-1-phosphatase and alkaline phosphatase in the periplasmic space . The mutation lies at min 26.2 of the linkage map, and does not affect expression of DsbA . Addition of oxidized glutathione to the growth medium restores the wild-type phenotype in the mutant while this is not the case in a dsbA strain . The product of this new gene dsbX is thus actually involved in the formation of disulfide bridges in the periplasmic space but can only operate if DsbA is functional.

Adv Exp Med Biol, 1993, 343, 319 - 26
IGFs and muscle differentiation; Florini JR et al.; The Role of IGFs in Myogenesis . Thus we are now convinced that the control of myogenesis by IGFs is a general phenomenon that occurs in all skeletal muscle cells, whether or not IGFs are added to the "differentiation" medium . We believe that several medium components contribute to the suppression of IGF-II expression in myoblasts incubated in high serum "growth" medium, and conclude that the IGF-I receptor mediates the feedback inhibition of IGF-II gene expression in muscle cells . Mechanism of Induction of Myogenesis by IGFs . The observations summarized here now permit a reasonably coherent overview of the stimulation of myogenic differentiation by the IGFs . It seems clear that all IGFs act by binding to the Type I IGF receptor, and that this process is inhibited to a significant extent by IGF binding proteins secreted by the target myoblasts . A major, but possibly not the only relevant effect of this binding is the induction of expression of the myogenin gene; this induction appears to require the presence of myf-5 protein, at least during the early part of the response . Cells capable of a mitogenic response undergo a round of division in response to IGF-I, thus delaying their entry into the final processes of postmitotic terminal differentiation . Other laboratories have shown that myogenin complexes with one or more widely occurring proteins such as E12 or E47 to form an active complex that interacts with CAnnTG elements in muscle specific genes, turning on expression of those genes and thus initiating the phenotype associated with terminally differentiated skeletal muscle.

Neurotoxicology, 1993 Winter, 14(4), 381 - 6
S9 liver fraction is cytotoxic to neurons in dissociated culture; Kohn J et al.; Methods to evaluate the neurotoxicity of chemicals in vitro must take into account that many xenobiotics exert their toxic effects through metabolites . S9 fraction of liver homogenate has been used in cultures of bacterial and mammalian cells as a system for metabolic activation . The suitability of the S9 activation system for long-term neurotoxicity studies in vitro was investigated in dissociated cultures of murine spinal cord-dorsal root ganglia . Exposure to amounts of S9 greater than 0.07 mg S9 protein/ml of culture medium for 4 days or longer was cytotoxic to all types of neurons in the cultures . Non-neuronal cells tolerated higher exposures, but contained numerous cytoplasmic inclusions when 0.35 mg S9 protein was included in the medium . It was demonstrated that cytotoxicity was caused by the particulate, microsomal fraction of S9 . It is concluded that direct incorporation of S9 fraction in the growth medium (0.07 mg S9 protein/ml or greater) is not a suitable method of generating metabolites in dissociated cultures of central nervous system when several days are required to elicit a biological response . Cytotoxicity can be prevented by using tissue culture inserts to separate cultured cells from S9 particulate fraction by a microporous membrane.

Arch Dermatol Res, 1993, 285(6), 347 - 51
Corticosteroids induce proliferation but do not influence TNF- or IL-1 beta-induced ICAM-1 expression of human dermal microvascular endothelial cells in vitro; Hettmannsperger U et al.; The effects of hydrocortisone, dexamethasone, betamethasone 17-valerate and clobetasol propionate at concentrations of 10(-5)-10(-12) M on the proliferation of human dermal microvascular endothelial cells (HDMEC) were studied in vitro . In addition, confluent HDMEC were treated with TNF (1000 U/ml) or IL-1 beta (1000 U/ml) alone or in combination with the corticosteroids (10(-5) M, 10(-6) M) for 24 h, and cytokine-induced expression of intercellular adhesion molecule-1 (ICAM-1) was assessed by immunocytochemistry . Controls were treated either with growth medium or with the corticosteroids alone . All tested corticosteroids stimulated HDMEC growth after 4 and 6 days of treatment in a dose-dependent manner, as assessed by 3H-thymidine incorporation and the 4-methyl-umbelliferyl heptanoate (MUH) assay . The minimal effective concentration was 10(-9) M for hydrocortisone, 10(-10) M for dexamethasone and betamethasone, and 10(-12) M for clobetasol . In untreated and in corticosteroid-treated cultures, less than 5% of the cells expressed ICAM-1 . TNF and IL-1 beta were strong inducers of ICAM-1 expression on 74% and 82% of the cells, respectively . None of the tested corticosteroids significantly influenced cytokine-induced ICAM-1 expression, suggesting that the anti-inflammatory effects of corticosteroids are not related to ICAM-1 modulation on HDMEC.

Drug Metab Dispos, 1993 Jan-Feb, 21(1), 56 - 61
Inducibility of enzyme activities associated with the cytochrome P-450 1A family, ethoxyresorufin O-deethylase, and methoxyresorufin O-demethylase in human hepatocyte lines derived from normal liver tissue; Roberts EA et al.; Three human hepatocyte cultures have been developed from specimens of normal human liver, in each case from an infant or child, by coculture with liver epithelial cells from 6-day-old rat pups in a complex growth medium . In the established cultures hepatocytes predominate and maintain typical hepatocellular morphology by light microscopy and albumin secretion into supernatant medium . The activity of ethoxyresorufin O-deethylase (EROD) and methoxyresorufin O-demethylase (MROD) basally and after treatment with polycyclic aromatic hydrocarbons was measured spectrofluorometrically in cell homogenates from each culture . Very low levels of EROD and MROD activity were found in each culture without induction {EROD: 1.78 +/- 0.71 (mean +/- SE) pmol/min/mg protein; MROD: 1.33 +/- 0.10 pmol/min/mg protein} . After treatment with 10 microM dibenz (a,h)anthracene x 48 hr, EROD and MROD activities rose approximately 20- to 50-fold . When the basal and induced enzyme activities were remeasured 2 months later, results were essentially the same . Incubation with 10 microM benz(a)anthracene x 48 hr also led to induction of EROD and MROD activities . We believe that these cultures can be regarded as human hepatocyte lines, which conserve human hepatic polycyclic aromatic hydrocarbon-inducible P-450s, most likely including P-450 1A2.

Eur J Immunol, 1993 Jan, 23(1), 103 - 9
Rapid re-expression of CD45RC on rat CD4 T cells in vitro correlates with a change in function; Sarawar SR et al.; Rat CD4+ T cells are divided phenotypically by the anti-CD45RC monoclonal antibody OX22 into subsets with contrasting functions . Stimulation of T cells in vitro is known to induce a change in isoform from CD45RC+ to CD45RC- . We have investigated the in vitro conditions which promote a switch in isoform in the opposite direction . We observed that a majority of CD45RC- CD4 T cells (> 90%) spontaneously re-expressed CD45RC during the first 1-3 days of culture in both the presence and absence of alloantigen . The T cells remained CD45RC+ when cultured for 7 days in serum-free growth medium . However, alloantigen-activated lymphocytes, expressing the interleukin-2 receptor (IL-2R), downregulated CD45RC by day 4 and remained CD45RC- during the course of the experiment . Using mixtures of allotype-marked CD45RC+ and CD45RC- T cells, it was demonstrated that each subset showed comparable survival, IL-2R expression and time courses of activation in response to alloantigen . The repertoire of neither subset was, therefore, deficient in terms of allorecognition . The rapid re-expression of CD45RC in culture was accompanied by a change in function: CD45RC+ "converts", obtained by overnight culture of CD45RC- T cells, induced significantly higher graft-versus-host responses . Thus, the transition in culture from CD45RC- to CD45RC+ reflects a major functional reprogramming of the cell and not a trivial modulation of a surface antigen.

Rom J Physiol, 1993 Jan-Jun, 30(1-2), 65 - 71
The effect of pulsed electromagnetic field (Diapulse) on cellular systems; Badea MA et al.; The effect of a 27.12 MHz pulsed electromagnetic field (Diapulse) on microbial growth is investigated . A strain of K 12 E . coli grown in complete Pennassay medium is subjected to Diapulse action for 30 min, at 8 hrs and 12 hrs of growth . In this experimental set-up, designed to be closed to the physiological conditions of open wounds, the Diapulse action does not promote any increase of cell population, indicating the safety of this type of therapy for wound healing process . The same K 12 E . coli strain grown in Pennassay medium for 2 hours is inoculated into a minimal growth medium and the lagless exponential growth thus obtained is followed by a spectrophotometric method . Diapulse field is applied to this lagless phase of cellular cultures at 30, 60, and 90 minutes after inoculation . A slight increase in the number of cells was observed at 2 and 4 hours after the Diapulse application, when the cultures were previously subjected to Diapulse action between the period of 60 and 90 minutes of their growth . A possible molecular mechanism for these effects is discussed.

Cytotechnology, 1993, 13(3), 185 - 93
Dielectric spectroscopy of mammalian cells . 1 . Evaluation of the biomass of HeLa- and CHO cells in suspension by low-frequency dielectric spectroscopy; Cerckel I et al.; The capacitance of suspensions of CHO and HeLa cells (0.5-3 x 10(6) cells/ml) has been measured between 0.2 and 10 MHz . As frequencies decrease, there is a continuous increase in capacitance of both the cell suspension and the spent growth medium free of cells, a phenomenon which is partially attributed to an increased polarisation of the electrodes . At a given frequency, subtraction of the capacitance of the spent medium from that of the cell suspension allows one to determine the capacitance of the cells only . The intensity of this signal varies linearly with the biomass and cell size . At low frequencies such as those used in this study (0.25 MHz), where sensitivity is the highest, concentrations as low as 0.5 x 10(6) cells/ml can be accurately measured . Suggestions are made how to make these measures on-line, non-invasive and in real time.

Mutat Res, 1993 Jan, 285(1), 13 - 8
The influence of growth medium on the yield of X-ray-induced chromatid exchanges in the presence and absence of aphidicolin; Moore RC et al.; The frequency of exchanges in JU56 cells irradiated in the G2 phase in the presence and absence of the polymerase inhibitor aphidicolin (APC), and in the presence of a range of concentrations of cysteine was measured . It was found that in the absence of cysteine, incubation for 2 h with APC had no effect on the yield . Addition of cysteine at concentrations of 50-250 mg/l reduced the frequency of exchanges, and at these concentrations the frequency was increased by incubation with APC . At higher concentrations, the yield was reduced and incubation with APC did not elevate it . In following experiments, it was found that incubation with cysteine for a period of longer than 10 minutes was necessary before APC affected the yield of exchanges.

Cytotechnology, 1993, 13(3), 195 - 202
Dielectric spectroscopy of mammalian cells . 2 . Simultaneous in situ evaluation by aperture impedance pulse spectroscopy and low frequency dielectric spectroscopy of the biomass of HTC cells on Cytodex 3; Degouys V et al.; Low-frequency dielectric spectroscopy has been used in situ, i.e . while the cells are still attached to their microsupport, to monitor the changes of biomass accompanying the growth of anchorage-dependent cells . This method, when compared to Aperture Impedance Pulse Spectroscopy (also called electronic sizing), is characterized by a somewhat lower degree of resolution . Suggestions are made on how to determine the capacitance of the spent growth medium alone, still keeping the probe inserted in the bioreactor . This will make dielectric spectroscopy the first truly in situ, on-line, in real time, non-invasive measure of the biomass.

Proc Natl Acad Sci U S A, 1992 Dec 15, 89(24), 11881 - 5
1H NMR studies on the catalytic subunit of aspartate transcarbamoylase; Cohen RE et al.; The 1H NMR spectrum of the catalytic subunit of Escherichia coli aspartate transcarbamoylase (EC 2.1.3.2) was simplified by using strains auxotrophic for the aromatic amino acids and a growth medium containing fully deuterated Trp, Phe, and His and partially deuterated Tyr . 1H resonances for Tyr in the catalytic trimer (M(r) = 10(5)) were partially resolved into five peaks at 27 degrees C, which above 50 degrees C were further resolved to give a distinct resonance for each of the eight Tyr residues in the polypeptide chain . Experiments on chemically modified catalytic subunits and on a mutant form in which Tyr-165 was converted to Ser-165 led to the assignment of resonances for Tyr-165, Tyr-240, and Tyr-185 . Binding of the substrate, carbamoyl phosphate, caused shifts of two of the unassigned resonances, and the subsequent binding of the aspartate analog succinate perturbed the resonances corresponding to Tyr-165 and Tyr-240 . The bisubstrate analog N-(phosphonacetyl)-L-aspartate produced a spectrum differing considerably from that caused by the combination of carbamoyl phosphate and succinate . The NMR spectrum for the Tyr-165-->Ser mutant trimer showed clearly that the single amino acid substitution caused conformational changes affecting the environment of residues remote from the position of the replacement . In contrast, the inactive mutant subunit in which Gly-128 was replaced by Asp exhibited a spectrum virtually identical to that of the wild-type protein . However, addition of the substrate carbamoyl phosphate caused a marked change in the spectrum of the mutant enzyme, whereas that of the wild-type trimer was altered only slightly, showing that the effect of the amino acid substitution was manifested in the NMR spectrum only with the liganded enzyme.

J Biol Chem, 1992 Dec 15, 267(35), 25264 - 73
Distinct sterol and nonsterol signals for the regulated degradation of 3-hydroxy-3-methylglutaryl-CoA reductase; Roitelman J et al.; The in vivo turnover rate of the endoplasmic reticulum protein 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase, the rate-limiting enzyme in the mevalonate (MVA) pathway, is accelerated when excess MVA or sterols are added to the growth medium of cells . As we have shown recently (Roitelman, J., Bar-Nun, S., Inoue, S., and Simoni, R . D . (1991) J . Biol . Chem . 266, 16085-16091), perturbation of cellular Ca2+ homeostasis abrogates the MVA-accelerated degradation of HMG-CoA reductase and HMGal . Here we show that, in contrast, the sterol-accelerated degradation of HMG-CoA reductase is unaffected by Ca2+ perturbation achieved either by Ca2+ ionophore or by inhibitors of the endoplasmic reticulum Ca(2+)-ATPase . The differential effects of Ca2+ perturbation can be attributed neither to global alteration in protein synthesis nor to inhibition of MVA conversion to sterols . Yet, such manipulations markedly reduce the incorporation of MVA into cellular macromolecules, including prenylated proteins . Furthermore, we directly demonstrate that MVA gives rise to at least two distinct signals, one that is essential to support the effect of sterols and another that operates independently of sterols . Our results indicate that the cellular signals operating in the MVA-accelerated turnover of HMG-CoA reductase are distinct from those involved in the sterol-regulated degradation . A working model for the degradation pathway is proposed.

Proc Natl Acad Sci U S A, 1992 Dec 1, 89(23), 11327 - 31
Stable heparin-producing cell lines derived from the Furth murine mastocytoma; Montgomery RI et al.; Stable cell lines that synthesize heparin have been established from the Furth murine mastocytoma . The parental line (MST) divides in suspension every 14-18 h in growth medium supplemented with fetal bovine serum or defined growth factors . Adherent subclones were selected by adhesion to plastic culture vessels . Both adherent and nonadherent cells contain about 0.4 micrograms of glycosaminoglycan hexuronic acid per 10(6) cells, composed of 80% heparin and 20% chondroitin sulfate E . Deaminative cleavage of MST heparin by HNO2 at pH 1.5 released disaccharides that were similar in composition to those obtained from commercial heparin, except that disaccharides containing 3,6-O-desulfated GlcN units were not found . Greater than 90% of the glycosaminoglycans were stored in cytoplasmic granules, and challenge of the cells with dinitrophenylated bovine serum albumin and anti-dinitrophenyl IgE released a portion of the stored material . Growth studies of subclones showed that MST cells tolerate a 10-fold variation in glycosaminoglycan content . Incubation of cells with sodium chlorate reduced glycosaminoglycan sulfation by > 95% without affecting cell growth . Thus, granule glycosaminoglycans appear to be nonessential for growth of MST cells.

Gene, 1992 Dec 1, 122(1), 219 - 23
An inducible expression system for the production of human lactoferrin in Aspergillus nidulans; Ward PP et al.; The production and secretion of human lactoferrin (hLF) in Aspergillus nidulans is described . The hLF cDNA was expressed under the control of the strong ethanol-inducible alcohol dehydrogenase (alcA) promoter . Recombinant hLF (re-hLF) is produced at levels up to 5 micrograms/ml . Approximately 30% of the re-hLF produced in this system is secreted into the growth medium . The re-hLF is indistinguishable from native hLF with respect to size and immunoreactivity . Furthermore, re-hLF is functional by the criterion of iron-binding capacity . The A . nidulans expression system offers an inexpensive, convenient method for the controlled production of mg amounts of biologically active mammalian glycoproteins.

J Invest Dermatol, 1992 Dec, 99(6), 683 - 90
HMEC-1: establishment of an immortalized human microvascular endothelial cell line; Ades EW et al.; The study of human microvascular endothelial cells has been limited, because these cells are difficult to isolate in pure culture, are fastidious in their in vitro growth requirements, and have a very limited lifespan . In order to overcome these difficulties, we have transfected human dermal microvascular endothelial cells (HMEC) with a PBR-322-based plasmid containing the coding region for the simian virus 40 A gene product, large T antigen, and succeeded in immortalizing them . These cells, termed CDC/EU.HMEC-1 (HMEC-1), have been passaged 95 times to date and show no signs of senescence, whereas normal microvascular endothelial cells undergo senescence at passages 8-10 . HMEC-1 exhibit typical cobblestone morphology when grown in monolayer culture, express and secrete von Willebrand's Factor, take up acteylated low-density lipoprotein, and rapidly form tubes when cultured on matrigel . HMEC-1 grow to densities three to seven times higher than microvascular endothelial cells and require much less stringent growth medium . HMEC-1 will grow in the absence of human serum, whereas microvascular endothelial cells require culture medium supplemented with 30% human serum . These cells express other cell-surface molecules typically associated with endothelial cells, including CD31 and CD36 and epitopes identified by monoclonal antibodies EN4 and PAL-E . They also express the cell adhesion molecules ICAM-1 and CD44 and following stimulation with interferon-gamma express major histocompatibility complex class II antigens . HMEC-1 specifically bind lymphocytes in cell adhesion assays . Thus HMEC-1 is the first immortalized human microvascular endothelial cell line that retains the morphologic, phenotypic, and functional characteristics of normal human microvascular endothelial cells.

J Bacteriol, 1992 Dec, 174(23), 7743 - 9
Escherichia coli is able to grow with negligible sodium ion extrusion activity at alkaline pH; Ohyama T et al.; The Escherichia coli mutant NM81, which is deficient in the nhaA gene for the sodium/proton antiporter, still has a sodium ion extrusion activity because of a second antiporter encoded by nhaB (E . Padan, N . Maisler, D . Taglicht, R . Karpel, and S . Schuldiner, J . Biol . Chem . 264:20297-20302, 1989) . By chance, we have found that E . coli pop6810 already contains a mutation affecting the sodium ion circulation, probably in or near nhaB, and that its delta nhaA mutant, designated RS1, has no sodium ion extrusion activity at alkaline pH . The growth of RS1 was inhibited completely by 0.1 M sodium, whereas growth inhibition of NM81 was observed only at sodium concentrations greater than 0.2 M . RS1 grew at a normal rate in an alkaline medium containing a low sodium concentration . Furthermore, RS1 grew with a negligible proton motive force in the alkaline medium containing carbonyl cyanide m-chlorophenylhydrazone . The transport activities for proline and serine were not impaired in RS1, suggesting that these transport systems could be driven by the proton motive force at alkaline pH . These findings led us to conclude that the operation of the sodium/proton antiporter is not essential for growth at alkaline pH but that the antiporter is required for maintaining a low internal sodium concentration when the growth medium contains a high concentration of these ions.

Yonsei Med J, 1992 Dec, 33(4), 344 - 50
Culture of melanocytes obtained from normal and vitiligo subjects; Im S et al.; The development of human melanocyte culture in vitro from normal adult skin and uninvolved skin of vitiligo patients is essential to investigate the mechanism of depigmentation in vitiligo and other pigmentary dermatoses . By using selective growth and long-term maintenance conditions, we selectively cultured melanocytes derived from normal foreskins and arm skins, and uninvolved foreskins and arm skins of vitiligo patients . The melanocytes of the arm skins were successfully cultured from the roofs of suction blisters . Melanocyte Growth Media (MGM) consisting of MCDB-153 formulation with basic fibroblast growth factor (bFGF), bovine pituitary extract (BPE), insulin, hydrocortisone, phorbol 12-myristate 13-acetate (PMA) and 10% human AB serum was sufficient to grow the melanocytes from normal and vitiligo donors . Melanocytes from uninvolved skin of vitiligo donors showed no different morphologic features, initial seeding capacity and population doubling time compared with those from normal skin . Melanocytes from both cell types grew without any lag period for more than 6 months (6-11 passages) . Melanocytes obtained from foreskins had higher initial seeding capacity and shorter population doubling time than those obtained from arm skins using suction-blistered roofs . Our results suggest that the culture method using suction blisters may be a simple and easy way to obtain melanocytes . In addition, vitiligo melanocytes can be successfully cultured with appropriate growth conditions and may show no defective growth patterns . This culture system will be applied to investigate the basic pathophysiology of vitiligo and other various pigmentary dermatoses.

Biofactors, 1992 Dec, 4(1), 47 - 9
Variable alpha-tocopherol stimulation and protection of glutathione peroxidase activity in established and malignant fibroblasts; Hu WL et al.; Studies on glutathione (GSH) metabolism in an established baby hamster kidney fibroblast cell line (BHK-21/C13) and in its polyoma virus-transformed counterpart (BHK-21/PyY) have revealed a significant stimulation of intracellular GSH peroxidase (GSHpx) activity (selenium-independent plus selenium-dependent) by alpha-tocopherol supplementation (14 microM) . This stimulation was found to be much greater in the transformed cells . Other GSH-requiring enzyme activities (i.e . GSH reductase and GSH S-transferase) were unaltered by alpha-tocopherol treatment, suggesting a degree of specificity in its action on GSHpx . In unsupplemented growth media, the GSHpx activity in both cell lines was significantly decreased by oxidative stress . However, the same stress applied to the alpha-tocopherol-supplemented cells had no effect on the stimulated GSHpx activity, suggesting that some protection was afforded by the alpha-tocopherol.

Melanoma Res, 1992 Dec, 2(5-6), 321 - 6
Potentiation of radiation lethality in mouse melanoma cells by mild hyperthermia and chloroquine; Djordevic B et al.; To test the hypothesis that radiosensitization by combined mild hyperthermia and chloroquine may be increased by the presence of melanin in treated cells, Cloudman melanotic mouse melanoma S91/6 cells, and the amelanotic S91/amel cells were incubated during a 3 h post-irradiation period with 0.03 mM chloroquine at 41 degrees C . A considerable increase in radiation lethality was observed (radiation potentiation factor > 1.6) in both cases . Addition of 0.1 mM isobutyl-methyl xanthine (IBMX), a promoter of melanin synthesis, to the growth medium of S91/6 cells 10 days before irradiation, did not further increase the lethality of radiation followed by combined heat and chloroquine treatment . Under these conditions, toxicity to unirradiated cells was slight . On the other hand, 10 microM chloroquine showed similar toxicity to unirradiated B-16 mouse melanoma cells, but did not increase radiation lethality . Factors other than melanin content therefore play a role in the potentiation of radiation lethality by mild hyperthermia and chloroquine.

Mutat Res, 1992 Dec, 298(2), 97 - 103
Mutagenesis and comutagenesis by lead compounds; Roy NK et al.; We have previously reported that lead(II) is weakly mutagenic to Chinese hamster V79 cells . A transgenic cell line G12 containing a single copy of the E . coli gpt gene was developed in this laboratory from Chinese hamster V79 cells . The gpt locus in the G12 cells is more mutable by radiation and oxidative agents compared with the endogenous hprt locus of wild-type V79 cells . We have investigated the mutagenicity of two lead compounds at the gpt locus in G12 cells . Only at a toxic dose is lead acetate significantly mutagenic to G12 cells . Lead nitrate is not significantly mutagenic at any dose . Although both compounds are water-soluble, lead acetate, but not lead nitrate, forms a fine white insoluble precipitate upon addition to growth medium . A nick translation assay on cells treated with lead compounds and then permeabilized indicated that lead nitrate and, to a greater extent, lead acetate causes the appearance of nicks in chromosomal DNA . Lead ions in the presence of hydrogen peroxide, but not alone, introduced nicks into supercoiled plasmid DNA in vitro, suggesting that lead ions can partake in a Fenton reaction and thereby damage DNA . At lower nonmutagenic concentrations, lead acetate enhances the mutagenicity of MNNG and ultraviolet light . DNA damage by ultraviolet light is not enhanced by lead ions in vitro . Our data support the concept that non-toxic concentrations of lead(II) can inhibit DNA repair . Thus, at biologically relevant doses, lead(II) could act as a comutagen and possibly a cocarcinogen, but is not likely to act as an initiating genotoxic carcinogen.

Proc Natl Acad Sci U S A, 1992 Nov 15, 89(22), 10807 - 11
Covalent modification of proteins by ligands of steroid hormone receptors; Takahashi N et al.; Retinoylation, acylation with retinoic acid (RA), is a covalent modification of proteins occurring in a variety of eukaryotic cell lines . In this study, we found that proteins in HL-60 cells were labeled by 17 beta-{3H}estradiol (E2), {3H}progesterone (Pg), 1 alpha,25-dihydroxy{3H}vitamin D3 {1,25(OH)2D3}, {125I}triiodothyronine (T3), {125I}thyroxine (T4), and {3H}prostaglandin E2 (PGE2) . All of these hormones, except PGE2, are ligands of the steroid hormone receptor family . Addition to the growth medium of 5 microM ketoconazole, an inhibitor of cytochrome P450-dependent enzymes, increased about 2-fold the labeling of proteins by T3, T4, 1,25(OH)2D3, and PGE2 . In contrast, ketoconazole did not change markedly the extent of labeling by RA, E2, or Pg . Alkaline methanolysis, which cleaves ester bonds, released variable percentages of the radioactive ligands bound to protein . These values were about 80% for RA and PGE2; 50% for T3, T4, and Pg; and 20% for E2 and 1,25(OH)2D3 . Treatment with thioether-cleavage reagents, iodomethane or Raney nickel catalyst, released < 2% of the covalently bound ligands . Two-dimensional polyacrylamide gel electrophoresis patterns of labeled proteins were unique for each ligand . Proteins of M(r) 47,000 and 51,000 were labeled by RA, E2, T3, and T4 . These proteins had the same mobilities as RI and RII, the cAMP-binding regulatory subunits of type I and type II cAMP-dependent protein kinases . 1,25(OH)2D3 also bound to proteins of M(r) 47,000 and 51,000 . However, these proteins had pI values different from those of RI or RII . These results suggest that some activities of ligands of the steroid hormone receptor family and of PGE2 may be mediated by their covalent modification of proteins.

J Biol Chem, 1992 Nov 15, 267(32), 23261 - 8
Processing and secretion of tumor necrosis factor alpha in endotoxin-treated Mono Mac 6 cells are dependent on phorbol myristate acetate; Pradines-Figueres A et al.; Lipopolysaccharide (LPS, endotoxin) is a potent stimulator of tumor necrosis factor alpha (TNF alpha) synthesis and secretion in mouse macrophage tumor cells (Golenbock, D . T., Hampton, R . Y., Qureshi, N., Takayama, K., and Raetz, C . H . R . (1991) J . Biol . Chem . 266, 19490-19498) . In contrast, addition of LPS (10 ng/ml) to human monomyelocytic (Mono Mac 6) cells induces very little production of TNF alpha, as judged by immunoassay of the growth medium . When 30 ng/ml 4-beta-phorbol-12-myristate 13-acetate (PMA) is added together with LPS, large amounts of TNF alpha are secreted . PMA alone is inactive . Maximal TNF alpha levels in the medium are achieved at 1 ng/ml of LPS . Protein kinase C inhibitors, such as H7 (1-(5-isoquinolinylsulfonyl)-2-methylpiperazine), staurosporine, and sphingosine, reduce TNF alpha secretion stimulated by PMA . The effect of PMA has been investigated at each stage of TNF alpha biogenesis . Treatment of Mono Mac 6 cells with LPS alone results in rapid, transient, and full expression of TNF alpha mRNA . Concomitant addition of PMA does not increase TNF alpha mRNA synthesis any further, but it prolongs the half-life of TNF alpha mRNA about 3-fold . However, mRNA stabilization does not account for the striking effect of PMA on TNF alpha secretion . Analysis of TNF alpha synthesis and secretion by immunoprecipitation indicates that LPS alone is fully effective in stimulating the formation of the intracellular 26-kDa TNF alpha precursor . LPS alone is not sufficient to allow processing of the precursor and secretion of mature 17-kDa TNF alpha . The rate of TNF alpha secretion observed immediately after the addition of PMA to LPS-pretreated cells is similar to the maximum rate from LPS/PMA-treated cells, but without the lag observed in cells after being exposed to LPS and PMA simultaneously . In summary, PMA is required for the completion of TNF alpha precursor processing and secretion in LPS-treated human Mono Mac 6 cells, whereas murine RAW cells are able to complete the terminal steps of TNF alpha processing in the absence of PMA.

J Chromatogr, 1992 Nov 6, 582(1-2), 253 - 7
High-performance liquid chromatographic monitoring of metabolic products resulting from the treatment of mouse hepatoma cells with N6-cycloalkylated nucleosides; Thedford R et al.; High-performance liquid chromatography was used to analyze cell lysates and growth medium of mouse hepatoma cells, separately treated with N6-cyclopropyl-, N6-cyclobutyl-, and N6-cyclopentyladenosines, in an effort to gain insight into the mechanism by which these modified nucleosides exert their cytotoxic effect(s) . The corresponding 5'-monophosphate of the respective modified nucleoside was detected in the separate cell lysate samples . Both the modified nucleoside and its corresponding 5'-monophosphate were detected in the separate growth medium samples and their relative concentrations therein were determined . These results indicate that the cytotoxicity of these N6-cycloalkylated nucleosides may be attributed to their 5'-monophosphates within the cells.

In Vitro Cell Dev Biol, 1992 Nov-Dec, 28A(11-12), 730 - 4
Relative promoter activity in human mammary epithelial cells assayed by transient expression; Huper G et al.; Chimeric DNA expression vectors containing regulatory sequences proximal to the 5' end of coding sequences for mammalian genes provide valuable tools to study gene expression . Genes coding for easily measured products (reporter genes) can be used to study promoter strength and regulation of gene expression after transient expression of promoter-reporter constructs in mammalian cells . To determine the strength of a variety of mammalian and viral promoter-enhancer sequences in primary cultures of human mammary epithelial cells (HMEC), these sequences were fused to the bacterial chloramphenicol acetyltransferase (CAT) gene and transfected into HMEC using strontium phosphate . The long terminal repeat (LTR) of the endogenous murine leukemia virus AKR-623 was the most potent promoter of transient CAT expression in HMEC . A number of commonly available promoter sequences displayed a wide range of activities in these cells . The glucocorticoid responsive LTR promoter from the murine mammary tumor virus modulated expression of CAT and was sensitive to the concentration of dexamethasone in the growth media . In a similar fashion, the regulatory sequences from the murine metallothionein-1 gene retained responsiveness to zinc concentration in the growth media.

Mol Gen Genet, 1992 Nov, 235(2-3), 242 - 6
Leucine and serine induce mecillinam resistance in Escherichia coli; Bouloc P et al.; We have previously shown that resistance to the beta-lactam mecillinam in Escherichia coli can be brought about by a high ppGpp pool, as observed under conditions of partial amino acid starvation and RelA-dependent induction of the stringent response . We show here that our E . coli wild-type strain, which is sensitive to mecillinam on minimal glucose plates, becomes resistant in the presence of L-leucine or L-serine (or cysteine, which inactivates the antibiotic) . The resistance, which is not a transient effect and does not depend on the physiological state of the cells when plated, is specific for mecillinam and is reversed by the presence of isoleucine and valine in the medium . At least in the case of serine, the resistance is RelA-dependent . We conclude that the presence of leucine and serine in the growth medium cause partial starvation for isoleucine/valine, leading to induction of the stringent response and concomitant resistance to mecillinam.

