Growth media

Br J Haematol, 1999 Feb, 104(2), 365 - 73
Syndecan-1 expression suppresses the level of myeloma matrix metalloproteinase-9; Kaushal GP et al.; ARH-77 human myeloma cells invade into type I collagen gels but become non-invasive when engineered to express syndecan-1, a heparan sulphate proteoglycan that promotes cell adhesion to collagen . To determine if syndecan-1 expression influences the activity of proteases that may facilitate invasion, we analysed media harvested from syndecan-1 expressing and non-expressing cells . High levels of a 92 kD gelatinase accumulated in serum-free growth medium of both parental and control-transfected ARH-77, but much less 92 kD gelatinase accumulated in the medium of ARH-77 transfectants expressing syndecan-1 . The gelatinase was identified as matrix metalloproteinase (MMP)-9 because its activity was immunoprecipitated with a MMP-9-specific monoclonal antibody . Gelatinase activity and Western blot analyses revealed 2-3-fold less MMP-9 in medium from syndecan-1 transfected cells than in medium from parental cells . Decreased MMP-9 was not due to increased association of MMP-9 with cells expressing syndecan-1 . An inverse correlation between the syndecan 1 level and the level of MMP-9 accumulation in the media was observed using a panel of ARH-77 transfectants expressing syndecan-1 . Investigation of six unrelated human myeloma cell lines confirmed that high gelatinase levels were recovered from conditioned media of those that did not express syndecan-1 (ARH-77, Mer and Col) and one line that expressed a low level of syndecan-1 (RPMI-8226), but low gelatinase levels were recovered from media of lines that expressed high levels of syndecan-1 (ARK and clone 2+) . Therefore syndecan-1 may play a dual role in inhibiting the metastasis of tumour cells by promoting cell adhesion to the extracellular matrix and suppressing the proteolytic activity needed for invasion.

Appl Environ Microbiol, 1999 Mar, 65(3), 1092 - 8
Enhanced bioaccumulation of heavy metal ions by bacterial cells due to surface display of short metal binding peptides; Kotrba P et al.; Metal binding peptides of sequences Gly-His-His-Pro-His-Gly (named HP) and Gly-Cys-Gly-Cys-Pro-Cys-Gly-Cys-Gly (named CP) were genetically engineered into LamB protein and expressed in Escherichia coli . The Cd2+-to-HP and Cd2+-to-CP stoichiometries of peptides were 1:1 and 3:1, respectively . Hybrid LamB proteins were found to be properly folded in the outer membrane of E . coli . Isolated cell envelopes of E . coli bearing newly added metal binding peptides showed an up to 1.8-fold increase in Cd2+ binding capacity . The bioaccumulation of Cd2+, Cu2+, and Zn2+ by E . coli was evaluated . Surface display of CP multiplied the ability of E . coli to bind Cd2+ from growth medium fourfold . Display of HP peptide did not contribute to an increase in the accumulation of Cu2+ and Zn2+ . However, Cu2+ ceased contribution of HP for Cd2+ accumulation, probably due to the strong binding of Cu2+ to HP . Thus, considering the cooperation of cell structures with inserted peptides, the relative affinities of metal binding peptide and, for example, the cell wall to metal ion should be taken into account in the rational design of peptide sequences possessing specificity for a particular metal.

J Cell Physiol, 1999 Feb, 178(2), 188 - 96
1,25 dihydroxyvitamin D3 enhances the calcium response of keratinocytes; Ratnam AV et al.; The steroid hormone 1,25 dihydroxyvitamin D3 (1,25(OH)2D3) regulates cell proliferation and differentiation . Intracellular calcium (Cai) concentrations play a crucial role in these events . From our previous studies, we have demonstrated a calcium receptor (CaR) in keratinocytes which appears to regulate the initial release of Cai from intracellular stores in response to extracellular calcium (Cao) and so is likely to participate in the differentiation process . In this study, we determined whether the ability of 1,25(OH)2D3 to enhance Ca++ -induced differentiation was mediated at least in part through changes in the CaR . Keratinocytes were grown in keratinocyte growth medium (KGM) with 0.03 mM, 0.1 mM, or 1.2 mM Ca and treated with 10(-8) M 1,25(OH)2D3 till harvest after 5, 7, 14, and 21 days . CaR mRNA levels were quantitated by polymerase chain reaction . The results were compared to the ability of 1,25(OH)2D3 to enhance calcium-stimulated increases in Cai . In cells grown in 0.03 mM Ca, the CaR mRNA levels decreased with time . 1,25(OH)2D3 stimulated the levels at 5 days and prevented the falloff over the subsequent 16 days . On the other hand, in cells grown in 0.1 or 1.2 mM Ca, the message levels remained high, and 1,25(OH)2D3 had no further effect . To study the functional relationship, we harvested cells after 5 and 7 days in culture following a 24 h treatment with 1,25(OH)2D3 or vehicle to measure the Cai response to 2 mM Cao . The preconfluent cells grown in 0.03 mM Ca showed a nearly twofold increase in the Cai response to Cao when pretreated with 1,25(OH)2D3, whereas the confluent cells and those grown in 1.2 mM Ca showed no enhancement by 1,25(OH)2D3 . Studies with 45Ca influx into keratinocytes revealed that 1,25(OH)2D3 enhanced the influx in preconfluent and confluent cells when grown in KGM containing 0.03 mM Ca but not in cells grown in 1.2 mM calcium . We conclude that 1,25(OH)2D3 maintains the CaR mRNA levels in cells grown in 0.03 mM Ca, thus maintaining their responsiveness to Cao and so ensuring their ability to differentiate in response to the calcium signal.

Biol Reprod, 1999 Mar, 60(3), 674 - 82
Ontogeny of expression of a receptor for platelet-activating factor in mouse preimplantation embryos and the effects of fertilization and culture in vitro on its expression; Stojanov T et al.; Platelet-activating factor (PAF; 1-o-alkyl-2-acetyl-sn-glycero-3-phosphocholine) is a potent ether phospholipid . It is one of the preimplantation embryo's autocrine growth/survival factors . It may act via a G protein-linked receptor on the embryo; however, the evidence for this is conflicting . The recent description of the intracellular form of the PAF:acetlyhydrolase enzyme as having structural homology with G proteins and Ras also suggests this as a potential intracellular receptor/transducer for PAF . This study used reverse transcription-polymerase chain reaction to examine the ontogeny of expression of the genes for these proteins in the oocyte and preimplantation-stage embryo . Transcripts for the G protein-linked PAF receptor were detected in the late 2-cell-stage embryo and in all stages from the 4-cell stage to blastocysts . They were also present in unfertilized oocytes and newly fertilized zygotes but only at relatively low levels . The incidence of expression was generally low and variable in late zygotes and early 2-cell embryos . Expression past the 2-cell stage was alpha-amanitin sensitive . The results indicated that mRNA for this receptor is a maternal transcript that was degraded during the zygote-2-cell stage . New expression of the receptor transcript required activation of the zygotic genome . Fertilization of embryos in vitro caused this transcript not to be expressed in the zygote . Culture of zygotes (irrespective of their method of fertilization) caused expression from the zygotic genome to be retarded by more than 24 h . This retardation did not occur if culture commenced at the 2-cell stage . The transcripts for the subunits of intracellular PAF:acetylhydrolase were not detected in oocytes or at any stage of embryo development examined, despite their being readily detected in control tissue . This study confirms the presence of the G protein-linked PAF receptor in the 2-cell embryo and describes for the first time its normal pattern of expression during early development . The adverse effects of in vitro fertilization (IVF) and embryo culture on the expression of this transcript may be a contributing factor for the poor viability of embryos produced in this manner . The reduced expression of PAF-receptor mRNA following IVF predicts that such embryos may have a deficiency in autocrine stimulation and also suggests that supplementation of growth media with exogenous PAF would be only partially beneficial . The effect of IVF and culture may also explain the conflicting literature.

Hum Exp Toxicol, 1999 Jan, 18(1), 1 - 11
Response of normal human keratinocytes to sulfur mustard (HD): cytokine release using a non-enzymatic detachment procedure; Arroyo CM et al.; Cytokines play a major role in both acute and chronic inflammatory processes, including those produced by sulfur mustard (HD) . This study describes responses of normal human epidermal keratinocyte (NHEK) cells to 2,2'-dichlorodiethyl sulfide, sulfur mustard (HD), defined by interleukin-1 beta (IL-1beta), interleukin-6 (IL-6), interleukin-8 (IL-8), and tumor necrosis factor-alpha (TNF-alpha) release . A new method for detaching cell to cell adhesion between keratinocytes has been applied . This method permits the characterization of endogenous fluid from cellular content that could be applied for the development of therapeutic intervention . NHEK (typical average cell density 4.4 x 10(6) cells/mL) were exposed to HD (100 and 300 microM) in keratinocyte growth medium (KGM) for 24 h at 37 C in humidified air . Commercially available enzyme-linked immunosorbent assay (ELISA) kits were used to measure the cytokine release in NHEK during exposure to 100 and 300 microM of HD . Exposure to 100 microM HD increased release of cytokines . IL-1beta (exposed: 1.41 x 10(-5) pg/ cell+/-1.60 x 10(-6) pg/cell: control 7.10 x 10(-6) pg/ cell+/-1.20 x 10(-6) pg/cell), TNF-alpha (exposed: 1.06 x 10(5) pg/cell+/-7.3 x 10(-7)pg/cell; control: 4.04 x 10(-6)+/-2.80 x 10(-7) pg/cell) and IL-8 (exposed: 3.71 x 10(-5) pg/ cell+/- 3.26 x 10(-6) pg/cell; control: 2.99 x 10(-6) pg/cell+/-8.80 x 10(-7) pg/cell) were significantly enhanced when NHEK cells were detached from culture flasks by non-enzymatic procedures . Cell suspensions of NHEK released low amounts of IL-6 when exposed to 100 microM for 24 h (exposed: 1.47 x 10(-6)+/-1.60 x 10(-7) pg/cell; control: 1.28 x 10(-6)+/-8.40 x 10(-8) pg/cell) . However, cell suspensions of NHEK increased levels of IL-6 after exposure to 300 microM HD (4.67 x 10(-5) pg/cell+/-3.90 x 10(-6) pg/cell; control: 3.99 x 10(-6) pg/cell+/-5.50 x 10(-7) pg/cell) . The amount of IL-8 and TNF-alpha present in cell suspensions increased up to 59-fold and fourfold, respectively, above control levels when NHEK cells were exposed to 300 microM HD . Exposure of NHEK to 300 microM HD had a highly variable effect on the release of IL-1beta, where sometimes the secretion of IL-1beta increased above baseline level and other times decreased in cell suspensions . Supernatants were collected from cell culture flasks 24 h after exposure of 100 and 300 microM and significantly increased levels of IL-6 were observed . IL-6 was released in a concentration-dependent manner, 3.6-fold up to 8.4-fold, respectively, in supernatant . These pro-inflammatory mediators IL-1beta, IL-8, TNF-alpha and IL-6 may play an important role in HD injury . The present findings suggest that cytokine changes detected could be used as potential biomarkers of cutaneous vesicant injury.

Biochem J, 1999 Mar 1, 338 ( Pt 2), 335 - 42
Co-expression of glutathione S-transferase with methionine aminopeptidase: a system of producing enriched N-terminal processed proteins in Escherichia coli; Hwang DD et al.; We describe here an Escherichia coli expression system that produces recombinant proteins enriched in the N-terminal processed form, by using glutathione S-transferase cGSTM1-1 and rGSTT1-1 as models, where c and r refer to chick and rat respectively . Approximately 90% of the cGSTM1-1 or rGSTT1-1 overexpressed in E . coli under the control of a phoA promoter retained the initiator methionine residue that was absent from the mature isoenzymes isolated from tissues . The amount of initiator methionine was decreased to 40% of the expressed cGSTM1-1 when the isoenzyme was co-expressed with an exogenous methionine aminopeptidase gene under the control of a separate phoA promoter . The recombinant proteins expressed were mainly methionine aminopeptidase . The yield of cGSTM1-1 was decreased to 10% of that expressed in the absence of the exogenous methionine aminopeptidase gene . By replacing the phoA with its natural promoter, the expression of methionine aminopeptidase decreased drastically . The yield of the co-expressed cGSTM1-1 was approx . 60% of that in the absence of the exogenous methionine aminopeptidase gene; approx . 65% of the initiator methionine residues were removed from the enzyme . Under similar conditions, N-terminal processing was observed in approx . 70% of the recombinant rGSTT1-1 expressed . By increasing the concentration of phosphate in the growth medium, the amount of initiator methionine on cGSTM1-1 was decreased to 14% of the overexpressed isoenzymes, whereas no further improvement could be observed for rGSTT1-1 . The initiator methionine residue does not affect the enzymic activities of either cGSTM1-1 or rGSTT1-1 . However, the epoxidase activity and the 4-nitrobenzyl chloride-conjugating activity of the purified recombinant rGSTT1-1 are markedly higher that those reported recently for the same isoenzyme isolated from rat livers.

J Cell Biochem, 1999 Feb 1, 72(2), 210 - 20
Cell adhesion and proliferation mediated through the G1 domain of versican; Yang BL et al.; We have demonstrated previously that versican stimulated cell proliferation through the G3 domain . In these experiments, we show that versican mini-gene-transfected cell lines exhibited decreased cell-substratum interaction and increased cell proliferation . Exogenous addition of growth medium containing the versican gene product produced the same results . Because the G1 domain of versican is structurally similar to the G1 domain of aggrecan and to link protein, both of which play role in cell adhesion, we hypothesized that versican's proliferative effects may be a consequence of its ability to reduce cell adhesion, and may be mediated through the G1 domain . To investigate this, we expressed a G1 construct in NIH3T3 cells and showed that it reduced cell adhesion and enhanced cell proliferation . We then demonstrated that deletion of the G1 domain from versican greatly, but not completely, reversed the effects of versican: G1-deletion mutants of versican show slightly reduced amounts of cell adhesion and slightly increased rates of proliferation . We concluded that versican can stimulate cell proliferation via two mechanisms: through two EGF-like motifs in the G3 domain which play a role in stimulating cell growth, and through the G1 domain, which destabilizes cell adhesion and facilitates cell growth . We purified the G1 product with an affinity column and demonstrated that it reduced cell adhesion and enhanced cell proliferation.

J Biol Chem, 1999 Feb 19, 274(8), 4863 - 8
Post-translation control of Nramp metal transport in yeast . Role of metal ions and the BSD2 gene; Liu XF et al.; The Saccharomyces cerevisiae SMF1 gene encodes a member of the well conserved family of Nramp metal transport proteins . Previously, we determined that heavy metal uptake by Smf1p was down-regulated by the product of the S . cerevisiae BSD2 gene . We now demonstrate that this regulation occurs at the level of protein stability . In wild type strains, the bulk of Smf1p is normally directed to the vacuole and is rapidly degraded by vacuolar proteases in a PEP4-dependent manner . In bsd2Delta mutants, Smf1p fails to enter the vacuole, and the Nramp protein is stabilized . Metal ions themselves play an important role in the post-translational regulation of Smf1p . The depletion of heavy metals from the growth medium effects stabilization of Smf1p and additionally results in accumulation of this transporter at the cell surface . Supplementation of manganese alone is sufficient to trigger rapid degradation of Smf1p in a Bsd2p-dependent manner . Together the action of Bsd2p and metal ions provide a rapid and effective means for controlling Nramp metal transport in response to environmental changes.

Hum Exp Toxicol, 1998 Dec, 17(12), 652 - 60
Protection by extracellular glutathione against sulfur mustard induced toxicity in vitro; Amir A et al.; 1 . The present study characterizes the role of extracellularly added glutathione in protection against sulfur mustard (HD) toxicity in a macrophage monocyte cell line J774 . 2 . Toxic effects of HD depend on dose and duration of exposure with an ED50 of 50 and 75 microM for dividing and confluent cells respectively . 3 . Exposure to HD, 100-200 microM caused approximately 15% decrease in the cellular glutathione (GSH) content 2 h after exposure, pretreatment with GSH, 0.2-10 mM, elevated cellular GSH approximately x 1.5 . 4 . GSH pretreatment increased cell viability after HD 2-3-fold . Similar protective effects of GSH treatment were found in a human epidermoid carcinoma cell line (KB) . 5 . Protection by post treatment with GSH was apparent even 60 min post HD exposure . 6 . No protection was afforded when the intracellular GSH concentration was elevated prior to exposure and the extracellular GSH had been washed out . However, GSH depleted cells were more sensitive to HD than normal cells, and were also protected by addition of GSH to the growth medium, although the intracellular GSH content remained low . 7 . We conclude that it is essential for the GSH to be present extracellularly in order to protect cells from HD toxicity . 8 . Our findings have therapeutic implications in particular for the protection of lungs after inhalation exposure to HD vapor.