Cell Prolif, 1992 Nov, 25(6), 643 - 50
Effects of growth media on cell cycle progression in CHO cells exposed to the radioprotector WR-1065; Murley JS et al.; WR-1065 (2-{(aminopropyl)amino}ethanethiol) reduces cytotoxic and mutagenic effects caused by exposure of cells to radiation and chemotherapeutic drugs, but the mechanisms involved are not fully known . We have observed an accumulation of cells in G2 in WR-1065 treated Chinese hamster ovary cells grown in alpha-minimal essential medium, while others have found no cell cycle effects in WR-1065 treated Chinese hamster ovary cells grown in McCoy's 5A medium . To determine if the two types of media had an effect on cells treated with WR-1065, we examined survival and cell cycle progression . Population doubling times of 12 h were observed for cells grown in both media . Incubation of AA8 cells grown in McCoy's 5A medium with 4 mM WR-1065 30 min prior to and during irradiation with 137Cs gamma-rays resulted in a protection factor of 2.2, in close agreement with the value of 2.0 we previously obtained for AA8 cells grown in alpha-minimal essential medium . Treatment with WR-1065 caused an alteration in the cell cycles of cells grown in both media . An increase in the G2 population and a decrease in the G1 population was observed in cells incubated up to 3 h in the presence of 4 mM WR-1065, with a redistribution of the cells throughout the cell cycle occurring following removal of the drug . These data suggest that exposure of cells to WR-1065 is the cause of perturbations in cell cycle progression, and is not affected by the type of medium the cells are grown in.

EMBO J, 1992 Nov, 11(11), 4175 - 85
Comparison of the expression of the seven ribosomal RNA operons in Escherichia coli; Condon C et al.; We have compared the expression of the seven ribosomal RNA operons (rrn) of Escherichia coli and their responses to a variety of physiological and genetic perturbations . We used a set of rrn promoter fusion constructs in their native chromosomal positions to examine effects of chromosomal location on rrn operon expression and the same set of fusions on lambda lysogens to assay intrinsic promoter strengths independent of chromosome context . In its native chromosomal location, expression of the rrnH operon was significantly lower than expected . This effect was not attributable to weak promoter activity and was dependent on the growth medium . The rrnE operon had reduced promoter activity relative to the other ribosomal operons in minimal medium and thus appears to have abnormal growth rate regulation . The ribosomal RNA operons showed varied responses to amino acid starvation; expression of rrnD was inhibited most . There was only a slight increase in rrn transcription in response to a temperature shift (30 degrees C to 42 degrees C) and the differences between individual operons was very small . The rrnG operon showed a significantly lower response than the other ribosomal RNA operons to a depletion of the rrn transcription activator, Fis, and thus appears to have decreased Fis-mediated transactivation . Finally, the chromosomal fusion strains were used to study the effect on growth rate of inactivating each rrn operon . In fast growth conditions, loss of certain rrn operons caused subtle decreases in growth rate on complex medium.

Mech Ageing Dev, 1992 Nov, 66(2), 159 - 71
Heavy metals and lipofuscinogenesis . A study on myocardial cells cultured under varying oxidative stress; Marzabadi MR et al.; The aim of this study was to examine the influence on lipofuscinogenesis of a number of transition and non-transition heavy metals in cultured post-mitotic cells (neonatal rat myocytes) at varying oxidative stress . The effects of Al, Cd, Cr, Cu, Hg, Pb and Zn, added to the medium as chlorides, were examined after 14 days in culture under 5, 20 and 40% ambient oxygen . Lipofuscin was quantified by microspectrofluorometry of individual cells . The addition of Al (40 microM), Cd (40 nM), Hg (30 microM) and Pb (40 microM) to the culture growth medium markedly increased the amount of intracellular lipofuscin, whereas Cr (40 microM), Cu (40 microM) and Zn (40 microM) had the opposite effect . Transmission electron microscopic examination of the myocytes showed greatly increased numbers of autophagic vacuoles in cells exposed to those heavy metals that increased lipofuscin formation . This effect was most pronounced when cells were grown at high (40%) oxygen tension . Possible explanations for the metal augmented pigment formation may be (i) inhibition of lysosomal enzymes, (ii) catalytic interference with peroxidative reactions, or (iii) general toxicity with unspecifically increased autophagocytosis . The decreased pigment accumulation after the addition of Zn, Cr and Cu may, at least partly, be related to the replacement of iron, which has catalytic activity in Fenton reactions.

Mol Microbiol, 1992 Nov, 6(22), 3427 - 37
A plasmid-encoded type IV fimbrial gene of enteropathogenic Escherichia coli associated with localized adherence; Donnenberg MS et al.; Enteropathogenic Escherichia coli (EPEC) form adherent microcolonies on the surface of tissue culture cells in a pattern termed localized adherence . Localized adherence requires the presence of a large EPEC adherence factor (EAF) plasmid . Recently a bundle-forming pilus has been described in EPEC possessing the EAF plasmid . An analysis of 22 non-invasive EPEC TnphoA mutants revealed that seven have insertions in the EAF plasmid and are incapable of localized adherence . We report here the mapping of the TnphoA insertions in these mutants . The nucleotide sequence of the gene interrupted in these TnphoA mutants (bfpA) was determined and found to correspond to the N-terminal amino acid sequence of the major structural protein of the bundle-forming pilus . The bfpA gene bears sequence similarities to members of the type IV fimbrial gene family and encodes a potential site for processing by a prepilin peptidase . A plasmid containing bfpA as the only open reading frame directs the synthesis of a protein recognized by antiserum raised against the bundle-forming pilus . TnphoA mutants at this locus are unable to synthesize BfpA, but synthesis is restored by introduction of a plasmid containing the cloned gene . The minimum fragment of DNA required to restore localized adherence is considerably greater than that required to restore BfpA synthesis . BfpA expression, as assessed by alkaline phosphatase activity in bfpA::TnphoA mutants, is affected by temperature and growth medium . These studies describe an EPEC plasmid-encoded fimbrial gene, a candidate for the elusive EPEC adherence factor responsible for localized adherence.

Mol Microbiol, 1992 Nov, 6(21), 3159 - 69
A putative anaerobic coproporphyrinogen III oxidase in Rhodobacter sphaeroides . I . Molecular cloning, transposon mutagenesis and sequence analysis of the gene; Coomber SA et al.; A mutant of Rhodobacter sphaeroides, N1, has been isolated which is incapable of photosynthetic growth and, instead of synthesizing bacteriochlorophyll, N1 excretes coproporphyrin III into the growth medium . Using conjugative gene transfer, several clones were isolated from a R . sphaeroides gene library which restored normal pigment synthesis and photosynthetic growth to N1 . Using transposon Tn5 mutagenesis, the gene was located to a 1.05 kb EcoRI fragment . Sequence and transcription analysis defined the position and expression of an open reading frame of approximately 920 bp, which is proposed as the anaerobic coproporphyrinogen III oxidase dedicated to bacteriochlorophyll biosynthesis.

Proc Natl Acad Sci U S A, 1992 Nov 1, 89(21), 10395 - 9
Zinc rapidly induces a metal response element-binding factor; Czupryn M et al.; Metal activation of metallothionein gene transcription is mediated by specific promoter sequences, termed metal regulatory elements (MREs) . Nuclear extracts prepared from various human cell lines were assayed for their capacity to bind to a synthetic human MREa (hMREa) oligomer . Electrophoretic mobility-shift assays with extracts from control cells detected a single hMREa-containing complex . Addition to the growth medium of zinc, cadmium, or copper--metals known to induce MT biosynthesis in vivo--resulted in the rapid but reversible appearance of a second distinct hMREa-protein complex in all cell lines studied . This result was not seen when the metals were added directly to the extracts from control cells . DNA-binding protein blotting, UV crosslinking, and electroelution experiments were used to characterize the two hMREa-binding factors, termed BF1 and BF2 . MRE-BF1 has an apparent molecular mass of approximately 86 kDa and binds to the hMREa in control cells, whereas MRE-BF2 consists of two molecules of approximately 28 kDa and binds to the hMREa in metal-treated cells . EDTA and o-phenanthroline inhibited binding of both factors to hMREa in a dose-dependent manner, indicating that a metal atom or atoms are essential for interaction of the factors with DNA.

J Dermatol, 1992 Nov, 19(11), 652 - 3
Stimulation of keratinocyte migration by growth factors; Tsuboi R et al.; Migration of keratinocytes from the wound edge is thought to be one of the critical features of reepithelialization . A quantitative migration assay was carried out using normal human keratinocytes . Keratinocytes, seeded on 12 well plates, were grown in serum free, keratinocyte growth medium (KGM, Curabo Co) with 0.08 mM Ca2+ . The medium was switched from KGM to keratinocyte basal medium (KBM) 6 h prior to the wounding . Half of the plate's confluent monolayer of keratinocytes was removed with razor blade, and the remaining keratinocytes were incubated in KBM for 16 hrs in the presence of indicated growth factors . After incubation, the cells were fixed and counted at 100 magnification . Migration was quantitated by counting the number of cells in ten successive 125-microns zones . Transforming growth factor alpha (TGF-alpha), acidic and basic fibroblast growth factor (aFGF, bFGF), keratinocyte growth factor (KGF), hepatocyte growth factor (HGF), and insulin-like growth factor-I (IGF-I) stimulated the migration of keratinocytes, while TGF-beta suppressed it.

Biochim Biophys Acta, 1992 Oct 30, 1128(2-3), 250 - 7
The phosphonic acid analog of phosphatidylglycerol phosphate: influence on Escherichia coli growth and physiology; Ke L et al.; At 20 microM, rac-3,4-dihydroxybutyl-1-phosphonate (DBP) has only a slight bacteriostatic effect on Escherichia coli . However, cells lose viability when the medium also contains either 20 mM magnesium or calcium ions . Magnesium ions stimulate the incorporation of DBP into (1,2-diacyl)-sn-glycerol-D-4'-phosphoryloxy-3'-hydroxybutyl-1'-pho sphonate, the phosphonate analog of phosphatidylglycerol phosphate . Much higher DBP concentrations are needed to block the growth of a pgsA3 mutant than to block the growth of an isogenic wild-type strain . The DBP-treated pgsA mutant also has a much higher survival rate when stored in the cold than does the DBP-treated wild-type strain . Furthermore, the pgsA3 mutant grows normally in the presence of DBP and magnesium ions . Treatment with DBP and magnesium ions does not appear to disrupt the cell's inner or outer membranes . However, it does block macromolecular and phosphoglyceride synthesis . A combination of 20 microM rac-DBP and 0.5 mM spermidine or 0.125 mM spermine is bacteriostatic . These studies indicate that the PGP analog contributes to DBP's bacteriostatic effect when the growth medium contains low concentrations of magnesium or calcium ions and is responsible for its bactericidal effect when the medium contains high concentrations of these ions.

FEBS Lett, 1992 Oct 12, 311(1), 60 - 2
Effect of thiamin on cordycepin sensitivity in Saccharomyces cerevisiae; Iwashima A et al.; The sensitivity of Saccharomyces cerevisiae to the antibiotic cordycepin (3'-deoxyadenosine) was found to be decreased by the addition of thiamin to the growth medium . A thiamin transport mutant of S . cerevisiae was also found to be resistant to the growth inhibitory effect of cordycepin . Not only the thiamin uptake but also adenosine uptake by this mutant cell was markedly reduced compared to those by the parent yeast cells . This strongly suggested that cordycepin, an analog of adenosine, is virtually taken up by the thiamin transport system of growing yeast cells; thus the drug sensitivity is decreased by the presence of thiamin in the growth medium.

J Cell Physiol, 1992 Oct, 153(1), 112 - 7
Apoptosis of murine BW 5147 thymoma cells induced by cold shock; Kruman II et al.; Exposure of thymoma BW 5147 cells to cold (0-2 degrees C) followed by rewarming at 37 degrees C (cold shock) resulted in internucleosomal DNA cleavage . Sensitivity to cold shock-induced cell death was critically dependent on the serum concentration in the medium and limited to serum-deficient medium (2% serum concentration), whereas cells in the complete growth medium (10%) were completely resistant . RNA/protein-synthesis inhibitors (cycloheximide and actinomycin D) had no effect on cold shock-induced DNA cleavage in BW 5147 cells . The DNA fragmentation seems to be independent of increase in the cytosolic Ca2+ level . Moreover, reduction in the calcium content of the external medium by EGTA induced DNA cleavage . Incubation of BW 5147 cells in the presence of colchicine and cytochalasin B led to the apoptosis . The latter suggests that the internucleosomal DNA cleavage induced by cold shock may be concerned with the disruption of some cytoskeletal network caused by cooling . The results are discussed in relation to cell proliferation.

J Cell Physiol, 1992 Oct, 153(1), 103 - 11
Heparin inhibition of autonomous growth implicates amphiregulin as an autocrine growth factor for normal human mammary epithelial cells; Li S et al.; Normal human mammary epithelial cells (HMECs) proliferate in a serum-free defined growth medium in the absence of epidermal growth factor (Li and Shipley, 1991) . Amphiregulin (AR) is a heparin-regulated, EGF-like growth factor . Our observation that one strain of HMECs produce AR mRNA (Cook et al., 1991 a) stimulated us to determine whether AR expression was a common phenomenon in HMECs and whether AR could act as an autocrine growth factor to support the EGF-independent growth of these cells . In this study, we detected high levels of AR expression in four separate HMEC strains while one immortal mammary cell line (HBL-100) and six mammary tumor-derived cell lines had low to undetectable levels of AR . The EGF-independent growth of HMECs was blocked by the addition of heparin or a monoclonal anti-EGF receptor antibody to the culture medium, implicating AR as an autocrine growth mediator . This hypothesis is further supported by the fact that medium conditioned by HMECs contains secreted AR protein . A mammary tumor-derived cell line, Hs578T, which proliferates in an EGF-independent manner, does not express detectable levels of AR and is not growth inhibited by heparin . Examination of the same cell types for expression of transforming growth factor type-alpha (TGF-alpha) mRNA revealed coordinate expression of AR and TGF-alpha in these cells . These data suggest that both AR and TGF-alpha mRNA are produced in much greater abundance by normal HMECs than in tumor-derived cells in culture, and that AR is an important autostimulatory factor for the growth of normal HMECs.

Int J Dev Neurosci, 1992 Oct, 10(5), 413 - 9
NMDA receptor levels in chronically depolarized long-term neonatal rat neocortical explants; Baker RE et al.; The levels of the N-methyl-D-aspartate subclass of glutamate receptor were determined in organotypic neocortical explants chronically exposed to a growth medium containing 25 mM potassium (K25) . Explants exposed to 25 mM potassium for 2-3 weeks evinced significantly less binding of the non-competitive N-methyl-D-aspartate receptor-associated channel antagonist 125I-MK801 than did age-matched controls . Surprisingly, cultures that were returned to control growth medium for a further 2 or 7 days showed even less binding of the ligand . The Kd values of binding were not affected and were similar to those of fresh postnatal cortex . The maximum number of binding sites did not vary between postnatal day 6 and 14 days in vitro control cultures, but were significantly less than those measured at postnatal day 20 (comparable age: 14 days in vitro) . Several conclusions can be drawn from these findings: (i) the density of the N-methyl-D-aspartate subclass glutamate receptor does not attain in vivo levels under the present culturing conditions, but remains at those levels associated with the stage of development at which the tissue was brought into culture, (ii) chronic depolarization results in a drastic reduction in N-methyl-D-aspartate receptor density, which is not compensated for after the return to normal growth conditions, (iii) depolarization selectively inhibits cellular maturation of the neocortex (but not survival, as shown previously), including neurotransmitter receptor production and/or insertion into membranes or assembly.

Glycoconj J, 1992 Oct, 9(5), 265 - 73
Ganglioside alterations in YAC-1 cells cultivated in serum-supplemented and serum-free growth medium; Muthing J et al.; Gangliosides of the 'GM1b-pathway' (GM1b and GalNAc-GM1b) have been found to be highly expressed by the mouse T lymphoma YAC-1 grown in serum-supplemented medium, whereas GM2 and GM1 ('GM1a-pathway') occurred only in low amounts {Muthing, J., Peter-Katalinic, J., Hanisch, F.-G., Neumann, U . (1991) Glycoconjugate J 8:414-23} . Considerable differences in the ganglioside composition of YAC-1 cells grown in serum-supplemented and in well defined serum-free medium were observed . After transfer of the cells from serum-supplemented medium (RPMI 1640 with 10% fetal calf serum) to serum-free medium (RPMI 1640 with well defined supplements), GM1b and GalNAc-GM1b decreased and only low amounts of these gangliosides could be detected in serum-free growing cells . The expression of GM1a was also diminished but not as strongly as that of GM1b and GalNAc-GM1b . These growth medium mediated ganglioside alterations were reversible, and the original ganglioside expression was achieved by readaptation of serum-free growing cells to the initial serum-supplemented medium . On the other hand, a 'new' ganglioside, supposed to represent GalNAc-GD1a and not expressed by serum-supplemented growing cells, was induced during serum-free cultivation, and increased strongly after readaptation . These observations reveal that the ganglioside composition of in vitro cultivated cells can be modified by the extracellular environment due to different supplementation of the basal growth medium.

Hum Antibodies Hybridomas, 1992 Oct, 3(4), 206 - 11
Growth of hybridoma cells and antibody production in agamma calf serum; Torres AR et al.; An agamma calf serum (ACS) was compared to fetal bovine serum (FBS) and calf serum supplemented growth media for both murine and human fusion partners and derived hybridoma cells . The variables analyzed were cloning efficiencies, growth characteristics, and MAb production levels . Cultures were established in each serum source, and supernatants were kept for analysis . For cloning efficiencies, the results indicate that although there is no clear advantage in the performance of FBS-supplemented medium, ACS-supplemented RPMI 1640 medium can be used to clone hybridomas and the fusion partners . When analyzed for MAb production in the various sera, the hybridoma cell lines used in this study appeared to produce up to twice as much immunoglobulin when grown in ACS as when grown in supplemented medium . IgG- and IgM-secreting hybridoma cultures should be checked independently for antibody production . Six out of eight murine and human hybridoma cell lines produced higher levels of MAbs when grown in ACS.

Vet Microbiol, 1992 Oct, 32(3-4), 363 - 74
In vitro stimulation of antibody production to Mycoplasma hyopneumoniae by porcine peripheral blood mononuclear cells; Wallgren P et al.; A method to stimulate and detect the in vitro production of antibodies to Mycoplasma hyopneumoniae by porcine peripheral blood mononuclear cells (PBMC) was established . PBMC were cultured in microtiter plates coated with a sonicated M . hyopneumoniae whole cell antigen and the amount of antibody bound to the coating antigen was determined by an enzyme linked immunosorbent assay (ELISA) . In addition, the amount of non-bound antibody was determined by testing the culture supernatants in the ELISA which detects porcine antibodies to M . hyopneumoniae . The production of antibodies, in terms of total absorbance values, was enhanced by including 2.5 ng pokeweed mitogen (PWM) per ml growth medium without altering the specificity of the assay . In a pilot experiment, the applicability of the method to follow the development of antigen-reactive cells during primary and secondary immunizations with M . hyopneumoniae was evaluated . Antigen-reactive cells, identified by their ability to produce antibodies to M . hyopneumoniae in vitro, were detected seven days after the primary immunization and reached their highest antigen reactivity one week later . In comparison, antigen-reactive cells could be detected three days after the booster immunization and remained in the circulation for 2 weeks.

Parasitology, 1992 Oct, 105 ( Pt 2), 193 - 202
Axenic cultivation and characterization of Leishmania mexicana amastigote-like forms; Bates PA et al.; A new method is described which has made possible the long-term axenic cultivation of Leishmania mexicana amastigote-like forms in Schneider's Drosophila medium supplemented with 20% (v/v) foetal calf serum . Unlike previous methods, it utilizes direct culture of parasites obtained from the lesions of infected animals rather than adaptation of promastigotes in vitro . Ultrastructural (possession of megasomes), biochemical (cysteine proteinase activity and gelatin SDS-PAGE banding pattern) and infectivity (in vivo) data are presented which show the close similarity of the cultured forms to lesion amastigotes . The axenically cultured forms grew optimally at a temperature of 32-33 degrees C, providing further evidence for their amastigote nature . It was found that adjustment of the pH of the growth medium to 5.4 was required in order to retain the amastigote morphology of the cultured parasites . This supports the notion that leishmanial amastigotes are acidophiles.

Neurosurgery, 1992 Oct, 31(4), 717 - 24; discussion 724
Protein kinase C activity correlates with the growth rate of malignant gliomas: Part II . Effects of glioma mitogens and modulators of protein kinase C; Couldwell WT et al.; The proliferation rates of gliomas may be modulated by the protein kinase C (PKC) signal transduction system . The present study was undertaken to further examine the role of PKC system in growth regulation of gliomas in vitro by measurement of PKC activity over various phases of tumor growth and by assessing its potential role as a signal transduction system induced by serum mitogens and the known glioma mitogens epidermal growth factor and fibroblast growth factor . All human glioma lines examined, and the rat glioma C6, displayed high PKC activity relative to nonmalignant glial cells, which correlated with their proliferation rates over their respective growth phase . Frozen surgical human malignant glioma specimens also displayed high PKC activity . The relatively selective PKC inhibitor staurosporine (SP) reduced PKC activity and corresponding growth rates in a dose-related manner . Stimulation of PKC with phorbol esters under different concentrations of serum in the growth medium indicated that the high PKC activity, which correlated with their rapid growth rates, is highly susceptible to down-regulation by these agents . Epidermal growth factor and fibroblast growth factor increased both PKC activity and the growth rate of glioma line A172; addition of SP reduced the growth rate to levels observed in SP-treated control tumors, indicating that PKC may be a common signal transduction system induced by these mitogens . These results implicate PKC as an important signal transduction system regulating glioma growth, and offers a potential target for tumor inhibition.

J Clin Endocrinol Metab, 1992 Oct, 75(4), 1081 - 6
Biosynthesis of interleukin-6 by cultured human chorion laeve cells: regulation by cytokines; Dudley DJ et al.; Intrauterine infection is an important cause of preterm labor and delivery and is characterized by increased production of inflammatory cytokines by gestational tissues . We evaluated the biosynthesis of the inflammatory cytokine interleukin-6 (IL-6) by human chorion laeve cells and its regulation by other cytokines essential to the inflammatory process . We found that cultured chorion cells secrete IL-6 in the presence of growth medium supplemented only with 10% fetal calf serum . IL-1 beta, tumor necrosis factor, and lipopolysaccharide all induced a significant concentration-dependent stimulation of IL-6 production by chorion cells . The concentration range of each cytokine tested (0.1-10 ng/mL) is within the range of values found in the amniotic fluid of women destined to deliver preterm due to infection of gestational tissues . Additionally, treatment of chorion cells with IL-1 beta in combination with actinomycin-D or cycloheximide attenuated the stimulatory action of IL-1 beta on IL-6 production . Northern blot analysis of total RNA from cultured chorion cells stimulated with IL-1 beta demonstrated that IL-6 mRNA increases over time . Our data suggest that IL-6 is produced by human fetal chorion in response to infection of maternal gestational tissues . In conjunction with other inflammatory mediators, fetally derived IL-6 may play a role in the pathophysiology of preterm labor due to infection.

J Bacteriol, 1992 Oct, 174(19), 6191 - 7
Selective synthesis and labeling of the polysialic acid capsule in Escherichia coli K1 strains with mutations in nanA and neuB; Vimr ER; The enzymes required for polysialic acid capsule synthesis in Escherichia coli K1 are encoded by region 2 neu genes of the multigenic kps cluster . To facilitate analysis of capsule synthesis and translocation, an E . coli K1 strain with mutations in nanA and neuB, affecting sialic acid degradation and synthesis, respectively, was constructed by transduction . The acapsular phenotype of the mutant was corrected in vivo by exogenous addition of sialic acid . By blocking sialic acid degradation, the nanA mutation allows intracellular metabolite accumulation, while the neuB mutation prevents dilution by the endogenous sialic acid pool and allows capsule synthesis to be controlled experimentally by the exogenous addition of sialic acid to the growth medium . Complementation was detected by bacteriophage K1F adsorption or infectivity assays . Polysialic acid translocation was observed within 2 min after addition of sialic acid to the growth medium, demonstrating the rapidity in vivo of sialic acid transport, activation, and polymerization and translocation of polysaccharide to the cell surface . Phage adsorption was not inhibited by chloramphenicol, demonstrating that de novo protein synthesis was not required for polysialic acid synthesis or translocation at 37 degrees C . Exogenous radiolabeled sialic acid was incorporated exclusively into capsular polysaccharide . The polymeric nature of the labeled capsular material was confirmed by gel permeation chromatography and susceptibility of sialyl polymers to K1F endo-N-acylneuraminidase . The ability to experimentally manipulate capsule expression provides new approaches for investigating polysialic acid synthesis and membrane translocation mechanisms.

Int J Biochem, 1992 Oct, 24(10), 1625 - 31
Modulation of ribonuclease P expression in Escherichia coli by polyamines; Panagiotidis CA et al.; 1 . The presence of polyamines in the growth medium of Escherichia coli can modulate the activity of the RNA-processing enzyme, ribonucleoprotein ribonuclease P (RNase P), by altering the expression of the rnpA and rnpB genes, which encode its C5 protein and M1 RNA subunits, respectively . 2 . Following growth in the presence of 1 mM spermidine the levels of C5 protein mRNA and catalytic M1 RNA were significantly elevated in the wild type E . coli K-12 strain MG1655 . 3 . The rnpA mRNA, together with the ribosomal protein L34 (rpmH) mRNA, was found to constitute a dicistronic rpmH-rnpA message whose half-life did not change upon Escherichia coli growth in the presence of spermidine . 4 . This suggests that the spermidine effect is on the transcriptional level . 5 . Increased expression of the rnpA and rnpB genes was reflected in the activity of RNase P, which almost doubled . 6 . These results identify yet another component of the protein synthetic machinery which is specifically affected by polyamines.

Endocrinology, 1992 Oct, 131(4), 2024 - 6
Screening for hybridomas producing antibodies to steroid hormone receptors: interference from phenol red in the hybridoma culture supernates; Raam S; The culture supernates from a variety of mouse hybridomas which were known to secrete antibodies to antigens unrelated to steroid hormone receptors were examined for routine use as experimental controls for monoclonal anti-oestrogen receptor (ER) antibodies . During this process, we made an observation which is important for all investigations on monoclonal anti-receptor antibodies . Our results indicated that the phenol red dye present in these hybridoma culture fluids by binding to the immunoglobulins (Ig) secreted by the hybridoma cells, may impart to the Ig-dye complex, a capacity to interact with ER/progesterone receptors(PR) which are present in the cytosols or in the sections of ER+ tumors . The Ig-dye may thus mimic anti-receptor antibody activity . Growth media containing phenol red and Ig-free bovine calf serum supplement or culture fluids from a non-Ig secreting hybridoma failed to show similar binding to ER+ tissues . This observation documents the need for Ig-dye complex formation to obtain such ER-Ig interaction . Complete dialysis of phenol red from the Ig positive supernates removed the observed interference of the dye . In this report we describe the nature of this interference and the results obtained before and after the removal of the dye from Ig+ culture fluids . In addition, we suggest some remedial measures that should be considered, while screening and testing the specificity of any hybridoma culture product for anti-receptor antibody activity (particularly, anti-ER or anti-PR antibodies).

Neurosci Lett, 1992 Sep 28, 145(1), 1 - 5
Simultaneous measurement by HPLC of the excitatory amino acid transmitter candidates homocysteate and homocysteine sulphinate supports a predominant astrocytic localisation; Grieve A et al.; Primary cultures of mouse cerebral cortex neurons, cerebellar granule cells and cortical astrocytes were maintained in vitro for respectively 8-10, 7-10 and 21-24 days . Following these times, amino acids were extracted from the cells by use of ice-cold 70% (v/v) ethanol and the extracts lyophilised . The lyophilised extracts when resuspended were subjected to reverse-phase high performance liquid chromatographic (HPLC) analysis for detection of free amino acids . Samples of cell culture growth medium and water blanks were treated in a similar manner . Identification of L-homocysteate (HCA) and L-homocysteine sulphinate (HCSA) was undertaken by matching retention times with regard to external standards and by 'spiking' cell extracts with authentic compounds . On this basis, HCA and HCSA were consistently detectable in astrocytes at levels of, respectively, 72.3 +/- 33.7 pmol/mg protein (n = 24) and 49.4 +/- 28.7 pmol/mg protein (n = 24) . However, in neurons, a peak corresponding to HCSA could not be detected above the background noise, while the area of the peak corresponding to HCA was always greater than, but not significantly different from, that of the background noise present in water blanks . HCA and HCSA were not detectable in the serum used for preparation of the cell culture growth medium . Taken together, these findings indicate a predominant localisation of HCA and HCSA in astrocytes which, at least in culture, appear to possess the metabolic machinery necessary for synthesising and storing these amino acids without any neuronal influence.

J Biol Chem, 1992 Sep 25, 267(27), 19054 - 9
Copper-mediated regulation of cytochrome c553 and plastocyanin in the cyanobacterium Synechocystis 6803; Zhang L et al.; In certain cyanobacteria and algae, cytochrome c553 or plastocyanin can serve to carry electrons from the cytochrome bf complex to photosystem I . The availability of copper in the growth medium regulates which protein is present . To investigate copper induced control of gene expression we isolated these proteins from the cyanobacterium Synechocystis 6803 . Using immunodetection and optical spectroscopy, the steady state levels of cytochrome c553 and plastocyanin were measured in cells grown at different copper concentrations . The results show that in cells grown in 20-30 nM copper, cytochrome c553 was present, whereas plastocyanin was not detected . The opposite behavior was observed in cells grown in the presence of 1 microM copper; plastocyanin was present, whereas cytochrome c553 could not be detected . Both proteins were present in cells grown in 0.3 microM copper . Northern analysis of total RNA, probed with a gene fragment for cytochrome c553 or the plastocyanin gene, showed that cells grown in the presence of 20-30 nM copper have message for cytochrome c553, but not for plastocyanin, whereas cells grown in 1 microM copper have message for plastocyanin, but not for cytochrome c553 . These results demonstrate that copper regulates expression of both of the genes encoding cytochrome c553 and plastocyanin prior to translation in Synechocystis 6803.

Biochim Biophys Acta, 1992 Sep 21, 1110(1), 51 - 8
Nucleoside transport-deficient mutants of PK-15 pig kidney cell line; Aran JM et al.; Previous studies indicated that PK-15 pig kidney cells express solely a nitrobenzylthioinosine-sensitive, equilibrative nucleoside transporter . In the present study, PK-15 cells were mutagenized by treatment with ICR-170 and nucleoside transport-deficient mutants selected in a single step in growth medium containing tubercidin and cytosine arabinoside at a frequency of about 2 x 10(6) . The mutants were simultaneously at least 100-times more resistant to tubercidin, cytosine arabinoside and 5-fluorodeoxyuridine than the wild-type parent cells . The mutants failed to transport thymidine and uridine and had lost all high affinity nitrobenzylthioinosine binding sites . Residual low level uptake of thymidine by the mutants was shown to be due to nonmediated permeation (passive diffusion), which explains the sensitivity of the mutants to growth inhibition by high concentrations of the nucleoside drugs . Passive diffusion of thymidine at a concentration of 16 microM was not rapid enough to support the growth of nucleoside transport-deficient mutant cells that had been made thymidine-dependent by treatment with methotrexate, whereas wild-type cells grew normally under these conditions . The nucleoside transport-deficient mutants exhibited about the same growth rate and plating efficiency (60-80%) as wild-type cells, but formed larger colonies than wild-type cells because of a more extensive spread of the cells on the surface of culture dishes . PK-15 cells adhere very strongly to the surface of culture dishes and have been transformed with high efficiency with plasmid DNA either via lipofection or electroporation.

FEMS Microbiol Lett, 1992 Sep 15, 75(2-3), 207 - 11
Metal ion-catalysed hydrolysis of ampicillin in microbiological growth media; Beard SJ et al.; Anodic stripping voltammetry of bacterial growth medium containing copper(II) and ampicillin shows that Cu(II) is complexed by the antibiotic and that this complex decomposes to give Cu(II) complexes with ligands derived from ampicillin . At pH 7, substantial decomposition of ampicillin occurs over a few minutes, and even the very low levels of Cu(II) in Chelex-extracted medium are able effectively to catalyse the decomposition . The significance of this observation was shown during the screening of an Escherichia coli cosmid library for clones exhibiting increased resistance to Zn(II), Co(II) or Cd(II); the unexpected growth of the ampicillin-sensitive host E . coli strain on Luria-Bertani plates containing ampicillin and any of these metals was attributed to metal ion-catalysed decomposition of ampicillin . The instability of ampicillin (and other beta-lactam antibiotics) to metal ion-catalysed hydrolysis means that great care must be taken to ensure that such reactions do not occur in growth media . Furthermore, it is clear that double selection for resistance to ampicillin and metals such as Cu(II), Zn(II), Co(II) and Cd(II) is impossible.