Planta, 1999 Jan, 207(3), 426 - 35
Salt stress in Mesembryanthemum crystallinum L . cell suspensions activates adaptive mechanisms similar to those observed in the whole plant; Vera-Estrella R et al.; A salt-tolerant stable cell-suspension culture from the halophyte Mesembryanthemum crystallinum L . has been established from calli generated from leaves of 6-week-old well-watered plants . Optimal cell growth was observed in the presence of 200 mM NaCl, and within 7 d cells were able to concentrate Na+ to levels exceeding those in the growth medium . Accumulation of Na+ was paralled by increases in the compatible solute pinitol and myo-inositol methyl transferase (IMT), a key enzyme in pinitol biosynthesis . Increasing concentrations of NaCl stimulated the activities of tonoplast and plasma-membrane H(+)-ATPases . Immunodetection of the ATPases showed that the increased activity was not due to changes in protein amount that could be attributed to treatment conditions . A specific role for these mechanisms in salt-adaptation is supported by the inability of mannitol-induced water stress to elicit the same responses, and the absence of enzyme activity and protein expression associated with Crassulacean acid metabolism in the cells . Results demonstrate that these M . crystallinum cell suspensions show a halophytic growth response, comparable to that of the whole plant, and thus provide a valuable tool for studying signaling and biochemical pathways involved in salt recognition and response.

Int J Cancer, 1999 Jan 29, 80(3), 431 - 8
Activation of the insulin-like growth factor-1 receptor promotes the survival of human keratinocytes following ultraviolet B irradiation; Kuhn C et al.; The ultraviolet B (UVB) component of sunlight causes non-melanoma skin cancers due to the damage it inflicts on genomic DNA . The response of epidermal keratinocytes to sunlight depends on the dose of UVB received and the severity of the damage to the DNA . Mild DNA damage typically induces DNA-repair pathways and cell survival, while severe DNA damage provokes apoptosis . Primary human keratinocytes grown in serum-free media respond in a similar manner to UVB irradiation . However, we observed that keratinocytes are exquisitely more susceptible to UVB-induced apoptosis if the growth medium is depleted of exogenous growth factors . Therefore, an exogenous growth factor could provide protection from UVB-induced apoptosis . We found that the only growth factor that provided protection from UVB-induced apoptosis was insulin and that the protective effect elicited by insulin was not due to binding the insulin receptor but, rather, to activation of the insulin-like growth factor-1 (IGF-1) receptor . Additionally, activation of the IGF-1 receptor in combination with UVB irradiation induced keratinocytes to become post-mitotic . This survival function of the IGF-1 receptor in response to UVB irradiation was influenced by activation of phosphatidylinositol-3 kinase and MAP kinase . Prior to UVB irradiation, insulin or IGF-1 had little to no effect on cell growth or viability . Therefore, activation of the IGF-1 receptor in conjunction with UVB irradiation promotes keratinocyte survival at the expense of cell proliferation.

Biotechnol Prog, 1999 Jan, 15(1), 43 - 50
GlaA promoter controlled production of a mutant green fluorescent protein (S65T) by recombinant aspergillus niger during growth on defined medium in batch and fed-batch cultures
Siedenberg D, Mestric S, Ganzlin M, Schmidt M, Punt PJ, van den Hondel CAMJJ, Rinas U.
The first successful expression of the Aequorea victoria green fluorescent protein (GFP) gene in Aspergillus niger is described . When the wild-type GFP gene was expressed in A . niger, neither the fluorescence nor the full translation product of the wtGFP gene was detectable . However, the expression of a mutant form of the green fluorescent protein (S65TGFP) gene resulted in the formation of a functional fluorescent polypeptide . The synthesis of S65TGFP was used to study glaA promoter controlled heterologous gene expression by recombinant A . niger in batch and fed-batch cultures using a defined growth medium . Cells were grown on xylose as noninducing carbon source, and the production of S65TGFP was accomplished by the addition of maltose . The recombinant protein accumulated up to 10 or 25 mg of S65TGFP g-1 cell dry weight using either a maltose pulse for induction or continuous addition of the inducing carbon source, respectively . Irrespective of the induction protocol, the recombinant protein started to accumulate 2 h after addition of the inducing carbon source and reached its maximum specific concentration 10 h after induction . Bright green fluorescing fungal pellets were first detectable by fluorescence microscopy 4-5 h after the onset of maltose addition.

Biotechnol Prog, 1999 Jan-Feb, 15(1), 140 - 5
Improvement of biomass yield and recombinant gene expression in Escherichia coli by using fructose as the primary carbon source; Aristidou AA et al.; The feasibility of substituting glucose with fructose as a carbon source in Escherichia coli fermentations was investigated . Glucose, the most commonly used sugar in bacterial cultivations, is well-known to pose a number of drawbacks; the most important of which is the Crabtree effect, which results in acidogenesis . Fructose, a glucose structural isomer, offers a reasonable alternative for glucose, since its uptake and utilization are more tightly regulated . Comparative fermentation studies indicate that lower acetate excretion and higher biomass yields were attained in fructose-supplemented growth media compared with those of glucose media . More specifically, cells grown in defined media supplemented with fructose do not excrete detectable amounts of acetate, while about 40 mM of acetate was detected extracellularly in similar glucose cultures . A reduction in the initial growth rate of about 20% was observed with fructose, but final cell densities were about 70% higher compared with glucose supplements . Growth in complex LB media supplemented with fructose again resulted in higher biomass yields (up to 40%) and lower acetate excretion (30-40%) than the comparable glucose media . In bioreactor studies using LB media, acetate levels were reduced from 90 to less than 6 mM, while achieving a 25% improvement in biomass yield . When using richer media, cell densities of more than 40 g L-1 dry cell weight were attained in batch cultivation using fructose compared with 30 g L-1 for glucose . These results have immense applicability in the area of recombinant protein processes . Recombinant E . coli, overexpressing beta-galactosidase under the control of the strong pH-inducible promoter, achieved a volumetric recombinant protein yield of 2.2 million U mL-1 (corresponding to approximately 1.5 g L-1) in batch fructose cultures . This represents a 65% recombinant protein yield enhancement when compared to similar glucose cultivations.

Plant Cell, 1999 Feb, 11(2), 263 - 72
Modulation of plasma membrane H+-ATPase activity differentially activates wound and pathogen defense responses in tomato plants; Schaller A et al.; Systemin is an important mediator of wound-induced defense gene activation in tomato plants, and it elicits a rapid alkalinization of the growth medium of cultured Lycopersicon peruvianum cells . A possible mechanistic link between proton fluxes across the plasma membrane and the induction of defense genes was investigated by modulating plasma membrane H+-ATPase activity . Inhibitors of H+-ATPase (erythrosin B, diethyl stilbestrol, and vanadate) were found to alkalinize the growth medium of L . peruvianum cell cultures and to induce wound response genes in whole tomato plants . Conversely, an activator of the H+-ATPase (fusicoccin) acidified the growth medium of L . peruvianum cell cultures and suppressed systemin-induced medium alkalinization . Likewise, in fusicoccin-treated tomato plants, the wound- and systemin-triggered accumulation of wound-responsive mRNAs was found to be suppressed . However, fusicoccin treatment of tomato plants led to the accumulation of salicylic acid and the expression of pathogenesis-related genes . Apparently, the wound and pathogen defense signaling pathways are differentially regulated by changes in the proton electrochemical gradient across the plasma membrane . In addition, alkalinization of the L . peruvianum cell culture medium was found to depend on the influx of Ca2+ and the activity of a protein kinase . Reversible protein phosphorylation was also shown to be involved in the induction of wound response genes . The plasma membrane H+-ATPase as a possible target of a Ca2+-activated protein kinase and its role in defense signaling are discussed.

Int J Biochem Cell Biol, 1998 Dec, 30(12), 1345 - 52
Glutathione modulates lipid composition of human colon derived HT-29 cells; Madesh M et al.; Glutathione (GSH) is important in maintaining intracellular thiol status . The present study looked at the effect of GSH depletion on lipid composition of colon-derived HT-29 cells . GSH was depleted in HT-29 cells by incubation either with buthionine-S, R-sulfoximine (BSO) or diethylmaleate (DEM) . GSH was restored during early periods of cells growth by supplementation of growth medium with either GSH ester or N-acetyl cysteine (NAC) . Lipids were analysed following GSH depletion and supplementation . Among the neutral lipids, an increase in free cholesterol and diacylglycerol and decrease in cholesteryl ester and triacylglycerol were seen in GSH-depleted cells as compared to control cells . There were no detectable free fatty acids either in control or GSH-depleted cells . Among the phospholipids, a decrease in phosphatidylcholine and phosphatidylinositol and an increase in phosphatidylethanolamine were observed . These changes were a completely reversed by supplementation of BSO-treated cells with GSH ester and partially reversed by N-acetyl cysteine . These results suggest that the GSH status of the cell plays an important role in the lipid composition of the cells.

Biochim Biophys Acta, 1998 Dec 10, 1448(2), 299 - 310
Characterisation of calcium signalling in DT40 chicken B-cells; Kubista H et al.; The chicken DT40 pre-B-cell line is becoming a potent experimental tool in the elucidation of higher organism cellular functions due to its unique genetic tractability . While several publications have described the effects of disruption of a range of genes in DT40 cells on calcium signalling, there has been no general overview of Ca2+ responses in wild-type cells . Here, we present experimental data comparing and contrasting the calcium responses to a range of agonists, such as alphaIgM, H2O2 and thapsigargin, applied singly or consecutively in the presence or absence of extracellular calcium . Briefly, we show that calcium release is from thapsigargin-sensitive and also -insensitive stores . This release results in, or is concomitant with, calcium entry across the plasma membrane through store-operated, receptor-operated and possibly L-type like Ca2+ channels . The agonists activate these pathways differentially producing a wide range of different sized and shaped Ca2+ signals . Furthermore, we report that Ca2+ responses in DT40 cells are dependent on the growth conditions . The presence of 1% chicken serum in the growth medium increased amplitudes of calcium responses and enhanced the sustained phase of the alphaIgM response, while 10 microM beta-mercaptoethanol in the medium (not, however, present during calcium measurements) resulted in more transient H2O2 responses and larger amplitude alphaIgM responses while failing to affect thapsigargin responses . The possible causes of these effects and their importance in comparing data from different studies on DT40 cells is discussed.

FEMS Microbiol Lett, 1999 Jan 1, 170(1), 265 - 70
An efficient microbiological growth medium for screening phosphate solubilizing microorganisms; Nautiyal CS; A novel defined microbiological growth medium, National Botanical Research Institute's phosphate growth medium (NBRIP), which is more efficient than Pikovskaya medium (PVK), was developed for screening phosphate solubilizing microorganisms . In plate assay the efficiency of NBRIP was comparable to PVK; however, in broth assay NBRIP consistently demonstrated about 3-fold higher efficiency compared to PVK . The results indicated that the criterion for isolation of phosphate solubilizers based on the formation of visible halo/zone on agar plates is not a reliable technique, as many isolates which did not show any clear zone on agar plates solubilized insoluble inorganic phosphates in liquid medium . It may be concluded that soil microbes should be screened in NBRIP broth assay for the identification of the most efficient phosphate solubilizers.

Mol Gen Genet, 1998 Dec, 260(5), 492 - 502
GRISEA, a copper-modulated transcription factor from Podospora anserina involved in senescence and morphogenesis, is an ortholog of MAC1 in Saccharomyces cerevisiae; Borghouts C et al.; The initial characterization of Grisea suggested that this gene codes for a transcription factor involved in the genetic control of cellular copper homeostasis in Podospora anserina . Here we demonstrate that GRISEA activates in vivo gene expression in Saccharomyces cerevisiae and is characterized by a modular organization . The DNA-binding domain was mapped to the first 168 N-terminal amino acids and the transactivation domain to the C-terminal half of the protein . Increased levels of copper in the growth medium lead to repression of the transactivation function possibly via intramolecular interactions between parts of the DNA-binding domain and the transactivation domain . The wild-type copy of Grisea was found to complement the phenotype of the mac1-1 mutant of S . cerevisiae . GRISEA is able to bind to the promoter of CTR1, a MAC1 target gene that encodes a high-affinity copper transporter . Taken together, the data reported here and in earlier investigations indicate that GRISEA is an ortholog of the yeast transcription factor MAC1 and suggest at least a partial conservation of the molecular machinery involved in the control of cellular copper homeostasis in eukaryotes . Remarkably, in P . anserina, the spectrum of phenotypes affected by this regulatory protein is much broader than that known in yeast and includes morphogenetic traits as well as lifespan and senescence.

Anal Biochem, 1999 Jan 1, 266(1), 58 - 65
13C-Isotopic enrichment of glutathione in cell extracts determined by nuclear magnetic resonance spectroscopy; Gamcsik MP; An NMR method was developed for measuring the isotopic enrichment of glutathione in extracts of cells fed a medium containing {3, 3'-13C2}cystine . Two sublines of human mammary adenocarcinoma MCF-7 cells were exposed to growth medium containing the labeled cystine for varying periods, treated with monobromobimane, harvested, and extracted with perchloric acid . The glutathione-bimane adduct was partially purified by solid-phase extraction before analysis by 1H NMR spectroscopy . The isotopic enrichment of the beta-carbon of the cysteinyl residue of glutathione was determined directly in the cell extracts without further purification . These isotopic enrichment data can be used to determine the rate of synthesis of glutathione in cell and tissue extracts .

Microbiology, 1998 Dec, 144 ( Pt 12), 3447 - 54
Mycoplasma penetrans infection of Molt-3 lymphocytes induces changes in the lipid composition of host cells; Salman M et al.; The AIDS-associated Mycoplasma penetrans is capable of inducing its own uptake by non-phagocytic cells . The ability of M . penetrans to both adhere to and invade Molt-3 lymphocytes was markedly increased in the presence of polyethylene glycol 8000 (PEG) . The effect of PEG was more pronounced in the more alkaline pH range, where the binding kinetics were much faster and almost unaffected by temperature (4-37 degrees C) . Incubation of {14C}oleic-acid-labelled Molt-3 cells with viable M . penetrans resulted in a substantial release of radioactive fatty acids, whereas treating the host cells with heat-inactivated mycoplasmas, isolated M . penetrans membrane preparations, or M . penetrans growth medium, had no effect . Total lipid analysis of Molt-3 lymphocytes infected by M . penetrans revealed an augmented level of the neutral lipid fraction that was associated with a decrease in the relative amounts of polar lipids, mainly a decrease in the amount of phosphatidylserine and diphosphatidylglycerol . Analysis of the neutral lipid fraction in the infected Molt-3 cells revealed a fivefold increase in the relative amount of diacylglycerol and a marked increase in the free fatty acid (FFA) fraction . The profile of the FFAs released was dominated by a relatively high concentration of the polyunsaturated fatty acid docosahexaenoic acid . The release of lipid intermediates suggests that the degradation of Molt-3 cell phospholipids induced by M . penetrans may initiate a signal transmission cascade in the host cell.

J Pharm Biomed Anal, 1998 Sep 1, 17(6-7), 1143 - 53
Separation and quantitation of monoclonal antibodies in cell growth medium using capillary zone electrophoresis; Dai HJ et al.; IgG1 is separated from its impurities in cell growth medium under simple CZE conditions without specific sample pretreatment . Linearity, limit of quantitation, limit of detection, precision and accuracy for the method are demonstrated . The quantitation for IgG1 in the cell growth medium is obtained by generating a calibration curve and by using standard additions . This CE method can offer a good alternative to conventional HPLC methods . Attempts are also made to separate the heterogeneous species in monoclonal antibodies using both CZE and MECC.

J Virol, 1999 Feb, 73(2), 1293 - 301
The YXXL sequences of a transmembrane protein of bovine leukemia virus are required for viral entry and incorporation of viral envelope protein into virions; Inabe K et al.; The cytoplasmic domain of an envelope transmembrane glycoprotein (gp30) of bovine leukemia virus (BLV) has two overlapping copies of the (YXXL)2 motif . The N-terminal motif has been implicated in in vitro signal transduction pathways from the external to the intracellular compartment and is also involved in infection and maintenance of high viral loads in sheep that have been experimentally infected with BLV . To determine the role of YXXL sequences in the replication of BLV in vitro, we changed the tyrosine or leucine residues of the N-terminal motif in an infectious molecular clone of BLV, pBLV-IF, to alanine to produce mutated proviruses designated Y487A, L490A, Y498A, L501A, and Y487/498A . Transient transfection of African green monkey kidney COS-1 cells with proviral DNAs that encoded wild-type and mutant sequences revealed that all of the mutated proviral DNAs synthesized mature envelope proteins and released virus particles into the growth medium . However, serial passages of fetal lamb kidney (FLK) cells, which are sensitive to infection with BLV, after transient transfection revealed that mutation of a second tyrosine residue in the N-terminal motif completely prevented the propagation of the virus . Similarly, Y498A and Y487/498A mutant BLV that was produced by the stably transfected COS-1 cells exhibited significantly reduced levels of cell-free virion-mediated transmission . Analysis of the protein compositions of mutant viruses demonstrated that lower levels of envelope protein were incorporated by two of the mutant virions than by wild-type and other mutant virions . Furthermore, a mutation of a second tyrosine residue decreased the specific binding of BLV particles to FLK cells and the capacity for viral penetration . Our data indicate that the YXXL sequences play critical roles in both viral entry and the incorporation of viral envelope protein into the virion during the life cycle of BLV.