Nucleic Acids Res, 1992 Sep 11, 20(17), 4515 - 23
Preparation of isotopically labeled ribonucleotides for multidimensional NMR spectroscopy of RNA; Batey RT et al.; A general method for large scale preparation of uniformly isotopically labeled ribonucleotides and RNAs is described . Bacteria are grown on isotopic growth medium, and their nucleic acids are harvested and degraded to mononucleotides . These are enzymatically converted into ribonucleoside triphosphates, which are used in transcription reactions in vitro to prepare RNAs for NMR studies . For 15N-labeling, E.coli is grown on 15N-ammonium sulfate, whereas for 13C-labeling, Methylophilus methylotrophus is grown on 13C-methanol, which is more economical than 13C-glucose . To demonstrate the feasibility and utility of this method, uniformly 13C-labeled ribonucleotides were used to synthesize a 31 nucleotide HIV TAR RNA that was analyzed by 3D-NMR . This method should find widespread use in the structural analysis of RNA by NMR.

Nucleic Acids Res, 1992 Sep 11, 20(17), 4499 - 505
Thiolation of transfer RNA in Escherichia coli varies with growth rate; Emilsson V et al.; We have used an affinity electrophoresis assay which when combined with Northern hybridization techniques permits us to estimate the degree of thiolation of individual tRNA species in Escherichia coli . We observe that the levels of 4-thio 2'(3')-uridine (4-thioU) in many but not all tRNAs varies dramatically at different bacterial growth rates: Five tRNAs are completely thiolated at all growth rates, while another eight tRNAs are incompletely thiolated and the fraction of the unthiolated form of these tRNA species increases as the growth rates increase . Transfer RNA(2Glu) contains 4-thioU as well as (methylamino)methyl-2-thio uridine (mnm(5)2-thioU) . The level of mnm(5)2-thioU of tRNA(2Glu) is invariant with growth rate . Surprisingly, none of the thirteen tRNA species that we have studied is completely unmodified in all growth media . In particular, at the slowest growth rates every tRNA class that we have studied contains a form that has 4-thioU residues.

J Bacteriol, 1992 Sep, 174(18), 5780 - 7
Cell surface modifications induced by calcium ion in the myxobacterium Stigmatella aurantiaca; Chang BY et al.; Calcium ion induces in the myxobacterium Stigmatella aurantiaca the ability to glide on solid surfaces and to become cohesive (D . F . Gilmore and D . White, J . Bacteriol . 161:113-117, 1985; B . J . Womack, D . F . Gilmore, and D . White, J . Bacteriol . 171:6093-6096, 1989) . The addition of calcium ion to the growth medium resulted in the formation of extracellular fibrils, the appearance in the membrane fractions of a 30-kDa protein, and the accumulation in a low-speed centrifugal pellet of 10 polypeptides that cross-reacted with affinity-purified antibody to one of the polypeptides . One of the polypeptides, a 55-kDa protein, was present in the membrane fraction of control cells not incubated with calcium ion and was apparently translocated to the extracellular matrix during incubation in medium containing calcium ion . The 55-kDa protein was immunologically related to a 65-kDa protein located on the fibrils of another myxobacterium, Myxococcus xanthus.

Chest, 1992 Sep, 102(3), 832 - 7
Proliferative characteristics of fibroblast lines derived from open lung biopsy specimens of patients with IPF (UIP); Mio T et al.; We compared the doubling time of fibroblasts derived from idiopathic pulmonary fibrosis (usual interstitial pneumonia) (IPF {UIP}) lung tissues and control fibroblasts, cultured in usual growth medium, and examined the response of these fibroblasts to platelet-derived growth factor (PDGF) and prostaglandin E2 (PGE2) . Ten fibroblast lines from open lung biopsy specimens of patients with IPF (UIP) and ten control fibroblast lines from surgically resected lung tissue of patients with limited lung diseases were established . The average doubling time of fibroblast lines was 32.0 +/- 6.0 h (mean +/- SD) in UIP and 33.2 +/- 10.4 h in controls, showing no difference between the two groups . To examine the responses of fibroblasts to PDGF and PGE2 and the differences between fibroblasts derived from fibrotic tissues with different intensity of fibrosis, lung specimens from five patients with IPF were subdivided into two groups, higher-intensity fibrotic lesions (H) and lower-intensity fibrotic lesions (L) . The fibroblast lines were established separately . 3H-thymidine uptake with or without PDGF or PGE2 was examined . Results were expressed as the index of thymidine incorporation into the fibroblasts . There were no differences in the doubling times and the responses to PDGF and PGE2 between H and L . There were no differences between control and H regarding their response to PDGF . In response to PGE2, the growth inhibition for H was significantly decreased compared with the control (p less than 0.05) . There was no difference in growth inhibition between H and L . The finding that PGE2 inhibits fibroblast proliferation less in UIP lung tissue suggests that fibroblasts from UIP were functionally altered cells or, to some extent, out of normal regulation . These results suggest an abnormal proliferation of fibroblasts observed in IPF (UIP).

Mol Cell Biol, 1992 Sep, 12(9), 3678 - 88
COT1, a gene involved in cobalt accumulation in Saccharomyces cerevisiae; Conklin DS et al.; The COT1 gene of Saccharomyces cerevisiae has been isolated as a dosage-dependent suppressor of cobalt toxicity . Overexpression of the COT1 gene confers increased tolerance to cobalt and rhodium ions but not other divalent cations . Strains containing null alleles of COT1 are viable yet more sensitive to cobalt than are wild-type strains . Transcription of COT1 responds minimally to the extracellular cobalt concentration . Addition of cobalt ions to growth media results in a twofold increase in COT1 mRNA abundance . The gene encodes a 48-kDa protein which is found in mitochondrial membrane fractions of cells . The protein contains six possible membrane-spanning domains and several potential metal-binding amino acid residues . The COT1 protein shares 60% identity with the ZRC1 gene product, which confers resistance to zinc and cadmium ions . Cobalt transport studies indicate that the COT1 product is involved in the uptake of cobalt ions yet is not solely responsible for it . The increased tolerance of strains containing multiple copies of the COT1 gene is probably due to increased compartmentalization or sequestration of the ion within mitochondria.

Diabetes, 1992 Sep, 41(9), 1050 - 5
Variation in sorbitol accumulation and polyol-pathway activity in cultured human proximal tubule cells; Flath MC et al.; The polyol pathway is present in tissues of several organs where its activation may participate in the development of diabetic complications . We measured the accumulation of polyol-pathway intermediates in HPT cells isolated from 21 different human kidneys from nondiabetic individuals . When exposed to 27.5 mM glucose in the growth media, cells isolated from approximately 75% of individuals (accumulators) accumulated sorbitol within 1-4 days, whereas 25% (nonaccumulators) accumulated only negligible amounts, even when the period of exposure was extended to 2 wk . Surprisingly, measurement of the activities of the polyol-pathway enzymes showed no difference in the levels of either AR or SDH between accumulators and nonaccumulators, even when the conversion of galactose to galactitol was used to measure AR activity in intact cells independently of SDH . Measurement of sorbitol in the growth media indicated that nonaccumulators were not releasing sorbitol into the growth media . Fructose levels in the conditioned growth media were 4 times higher in the sorbitol-accumulating cells . Together, these results indicate that the tendency of cells from an individual to accumulate significant amounts of sorbitol may reflect the cells' ability to metabolize sorbitol in steps subsequent to the polyol pathway.

Neurobiol Aging, 1992 Sep-Oct, 13(5), 543 - 51
Effect of beta amyloid peptides on neurons in hippocampal slice cultures; Malouf AT; Several investigators have described the neurotrophic and neurotoxic effects of beta amyloid peptide fragments on dissociated hippocampal neurons in culture . In these prior studies, the peptides were added to dissociated cultures between day 0 and day 4 in vitro, before hippocampal neurons are fully mature . We have analyzed the neurotrophic and neurotoxic effects of beta amyloid fragments beta 1-28, beta 25-35 and beta 1-40 on hippocampal slice cultures, whose physiology and morphology resembles the intact hippocampus . Addition of beta 1-28 or beta 25-35 to the growth medium did not produce significant changes in dendritic length or number of branches . Nerve growth factor, previously reported to enhance the neurotoxic effects of beta 1-40 on dissociated hippocampal neurons in culture, did not significantly enhance the neurotrophic effects of beta 1-28 . To achieve high local concentrations of peptides and to avoid potential access problems in the cultures, we injected beta 1-28, beta 25-35, and beta 1-40 directly into the cultures . Amyloid-mediated neurotoxicity was not observed for beta 1-28 or beta 25-35, but beta 1-40 appeared to produce neurodegeneration around the site of injection.

Anticancer Res, 1992 Sep-Oct, 12(5), 1559 - 63
Effect of calcium-modifying agents on the growth of human gastric carcinoma cells in vitro; Piontek M et al.; The antiproliferative effects of various calcium-modifying agents were investigated in human AGS gastric carcinoma cells in culture . Variation of the extracellular calcium concentration, achieved by addition of calcium to the growth medium or binding of calcium to the calcium-chelating agent EDTA, appeared to have little influence on growth of the tumor cells . In contrast, the calcium antagonist verapamil, the calcium ionophore A 23.187 and the calmodulin antagonist W 7, agents supposed to interfere with the regulation of the intracellular calcium concentration, all exerted marked growth inhibiting effects . Our results provide evidence for an important role of intracellular calcium-dependent mechanisms in growth regulation of the human gastric adenocarcinoma cell line AGS.

Cell Biochem Funct, 1992 Sep, 10(3), 217 - 24
Modifications of adhesion properties and proteoglycan structure in rat embryo fibroblast cultures with increasing passages; Pippia P et al.; Adhesion properties of rat embryo fibroblast cultures and proteoglycans (PGs) produced both in the growth medium and in the cell layer were investigated with increasing passages . Both cell-cell and cell-substrate adhesion increased with increasing subculture number . Cell adhesion properties were improved by cell treatment with chondroitinase ABC . The increase in subculture number was coupled with a constant increase of PG molecular size, which was particularly evident in cell layer extracts . The ratio HS-PGs/DS-PGs increased with increasing passages . PG modifications are likely to represent evidence of changes in extracellular matrix organization and could play a role in the increase of cell adhesion properties.

Exp Eye Res, 1992 Sep, 55(3), 469 - 78
Immunohistochemical localization of Na,K-ATPase in the in situ lens, cultured human lens epithelium and lentoid; Tsuji T et al.; The localization of Na,K-ATPase in the lens is quite controversial . We explored this problem through immunoelectron microscopic examination of rat and human lens . Unlike previously reported results, we have found that Na,K-ATPase is localized in the basal plasma membrane, but not in the lateral or apical plasma membrane of both rat and human lens epithelium . The lens fiber lacked immunoreaction . Localization of Na,K-ATPase was also investigated in the cultured human lens epithelium and in lentoid . Immunoreaction was detected in the apical (facing the media) plasma membrane of the lens epithelium cultured on the lens capsule, whereas the reaction was observable in both apical and basal plasma membrane of the lens epithelium cultured on the biopore membrane filters . Immunoreaction in lentoid was observed in the surface plasma membrane . These data indicate that the polarized distribution seen in the in situ lens epithelium changes when these cells are cultured, and that Na,K-ATPase in the cultured lens cells including lentoid is located in the plasma membrane which is in contact with the growth media . This change in polarity of Na,K-ATPase distribution in cultured epithelial cells may be dictated by the need to maintain ion homeostasis by extrusion of sodium ions across the cell membrane facing the media.

Cell Tissue Res, 1992 Sep, 269(3), 535 - 45
Functional and morphological changes associated with the ageing of primary cultures of embryonic adrenal gland cells derived from the Pekin duck; Cronshaw J et al.; The morphological and functional changes associated with ageing were studied in adrenal steroidogenic cells derived from duck embryos . Cells grown for not more than three days had structural characteristics similar to their counterparts in vivo; they contained numerous lipid droplets and mitochondria, an abundant smooth endoplasmic reticulum, an even network of microtubules, and microfilaments that formed extensive and elaborate systems of parallel stress fibers . After the 3rd day of growth in culture, many of the cells started to decrease in size and become elongated; the older cells showed less well-defined actin filaments and contained elongated mitochondria, fewer lipid droplets, less smooth endoplasmic reticulum, and swollen cisternae of rough endoplasmic reticulum . The proliferative capacity of the cells was the same when they were cultured in either the presence or the absence of 1-24 ACTH . After the first day of growth in culture, the steroidogenic capacity of the cells declined and the addition of 1-24 ACTH to the growth medium did not prevent changes in their structure and function . The decline in steroidogenic capacity occurred both in terms of the amount of hormone released into the culture medium and in the ability of the cells to respond when incubated in buffer containing 1-24 ACTH . Since the basal unstimulated rates of corticosteroid production also declined as the cells aged, it is probable that the steroidogenic deficiency occurs at a site distal to the corticotropin receptor; this is also consistent with the ultrastructural observations that suggest a relationship between the morphological changes and the decline in steroidogenic capacity as the cells age.

J Gen Microbiol, 1992 Sep, 138 ( Pt 9), 1829 - 35
The regulation of expression of the porin gene ompC by acid pH; Thomas AD et al.; The regulation of expression of the porin genes of Escherichia coli by acid pH was investigated using reporter gene fusions . The ompC-lacZ gene fusion was expressed in response to acidification of the external medium . The kinetics of beta-galactosidase synthesis under acid-induction differed significantly from those obtained under conditions of osmotic stress . The latter led to rapid induction without a lag, followed by establishment of a rate that was equal to the growth rate; acid-induction was frequently preceded by a short lag period, was relatively slow and did not achieve a rate that was in balance with the growth rate . Further, induction of the ompC gene at acid external pH was dependent upon the presence of glucose as sole carbon source; growth with either glycerol or succinate as sole carbon source reduced induction of ompC at acid pH . Osmotic induction was independent of carbon source . The induction of the ompC gene at acid pH was also reduced by addition of cAMP to the growth medium . The porins are known to be subject to catabolite repression and our data are consistent with the exposure to acidic pH resulting in progressive changes in the state of catabolite repression . Acidification of the cytoplasm also provoked a rapid induction of the ompC-lacZ gene fusion . The kinetics of induction resembled the response to osmotic upshock . This response was independent of the identity of the carbon source supplied for growth . The contribution of changes in cytoplasmic pH to the induction of ompC at acid pH is discussed.

J Bacteriol, 1992 Aug, 174(16), 5265 - 71
Cloning, characterization, and expression of the dapE gene of Escherichia coli; Bouvier J et al.; The dapE gene of Escherichia coli encodes N-succinyl-L-diaminopimelic acid desuccinylase, an enzyme that catalyzes the synthesis of LL-diaminopimelic acid, one of the last steps in the diaminopimelic acid-lysine pathway . The dapE gene region was previously purified from a lambda bacteriophage transducing the neighboring purC gene (J . Parker, J . Bacteriol . 157:712-717, 1984) . Various subcloning steps led to the identification of a 2.3-kb fragment that complemented several dapE mutants and allowed more than 400-fold overexpression of N-succinyl-L-diaminopimelic acid desuccinylase . Sequencing of this fragment revealed the presence of two closely linked open reading frames . The second one encodes a 375-residue, 41,129-M(r) polypeptide that was identified as N-succinyl-L-diaminopimelic acid desuccinylase . The first one encodes a 118-residue polypeptide that is not required for diaminopimelic acid biosynthesis, as judged by the wild-type phenotype of a strain in which this gene was disrupted . Expression of the dapE gene was studied by monitoring amylomaltase activity in strains in which the malPQ operon was under the control of various fragments located upstream of the dapE gene . The major promoter governing dapE transcription was found to be located in the adjacent orf118 gene, while a minor promoter allowed the transcription of both orf118 and dapE . Neither of these two promoters is regulated by the lysine concentration in the growth medium.

J Bacteriol, 1992 Aug, 174(16), 5258 - 64
The essential Escherichia coli msgB gene, a multicopy suppressor of a temperature-sensitive allele of the heat shock gene grpE, is identical to dapE; Wu B et al.; The grpE gene product is one of three Escherichia coli heat shock proteins (DnaK, DnaJ, and GrpE) that are essential for both bacteriophage lambda DNA replication and bacterial growth at all temperatures . In an effort to determine the role of GrpE and to identify other factors that it may interact with, we isolated multicopy suppressors of the grpE280 point mutation, as judged by their ability to reverse the temperature-sensitive phenotype of grpE280 . Here we report the characterization of one of them, designated msgB . The msgB gene maps at approximately 53 min on the E . coli chromosome . The minimal gene possesses an open reading frame that encodes a protein with a predicted size of 41,269 M(r) . This open reading frame was confirmed the correct one by direct amino-terminal sequence analysis of the overproduced msgB gene product . Genetic experiments demonstrated that msgB is essential for E . coli growth in the temperature range of 22 to 37 degrees C . Through a sequence homology search, MsgB was shown to be identical to N-succinyl-L-diaminopimelic acid desuccinylase (the dapE gene product), which participates in the diaminopimelic acid-lysine pathway involved in cell wall biosynthesis . Consistent with this finding, the msgB null allele mutant is viable only when the growth medium is supplemented with diaminopimelic acid . These results suggest that GrpE may have a previously unsuspected function(s) in cell wall biosynthesis in E . coli.

FEMS Microbiol Lett, 1992 Aug 1, 74(1), 95 - 8
Nitrosating activity in Escherichia coli; O'Donnell CM et al.; Nitrosation activity was measured in Escherichia coli isolates and a range of nitrite reductase (nir) mutants . Activity was only detected in intact cells and could be inhibited by a number of treatments such as sonication and osmotic shock . Aerobically-grown cells had highest nitrosation activity compared to oxygen-limited ones . Inclusion of nitrite in growth media induced high activities of nitrite reductase and for some isolates, nitrosation . Analysis of nir mutants identified two which were unable to nitrosate . This result suggested that NADH-dependent nitrite reductase was implicated either directly or indirectly in nitrosation.

Biol Trace Elem Res, 1992 Aug, 34(2), 177 - 83
Copper organo-chelators in Aspergillus fumigatus and Penicillium chrysogenum; el-Meleigy MA; The presence of copper in the growth medium induces the biosynthesis of several low-mol-wt copper ogano-chelators in Aspergillus fumigatus and Penicillium chrysogenum . By use of P . chrysogenum, we were able to prepare several low-mol-wt metallothionein chelators, as well as several short-chain copper chelatins . The amino acid composition of a chelatin of mol wt approx 799 Da revealed the presence of cysteine, glutamic acid, glycine, and leucine . Use of A . fumigatus led to fewer copper chelators and one very short-chain peptide: a chelatin of mol wt approx 624 Da . composed of four amino acids, asparagine, glutamic acid, cysteine, and glycine . These results may confirm our assumption that fungi detoxify the deleterious action of toxic elements by producing high levels of heavy metal chelators/chelatins.

Genetika, 1992 Aug, 28(8), 46 - 51
{Role of fructose-1-phosphate kinase in expression of PEP-synthase in Escherichia coli K-12}; Molchanova ML et al.; Mutational damage of the fruK gene coding for fructose-1-phosphate kinase leads to 2-6-fold (depending on the strain) decrease in FEP synthase activity in Escherichia coli . The fruK mutants were unable to utilize lactate as well as fructose and fructose-1-phosphate, acquiring, in addition, sensitivity to mannose in their growth medium . Reversions back to FruK+ phenotype or introduction of an intact fruK allele resulted in restoration of both FEP synthase activity and the ability to grow on lactate.

Cancer Lett, 1992 Jul 31, 65(1), 61 - 71
Involvement of p53 mutation in the development of human salivary gland pleomorphic adenomas; Azuma M et al.; We examined the status of the p53 mutation, a putative tumor suppressor gene, as well as the expressions of myc and mos oncogene products in human salivary gland pleomorphic adenoma cells in culture derived from four individuals using techniques which enabled selective and favourable growths of tumor cells . Culture techniques empolyed in this study consisted of type I collagen gel-coated dishes and serum-free medium as substrates and growth medium, respectively . Cells grown under above conditions were subjected to the analyses of p53, myc and mos expression . When analyzed by both immunocytochemical staining and immunoblot, mutant forms of p53 specifically detected by PAb240 were observed in three of 4 cases . However, none of the 4 cases expressed myc and mos oncogene products . These results may imply a role for p53 mutation in the development of human salivary gland pleomorphic adenomas.

Biochem Pharmacol, 1992 Jul 22, 44(2), 243 - 50
Inhibition of Trichomonas vaginalis ornithine decarboxylase by amino acid analogs; Yarlett N et al.; Ornithine decarboxylase (ODC) from Trichomonas vaginalis was inhibited irreversibly by several substrate analogs . Of these, DL-alpha-monofluoromethyldehydroornithine (MFMDO) and DL-alpha-monofluoromethylornithine (MFMO) were the most potent . The enzyme was unaffected by putrescine analogs suggesting that differences exist between the regulation of the trichomonad enzyme and that in other eukaryotes . In culture the ornithine analogs strongly inhibited putrescine synthesis and increased the generation time after 24 hr of exposure . In a semi-defined growth medium MFMDO methyl and ethyl esters increased the generation time from 4.5 hr to 9.0 and 8.2 hr, respectively . In standard undefined growth medium the trichomonad ODC was fully induced only after 15 hr (late log) and had an extended half-life of greater than 8 hr.

FEBS Lett, 1992 Jul 20, 306(2-3), 199 - 202
Involvement of a d-type oxidase in the Na(+)-motive respiratory chain of Escherichia coli growing under low delta mu H+ conditions; Avetisyan AV et al.; An attempt has been made to find out which of the two terminal oxidases, the d-type or the o-type, operates as a Na+ pump in Escherichia coli grown at low delta mu H+ conditions . For this purpose, mutants lacking either d or o oxidase have been studied . It is shown that a d-,o+ mutant grows slowly or does not grow at all under low delta mu H+ conditions (alkaline or protonophore-containing growth media were used) . Inside-out subcellular vesicles from the d-,o+ mutant cannot oxidize ascorbate and TMPD, and cannot transport Na+ when succinate is oxidized in the presence of a protonophore . The same vesicles are found to transport Na+ when NADH is oxidized as if the Na(+)-motive NADH-quinone oxidase were operative . On the other hand, a mutant lacking o oxidase (d+,o-) grows at low delta mu H+ conditions as fast as the maternal E . coli strain containing both d and o oxidases . Corresponding vesicles oxidize ascorbate and TMPD as well as succinate, the oxidations being coupled to the protonophore-stimulated Na+ transport . Growth in the presence of a protonophore is found to induce a strong increase in the d oxidase level in the maternal d+,o+ E.coli strain . It is concluded that oxidase of the d-type, rather than of the o-type, operates as a Na+ pump in E . coli grown under conditions unfavorable for the H+ cycle.

Gene, 1992 Jul 15, 116(2), 159 - 64
Sodium butyrate selectively induces transcription of promoters adjacent to the MoMSV viral enhancer; Yeivin A et al.; The long terminal repeat region of the Moloney murine sarcoma virus (MoMSV) was cloned upstream from the Chinese hamster ovary adenine phosphoribosyltransferase (APRT)-encoding gene (APRT) in order to enhance synthesis of the APRT protein . The replacement of the native promoter with the viral enhancer-promoter increased the enzymatic activity of APRT two- to threefold . Addition of sodium butyrate (NaBu) to the cell growth medium induced APRT activity ten- to 20-fold above wild-type levels in both transient and stable transfectants . The introduction of the APRT native promoter between the MoMSV enhancer-promoter and structural gene reduced the magnitude of the NaBu response . The bacterial cat gene was also stimulated by NaBu when linked to the viral enhancer-promoter . No NaBu response was found in constructs lacking the MoMSV enhancer region . Northern analysis and nuclear run-on experiments indicated that NaBu enhanced transcription of APRT mRNA in both transiently and stably transfected cells, but not in cells inhibited by cycloheximide . Thus, a butyrate-response element (BRE) is associated with the MoMSV enhancer and the action of the MoMSV BRE is promoter-dependent.

J Biol Chem, 1992 Jul 5, 267(19), 13278 - 83
Characterization of the zinc binding site of bacterial phosphotriesterase; Omburo GA et al.; The bacterial phosphotriesterase has been found to require a divalent cation for enzymatic activity . This enzyme catalyzes the detoxification of organophosphorus insecticides and nerve agents . In an Escherichia coli expression system significantly higher concentrations of active enzyme could be produced when 1.0 mM concentrations of Mn2+, Co2+, Ni2+, and Cd2+ were included in the growth medium . The isolated enzymes contained up to 2 equivalents of these metal ions as determined by atomic absorption spectroscopy . The catalytic activity of the various metal enzyme derivatives was lost upon incubation with EDTA, 1,10-phenanthroline, and 8-hydroxyquinoline-5-sulfonic acid . Protection against inactivation by metal chelation was afforded by the binding of competitive inhibitors, suggesting that at least one metal is at or near the active site . Apoenzyme was prepared by incubation of the phosphotriesterase with beta-mercaptoethanol and EDTA for 2 days . Full recovery of enzymatic activity could be obtained by incubation of the apoenzyme with 2 equivalents of Zn2+, Co2+, Ni2+, Cd2+, or Mn2+ . The 113Cd NMR spectrum of enzyme containing 2 equivalents of 113Cd2+ showed two resonances at 120 and 215 ppm downfield from Cd(ClO4)2 . The NMR data are consistent with nitrogen (histidine) and oxygen ligands to the metal centers.

J Clin Microbiol, 1992 Jul, 30(7), 1625 - 30
Efficient culture of Chlamydia pneumoniae with cell lines derived from the human respiratory tract; Wong KH et al.; Two established cell lines, H 292 and HEp-2, originating from the human respiratory tract, were found to be significantly more efficient and practical than the currently used HeLa 229 cells for growth of Chlamydia pneumoniae . Six strains of C . pneumoniae recently isolated from patients with respiratory ailments were used as test cultures . The H 292 and HEp-2 cells yielded much higher inclusion counts for all the test strains than did HeLa 229 cells . When they were compared with each other, H 292 cells yielded more inclusions than did HEp-2 cells, and the differences were statistically significant in 10 of 18 test sets . A simple system with these two cell lines appeared to be very efficient for culturing C . pneumoniae . It does not require treatment of tissue cells with DEAE-dextran before infection, and it may eliminate the need for serial subpassages of specimens to increase culture sensitivity . Monolayers of these cells remained intact and viable in the Chlamydia growth medium so that reinfection could take place, resulting in greatly increased inclusion counts for specimens containing few infectious units . This system may make it more practical for laboratories to culture for C . pneumoniae for treatment of infections and outbreak intervention and will facilitate studies on this recently recognized pathogen.

J Bacteriol, 1992 Jul, 174(14), 4683 - 8
Immunochemical differences among Methanosarcina mazei S-6 morphologic forms; Yao R et al.; Methanosarcinae are the only archaeobacteria known to undergo major morphologic changes during growth involving unicellular and multicellular forms, and Methanosarcina mazei S-6 is the only strain for which three distinct forms, packets, single cells, and lamina, have so far been observed . It is reported that two pairs of these forms, either packets and single cells or single cells and lamina, grew and interconverted in medium with the same composition, Ca2+ and Mg2+ concentrations, and growth substrate, and that the two forms in each pair displayed distinctive differences revealed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and immunoblotting, the same growth medium-substrate notwithstanding.

J Bacteriol, 1992 Jul, 174(13), 4223 - 9
Characterization of a Bradyrhizobium japonicum ferrochelatase mutant and isolation of the hemH gene; Frustaci JM et al.; A Tn5-induced mutant of Bradyrhizobium japonicum, strain LORBF1, was isolated on the basis of the formation of fluorescent colonies, and stable derivatives were constructed in backgrounds of strains LO and I110 . The stable mutant strains LOek4 and I110ek4 were strictly dependent upon the addition of exogenous hemin for growth in liquid culture and formed fluorescent colonies . The fluorescent compound was identified as protoporphyrin IX, the immediate precursor of protoheme . Cell extracts of strains LOek4 and I110ek4 were deficient in ferrochelatase activity, the enzyme which catalyzes the incorporation of ferrous iron into protoporphyrin IX to produce protoheme . Mutant strain I110ek4 could take up 55Fe from the growth medium, but, unlike the parent strain, no significant incorporation of radiolabel into heme was found . This observation shows that heme was not synthesized in mutant strain I110ek4 and that the heme found in those cells was derived from exogenous hemin in the growth medium . The putative protein encoded by the gene disrupted in strain LORBF1 and its derivatives was homologous to ferrochelatases from eukaryotic organisms . This homology, along with the described mutant phenotype, provides strong evidence that the disrupted gene is hemH, that which encodes ferrochelatase . Mutant strain I110ek4 incited nodules on soybean that did not fix nitrogen, contained few viable bacteria, and did not express leghemoglobin heme or apoprotein . The data show that B . japonicum ferrochelatase is essential for normal nodule development.

Curr Genet, 1992 Jul, 22(1), 9 - 11
6-Azauracil inhibition of GTP biosynthesis in Saccharomyces cerevisiae; Exinger F et al.; The addition of 6-azauracil to the growth medium causes a strong reduction of the GTP level in the nucleotide pool of Saccharomyces cerevisiae . In-vitro experiments show a strong inhibition of IMP dehydrogenase activity by 6-azaUMP explaining the preceding effect . PPR2 mutants, previously characterized by an increased sensitivity to 6-azauracil compared to the wild-type, are specifically susceptible to the lowering of the GTP pool, and are able to grow in presence of 6-azauracil when guanine is added to the medium.

Mol Biochem Parasitol, 1992 Jul, 53(1-2), 97 - 103
Arginine metabolism during culture of Giardia intestinalis; Edwards MR et al.; The effect of arginine on the growth and metabolism of Giardia intestinalis trophozoites was determined . Supplementation of the normal growth medium (Diamond's TYI-S-33) with 5 or 10 mM arginine accelerated trophozoite growth over the first 2 days . There was a corresponding rapid utilisation of arginine, with none being detectable after this time . The decrease was associated with the appearance in the growth medium of 1 mol of ornithine and 2 mol of ammonia per mol of arginine utilised, the stoichiometry being consistent with the operation of the arginine dihydrolase pathway . Subsequently, there was a decrease in the ammonia concentration in the medium . Removal of arginine from the medium by pretreatment with arginase substantially decreased cell growth . In TYI-S-33 medium containing no added glucose, instead of the normal 50 mM glucose concentration, arginine supplementation also increased cell growth over the first 2 days, with concurrent stoichiometric production of ornithine and ammonia . However, in these conditions, the ammonia concentration remained elevated . This suggests that under normal conditions there is re-uptake of ammonia, which is glucose dependent . The observations confirm the operation of a functional arginine dihydrolase pathway in G . intestinalis . The concordance of cessation of rapid growth with the depletion of arginine, and the beneficial effect on growth of arginine supplementation suggests that arginine availability is a limiting factor during the initial stages of rapid growth . It would appear that arginine is a major potential energy source during the initial stages of giardial growth, and that supplementation of Diamond's TYI-S-33 medium with additional arginine may provide an improved in vitro culture medium.

Mol Biochem Parasitol, 1992 Jul, 53(1-2), 71 - 8
Sulfate metabolism in Entamoeba histolytica; Bakker-Grunwald T et al.; Sulfate fluxes and sulfate metabolites in Entamoeba histolytica were characterized employing {35S}sulfate as a marker . Sulfate was taken up both across the plasma membrane and by pinocytosis; in growth medium (sulfate concentration, 1.1 mM) total uptake was 1.5 mumol h-1 (5 x 10(7) cells)-1 . The fate of sulfate within the cells was investigated by thin-layer chromatography . Major metabolites (together greater than 3 mumol (5 x 10(7) cells)-1) were monoethyl sulfate and 3-cholesteryl sulfate; both these products were released into the growth medium . As minor components we identified the activated sulfate derivatives, adenosine-5'-phosphosulfate and 3'-phosphoadenosine-5'-phosphosulfate . In addition, up to 10% of the sulfate taken up was incorporated into high-molecular weight material (possibly proteoglycans) . We propose that sulfurylation of cholesterol may play a role in controlling membrane sterol content.