Int J Radiat Biol, 1998 Dec, 74(6), 755 - 64
Evidence for a role of delayed death and genomic instability in radiation-induced neoplastic transformation of human hybrid cells; Mendonca MS et al.; HeLa x skin fibroblast human hybrid cells have been developed into a model of radiation-induced neoplastic transformation . The authors' studies indicate that the loss of putative tumour suppressor loci on fibroblast chromosomes 11 and 14 is evident after radiation-induced neoplastic transformation . How these fibroblast chromosomes/putative tumour suppressor loci are lost after radiation exposure is currently being investigated . It has been shown that the appearance of transformed foci correlates with the onset of the delayed reduction in plating efficiency or delayed death . This delayed death appears to be the result of the onset of a novel delayed apoptosis in the irradiated progeny beginning around day 8 post-irradiation . It was proposed that the reduction in plating efficiency and subsequent neoplastic transformation are all the result of a radiation-induced genomic instability . The instability process has two relevant outcomes: (1) cell death due to the induction of a delayed apoptosis in cells; and (2) neoplastic transformation of a small subset of survivors that have lost fibroblast chromosomes 11 and 14 (tumour suppressor loci) but either have not acquired enough genetic damage to induce the apoptotic response or have undergone molecular changes allowing them to bypass apoptosis . Data from the genomic instability and delayed death literature will be reviewed in terms of relevance to radiation-induced neoplastic transformation . New data are presented which demonstrate that use of growth media supplemented with a specific lot of calf serum was found to increase the number of cells undergoing radiation-induced neoplastic transformation, compared with standard serum after a fixed dose of radiation . This correlates with an increase in delayed death in the irradiated progeny which the authors propose is the result of increased genomic instability post-irradiation of cells grown in this serum . Preliminary data are presented indicating that a delayed apoptosis is also seen after high-energy He- particle exposure in this system.

J Biol Chem, 1999 Jan 15, 274(3), 1691 - 7
Enzymes in the extracellular matrix of Volvox: an inducible, calcium-dependent phosphatase with a modular composition; Hallmann A; The volvocine algae provide the unique opportunity for exploring development of an extracellular matrix . Volvox is the most advanced member of this family and represents the simplest multicellular organism, with differentiated cells, a complete division of labor, and a complex extracellular matrix, which serves structural and enzymatic functions . In Volvox carteri a glycosylated extracellular phosphatase was identified, which is partially released from the extracellular matrix into the growth medium . The phosphatase is synthesized in response to inorganic phosphate starvation and is strictly calcium-dependent . The metalloenzyme has been purified to homogeneity and characterized . Its gene and cDNA have been cloned . Comparisons of genomic and cDNA sequences revealed an extremely intron-rich gene (32 introns) . With an apparent molecular mass of 160 kDa the Volvox extracellular phosphatase is the largest phosphatase cloned, with no sequence similarity to any other phosphatase . This enzyme exhibits a modular composition . There are two large domains and a small one . The large domains are highly homologous to each other and therefore most likely originated from gene duplication and fusion . At least one EF-hand motif for calcium binding was identified in this extracellular protein . Volvox extracellular phosphatase is the first calcium-dependent extracellular phosphatase to be cloned.

Plant Physiol, 1999 Jan, 119(1), 305 - 12
Cellular compartmentation of zinc in leaves of the hyperaccumulator thlaspi caerulescens
K pper H, Jie Zhao F, McGrath SP.
Cellular compartmentation of Zn in the leaves of the hyperaccumulator Thlaspi caerulescens was investigated using energy-dispersive x-ray microanalysis and single-cell sap extraction . Energy-dispersive x-ray microanalysis of frozen, hydrated leaf tissues showed greatly enhanced Zn accumulation in the epidermis compared with the mesophyll cells . The relative Zn concentration in the epidermal cells correlated linearly with cell length in both young and mature leaves, suggesting that vacuolation of epidermal cells may promote the preferential Zn accumulation . The results from single-cell sap sampling showed that the Zn concentrations in the epidermal vacuolar sap were 5 to 6.5 times higher than those in the mesophyll sap and reached an average of 385 mM in plants with 20,000 &mgr;g Zn g-1 dry weight of shoots . Even when the growth medium contained no elevated Zn, preferential Zn accumulation in the epidermal vacuoles was still evident . The concentrations of K, Cl, P, and Ca in the epidermal sap generally decreased with increasing Zn . There was no evidence of association of Zn with either P or S . The present study demonstrates that Zn is sequestered in a soluble form predominantly in the epidermal vacuoles in T . caerulescens leaves and that mesophyll cells are able to tolerate up to at least 60 mM Zn in their sap.

Appl Environ Microbiol, 1999 Jan, 65(1), 175 - 80
Production of biogenic Mn oxides by leptothrix discophora SS-1 in a chemically defined growth medium and evaluation of their Pb adsorption characteristics
Nelson YM, Lion LW, Ghiorse WC, Shuler ML.
Biogenic Mn oxides were produced by the bacterium Leptothrix discophora SS-1 (= ATCC 3182) in a chemically defined mineral salts medium, and the Pb binding and specific surface area of these oxides were characterized . Growth of SS-1 in the defined medium with pyruvate as a carbon and energy source required the addition of vitamin B12 . Complete oxidation of Mn(II) within 60 h required the addition of >/=0.1 &mgr;M FeSO4 . Pb adsorption isotherms were determined for the biogenic Mn oxides (and associated cells with their extracellular polymer) and compared to the Pb adsorption isotherms of cells and exopolymer alone, as well as to abiotic Mn oxides . The Pb adsorption to cells and exopolymer with biogenic Mn oxides (0.8 mmol of Mn per g) at pH 6.0 and 25 degreesC was 2 orders of magnitude greater than the Pb adsorption to cells and exopolymer alone (on a dry weight basis) . The Pb adsorption to the biogenic Mn oxide was two to five times greater than the Pb adsorption to a chemically precipitated abiotic Mn oxide and several orders of magnitude greater than the Pb adsorption to two commercially available crystalline MnO2 minerals . The N2 Brunauer-Emmet-Teller specific surface areas of the biogenic Mn oxide and fresh Mn oxide precipitate (224 and 58 m2/g, respectively) were significantly greater than those of the commercial Mn oxide minerals (0.048 and 4 . 7 m2/g) . The Pb adsorption capacity of the biogenic Mn oxide also exceeded that of a chemically precipitated colloidal hydrous Fe oxide under similar solution conditions . These results show that amorphous biogenic Mn oxides similar to those produced by SS-1 may play a significant role in the control of trace metal phase distribution in aquatic systems.

J Biol Chem, 1999 Jan 8, 274(2), 735 - 8
Phospholipase D mediates matrix metalloproteinase-9 secretion in phorbol ester-stimulated human fibrosarcoma cells; Williger BT et al.; Phospholipase D (PLD) has been implicated in vesicle trafficking in the Golgi and hence secretion . In this study, we show that the secretion of matrix metalloproteinase-9 (MMP-9) from HT 1080 human fibrosarcoma cells was stimulated by phorbol 12-myristate 13-acetate in a time- and dose-dependent manner that involved protein kinase C . The phorbol ester also increased PLD activity in the cells . Evidence that PLD was involved in the stimulation of MMP-9 secretion was provided by the observations that the secretion of MMP-9 was stimulated by the introduction of short-chain phosphatidic acid (PA) into the growth medium and that inhibition of PA production by 1-propanol inhibited secretion . Using a short-chain diacylglycerol we excluded the possibility that MMP-9 secretion was induced by diacylglycerol formed from PA by phosphatidic acid phosphatase . Furthermore, propranolol, an inhibitor of this enzyme, had no effect on secretion induced by either phorbol 12-myristate 13-acetate or PA . The data presented here indicate that activation of protein kinase C increases MMP-9 secretion in HT 1080 cells and implicate PLD and PA formation in the effect.

Folia Microbiol (Praha), 1998, 43(5), 453 - 8
Alteration of cell-wall composition of Fusarium oxysporum by copper stress; Hefnawy MA et al.; A strain of Fusarium oxysporum tolerated copper in the growth medium at concentrations up to 600 mg/L . The optimum growth was obtained at 200 mg Cu/L . The mycelium acquired a blue color in the presence of copper . The copper content of isolated cell walls obtained from mycelium grown in the presence of 600 mg Cu/L was 1.5 times higher than that of cell walls obtained from mycelium grown at 200 mg Cu/L and it contained 2.2 and 3.3% copper at 200 and 600 mg Cu/L, respectively . The amount of protein and total sugars increased in both the mycelium and its isolated cell walls in the presence of copper in the growth medium, chitin was also increased in the cell wall, reaching its maximum amount at 200 mg Cu/L--about 2.4 times higher than without copper . Most of amino acid concentrations in the cell wall were increased in the presence of 200 mg Cu/L and decreased above this concentration . Isoleucine, leucine, tyrosine, phenylalanine, and arginine showed the highest increase at this concentration . The altered cell walls obtained from mycelium grown at 200 and 400 mg Cu/L could rebind individual metals more than the control cell walls could . Rebinding of individual metals was in the order Zn > Fe > Ni > Cu > Co . Rebinding of copper by isolated cell walls depended on pH and temperature.

Biol Chem, 1998 Nov, 379(11), 1349 - 54
The glutamine synthetase from the hyperthermoacidophilic crenarcheon Sulfolobus acidocaldarius: isolation, characterization and sequencing of the gene; Yin Z et al.; The glutamine synthetase (EC 6.3.1.2) from the hyperthermoacidophilic crenarcheon Sulfolobus acidocaldarius (DSM 639) was purified to homogeneity, characterized and the glnA gene isolated and sequenced . The amount of enzyme present in the cytosolic fraction from Sulfolobus cells showed a strong variation depending on the carbon and nitrogen sources in the growth medium . The enzyme was found to be a dodecameric protein composed of identical subunits of 52 kDa . It was stable at 78 degrees C in the presence of Mn2+ ions . The catalytic activity was regulated solely by feed-back inhibition through L-alanine and glycine and not by adenylylation . No evidence for the presence of isoenzymes was found . Sequence comparison showed that the Sulfolobus protein is most closely related to the glutamine synthetases of the I-beta type despite its regulatory properties and the finding that the known euryarcheal glutamine synthetase sequences belong to the I-alpha subgroup of these enzymes . Our phylogenetic analysis suggests that the gene duplication leading to the development of the I-alpha and I-beta enzymes preceded the separation of the archea and the bacteria.

Hepatology, 1999 Jan, 29(1), 90 - 100
Morphogenetic events in mixed cultures of rat hepatocytes and nonparenchymal cells maintained in biological matrices in the presence of hepatocyte growth factor and epidermal growth factor; Michalopoulos GK et al.; Hepatocytes were grown in chemically defined hepatocyte growth medium (HGM) containing hepatocyte growth factor (HGF) and epidermal growth factor (EGF) on collagen-coated polystyrene beads in roller bottle cultures, forming clusters of beads, and proliferating hepatocytes and nonparenchymal cells, including fenestrated endothelium-forming vascular structures . Desmin-positive cells surrounded hepatocytes . Collagen types I and III were deposited in a diffuse manner whereas collagen type IV surrounded the clusters of the epithelial cells, forming a basement membrane . When the mixed cell clusters were implanted in Matrigel (Collaborative Research, Bedford, MA), hepatocytes grew in three dimensions, forming plates and ducts . Many single, long plates of hepatocytes were seen, suggesting progressive linear assembly guided by hepatocyte specific structural parameters . HGF, EGF, and transforming growth factor-alpha (TGF-alpha) enhance these phenomena . HGF plus EGF elicited maximal response . TGF-beta1 suppressed formation of the ducts and plates . Within three months in Matrigel, the cultures established monolayers composed of plates, ducts, and a well-delineated canalicular network . The mixed cultures expressed albumin, A1AT, AFP, transferrin, and CYPIIB1 . Following implantation of the cell clusters in Matrigel, there was decreased expression of c-met, urokinase, urokinase receptor, and TGF-beta1 . Electron microscopy showed differentiated hepatocytes with nearly normal ultrastructure . The proliferating cell nuclear antigen (PCNA) labeling index was high (more than 80%) whereas the Bromo-deoxyaridine labeling index of ongoing DNA synthesis varied from 10% to 15% . These results show that the mixed cultures of proliferating hepatocytes and nonparenchymal cells can reproduce the hallmark structures of hepatic histological architecture while maintaining differentiation and the capacity to proliferate . (HEPATOLOGY 1999;29:90-100.)

J Bacteriol, 1998 Dec, 180(24), 6476 - 83
The EIIGlc protein is involved in glucose-mediated activation of Escherichia coli gapA and gapB-pgk transcription; Charpentier B et al.; The Escherichia coli gapB gene codes for a protein that is very similar to bacterial glyceraldehyde-3-phosphate dehydrogenases (GAPDH) . In most bacteria, the gene for GAPDH is located upstream of the pgk gene encoding 3-phosphoglycerate kinase (PGK) . This is the case for gapB . However, this gene is poorly expressed and encodes a protein with an erythrose 4-phosphate dehydrogenase activity (E4PDH) . The active GAPDH is encoded by the gapA gene . Since we found that the nucleotide region upstream of the gapB open reading frame is responsible for part of the PGK production, we analyzed gapB promoter activity in vivo by direct measurement of the mRNA levels by reverse transcription . We showed the presence of a unique transcription promoter, gapB P0, with a cyclic AMP (cAMP) receptor protein (CRP)-cAMP binding site centered 70.5 bp upstream of the start site . Interestingly, the gapB P0 promoter activity was strongly enhanced when glucose was used as the carbon source . In these conditions, deletion of the CRP-cAMP binding site had little effect on promoter gapB P0 activity . In contrast, abolition of CRP production or of cAMP biosynthesis (crp or cya mutant strains) strongly reduced promoter gapB P0 activity . This suggests that in the presence of glucose, the CRP-cAMP complex has an indirect effect on promoter gapB P0 activity . We also showed that glucose stimulation of gapB P0 promoter activity depends on the expression of enzyme IIGlc (EIIGlc), encoded by the ptsG gene, and that the gapA P1 promoter is also activated by glucose via the EIIGlc protein . A similar glucose-mediated activation, dependent on the EIIGlc protein, was described by others for the pts operon . Altogether, this shows that when glucose is present in the growth medium expression of the E . coli genes required for its uptake (pts) and its metabolism (gapA and gapB-pgk) are coordinately activated by a mechanism dependent upon the EIIGlc protein.

Eur J Pharmacol, 1998 Nov 13, 361(1), 151 - 5
Furosemide and digoxin inhibit thiamine uptake in cardiac cells; Zangen A et al.; Heart cells in culture were used to clarify whether furosemide or digoxin cause thiamine deficiency and if so, by what mechanism . The intracellular level of thiamine pyrophosphate gradually decreased, with a half-life of 16-19 days, after treatment of cardiac cells with furosemide or digoxin . When thiamine was excluded from the growth medium, thiamine pyrophosphate levels gradually decreased, with a half-life of 5-6 days . No additive effect was observed in the presence of the above drugs when thiamine was excluded from the medium . Thiamine uptake by cardiac cells grown in a thiamine-free medium for 7 days decreased significantly in the presence of furosemide or digoxin . The effect of furosemide or digoxin on thiamine uptake was found to be dose dependent . Co-administration of furosemide and digoxin to the cardiac cell cultures resulted in an additive effect on thiamine uptake . Our results demonstrate that furosemide and digoxin inhibit thiamine uptake by cardiac cells in culture and may therefore cause thiamine deficiency in patients undergoing chronic treatment with these drugs.

FEMS Microbiol Lett, 1998 Dec 1, 169(1), 111 - 6
Molybdate-dependent transcription of hyc and nar operons of Escherichia coli requires MoeA protein and ModE-molybdate; Hasona A et al.; In Escherichia coli, ModE-molybdate, a repressor of modABCD operon (molybdate transport), was previously shown to be an additional transcriptional activator of hyc operon (formate hydrogenlyase) and narGHJI operon (respiratory nitrate reductase) . However, in a modE mutant, both operons were expressed at about 50% of the wild-type level in a molybdate-dependent manner . This ModE-independent, molybdate-dependent, expression of hyc, narG and narK operons required MoeA protein . An E . coli modE, moeA double mutant failed to produce formate hydrogenlyase or respiratory nitrate reductase activity irrespective of the growth medium . Tungstate substituted for molybdate in the activation of transcription of hyc and nar operons by ModE could not replace molybdate for MoeA-dependent expression . It is proposed that the MoeA-catalyzed product, an activated form of molybdate, interacts with a transcriptional activator/regulator other than ModE and regulates hyc and nar operons.

J Periodontol, 1998 Nov, 69(11), 1263 - 70
Effects of Porphyromonas gingivalis 2561 extracts on osteogenic and osteoclastic cell function in co-culture; Loomer PM et al.; This study was undertaken to determine the direct effects of extracts derived from Porphyromonas gingivalis on bone formation and mineral resorption in an osteogenic/osteoclastic cell in vitro co-culture model . Osteogenic bone marrow derived stromal cells were isolated from 18-day old embryonic chickens, while osteoclastic cells were isolated from laying white Leghorn hens on calcium deficient diets . Osteoclastic cells (5 x 10(5)) were seeded onto mineral thin films and suspended above osteogenic cells (1 x 10(4)) already plated on the bottoms of tissue culture plate wells . Sonicated P . gingivalis 2561 extracts were prepared from whole bacterial cells and added in varying proportions (0 to 2 microg/ml) to the co-culture growth medium . These co-cultures, and appropriate mono-culture controls, were incubated for a further 4 days . Parameters of bone forming cell activity including alkaline phosphatase activity, calcium and inorganic phosphate accumulation were performed on the osteogenic cells . Mineral substrate resorption by osteoclastic cells was assessed morphometrically . In their respective mono-cultures, the addition of P . gingivalis sonicate to the culture medium had no effect on osteoclastic mineral resorption, but significantly inhibited osteogenesis (up to 45%; P <0.05) . In co-cultures, however, the sonicate induced significant increases in mineral resorption (up to 70%; P <0.05), whereas bone forming cell activity was still inhibited, although to a significantly lesser extent than in mono-cultures (up to 25%; P <0.05) . These results suggest that P . gingivalis sonicate induced up-regulation of mineral resorption may be mediated via osteogenic cells.

J Colloid Interface Sci, 1998 Aug 15, 204(2), 237 - 46
Interaction of Sulfate-Reducing Bacteria with Molybdenum Dissolved from Sputter-Deposited Molybdenum Thin Films and Pure Molybdenum Powder; Chen G et al.; When sputter-deposited Mo thin films were exposed to sulfate-reducing bacterium Desulfovibrio desulfuricans, dissolved Mo markedly delayed the culture growth and reduced the rate of sulfate reduction . The interaction led to an orange coloration of the culture liquid . X-ray photoelectron spectroscopy of dried culture droplets revealed that Mo dissolution products existed mostly in pentavalent state, and a smaller amount of molybdate and molybdenum disulfide . In contrast, Mo dissolution in uninoculated medium was negligible . Subsequently, different concentrations of molybdate, ranging from 0.1 to 20 mM, were added to the growth medium and it was found that a low concentration of molybdate (1 mM) was able to reduce the culture growth rate and sulfate reduction by forming Mo(V)-S complexes . In order to study the dependence of the degree of interaction upon microbial activity and growth-dependent metabolic products, 1.0 g/L Mo powder was added to (a) the growth medium, (b) a 3-day-old culture, and, (c) the supernatants of 2 h to 5-day-old cultures . Ultraviolet-visible spectroscopy indicated that the Mo(V)-S complexes consisted of a Mo-S compound analogous to a binuclear dioxobridged Mo(V)-cysteine complex (314 nm) and Mo(V)-containing molybdenyl thiocyanate (468 nm) . Dissolution of Mo was induced by H2S, a product of the bacterial sulfate reduction, and was further increased probably by sulfur-containing amino groups and proteins .

EMBO J, 1998 Dec 1, 17(23), 6942 - 51
Efficient transition to growth on fermentable carbon sources in Saccharomyces cerevisiae requires signaling through the Ras pathway; Jiang Y et al.; Strains carrying ras2(318S) as their sole RAS gene fail to elicit a transient increase in cAMP levels following addition of glucose to starved cells but maintain normal steady-state levels of cAMP under a variety of growth conditions . Such strains show extended delays in resuming growth following transition from a quiescent state to glucose-containing growth media, either in emerging from stationary phase or following inoculation as spores onto fresh media . Otherwise, growth of such strains is indistinguishable from that of RAS2(+) strains . ras2(318S) strains also exhibit a delay in glucose-stimulated phosphorylation and turnover of fructose-1,6-bisphosphatase, a substrate of the cAMP-dependent protein kinase A (PKA) and a key component of the gluconeogenic branch of the glycolytic pathway . Finally Tpk(w) strains, which fail to modulate PKA in response to fluctuations in cAMP levels, show the same growth delay phenotypes, as do ras2(318S) strains . These observations indicate that the glucose-induced cAMP spike results in a transient activation of PKA, which is required for efficient transition of yeast cells from a quiescent state to resumption of rapid growth . This represents the first demonstration that yeast cells use the Ras pathway to transmit a signal to effect a biological change in response to an upstream stimulus.

Acta Otolaryngol, 1998 Sep, 118(5), 651 - 9
Basilar papilla explants: a model to study hair cell regeneration-repair and protection; Frenz DA et al.; Explants of basilar papillae from 6-7 days posthatch chicks were cultured in growth medium for a period of 1-8 days . Hair cells were counted following staining of stereocilia bundles with FITC-phalloidin, and the percentage of hair cell survival was determined by comparison to control (i.e . uncultured) specimens . Hair cell integrity was evaluated by scanning electron microscopy . Although previous studies have utilized organotypic culture of the basilar papilla to assess cell proliferation and ototoxicity, viability and integrity of hair cells was documented for periods of up to only 2 3 days . Our results demonstrate substantive auditory hair cell viability for a period of 7 days in vitro . We describe a pattern of natural hair cell loss in organotypic culture that progresses along a proximal-distal, abneural-neural gradient, mimicking the pattern of hair cell loss that occurs following ototoxic insult to the chick basilar papilla in vivo and the pattern we observed during a 48-h period of exposure of basilar papilla explants to an ototoxic dose of neomycin . Our results provide an important quantitative step for the use of organotypic culture of the chick basilar papilla as a purposeful model to investigate the process of hair cell regeneration-repair in the avian auditory system.

Mediators Inflamm, 1998, 7(2), 99 - 103
Effect of platelet-activating factor on the growth of human erythroid and myeloid CD34+ progenitors; Dupuis F et al.; We have assessed the effect of platelet-activating factor (PAF), a biologically active phospholipid present in the human marrow, on the growth of human marrow and blood CD34+ progenitors . While the metabolization rate of PAF by CD34+ cells is low (weak acetylhydrolase and acylation processes) it is readily catabolized by the acetylhydrolase activity present in the growth medium (10% fetal calf serum + 10% 5637-conditioned medium) . Treatment of marrow CD34+ cells with the non-metabolizable PAF agonist C-PAF (1 nM to 100 nM) immediately before semi-solid culture significantly (P < 0.01) decreased the number of BFU-E but not of CFU-GM colonies . Treatment of marrow or blood CD34+ cells with C-PAF (10-100 nM) for 3 days in liquid medium before semi-solid culture significantly (P < 0.01) decreased the number of BFU-E and CFU-GM colonies . Treatment of blood CD34+ cells with the two PAF receptor antagonists CV 3988 and BN 52021 (1 microM) had no significant effect on the number of BFU-E and CFU-GM colonies suggesting no role of endogenous PAF in these processes . These results show that exogenous PAF downregulates human erythropoiesis and myelopoiesis, a result that might be of importance during inflammatory states.

Proc West Pharmacol Soc, 1998, 41, 85 - 6
Uptake and metabolism of {3H}testosterone by Penicillium crustosum maintained in cultures at different pHs; Gutierrez E et al.; In order to determine the uptake and the metabolism of {3H}T by the mycelium of Penicillium crustosum, cultures of the fungi containing radiolabeled testosterone were developed at different pHs . Also, the metabolism of this androgen in the growth medium of the fungus was determined . Results show that the {3H}T was taken up by the mycelium at pHs 6, 7, 8 and 9 . In addition, the presence of {3H}DHT in the incubation medium indicated the participation of 5 alpha-reductase . The maximal activities of this enzyme were determined at pHs 6 and 8, suggesting the presence of two isozymes: type 1 (pH 8), and type 2 (pH 6) . Into the mycelium only {3H}T was identified, indicating that isozymes are produced by the fungus under testosterone stimuli.

Appl Environ Microbiol, 1998 Dec, 64(12), 5042 - 5
Effects of halides on plasmid-mediated silver resistance in Escherichia coli; Gupta A et al.; Silver resistance of sensitive Escherichia coli J53 and resistance plasmid-containing J53(pMG101) was affected by halides in the growth medium . The effects of halides on Ag+ resistance were measured with AgNO3 and silver sulfadiazine, both on agar and in liquid . Low concentrations of chloride made the differences in MICs between sensitive and resistant strains larger . High concentrations of halides increased the sensitivities of both strains to Ag+.

Ultrasound Med Biol, 1998 Oct, 24(8), 1209 - 13
Sonochemicals increase the mutation frequency of V79 cells in vitro; Doida Y et al.; Phosphate buffered saline (PBS) was insonated or sham-insonated (1 MHz, 35 W/cm2, continuous wave, 30 min) in rotating (200 rpm) sterile polystyrene culture tubes . After treatment, the PBS was used immediately to suspend washed Chinese hamster V79 cells in vitro . Cells were incubated in the PBS at 37 degrees C for 15 min and then transferred to complete growth medium . Some insonation regimens also involved the inclusion of Albunex (ALX; an ultrasound microbubble contrast agent) to enhance ultrasound-induced inertial cavitation . Following exposure to the pretreated PBS and 6 d of subculture in complete medium, the cells were assayed for plating efficiencies and mutation frequencies (resistance to 6-thioguanine) . X-rays (3 Gy) served as a positive control . Cells exposed to insonated PBS with or without ALX or x-rays had statistically significantly elevated mean mutation frequencies (4.37+/-0.97, 4.54+/-1.00, and 24.28+/-3.83 mutant colonies/10(6) viable cells, respectively) relative to corresponding control regimens (ultrasound sham, 2.44+/-0.56; x-ray sham, 2.96+/-0.88 mutant colonies/10(6) viable cells . The data supported the hypothesis that sonochemicals resulting from inertial cavitation have mutagenic potential.

Membr Cell Biol, 1998, 12(1), 67 - 78
Regulation of intracellular pH and proton-potassium exchange in fermenting Escherichia coli grown anaerobically in alkaline medium; Trchounian A et al.; Fermenting Escherichia coli wild type cells, grown anaerobically at alkaline pH (pH 8.3-8.6), upon transfer into the medium at pH 7.5-7.8 were shown to maintain intracellular pH at 7.5, acidify medium, take in K+, generate membrane potential of -160 mV and produce molecular hydrogen . Proton-potassium exchange proceeded in one step, was inhibited by the N,N'-dicyclohexylcarbodiimide (DCCD) and protonophore CCCP . H+ secretion was sensitive to osmotic shock, and K+ uptake up to the potassium gradient between the cytoplasm and the medium of more than 2 x 10(3) occurred at Km 3.0 mM and was carried out upon upshock or downshock . The stoichiometry of DCCD-inhibited cation fluxes was unstable upon change of experimental conditions . This H+,K+ exchange was not observed in E . coli mutants with the defect in the alpha-subunit of H(+)-ATPase F0F1 complex (uncA) or in the TrkA system of K+ uptake (trkA trkD) . The DCCD-inhibited ATPase activity of membrane vesicles did not show any significant dependence on K+ activity in the medium . We suggest that proton and potassium transport systems are involved in the regulation of intracellular pH in E . coli . K+ uptake in the bacteria grown anaerobically at alkaline pH is carried out by the TrkA system, which functions as uniporter, interacts with the F0F1 proton pump by means of transmembrane electrochemical gradient for H+ which is used as the driving force . Growth medium pH, probably, determines the character of interaction of the TrkA with the F0F1.

Proc Natl Acad Sci U S A, 1998 Nov 24, 95(24), 14071 - 5
Overexpression of peptide-methionine sulfoxide reductase in Saccharomyces cerevisiae and human T cells provides them with high resistance to oxidative stress; Moskovitz J et al.; The yeast peptide-methionine sulfoxide reductase (MsrA) was overexpressed in a Saccharomyces cerevisiae null mutant of msrA by using a high-copy plasmid harboring the msrA gene and its promoter . The resulting strain had about 25-fold higher MsrA activity than its parent strain . When exposed to either hydrogen peroxide, paraquat, or 2,2'-azobis-(2-amidinopropane) dihydrochloride treatment, the MsrA overexpressed strain grew better, had lower free and protein-bound methionine sulfoxide and had a better survival rate under these conditions than did the msrA mutant and its parent strain . Substitution of methionine with methionine sulfoxide in a medium lacking hydrogen peroxide had little effect on the growth pattern, which suggests that the oxidation of free methionine in the growth medium was not the main cause of growth inhibition of the msrA mutant . Ultraviolet A radiation did not result in obvious differences in survival rates among the three strains . An enhanced resistance to hydrogen peroxide treatment was shown in human T lymphocyte cells (Molt-4) that were stably transfected with the bovine msrA and exposed to hydrogen peroxide . The survival rate of the transfected strain was much better than its parent strain when grown in the presence of hydrogen peroxide . These results support the proposition that the msrA gene is involved in the resistance of yeast and mammalian cells to oxidative stress.

J Dent Res, 1998 Nov, 77(11), 1896 - 903
Toxicity of formaldehyde to human oral fibroblasts and epithelial cells: influences of culture conditions and role of thiol status; Nilsson JA et al.; The toxicity of formaldehyde, a monomer released from certain polymeric dental materials, was studied in cultured human oral fibroblasts and epithelial cells . The influences of growth conditions were evaluated for both cell types, as well as the role of the internal and external thiol states . A one-hour exposure to formaldehyde decreased the colony-forming efficiency (CFE) of both cell types in a concentration-dependent manner, although the toxicity varied up to 100-fold with the conditions . Clearly, the presence of serum and the thiol cysteine counteracted the toxicity in fibroblasts . Similarly, pituitary extract and cysteine, or a mixture of amino acids and ethanolamines, counteracted the formaldehyde toxicity in serum-free cultures of epithelial cells . In contrast, a growth-promoting surface matrix of fibronectin and collagen did not influence the formaldehyde toxicity, as shown by both the CFE assay and a dye reduction assay . Further, a short-term change to the various growth media per se with or without the supplements serum or cysteine did not significantly alter the CFE . Analysis of the thiol state demonstrated significant differences between epithelial cells and fibroblasts, i.e., comparatively lower cellular levels of the free low-molecular-weight thiols glutathione and cysteine in fibroblasts . This result correlated to significantly higher formaldehyde toxicity in the fibroblasts than in the epithelial cells . Taken together, the results indicated the cytoprotective function of both intracellular and extracellular thiols toward formaldehyde, as well as the usefulness of thiol-free and chemically defined conditions for toxicity assessments in oral epithelial cells and fibroblasts . We conclude that the combined use of a controlled external milieu and the presumed target cell type may be advantageous in evaluations of oral toxicity mechanisms or the toxic potency of dental materials, particularly those which, like formaldehyde, may react with thiols or amines.

Diagn Microbiol Infect Dis, 1998 Oct, 32(2), 81 - 4
Performance of six cell lines in the suspension-infection test used for the detection of herpes simplex virus; Rich T et al.; Six cell lines were assessed in the suspension-infection (SI) test for suitability for the rapid culture of herpes simplex virus (HSV) . For the SI test, the specimen was combined in growth medium with trypsinized, suspended culture cells before allowing the cells to settle into a monolayer growth pattern . The cells tested in the SI assay were MV1-Lu, vero C1008, BSC-1, RD, and MRC-9 cells . Fifty clinical specimens composed of 7 HSV-1-positive samples, 9 HSV-2-positive samples, 12 positive for HSV (not typed), and 22 HSV-negative samples were tested . For the detection of HSV in clinical specimens, MV1Lu cells were most sensitive and demonstrated large, darkly stained foci of virus infection.

Intervirology, 1998, 41(2-3), 120 - 6
Environmental influence on immune inhibition of release of herpes simplex virus from cells; Benitez J et al.; Immune inhibition of virus release (IVR) of herpes simplex virus type 1 (HSV-1) from baby hamster kidney cells (BHK-21) was mediated by antisera against BHK cells, HSV-1, human fibronectin and mouse heparan sulphate proteoglycan and was irreversible for at least 24 h following removal of antiserum . Enhancement of IVR by calf serum depletion of growth media was obtained in varying measure using each of these antisera and also by treatment of virus-infected cells by the lectin concanavalin A . Enhancement was reversible by replenishment of growth media with bovine serum components larger than 12 kD but this only occurred when replenishment was instituted prior to virus infection . There was also reversibility to varying degree following replenishment by ovine, equine and human serum which indicates that this phenomenon is not species specific . In addition to the presence of relevant antigens on the cell surface, IVR may also require an alteration in the cell membrane; this is evidenced by the absence of anticellular serum-mediated IVR when treatment was introduced less than 6 h after virus infection, suggesting that a certain level of alteration or possibly cell damage - in this case virus induced - is necessary . Enhancement of IVR by calf serum depletion would seem to operate through a specific alteration in the virus-infected cell membrane as serum-depleted cells did not show histological alteration and were able to replicate HSV-1 to usual titres; it is possible that this enhancement may represent an as yet unidentified host defence mechanism whereby extracellular release of virus will be reduced in ischaemic or necrotic tissue in the course of infectious inflammatory processes.