Biochem Int, 1992 Jul, 27(3), 449 - 56
Destruction of vitamin K1 of cultured carrot cells by ultraviolet radiation and its effect on plasma membrane electron transport reactions; Barr R et al.; The effect of ultraviolet radiation on plasma membrane electron transport reactions was studied in cultured carrot cells . It was found that a 90 min treatment inhibited transmembrane hexacyanoferrate reduction greater than 50% . Extraction of lipophilic quinones from irradiated cells showed that vitamin K1 and coenzyme Q were totally destroyed, while control unirradiated cells showed the presence of 0.4 mumole vitamin K1 g dry wt.-1 . The addition of exogenous vitamin K1 in concentrations of 1-10 microM partially restored plasma membrane electron transport with impermeable hexacyanoferrate as the electron acceptor . Total restoration of activity was given by growing irradiated cells in vitamin K1 supplemented growth media for 6 days . This shows that vitamin K1 may function as a member of the transplasma membrane electron transport chain in cultured carrot cells.

Hum Antibodies Hybridomas, 1992 Jul, 3(3), 123 - 8
Expression and secretion of an assembled tetrameric CH2-deleted antibody in E . coli; Lo KM et al.; We have expressed in E . coli a functional assembled antibody variant that is secreted into the media . The antibody variant is a CH2-deleted chimeric antibody 14.18, which was previously shown to be a potentially useful reagent for radioimmunodetection of human tumors . The bacterial expression vector contains a dicistronic unit comprised of a L-chain cDNA and a CH2-deleted H-chain cDNA . For translocation across the bacterial membranes, we have replaced the natural signal peptides of the H and L chains with the signal peptide of the bacterial protein pectate lyase B . When expressed in the JM105 host under the control of the trp-lac promoter, the products were secreted into the M9 growth media to about 350 micrograms/L . The secreted antibody, which can be readily purified from the media without any denaturation or renaturation steps, retains antigen-binding activity . SDS-PAGE and nondenaturing size exclusion high-pressure liquid chromatography showed that it is a mixture of assembled HL and fully assembled H2L2 . In H2L2, inter-H chain disulfide bonds are not formed, and the two HL half-molecules are likely held together by the trans interaction between the two CH3 domains.

Genes Chromosomes Cancer, 1992 Jul, 5(1), 14 - 20
Improved technique for short-term culture and cytogenetic analysis of human breast cancer; Pandis N et al.; Various growth media and procedures for tissue disaggregation and culturing were tested with regard to cell attachment, the type of cells to grow out, and the emergence of cytogenetically abnormal clones in cultures of 20 primary breast carcinomas . Clonal chromosome abnormalities were detected in 16 cases (80%) . Our findings allow us to suggest a series of modifications of existing culturing and chromosome preparation techniques for breast cancer cytogenetic analysis . The improvements include: (1) combined mechanical and enzymatic disaggregation of the tumor samples, (2) initiation of short-term cultures in plastic flasks that have a Primaria-modified tissue culture surface or have been coated with Vitrogen 100, (3) use of serum-free growth medium, CDM-5, but with temporary (24 hours) enrichment with 20% FBS if rapid cell attachment is not achieved, (4) partial and sequential harvesting of the cultures, and (5) use of minimal volumes of hypotonic and fixative solutions during harvesting.

Mutat Res, 1992 Jul, 280(1), 17 - 27
Evaluation of genotoxicity of tert.-butylhydroquinone in an hepatocyte-mediated assay with V79 Chinese hamster lung cells and in strain D7 of Saccharomyces cerevisiae; Rogers CG et al.; tert.-Butylhydroquinone (TBHQ) has been reported to be genotoxic in some short-term assays but non-genotoxic in others . We have examined cytotoxicity and genotoxicity of TBHQ, a principal metabolite of the phenolic antioxidant 2(3)-tert.-butyl-4-hydroxyanisole (BHA), in an hepatocyte-mediated assay with V79 Chinese hamster lung cells including both sister-chromatid exchange (SCE) and thioguanine-resistance (TGR) endpoints . The ability of BHA and of TBHQ to elicit a genotoxic response in Saccharomyces cerevisiae strain D7 was also investigated . In V79 cytotoxicity tests, TBHQ without hepatocytes produced a 50% reduction in colony formation at 4.2 micrograms/ml and was lethal to 100% of the cells at concentrations above 5 micrograms/ml . At partially cytotoxic dose levels, (0.17-3.4 micrograms/ml of medium), TBHQ sometimes increased significantly the frequency of SCE . TBHQ also produced sporadic statistically significant increases in the mutation frequency at the HGPRTase (TGR) gene locus when tested alone or with activation by rat or hamster hepatocytes . Mitotic gene conversion and reverse mutation were not induced in strain D7 of Saccharomyces cerevisiae by exposure to BHA or to TBHQ for 4 h at concentrations as high as 200 micrograms/ml for BHA or 500 micrograms/ml for TBHQ, either alone or with activation by rat-liver S9 . Incubation of the yeast cells with BHA or TBHQ for 24 h in growth medium without activation also did not induce genotoxic activity . The slight and sporadic response to TBHQ in the V79 test system may indicate weak genotoxicity which is sensitive to slight differences in test conditions . The classification and test strategies adopted for compounds such as TBHQ could have important implications for regulatory decisions and for the validation of short-term tests.

Curr Microbiol, 1992 Jul, 25(1), 57 - 9
Change of speciation of Cu(II) in growth medium due to uptake of ammonia by Pseudomonas testosteroni during growth; Ahmad A et al.; Growth of Pseudomonas testosteroni in a medium containing 1 mM Cu(II) causes a color change from blue to green . The spectrum of the supernatant solution from the blue culture shows an absorption at 660 nm, identical to that of 1 mM {Cu(II)} in the medium . The green supernatant solution shows a UV absorption, which tails into the visible and so is responsible for the green color, and a d-d absorption at 720 nm . The absorption at 660 nm for the blue supernatant solution is probably due to {Cu(NH3)3(H2O)3}2+ . Growth of the organism causes loss of ammonia and a speciation change to {CU(NH3)2(H2O)4}2+, with a shift in absorption maximum from 660 to 720 nm . These conclusions are based upon the spectra of known aquaammine complexes of Cu(II) and calculations of the speciation of Cu(II) before and after growth . Change in metal speciation owing to nutrient uptake by an organism does not appear to have been recognized previously.

Biotechnology (N Y), 1992 Jul, 10(7), 784 - 9
Production of biologically active recombinant human lactoferrin in Aspergillus oryzae; Ward PP et al.; We report the production of recombinant human lactoferrin in Aspergillus oryzae . Expression of human lactoferrin (hLF), a 78 kD glycoprotein, was achieved by placing the cDNA under the control of the A . oryzae alpha-amylase promoter and the 3' flanking region of the A . niger glucoamylase gene . Using this system, hLF is expressed and secreted into the growth medium at levels up to 25 mg/l . The recombinant lactoferrin is indistinguishable from human milk lactoferrin with respect to its size, immunoreactivity, and iron-binding capacity . The recombinant protein appears to be appropriately N-linked glycosylated and correctly processed at the N-terminus by the A . oryzae secretory apparatus . Lactoferrin is the largest heterologous protein and the first mammalian glycoprotein expressed in the Aspergillus system to date . Hence, this expression system appears suitable for the large-scale production and secretion of biologically active mammalian glycoproteins.

Nucleic Acids Res, 1992 Jun 25, 20(12), 3029 - 36
Different effects of mioC transcription on initiation of chromosomal and minichromosomal replication in Escherichia coli; Lobner-Olesen A et al.; The mioC gene, which neighbors the chromosomal origin of replication (oriC) in Escherichia coli, has in a number of studies been implicated in the control of oriC initiation on minichromosomes . The present work reports on the construction of cells carrying different mioC mutations on the chromosome itself . Flow cytometry was employed to study the DNA replication control and growth pattern of the resulting mioC mutants . All parameters measured (growth rate, cell size, DNA/cell, number of origins per cell, timing of initiation) were the same for the wild type and all the mioC mutant cells under steady state growth and after different shifts in growth medium and after induction of the stringent response . It may be concluded that the dramatic effects of mioC mutations reported for minichromosomes are not observed for chromosomal replication and that the mioC gene and gene product is of little importance for the control of initiation . The data demonstrate that a minichromosome is not necessarily a valid model for chromosomal replication.

J Appl Bacteriol, 1992 Jun, 72(6), 493 - 9
Myceloid cell formation in Arthrobacter globiformis during osmotic stress; Deutch CE et al.; Arthrobacter globiformis was grown in a semi-defined liquid medium containing added solutes to determine the effects of osmotic stress on its reproduction and cell morphology . There was a progressive reduction in the specific growth rate during exponential phase as the concentration of NaCl was increased, although the final yields of the cultures during stationary phase were not affected . Clusters of branching myceloid cells rather than the typical bacillary forms predominated during exponential phase . These myceloids did not undergo complete septation and persisted into stationary phase . Similar responses were observed with potassium sulphate as the exogenous solute but less dramatic morphological effects were found with added polyethylene glycol or sucrose . The myceloids formed in response to osmotic stress could not be disrupted mechanically but were more sensitive than normal cells to lysozyme, particularly during stationary phase . Addition of osmoprotective compounds such as proline, glutamate, glycine betaine, or trehalose to the growth medium did not significantly relieve the effects of osmotic stress on growth rate or morphology . A . simplex also formed myceloid cells during osmotic stress but A . crystallopoietes did not . These results indicate that arthrobacters exhibit characteristic responses to osmotic stress and suggest these bacteria may contain novel osmoprotective compounds.

Brain Res Bull, 1992 Jun, 28(6), 975 - 8
Effects of spontaneous bioelectric activity and gangliosides on cell survival in vitro; Baker RE et al.; Chronic suppression of spontaneous bioelectric activity in spinal cord explants in the presence of tetrodotoxin (TTX) during network formation caused a large reduction in cell number (lowered DNA levels) . The addition of gangliosides failed to protect against this cell loss . Conversely, the omission of galactose from the growth medium had no effect on DNA levels . It was concluded that the presence or absence of afferent selectivity is unlikely to require the survival of a regionally specific subpopulation of preferred dorsal root ganglion target cells . Neocortical explants also showed a large reduction in DNA levels following chronic TTX treatment, and morphometric analysis confirmed that neuronal survival was affected to the same degree . Chronic ganglioside supplementation failed to influence DNA and cell counts in either control or TTX-treated explants, but one of the added gangliosides (GD1a) stimulated extensive neuritic outgrowth in electrically silenced cultures . Particular ganglioside species, therefore, may exert a growth stimulating influence that can partially compensate for the absence of bioelectric self-stimulation during early development.

J Neurol Sci, 1992 Jun, 109(2), 168 - 72
Effects of astrocytes, insulin and insulin-like growth factor I on the survival of motoneurons in vitro; Ang LC et al.; We isolated motoneurons from E15 dissociated mouse spinal cord by density centrifugation and planted them onto poly-ornithine-coated coverslips in a growth medium (DMEM/F12) supplemented with progesterone, transferrin, selenium, horse serum and muscle extract . Under these conditions only 28% of the motoneurons survived for 8 days . When living astrocytes on a separate coverslip were introduced into dishes containing motoneurons, there was a two-fold increase in neuronal survival . The addition of insulin and insulin-like growth factor I (IGF-I) to such cultures alone or together, still further increased motoneuron survival, but this did not happen in the absence of astrocytes . We conclude that (a) astrocytes exert a trophic role in the survival of spinal motoneurons, (b) the effect does not require physical contact of the cells, and (c) insulin and IGF-1 have neurotrophic activity for motoneurons, an effect possibly mediated by living astrocytes.

Clin Immunol Immunopathol, 1992 Jun, 63(3), 214 - 20
The atherogenic effect of lupus sera: systemic lupus erythematosus-derived immune complexes stimulate the accumulation of cholesterol in cultured smooth muscle cells from human aorta; Kabakov AE et al.; The influence of systemic lupus erythematosus (SLE) patients' sera on lipid accumulation in the cultured smooth muscle cells (SMC) from unaffected human aortic intima was examined . It was demonstrated that the cholesterol uptake in the SMC cultured in the presence of SLE sera is 1.5- to 6-fold higher than in the cells cultured with normal human sera (NHS) obtained from healthy donors . Incubation of the SMC with circulating immune complexes (CIC) isolated from lupus sera by precipitation with 2.5% polyethylene glycol 6000 (PEG) caused a 3- to 4-fold rise in the intracellular cholesterol level . The atherogenic effect of lupus sera, as well as isolated CIC, strongly correlated (r = 0.98) with the low density lipoprotein (LDL) content in the PEG-precipitated CIC . The cholesterol level in cultured SMC also increased 2- to 3-fold when growth medium was supplemented with LDL, DNA, and anti-DNA autoantibodies (IgG) affinity isolated from lupus sera . Using immunofluorescent staining, it was shown that the addition of a DNA-anti-DNA IgG mixture to the growth medium, together with NHS, stimulated LDL incorporation in the SMC . The results of double-label staining suggest the formation of LDL-DNA-IgG complexes which seem to be entrapped in cells more actively than free LDL . The composition of PEG-precipitated CIC was studied by electrophoresis and immunoblotting . Significant amounts of apolipoprotein B, as well as low molecular weight DNA and immunoglobulins, were found in SLE-derived CIC . The data obtained suggest that the atherogenic effect of human lupus sera in vitro is generally due to the appearance of LDL-containing immune complexes . Different mechanisms possibly involved in the lupus atherogenesis are discussed.

Scanning Microsc, 1992 Jun, 6(2), 543 - 55; discussion 556-9
Detection of X-ray damage repair by the immediate versus delayed plating technique is dependent on cell shape and cell concentration; Reddy NM et al.; A method commonly used to measure the ability of cells to repair potentially lethal damage (PLD) is to compare immediate plating (IP) and delayed plating (DP) survival . Lower cell survival under IP conditions relative to that after DP conditions has been interpreted to indicate a higher ability of cells to repair potentially lethal damage (PLD) under DP conditions . However, this IP radiosensitization has not been observed in several cell lines and tumor models . IP conditions involve treatment of cells with trypsin and plating them into fresh growth medium . We have investigated the possibility that radiosensitization under IP conditions may be related to both the cell-shape and the nutrient concentration in growth medium (GM, MEM + 15% serum) . This idea predicts that the IP and DP survival of spheroids will show a response similar to the IP survival of cells in monolayers and that the IP and DP survival of crowded monolayer cells in high densities will be the same . Chinese hamster V79 cells grown in monolayers (spread cells) and spheroids (clumps of round cells) were used . The IP survival was lower than the DP survival for spread log phase monolayer cells but not for round log phase cells in spheroids . Radiosensitization of cells by fresh (as opposed to spent) growth medium was absent for high density plateau phase cells in monolayers at or above 2 x 10(6) cells/ml . However, PLD repair could be demonstrated in spheroid cells and in high density plateau phase cultures by exposing cells to hyperthermia or hypertonic saline . Comparison of immediate plating versus delayed plating survival detects PLD repair only in well spread low density monolayer cells, but not in round spheroid cells nor in dense monolayer cells at > 10(7) cells/25 cm2 flask/5 ml medium . The absence of a difference between IP and DP cell survival does not mean that PLD repair is absent . Incorrect prediction of tumor response to radiotherapy can occur when PLD repair capacity is assayed as a ratio of DP/IP survival . More than one method must be used to measure the capacity of cells to repair their PLD.

J Bone Miner Res, 1992 Jun, 7(6), 675 - 81
Adaptive regulation of ascorbate transport in osteoblastic cells; Dixon SJ et al.; Osteoblasts possess a concentrative L-ascorbate (vitamin C) uptake mechanism involving a Na(+)-dependent ascorbate transporter located in the plasma membrane . The transporter is specific for ascorbate and stereoselective for L-ascorbate over D-isoascorbate . The present study examined the effects of ascorbate supplementation and deprivation on the activity of this transport system . L-ascorbate transport activity was determined by measuring uptake of the vitamin by ROS 17/2.8 osteosarcoma cells during 1 minute incubations with 5 microM L-{14C}ascorbate . The initial rate of L-{14C}ascorbate uptake by ROS 17/2.8 cells grown for 18 h in L-ascorbate-replete medium was 89 +/- 8 nmol/g protein per minute . Following removal of L-ascorbate from the growth medium, the initial rate of uptake increased within 6 h to 126 +/- 13 nmol/g protein per minute . Conversely, the initial rate of uptake by cells grown in ascorbate-free medium decreased following the addition of L-ascorbate, but not D-isoascorbate, to the medium . The effect of ascorbate pretreatment was specific for ascorbate transport in that preincubation of cultures with L-ascorbate did not affect uptake of 2-deoxy-D-glucose . Kinetic analysis revealed that modulation of ascorbate transport arose from changes in the apparent maximum rate of transport (Vmax) without changes in the affinity of the transport system for L-ascorbate . These experiments are the first to show that ascorbate transport by osteoblastic cells responds to vitamin C deprivation and supplementation . Adaptation of transport activity to substrate availability may play an important role in the physiological regulation of intracellular ascorbate levels.

Protein Expr Purif, 1992 Jun, 3(3), 185 - 95
Characterization of recombinant antistasin secreted by Saccharomyces cerevisiae; Przysiecki CT et al.; Secretion from recombinant yeast was used as a potential source of large quantities of the leech protein antistasin (ATS), a potent and highly specific inhibitor of the serine protease coagulation factor Xa . Mature recombinant ATS (r-ATS) is obtained after intracellular cleavage by the yscF protease of the mating factor alpha-1 pre-proleader from the fusion protein at the Lys-Arg sequence junction . Production levels are relatively low (ca . 1 mg/liter) . Purification of the secreted product from a complex growth medium involved cell removal by microfiltration and diafiltration, cation-exchange capture and concentration on S-Sepharose Fast Flow, C-4 reverse-phase high-performance liquid chromatography (RP-HPLC), and HPLC cation-exchange chromatography step, and RP-HPLC concentration and desalting . The process was scaled up from the 16- to the 250-liter level with a corresponding increase in amount of r-ATS . From the 250-liter fermentation two major forms, r-ATS-I and r-ATS-II, distributed approximately 60:40, and a minor form, r-ATS-minor (ca . 1% of the purified r-ATS), were characterized . Limited N-terminal sequence analysis by Edman degradation indicated that r-ATS-I has the predicted mature N-terminus starting with Gln, that r-ATS-II is N-terminally blocked with pyroglutamate, and that r-ATS-minor is an incompletely processed form . RP-HPLC, hydrophilic-interaction HPLC, cation-exchange HPLC analysis, and electrophoresis results are consistent with the differences observed by sequencing . Preliminary in vitro characterization by intrinsic Ki determination for factor Xa inhibition indicated that the yeast r-ATS forms are indistinguishable from each other as well as from r-ATS expressed by the insect baculovirus host-vector system . Nevertheless, r-ATS-I and r-ATS-II appear less potent than insect-derived r-ATS in the activated partial thromboplastin time clotting assay . Further characterization indicated that C-terminal cleavage at Pro-116 had occurred in r-ATS-I and r-ATS-II as well as oxidation of methionine residues to methionine sulfoxide . The possible role of the C-terminus in inhibition of the prothrombinase complex is discussed.

Biotechnol Appl Biochem, 1992 Jun, 15(3), 314 - 20
Effect of 5,7-unsaturated sterols on ethanol tolerance in Saccharomyces cerevisiae; Novotny C et al.; Structural membrane lipids are known to contribute to the high ethanol resistance of Saccharomyces cerevisiae (2, 4, 17) . By manipulating the yeast cellular sterol level by changing the carbon-to-nitrogen source ratio in the chemostat growth medium, high delta 5,7-sterol levels were found to increase the resistance of yeast populations to ethanol-induced death . The resistance of the erg2 (delta 8----delta 7-sterol isomerase) mutant to ethanol-induced death was generally comparable with that of the delta 5,7-sterol-synthesizing strain . In contrast, the sensitivity of anaerobic growth to inhibition by ethanol was higher in the erg2 mutant in comparison with the delta 5,7-sterol-synthesizing strains but a high level of those sterols increased the vulnerability of anaerobic growth to ethanol inhibition.

J Bacteriol, 1992 Jun, 174(12), 4148 - 56
Proline biosynthesis in Saccharomyces cerevisiae: molecular analysis of the PRO1 gene, which encodes gamma-glutamyl kinase; Li W et al.; The PRO1 gene of Saccharomyces cerevisiae encodes the 428-amino-acid protein gamma-glutamyl kinase (ATP:L-glutamate 5-phosphotransferase, EC 2.7.2.11), which catalyzes the first step in proline biosynthesis . Amino acid sequence comparison revealed significant homology between the yeast and Escherichia coli gamma-glutamyl kinases throughout their lengths . Four close matches to the consensus sequence for GCN4 protein binding and one close match to the RAP1 protein-binding site were found in the PRO1 upstream region . The response of the PRO1 gene to changes in the growth medium was analyzed by measurement of steady-state mRNA levels and of beta-galactosidase activity encoded by a PRO1-lacZ gene fusion . PRO1 expression was not repressed by exogenous proline and was not induced by the presence of glutamate in the growth medium . Although expression of the PRO1 gene did not change in response to histidine starvation, both steady-state PRO1 mRNA levels and beta-galactosidase activities were elevated in a gcd1 strain and reduced in a gcn4 strain . In addition, a pro1 bradytrophic strain became completely auxotrophic for proline in a gcn4 strain background . These results indicate that PRO1 is regulated by the general amino acid control system.

J Cell Physiol, 1992 Jun, 151(3), 579 - 87
1,25-Dihydroxy-vitamin-D3 enhances antiproliferative effect and transcription of TGF-beta1 on human keratinocytes in culture; Kim HJ et al.; Both TGF-beta and 1,25-dihydroxy-vitamin-D3 (1,25(OH)2D3) have been reported to decrease the proliferation of normal human keratinocytes . The effect and expression of TGF-beta in keratinocytes treated with 1,25(OH)2D3 was investigated . Human keratinocytes were grown in the presence of various concentrations of TGF-beta and/or 1,25(OH)2D3 prior to enumeration . TGF-beta, alone, has a half maximal dose of inhibition (ED50) of approximately 750 pg/ml after seven days in culture in Keratinocyte Growth Medium (KGM; Clonetics) supplemented with 1.5 mM calcium . When 1,25(OH)2D3 (10(-7)M) was also added to cultures with various concentrations of TGF-beta, the ED50 shifted an average of 2-fold less . The presence of TGF-beta (10 pg/ml) augmented the potency of 1,25(OH)2D3 by at least 10-fold . In keratinocyte cultures, the antiproliferative effect of the two compounds together is synergistic . In keratinocytes grown for 1 week in the presence of 1,25(OH)2D3 at 10(-6)M, the TGF-beta 1 message increased approximately 5-fold . An increase is detected within 2 hours of exposure to 1,25(OH)2D3 . There was only a 50% increase in the levels of TGF-beta 2 and no detection of TGF-beta 3 . When keratinocyte cultures were treated with 1,25(OH)2D3 and neutralizing antibodies to TGF-beta, the induced-antiproliferative activity was blocked by more than 50% . The keratinocytes produced more active than latent TGF-beta after growth with high doses of 1,25(OH)2D3.

Int J Cancer, 1992 May 28, 51(3), 439 - 44
Pleiotropic actions of suramin on the proliferation of human breast-cancer cells in vitro; Foekens JA et al.; Suramin, a non-specific growth factor antagonist, is currently under investigation for treatment of cancer patients . We studied its action on 6 different human breast-cancer cell lines in vitro . In complete growth medium, pleiotropic effects were observed with respect to cell proliferation, i.e . suramin is stimulatory at low concentrations and inhibitory at higher concentrations, for 4 of the 6 cell lines studied . The various cell lines showed marked differences with respect to the antiproliferative action of suramin, the Evsa-T cells being by far the most sensitive ones . A suramin concentration of 100 micrograms/ml brought about a 100% stimulation of the proliferation of ZR/HERc cells, ZR 75.1 cells ectopically expressing a human epidermal growth factor receptor (EGF-R) cDNA . Although less pronounced (10 to 60% stimulation), a similar response was observed for the parent ZR 75.1 cells, as well as for T-47D and MDA-MB-231 cells . The non-specificity of the action of suramin was established by the observation that suramin-induced inhibition of cell proliferation could be abolished by insulin-like growth factor-I (IGF-I) or basic fibroblast growth factor (bFGF), and even by estradiol, both in complete growth medium and under defined serum-free conditions . Our data indicate that suramin exerts pleiotropic effects on the proliferation of human breast cancer cells in vitro, and confirm the non-specific nature of its action . The stimulatory effect of low concentrations of suramin on the proliferation of breast cancer cells may have important consequences for breast cancer patients treated with suramin.

J Biol Chem, 1992 May 25, 267(15), 10866 - 73
A recombinant ectodomain of the receptor for the stem cell factor (SCF) retains ligand-induced receptor dimerization and antagonizes SCF-stimulated cellular responses; Lev S et al.; The stem cell factor (SCF) is a polypeptide ligand that is essential for the development of germ cells, hematopoietic progenitor cells, and melanocyte precursors . It binds to a tyrosine kinase membrane receptor that is encoded by the c-kit proto-oncogene . We have constructed an expression vector that directs the synthesis of the entire extracellular ligand-binding domain of the Kit/SCF receptor . When expressed and amplified in Chinese hamster ovary cells, a secreted 90-kDa glycoprotein could be harvested from the growth medium of the cells in a soluble form . This extracellular portion of the Kit/SCF receptor, denoted Kit-X, was recognized by antibodies specific to the SCF receptor; and when injected into animals, it raised antibodies that were reactive with the complete membrane form of the receptor . Direct binding and covalent cross-linking of radiolabeled SCF showed that Kit-X fully retained high affinity ligand binding and also underwent efficient dimerization in the presence of the ligand . The capacity of Kit-X to act as an antagonist of SCF was assayed on cultured cells that overexpress the receptor . Simultaneous addition of SCF and Kit-X to these cells resulted in a stoichiometric inhibition of SCF binding and a consequent decrease in autophosphorylation of the SCF receptor on tyrosine residues . The inhibition extended to later SCF-mediated responses, including the association of the receptor with phosphatidylinositol 3'-kinase and coupling to the Raf1 protein kinase . These results indicate that the recombinant ectodomain of the Kit-SCF receptor can be used as a specific antagonist of SCF actions and may enable detailed molecular analysis of ligand-receptor interactions.

J Biol Chem, 1992 May 25, 267(15), 10810 - 5
Inhibition of myogenesis by okadaic acid, an inhibitor of protein phosphatases, 1 and 2A, correlates with the induction of AP1; Park K et al.; Recently, we demonstrated that okadaic acid, an inhibitor of protein phosphatases 1 and 2A, inhibits myogenesis by extinguishing the expression of MyoD1 and inducing the expression of Id . Since it has been reported that transformation by c-fos also inhibits myogenesis through inhibition of MyoD1 expression, we examined the effects of okadaic acid on the activation of the c-fos and jun family of proto-oncogenes in an attempt to understand the mechanism by which okadaic acid inhibits the myogenic differentiation . Treatment of C2C12 cells in growth medium with okadaic acid increased expression of the mRNAs for the c-fos family continuously and for the jun family to a lesser extent . In contrast, in differentiation medium, the induction of c-fos, c-jun, and fos B mRNAs by okadaic acid was transient, whereas fra-1, jun D, and jun B mRNAs were induced continuously, suggesting that okadaic acid regulates the expression of the c-fos and jun family through complex regulatory mechanisms depending on the state of differentiation of the cells . Transfection of c-jun and c-fos promoter-chloramphenicol acetyltransferase constructs demonstrated that the effects of okadaic acid on the induction of c-fos and c-jun are mediated through the activation of promoter elements . These results suggest that some of the targets of protein phosphatases 1 and 2A may include transcription factors capable of forming AP1 complexes and that these factors may play an important role during myogenic differentiation.

J Biol Chem, 1992 May 5, 267(13), 9140 - 5
Transcriptional and maturational effects of manganese and iron on the biosynthesis of manganese-superoxide dismutase in Escherichia coli; Privalle CT et al.; Anaerobically grown Escherichia coli contain an enzymatically active iron superoxide dismutase (Fe2-FeSOD) and an inactive iron-substituted manganese superoxide dismutase (Fe2-MnSOD) . The anaerobic electron sink, nitrate plus paraquat, enhanced biosynthesis of the MnSOD polypeptide, with accumulation of inactive Fe2-MnSOD . The oxidant, diamide, in contrast, allowed anaerobic production of the active forms of MnSOD, i.e . Mn2-MnSOD and Mn/Fe-MnSOD . Nutritional supplementation with Mn(II) favored occupancy of the MnSOD active site with manganese and allowed anaerobic accumulation of Mn2-MnSOD in the absence of diamide . Enrichment of the anaerobic growth medium with Fe(II) both suppressed biosynthesis of the MnSOD polypeptide and inhibited formation of the active manganese-containing forms . A tac-sodA operon fusion was used to examine the effects of chelating agents and metals on maturation of nascent MnSOD, independent from the transcriptional effects these agents impose . Isopropyl-1-thio-beta-D-galactopyranoside (IPTG) elicited anaerobic biosynthesis of MnSOD, which accumulated as the inactive Fe2-MnSOD . Diamide, with IPTG, allowed formation of active Mn/Fe-MnSOD while 1,10-phenanthroline with IPTG resulted in accumulation of Mn2-MnSOD . These results suggest that iron participates in the redox-sensitive control of the formation of active MnSOD at two levels, i.e . that of transcription as well as that of maturation . During maturation of the nascent MnSOD polypeptide, iron and manganese compete for the metal-binding site; anaerobic conditions favor iron-binding, whereas oxidants, such as dioxygen or diamide, favor binding of manganese.

Z Lebensm Unters Forsch, 1992 May, 194(5), 465 - 8
Action of gamma-irradiated patulin on Saccharomyces cerevisiae; Zegota H et al.; The action of gamma-irradiated patulin on Saccharomyces cerevisiae LOCK 2144 in liquid culture was studied . Patulin irradiated in an aqueous solution with doses ranging over 0.34-1.36 kGy inhibited the yeast growth in a proportion to the concentration of undestroyed toxin . Patulin disappearance in the growth medium occurred between 12-72 h of incubation at 30 degrees C . The patulin content did not essentially change in the period of log phase of yeast growth which is accompanied by rapid glucose uptake.

Chem Biol Interact, 1992 May, 82(3), 295 - 316
Changes in the cytoskeleton pattern of tumor cells by cisplatin in vitro; Kopf-Maier P et al.; The influence of the antitumor drug cis-diamminedichloroplatinum(II) ('cisplatin') upon the structural pattern of the main cytoskeletal components, i.e . microtubules, intermediate filaments and microfilaments, was investigated in squamous carcinoma cells derived from the mouse stomach (G 22) or the human lung (L 266) and growing in vitro as monolayer cultures . The studies were performed by the indirect immunofluorescence technique using monoclonal antibodies against alpha-tubulin, type 19 cytokeratin and actin at the end of a 90-min exposure to 2.5 x 10(-6), 5 x 10(-6) or 10(-5) mol cisplatin/l and a subsequent 24-h recovery period . Under the influence of cisplatin, the cytoskeletal tubules and filaments, which were distributed in untreated cells as a finely organized network spreading through the whole cytoplasm like a spider's web, collapsed and aggregated to dense and circularly arranged bands of bright immunofluorescence around the nucleus or to cap-like structures apposing the nucleus . These phenomena developed in clear dependence upon the dose of cisplatin applied and were observable in a modified manner and to a different degree with the three structural elements of the cytoskeleton . During the subsequent 24-h interval, during which the cells were allowed to recover in drug-free growth medium, the before-mentioned collapse of the cytoskeletal network was only partially reversible following previous treatment with the medium (5 x 10(-6) mol/l) and the high (10(-5) mol/l) dose of cisplatin and restored totally to the normal structural pattern of untreated control cells when the low dose of 2.5 x 10(-6) mol cisplatin/l had been administered before . These results give evidence that the DNA cannot be the only cellular target for the antitumor drug cisplatin, but that it also effects other intracellular lesions which cause structural alterations of cellular organelles independently of the primary molecular attack at nuclear DNA strands . Probably, these additional interactions fortify the antiproliferative effect and contribute to the achievement of important biological and cytological effects of cisplatin such as growth inhibition or giant cell formation.