J Bacteriol, 1998 Nov, 180(22), 5921 - 7
Adherence of the gram-positive bacterium Ruminococcus albus to cellulose and identification of a novel form of cellulose-binding protein which belongs to the Pil family of proteins; Pegden RS et al.; The adherence of Ruminococcus albus 8 to crystalline cellulose was studied, and an affinity-based assay was also used to identify candidate cellulose-binding protein(s) . Bacterial adherence in cellulose-binding assays was significantly increased by the inclusion of either ruminal fluid or micromolar concentrations of both phenylacetic and phenylpropionic acids in the growth medium, and the addition of carboxymethylcellulose (CMC) to assays decreased the adherence of the bacterium to cellulose . A cellulose-binding protein with an estimated molecular mass following sodium dodecyl sulfate-polyacrylamide gel electrophoresis of approximately 21 kDa, designated CbpC, was present in both cellobiose- and cellulose-grown cultures, and the relative abundance of this protein increased in response to growth on cellulose . Addition of 0.1% (wt/vol) CMC to the binding assays had an inhibitory effect on CbpC binding to cellulose, consistent with the notion that CbpC plays a role in bacterial attachment to cellulose . The nucleotide sequence of the cbpC gene was determined by a combination of reverse genetics and genomic walking procedures . The cbpC gene encodes a protein of 169 amino acids with a calculated molecular mass of 17,655 Da . The amino-terminal third of the CbpC protein possesses the motif characteristic of the Pil family of proteins, which are most commonly involved with the formation of type 4 fimbriae and other surface-associated protein complexes in gram-negative, pathogenic bacteria . The remainder of the predicted CbpC sequence was found to have significant identity with 72- and 75-amino-acid motifs tandemly repeated in the 190-kDa surface antigen protein of Rickettsia spp., as well as one of the major capsid glycoproteins of the Chlorella virus PBCV-1 . Northern blot analysis showed that phenylpropionic acid and ruminal fluid increase cbpC mRNA abundance in cellobiose-grown cells . These results suggest that CbpC is a novel cellulose-binding protein that may be involved in adherence of R . albus to substrate and extends understanding of the distribution of the Pil family of proteins in gram-positive bacteria.

Plant Physiol, 1998 Nov, 118(3), 793 - 801
petit1, a conditional growth mutant of Arabidopsis defective in sucrose-dependent elongation growth; Kurata T et al.; The hypocotyl of Arabidopsis is well suited for the analysis of cell elongation because it elongates without cell division . We have isolated a new class of recessive mutants, petit1 (pet1), which are defective in aspects of hypocotyl elongation . The short-hypocotyl phenotype of pet1 is caused by shortened cells . The cells of the elongation zone of the hypocotyl are often deformed . pet1 also shows defects in elongation of the roots, flower stalk, leaves, petals, pedicels, and siliques, and these defects cannot be repaired by the application of auxin, gibberellin, brassinolide, or an inhibitor of ethylene biosynthesis . The short-hypocotyl phenotype of pet1 is pronounced only in growth medium supplemented with sucrose, which has promotive effects on hypocotyl elongation . In pet1 this effect is much reduced, causing the sucrose-dependent short-hypocotyl phenotype of pet1 . pet1 accumulates more soluble sugars than the wild type and also shows more intensive iodo-starch staining in the cotyledon and hypocotyl . These results indicate that PETIT1 is involved in a sugar-dependent elongation process that may include correct assembly of expanding cell wall architecture.

Vet Parasitol, 1998 Oct, 79(2), 135 - 41
Successful long-term in vitro cultivation of Theileria annulata schizonts in media supplemented with homologous and heterologous sera; Sharma G et al.; The efficacy of medium RPMI-1640 supplemented with either foetal bovine, normal bovine, goat or sheep sera was compared for prolonged in vitro propagation of Theileria annulata (Hisar) schizonts . Medium RPMI-1640 supplemented with 20% foetal bovine serum (standard growth medium) resulted in optimum growth of T . annulata (Hisar) schizonts in vitro . Comparable viability and non-viability counts were observed in growth media supplemented with normal bovine or goat sera . However, viability counts in medium supplemented with sheep serum were significantly lower than that of the standard medium . Mitotic indices of cultures of T . annulata (Hisar) schizonts were directly related to the extent of cell growth and were lower in various growth media supplemented with normal bovine, goat or sheep sera than in that of the standard medium . The results suggested that normal bovine and goat sera could be successfully used in place of foetal bovine serum in the growth medium for long-term in vitro propagation of T . annulata schizonts . The study will help in reducing the cost of large-scale in vitro propagation of T . annulata aimed at mass production of the cell culture vaccine.

Int J Cancer, 1998 Nov 9, 78(4), 491 - 5
Inhibition of gap junctional intercellular communication by perfluorinated fatty acids is dependent on the chain length of the fluorinated tail; Upham BL et al.; Perfluorinated fatty acids (PFFAs), such as perfluorooctanoic acid (PFOA) and perfluorodecanoic acid (PFDA), are known peroxisome proliferators and hepatocarcinogens . A causal link between an increase in the oxidative stress by peroxisomes and tumor promotion has been proposed to explain the hepatocarcinogenicity of PFOA and PFDA . However, the down-regulation of gap junctional intercellular communication (GJIC) has also been linked to the tumor-promoting properties of many carcinogens . Therefore, the effect of PFFAs on GJIC in WB-rat liver epithelial cells was determined . The chain length of the PFFAs tested for an effect on GJIC ranged from 2 to 10, 16 and 18 carbons . Carbon lengths of 7 to 10 inhibited GJIC in a dose-response fashion, whereas carbon lengths of 2 to 5, 16 and 18 did not appreciably inhibit GJIC . Inhibition occurred within 15 min and was reversible, with total recovery from inhibition occurring within 30 min after the removal of the compound from the growth medium . This short time of inhibition suggests that GJIC was modified at the post-translational level . Also, this short time period was not long enough for peroxisome proliferation . The post-translational modification of the gap junction proteins was not a consequence of altered phosphorylation as determined by Western blot analysis . Perfluorooctanesulfonic acid also inhibited GJIC in a dose-response fashion similar to PFDA, indicating that the determining factor of inhibition was probably the fluorinated tail, which required 7-10 carbons . Our results suggest that PFFAs could potentially act as hepatocarcinogens at the level of gap junctions in addition to or instead of through peroxisome proliferation.

Biochem Biophys Res Commun, 1998 Oct 20, 251(2), 482 - 7
Enhancement of cell death due to decrease in Mg2+ uptake by OmpC (cation-selective porin) deficiency in ribosome modulation factor-deficient mutant; Apirakaramwong A et al.; Ribosome modulation factor (RMF) is involved in stabilization of ribosomes during the transition from exponential growth to the stationary growth phase in Escherichia coli . A deficiency of RMF is known to reduce cell viability . Overaccumulation of spermidine also leads to a decrease in cell viability and to a decrease in the synthesis of RMF and of the cation-selective porin OmpC . Thus, a decrease in RMF levels may be involved in the decreased cell viability caused by excess spermidine . Because spermidine also influences the expression of OmpC, we examined whether OmpC deficiency enhances the cell death caused by RMF deficiency . The ompC mutant by itself did not affect protein synthesis or cell viability, but the double rmf ompC mutant produced a much larger decrease in protein synthesis and cell viability than did the single rmf mutant . There was also a decrease in the amount of ribosomes and in the Mg2+ content in the double rmf ompC mutant, and cell viability could be partially restored by the addition of Mg2+ to the growth medium . RMF deficiency was found to inhibit the synthesis of another cation-selective porin OmpF . Thus, the double rmf ompC mutant is deficient in both OmpC and OmpF, which probably accounts for the pronounced decrease in Mg2+ uptake in this mutant . The results indicate that both RMF and Mg2+, acting through stabilization of ribosomes, are important for cell viability at the stationary growth phase .

J Gastroenterol Hepatol, 1998 Sep, 13 Suppl, S78 - 82
Dose-dependent biphasic effects of phenobarbital on growth and differentiation of primary culture rat hepatocytes; Miyazaki M et al.; The actions of phenobarbital, a liver tumour promoter, on growth and differentiation of primary culture normal rat hepatocytes change biphasically as a function of its concentration . At low concentrations of 0.5-2 mmol/L, phenobarbital enhances DNA synthesis of normal adult rat hepatocytes in the presence of epidermal growth factor (EGF) and/or dexamethasone . This is also true for normal suckling (1-2-week-old) rat hepatocytes, without added growth factor(s), in serum-free primary culture . Contrarily, phenobarbital at high concentrations (3-4 mmol/L) suppresses DNA synthesis of suckling rat hepatocytes . Furthermore, phenobarbital inhibits DNA synthesis of transforming growth factor-alpha-stimulated primary hepatocytes from normal adult rats in a dose-dependent manner within a concentration range of 3-6 mmol/L . When normal adult rat hepatocytes are led to undergo multiple proliferative cycles upon stimulation with hepatocyte growth factor (HGF) and EGF in the chemically defined hepatocyte growth medium (HGM), 3 mmol/L phenobarbital also remarkably suppresses DNA synthesis . Phenobarbital at 3 mmol/L effectively keeps these hepatocytes morphologically differentiated and accelerates restoration of the expression of markers characteristic of differentiated cells after the initial cellular growth phase . In addition, phenobarbital efficiently supports prolonged survival of the hepatocytes.

J Bacteriol, 1998 Nov, 180(21), 5612 - 8
Redox-dependent gene regulation in Rhodobacter sphaeroides 2.4.1(T): effects on dimethyl sulfoxide reductase (dor) gene expression; Mouncey NJ et al.; The ability of Rhodobacter sphaeroides 2.4.1(T) to respire anaerobically with the alternative electron acceptor dimethyl sulfoxide (DMSO) or trimethylamine N-oxide (TMAO) is manifested by the molybdoenzyme DMSO reductase, which is encoded by genes of the dor locus . Previously, we have demonstrated that dor expression is regulated in response to lowered oxygen tensions and the presence of DMSO or TMAO in the growth medium . Several regulatory proteins have been identified as key players in this regulatory cascade: FnrL, DorS-DorR, and DorX-DorY . To further examine the role of redox potentiation in the regulation of dor expression, we measured DMSO reductase synthesis and beta-galactosidase activity from dor::lacZ fusions in strains containing mutations in the redox-active proteins CcoP and RdxB, which have previously been implicated in the generation of a redox signal affecting photosynthesis gene expression . Unlike the wild-type strain, both mutants were able to synthesize DMSO reductase under strictly aerobic conditions, even in the absence of DMSO . When cells were grown photoheterotrophically, dorC::lacZ expression was stimulated by increasing light intensity in the CcoP mutant, whereas it is normally repressed in the wild-type strain under such conditions . Furthermore, the expression of genes encoding the DorS sensor kinase and DorR response regulator proteins was also affected by the ccoP mutation . By using CcoP-DorR and CcoP-DorY double mutants, it was shown that the DorR protein is strictly required for altered dor expression in CcoP mutants . These results further demonstrate a role for redox-generated responses in the expression of genes encoding DMSO reductase in R . sphaeroides and identify the DorS-DorR proteins as a redox-dependent regulatory system controlling dor expression.

Acta Anat (Basel), 1998, 162(1), 1 - 15
Identification of an autocrine signaling pathway that amplifies induction of endocardial cushion tissue in the avian heart; Ramsdell AF et al.; Endocardial cushion tissue is formed by an epithelial-mesenchymal transformation of endocardial cells, a process which results from an inductive interaction between the myocardium and endocardium within the atrioventricular (AV) and outflow tract (OT) regions of the heart . We report here that a protein previously found to be required for myocardially induced transformation of endocardial cells in vitro, ES/130, is highly expressed within the AV and OT regions not only by myocardial cells, but also by the endocardium and its mesenchymal progeny . Given these findings and others, we have tested the hypothesis that endocardial cushion tissue secretes factors which autoregulate its transformation to mesenchyme . Endocardial cushion tissue was cultured and its conditioned growth medium was harvested and applied to nontransformed endocardial cells maintained in the absence of the inductive myocardium . This treatment resulted in endocardial cell invasion into three-dimensional collagen gels plus increased expression of proteins associated with endocardial cell transformation in vivo . Whereas endocardial cushion tissue was found to express ES/130 protein in vivo and in vitro, minimal detection of ES/130 in its conditioned growth medium was observed in immunoblots . Attempts to inhibit the mesenchyme-promoting activity of the conditioned medium with ES/130 antisense were unsuccessful . However, strong intracellular ES/130 expression was detected in endocardial cells, and this expression correlated with the ability of endocardial cells to transform . For example, the minority of endocardial cultures that failed to transform in response to conditioned medium treatment also failed to undergo increased expression of ES/130 . These observations are interpreted to suggest that (i) endocardial cushion tissue secretes factors that promote its transformation to mesenchyme, and (ii) while endocardial cushion tissue appears to signal through secretion of factors other than or in addition to ES/130, intracellular ES/130 expression nevertheless may be a target endocardial cell response required for endocardial cell transformation.

Infect Immun, 1998 Nov, 66(11), 5147 - 56
Expression of the tpr protease gene of Porphyromonas gingivalis is regulated by peptide nutrients; Lu B et al.; The Tpr protease of Porphyromonas gingivalis W83 is a membrane-associated enzyme capable of hydrolyzing chromogenic substrates for trypsin and bacterial collagenases . A previous study by us indicated that Tpr expression was increased under conditions of nutrient limitation . In the present study, we further characterized expression of the tpr gene using a tpr::lacZ reporter gene construct under a range of nutrient conditions . In P . gingivalis, transcription of tpr was initiated 215 bp upstream of the coding region and regulation of tpr expression was at the level of transcription . Deletion mutations in the tpr upstream region identified the promoter region immediately upstream of the transcription start site, determined by primer extension analysis . Three identical 17-bp direct repeats identified within the 5' end of tpr mRNA were involved in tpr regulation . In an Escherichia coli background, tpr transcription was initiated after an AT-rich region upstream of tpr but not at the P . gingivalis start site . Tpr expression in P . gingivalis was suppressed by the addition of peptide and protein nutrients to a peptide-limited growth medium but was only slightly affected by addition of free amino acids . Low-molecular-weight fractions of brain heart infusion rich in phenylalanine, proline, and alanine had the greatest inhibitory effects on expression of the tpr::lacZ construct . Addition of the dipeptide phenylalanyl-phenylalanine to the growth medium resulted in a 10-fold decrease in tpr expression . This suggests that specific phenylalanine-containing peptides are a major factor controlling Tpr expression . Neither hemin starvation, heat shock, nor pH change had significant effects on Tpr expression.

Cell Mol Neurobiol, 1998 Oct, 18(5), 477 - 86
Pyrimidine nucleotide-stimulated thromboxane A2 release from cultured glia; Langley D et al.; 1 . Uridine triphosphate (UTP), uridine diphosphate (UDP), cytidine triphosphate (CTP), and deoxythymidine triphosphate (TTP) caused concentration-dependent increases in the release of thromboxane A2 (TXA2) from cultured glia prepared from the newborn rat cerebral cortex . Although each of the pyrimidine nucleotides displayed similar potencies, CTP and TTP were considerably less effective than either UTP or UDP . The purine nucleotide ATP was equally as potent as the pyrimidine nucleotides but was marginally less effective than either UTP or UDP . 2 . The ability of UTP, UDP, TTP, and CTP to promote TXA2 release from cultured glia was inhibited in a concentration-dependent manner by suramin and was markedly reduced when incubations were performed either in Ca(2+)-free medium or on cultures which had been maintained in serum-free growth medium for 4 days prior to experimentation . 3 . Challenges with UTP and UDP in combination were found to elicit a response which was no different from the effects of these nucleotides alone; in addition, their effects were reversed by the phospholipase A2 inhibitor ONO-RS-082 . A slight reduction in UTP- and UDP-stimulated TXA2 release was observed in cultures grown in the presence of leucine methyl ester, a treatment reported to limit microglial survival . 4 . These results suggest that glia are targets for extracellular pyrimidine nucleotides and that their ability to release eicosanoids from these cells may be important in the brain's response to damage.

Exp Cell Res, 1998 Oct 10, 244(1), 93 - 104
Differential expression and distribution of focal adhesion and cell adhesion molecules in rat hepatocyte differentiation; Kim TH et al.; Hepatocytes in primary culture enter into clonal proliferation in the chemically defined hepatocyte growth medium in the presence of hepatocyte growth factor and epidermal growth factor . Hepatocyte proliferation is associated with loss of differentiated gene expression . Overlay of matrix derived from Engelbreth-Holm-Swarm mouse sarcoma (Matrigel) on proliferating hepatocytes induces reexpression of the hepatic differentiation marker genes . To explore the role of matrix in the differentiation process of hepatocytes, we examined the mRNAs of fibronectin, vitronectin, and entactin in proliferating hepatocytes and Matrigel-treated hepatocytes . Fibronectin mRNA increased in proliferating hepatocytes at days 2-10 and then decreased; however, vitronectin mRNA disappeared in proliferating hepatocytes and was reexpressed in Matrigel-treated hepatocytes . We also found that focal adhesion kinase and paxillin were strongly increased in Matrigel-treated hepatocytes, and E-cadherin and beta-catenin slightly increased in Matrigel-treated hepatocytes, suggesting that both cell-to-extracellular matrix and cell-to-cell interactions may be an essential part of hepatocyte differentiation . To evaluate the distribution of focal adhesion associated molecules and cell-to-cell adhesion molecules, Triton X-100 soluble and insoluble fractions were examined at days 8, 9, 10, and 11 in proliferating hepatocytes and Matrigel-treated cells . We found that E-cadherin in Triton X-100 insoluble fractions dramatically decreased in Matrigel-treated hepatocytes; however, beta-catenin strongly increased in Triton X-100 soluble fractions of Matrigel-treated hepatocytes . These results suggest that the distribution of both focal adhesion associated molecules and cell adhesion molecules are reorganized during the process of differentiation induced by overlay of Matrigel .

FEMS Microbiol Lett, 1998 Sep 15, 166(2), 283 - 8
Extracellular proteins and other components as obligate intermediates in the induction of a range of acid tolerance and sensitisation responses in Escherichia coli; Rowbury RJ et al.; Several acid tolerance responses of Escherichia coli were associated with secretion into the growth media of components (frequently proteins) which altered acid tolerance of other cultures . First, medium filtrates from cultures induced to acid tolerance by several conditions converted pH 7.0-grown organism to tolerance and, for most such responses, filtrate proteins were needed for full induction . Secondly, filtrates from cultures induced to acid sensitivity at alkaline pH produced sensitisation of resistant cultures . Thirdly, filtrates from inherently tolerant or sensitive strains altered tolerance or sensitivity of normal strains . In many cases, filtrate components were essential for the original response, e.g . acid habituation at pH 5.0 . Extracellular components may function as intermediates only in stress tolerance responses, but other adaptive responses must be tested as such components may function in other inducible processes.

In Vitro Cell Dev Biol Anim, 1998 Sep, 34(8), 636 - 9
Organotypic culture of human ovarian surface epithelial cells: a potential model for ovarian carcinogenesis; Gregoire L et al.; The objective of this work was to establish an in vitro multidimensional culture system for human ovarian surface epithelial (HOSE) cells as a model for ovarian carcinogenesis . The epithelial origin of cell outgrowth from cells obtained from the ovarian surface was confirmed by keratin staining . Two cultures from two different patients were established, HOSE-A and HOSE-B . Cultures were infected with a retrovirus expressing human papillomavirus genes E6 and E7 to extend their life span . HOSE cells were seeded onto collagen gels containing NIH3T3-J2 fibroblasts as feeder cells and grown to confluence submerged in growth medium . The collagen bed was then raised to the air-medium interface for 7 d (organotypic culture) . Microscopically, fixed cultures revealed a single layer of flat cells growing on the collagen surface, reminiscent of HOSE cells in vivo . Infected HOSE-A and HOSE-B cells exhibited aberrant growth because they stratified . In addition, established ovarian cancer lines grown in this fashion stratified and showed malignant phenotypes . Thus, cells grown in organotypic culture resemble their in vivo counterparts, providing a basis for establishing a system to study growth, proliferation, differential gene expression, and perhaps malignant transformation of HOSE cells.

Mol Microbiol, 1998 Aug, 29(4), 937 - 43
FadR, transcriptional co-ordination of metabolic expediency; Cronan JE Jr et al.; FadR is an Escherichia coli transcriptional regulator that optimizes fatty acid metabolism in response to exogenously added fatty acids . Many bacteria grow well on long-chain fatty acids as sole carbon source, but at the expense of consuming a useful structural material . Exogenous fatty acids are readily incorporated into membrane phospholipids in place of the acyl chains synthesized by the organism, and phospholipids composed of any of a large variety of exogenously derived acyl chains make biologically functional membranes . It would be wasteful for bacteria to degrade fatty acids to acetyl-CoA and then use this acetyl-CoA to synthesize the same (or functionally equivalent) fatty acids for phospholipid synthesis . This line of reasoning suggests that bacteria might shut down endogenous fatty acid synthesis on the addition of long-chain fatty acids to the growth medium . Moreover, this shutdown could be closely coupled to fatty acid degradation, such that a bacterial cell would use a portion of the exogenous fatty acid for phospholipid synthesis while degrading the remainder to acetyl-CoA . To a degree, the bacterium could both have its cake (the acyl chains for phospholipid synthesis) and eat it (to form acetyl-CoA) . This scenario turns out to be true in E . coli . The key player in this regulatory gambit is FadR, a transcription factor that acts both as a repressor of the fatty acid degradation and as an activator of fatty acid biosynthesis.

Plant Physiol, 1998 Oct, 118(2), 651 - 9
Rapid Up-regulation of HKT1, a high-affinity potassium transporter gene, in roots of barley and wheat following withdrawal of potassium
Wang TB, Gassmann W, Rubio F, Schroeder JI, Glass AD.
High-affinity K+ uptake in plant roots is rapidly up-regulated when K+ is withheld and down-regulated when K+ is resupplied . These processes make important contributions to plant K+ homeostasis . A cDNA coding for a high-affinity K+ transporter, HKT1, was earlier cloned from wheat (Triticum aestivum L.) roots and functionally characterized . We demonstrate here that in both barley (Hordeum vulgare L.) and wheat roots, a rapid and large up-regulation of HKT1 mRNA levels resulted when K+ was withdrawn from growth media . This effect was specific for K+; withholding N caused a modest reduction of HKT1 mRNA levels . Up-regulation of HKT1 transcript levels in barley roots occurred within 4 h of removing K+, which corresponds to the documented increase of high-affinity K+ uptake in roots following removal of K+ . Increased expression of HKT1 mRNA was evident before a decline in total root K+ concentration could be detected . Resupply of 1 mM K+ was sufficient to strongly reduce HKT1 transcript levels . In wheat root cortical cells, both membrane depolarizations in response to 100 &mgr;M K+, Cs+, and Rb+, and high-affinity K+ uptake were enhanced by K+ deprivation . Thus, in both plant systems the observed physiological changes associated with manipulating external K+ supply were correlated with levels of HKT1 mRNA expression . Implications of these findings for K+ sensing and regulation of the HKT1 mRNA levels in plant roots are discussed.

Mol Microbiol, 1998 Aug, 29(4), 1091 - 9
Transcription of rpoH, encoding the Escherichia coli heat-shock regulator sigma32, is negatively controlled by the cAMP-CRP/CytR nucleoprotein complex; Kallipolitis BH et al.; In Escherichia coli, the rpoH gene encoding the essential heat-shock regulator sigma32, is expressed in a complex manner . Transcription occurs from four promoters (P1, P3, P4 and P5) and is modulated by several factors including (i) two sigma factors (sigma70 and sigmaE); (ii) the global regulator CRP; and (iii) the DnaA protein . Here, a further dissection of the rpoH regulatory region has revealed that an additional transcription control exists that appears to link rpoH expression to nucleoside metabolism . The cAMP-CRP complex and the CytR anti-activator bind co-operatively to the promoter region forming a repression complex that overlaps the sigmaE-dependent P3 promoter and the sigma70-dependent P4 and P5 promoters . During steady-state growth conditions with glycerol as the carbon and energy source, transcription from P3, P4 and P5 is reduced approximately threefold by CytR, whereas transcription from the upstream promoter, P1, appears to be unaffected . Furthermore, in strains that slightly overproduce CytR, transcription from P3, P4 and P5 is reduced even further (approximately 10-fold), and repression can be fully neutralized by the addition of the inducer cytidine to the growth medium . In the induced state, P4 is the strongest promoter and, together with P3 and P5, it is responsible for most rpoH transcription (65-70%) . At present, CytR has been shown to 'fine tune' transcription of two genes (rpoH and ppiA) that are connected with protein-folding activities . These findings suggest that additional assistance in protein folding is required under conditions in which CytR is induced (i.e . in the presence of nucleosides).

J Antibiot (Tokyo), 1998 Aug, 51(8), 708 - 14
Hongoquercins, new antibacterial agents from the fungus LL-23G227: fermentation and biological activity; Abbanat DA et al.; Two new antibiotics, hongoquercins A and B, were isolated from fermentation extracts of the unidentified fungus LL-23G227 . In the optimum medium, titers of the A and B components reached approximately 2.1 g/liter and 0.02 g/liter, respectively . The optimum temperature for antibiotic production was approximately 22 degrees C . Growth was delayed at 15 degrees C but appeared to reach higher levels than was observed at 22 degrees C . Addition of dextrose to growth media increased hongoquercin B production . Hongoquercin A exhibited moderate activity against Gram-positive bacteria . Mechanistic studies conducted in an E . coli imp strain suggested membrane damage as the primary mode of bactericidal action . These compounds also lysed human red blood cells, suggesting a similar mode of action on eukaryotic cells.

Res Microbiol, 1998 Jan, 149(1), 65 - 72
Heterotrophic growth on phenolic mixtures by Ochromonas danica; Semple KT; Because phenols are one of the most common groups of organic pollutants in the aquatic environment, heterotrophic growth-linked biodegradation of phenol and its methylated homologues by the eukaryotic alga Ochromonas danica (CCAP 933/2B) was investigated . The alga grew heterotrophically on phenol and mixtures of phenol with o- or p-cresols, or with 2,5-, 2,6-, 3,4- or 3,5-xylenols as the sole sources of carbon in the dark at 25 degrees C . Commensurate with growth, the alga removed phenol, both cresol isomers and 2,5- and 3,4-xylenols from the growth media over the incubation periods . In every case, phenol was removed preferentially to the methylated cosubstrates, but the rates of removal for phenol were slower than in incubations where phenol was the sole carbon source.

Thromb Haemost, 1998 Sep, 80(3), 481 - 7
Heparin regulates ICAM-1 expression in human endothelial cells: an example of non-cytokine-mediated endothelial activation; Miller SJ et al.; Activated endothelial cells up-regulate the expression of several molecules on their plasma membranes, including intercellular adhesion molecule-1 (ICAM-1) . The role of heparin in regulating endothelial cell gene expression is unclear . We thus have investigated the ability of heparin to regulate ICAM- gene expression by using flow cytometry and the ribonuclease protection assay with human umbilical vein and aortic endothelial cells cultured in growth medium supplemented with 90 {microg/ml heparin (heparin-sufficient, HS) or in growth medium without added heparin (heparin-deficient, HD) . We found that HD medium increased plasma membrane protein and mRNA for ICAM-1 but not for HLA-DR, even though both ICAM-1 and HLA-DR protein and mRNA were inducible by gamma interferon (IFN-gamma) . In addition, phorbol ester and IFN-gamma increased the expression of plasma membrane ICAM-1 or ICAM-1 and HLA-DR, respectively, more in HD medium than in HS medium . We found that the HD-mediated increase of ICAM- mRNA was reversible by the addition of heparin, and that the half-life of ICAM-1 mRNA was the same in both HS- and HD-treated cells . Also, heparin was found to suppress increases in ICAM-1 mRNA at a concentration as low as 5 microg/ml . These findings indicate that heparin deficiency induces endothelial activation characterized by increased ICAM-1, and that such induction is not dependent on cytokines or endotoxin . The modulation of ICAM-1 expression by heparin appears to occur at the transcriptional level . Thus, heparin may have a role in regulating endothelial function by affecting the expression of ICAM-1, thereby impacting upon the trans-endothelial trafficking of leukocytes.

Protein Expr Purif, 1998 Oct, 14(1), 13 - 22
TolAIII co-overexpression facilitates the recovery of periplasmic recombinant proteins into the growth medium of Escherichia coli; Wan EW et al.; Overproduction of the third topological domain of the transmembrane protein TolA (TolAIII) in the periplasm of Escherichia coli confers a "leaky" phenotype to host cells by disrupting the integrity of the outer membrane and causing periplasmic proteins to leach into the growth medium . To examine the physiological consequences of TolAIII overexpression in more detail and assess the usefulness of this strategy for the release of periplasmic recombinant proteins into the extracellular fluid, we constructed a ColE1-compatible plasmid encoding a fusion between the ribose binding protein signal sequence and TolAIII under T7lac transcriptional control . About half of the total TolAIII synthesized in IPTG-induced cells aggregated in a precursor form in the cytoplasm . However, the majority of the mature protein was soluble and located in the extracellular fluid . TolAIII-overproducing cultures exhibited only slight growth defects upon entry into stationary phase but underwent extensive lysis when treated with 0.1% (w/v) SDS, and were unable to divide when supplemented with 0.02% SDS . The loss of outer membrane integrity resulted in long-term damage since cell viability was reduced by three orders of magnitude compared to control or uninduced cells . Overexpression of TolAIII did not significantly interfere with the translocation and processing of a plasmid-encoded fusion between the OmpA signal sequence and TEM-beta-lactamase but led to the release of most periplasmic proteins and 90% of the active enzyme into the extracellular fluid . Although the total levels of beta-lactamase accumulation in TolAIII-overproducing cultures was only 1.5- to 2-fold less than in control cells, the formation of periplasmic inclusions bodies was completely suppressed . A threshold concentration of TolAIII was necessary for efficient release of periplasmic proteins since the viability and detergent sensitivity of uninduced cells was comparable to that of control cultures and 80% of the beta-lactamase synthesized remained confined to the periplasm .

J Biol Chem, 1998 Oct 9, 273(41), 27017 - 25
Structural requirements for in vivo myosin I function in Aspergillus nidulans; Osherov N et al.; We have investigated the minimal requirements of the tail region for myosin I function in vivo using the filamentous fungus Aspergillus nidulans . The CL3 strain (McGoldrick, C . A., Gruver, C., and May, G . S . (1995) J . Cell Biol . 128, 577-587) was transformed with a variety of myoA constructs containing mutations in the IQ, TH-1-like, SH3, and proline-rich domains by frameshift or in-frame deletions of the tail domains . The resulting strains contained wild type myoA driven by the alcA promoter and a mutant myoA driven by its endogenous promoter . This strategy allowed for selective expression of the wild type and/or mutant form of MYOA by the choice of growth medium . Proper septation and hyphal branching were found to be dependent on the interaction of the IQ motifs with calmodulin, as well as, the presence of its proline-rich domain . Additionally, a single proline-rich motif was sufficient for nearly wild type MYOA function . Most surprisingly, the SH3 domain was not essential for MYOA function . These studies expand our previous knowledge of the function of MYOA to include roles in hyphal morphogenesis, septal wall formation, and cell polarity, laying the groundwork for more detailed investigations on the function of the various tail domains in MYOA.

Eur J Orthod, 1998 Aug, 20(4), 357 - 67
Enhanced angiogenesis induced by diffusible angiogenic growth factors released from human dental pulp explants of orthodontically moved teeth; Derringer KA et al.; The aim of this study was to determine if diffusible angiogenic growth factors were released in human dental pulp during orthodontic tooth movement . These factors, if diffusible, could induce angiogenesis in other tissues, and may then be isolated and identified . The pulps from 14 premolar teeth treated with straight wire fixed orthodontic appliances for 2 weeks were compared with those of 14 untreated control premolar teeth from the same subjects . Following tooth extraction and sectioning, 1-mm horizontal sections of pulp tissue were embedded in collagen with 1-mm sections of rat aorta and co-cultured in growth media for up to 4 weeks . Sections of rat aorta alone were also cultured . Angiogenic changes in the form of microvessel growth were observed by light microscopy . Microvessel identification was confirmed by electron microscopy and by immunohistochemistry using staining for factor VIII-related antigen marker for endothelial cells . When compared at days 5, 10, and 14 of co-culture, the number of microvessels was significantly greater in the pulps from orthodontically moved teeth than in those from the control teeth . The number of rat aorta microvessels was also significantly greater when co-cultured with pulp from orthodontically moved teeth than with pulp from control teeth and when compared with control cultures of rat aorta alone . There were no significant differences in microvessel numbers between the rat aorta co-cultured with pulp from control teeth and control cultures of rat aorta alone . These results indicate an increase in angiogenic growth factors in the pulp of orthodontically moved teeth, and the enhanced response of the rat aorta when co-cultured with this pulp shows that these factors appear to be diffusible.

PDA J Pharm Sci Technol, 1998 Jul-Aug, 52(4), 165 - 9
Comparative mold and yeast recovery analysis (the effect of differing incubation temperature ranges and growth media); Marshall V et al.; Environmental monitoring methodology for recovering fungal organisms often dictates the use of a selective medium incubated at ambient temperatures (20-25 degrees C) for as many as seven days incubation to ensure reliable recovery . However, these methods (which must remain standardized for identification purposes) are not the only avenue environmental monitoring programs may follow . This study comparatively analyzed recovery rates of fungal organisms cultured on both a general purpose bacteriological nutrient medium (Tryptic Soy Agar supplemented with Lecithin and Polysorbate 80), and on a medium selective for the growth of yeasts and molds (Sabouraud Dextrose Agar) . The bacteriologic medium was incubated at elevated temperatures (30-35 degrees C) for 70-72 hours then transferred to ambient temperatures for another 70-72 hours incubation . The control case selective medium was incubated solely at 20-25 degrees C for 110-130 hours . Additionally, in a separate test the selective medium was incubated at the same temperature and time specifications as the bacteriologic medium to analyze recovery capabilities . Equivalent or better recovery was obtained for all test panel organisms of yeasts and molds using Tryptic Soy Agar supplemented with Lecithin and Polysorbate 80 incubated at 30-35 degrees C . Equivalent or better recovery was obtained for eight of the nine test panel organisms of yeasts and molds incubated at elevated temperatures on Sabouraud Dextrose Agar versus recovery on Agar versus recovery on Sabouraud Dextrose Agar incubated solely at 20-25 degrees C . No inhibition of growth was observed at the elevated temperature range of 30-35 degrees C . Three days incubation at elevated temperatures was a sufficient incubation period to detect the test organisms cultured on Tryptic Soy Agar supplemented with Lecithin and Polysorbate 80.