Int J Biochem, 1992 May, 24(5), 751 - 8
Effects of growth medium, electrical stimulation and paralysis on various enzyme activities in cultured rat muscle cells . Comparison with activities in rat muscles in vivo; Jacobs AE et al.; 1 . Replacement of fetal calf serum and chicken embryo extract by Ultroser G and rat brain extract during the proliferation phase resulted in a higher maturation grade of cultured rat muscle cells after 7 days of differentiation, on base of the percentage of the muscle specific isoenzyme of creatine kinase (CK-MM) . 2 . Furthermore, the activities of creatine kinase, citrate synthase, cytochrome c oxidase and hexokinase were significantly higher . 3 . Compared to the enzyme activities in m . quadriceps of 10 day-old rat and m . quadriceps, m . soleus and m . extensor digitorum longus of young adult rats, the metabolic capacity of cultured myotubes most closely resembles that of the first muscle . 4 . Paralysis with tetrodotoxin caused a slight decrease of the creatine kinase activity and the percentage of CK-MM of cultured myotubes and an increase of the activities of hexokinase, phosphorylase and AMP deaminase . 5 . Electrical stimulation performed at different frequencies and time periods had no effect on the enzyme activities of cultured rat muscle cells . 6 . Only the AMP deaminase activity was decreased after intense electrical stimulation.

Eur J Immunol, 1992 May, 22(5), 1207 - 13
Sublytic complement attack protects tumor cells from lytic doses of antibody and complement; Reiter Y et al.; Sublytic doses of the membrane attack complex (MAC) of complement are known to exert multiple stimulatory effects on metabolically active cells . Results presented herewith demonstrate that pretreatment of the human leukemic cells K562 and HL-60 with sublytic doses of antibody and normal human serum protects them from lytic complement concentrations, a phenomenon proposed to be called "complement-induced protection" . C7- and C8-deficient human sera are ineffective in inducing resistance unless they are reconstituted with purified human C7 and C8, respectively . The complement-induced protection is inhibitable by actinomycin D and cycloheximide indicating that the increased complement resistance depends on RNA and protein synthesis triggered by the sublytic complement doses . Free extracellular Ca2+ is also required to achieve maximal protection, indicating a role for Ca2+ ions in the cell stimulatory events which culminate in increased complement resistance . Quantitative analysis of bound complement components indicated that similar amounts of C3 and C9 molecules are deposited on "protected" and control cells during complement activation . The "protected" K562 and HL-60 cells regain sensitivity to lytic MAC doses after about 8 or 3 h, respectively, of culture in growth medium, in the absence or presence of actinomycin D and cycloheximide . The "induced protection" is not species restricted and protection from human complement can be induced in K562 cells by treatment with sublytic doses of antibody and rabbit or guinea pig sera.

J Surg Res, 1992 May, 52(5), 510 - 7
Basement membrane components enhance isolated enterocyte growth; Nguyen BL et al.; Isolated enterocyte transplantation may have a potential role in increasing intestinal surface area . However, enterocytes are notoriously difficult to grow . Basement membrane components (BMC) promote adherence, migration, and differentiation of enterocytes . Our aim was to determine if BMC enhance enterocyte growth . Twenty-nine rabbits had 5-cm ileal segments resected and serosal pouches (n = 22) created from the serosal surface of the colon . Harvesting of enterocytes by warm trypsinization resulted in 92 +/- 6% cell viability and yielded 5.0 +/- 2.4 10(6) enterocytes/cm intestine . Enterocytes (10(5) were cultured in vitro (n = 7) in 10 ml growth media in plain flasks and flasks coated with laminin alone or Matrigel (an extract of mouse basement membrane containing laminin plus Type IV collagen and heparan sulfate) . The cells were subcultured at 2 weeks and examined after Geimsa staining at 4 weeks . Epithelial growth was confirmed by light microscopy and staining for cytokeratin and quantitated by image analysis (JAVA) . Epithelial coverage of the flasks was greater with Matrigel (80 +/- 15%) than laminin (66 +/- 15%) which was greater than control (44 +/- 20%) (P less than 0.01) . For in vivo studies 10(5) harvested cells were infused into the serosal pouches either in growth media (n = 9) or media + Matrigel (n = 13) . Epithelial growth in the pouch was evaluated by qualitative scoring of cytokeratin staining . Cytokeratin staining was similar on control colon serosa (n = 5) and serosa after infusion of cells in media alone . However, Matrigel significantly enhanced the area, depth, and intensity of staining.(ABSTRACT TRUNCATED AT 250 WORDS)

Mutat Res, 1992 May, 282(1), 55 - 60
Influence of cinnamaldehyde on UV-induced gene conversion and point mutation in yeast: effect on protein synthesis; Galli A et al.; The activity of cinnamaldehyde (CIN), a bioantimutagen in bacterial systems, was tested in the D7 strain of yeast Saccharomyces cerevisiae . Yeast cells were UV-irradiated and post-incubated in liquid growth medium for 2 and 4 h with different concentrations of cinnamaldehyde . During the post-incubation period, DNA-damage-specific functions may be induced . This in turn may affect the genotoxicity and in fact a weak decrease in UV-induced convertant and revertant frequencies was observed after 4 h of post-incubation . The presence of CIN in the growth medium increased the UV-induced gene conversion and reversion . The addition of cycloheximide abolished this effect . To evaluate the CIN effect on protein synthesis, extracts of cells UV-treated and post-incubated for 2 h in the presence of 35S-methionine were performed . SDS-gel electrophoresis demonstrated the inhibitory effect of CIN on a UV-specific protein . This work suggests that CIN might interfere with DNA-damage-inducible systems although it did not exert an antimutagenic activity in our experimental conditions.

Exp Cell Res, 1992 May, 200(1), 175 - 85
Gene expression in hepatocyte-like lines established by targeted carcinogenesis in transgenic mice; Antoine B et al.; New hepatocyte-like cell lines (mhAT) were derived from the liver of a transgenic mouse expressing SV40 early genes under the direction of the liver-specific antithrombin III gene promoter (ATIII-TSV40) . Their differentiated phenotypes were improved and stabilized by the use of liver-specific growth media (arginine-free, glucose-free, or low-fructose/glucose-free medium) . The best differentiated lines display a very high level of albumin, transferrin, and L-type pyruvate kinase (L-PK) gene expression that is comparable to that observed in the mouse liver . Abundance of the aldolase B and phosphoenolpyruvate carboxykinase (PEPCK) transcripts varied from 5 to 35% of the in vivo concentrations while abundance of the alpha-fetoprotein and phenylalanine hydroxylase transcripts remained very low . Hormonal (cAMP and insulin) and nutritional (glucose) gene controls of PEPCK and L-PK were, at least partially, conserved . mhAT cells are readily transfectable by the calcium phosphate coprecipitation technique and exhibit a liver-specific pattern of expression of exogenous genes . Thus, mhAT cells seem suitable for the analysis of the regulatory regions involved in the tissue-specific transcription of genes . This work demonstrates, therefore, the great efficiency of targeted carcinogenesis in transgenic mice to create new differentiated cell lines . The availability of various lines of liver-specific cells with different phenotypes will constitute useful tools to establish correlations between expression of trans-acting factors and control of the phenotype.

J Invest Dermatol, 1992 May, 98(5), 730 - 3
Minoxidil sulfotransferase, a marker of human keratinocyte differentiation; Johnson GA et al.; The sulfation of minoxidil is catalyzed by a sulfotransferase activity in a number of tissues including skin . To investigate further the nature of the minoxidil sulfotransferase activity in epithelial tissue and to compare this activity to that of cholesterol sulfotransferase, which has already been shown to be induced during the differentiation of epithelial cells, we cultured normal human epidermal keratinocytes in a keratinocyte growth medium for 4 d, after which the media were replaced with either the same growth media or media with increasing Ca++ concentrations . Cholesterol sulfotransferase, minoxidil sulfotransferase, and transglutaminase were determined during the differentiation of the cells in the three media . Time-activity curves that suggested two different sulfotransferase activities were induced during the differentiation process . U-77581, a competitive inhibitor of minoxidil sulfotransferase activity, inhibited the sulfation of minoxidil sulfotransferase activity in the keratinocyte homogenates, but it did not inhibit the sulfation of cholesterol . These data indicate that at least two sulfotransferase activities are induced during the differentiation of epithelial keratinocytes and minoxidil sulfotransferase is an early marker of that differentiation.

Differentiation, 1992 May, 50(1), 25 - 33
Selection of variant hepatoma cells in liver-specific growth media: regulation at the mRNA level; Armbruster L et al.; Three liver-specific growth media, respectively free of arginine (Arg-), tyrosine (Tyr-) and glucose (G-), have been used to characterize cells of the rat H4IIEC3, human HepG2 and mouse BW hepatoma lines . Cells of clone FaO, a derivative of line H4IIEC3, freely grew in Tyr- and G- media, and gave rise to stable variants in Arg- conditions . Cells of line HepG2 and clone BWTG3, a derivative of line BW, degenerated in all three media . Arg and tyr variants were however derived from HepG2 cells; their genesis appeared to be pathway specific, illustrating the complexity of the regulatory loops that are implicated in the control of the differentiated state . No variant was ever obtained with BWTG3 cells, demonstrating the stability of their deficiency in the post-natal hepatic functions that are involved in Arg-, Tyr- and G- selections . Variant clones of HepG2 and FaO cells that have been isolated in Arg- medium were characterized in details for liver-specific urea-cycle enzyme activities and mRNA . These variants were shown to be controlled at the mRNA level, most likely at transcription . Isolation of stable FaO and HepG2 variant clones as well as the converse demonstration of the stable deficiency of BWTG3 cells in post-natal hepatic functions were aimed at expression cloning . Our results are thus discussed in terms of transfection with full-length cDNA expression libraries and cloning of regulatory genes that could activate or extinguish liver specific genes.

J Virol, 1992 May, 66(5), 2792 - 7
Formation of heterotrimers between the membrane-integrated and the soluble glycoproteins of vesicular stomatitis virus leads to their intracellular cotransport; Schmidt C et al.; BHK cells infected with vesicular stomatitis virus serotype Indiana generate intracellularly two different types of glycoproteins: the authentic membrane-integrated G protein of virions and a smaller soluble Gs protein lacking the transmembrane and cytoplasmic domains which is secreted into the growth medium . A Gs1 protein species which is formed during or shortly after translation in the endoplasmic reticulum lumen is modified in the same way as the G1 protein by endoglycosidase H-sensitive oligosaccharides of the high-mannose type . Both G1 and Gs1 are almost simultaneously transported, trimmed, and processed into G2 and Gs2 species which possess carbohydrate side chains of the complex type, making both glycoproteins resistant to endoglycosidase H cleavage . Secretion of Gs2 protein into the growth medium and arrival of G2 protein on the cell surface occur concomitantly . Membrane-integrated G protein and the soluble Gs protein molecules oligomerize intracellularly into heterotrimers which can be immunoprecipitated after chemical cross-linking . Gs protein seems to contain sufficient structural information for the formation of heterotrimers which are efficiently transported to the cell surface . Heterotrimer formation between G and Gs proteins explains the rapid secretion of Gs molecules.

Int J Cancer, 1992 Apr 22, 51(1), 99 - 107
An organoid culture assay (OCA) for determining the drug sensitivity of human tumors; Kopf-Maier P et al.; A model for testing chemotherapeutic agents in vitro is described . It is based on an organoid culture method which allows human carcinomas to grow in vitro and to maintain many typical in vivo properties, including 3-dimensional architecture, growth of multiple cell types, expression of morphological differentiation and formation of histotypical structures . The preservation of drug sensitivity and resistance under the conditions of our organoid culture assay (OCA) was demonstrated by investigating 3 strains of a human hypopharynx carcinoma which differed by different sensitivity to cisplatin in in vivo conditions . These differences were retained in vitro and the modified neutral-red (NR) assay was especially suitable for revealing drug-induced cytotoxic damage in OCA . On the basis of our findings, the following approach is proposed for the in vitro testing of cytostatic drugs before they are administered to patients . (1) Removal of carcinomas from patients; (2) dense cell suspensions of these carcinomas to be dropped on membrane filters at the air-medium interface, resulting in growth of solid nodules of organized carcinoma tissues; (3) addition of cytostatic drugs to the growth medium for 2 or 3 days; (4) detachment and bisection of the culture nodules; (5) determination of viable cells by NR uptake and total cell mass by the sulforhodamin B (SRB) assay; (6) determination of quotient NR:SRB absorbance, related as percentage to control value: this indicates the fraction of viable cells and gives a measure of the cytotoxic injury caused by the applied cytotoxic drug . Thus, the OCA seems to be suitable for defining the patterns of drug sensitivity and resistance of individual human carcinomas in vitro within a few days.

Proc Natl Acad Sci U S A, 1992 Apr 15, 89(8), 3571 - 5
Lipocortin 1 mediates dexamethasone-induced growth arrest of the A549 lung adenocarcinoma cell line; Croxtall JD et al.; The synthetic glucocorticoid dexamethasone (1 microM to 1 pM) strongly (maximum greater than 80%) inhibits proliferation of the A549 human lung adenocarcinoma line (EC50 greater than 1 nM) and leads to the appearance, or a further increase (approximately 3-fold) in the expression on the cell surface, of the calcium and phospholipid binding protein lipocortin (annexin) 1 . Both these effects, which are shared by hydrocortisone (1 microM) but not by progesterone or aldosterone (1 microM), are inhibited by the antiglucocorticoids RU38486 and RU43044 (1 microM) . The nonsteroidal antiinflammatory drugs indomethacin (1 microM) and naproxen (10 microM) and human recombinant lipocortin 1 (0.05-5.0 micrograms/ml) also produce growth arrest in this cell line . During proliferation A549 cells spontaneously release prostaglandin E2 {10-20 ng (28-57 pmol) per ml per 5-day period} into the growth medium . In concentrations that cause growth-arrest, dexamethasone, indomethacin, and lipocortin 1 abolish the generation of this eicosanoid by A549 cells . Prostaglandin E2 itself (0.01-1 pM) stimulates cell growth and partially reverses (approximately 50%) the inhibition of growth caused by dexamethasone and indomethacin . Addition of the neutralizing anti-lipocortin 1 monoclonal antibody 1A (5 micrograms/ml), but not the nonneutralizing anti-lipocortin monoclonal antibody 1B, substantially reversed (greater than 80%) the inhibitory activity of dexamethasone on both growth and prostaglandin E2 synthesis . The generation of prostaglandin E2 by A549 cells seems to be an important regulator of cell proliferation in vitro and the dexamethasone-induced suppression of proliferation in this model is attributable to eicosanoid inhibition caused by lipocortin 1.

FEBS Lett, 1992 Apr 6, 300(3), 254 - 8
Escherichia coli leucine-responsive regulatory protein (Lrp) controls lysyl-tRNA synthetase expression; Gazeau M et al.; Using random Tn10 insertion mutagenesis, we isolated an Escherichia coli mutant strain affected in the regulation of lysU, the gene encoding the inducible form of lysyl-tRNA synthetase . The transposon giving rise to the altered expression of lysU was found inserted within lrp . The latter gene codes for the leucine-responsive regulatory protein (Lrp) which mediates a global response of the bacterium to leucine . An involvement of Lrp in the regulation of lysU was searched for by using a lysU-lacZ operon fusion . The following conclusions were reached: (i) inactivation of lrp causes an increased activity of the lysU promoter, whatever the growth conditions assayed, (ii) insertion of a wild-type lrp gene into a multi-copy plasmid significantly reduces lysU expression, and (iii) sensitivity of the lysU promoter to the presence of leucine in the growth medium is abolished in the lrp context.

J Gen Microbiol, 1992 Apr, 138 ( Pt 4), 825 - 30
Control and location of acyl-hydrolysing phospholipase activity in pathogenic mycobacteria; Wheeler PR et al.; Phospholipase activities releasing fatty acyl moieties from phosphatidylcholine and phosphatidylethanolamine and lysophospholipase activity releasing fatty acid from lyso-phosphatidylcholine were detected in both Mycobacterium microti and Mycobacterium avium . Fatty acyl groups were released from both the 1- and 2-positions of phosphatidylcholine . Generally, phospholipase activities of M . avium were cryptic while phospholipase activities of M . microti were located on the bacterial surface . However, intact M . microti did not release fatty acids from phospholipids faster than M . avium . Neither Mycobacterium secreted acyl-hydrolysing phospholipase activity . All phospholipase activities were stimulated by including phospholipids in growth media: generally, cell extracts contained 6- to 15-fold higher specific activities than extracts from mycobacteria grown in media without added phospholipid . However, not all phospholipase activities were stimulated to the same degree in any given set of conditions, suggesting the existence of more than one phospholipase gene in each Mycobacterium.

Carcinogenesis, 1992 Apr, 13(4), 643 - 9
Abrogation of killing and neoplastic transformation of C3H10T1/2 cells due to 7,12-dimethylbenz{a}anthracene; Wells RL et al.; The combined action of 7,12-dimethylbenz{a}anthracene (DMBA) and alpha-naphthoflavone (alpha NF) on the survival and neoplastic transformation of C3H10T1/2 mouse embryo fibroblasts has been examined and correlated with DNA adduct formation and removal . When a 24 h DMBA treatment of asynchronously growing cells was followed for the next 24 h by a treatment with alpha NF + DMBA, both killing and transformation per viable cell were abrogated to a large extent . In some instances, transformation was completely abrogated--i.e . reduced to control frequencies--even at nontoxic concentrations of DMBA, indicating that changes in survival were not the reason for the reduction in transformation . Even at toxic concentrations of DMBA, post-treatment with alpha-NF + DMBA resulted in 10-fold reductions in transformation frequency . 3-Methylcholanthrene (3MC) also reversed DMBA cytotoxicity but with a dependence on 3MC concentration that was qualitatively different from that for alpha NF . The abrogation of cell killing occurred at lower molar ratios of alpha NF:DMBA than the abrogation of transformation; less than or equimolar concentrations resulted in maximal abrogation of killing, but about equal concentrations were required to abrogate transformation . Although the preceding findings suggest that different mechanisms may be involved in these endpoints, taken together they suggest that second treatments make apparent the repair of lesions due to a first treatment with DMBA alone . To test this hypothesis, the formation and removal of DMBA-DNA adducts were measured . Adducts were not removed when the second treatment was growth medium alone, but enhanced removal was observed when second treatments consisted of DMBA alone or DMBA plus one of several other polycyclic aromatic hydrocarbons (PAHs) . Relative to killing and neoplastic transformation, these results suggest DMBA induces a repair process that limits its own effectiveness--a process that can be sustained by other PAHs.

Biochem J, 1992 Apr 1, 283 ( Pt 1), 137 - 44
Secretion of mammalian ribonucleases from Escherichia coli using the signal sequence of murine spleen ribonuclease; Schein CH et al.; A nucleotide sequence identical with that of the recently identified murine pancreatic ribonuclease (RNAase) was isolated from a murine spleen cDNA library . Active RNAase was expressed and secreted from Escherichia coli lon-htpr- transformed with a plasmid containing the E . coli trp promoter followed by the murine RNAase gene sequence, including the original eukaryotic 26-amino-acid signal sequence . Approx . 1 mg of properly matured RNAase protein/litre was secreted into the medium of a fermentor culture after the promotor was induced by tryptophan starvation . When the signal sequence was deleted from the plasmid, intracellular RNAase activity was very low and there was no significant supernatant RNAase activity . Even higher RNAase yields were obtained with a synthetic gene for bovine pancreatic ribonuclease cloned after the signal sequence of the murine gene . About 2 mg of correctly processed RNAase A/litre was isolated from the growth medium, and a further 8-10 mg of correctly processed RNAase/litre could be isolated from the soluble fraction of the cells . Thus this eukaryotic signal sequence is both recognized by the E . coli transport and processing apparatus and gives efficient secretion, as well as export, of active, mature mammalian RNAases.

Gene, 1992 Apr 1, 113(1), 129 - 33
Isolation and analysis of the Penicillium chrysogenum phoA gene encoding a secreted phosphate-repressible acid phosphatase; Haas H et al.; We have isolated the genomic sequence encoding a secreted phosphate-repressible acid phosphatase (PHOA) from Penicillium chrysogenum using synthetic oligodeoxyribonucleotide probes . Nucleotide sequence data revealed that this gene consists of two exons of 192 and 1047 bp separated by an intron of 52 bp in length . A sequence encoding a putative signal peptide, resembling known signal sequences of fungi, was identified at the 5'-end of the coding sequence . Northern blot analysis of total cellular RNA indicated that the phoA gene codes for a 1.6-kb transcript . The expression of this gene is regulated at the transcriptional level and is markedly affected by the inorganic phosphate concentration of the growth medium.

J Cell Biol, 1992 Apr, 117(2), 347 - 56
Regulation of intracellular actin polymerization by prenylated cellular proteins; Fenton RG et al.; Posttranslational modification by covalent attachment of polyisoprene intermediates to a carboxyterminal CAAX-box motif is required for the biologic function of proteins such as p21ras, the supergene family of ras-related proteins, nuclear lamins, and subunits of heterotrimeric G-proteins . Cells grown in the presence of lovastatin, which inhibits HMG-CoA reductase and prevents synthesis of intermediates required for protein prenylation, develop a round, refractile morphology . Our data indicate that this is due to the selective loss of actin cables without gross changes in the microtubular lattice or intermediate filament structure . Microinjection of a competitive peptide inhibitor of protein prenyltransferases into the cytoplasm of cells induces an identical change in morphology with loss of actin cables . Mevalonate (MVA) reverses the lovastatin-induced morphologic change by inducing a rapid repolymerization of actin cables with coincident reversion to the flat morphology . Furthermore, microinjection of farnesyl-pyrophosphate or geranylgeranyl-pyrophosphate into lovastatin-treated cells also results in rapid morphologic reversion . The morphologic reversion induced by MVA requires the presence of serum, and is independent of extracellular calcium . The addition of cycloheximide to the growth medium prevents lovastatin-induced loss of actin cables, and causes morphologic reversion of lovastatin-treated cells by a mechanism that is independent of MVA . A1F4- induces morphologic reversion in a manner indistinguishable from MVA . These data indicate that prenylated protein(s) play a critical role in regulating the state of intracellular actin, and that GGPP can rescue the lovastatin-induced morphologic phenotype in the absence of upstream intermediates of cholesterol biosynthesis . We have begun to dissect the signaling events that mediate this pathway.

Invest Ophthalmol Vis Sci, 1992 Apr, 33(5), 1690 - 5
Characterization of cholinesterase activities in primary cultures of retinal pigment epithelium; Salceda R et al.; This report presents a comparative description of the acetylcholinesterase and butyrylcholinesterase activities and their molecular forms in primary cultures of retinal pigment epithelium (RPE) . Acetylcholinesterase activity increases during differentiation of the cells . Sucrose sedimentation analysis of acetylcholinesterase and butyrylcholinesterase molecular forms revealed the presence of A12, G4, G2, and G1 and A8, G4, G2 and G1, respectively . RPE cells in culture release both cholinesterases into the growth medium, sedimenting as the G4 molecular form . Changes in the molecular forms of both enzymes were observed during differentiation . The results suggest a possible relationship between butyrylcholinesterase activity and cell proliferation and acetylcholinesterase activity and cell differentiation.

Eur J Biochem, 1992 Apr 1, 205(1), 133 - 8
Evidence that cellobiose oxidase from Phanerochaete chrysosporium is primarily an Fe(III) reductase . Kinetic comparison with neutrophil NADPH oxidase and yeast flavocytochrome b2; Kremer SM et al.; Kinetic measurements were made for purified cellobiose oxidase in 100 mM acetate (pH 4.0) at 30 degrees C, with excess cellobiose as substrate and O2 or Fe(III) as acceptor . With O2 at 230 microM as sole electron acceptor, the O2 uptake rate corresponded to a one-electron turnover number of 0.13 +/- 0.01 s-1 . Measurements at different O2 concentrations indicated Km(O2) greater than 120 microM . In separate experiments, the reduction of Fe(III) acetate was monitored at 340 nm in the absence of oxygen . The maximum velocity of Fe(III)-acetate reduction (Vmax) was 4.5 +/- 0.7 s-1, while Km{Fe(III) acetate} was 34 +/- 12 microM . With ferricyanide in place of Fe(III) acetate, the corresponding values were 6.9 +/- 0.7 s-1 and 23 +/- 5 microM . Redox titrations established the potential of the haem prosthetic group of the oxidase at pH 4.0 as +165 mV . The midpoint potential for Fe(III)/Fe(II) acetate at pH 4.0 is much higher, a value of +535 mV being obtained with 200 microM Fe . Cellobiose oxidase resembles yeast flavocytochrome b2 and differs from the neutrophil NADPH oxidase in having the potential of its haem group far above the potential for one-electron reduction of O2 to superoxide (Em,4 = -110 mV) . A kinetic comparison led to the conclusion that the role of cellobiose oxidase is as an Fe(III) reductase . Fe(II) may have a biological importance as a component of Fenton's reagent {Fe(II)/H2O2} . The concentration of cellobiose oxidase in the growth medium at harvest (0.3 microM) can provide a far higher flux of Fe(II) than a non-enzymic proposal in the literature.

Dig Dis Sci, 1992 Apr, 37(4), 532 - 6
Tissue culture of epithelial cells from esophageal specialized columnar epithelium (Barrett's esophagus); Garewal HS et al.; A procedure is described for the tissue culture of epithelial cells from Barrett's specialized columnar epithelium . The method employs use of nonenzymatic disaggregation techniques and use of a specialized growth medium, M-19 . Successful growth can be achieved in 60-70% of cases . Characteristics of four cultures are presented including PAS, Alcian blue, and cytokeratin staining properties . Ultrastructural studies showed the presence of distended rough endoplasmic reticulum, abundant Golgi apparatus, glycogen granules, and cell-cell junctional complexes . Colony formation in anchorage-independent growth in soft agar was not observed in any culture . The cultured cells were not capable of indefinite growth . Thus, they do not behave as fully transformed cells . Availability of this culture system should be of use to the study of the biology of this metaplastic premalignant lesion.

J Clin Endocrinol Metab, 1992 Apr, 74(4), 884 - 9
Decidual cell biosynthesis of interleukin-6: regulation by inflammatory cytokines; Dudley DJ et al.; Intrauterine infection is an important cause of preterm labor and delivery and is characterized by increased production of inflammatory cytokines by gestational tissues . We have evaluated the biosynthesis of the inflammatory cytokine, interleukin-6 (IL-6), by human decidua and its regulation by other cytokines essential to the inflammatory process . We found that decidual cells secrete small amounts of IL-6 in the presence of growth medium supplemented only with 10% fetal calf serum . Interleukin 1 (alpha and beta) and tumor necrosis factor (TNF) all induced a significant concentration-dependent stimulation of IL-6 production by decidual cells . Treatment of decidual cells with actinomycin D or cycloheximide abrogated the increase in IL-6 production induced by IL-1 beta . Northern blot analysis of cultured decidual cells revealed an increase in IL-6 messenger RNA (mRNA) over time in response to IL-1 beta . These data indicate that IL-1 beta stimulates an increase in IL-6 mRNA and protein production, reflecting either direct gene activation or stabilization of IL-6 mRNA . The concentration range tested (0.1 to 10 ng/mL) of each cytokine is within the range of values found in the amniotic fluid of women destined to deliver preterm due to infection of gestational tissues . Our data suggest that IL-6 is produced by human decidua in response to inflammation and, in conjunction with other inflammatory mediators, may play a role in the pathophysiology of preterm labor due to infection.

Cell Immunol, 1992 Apr, 140(2), 304 - 18
Tumor-stimulated release of tumor necrosis factor-alpha by human monocyte-derived macrophages; DeMarco R et al.; Tumor necrosis factor-alpha (TNF) release by monocytes and macrophages may be an important determinant of the physiologic response of the host to neoplastic disease; however, the mechanisms which regulate TNF release by macrophages in hosts with neoplastic diseases are poorly understood . The purpose of this study was to determine if cell membranes and growth medium from human leukemia cell lines and solid tumor cell lines induced TNF release by cultured human blood monocyte-derived macrophages . The capacity for TNF release and direct tumor killing was highest in monocytes cultured for 7 to 11 days . Cell membranes and culture media from K562 erythroleukemia and several small cell lung carcinoma cell lines, including H82, induced the release of up to 1500 TNF units per 10(6) macrophages over 24 hr . By contrast, allogeneic peripheral blood lymphocytes, cell membranes from normal mixed donor peripheral blood leukocytes, or growth medium from normal embryonic lung fibroblasts induced the release of little or no TNF during culture up to 24 hr, suggesting that this macrophage response was specific for tumor cells . Release of TNF by tumor-stimulated macrophages was gradual, peaking 24 hr following the addition of stimuli . Induction of macrophage TNF release was concentration dependent, with half-maximal TNF levels induced by 12.5 and 25 micrograms/ml cell membranes prepared from K562 and H82, respectively . Pretreatment of tumor cell membranes with polymixin B, which inhibits many of the actions of endotoxin, failed to neutralize tumor induction of TNF, suggesting that endotoxin was not responsible for this activity . Depletion of macrophages by treatment with 3C10 monoclonal antibody and complement abrogated tumor-induced TNF release, indicating that macrophages were the source of the secreted TNF . HPLC analysis of H82 growth medium demonstrated a single peak of macrophage activating activity with approximate 40-kDa molecular weight . We have demonstrated that cell membranes and growth medium from some human leukemia and solid tumor cell lines, but not from normal human cells, induce human peripheral blood monocytes and monocyte-derived macrophages to release functionally active TNF . This process may contribute to the host response to some neoplastic diseases.

Curr Genet, 1992 Apr, 21(4-5), 339 - 44
Complementation of the Saccharomyces cerevisiae srb1-1 mutation: an autoselection system for stable plasmid maintenance; Rech SB et al.; The autonomously replicating plasmid YEpSS1, containing the S . cerevisiae SOD1 and SRB1 genes, was highly unstable in a wild-type strain . When transformed into a fragile srb1-1 mutant host, the same plasmid displayed different characteristics depending on the growth medium used . Both batch and continuous culture experiments demonstrated that the plasmid was very unstable when the transformed strain SLU15 was grown in the presence of an osmotic stabiliser (10% w/v sorbitol) . However, in the absence of the osmoticum, nearly 100% of the cells retained the plasmid and produced the Sod1 protein after 80 generations of growth.

J Protein Chem, 1992 Apr, 11(2), 201 - 11
An evaluation of different enzymatic cleavage methods for recombinant fusion proteins, applied on des(1-3)insulin-like growth factor I; Forsberg G et al.; Different enzymatic methods for cleavage of recombinant fusion proteins were compared . To find an efficient cleavage method, five different fusion proteins were produced . The fusion proteins differed only in the linker region between the fusion partner and the desired product, human des(1-3)insulin-like growth factor I . A cleavage study was performed with enterokinase, plasmin, thrombin, urokinase, and recombinant H64A subtilisin . Significant cleavage was obtained using thrombin, H64A subtilisin, and enterokinase . Thrombin cleavage was studied on a larger scale and des(1-3)IGF-I was recovered at a final yield of 3 mg/L growth medium . Thrombin and enterokinase were also studied as immobilized proteases and they cleaved the fusion proteins with retained activity . To further improve thrombin cleavage, a continuous reactor was constructed, consisting of a closed system with a thrombin column and an ion exchange column in series . Here, the fusion protein circulated while free des(1-3)IGF-I was bound to the ion exchange column after release from the fusion protein . In the reactor, thrombin was as efficient as the free enzyme but gave a diminished rate of product degradation.

J Biotechnol, 1992 Apr, 23(2), 167 - 82
Characterization of glutamine metabolism in two related murine hybridomas; Jenkins HA et al.; Aspects of glutamine metabolism were examined in two related hybridomas, a high-producing line (PQX B1/2) and a low-producing line (PQX B2/2) . The growth and metabolic characteristics of both cell lines were identical or very similar . During batch growth glutamine was completely exhausted from the medium and an examination of the fate of {14C}glutamine highlighted the importance of this amino acid as an energy source . The relative enzyme activities and the amount of ammonia produced during growth indicated that glutamine is oxidized preferentially via the transamination pathway . The overall rate of glutamine utilization from the growth medium was similar to the rate of {14C}glutamine uptake which suggests that transport may regulate glutamine metabolism.