J Appl Microbiol, 1998 Aug, 85(2), 231 - 7
Oxygen sensitivity of heated cells of Escherichia coli O157:H7; Bromberg R et al.; Following defined heat treatments (55 degrees C for 100 min, 50 degrees C for 5 min, 61 degrees C for l min), a 6 decimal (6-D) reduction was obtained when cells of Escherichia coli O157:H7 were enumerated in aerobic growth medium . Part of this reduction (3-D) was due to thermal inactivation (as determined when cells were enumerated in anaerobic growth medium), and part (3-D) was due to the inability of sub-lethally heat-injured cells of E . coli O157:H7 to grow in the presence of oxygen . When held anaerobically, the injured cells regained their ability to grow in the presence of oxygen . Following heating at 59 degrees C for 5 min, repair took 4 h at 30 degrees C, 48 h at 20 degrees C, 95 h at 10 degrees C, but did not occur in 816 at 5 degrees C . Recovery from sub-lethal heat injury was not influenced by heat shock . These findings are relevant to the safety of minimally-heated foods.

J Bacteriol, 1998 Oct, 180(19), 5269 - 72
Growth medium-dependent regulation of Myxococcus xanthus fatty acid content is controlled by the esg locus; Bartholomeusz G et al.; We compared the cellular fatty acid profiles of Myxococcus xanthus cells grown in either a Casitone-based complex medium or a chemically defined medium . The cells grown in the complex medium had a much higher content of the abundant branched-chain fatty acid iso-15:0 and several other branched-chain species . The higher branched-chain fatty acid content of the cells grown in the complex medium was dependent on the esg locus, which encodes the E1alpha and E1beta components of a branched-chain keto acid dehydrogenase (BCKAD) multienzyme complex involved in branched-chain fatty acid biosynthesis . Cells grown in the complex medium were also found to have a higher level of esg transcription and more BCKAD enzyme activity than cells from the chemically defined medium . The level of esg transcription appears to be an important factor in the growth medium-dependent regulation of the M . xanthus branched-chain fatty acid content.

J Bacteriol, 1998 Oct, 180(19), 5218 - 26
Carbon-source-dependent expression of the PalkB promoter from the Pseudomonas oleovorans alkane degradation pathway; Yuste L et al.; Pseudomonas oleovorans GPo1 can metabolize medium-chain-length alkanes by means of an enzymatic system whose induction is regulated by the AlkS protein . In the presence of alkanes, AlkS activates the expression of promoter PalkB, from which most of the genes of the pathway are transcribed . In addition, expression of the first enzyme of the pathway, alkane hydroxylase, is known to be influenced by the carbon source present in the growth medium, indicating the existence of an additional overimposed level of regulation associating expression of the alk genes with the metabolic status of the cell . Reporter strains bearing PalkB-lacZ transcriptional fusions were constructed to analyze the influence of the carbon source on induction of the PalkB promoter by a nonmetabolizable inducer . Expression was most efficient when cells grew at the expense of citrate, decreasing significantly when the carbon source was lactate or succinate . When cells were grown in Luria-Bertani rich medium, PalkB was strongly down-regulated . This effect was partially relieved when multiple copies of the gene coding for the AlkS activator were present and was not observed when the promoter was moved to Escherichia coli, a heterologous genetic background . Possible mechanisms responsible for PalkB regulation are discussed.

Infect Immun, 1998 Oct, 66(10), 4777 - 82
Contact-dependent protein secretion in Porphyromonas gingivalis; Park Y et al.; Porphyromonas gingivalis can induce its uptake by host epithelial cells; however, the nature and role of the P . gingivalis molecules involved in this invasion process have yet to be determined . In this study, modulation of secreted P . gingivalis proteins following association with gingival epithelial cells was investigated . Western immunoblot analysis showed that contact with epithelial cells or epithelial cell growth media induces P . gingivalis 33277 to secrete several proteins with molecular masses between 35 and 95 kDa . Secretion of the Arg-gingipain and Lys-gingipain proteases was repressed under these conditions . The contact-induced secreted protein profile was altered in Arg-gingipain-deficient and Lys-gingipain-deficient mutants, indicating a possible role for these proteases in the secretion pathway . The P . gingivalis contact-dependent protein secretion pathway differs to some extent from type III protein secretion pathways in enteric pathogens, as a gene homologous to the invA family genes was not detected in P . gingivalis . The secreted proteins of P . gingivalis may play a role in the interactions of the organism with host cells.

Eur J Biochem, 1998 Aug 1, 255(3), 755 - 65
Effect of molybdate and tungstate on the biosynthesis of CO dehydrogenase and the molybdopterin cytosine-dinucleotide-type of molybdenum cofactor in Hydrogenophaga pseudoflava; Hanzelmann P et al.; The molybdenum-containing iron-sulfur flavoprotein CO dehydrogenase is expressed in a catalytically fully competent form during heterotrophic growth of the aerobic bacterium Hydrogenophaga pseudoflava with pyruvate plus CO . We have adopted these conditions for studying the effect of molybdate (Mo) and tungstate (W) on the biosynthesis of CO dehydrogenase and its molybdopterin (MPT) cytosine-dinucleotide-(MCD)-type molybdenum cofactor . W was taken up by the Mo transport system and, therefore, interfered with Mo transport in an antagonistic way . Depletion of Mo from the growth medium as well as inclusion of excess W both resulted in the absence of intracellular Mo and led to the biosynthesis of CO dehydrogenase species of proper L2M2S2 subunit structure that carried the two 2Fe:2S type-I and type-II centers and two FAD molecules . EPR, ultraviolet/visible and CD spectroscopies established the full functionality of the cofactors . Due to the absence of the Mo-MCD cofactor, the enzyme species were catalytically inactive . Unexpectedly, the following cytidine nucleotides were present in inactive CO dehydrogenase: CDP, dCDP, CMP, dCMP, CTP or dCTP . The sum of cytidine nucleotides was two/mol enzyme . The binding specificities of inactive CO dehydrogenase for cytidine nucleotides (oxy > deoxy; diphosphate > monophosphate > triphosphate), and the absence of MPT suggest that, in active CO dehydrogenase, the cytidine diphosphate moiety of Mo-MCD provides the strongest interactions with the protein and determines the specificity for the type of nucleotide . In H . pseudoflava, the biosynthesis of MPT (identified as form A) was independent of Mo . Mo was, however, strictly required for the conversion of MPT to MCD (identified as form-A-CMP) as well as the insertion of Mo-MCD into CO dehydrogenase . These data support a model for the involvement of Mo in the biosynthesis of the Mo-MCD cofactor and of fully functional CO dehydrogenase in which the synthesis and insertion of Mo-MCD require Mo, and protein synthesis including integration of the FeS-centers and FAD are independent of Mo.

J Pharmacol Exp Ther, 1998 Sep, 286(3), 1129 - 39
Pharmacological characterization of human m1 muscarinic acetylcholine receptors with double mutations at the junction of TM VI and the third extracellular domain; Huang XP et al.; A mutant human m5 receptor containing the mutations of Ser465 to Tyr and Thr466 to Pro showed constitutive activity . By replacing the equivalent Ser388 with Tyr and Thr389 with Pro, we created a mutant human m1 (Hm1) receptor with comparable double mutations . The mutant receptor, Hm1(Ser388Tyr, Thr389Pro), was stably expressed in A9 L cells and displayed enhanced responses to classical muscarinic agonists with significantly increased potencies . Choline, a normal component of growth media, showed an efficacy comparable to acetylcholine and carbachol at Hm1(Ser388Tyr, Thr389Pro) receptors . Methylcarbachol, a selective nicotinic agonist, exhibited partial agonist activity at human m1 wild-type receptors and full agonist activity at Hm1(Ser388Tyr, Thr389Pro) receptors . l-Hyoscyamine inhibited the activities of choline and methylcarbachol . Muscarinic antagonists displayed small reductions in binding affinities, although muscarinic agonists showed greatly increased binding affinities for Hm1(Ser388Tyr, Thr389Pro) receptors . All agonists, including choline and methylcarbachol, showed multiple affinity states at Hm1(Ser388Tyr, Thr389Pro) receptors in the absence of GppNHp . The high affinity binding sites for acetylcholine, arecoline and choline were shifted in the presence of GppNHp . These results suggest that Hm1(Ser388Tyr, Thr389Pro) is conformationally favorable for agonist binding and receptor activation.

J Cell Physiol, 1998 Oct, 177(1), 130 - 6
Possible involvement of p21/waf1 in the growth inhibition of HepG2 cells induced by hepatocyte growth factor; Shima N et al.; Hepatocyte growth factor (HGF) is a potent mitogen for a variety of cell types, but it is also known as an antimitogenic factor for several types of tumor cell lines . The biological processes by which HGF inhibits tumor cell growth remain poorly understood . Here we report a comparative study of HGF-mediated signal transduction events between two opposite responding types of human hepatoblastoma cell lines, HuH6 and HepG2 . Following serum starvation, both cell lines were cultured in hepatocyte growth medium (HGM), a chemically defined medium, in the presence or absence of HGF . Under these culture conditions, cell growth in HuH6 was promoted by HGF, while it was inhibited in HepG2 . Phosphorylation of p42/mitogen-activated protein (MAP) kinase was observed within 10 min after HGF stimulation in both cell lines . The level of phosphorylated MAP kinase in HuH6 declined to basal levels after 2 hr . However, in HepG2 the phosphorylated form was detectable at 6 hr . p21/waf1 was induced in both cell lines where levels peaked 4-6 hr after HGF stimulation . In HuH6, a marked decrease of p21/waf1 was observed at 8-12 hr, while a high level of p21/waf1 was sustained for at least 24 hr in HepG2 . HGF treatment depressed cdk2 activity in a time-dependent manner in HepG2 while the activity increased in HuH6 . When serum-starved HepG2 was growth stimulated with serum in the presence or absence of HGF, the cells treated with HGF underwent growth inhibition correlating with a sustained induction of p21/waf1 and a decrease of cdk2 activity . Immunoprecipitation analysis revealed accumulation of cdk2-associated p21/waf1 in the HGF-treated HepG2 . Together, the results suggest that sustained induction of p21/waf1 mediates growth inhibition in HepG2 in the presence of HGF.

Appl Environ Microbiol, 1998 Sep, 64(9), 3305 - 12
A new intermediate in the mineralization of 3,4-dichloroaniline by the white rot fungus Phanerochaete chrysosporium; Sandermann H Jr et al.; Phanerochaete chrysosporium ATCC 34541 has been reported to be unable to mineralize 3,4-dichloroaniline (DCA) . However, high mineralization is now shown to occur when a fermentation temperature of 37 degrees and gassing with oxygen are used . Mineralization did not correlate with lignin peroxidase activity . The latter was high under C limitation and low under N limitation, whereas the reverse was true for mineralization . The kinetics of DCA metabolism was studied in low-N and low-C and C- and N-rich culture media by metabolite analysis and 14CO2 determination . In all cases, DCA disappeared within 2 days, and a novel highly polar conjugate termed DCAX accumulated in the growth medium . This metabolite was a dead-end product under C and N enrichment . In oxygenated low-C medium and in much higher yield in oxygenated low-N medium, DCAX was converted to DCA-succinimide and then mineralized . DCAX was purified by high-performance liquid chromatography and identified as N-(3,4-dichlorophenyl)-alpha-ketoglutaryl-delta-amide by high-performance liquid chromatography and mass spectroscopy, gas chromatography and mass spectroscopy, and nuclear magnetic resonance spectroscopy . The formation of conjugate intermediates is proposed to facilitate mineralization because the sensitive amino group of DCA needs protection so that ring cleavage rather than oligomerization can occur.

Protein Eng, 1998 Jun, 11(6), 489 - 94
Selection of cadmium specific hexapeptides and their expression as OmpA fusion proteins in Escherichia coli; Mejare M et al.; In searching for novel peptides with affinity for cadmium, the phage display technique was utilized . In the selection procedure, cadmium ions were immobilized on a metal chelating Sepharose gel . The peptides selected from a hexapeptide library showed no homology to naturally occurring metallothioneins . From the phage clones selected in the biopanning process, phages with affinity for Cd-109 in free solution were identified . The peptide His-Ser-Gln-Lys-Val-Phe, which was found to exhibit the strongest relative affinity for Cd-109, was cloned into Escherichia coli as a fusion to the cell surface exposed area of the outer membrane protein OmpA . Escherichia coli cells expressing this peptide showed increased survival in growth media containing up to 1.2 mM CdCl2 when compared with cells not expressing this peptide on their surface.

J Biol Chem, 1998 Sep 4, 273(36), 23274 - 82
Spectral and kinetic properties of the Fet3 protein from Saccharomyces cerevisiae, a multinuclear copper ferroxidase enzyme; Hassett RF et al.; High affinity iron uptake in Saccharomyces cerevisiae requires Fet3p . Fet3p is proposed to facilitate iron uptake by catalyzing the oxidation of Fe(II) to Fe(III) by O2; in this model, Fe(III) is the substrate for the iron permease, encoded by FTR1 . Here, a recombinant Fet3p has been produced in yeast that, lacking the C-terminal membrane-spanning domain, is secreted directly into the growth medium . Solutions of this Fet3p at >1 mg/ml have the characteristic blue color of a type 1 Cu(II)-containing protein, consistent with the sequence homology that placed this protein in the class of multinuclear copper oxidases that includes ceruloplasmin . Fet3p has an intense absorption at 607 nm (epsilon = 5500 M-1 cm-1) due to this type 1 Cu(II) and a shoulder in the near UV at 330 nm (epsilon = 5000 M-1 cm-1) characteristic of a type 3 binuclear Cu(II) cluster . The EPR spectrum of this Fet3p showed the presence of one type 1 Cu(II) and one type 2 Cu(II) (A parallel = 91 and 190 x 10(-4) cm-1, respectively) . Copper analysis showed this protein to have 3.85 g atom copper/mol, consistent with the presence of one each of the three types of Cu(II) sites found in multinuclear copper oxidases . N-terminal analysis demonstrated that cleavage of a signal peptide occurred after Ala-21 in the primary translation product . Mass spectral and carbohydrate analysis of the protein following Endo H treatment indicated that the preparation was still 15% (w/w) carbohydrate, probably O-linked . Kinetic analysis of the in vitro ferroxidase reaction catalyzed by this soluble Fet3p yielded precise kinetic constants . The Km values for Fe(II) and O2 were 4.8 and 1.3 microM, respectively, while kcat values for Fe(II) and O2 turnover were 9.5 and 2.3 min-1, consistent with an Fe(II):O2 reaction stoichiometry of 4:1.

Eur J Obstet Gynecol Reprod Biol, 1998 Aug, 79(2), 205 - 10
Morphological differentiation of cytotrophoblasts cultured in Medium 199 and in keratinocyte growth medium; Starreveld JS et al.; In culture, cytotrophoblast cells differentiate biochemically as well as morphologically into syncytiotrophoblast-like structures . Morphological and biochemical differentiation can be affected by the composition of the culture medium . The aim of this study was to analyze the morphological differentiation (syncytium formation) of cytotrophoblasts cultured in Medium 199 (M199) and keratinocyte growth medium (KGM) . Term human cytotrophoblast cells were cultured in either M199 or KGM with daily refreshment of the media . Both media induced biochemical differentiation, as monitored by measuring hCG secretion . Syncytium formation was visualized by immunocytochemical staining of desmosomes (cell membranes) . Cytotrophoblasts rapidly formed aggregates; however, single cells were seen throughout culture . Though the aggregates developed into syncytia, approximately 15% of the nuclei were still found in cell aggregates at the end of the culture period (4 days) . The final percentage of nuclei in syncytia (60-70%) did not differ between the culture media used . Syncytium formation occurred more rapidly in KGM medium . Approximately 50% of the nuclei were found in syncytia after 40 and 50 h in KGM and M199, respectively . The number of nuclei per syncytium was slightly higher in M199, but the average surface area of the syncytia was larger in KGM cultured cells (162-132 mm2) . These differences did not reach significance . We conclude that there is no major difference in morphological differentiation between cytotrophoblast cells in KGM or M199 . Moreover, both media sustain equal rates of hCG secretion.

Hokkaido Igaku Zasshi, 1998 May, 73(3), 239 - 52
{Studies on bovine leukemia virus envelope glycoprotein gp30 YXXL sequences in virus infection and fusogenic activity}; Inabe K; The bovine leukemia virus (BLV) envelope transmembrane protein (gp30) contains three YXXL sequences at its cytoplasmic tail . It is known that N-terminal two of these sequences participate in the induction of B cell activation when chimeric proteins in which cytoplasmic domain of CD8-alpha has been replaced with that of BLV gp30 are stimulated by anti-CD8-alpha antibody . In addition to such signal transduction activity, the two tyrosines in the YXXL sequences also appear to involve infection with high viral loads and their maintenance in the sheep experimentally infected with BLV . To analyze detailed biological relevance of these sequences in vitro, we constructed a full-length BLV-infectious molecular clone with two copies of long terminal repeats (LTRs), designated pBLV-IF, and then changed residues Y487, L490, Y498, L501 or Y487 plus Y498 in gp30 cytoplasmic tail to Ala by site-directed mutagenesis . Introduction of molecular clones of wild-type and mutants into COS-1 cells revealed that all mutated molecular clones synthesized matured envelope proteins and released virus particles into growth medium . Serial passages of transient transfectants with the molecular clones and cell-free inoculation resulted in the reduction of infectivity by mutation of Y498 and Y487 plus Y498 . In viral penetration, but not specific virus-cell binding, mutations of Y498 and Y487 plus Y498 substantially reduced the potential . Mutations of Y487, Y498 and Y487 plus Y498 increased syncytium-forming potential with increasing expression of envelope proteins on cell surface . In contrast, mutations of L490 and L501 affected neither infectivity nor syncytium-formation . Altogether, our data indicate that the YXXL sequences play a critical role during virus penetration and fusogenesity in the life cycle of BLV and regulate surface expression of envelope proteins.