J Cell Physiol, 1992 Apr, 151(1), 94 - 102
Identification of a new squamous cell differentiation marker and its suppression by retinoids; Lotan R et al.; Rabbit tracheobronchial epithelial cells (RbTE) can undergo squamous cell differentiation under defined culture conditions and, therefore, have been used as a model to study the regulation of squamous cell differentiation markers . In the present study, we identified a 20-kDa protein, designated rSQ20, in the serum-free growth medium conditioned by RbTE cells undergoing squamous cell differentiation . The protein was also found in extracts of squamous differentiated cells . rSQ20 was labeled by cells incubated with {35S}methionine but not with {3H}glucosamine, suggesting that it is not a glycoprotein . Undifferentiated cells did not produce this protein . rSQ20 was detected in the conditioned medium of RbTE cells after they reached a confluent and growth-arrested state, and thereafter its level increased markedly and concurrently with an increase in type I (epidermal) transglutaminase, an established marker of squamous cell differentiation . rSQ20 found in concentrated conditioned medium of squamous differentiated RbTE cells was eluted from a gel filtration column as a protein of 20 kDa, similar to that found by gel electrophoresis under denaturing conditions, suggesting that it is not a multimeric protein . A protein with an apparent molecular weight of 16 kDa (rSQ16), probably the product of partial proteolysis of rSQ20, was often found in various amounts in the conditioned medium of differentiated RbTE cells . beta-All-trans retinoic acid and other vitamin A analogues (retinoids), which suppress squamous cell differentiation, inhibited the expression of rSQ20 in RbTE cells . RbTE cells immortalized by transfection with SV40 large T antigen as well as malignantly transformed derivatives obtained from the immortalized cells by further transfection with v-Ha-ras secreted SQ20 and SQ16 when grown to high cell densities although their squamous differentiation was impaired . An analogous protein with an apparent molecular weight of 16 kDa, designated hSQ16, was detected in the medium of differentiated normal human bronchial epithelial (NHBE) cells and normal human epidermal keratinocytes (NHEK) . No such protein could be detected in the medium in which undifferentiated NHBE or NHEK cells were grown . These results suggest that rSQ20 and hSQ16 are new markers of squamous cell differentiation.

Can J Microbiol, 1992 Apr, 38(4), 339 - 42
Correlation between calmodulin-like protein, phospholipids, and growth in glucose-grown Mycobacterium phlei; Reddy PH et al.; In Mycobacterium phlei TMC 1548 supplementation of growth medium containing 2% v/v glycerol with glucose (up to 5% w/v) resulted in an increase in growth (yield of cells), in amount of total phospholipids, and in each of the individual phospholipids (cardiolipin, phosphatidylethanolamine, phosphatidylinositol and its mannosides, and phosphatidylglycerol) . However, when the medium was supplemented with a higher concentration (7.5% w/v) of glucose, both growth and phospholipid levels decreased to near control values (2% v/v glycerol alone) . Cyclic AMP levels, which decreased at all concentrations of glucose, had no relation to phospholipid content or growth . The presence of a protein that possesses the property of stimulating c-AMP phosphodiesterase activity was recently demonstrated in Mycobacterium smegmatis (Falah et al . 1988 . FEMS Microbiol . Lett . 56: 89-93) . In M . phlei the level of this calmodulin-like protein (assayed by radioimmunoassay) changed with different concentrations of glucose in the growth medium in a manner identical with that of phospholipids . We suggest that in mycobacteria (i) intracellular calmodulin-like protein levels are affected by glucose concentration in the growth medium and (ii) there is a positive correlation between the levels of calmodulin-like protein, total and individual phospholipids, and growth (yield of cells) in glucose-grown M . phlei.

J Biol Chem, 1992 Mar 25, 267(9), 5931 - 6
Specificity of unsaturated fatty acid-regulated expression of the Saccharomyces cerevisiae OLE1 gene; McDonough VM et al.; The Saccharomyces cerevisiae OLE1 gene encodes the delta-9 fatty acid desaturase, an enzyme which forms the monounsaturated palmitoleic (16:1) and oleic (18:1) fatty acids from palmitoyl (16:0) or stearoyl (18:0) CoA . Previous studies demonstrated that OLE1 mRNA levels and desaturase enzyme activity are repressed when either 16:1 delta-9 and 18:1 delta-9 are added to the growth medium (1) . The polyunsaturate, linoleic acid (18:2, delta-9,12), which is not a product of the enzyme, is also a strong repressor . The specificity of the OLE1 transcriptional regulatory sensor was examined by testing the response of OLE1 promoter-lacZ fusion reporter genes to fatty acids that differ in chain length, degree of unsaturation and double bond positions . Monounsaturated and polyunsaturated fatty acids that contain a delta-9 double bond are strong repressors of reporter gene activity and native OLE1 mRNA levels . Monounsaturated fatty acids containing double bonds in the delta-10, delta-11, or delta-5 positions showed no repression of reporter enzyme levels although they were rapidly incorporated into membrane lipids and some supported growth of an OLE1 gene disrupted strain . Although 17:1 delta-10 does not repress OLE1 transcription, lipid analysis showed that it replaces almost all of the endogenous 16:1 delta-9 and 18:1 delta-9 in cellular lipids and OLE1 mRNA levels are strongly repressed . This suggests that additional systems regulate desaturase activity by post-transcriptional mechanisms that differ from the transcriptional sensor in their responses to specific fatty acids.

Nucleic Acids Res, 1992 Mar 25, 20(6), 1411 - 8
Cis and trans regulatory elements required for regulation of the CHO1 gene of Saccharomyces cerevisiae; Bailis AM et al.; A 34 base-pair (bp) fragment spanning sequences -154 to -120 of the promoter of the CHO1 gene (structural gene for phosphatidylserine synthase) from the yeast Saccharomyces cerevisiae has been shown to place transcription of a promoter-less Escherichia coli lacZ gene under control of the phospholipid precursors inositol and choline . Furthermore, in deletion experiments the CHO1 UASINO was localized to sequences between -151 and -123 of the CHO1 promoter . A nine bp sequence was identified in the promoter region of the CHO1 gene that shares an eight out of nine bp match with a sequence (consensus 5' ATGTGAAAT 3') that is repeated a total of 23 times upstream from several coregulated phospholipid biosynthetic genes . This sequence is contained within the -151 to -123 region to which the CHO1 UAS has been localized . The nine bp repeated element is believed to be involved in the control of phospholipid biosynthetic gene transcription in response to changing levels of inositol and choline in the growth medium . This control has been shown to require activities encoded by the products of the three regulatory genes: INO2, INO4, and OPI1 . A mutation in any of these regulatory genes results in aberrant CHO1-lacZ gene regulation, and affects regulation of the construct containing the 34 bp (-154 to -120) CHO1 fragment demonstrating that the regulatory signal generated by these genes interacts with the 5' end of the CHO1 gene.

J Bacteriol, 1992 Mar, 174(5), 1586 - 95
Origins of the osmoprotective properties of betaine and proline in Escherichia coli K-12; Cayley S et al.; The amounts of cytoplasmic water and of all osmotically significant cytoplasmic solutes were determined for Escherichia coli K-12 grown in 3-(N-morpholino)propane sulfonate (MOPS)-buffered glucose-minimal medium containing 0.5 M NaCl in the presence and absence of the osmoprotectants betaine and proline . The goal of this work is to correlate the effects of osmoprotectants on the composition of the cytoplasm with their ability to increase the growth rate of osmotically stressed cells . At a concentration of 1 mM in the growth medium, betaine increases the growth rate more than does proline; choline, which is converted to betaine by E . coli, appears to have an intermediate effect on growth rate . The accumulation of either betaine or proline reduces the cytoplasmic amounts of K+, glutamate, trehalose, and MOPS (the major cytoplasmic osmolytes accumulated in the absence of osmoprotectants), so that at this external osmolarity the total amount of cytoplasmic solutes is essentially the same in the presence or absence of either osmoprotectant . More betaine than proline is accumulated, so the extent of replacement of cytoplasmic solutes is greater for betaine than for proline . Accumulation of these osmoprotectants is accompanied by a large (20 to 50%) increase in the volume of cytoplasmic water per unit of cell dry weight (Vcyto) . This effect, which appears to result from an increase in the volume of free water, Vf (as opposed to water of hydration, or bound water), is greater for betaine than for proline . Taken together, these results indicate that the molar effects of betaine and proline on water activity and on the osmotic pressure of the cytoplasm must be significantly larger than those of the solutes they replace . Cayley et al . (S . Cayley, B . A . Lewis, H . J . Guttman, and M . T . Record, Jr., J . Mol . Biol . 222:281-300, 1991) observed that, in cells grown in the absence of osmoprotectants, both growth rate and Vcyto decreased, whereas the amount of cytoplasmic K+ (nK+) increased, with increasing external osmolarity . We predicted that the observed changes in nK+ and Vcyto would have large and approximately compensating effects on key protein-nucleic acid interactions of gene expression, and we proposed that Vf was the fundamental determinant of growth rate in osmotically stressed cells . The properties of cells cultured in the presence of betaine and proline appear completely consistent with our previous work and proposals . Accumulation of betaine and, to a lesser extent, proline shifts the set of linked physiological parameters (nK+, Vcyto, growth rate) to those characteristic of growth at lower osmolarity in the absence of osmoprotectants . Models for the thermodynamic basis and physiological consequences of the effect of osmoprotectants on Vcyto and Vf are discussed.

Infect Immun, 1992 Mar, 60(3), 958 - 64
Characterization of the outer membrane proteins of Bordetella avium; Leyh R et al.; The outer membrane proteins of Bordetella avium were examined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis . Sarkosyl-insoluble outer membrane protein-enriched profiles from 50 virulent B . avium isolates, containing major 21,000- and 37,000-molecular-weight proteins (21K and 37K proteins, respectively) and at least 13 less intensely stained proteins with molecular weights ranging from 13,500 to 143,000, were very similar . The 21K, 27K, 31K, and 37K outer membrane proteins were shown to be associated noncovalently with the underlying peptidoglycan layer . It was necessary to treat cell envelopes with 2% sodium dodecyl sulfate and at temperatures in excess of 60 degrees C for 15 min to release these proteins . Exposure of proteins on the cell surface of B . avium was assessed by labeling with 125I followed by electrophoresis . As many as 13 bands were present in profiles from labeled whole cells . Of the surface-labeled bands, eight corresponded to bands in a radiolabeled outer membrane preparation . The outer membrane protein profile of B . avium was compared with profiles from other Bordetella spp., including 20 B . avium-like and 16 B . bronchiseptica strains isolated from turkeys . The outer membrane protein profile of B . avium was distinctly different from those of the other bordetella . The effect of variations in the growth medium on the expression of outer membrane proteins of B . avium was examined . Expression of 22K, 26K, 56K, and 73K proteins was decreased or eliminated by addition of 50 mM MgSO4 to the medium.

J Bacteriol, 1992 Mar, 174(5), 1505 - 14
Identification of intrinsic high-level resistance to rare-earth oxides and oxyanions in members of the class Proteobacteria: characterization of tellurite, selenite, and rhodium sesquioxide reduction in Rhodobacter sphaeroides; Moore MD et al.; We have identified intrinsic high-level resistance (HLR) to tellurite, selenite, and at least 15 other rare-earth oxides and oxyanions in the facultative photoheterotroph Rhodobacter sphaeroides grown either chemoheterotrophically or photoheterotrophically . Other members of the class Proteobacteria, including members of the alpha-2 and alpha-3 phylogenetic subgroups, were also shown to effect the reduction of many of these compounds, although genera from the alpha-1, beta-1, and gamma-3 subgroups did not express HLR to the oxyanions examined . Detailed analyses employing R . sphaeroides have shown that HLR to at least one class of these oxyanions, the tellurite class (e.g., tellurate, tellurite, selenate, selenite, and rhodium sesquioxide), occurred via intracellular oxyanion reduction and resulted in deposition of metal in the cytoplasmic membrane . The concomitant evolution of hydrogen gas from cells grown photoheterotrophically in the presence of these oxyanions was also observed . HLR to tellurite class oxyanions in R . sphaeroides was not affected by exogenous methionine or phosphate but was reduced 40-fold by the addition of cysteine to growth media . In contrast HLR to the periodate class oxyanions (e.g., periodate, siliconate, and siliconite) was inhibited by extracellular PO4(3-) but did not result in metal deposition or gas evolution . Finally, we observed that HLR to arsenate class oxyanions (e.g., arsenate, molybdate, and tungstate) occurred by a third, distinct mechanism, as evidenced by the lack of intracellular metal deposition and hydrogen gas evolution and an insensitivity to extracellular PO4(3-) or cysteine . Examination of a number of R . sphaeroides mutants has determined the obligate requirement for an intact CO2 fixation pathway and the presence of a functional photosynthetic electron transport chain to effect HLR to K2TeO3 under photosynthetic growth conditions, whereas functional cytochromes bc1 and c2 were required under aerobic growth conditions to facilitate HLR . Finally, a purification scheme to recover metals from intact bacterial cells was developed.

J Steroid Biochem Mol Biol, 1992 Mar, 41(3-8), 349 - 60
Steroids inversely affect the biosynthesis and secretion of human prostatic acid phosphatase and prostate-specific antigen in the LNCaP cell line; Henttu P et al.; In order to elucidate the mechanism of androgen-regulation of genes expressed only in the prostate gland, the effects of steroid hormones on the biosynthesis and secretion of human prostatic acid phosphatase (PAP) and prostate-specific antigen (PSA) were studied in the human prostatic carcinoma cell line, LNCaP . This cell line produces PAP and PSA, both of which were found to be similar to the proteins purified from and located in human prostatic tissue, as shown by Western blot analysis . The synthetic androgen, R1881, regulated the biosynthesis of these two important tumour marker proteins inversely: the amount of PSA released into the medium was increased to 506% +/- 100 of the control levels, while that of PAP was decreased to 26% +/- 3, in 7 days . These effects were dependent on the concentration of the steroid in the growth medium . The androgen-dependent changes observed in the amounts of the secreted proteins were correlated with alterations in their intra-cellular levels . LNCaP cells were found to have very different capacities for secreting PAP and PSA . Whereas the measurable, cellular amounts of PSA and PAP were of similar magnitudes, much larger amounts of PSA than PAP were secreted into the medium . PSA was also found to be more stable than PAP in the culture medium of the LNCaP cells . Other steroids could elicit effects on PAP and PSA biosynthesis similar to those induced by R1881, and the combined effects of effective concentrations of these steroids were undistinguishable from those caused by each one of them separately, suggesting that all these compounds compete for binding to the same modified androgen receptors of the LNCaP cells . Thus, our results confirm the observations of the altered nature of the LNCaP androgen receptors, and demonstrate the ability of these ligands to produce changes in the expression of androgen-dependent prostatic genes . The fact that the changes observed at the protein level were accompanied by increased levels of PSA mRNAs and by decreased levels of PAP mRNA in steroid-treated cells, suggests that one of the targets of androgen and steroid action in the regulation of these genes is at the mRNA level.

Infect Immun, 1992 Mar, 60(3), 804 - 9
Characterization of an antigenically conserved heat-modifiable major outer membrane protein of Branhamella catarrhalis; Sarwar J et al.; Branhamella catarrhalis is a common cause of otitis media in children and of respiratory infections in adults with chronic bronchitis . Little is known about the antigenic structure of the outer membrane proteins (OMPs) . In this study, two murine monoclonal antibodies, 7D6 and 5E8, were developed and used to characterize the major heat-modifiable OMP (OMP C/D) of B . catarrhalis . Immunoblot assays indicated that OMP C/D is heat modifiable, having a molecular mass of 55 kDa at room temperature and a mass of 60 kDa when heated under reducing conditions . Expression of the epitopes is independent of growth phase and growth media . Both epitopes are present in 51 of 51 strains of B . catarrhalis tested and are highly specific for Branhamella strains, being absent from a variety of other gram-negative species . Antibody 5E8 recognizes an epitope which is expressed on the surface of the intact bacterium . We conclude that OMP C/D is a major, heat-modifiable OMP antigen that expresses at least one stable, conserved epitope on the surface of B . catarrhalis . Future studies should focus on the role of OMP C/D in pathogenesis and on its potential role as a vaccine antigen.

Biochem Int, 1992 Mar, 26(3), 469 - 76
Inhibition of cellular glutathione biosynthesis by rifampicin in Mycobacterium smegmatis; Kumar S et al.; This study was carried out to investigate the mechanism of Rifampicin (RIF) induced glutathione (GSH) depletion in M . smegmatis . RIF at various concentrations decreased the activities of gamma glutamyl cysteine synthetase (GGCS) and GSH synthetase . Maximum decrease in the activities of biosynthetic enzymes of GSH was observed when 15 micrograms RIF ml-1 medium was incorporated in the growth medium before performing inoculations . The activity of GGCS was also decreased when three day grown M . smegmatis was exposed to 60 micrograms RIF ml-1 medium for a period of 6 h and 9 h . RIF did not alter the activity of gamma glutamyl transferase . The results of the present study demonstrate that the depletion caused by RIF in cellular GSH is due to its decreased biosynthesis whereas its degradation is not affected in M . smegmatis.

J Antibiot (Tokyo), 1992 Mar, 45(3), 394 - 9
Biosynthesis of griseolic acids: incorporation of 13C-labeled compounds into griseolic acid A; Miyakoshi S et al.; Biosynthesis of griseolic acids, competitive inhibitors of cyclic nucleotide phosphodiesterase, was investigated with the culture of a producing strain of Streptomyces griseoaurantiacus . 13C-Labeled and 15N-labeled compounds were added into the culture, and 13C-enriched and 15N-enriched griseolic acid A was isolated from the culture medium and analyzed by 13C NMR and 15N NMR spectroscopy . The compounds added to growth medium were {2-13C}acetate, {1,2-13C}acetate, {1,4-13C}succinate, {1-13C}glucose, {6-13C}glucose, {2-13C}ribose, and {1-13C, 15N}glycine . The results suggest that adenosine, which is formed from amino acids and sugars contributes the adenine and ribose moieties to griseolic acid A . The data also suggest that a dicarboxylic acid from the Krebs tricarboxylic acid cycle contributes to the dicarboxylic part of the compound.

J Neurosci, 1992 Mar, 12(3), 793 - 9
Acetylcholine synthesis and release is enhanced by dibutyryl cyclic AMP in a neuronal cell line derived from mouse septum; Blusztajn JK et al.; Cholinergic properties of the SN56.B5.G4 cell line derived from the fusion of neurons of the mouse postnatal day 21 septum and the murine neuroblastoma cell line N18TG2 were investigated and correlated with morphological differentiation . In basal serum-containing growth medium, few cells developed neurites . Neurite extension occurred in cells grown for 2 d with forskolin or dibutyryl cAMP (dbcAMP) but not with butyrate . In cells treated with these compounds, the activity of ChAT and ACh content were two- to threefold higher relative to controls . The cells synthesized ACh from choline taken up by the sodium-dependent high-affinity transport . Forskolin-, dbcAMP-, and butyrate-treated cells (but not the controls) were capable of spontaneous and depolarization-evoked ACh release . The results indicate that the morphological and the neurochemical aspects of SN56.B5.G4 cell differentiation are independently regulated.

FEBS Lett, 1992 Feb 3, 297(1-2), 155 - 8
Cloning and characteristics of a positive regulatory gene, THI2 (PHO6), of thiamin biosynthesis in Saccharomyces cerevisiae; Nishimura H et al.; A thi2(pho6) mutant of Saccharomyces cerevisiae, defective in the expression of structural genes for thiamin-repressible acid phosphatase and enzymes involved in thiamin biosynthesis, was found to retain sufficient thiamin transport activity . The transport activity was repressed by thiamin in growth medium . We isolated from a S . cerevisiae genomic library two hybrid plasmids, pTSR1 and pTSR2, containing 10.2- and 12.0-kilobase (kb) DNA fragments, respectively, which complement the thi2(pho6) mutation of S . cerevisiae . This gene was localized on a 6.0-kb ClaI-ClaI fragment in the subclone pTSR3 . Complementation of the enzyme activities for thiamin metabolism in the thi2(pho6) mutant transformed by some plasmids with the TH12(PHO6) gene was also examined.

Infect Immun, 1992 Feb, 60(2), 353 - 9
Influence of calcium on secretion and activity of the cytolysins of Actinobacillus pleuropneumoniae; van Leengoed LA et al.; In vitro production of a secreted hemolytic cytolysin of Actinobacillus pleuropneumoniae has been reported to be dependent on the presence of calcium in culture media . This is not the case with Escherichia coli hemolysins, however, where calcium has been shown to be required only for activation and binding to target cells . Because the cytolysins of A . pleuropneumoniae have structural and functional similarities to those of hemolytic E . coli, we sought to reexamine the role that calcium plays in the secretion and activity of A . pleuropneumoniae cytolysins . A . pleuropneumoniae hemolytic strain S4074 secreted two major proteins into culture supernatants independent of the presence of calcium in growth medium . These proteins were identified with murine monoclonal antibodies as the 105-kDa cytolysin I and the 103-kDa cytolysin II . It was found that both cytolysins required calcium for binding to erythrocyte membranes . Culture fluids from bacteria grown with calcium lysed porcine erythrocytes even after free calcium in the fluid was removed prior to the hemolytic assay . When bacteria were grown in medium depleted of calcium, no lysis of erythrocytes was detected unless calcium was added to assay buffers . Culture supernatants from A . pleuropneumoniae nonhemolytic strain 1421 grown with or without calcium contained two predominant proteins, which were identified with mouse monoclonal antibodies as the 103-kDa cytolysin II and the 120-kDa cytolysin III . Binding to erythrocytes (without hemolysis) by cytolysin II was dependent on calcium . Cytolysin III did not bind to erythrocytes . These results indicate that the ability of strain S4074 to lyse swine erythrocytes (and the inability of strain 1421 to do so) was directly correlated with the presence of cytolysin I . Cytolysins I, II, and III bound to swine neutrophils and purified neutrophil membranes only in the presence of calcium . When calcium was depleted, cytolysin I of strain S4074 had a reduced binding and cytolysis II and III of strain 1421 did not bind at all . The data suggest that regardless of the target cell involved, calcium plays an integral role in the function but not the production of A . pleuropneumoniae cytolysins.

Appl Environ Microbiol, 1992 Feb, 58(2), 485 - 95
Effects of chlorobenzoate transformation on the Pseudomonas testosteroni biphenyl and chlorobiphenyl degradation pathway; Sondossi M et al.; Bacterial conversion of biphenyl (BP) and chlorobiphenyls (CBPs) to benzoates and chlorobenzoates (CBAs) proceeds by introduction of molecular oxygen at the 2,3 position, followed by a 1,2-meta cleavage of the molecule . Complete mineralization of CBPs requires the presence of two sets of genes, one for the transformation fo CBPs into CBAs and a second for the degradation of CBAs . It has been shown previously that removal of the CBAs produced from the degradation of CBPs is essential for efficient degradation of CBPs . In this study we confirmed that CBAs inhibit BP and CBP transformation in Pseudomonas testosteroni B-356 . Among the three monochlorobenzoates tested, 3-chlorobenzoate was the most effective inhibitor . Furthermore, we found that in strain B-356, CBA transformation is controlled by BP-induced oxygenases that are not present in benzoate-grown cells . We found that this BP-linked CBA transformation pathway transformed CBAs produced from CBPs into several metabolites, including chlorocatechols and corresponding muconic semialdehydes . These metabolites inhibited the 2,3-dihydroxybiphenyl 1,2-dioxygenase, while CBAs by themselves had no effect on this enzyme . Therefore, on the basis of this and other observations, it appears that when CBAs produced from CBPs accumulate in the growth medium, they are converted into unproductive metabolites that reduce the flux of the BP and CBP degradation pathway . The practical implications of these interactions on the microbial degradation of polychlorinated BPs are also discussed.

Appl Environ Microbiol, 1992 Feb, 58(2), 444 - 9
Cell surface redox potential as a mechanism of defense against photosensitizers in fungi; Sollod CC et al.; The phytotoxin cercosporin, a singlet oxygen-generating photosensitizer, is toxic to plants, mice, and many fungi, yet the fungi that produce it, Cercospora spp., are resistant . We hypothesize that resistance to cercosporin may result from a reducing environment at the cell surface . Twenty tetrazolium dyes differing in redox potential were used as indicators of cell surface redox potential of seven fungal species differing in resistance to cercosporin . Resistant fungi were able to reduce significantly more dyes than were sensitive fungi . A correlation between dye reduction and cercosporin resistance was also observed when resistance levels of Cercospora species were manipulated by growth on different media . The addition of the reducing agents ascorbate, cysteine, and reduced glutathione (GSH) to growth media decreased cercosporin toxicity for sensitive fungi . None of these agents directly reduced cercosporin at the concentrations at which they protected fungi . Spectral and thin-layer chromatographic analyses of cercosporin solutions containing the different reducing agents indicated that GSH, but not cysteine or ascorbate, reacted with cercosporin . Resistant and sensitive fungi did not differ in endogenous levels of cysteine, GSH, or total thiols . On the basis of data from this and other studies, this report presents a model which proposes that cercosporin resistance results from the production of reducing power at the surfaces of resistant cells, leading to transient reduction and detoxification of the cercosporin molecule.

Regul Toxicol Pharmacol, 1992 Feb, 15(1), 3 - 9
Toxicological considerations for protein components of biological pesticide products; Sjoblad RD et al.; The toxicity of protein components of microbial pesticide products is evaluated at EPA by requiring that pesticide manufacturers conduct a thorough taxonomic evaluation of the active microbial ingredient . The requirement for acute toxicity testing by dosing laboratory animals with the active microbial ingredient and with fermentation growth medium materials provides additional information on the toxicity of protein components of microbial pesticides . The potential for toxicity from proteins associated with contaminating organisms is addressed by use of appropriate quality control procedures to minimize or prevent growth of contaminants and by screening of fermentation batches for known human/mammalian pathogens . These considerations also would apply to any biochemical pesticide that is formed via the growth of a microorganism . If a protein itself is intended for commercial use as an active pesticide ingredient, acute exposure studies and in vitro digestibility studies could be done to answer potential concerns regarding toxicity.

Immunol Invest, 1992 Feb, 21(1), 25 - 38
Embryo associated immunosuppressor factor(s) secreted by preembryo and serum estradiol levels are predictive of pregnancy outcome: effect of gonadotropin releasing hormone agonist (GnRHa) treatment of patients undergoing in vitro fertilization and embryo transfer (IVF-ET); Bose R et al.; The aim of the present study was to determine whether (i) the secretion of embryo associated immunosuppressor factor (EASF) by preimplantation embryo and serum estradiol (E2) play a cooperative role in pregnancy outcome and (ii) whether this association was affected by gonadotropin releasing hormone agonist (GnRHa) pretreatment of IVF-patients for ovarian stimulation . EASF activity was measured using concanavalin A-induced lymphocyte proliferation assay in 251 IVF-culture media obtained from 59 patients undergoing IVF-ET . EASF activity in embryo growth media, cycle day 3 serum E2 level and peak E2 levels were correlated with pregnancy outcome . Results indicate that in GnRHa non-treated patients (i) the immunosuppressive activity of embryo growth media significantly (p less than 0.05) differed between the patients with varying concentration of day 3 serum E2, (ii) the presence of immunosuppressive activity in the embryo growth media was associated with success of pregnancy in patients with day 3 serum E2 level of 21-40 pg/ml (p less than 0.005) and in patients with peak E2 level in serum of less than = 1500 pg/ml (p less than 0.05) . No association in immunosuppressive activity, serum E2 level and successful pregnancy was seen in GnRHa treated patients . This preliminary study suggests that the concentration of serum hormones and factors that are altered due to GnRHa pretreatment of IVF-patients may affect the cooperative role played by EASF, E2 and other factors in the success of pregnancy.

In Vitro Cell Dev Biol, 1992 Feb, 28A(2), 109 - 19
Effects of subcultivation and culture medium on differentiation of human fetal cardiac myocytes; Goldman BI et al.; Previous work has suggested that subcultivated human fetal heart muscle cell cultures contain immature cardiac muscle cells capable only of limited differentiation after mitogen withdrawal . We studied several human fetal heart cultures (14-15 wk gestation) at several passage levels using immunocytochemistry, autoradiography, and Northern blot analysis . Characteristics in high-mitogen (growth) medium were compared with those after serum withdrawal . Cultured cells from one heart, expanded through 2 passages in growth medium, did not beat; however, 75% of cells did beat after subsequent culture for 24 days in low-serum (differentiation) medium containing insulin . In confluent cultures after 1 passage, there was no detectable difference in the number of cardiac myocytes present in growth medium compared with that 7 days after serum withdrawal . After 4 passages, however, serum withdrawal increased the number of cells expressing immunoreactive sarcomeric myosin heavy chain by 100-fold; expression of immunoreactive sarcomeric actin and alpha-cardiac actin mRNA also increased in the same cultures . Similar results were obtained in cultures kept in differentiation medium for 20 days before passage and expansion in growth medium . Using isopycnic centrifugation, a high-density cell fraction was isolated which contained no immunostained myocytes in growth medium but numerous myocytes after serum withdrawal . Combined immunocytochemistry/autoradiography showed that myocytes synthesize DNA in growth medium and in serum-free medium containing fibroblast growth factor, but not in serum-free medium alone . The results indicate that a) human fetal cardiac muscle cells proliferate in vitro and can maintain a phenotype characteristic of fetal myocytes after multiple subcultivations followed by serum withdrawal; b) after subcultivation in growth medium, some myocytes modulate their phenotype into one in which detectable levels of cardiac contractile proteins are expressed only after mitogen withdrawal, and c) the phenotype attained after serum withdrawal is in part dependent on passage level . Cultured human fetal myocardial cells may provide a useful experimental system for the study of human cardiac muscle cell biology.

Mol Chem Neuropathol, 1992 Feb-Apr, 16(1-2), 33 - 44
Primary cultures of astrocytes from rat as a model for biotin deficiency in nervous tissue; Rodriguez-Pombo P et al.; The activities and biotin-dependence of the three mitochondrial biotin-dependent carboxylases: pyruvate carboxylase, propionyl CoA carboxylase, and beta-methylcrotonyl CoA carboxylase of primary culture of astrocytes have been examined . An increase of the three mitochondrial carboxylase activities was observed during cell growth, as was the case for developing rat brain . Mitochondrial carboxylase activities from 3-wk-old primary cultures of astrocytes were higher than those in the neonatal rat brain . When astrocytes were grown in a 10% serum-enriched medium supplemented with avidin to bind biotin, the mitochondrial carboxylase activities were reduced to 15% of control value . Consistent with these results, after 3 wk in culture, the 3-hydroxyisovaleric acid concentration in the growth medium was tenfold higher than the controls . In this culture condition, cellular growth and the nonbiotin-dependent enzyme, glutamine synthetase, were not modified with respect to control . Primary cultures from newborn rat brain hemispheres are suggested as an experimental approach to the study of biotin deficiency in nervous tissue.

J Cell Physiol, 1992 Feb, 150(2), 416 - 25
Mitogenic, melanogenic, and cAMP responses of cultured neonatal human melanocytes to commonly used mitogens; Abdel-Malek Z et al.; The following studies have been undertaken to compare and correlate the effects of 12-O-tetradecanoylphorbol acetate (TPA), basic fibroblast growth factor (bFGF), cholera toxin (CT), and isobutyl methylxanthine (IBMX) on neonatal human melanocyte (NHM) proliferation, tyrosinase activity, and cyclic adenosine monophosphate (cAMP) concentration . NHM proliferated at a maximal rate in medium containing 8 nM TPA, 200 ng/ml CT, and 10(-4) M IBMX . TPA alone did not result in optimal melanocyte proliferation, and, as previously shown, its mitogenic effect was greatly enhanced by the addition of CT and IBMX individually or concomitantly . Human recombinant (hr) bFGF could replace TPA in the NHM growth medium . Maximal proliferation was achieved using 3 ng/ml hrbFGF, 20 ng/ml CT, and 10(-4) M IBMX . The mitogenic effect of 1.2 ng/ml hrbFGF was potentiated in the concomitant but not individual presence of CT and IBMX . TPA alone in the absence of CT and IBMX caused a dose-dependent stimulation of tyrosinase activity . Maximal tyrosinase activity was obtained in the presence of 0.8 nM TPA, 20 ng/ml CT, and 10(-4) M IBMX . Unlike TPA, hrbFGF alone resulted in inhibition of tyrosinase activity . In the presence of hrbFGF, tyrosinase activity was potentiated by CT and IBMX, but not by CT alone . Neither TPA nor hrbFGF alone could increase intracellular cAMP levels . The effects of CT and IBMX on intracellular cAMP concentration were enhanced to a greater extent by TPA than by hrbFGF . Under our experimental conditions, in the presence of hrbFGF, CT but not IBMX resulted in a dose-dependent increase in cAMP concentration . Further studies on NHM will be aimed at determining the exact role of protein kinase C (PKC) in regulating proliferation and melanogenesis and the mechanism(s) activated by hrbFGF.

Int J Biochem, 1992 Feb, 24(2), 215 - 22
Biosynthesis of inter-alpha-trypsin inhibitor and alpha 1-microglobulin in a human hepatoma cell line; Odum L; 1 . Biosynthesis of alpha 1-microglobulin and inter-alpha-trypsin inhibitor was investigated in a human hepatoma cell line HepG-2 . 2 . alpha 1-Microglobulin was translated as a precursor common with the light chain of inter-alpha-trypsin inhibitor . 3 . alpha 1-Microglobulin was synthesized and secreted into the growth medium within 30 min . 4 . Processing of inter-alpha-trypsin-inhibitor-related proteins appeared slow and incomplete . The light chain was connected via a chondroitinsulphate to a heavy chain to form a 125,000-Mr protein and secreted within 1-4 hr.

Appl Microbiol Biotechnol, 1992 Feb, 36(5), 598 - 603
Production of androsta-1,4-diene-3,17-dione from cholesterol using immobilized growing cells of Mycobacterium sp . NRRL B-3683 adsorbed on solid carriers; Lee CY et al.; Living cells of Mycobacterium sp . NRRL B-3683 were immobilized by adsorption on different types of solid carriers in order to produce androsta-1,4-diene-3,17-dione (ADD) from cholesterol . Activated alumina proved to be the most preferred carrier for long-term operation when glucose and peptone were added to the reaction medium . In a repeated-batch process, the maximum productivity of ADD was about 0.19 g/l per day with a molar conversion rate of 77% when 1.0 g/l of cholesterol was added to the reaction medium . The half-life of the immobilized cells was more than 45 days and the system could be reactivated by incubating the immobilized cells in a cell growth medium.

J Bacteriol, 1992 Feb, 174(4), 1240 - 7
Isolation and characterization of the Escherichia coli htrD gene, whose product is required for growth at high temperatures; Delaney JM et al.; Those genes in Escherichia coli defined by mutations which result in an inability to grow at high temperatures are designated htr, indicating a high temperature requirement . A new htr mutant of E . coli was isolated and characterized and is designated htrD . The htrD gene has been mapped to 19.3 min on the E . coli chromosome . Insertional inactivation of htrD with a mini-Tn10 element resulted in a pleiotropic phenotype characterized by a severe inhibition of growth at 42 degrees C and decreased survival at 50 degrees C in rich media . Furthermore, htrD cells were sensitive to H2O2 . Growth rate analysis revealed that htrD cells grow very slowly in minimal media supplemented with amino acids . This inhibitory effect has been traced to the presence of cysteine in the growth medium . Further studies indicated that the rate of cysteine transport is higher in htrD cells relative to the wild type . All of these results, taken together, indicate that the htrD gene product may be required for proper regulation of intracellular cysteine levels and that an increased rate of cysteine transport greatly affects the growth characteristics of E . coli.

Protein Sci, 1992 Feb, 1(2), 271 - 7
Yeast ferrochelatase: expression in a baculovirus system and purification of the expression protein; Eldridge MG et al.; The terminal step of the heme biosynthetic pathway is catalyzed by the enzyme ferrochelatase (EC 4.99.1.1) . In eukaryotes this enzyme is bound to the inner mitochondrial membrane with its active site facing the matrix side of the membrane . Previously this laboratory has characterized this enzyme via kinetic and protein chemical modification techniques, and with the recent cloning of the enzyme from yeast, mouse, and human sources it now becomes possible to approach structure-function questions by using site-directed mutagenesis . Of primary significance to this is the development of an efficient expression vector . This is of particular significance for ferrochelatase, as it is a low-abundance protein whose DNA coding sequence has a very low codon bias . In the current work we describe the production of yeast ferrochelatase in a baculovirus system . This system is shown to be an excellent one in which to produce large quantities of active ferrochelatase . The expressed enzyme is membrane associated and is not released into the growth medium either during or after virus development and cell lysis . The expressed protein can be purified in a procedure that requires only 1 day and makes use of a Pharmacia Hi Trap blue affinity column . The measured Km's for the substrates mesoporphyrin and iron are the same as those reported previously for the yeast enzyme . To our knowledge this is the first example of a mitochondrial membrane protein that has been expressed in a baculovirus system.

Biochem Pharmacol, 1992 Jan 22, 43(2), 213 - 7
Effects of calcium and calcium-channel blocker methoxyverapamil on the beta-adrenoceptors in myocardial cells in vitro; Disatnik MH et al.; The possible relationship between methoxyverapamil (D600) as a calcium-channel blocker and the beta-adrenoceptors was investigated on heart cells grown in culture, using {3H}CGP-12177 as a radioligand . Treatment with D600 (20 micrograms/mL) for 24 hr caused a decrease of 30% in the {3H}CGP-12177 binding sites . Scatchard analysis showed that the Bmax is similar in control and D600-treated cells, but the Kd in D600-treated cells increases . The effect of D600 on the isoproterenol-induced adenylate cyclase activation was examined and it was found that the D600 prevented the increase in cAMP obtained by isoproterenol treatment . These results indicate that the action of D600 on the beta-adrenoceptors is a competitive inhibition of the {3H}CGP-12177 binding sites . We investigated the effect of Ca2+ in the growth medium on the level of beta-adrenoceptors . Heart cells grown for 24 hr in Ca(2+)-free medium showed a decrease of 36% in the {3H}CGP-12177 binding sites without changing the dissociation constant . This decrease is probably a result of reduction in synthesis of the receptors . The level of receptors returned to control values following replenishment with normal growth medium . These results show that calcium is essential for the development of the beta-adrenoceptors in heart cells in vitro.

Brain Res Dev Brain Res, 1992 Jan 17, 65(1), 57 - 64
Spontaneous firing as an epigenetic factor in brain development--physiological consequences of chronic tetrodotoxin and picrotoxin exposure on cultured rat neocortex neurons; Corner MA et al.; Functional consequences of either suppressing or intensifying spontaneous neuronal firing have been studied in developing rat cerebral cortex cultures using, respectively, tetrodotoxin (TTX) and picrotoxin (PTX) added chronically to the growth medium . Simple measures derived from the interspike interval histogram were able to powerfully discriminate between age and treatment groups . After return to control medium, most TTX-treated neurons spontaneously displayed stereotyped clustering of action potentials ('phasic' firing) which closely resembled the characteristic firing patterns seen acutely in the presence of PTX . The 'TTX-syndrome' thus suggests that GABAergic synaptic inhibition is ineffective in cortical networks grown under conditions which prevent the expression of bioelectric activity . In contrast, after return to control medium, neurons which had been partially disinhibited throughout development (by continuous exposure to PTX) had even less phasic firing than was measured in age-matched controls . Based upon these and previous findings, a two (main) factor model is put forth which can economically account for the major effects . The working hypothesis embodied in this model is that phasic neuronal discharges not only accelerate the maturation of excitatory connections within the neocortex but, even more important, are crucial for the development of adequate inhibitory synaptic transmission.

J Biol Chem, 1992 Jan 15, 267(2), 774 - 9
Cloning and mutational analysis of the gene encoding subunit C of yeast vacuolar H(+)-ATPase; Beltran C et al.; A DNA fragment containing the gene encoding subunit C of vaculor H(+)-ATPase (V-ATPase) was cloned from a yeast library . The predicted amino acid sequence indicated that the C subunit consists of 373 amino acids with a calculated molecular mass of 42,287 Da . The protein from yeast is 37% identical in its amino acid sequence to the C subunit of bovine V-ATPase . The DNA fragment that was cloned in this study contained two additional reading frames . At the 5' end an amino acid sequence that is homologous to Artemia elongation factor 1 was detected . At the 3' end the N-terminal part of a kinesin-like protein was observed . The gene encoding subunit C of the V-ATPase was interrupted, and the resulting mutant could not grow at high pH and was sensitive to low and high Ca2+ concentrations in the growth medium . Transformation of the mutant by a plasmid containing the gene encoding subunit C repaired the phenotype of the mutant . Substitution of more than half of the coding region by a corresponding DNA fragment encoding the bovine subunit C resulted in a phenotype indistinguishable from wild type . Immunological studies with the disruptant mutant revealed that subunit C is necessary for the assembly of the catalytic sector of the enzyme.

Mol Cell Biochem, 1992 Jan 15, 109(1), 51 - 60
Heparin releasable and nonreleasable forms of heparan sulfate proteoglycan are found on the surfaces of cultured porcine aortic endothelial cells; Lowe-Krentz LJ et al.; Evidence suggests that endothelial cell layer heparan sulfate proteoglycans include a variety of different sized molecules which most likely contain different protein cores . In the present report, approximately half of endothelial cell surface associated heparan sulfate proteoglycan is shown to be releasable with soluble heparin . The remaining cell surface heparan sulfate proteoglycan, as well as extracellular matrix heparan sulfate proteoglycan, cannot be removed from the cells with heparin . The heparin nonreleasable cell surface proteoglycan can be released by membrane disrupting agents and is able to intercalate into liposomes . When the heparin releasable and nonreleasable cell surface heparan sulfate proteoglycans are compared, differences in proteoglycan size are also evident . Furthermore, the intact heparin releasable heparan sulfate proteoglycan is closer in size to proteoglycans isolated from the extracellular matrix and from growth medium than to that which is heparin nonreleasable . These data indicate that cultured porcine aortic endothelial cells contain at least two distinct types of cell surface heparan sulfate proteoglycans, one of which appears to be associated with the cells through its glycosaminoglycan chains . The other (which is more tightly associated) is probably linked via a membrane intercalated protein core.

FASEB J, 1992 Jan 6, 6(2), 777 - 85
Transcriptional derepression of the murine Cyp1a-1 gene by mevinolin; Puga A et al.; In mouse hepatoma Hepa-1c1c7 cultures, polycyclic aromatic compounds such as benzol{a}pyrene and 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD; dioxin) activate the Cyp1a-1 (cytochrome P(1)450) and Nmo-1{NAD(P)H:menadione-oxidoreductase} genes, two members of the aromatic hydrocarbon (Ah)-responsive gene battery . Mevinolin is known to inhibit 3-hydroxy-3-methylglutaryl CoA (HMG-CoA) reductase (EC 1.1.1.34), the rate-limiting step in cholesterol biosynthesis . We show here that in the absence of TCDD, mevinolin markedly increases Cyp1a-1 transcription, CYP1A1 mRNA and protein levels and enzyme activity, and NMO1 mRNA concentrations . Addition of mevalonate, the product of HMG-CoA reductase activity, fails to reverse the effects of mevinolin . In fact, when used at high concentrations, mevalonate activates Cyp1a-1 transcription . Mevinolin-induced Cyp1a-1 gene activation: (1) occurs independently of the lipid content of the growth medium, (2) is not suppressed by adding 25-hydroxycholesterol, which blocks MHG-CoA reductase activity, and (3) requires a functional Ah receptor and unimpaired nuclear translocation of the receptor . It is possible that an unknown metabolite (or metabolites) of mevinolin activates Cyp1a-1 expression and that high concentrations of mevalonate act via the same mechanism . Using chimaeric plasmids that contain different lengths of Cyp1a-1 5' flanking regions fused to the bacterial neomycin (neo) gene, we find that the mevinolin effect on Cyp1a-1 induction requires the 5' flanking sequences between -1647 and -824, which are also needed for TCDD induction . Mevinolin, however, is not a ligand for the Ah receptor . Gel mobility shift assays revealed that Cyp1a-1 activation caused by mevinolin does not involve the ligand-dependent formation of a functional Ah receptor-dependent DNA-binding complex, but instead appears to be correlated with release of a putative repressor from its cognate DNA site . Our results suggest that the basel level of Cyp1a-1 transcription is maintained by an unknown negative regulatory factor . We propose that Cyp1a-1 transcriptional activation can result not only from induction by polycyclic aromatic compounds but also from derepression by mevinolin, independent of HMG-CoA reductase inhibition.

J Bacteriol, 1992 Jan, 174(1), 108 - 15
Lrp, a leucine-responsive protein, regulates branched-chain amino acid transport genes in Escherichia coli; Haney SA et al.; We investigated the relationship between two regulatory genes, livR and lrp, that map near min 20 on the Escherichia coli chromosome . livR was identified earlier as a regulatory gene affecting high-affinity transport of branched-chain amino acids through the LIV-I and LS transport systems, encoded by the livJ and livKHMGF operons . lrp was characterized more recently as a regulatory gene of a regulon that includes operons involved in isoleucine-valine biosynthesis, oligopeptide transport, and serine and threonine catabolism . The expression of each of these livR- and lrp-regulated operons is altered in cells when leucine is added to their growth medium . The following results demonstrate that livR and lrp are the same gene . The lrp gene from a livR1-containing strain was cloned and shown to contain two single-base-pair substitutions in comparison with the wild-type strain . Mutations in livR affected the regulation of ilvIH, an operon known to be controlled by lrp, and mutations in lrp affected the regulation of the LIV-I and LS transport systems . Lrp from a wild-type strain bound specifically to several sites upstream of the ilvIH operon, whereas binding by Lrp from a livR1-containing strain was barely detectable . In a strain containing a Tn10 insertion in lrp, high-affinity leucine transport occurred at a high, constitutive level, as did expression from the livJ and livK promoters as measured by lacZ reporter gene expression . Taken together, these results suggest that Lrp acts directly or indirectly to repress livJ and livK expression and that leucine is required for this repression . This pattern of regulation is unusual for operons that are controlled by Lrp.

Adv Biochem Eng Biotechnol, 1992, 46, 35 - 62
Use of regulated secretion in protein production from animal cells: an overview; Grampp GE et al.; Traditional industrial cell culture processes require extensive downstream product refining due to low product titer and purity in the spent growth medium . A controlled secretion process incorporating cells derived from endocrine or exocrine organs could potentially alleviate this processing burden by dynamically decoupling product recovery from cell growth and product biosynthesis . In addition, such specialized secretory cells may be uniquely capable of performing desirable post-translational processing of the secretory product . We briefly review the biology of regulated protein secretion as well as the biology and biochemistry of the signal transduction mechanisms by which regulated systems respond to environmental stimuli . Drawing on these and other basic principles from cell biology and bioengineering, we describe the important features of a controlled secretion process . Among other issues we discuss the choice of cell lines, expression systems, cell culture methods, and bioreactor configurations . We extensively analyze the kinetics of regulated secretion in the context of a controlled secretion process . This discussion is illustrated with experimental results from two model cell lines, recombinant AtT-20 and beta TC3, expressing recombinant human endocrine hormones or native murine insulin respectively.

EXS, 1992, 61, 198 - 204
Possible mechanisms of type I collagen-induced vascular tube formation; Jackson CJ et al.; We investigated the effect of type I collagen on endothelial behaviour following its contact with the apical versus basal surface of cultured human endothelial cells . When endothelial cells were plated onto type I collagen they attached via their basal surface and formed a confluent monolayer . However, when type I collagen (100 micrograms/ml) was added directly to the growth medium, so that it made contact with the apical surface of endothelial cells, it induced rapid capillary-like tube formation . Possible mechanisms were assessed using a) polyclonal (anti-VLA-2) and monoclonal (AK7) antibodies to different epitopes on the alpha 2 beta 1 integrin receptor for collagen and b) drugs (chlorpromazine and trifluoperazine) that inhibit protein kinase C activity . Both anti-VLA-2 and AK7 (1-50 micrograms/ml) showed a dose-dependent inhibition of tube formation and cell attachment . At 50 micrograms/ml, anti-VLA-2 completely inhibited tube formation whereas AK7 caused only partial inhibition (less than 50%) . By contrast, AK7 was a more potent inhibitor of cell attachment than anti-VLA-2 . Both chlorpromazine and trifluoperazine prevented tube formation . Conclusions: 1) The alpha 2 beta 1 integrin receptor plays a role in both endothelial cell attachment and the induction of tube formation by type I collagen . 2) Protein kinase C may be involved in collagen-induced tube formation.

Dev Neurosci, 1992, 14(1), 53 - 60
Effect of colchicine on ganglioside composition of rat primary culture neurons; Ogiso M et al.; Developmental changes in gangliosides in the course of neurite outgrowth were examined in dissociated fetal rat cerebral neurons in culture . About a 2-fold increase in ganglioside levels was seen with the progression of neurite formation for up to 24 h in predominantly neuronal cultures . Ganglioside patterns appeared to be unchanged during the first 24 h, subsequently consisted of higher amounts of GD3 and b-series gangliosides (such as GD1b, GT1b, and GQ1b), and lower amounts of a-series gangliosides (GM1 and GD1a) . Although the addition of colchicine to the cell growth medium inhibited neurite outgrowth in developing neurons, little if any differences in ganglioside patterns were found between control and colchicine-treated cells . Ganglioside levels decreased slightly in colchicine-treated cells in agreement with the decrease in cell attachment to culture dishes . Although colchicine treatment 8 h after plating caused complete retraction of formed neurites, the ganglioside level of the cells continued to increase during the following 16-hour incubation . Thus, the data suggest that ganglioside synthesis in differentiating neurons does not primarily accompany the expansion in cell surfaces due to neurite formation, and raises the possibility that a large proportion of gangliosides is retained in intracellular compartments.

Mol Plant Microbe Interact, 1992 Jan-Feb, 5(1), 55 - 61
Effect of a virus on accumulation of a tissue-specific cell-surface protein of the fungus Cryphonectria (Endothia) parasitica; Carpenter CE et al.; Hypovirulence and decreased sporulation of the plant pathogenic fungus Cryphonectria (Endothia) parasitica is caused by double-stranded (ds)RNAs . These symptoms of dsRNA infection are correlated with down-regulation of at least nine major fungal polypeptides . One of the regulated polypeptides was purified to homogeneity and antibody to it was prepared . This polypeptide (cryparin) has a -glycine-serine-repeating sequence near the amino-terminal end that is typical of structural proteins and has properties of a lectin . Antibody-staining showed that this 18.6-kDa polypeptide is specific to aerial hyphae and fruiting bodies and that it accumulates in large amounts on hyphal cell surfaces . The dsRNA affects accumulation of this protein, both in the fugal hyphae and in the growth medium . Cryparin is similar in physical properties to those of the putative phytotoxin cerato-ulmin produced by the Dutch elm disease fungus . Toxicity of cryparin is not detectable, but the striking similarities between the physical properties and locations of accumulation of cryparin and cerato-ulmin in fungal fruiting structures suggest either conservation of structure or convergent evolution in function of these two proteins.

Physiol Chem Phys Med NMR, 1992, 24(1), 21 - 7
Membrane lipids of Mycoplasma orale: lipid composition and synthesis of phospholipids; Hirai Y et al.; Lipid composition of Mycoplasma orale was examined and compared with that of horse serum added to the growth medium . Ratios of cholesterol/cholesterol ester and sphingomyelin/phosphatidylcholine were much higher in M . orale than in the horse serum, indicating the organism incorporates selectively cholesterol and sphingomyelin . A distinct difference between the lipids from the two sources was that in phospholipids of M . orale almost all (greater than 95%) of the fatty acyl residues were saturated whereas nearly half of the residues were unsaturated in horse serum phospholipids . Approximately one third of M . orale phospholipids was phosphatidylglycerol, which was synthesized by the organism as was demonstrated by 32P-labeling experiment . Its acyl residues consisted mainly of C16:0 and were efficiently labeled with 14C-palmitate but not with 14C-acetate . These results clearly indicate the de novo synthesis of phosphatidylglycerol by M . orale is through acylation with exogenous saturated fatty acids . On the other hand, all the phosphatidylcholine and sphingomyelin of M . orale were derived from the medium . The 14C-labeling experiment demonstrates that no fatty acid synthesis takes place nor exogenous fatty acid can be incorporated so efficiently as phosphatidylglycerol, suggesting that extremely high proportion of saturated fatty acyl residues in these phospholipids is the consequence of saturation directed to the acyl chains of the incorporated phospholipids.

Life Sci, 1992, 50(22), 1711 - 8
A new approach for realizing the "antioncogram"; Kopf-Maier P; On the basis of an organoid culture method which allows organoid reorganization and histotypical growth of human carcinomas under in vitro conditions, we propose in the present study an organoid culture assay (OCA) as "antioncogram", i.e., as in vitro model for testing the drug sensitivity and resistance of individual patients' carcinomas previous to clinical chemotherapy . At the beginning of the assay, specimens of human carcinomas are disaggregated to dense single cell suspensions and dropped on a filter sheet at the air-medium interface . Organoid culture nodules develop within several days to weeks . They are exposed to cytostatics by adding the drugs for 2-3 days to the growth medium below the filter sheet . At the end of the exposure period, the cytotoxic effects are estimated by determining the fraction of viable cells, measured by the uptake of neutral red in relation to the total cell mass . In the present study we could show that this assay actually reflects the different levels of experimentally induced resistance of three human carcinoma strains of an epidermoid hypopharynx carcinoma to the cytostatic drug cisplatin and is obviously suited to predict the response of individual human carcinomas to chemotherapy in a rapid and feasible manner.

Arch Microbiol, 1992, 157(4), 305 - 10
Saccharomyces cerevisiae and Neurospora crassa contain heavy metal sequestering phytochelatin; Kneer R et al.; In fungi, cellular resistance to heavy metal cytotoxicity is mediated either by binding of metal ions to proteins of the metallothionein type or by chelation to phytochelatin-peptides of the general formula (gamma-Glu-Cys)n-Gly . Hitherto, only one fungus, Candida glabrata has been shown to contain both metal inactivating systems . Here we show by unambiguous FAB-MS analysis that both a metallothionein-free mutant of Saccharomyces cerevisiae as well as a wildtype strain synthesize phytochelatin (PC2) upon exposure to 250 microM Cd2+ ions . The presence of Zn and/or Cu ions in the nutrient broth also induces PC2 synthesis in this organism . By 109Cd exchange and subsequent monobromobimane fluorescence HPLC, it could be shown that the presence of Cd2+ in the growth medium also induces phytochelatin synthesis in Neurospora crassa, which contains metallothioneins.

Res Exp Med (Berl), 1992, 192(2), 115 - 22
Effect of caffeine and zinc on DNA and protein synthesis of neonatal rat cardiac muscle cell in culture; Kanemaru Y et al.; The effect of caffeine and/or zinc on DNA and protein synthesis of purified neonatal-rat ventricular cardiac myocytes was studied . Caffeine (0.2-2 mM) inhibited both DNA and protein synthesis of the cells . Addition of EDTA in the growth medium inhibited both DNA and protein synthesis . Without caffeine and in the presence of lower concentrations of caffeine (0.2 mM) in the growth medium, 10 microM of zinc concentration reversed DNA synthesis, which was inhibited by the chelating agent (EDTA) . Higher concentrations of caffeine (2 mM) in the growth medium completely abolished sensitivity of cardiac myocytes to zinc . Additional zinc supplementation to the growth medium of cardiac myocytes did not alter the rate of protein synthesis . The present results suggest that the effect of caffeine on cardiac myocytes may be associated with the zinc-dependent enzymes involved in DNA synthesis.

Microbiol Immunol, 1992, 36(5), 465 - 78
Definition and application of a histopathological scoring scheme for an animal model of acute Mycoplasma pneumoniae pulmonary infection; Cimolai N et al.; A histopathological scoring system was developed to assess the pathology of acute Mycoplasma pneumoniae pulmonary infection in a hamster model . A final score per animal (ranging 0-26) is obtained by averaging scores from each lung which have been accumulated by the addition of subscores from the assessments of quantity and quality of peribronchiolar and peribronchial infiltrates, luminal exudates, perivascular infiltrates, and parenchymal pneumonia . The scoring scheme was then applied to test the ability of a heat-killed inoculum to induce pulmonary pathology and to the trial of a 43 kDa protein-associated antigen as a vaccine immunogen . A heat-killed inoculum delivered by both intratracheal and intranasal routes did not induce pulmonary pathology compared to a live inoculum (respective mean scores 0.1, 6.7; P less than 0.01) . Animals prevaccinated with the 43 kDa antigen developed an accentuated pathological response after live challenge compared to those unvaccinated (respective mean scores 16.8, 5.8; P = 0.00007) . Hypersensitization to growth medium components may, however, have contributed to the accentuated disease since the lungs of vaccinated animals challenged with culture-negative media also were affected (mean score 5.4) . Reproducibility of the scoring system was measured by duplicate reading of histology slides which were randomized to the observer upon the second reading (r = 0.93; P = 0.000009) . The scoring system has the ability to differentiate disease severity in small groups of animals.

Placenta, 1992 Jan-Feb, 13(1), 43 - 53
Expression of transferrin receptors during differentiation of human placental trophoblast cells in vitro; Kennedy ML et al.; In most cell types, transferrin receptor expression is correlated with the proliferation rate, being increased by growth stimulation, or decreased by induction of terminal differentiation . In the human placenta the multinucleated syncytiotrophoblast, in direct contact with maternal blood, is derived by differentiation from mononucleated cytotrophoblast . In this study we examined changes in transferrin receptor expression during in vitro differentiation of trophoblast . Cells cultured in Ham's/Waymouth's medium (HWM) remained primarily mononuclear throughout the study, whereas incubation in keratinocyte growth medium (KGM) led to formation of multinucleate masses within 2-3 days of culture . Cell surface binding of 125I-labelled transferrin increased fivefold between days 1-5 of culture in both media and surface receptors were saturated at 7-14 micrograms/ml (90-200 fM) . At saturation, the amount of transferrin bound to syncytiotrophoblast was 37 per cent lower than in cytotrophoblast . Scatchard analysis revealed a reduction in the number of surface transferrin receptors in syncytiotrophoblast compared to cytotrophoblast . A corresponding 29 per cent reduction in the binding of transferrin to intracellular sites was observed in syncytiotrophoblast . Distribution of receptors between surface and intracellular sites was therefore similar in both cytotrophoblast and syncytiotrophoblast . The affinity of transferrin for transferrin receptors was 3.7-fold higher in syncytiotrophoblast when compared to cytotrophoblast . Observed differences between the two cell types were not due to the presence of growth factors or higher iron levels in KGM . Expression of a high number of surface transferrin receptors in syncytiotrophoblast (1.5 x 10(12)/mg protein), along with a high affinity of these receptors for iron-saturated transferrin, could help explain the efficient transport of large amounts of iron from mother to fetus.

Cancer Detect Prev, 1992, 16(5-6), 305 - 19
Cancer-associated SCM-recognition, immunedefense suppression, and serine protease protection peptide . Part I . Isolation, amino acid sequence, homology, and origin; Cercek L et al.; The isolation of a cancer-associated, SCM-recognition, immunedefense-suppressing, and serine protease-protecting (CRISPP) peptide from the blood plasma of cancer patients is described . The amino acid sequences were determined on preparations from 12 different cancers . The peptide is composed in 9 cancers of 29 and in 3 cancers of 35 amino acid residues with molecular weights of 3410 and 4007 Da, respectively . A consensus, synthetic 29 amino acid CRISPP peptide (CRISPPs) has the same cancer SCM-recognition (CR) activity and SCM-response modifying effects as the natural peptide . The "cancer SCM-recognition epitope" of the CRISPP peptide was determined . Anti-CRISPPs antibodies were raised and used in immunoassays to confirm the presence of the CRISPP peptides in cancer blood plasmas, in supernatants of cancer cell growth media and in cultured human cancer cells . The amino terminal end sequences of peptides isolated from growth media of cultured breast and colon cancer cells corresponded to amino acid sequences of CRISPP peptides isolated from cancer blood plasmas of subjects with the respective cancers . The CRISPP peptides are between 83 to 100% homologous to the alpha 1-protease inhibitor amino acid sequence located at the carboxy terminal end between residues 358 and 393 . The genetic origin of the CRISPP peptides and their selective advantage to cancer cell survival are discussed.

Acta Anat (Basel), 1992, 145(2), 119 - 26
Degranulation of rough endoplasmic reticulum in M phase and adenosine-triphosphate-treated interphase cells is reversible; Sit KH et al.; In the preferential harvesting of rounded mitotic (M phase) cells of human Chang liver monolayer cultures by mechanical agitation in Ca(2+)-free phosphate-buffered saline, degranulation of endoplasmic reticulum (ER) was observed . Mitotic cells are known to have a series of Ca2+ transients and, without being subjected to Ca(2+)-free washings, did not have degranulated ER . Quiescent cells incubated with 0.7 mM adenosine 5'-triphosphate (ATP) in Ca(2+)-free HEPES-buffered saline produced very similar ER degranulations . Confocal argon laser imaging of fluo-3-loaded cells showed a Ca2+ transient peaking at 2 min after ATP treatment . In the absence of extracellular Ca2+, transients of Ca2+ elevation in the cytosol would exit the cell in a down-gradient, draining the ER Ca2+ stores . Substituting ATP with 1 microM brominated A23187 calcium ionophore in the incubation that contained 1-100 mM CaCl2, respectively, did not produce ER degranulation, thereby excluding raised cytosolic Ca2+ per se as the cause of ER degranulation . In fact, incubation with 0.7 mM ATP in the presence of 1-5 mM CaCl2 failed to produce ER degranulation . ER degranulated cells, from treatment with ATP without extracellular Ca2+ as well as from Ca(2+)-free washings at M phase, could be rescued by subsequent incubation in growth medium that contains Ca2+ whereupon the rounded cells re-flatten (a round-to-flat change) and have well-defined rough ER . It therefore seems possible for Ca2+ depletion, or at least a reduction, to be causally related to ER degranulation . If that were the case, ER granularity would appear to be a facultative rather than a constitutive state.

Scand J Rheumatol, 1992, 21(5), 215 - 9
Morphological evidence for biological control of urate crystal formation in vivo and in vitro; McGill NW et al.; Urate crystal formation, and subsequent gout, occurs in only a minority of hyperuricaemic subjects indicating that factors other than hyperuricaemia are involved . Biological substances (especially proteins) alter crystal growth in vitro independently of uric acid concentration . Physiological crystal formation, known to be under biological control, is characterised morphologically by uniformity of size and constraint of crystal shape . To determine whether pathological urate crystal formation is influenced by the surrounding biological milieu we examined, using scanning electron microscopy, the morphology of urate crystals formed either in vivo or in vitro . We found morphological evidence of biological control of in vivo urate crystal formation and demonstrated that those characteristics could be induced in vitro by the addition of serum, synovial fluid and certain serum proteins to the growth medium during crystal formation . Urate crystal formation is dependent, not only on the degree of hyperuricaemia, but also on the surrounding biological milieu.

Int Arch Allergy Immunol, 1992, 98(3), 211 - 9
Factors affecting the growth and maintenance of human skin mast cells in cell culture; Rein G et al.; The isolation and kinetics of survival of human mast cells from newborn and adult skin is described . Recombinant human interleukins and conditioned medium from several human cell lines were tested for their ability to maintain mast cells in vitro . Growth medium supplemented with IL-2, IL-4 and conditioned medium from a mixed lymphocyte culture enhanced mast cell survival resulting in a 30-fold increase in survival (relative to that obtained with non-supplemented medium) at 7 days, and a 15-fold increase at 15 days . Cell survival for time periods longer than 21 days was not observed . Inclusion of cAMP, agents that elevate cAMP, insulin, and epidermal growth factor in supplemented growth medium prevented the enhanced survival by 40-70% . Incorporation of bromodeoxyuridine (BrdU) into mast cells in 3-day cultures demonstrated that 15% of the mast cell population was capable of proliferation . At 21 days, no incorporation of BrdU could be detected . After 3 days in culture mast cells released 16% of their histamine stores in response to A23187 and 10% in response to anti-human IgE . Electron microscopy of cultured cells at 3 days revealed cells with both intact and empty mast cell granules . These results demonstrate that human skin mast cells proliferate in response to cytokines and release histamine when stimulated with classical secretagogues . Since human skin mast cells retain these basic properties in vitro, they may be useful in further functional studies involving their proliferation and secretion.

J Neuroimmunol, 1992 Jan, 36(1), 41 - 55
Tenascin expression in human glioma cell lines and normal tissues; Ventimiglia JB et al.; Tenascin expression was evaluated in 21 human glioma cell lines and in normal adult tissue extracts by Western and Northern blotting . The cell lines differed in their relative expression of tenascin in the cell-associated and supernatant compartments . Glioma cell line tenascin production was not uniformly stimulated by changes in fetal bovine serum concentration in the growth media . In most glioma cell lines and normal tissue extracts, reducing Western blots and Northern blots revealed two tenascin species, respectively: a major 340 kDa polypeptide and a 9 kb RNA transcript accompanied by a less intense 250 kDa polypeptide and 7 kDa RNA species . In U-87 MG and in normal adult kidney extracts, however, the 250 kDa band and 7 kb transcript were more prominent . Quantitation of tenascin in the glioma lines revealed variable levels that were significantly higher than those in the tissue extracts.

Mutat Res, 1992 Jan, 265(1), 83 - 101
Involvement of RecB-mediated (but not RecF-mediated) repair of DNA double-strand breaks in the gamma-radiation production of long deletions in Escherichia coli; Sargentini NJ et al.; Experiments were designed to determine the association between the repair of gamma-radiation-induced DNA double-strand breaks (DSB) and the induction of 700-1000 bp long deletions (Lac(-)----Lac+), base substitutions (leuB19----Leu+), and frameshifts (trpE9777----Trp+) in Escherichia coli K-12 . Over the range of 2.5-20 krad, deletions were induced with linear kinetics, as has been shown for the induction of DSB, while the induction kinetics of base substitutions and frameshifts were curvilinear . Like the repair of DSB, deletion induction showed an absolute requirement for an intact recB gene as well as a dependency on the type of preirradiation growth medium; these requirements were not seen for base substitutions or frameshifts . In addition, about 80% of the spontaneous deletions were absent in the recB21 strain . A recC1001 mutation, which confers a 'hyper-Rec' phenotype, increased the rate of gamma-radiation-induced deletions as well as the low-dose production of base substitutions and frameshifts . A recF143 mutation increased the yield of gamma-radiation-induced deletions without increasing base substitutions or frameshifts . A mutS mutation markedly enhanced the gamma-radiation induction of frameshifts, and had a slight effect on base substitutions, but did not affect the induction of deletions . Resistance to gamma-irradiation and the capacity to repair DSB (albeit at about half the normal rate) were restored to the radiosensitive recB21 strain by the addition of the sbcB21 and sbcC201 mutations . However, the radioresistant recB sbcBC strain, which is recombination proficient via the RecF pathway, was still grossly deficient in the ability to produce deletions . A model for deletion induction as a by-product of the recB-dependent (Chi-dependent) repair of gamma-radiation-induced DSB is discussed, as is the inability to detect deletions in cells that use only the recF-dependent (Chi-independent) mechanism to repair DSB.

Cytotechnology, 1992, 8(3), 195 - 205
An easy-to-handle semi-automated method for media development using a colorimetric viability assay and fractional factorial designs; Rexen P et al.; A rapid and effective semi-automated screening method has been developed for the development of growth media for mammalian cell culture . The method has proven to be a powerful tool for preliminary evaluation and comparison of new media formulations, but has some inherent disadvantages, which are important to recognise in order to interpret the results.

Biotechnology (N Y), 1992 Jan, 10(1), 82 - 5
Saccharomyces cerevisiae cells secreting an Aspergillus niger beta-galactosidase grow on whey permeate; Kumar V et al.; We describe the construction of a lactose-utilizing Saccharomyces cerevisiae that expresses the cDNA for a secreted, thermostable beta-galactosidase (lacA) from Aspergillus niger . Yeast cells expressing the lacA gene from the yeast ADH1 promotor on a multicopy plasmid secrete up to 40% of the total beta-galactosidase activity into the growth medium . The secreted product is extensively N-glycosylated, and cells expressing the lacA gene grow on whey permeate (4% w/v lactose) with a doubling time of 1.6 hours . Such strains may offer a solution to the increasing problem of waste whey disposal.

Biotechnol Prog, 1992 Jan-Feb, 8(1), 25 - 9
Protein release in recombinant Escherichia coli using bacteriocin release protein; Yu P et al.; The effect of induction level of the bacteriocin release protein (BRP) on cell growth characteristics, protein expression, and protein release in a recombinant strain of Escherichia coli RR1 was investigated . Mitomycin C, the inducing agent, when added to the growth medium in moderate amounts (up to 200 ng/mL) was observed to enhance the release of periplasmic proteins from the cell to the fermentation broth substantially . The percentages of release of the proteins alpha-amylase and beta-lactamase were increased by factors of about 7 and 3, respectively, upon induction of the BRP . The percentage of alpha-amylase released into the broth increased from only about 5% to almost 50% with the aid of BRP . The cell growth curve and low extracellular activity of the cytoplasmic protein beta-galactosidase were indicative that cell lysis did not occur in an appreciable amount at a low induction level, with a mitomycin C concentration of less than 300 ng/mL.

J Cell Physiol, 1992 Jan, 150(1), 104 - 15
Density-dependent regulation of cell surface gamma-glutamyl transpeptidase in cultured glial cells; Morgenstern K et al.; A decline in cell surface gamma-glutamyl transpeptidase specific activity was previously observed to be concomitant with C6 glial cell proliferation . To elucidate the underlying factor(s) mediating gamma-glutamyl transpeptidase down-regulation, the effects of C6 cell density and culture conditions on cell surface transpeptidase activity levels were investigated . After 24 h of culture, the transpeptidase specific activities were inversely related to the initial plating densities . The lower-density cultures showed an induction within 24 h of plating . As the cultures proliferated, the specific transpeptidase activities declined to a common low level at post-confluency . The gamma-glutamyl transpeptidase down-regulation was unrelated to cell growth rate and was most pronounced during logarithmic proliferation . Induction and down-regulation of gamma-glutamyl transpeptidase activity at low cell densities were not a result of trypsinization . Supplementation of low-density cultures with conditioned medium, use of matrix-coated wells, or periodic replacement of growth media to prevent conditioning had minor effects on the decline of cell surface activity . Kinetic analysis showed that the Michaelis constants and the reaction mechanism were unaltered by cell density, indicating that down-regulation was not due to allosteric factors or an alteration in enzyme character . A reduction in the maximal velocity of cell surface transpeptidation at higher cell densities suggested that gamma-glutamyl transpeptidase down-regulation is related to the concentration of enzyme at the cell surface . Immunocytochemical localization of gamma-glutamyl transpeptidase demonstrated that gamma-glutamyl transpeptidase antigen levels decrease as C6 cell density increases . These results led us to propose that cell-cell contact stimulates the disappearance of gamma-glutamyl transpeptidase from the surface of cultured C6 glial cells.

Cell Transplant, 1992, 1(6), 383 - 90
Cell transplantation for myocardial repair: an experimental approach; Marelli D et al.; Myocardium lacks the ability to regenerate following injury . This is in contrast to skeletal muscle (SKM), in which capacity for tissue repair is attributed to the presence of satellite cells . It was hypothesized that SKM satellite cells multiplied in vitro could be used to repair injured heart muscle . Fourteen dogs underwent explantation of the anterior tibialis muscle . Satellite cells were multiplied in vitro and their nuclei were labeled with tritiated thymidine 24 h prior to implantation . The same dogs were then subjected successfully to a myocardial injury by the application of a cryoprobe . The cells were suspended in serum-free growth medium and autotransplanted within the damaged muscle . Medium without cells was injected into an adjacent site to serve as a control . Endpoints comprised histology using standard stains as well as Masson trichrome (specific for connective tissue), and radioautography . In five dogs, satellite cell isolation, culture, and implantation were technically satisfactory . In three implanted dogs, specimens were taken within 6-8 wk . There were persistence of the implantation channels in the experimental sites when compared to the controls . Macroscopically, muscle tissue completely surrounded by scar tissue could be seen . Masson trichrome staining showed homogeneous scar in the control site, but not in the test site where a patch of muscle fibres containing intercalated discs (characteristic of myocardial tissue) was observed . In two other dogs, specimens were taken at 14 wk postimplantation . Muscle tissue could not be found . These preliminary results could be consistent with the hypothesis that SKM satellite cells can form neo-myocardium within an appropriate environment . Our specimens failed to demonstrate the presence of myocyte nuclei.(ABSTRACT TRUNCATED AT 250 WORDS)

Cell Transplant, 1992, 1(4), 299 - 306
Factors affecting in vitro growth of harvested enterocytes; Santos M et al.; Selective enterocyte transplantation may be an alternative to whole organ transplantation for increasing absorptive capacity . Our aim was to determine the effect of initial cell number and viability, proportion of intact crypts, and basement membrane components (BMC) on the in vitro growth of rabbit enterocytes . Enterocytes were harvested using warm trypsinization from ileal segments in 40 rabbits . Initial cell viability was 92 +/- 4% (mean +/- SD), cell yield was 7.7 +/- 3.6 x 10(6) cells/cm, and there were 0.51 +/- 0.33 crypts/100 cells . Initial cell viability correlated with cell yield (r = -0.508, p < 0.001) and % crypts (r = 0.313, p < 0.05) . Cell yield also correlated with % crypts (r = -0.645, p < 0.001) . Enterocytes (5 x 10(6)) were incubated in growth media in plain or BMC coated growth culture vessels for 14 days . There was a correlation between both number of cells seeded (r = 0.824, p < 0.001) and cell viability (r = -0.696, p < 0.01) and % growth colonies containing epithelial cells at 14 days . Both total growth colonies (r = -0.565, p < 0.05) and colonies with epithelial cells (r = -0.589, p < 0.05) had a negative correlation with % crypts . Incubating cells in BMC coated vessels (n = 6) resulted in significantly more dispase liberated cells after 14 days than in plain vessels (n = 6) (6.6 +/- 1.1 vs . 3.8 +/- 1.0 x 10(6), p < 0.05) but viability was similar (97 +/- 2% vs . 96 +/- 2%).(ABSTRACT TRUNCATED AT 250 WORDS)

Nauchnye Doki Vyss Shkoly Biol Nauki, 1992, (2), 103 - 7
{The biochemical aspects of the stimulating action of exogenous RNAse}; Kolpakov AI et al.; A sequence of biochemical reactions in yeast from moment of RNAse interaction with cell membrane to cell division has been studied . RNAse addition in growth medium causes the increase of Ca2+ entering rate in cells in 2.4 times . Under this condition the increase of membrane enzyme adenylate cyclase correlating with the growth of cAMP content in cell and rise of cAMP-dependent protein kinase activity have been observed . A hypothetic scheme of cellular response on the RNAse effect is suggested.

EXS, 1992, 62, 251 - 6
Variable alpha-tocopherol stimulation and protection of glutathione peroxidase activity in non-transformed and transformed fibroblasts; Goldring CE et al.; Studies on glutathione metabolism in an established baby hamster kidney cell line (BHK-21/C13) and in its polyoma virus-transformed counterpart (BHK-21/PyY), have revealed a significant stimulation of intracellular glutathione peroxidase activity (Se-independent plus Se-dependent) by alpha-tocopherol supplementation (14 microM) . This stimulation was found to be much greater in the transformed cells . Other GSH-requiring enzyme activities (namely glutathione reductase and glutathione transferase) were unaltered by alpha-tocopherol treatment, suggesting a degree of specificity in its action on GSHpx . In unsupplemented growth media, the GSHpx activity in both cell lines was significantly decreased by an oxidative stress . However, the same stress applied to the alpha-tocopherol-supplemented cells had no effect on the stimulated GSHpx activity, suggesting a protection afforded by the alpha-tocopherol.

Acta Physiol Scand, 1992 Jan, 144(1), 83 - 8
Growth inhibition of human hepatoma cells (HepG2) by polyunsaturated fatty acids . Protection by albumin and vitamin E; Hostmark AT et al.; Albumin carries fatty acids and has also been suggested to act as an antioxidant . In the present work, polyunsaturated fatty acids (linoleic, arachidonic, eicosapentaenoic and docosahexaenoic acids)--but not palmitic and oleic acid--inhibited growth of human hepatoma cells in low albumin concentration (0.5%) . Growth inhibition by polyunsaturated fatty acids was prevented by albumin in a dose-related manner in the range 0.7-5.0% . Albumin also protected against growth inhibition following catabolism (by lipoprotein lipase) of very low density lipoproteins . Vitamin E strongly counteracted the inhibitory effect of polyunsaturated fatty acids . Vitamin E and albumin appeared to have additive effects in protecting against growth inhibition by polyunsaturated fatty acids . Indomethacin did not greatly modify the polyunsaturated fatty acids effect . Growth inhibition by polyunsaturated fatty acids, as well as the level of thiobarbituric acid reacting substances (a measure of lipid peroxidation) in growth media, increased with increasing number of fatty acids double bonds . Vitamin E and albumin prevented both thiobarbituric acid reacting substances formation and growth inhibition by polyunsaturated fatty acids . The results suggest that the concentrations of albumin and vitamin E in the incubation medium are essential when studying polyunsaturated fatty acids effects on cell growth.

Cancer Invest, 1992, 10(2), 111 - 27
Effect of dibutyryl cyclic adenosine 3',5'-monophosphate on morphological features and biological markers of human salivary myoepithelial cell line in culture; Kawamata H et al.; We have found the emergence of myoepithelial cells (HSG-AZA1) in neoplastic human salivary intercalated duct cell line HSG in culture after treatment with 5-azacytidine . When HSG-ZAZ1 cells were cultured in the presence of N6,O2-dibutyryl cyclic adenosine 3',5'-monophosphate (dB-cAMP), they formed long cytoplasmic processes which were densely packed with ample microfibrils in addition to microtubule bundles . The expression of neuron-specific enolase, synaptophysin, and catecholamine as well as neurofilaments in the treated HSG-AZA1 cells was found by the immunofluorescence staining technique, immunoblotting, immunoelectron microscopy, or catecholamine fluorescence . Both the anchorage-independent and anchorage-dependent growths of HSG-AZA1 cells were suppressed in the presence of dB-cAMP . After the removal of dB-cAMP from the culture, the treated cells returned rapidly to the phenotype and growth rate of the untreated cells . These findings indicate that reversible differentiation into the neuron-like cells of HSG-AZA1 cells occurs in growth medium containing dB-cAMP.

Mol Microbiol, 1992 Jan, 6(1), 123 - 32
Iron regulates growth of Trichomonas vaginalis and the expression of immunogenic trichomonad proteins; Lehker MW et al.; Iron is an essential nutrient for Trichomonas vaginalis and is acquired via highly specific receptor-mediated mechanisms from the host . Responses of T . vaginalis to conditions of iron limitation or iron excess were analysed in order to determine whether iron levels in the growth medium regulate certain properties of the parasite . When compared with organisms grown in excess iron, iron limitation resulted in greater than or equal to 80% lower rates of protein synthesis and greater than or equal to 3-fold decreases in cell densities . These parasites also exhibited generation times of approximately 10 hours, 2.5-fold longer than organisms grown in the usual complex medium . Iron-restricted growth also resulted in increased binding of lactoferrin by trichomonads, which paralleled elevated expression of the lactoferrin-binding receptor protein having a relative molecular mass of 136,000 daltons (136 kDa) . A Mr 126 kDa protein was concomitantly repressed in low-iron-grown parasites . The greater amounts of lactoferrin bound by iron-depleted T . vaginalis organisms corresponded with both the expression of additional receptors onto trichomonal surfaces and increased affinity of the receptor for the lactoferrin molecule . Finally, immunoblot analysis of parasites grown under high- and low-iron conditions using sera from patients with trichomoniasis further revealed the synthesis by T . vaginalis of at least 19 iron-regulated immunogens, and patients' sera also detected the lactoferrin receptor . These data not only show the overall importance of iron to the biology of this protozoan, but illustrate the in vivo iron modulation of gene expression of the biofunctional lactoferrin receptor and other immunogens.

Invasion Metastasis, 1992, 12(3-4), 218 - 32
Polyclonal natural antitumor antibody binding dynamics: preferential release of surface membrane molecules and increased metastasis; Chow DA et al.; Flow cytometry revealed the dynamic nature of polyclonal whole serum naturally occurring IgG and IgM antibody binding to the syngeneic murine T cell lymphomas SL2-5, L5178Y-F9 and the in vitro selected high natural antibody binding variant L5178Y-F9 TPA/NAb+3 . This was particularly evident at physiological conditions where the temperature was 37 degrees C and the concentration of reactive serum natural antibodies (NAb) was high . Lower binding was observed at 37 versus 4 degrees C, or after raising the temperature from 4 to 37 degrees C, a procedure which was associated with an augmented loss of 125I-surface-labelled material from cells incubated in NAb compared to cells exposed to growth media . Even at 4 degrees C, NAb binding exhibited biphasic kinetics suggesting a loss of surface-bound NAb and a subsequent cycle of NAb uptake . The increased intravenous liver metastasis potential of the high NAb binding L5178Y-F9 TPA/NAb+3 corresponded with its higher total loss of 125I-surface-labelled material when incubated in NAb at 37 degrees C, and with its extensive loss of NAb binding when the temperature was raised from 4 to 37 degrees C . These observations are consistent with the idea that molecules released from the cell may contribute to the higher metastasis . This thinking was supported by the increased metastasis of tumor cells injected intravenously, either with serum in which they had been preincubated at 37 degrees C or into mice treated with supernatants from tumor cells incubated in NAb.

Gene, 1991 Dec 20, 109(1), 107 - 13
Cloning, expression and characterization of a cDNA encoding a lipase from Rhizopus delemar; Haas MJ et al.; A lambda gt11 cDNA library was constructed in Escherichia coli using poly(A)-selected mRNA from the fungus, Rhizopus (Rp.) delemar . Lipase-producing members of the library were identified by means of a phenotypic score wherein the release of fatty acids by lipase causes a characteristic color change in the growth medium . One such isolate contained a 1287-bp insert (LIP cDNA) which hybridizes to 1.25- to 1.35-kb mRNA species from Rp . delemar . The lipase produced in E . coli containing the LIP cDNA exhibits the same substrate selectivity as the authentic fungal enzyme, hydrolyzing ester bonds at the stereospecific numbering (sn) sn-1 and sn-3, but not the sn-2, positions of triglycerides . The complete nucleotide sequence of the LIP cDNA was determined . By reference to the N-terminal sequence of authentic Rp . delemar lipase, the lipase-encoding region was identified within this fragment . The LIP cDNA encodes a putative preprolipase consisting of a 26-amino-acid(aa) signal sequence, a 97-aa propeptide, and a 269-aa mature enzyme . The predicted mature lipase has the same molecular weight and aa composition as that of Rp . delemar, is highly homologous to that produced by the fungus Rhizomucor miehei, and contains the consensus pentapeptide (Gly-Xaa-Ser-Yaa-Gly) which is conserved among lipolytic enzymes . It is concluded that the LIP cDNA is an essentially full-length analogue of the lipase-encoding gene of Rp . delemar . The lipase encoded by the LIP cDNA occupies a cytoplasmic location when synthesized in E . coli . Unprocessed forms of the lipase accumulate in E . coli.

Proc Natl Acad Sci U S A, 1991 Dec 15, 88(24), 11177 - 81
Identification of a 170-kDa protein associated with the vacuolar Na+/H+ antiport of Beta vulgaris; Barkla BJ et al.; The effect of the addition of amiloride to the growth medium was tested on the Na+/H+ antiport activity of tonoplast vesicles isolated from sugar beet (beta vulgaris L.) cell suspensions . Cells grown in the presence of NaCl and amiloride displayed an increased antiport activity . Analysis of the kinetic data showed that while the affinity of the antiport for Na+ ions did not change, the maximal velocity of the Na+/H+ exchange increased markedly . These results suggest the addition of more antiport molecules to the tonoplast and/or an increase in the turnover rate of the Na+/H+ exchange . The increase in activity of the antiport by the presence of amiloride was correlated with the enhanced synthesis of a tonoplast 170-kDa polypeptide . The increased synthesis of this polypeptide was detected not only upon exposure of the cells to amiloride but also when the cells were exposed to high NaCl concentrations . Polyclonal antibodies against the 170-kDa polypeptide almost completely inhibited the antiport activity . These results suggest the association of the 170-kDa polypeptide with the vacuolar Na+/H+ antiport.

Scanning Microsc, 1991 Dec, 5(4), 1019 - 26; discussion 1026-7
Calcium oxalate crystal growth in the presence of mucin; Akbarieh M et al.; Using interfacially-controlled crystallization and gel diffusion crystallization methods, calcium oxalate monohydrate (COM), dihydrate (COD) and trihydrate (COT) crystals were grown by the slow diffusion of reacting ions in the presence of mucin . It was demonstrated that mucin in the growth media dramatically affected the size, habit, surface structure, thermodynamic stability and phase transition kinetics of hydrated calcium oxalate crystals . The results obtained revealed that mucin as a glycoprotein model controlled the growth of COT and COD single crystals as well as cluster formation . Growth inhibition of specific crystal faces and phase transition retardation occurred in its presence . The data confirmed that glycoproteins are more than just adhesive materials, enhancing crystal aggregation in stone formation.

J Radiat Res (Tokyo), 1991 Dec, 32(4), 352 - 65
Effects of post-treatment incubation on recombinogenesis in incision-proficient and incision-deficient strains of Saccharomyces cerevisiae: I . Recombinogenesis after UV- and gamma-ray-irradiation; Saeki T et al.; After UV-irradiation of G1 phase cells, most gene conversion and crossing-over took place in cells of incision-proficient strains without post-irradiation incubation . In contrast, incision-deficient cells markedly induced both of these recombinational events only after irradiated cells were incubated for several hours in liquid growth medium before selection . These results indicate that when G1 cells are irradiated with UV recombinational events are initiated during that G1 phase in incision-proficient strains; whereas, they are not initiated in irradiated incision-deficient strains before the cells reach the S/G2 phase . Experimental gamma-ray results also suggest that post-irradiation incubation is not required for incision-proficient and -deficient strains to initiate mitotic recombinational events . Taken together, these results show that the formation of incision nicks and of post-replication gaps in DNA appears to be necessary for the initiation of recombinational events after UV treatment; whereas, it is not required after gamma-irradiation.

Indian J Exp Biol, 1991 Dec, 29(12), 1134 - 9
Action of DL-alpha-difluoromethyl ornithine on Acanthamoeba culbertsoni; Kishore P et al.; The multiplication of A . culbertsoni in the peptone medium was not inhibited by 10-20 mM concentration of alpha-difluoromethyl ornithine (DMFO) while a partial and transient inhibition of cell multiplication was observed by 10-20 mM DFMO in proteose peptone, yeast extract, glucose (PYG) medium . Ornithine decarboxylase (ODC) activity in the cells and cell free extracts was strongly inhibited by DFMO, excluding enzyme refractoriness and impermeability of cells for DFMO as the possible causes of DFMO resistance . The presence of polyamines in the peptone and PYG media as well as uptake of polyamines by the amoebae has been demonstrated . The growth and multiplication of A . culbertsoni in chemically defined medium was not affected by 1-5 mM DFMO while 10-20 mM DMFO yielded partial inhibition . A lowering of diaminopropane levels and enhancement of spermidine levels was observed in DFMO inhibited cells and level of ODC was drastically reduced in the inhibited cultures . Uptake of polyamines from the growth media may partly account for DFMO resistance of A . culbertsoni . Alternative mechanisms for DFMO resistance are indicated.

J Am Acad Dermatol, 1991 Dec, 25(6 Pt 1), 1054 - 8
Human wound fluid from acute wounds stimulates fibroblast and endothelial cell growth; Katz MH et al.; One proposed mechanism for the beneficial effect of occlusive dressings on healing is the maintenance of contact between the wound bed and accumulated wound fluid, which is thought to contain growth stimulatory substances . We have examined the effect of human wound fluid on the in vitro growth of human dermal fibroblasts and umbilical vein endothelial cells . Acute wound fluid was collected from six patients undergoing split-thickness skin grafting . The acute wound fluid was sterilely collected daily from underneath a vapor-permeable membrane applied to the donor site and changed every 24 hours for 3 days postoperatively . After seeding in optimal growth media (control) on day 0, cultures of human dermal fibroblasts and umbilical vein endothelial cells were supplemented with or without acute wound fluid on the next day (day 1) and on day 3 . As determined by cell counts, 2% acute wound fluid stimulated the growth of human dermal fibroblasts (p less than 0.05) and umbilical vein endothelial cells (p less than 0.01) when these cells were cultured in 2% fetal bovine serum and endothelial growth medium, respectively . Wound fluid from postoperative days 1 or 3 caused the same level of stimulation . The addition of an anti-platelet-derived growth factor antibody to wound fluid resulted in a 45% mean reduction in its stimulatory effect on fibroblast growth (p less than 0.02), suggesting that platelet-derived growth factor contributes to the observed effect.(ABSTRACT TRUNCATED AT 250 WORDS)

J Parasitol, 1991 Dec, 77(6), 974 - 81
In vitro encystation of Giardia lamblia: large-scale production of in vitro cysts and strain and clone differences in encystation efficiency; Kane AV et al.; A method for obtaining large numbers of Giardia lamblia cysts in vitro was developed based on modification of earlier methods of in vitro encystation . Maximal numbers of cysts were obtained by growing trophozoites to confluence in TYI-S-33 growth medium containing 0.5 mg/ml of bovine bile, followed by incubation in medium containing 10 mg/ml of bovine bile, at pH 7.8 for 96 h at 37 C . Up to 4 x 10(5) cysts were obtained per milliliter of encystation medium . Cysts thus obtained were similar in structure to those in vivo, were resistant to hypotonic lysis, and reacted with a cyst-specific monoclonal antibody . Further modification of this method by returning the trophozoites to growth medium after 24 hr of exposure to encystation medium resulted in production of cysts that were shown to be viable by fluorogenic dye staining and ability to excyst . This method was scaled up using roller bottles, which resulted in production of up to 1.6 x 10(8) cysts per roller bottle . In addition, of 4 strains tested, the LT strain yielded the highest number of cysts . Of 4 clones of the WB strain, clone A consistently produced the largest number of cysts.

Neurosurgery, 1991 Dec, 29(6), 880 - 6; discussion 886-7
Enhanced protein kinase C activity correlates with the growth rate of malignant gliomas in vitro; Couldwell WT et al.; Direct measurement of protein kinase C (PKC) activity in vitro revealed a significant increase in the activity of the enzyme in all human malignant glioma lines examined and the rat C6 tumor in comparison with control nonneoplastic astrocyte and mixed glial cultures . The total and particulate PKC activity in these cell types correlated strongly {r = 0.98 (P less than 0.001) and 0.94 (P = 0.002), respectively} with the maximal growth rates as measured by 3H-thymidine incorporation in each of the samples . An alteration in the growth rate of an individual glioma line (A172) by varying the serum concentration in the growth medium produced comparative changes in the measured PKC activity . The addition of the phorbol ester phorbol-12-myristate-13-acetate to this tumor line under high serum conditions produced down-regulation of the enzyme, which was accompanied by a corresponding reduction in thymidine incorporation . The administration of the PKC inhibitor staurosporine produced a dose-related decrease in the basal proliferation rate of glioma lines A172 and C6, as measured by 3H-thymidine uptake and confirmed by flow cytometry, indicating that the high intrinsic PKC activity is amenable to pharmacological manipulation . Cytofluorometric deoxyribonucleic acid cell cycle analysis of the tumors treated with PKC modulators demonstrated that reduced proliferation rates were caused by an inhibition of entrance into the deoxyribonucleic acid synthesis (S) phase (decrease in proliferative index), supporting the evidence that these modulators are not slowing the tumor growth in a nonspecific cytotoxic manner.(ABSTRACT TRUNCATED AT 250 WORDS)

In Vitro Cell Dev Biol, 1991 Dec, 27A(12), 914 - 20
A method for the harvest, culture, and characterization of human adult atrial myocardial cells: correlation with age of donor; Smith DA et al.; Myocardial cell culture methods are now well established for animal and fetal human tissue . We present here a method for harvesting and culturing adult human atrial myocardiocytes . Cells are obtained from fresh atrial tissue normally discarded after being removed to cannulate the right atrium during open heart surgery . The atrial tissue is minced and then digested using collagenase . The single cell suspension is initially cultured in serum-containing growth medium, then transferred to defined medium, selective for myocardial cell growth . The cells are characterized by immunoperoxidase stains and transmission electron microscopy . The cultured cells stain positive for myoglobin, whereas control cultured fibroblasts and endothelial cells do not . Electron microscopy shows the presence of numerous myofibrils, Z-bodies, pleomorphic mitochondria, and secretory granules . The chronological age of the donor was an important factor in culturing the adult tissue, the younger tissue correlated with a higher success rate . This method provides a means for in vitro study of human adult myocardial cells and provides guidelines for appropriate atrial tissue to use.

J Bacteriol, 1991 Dec, 173(24), 7790 - 801
13C nuclear magnetic resonance and gas chromatography-mass spectrometry studies of carbon metabolism in the actinomycin D producer Streptomyces parvulus by use of 13C-labeled precursors; Inbar L et al.; Fructose and glutamate metabolism was monitored in cell suspensions of streptomyces parvulus by 13C nuclear magnetic resonance . The experiments were performed for cells grown with various 13C sources in a growth medium containing D-{U-13C}fructose, L-{13C}glutamate, or L-{U-13C}aspartate and with nonlabeled precursors to compare intracellular pools in S . parvulus cells at different periods of the cell life cycle . The transport of fructose into the cells was biphasic in nature; during rapid transport, mannitol, fructose, and glucose 6-phosphate were accumulated intracellularly, whereas during the passive diffusion of fructose, the intracellular carbohydrate pool comprised mainly trehalose (1,1'-alpha-alpha-D-glucose) . The regulation of fructokinase activity by the intracellular intermediates may play an important role in fructose catabolism in S . parvulus . Transaldolase activity in S . parvulus was determined from the 13C nuclear magnetic resonance labeling pattern of trehalose carbons obtained from cells grown in medium containing either L-{U-13C}aspartate or L-{U-13C}glutamate . Only carbons 4, 5, and 6 of the disaccharide were labeled . Isotopomer analysis of the trehalose carbons led us to conclude that the flux through the reverse glycolytic pathway, condensation of glyceraldehyde 3-phosphate with dihydroxyacetone phosphate, makes at best a minor contribution to the 13C-labeled glucose units observed in trehalose . The pentose pathway and transaldolase activity can explain the labeling pattern of 4,5,6-13C3 of trehalose . Moreover, the transfer of the 13C label of L-{U-13C}aspartate into the different isotopomers of trehalose C4, C5, and C6 by the transaldolase activity allowed us to calculate the relative fluxes from oxaloacetate via gluconeogenesis and through the tricarboxylic acid cycle . The ratio of the two fluxes is approximately 1 . However, the main carbon source for trehalose synthesis in S . parvulus is fructose and not glutamate or aspartate . The 13C enrichment and isotopomer population, measured by nuclear magnetic resonance and gas chromatography-mass spectrometry, of the actinomycin D peptide ring enabled us to specify the origins of the five amino acids of actinomycin D . Threonine and proline exhibited isotopomer populations similar to that of the extracellular L-{13C}glutamate, indicating that protein catabolism is the origin of their 13C label, whereas the isotopomer populations of sarcosine and N-methylvaline were similar to those of the new intracellular pool of S . parvulus that originated from D-{U-13C}fructose during the production of actinomycin D.

Toxicol Appl Pharmacol, 1991 Dec, 111(3), 496 - 503
2,3,7,8-Tetrachlorodibenzo-p-dioxin causes unbalanced growth in 5L rat hepatoma cells; Gottlicher M et al.; 5L cells, dedifferentiated descendents of the rat hepatoma line H4IIEC3, constitute one of the rare continuous lines which are sensitive to the toxic effects of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) . In the present study we investigated the nature of TCDD toxicity in these cells . The following results were obtained: (1) Exposure to 0.1 nM TCDD for 48 hr inhibits the proliferation of 5L cells by more than 50%, as determined by the increase in the number of cells and the amount of DNA per culture . (2) TCDD doubles the amount of protein and the uptake of neutral red per cell during the 48-hr exposure period . (3) TCDD restores neither constitutive levels of tyrosine aminotransferase, a marker of liver-specific functions, nor its inducibility by dexamethasone . (4) The effects of TCDD are reversible when TCDD-containing growth media are replaced by TCDD-free medium . (5) 5L cells grown at 2% of fetal bovine serum are considerably more sensitive to TCDD than those grown at 10% serum . These results indicate that TCDD inhibits the proliferation of 5L cells without retarding the rate of growth or overtly changing the status of differentiation . The dioxin possibly causes unbalanced cell growth by interfering with the action of hormones or factors contained in the growth medium.

Asian Pac J Allergy Immunol, 1991 Dec, 9(2), 153 - 7
Establishment and characterization of a cholangiocarcinoma cell line from a Thai patient with intrahepatic bile duct cancer; Sirisinha S et al.; A new human cholangiocarcinoma cell line (HuCCA-1) was established from cholangiocarcinoma (CCA) tissue fragments surgically removed from a Thai patient with intrahepatic bile duct cancer . The growth medium used for the primary cell culture was Ham's F12 supplemented with 10% fetal bovine serum (FBS) and 10 ng/ml epithelial growth factor (EGF) . Approximately one month later, the cells were subcultured in Ham's F12 supplemented with only 10% FBS . The population doubling time was approximately 55 hr . Staining of the cells for cytokeratin and mucin confirmed that the cells were mucin-secreting tumor of epithelial cell origin . The supernatant fluid secreted a number of non-specific tumor markers including CA125 and traces of MCA and AFP . The ability of the HuCCA-1 cell line to synthesize specific marker that may have potential in the diagnosis of cholangiocarcinoma is now being investigatedPublication Types:

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