Medicine (Baltimore), 1998 Jul, 77(4), 255 - 67
Nocardia bacteremia . Report of 4 cases and review of the literature; Kontoyiannis DP et al.; Bacteremic nocardiosis is reported rarely . We discuss 4 recent cases seen at our institution and 32 other cases described in the English literature . We found that patients with bacteremic nocardiosis were similar in presentation, risk factors, course, and therapeutic outcome to nonbacteremic patients with nocardiosis . The presence of endovascular foreign bodies appeared to be the only unique risk factor associated with bacteremic illness . Seeding of the central nervous system appeared to be relatively uncommon . Thirty percent of patients with nocardemia had concomitant bacteremia with other pathogens, mostly Gram-negative organisms . Nocardia grew in a variety of growth media, and the median incubation time to detection was 4 days . Fifty percent of patients with Nocardia bacteremia died . Positive blood cultures were a preterminal finding in the fatal, acute cases and occurred relatively early in the subacute, nonfatal cases . Poor outcome seemed to correlate with acute onset of nocardiosis (duration less than 1 month), late identification of nocardemia, involvement of more than 2 sites, and the lack of treatment with a sulfonamide-containing regimen.

J Food Prot, 1998 Aug, 61(8), 934 - 8
Evaluation of an enzyme-linked immunosorbent assay, direct immunofluorescent filter technique, and multiplex polymerase chain reaction for detection of Escherichia coli O157:H7 seeded in beef carcass wash water; Fratamico PM et al.; In commercial beef processing, carcasses are customarily washed with water to remove physical and microbial contamination . Assaying the water that is shed from the carcasses after washing is a convenient method to determine whether the carcass is contaminated with Escherichia coli O157:H7 or other bacterial pathogens . E . coli O157:H7 was inoculated into carcass wash water at various levels and the bacteria were then concentrated by filtration . After collection of bacteria in the filter units, the nylon membranes were cut out and placed in tubes containing growth medium, and the tubes were mixed vigorously to dislodge the bacteria from the membranes . Prior to enrichment, samples were removed for testing by a multiplex polymerase chain reaction (PCR) and a direct immunofluorescent filter technique (DIFT) . The remaining samples were subjected to 4-h enrichment culturing at 37 degrees C, after which aliquots were removed for testing by multiplex PCR, DIFT, and an enzyme-linked immunosorbent assay (ELISA) . Following 4-h enrichment culturing, E . coli O157:H7 was detected in wash water samples initially inoculated with ca . 100, 0.1, and 1 CFU/ml by ELISA, DIFT, and multiplex PCR, respectively . Testing of the wash water using the ELISA and the DIFT can be accomplished in less than 8 h . On the basis of these results, assaying carcass wash water by ELISA, DIFT, or multiplex PCR can be useful for detection of E . coli O157:H7 beef carcass contamination and can potentially be employed to identify carcasses for further processing to inactivate the organism.

J Mol Cell Cardiol, 1998 Jul, 30(7), 1449 - 55
Regulation of Kv4.2 and Kv1.4 K+ channel expression by myocardial hypertrophic factors in cultured newborn rat ventricular cells; Guo W et al.; Postnatal development and myocardial hypertrophy are associated with alterations in cardiac voltage-gated K+ channels . To investigate mechanisms underlying this K+ channel remodeling, expression of Kv4.2 and Kv1.4 K+ channel alpha-subunits was examined in cultured newborn rat ventricular myocytes by Western blot analysis using polyclonal antibodies against each of the subunits . At day 5 of cell culture, Kv1.4 protein was expressed at higher level than Kv4.2; as the age of culture progressed, Kv1.4 was significantly diminished while Kv4.2 increased with time in culture and became the predominant K+ channel protein . Such K+ channel isoform switch from Kv1.4 to Kv4.2 resembles that of the development in vivo . A 72-h treatment with exogenous triiodothyronine (T3, 0.1 microM) to cultured neonatal myocytes enhanced the expression of Kv4.2 by 73% and decreased the Kv1.4 expression by 22% . The effects of T3 were associated with an increase in the protein-to-DNA ratio indicating myocyte hypertrophy . On the other hand, a 72-h treatment with cardiac non-myocyte cell (NMC)-conditioned growth medium (NCGM) or phenylephrine (20 microM) induced similar cell hypertrophy, but in sharp contrast to T3, both markedly suppressed the Kv4.2 channel protein level . In addition, the trophic and the Kv4.2-downregulating effects of NCGM could be mimicked by exogenous endothelin-1 (0.1 microM), a paracrine factor secreted from cardiac NMCs . Our observations for the first time suggest that cardiac Kv4.2 and Kv1.4 K+ channel alpha-subunits are differentially regulated by a variety of myocardial hypertrophic factors . That T3 accelerated the developmental K+ channel isoform switch from Kv1.4 to Kv4.2 in vitro indicates the critical importance of thyroid hormone in postnatal K+ channel remodeling . Cardiac NMCs and alpha-adrenoceptor activation may contribute to the reduced outward K+ channel density in hypertrophied cardiomyocytes.

Int J Food Microbiol, 1998 Jun 30, 42(1-2), 63 - 9
Enhancement of bacteriocin production by Carnobacterium divergens AS7 in the presence of a bacteriocin-sensitive strain Carnobacterium piscicola; Sip A et al.; The effect of Carnobacterium piscicola in the growth medium of Carnobacterium divergens on divercin production was studied . C . piscicola cultures were added in the form of living cultures, thermally inactivated cultures and pretreated autolyzed cultures . Each form was applied as whole culture comprising growth medium with cells, culture supernatants and cell pellets . It was found that the divercin-sensitive bacterium enhanced significantly the divercin production by C . divergens . The highest stimulating effect was shown by C . piscicola culture autoclaved at 121 degrees C . It enhanced the divercin activity about 64-times compared to the control . The nonautolyzed cultures stimulated divercin biosynthesis to a greater extent than autolyzed cultures, independent of the culture pretreatment . The form of addition was the main external factor affecting divercin production . The possible biochemical mechanisms involved in this enhancement of production are examined.

Biochem Biophys Res Commun, 1998 Jul 30, 248(3), 458 - 63
Expression of Bcl-2 increases intracellular glutathione by inhibiting methionine-dependent GSH efflux; Meredith MJ et al.; Overexpression of Bcl-2 and related anti-apoptotic gene products has been shown to increase the intracellular concentration of the antioxidant tripeptide glutathione in neuronal and hematopoietic cells . A similar examination of HeLa cells that stably overexpress Bcl-2 (Bcl-2/HeLa) demonstrated that the reduced form of glutathione (GSH) was increased by 60% compared to control cells (80 nmol GSH/mg protein compared to 50 nmol GSH/mg) . Expression of gamma-glutamylcysteine synthetase, the rate limiting enzyme for glutathione synthesis was found to be independent of Bcl-2 overexpression, as determined by Northern blot analysis and immunoprecipitation of {35-S}-labeled enzyme . Bcl-2 overexpression did not alter the rate of GSH biosynthesis, measured under steady state conditions . Thus, the increase in GSH concentration was not the result of increased synthesis . Two activities have been described which govern efflux of reduced glutathione (GSH), RsGshT known as the sinusoidal transporter and RcGshT, known as the canalicular transporter . Both are low affinity, bidirectional, ATP and Na-independent . Consistent with expression of sinusoidal activity, DTT was found to stimulate GSH efflux while the amino acid methionine inhibited efflux in both HeLa and Bcl-2/HeLa cells . However, methionine-dependent inhibition of efflux was found to be significantly increased by expression of Bcl-2 . To test the prediction that the increase in GSH observed in Bcl-2/HeLa cells was mediated by methionine; Bcl-2/HeLa cells were cultured for 24 hrs in methionine-free growth medium . Under these conditions, the GSH concentration of the Bcl-2/HeLa cells dropped to the level observed in HeLa cells (50 nmol GSH/mg protein) . These studies suggest that overexpression of Bcl-2 increases GSH levels by altering methionine-dependent GSH efflux, an activity associated in HeLa cells with expression of the RsGshT transporter.

Vopr Med Khim, 1998 May-Jun, 44(3), 229 - 40
{Comparative evaluation of the cytotoxic effect of hydrogen peroxide and tumor necrosis factor alpha on nonischemic and ischemic endothelial cells}; Bilenko MV et al.; We studied cytotoxic effects (CTE) induced in confluent cultures of human umbilical vein endothelial cells (HUVEC) by initiators of free-radical reactions (FRR): H2O2 (10(-6)-10(-9) M), recombinant human tumor necrosis factor-{symbol; see text} (TNF-alpha, 0.05-100 ng/ml), and a combination of TNF-alpha with low-density lipoproteins (LDL, 100 microgram/ml) . HUVEC were incubated with these substances for 6 or 24 h in parallel tests performed under aerobic (CO2-incubator) and ischemic conditions (a mixture of 95% N2 + 5% CO2 in RPMI-1640 medium containing no substrate additives, growth factor or protein) . HUVEC viability was determined by counting cells adherent to the bottom of wells after 24 h of reincubation under aerobic conditions in the growth medium (Plating Efficiency Index) . The data showed that: 1) CTE of these compounds were dose-dependent (H2O2 and TNF-alpha) and time-dependent (TNF-alpha); 2) CTE of FRR initiators and CTE of ischemia were synergistic, that is, their combination produced a greater decrease HUVEC viability than any substance examined or ischemia alone; 3) CTE of TNF-alpha observed in experiments in substrate-deficient, protein-free medium was considerably stronger than in the growth medium; 4) a combination of TNF-a and LDL caused a stronger CTE on HUVEC than either factor alone, and this synergism was more pronounced during incubation under ischemic conditions . Thus, the data indicate that FRR initiators and TNF-alpha + LDL particularly increase the severity of ischemic injuries of EC and therefore they can be factors which in hypercholesterolemic patiens predispose vascular wall to atherosclerosis.

J Cell Physiol, 1998 Sep, 176(3), 588 - 94
Riboflavin uptake by the human-derived liver cells Hep G2: mechanism and regulation; Said HM et al.; The water-soluble vitamin riboflavin (RF) plays a critical role in many metabolic reactions, and thus, is essential for normal cellular functions and growth . The liver plays a central role in normal RF metabolism and is the site of maximal utilization of the vitamin . The mechanism of liver uptake of RF has been studied in animals, but no information is available describing the mechanism of the vitamin uptake in the human situation and its cellular regulation . In this study, we used the human-derived liver cells Hep G2 as an in vitro model system to address these issues . Uptake of RF by Hep G2 cells was found to be temperature- and energy-dependent but Na+-independent in nature . Uptake seemed to involve a carrier-mediated process as indicated by the saturation as a function of substrate concentration (apparent Km 0.41 +/- 0.08 microM), and by the ability of the structural analogs lumiflavin and lumichrome to inhibit the uptake process {inhibition constant (K) of 1.84 and 6.32 microM, respectively} . RF uptake was energy dependent, and was inhibited by the -SH group blocker p-chloromercuriphenylsulfonate (p-CMPS) (Ki of 0.10 mM) . Specific modulators of intracellular protein kinase A (PKA)-, protein kinase C (PKC)-, and protein tyrosine kinase (PTK)-mediated pathways did not affect RF uptake by Hep G2 cells . On the other hand, specific inhibitors of Ca2+/calmodulin-mediated pathway significantly inhibited the uptake process; this effect seemed to be mediated through a decrease in the Vmax of the substrate uptake process . Maintaining Hep G2 cells in a RF-deficient growth medium was associated with a significant up-regulation in the substrate uptake; this effect was specific for RF and was mediated mainly by means of an increase in the Vmax of the uptake process . These results describe, for the first time, the mechanism and cellular regulation of RF uptake by a human-derived liver cellular preparation, and shows the involvement of a carrier-mediated system in the uptake process . Furthermore, the uptake process seems to be regulated by an intracellular Ca2+/calmodulin-mediated pathway and by extracellular substrate levels.

Plant Cell Physiol, 1998 Jun, 39(6), 660 - 4
Differential effects of 1-naphthaleneacetic acid, indole-3-acetic acid and 2,4-dichlorophenoxyacetic acid on the gravitropic response of roots in an auxin-resistant mutant of arabidopsis, aux1; Yamamoto M et al.; The agravitropic nature of root growth of an auxin-resistant mutant of Arabidopsis, aux1, was restored when the synthetic auxin 1-naphthaleneacetic acid (NAA) was added to the growth medium; aux1 roots were not resistant to NAA . Neither indole-3-acetic acid nor, 2,4-dichlorophenoxyacetic acid had the same effects as NAA . These differential effects of the three auxins on aux1 defects suggest that AUX1 may encode the auxin influx carrier according to the model proposed by Delbarre et al.

J Pharmacol Toxicol Methods, 1998 Mar, 39(2), 117 - 23
An in vitro test system for differentiation between antiproliferative and toxic effects in vascular smooth muscle cells; Herrmann C et al.; We describe an in vitro test system looking for four endpoints in vascular smooth muscle cells (SMC): toxicity (cell number), DNA synthesis, reversibility of effects, and specificity of effects for SMC . SMC cultures at a low cell density, either actively proliferating or arrested by serum starvation and stimulated with 10% or 2% fetal calf serum (FCS) in parallel to treatment, are subject to a 52-h treatment phase with a test compound . Cultures were labeled with bromodeoxyuridine during the last 4 h of the treatment phase . Thereafter, the cultures are divided into two groups . In the first group, directly following treatment with a test compound, the cell number of the cultures is determined indirectly by using the vital dye neutral red . Subsequently, in the same cultures DNA synthesis is measured with an antibody directed to bromodeoxyuridine . The second group of cultures is reincubated with normal growth medium without test drug for a further 24 h . This recovery period is followed by determination of cell number and DNA synthesis as described above . This procedure allows determination of reversibility of effects observed directly after treatment giving important information for differentiation between toxic and antiproliferative mechanisms resulting in the reduction of DNA synthesis.

Biotechnol Appl Biochem, 1998 Aug, 28 ( Pt 1), 47 - 54
Purification and characterization of the constitutive form of laccase from the basidiomycete Coriolus hirsutus and effect of inducers on laccase synthesis; Koroljova-Skorobogat'ko OV et al.; An isolate of Coriolus hirsutus constitutively expresses substantial amounts of extracellular laccase on a defined growth medium . The most efficient inducer of extracellular laccase synthesis was syringaldazine, which increased the enzyme yield by 1000% at a concentration of 0.11 microM . The constitutive form of the enzyme was purified 312-fold . Laccase from C . hirsutus, with an estimated molecular mass of 55 kDa and pI of 4.0, is a monomeric glycoprotein containing 12% carbohydrate consisting of mannose and N-acetylglucosamine . The laccase was found to contain 3.9-4.1 copper atoms per molecule . The absorption spectrum shows a maximum at 610 nm and a shoulder at 330 nm, which is typical of laccase possessing type 1 and type 3 copper atoms . The parameters of the first type of copper were determined by EPR as g perpendicular=2.046 and g parallel=2.200, A parallel=8.103 x 10(-3) cm-1 . Laccase was found to be a pH-stable and thermostable enzyme . With organic substrates it exhibits a pH optimum of 4.5, but with the inorganic substrate K4{Fe(CN)6} this decreased to 3.5 . The highest efficiency of catalysis was observed with sinapinic acid as the substrate . The kinetic constants kcat and Km of this reaction were 578 s-1 and 24 microM respectively . It was established that the kinetics of the assayed reaction shows a Ping Pong mechanism.

EMBO J, 1998 Aug 3, 17(15), 4370 - 8
Regulation of the Cln3-Cdc28 kinase by cAMP in Saccharomyces cerevisiae; Hall DD et al.; The yeast Saccharomyces cerevisiae grows at widely varying rates in different growth media . In order to maintain a relatively constant cell size, yeast cells must regulate the rate of progress through the cell cycle to match changes in growth rate, moving quickly through G1 in rich medium, and slowly in poor medium . We have examined connections between nutrients, and the expression and activity of Cln3-Cdc28 kinase that regulates the G1-S boundary of the cell cycle in yeast, a point referred to as Start . We find that Cln3 protein levels are highest in glucose and lower in poorer carbon sources . This regulation involves both transcriptional and post-transcriptional control . Although the Ras-cAMP pathway does not appear to affect CLN3 transcription, cAMP increases Cln3 protein levels and Cln3-Cdc28 kinase activity . This regulation requires untranslated regions of the CLN3 message, and can be explained by changes in protein synthesis rates caused by cAMP . A model for CLN3 regulation and function is presented in which CLN3 regulates G1 length in response to nutrients.

Arch Microbiol, 1998 Sep, 170(3), 141 - 6
Acetyl phosphate and the phosphorylation of OmpR are involved in the regulation of the cell division rate in Escherichia coli; Pruss BM; Carbon sources that can be converted to acetate were added to the growth medium of Escherichia coli wild-type cells . Cells responded with an increased cell division rate . The addition of acetate also caused a decreased synthesis of flagella . Mutants in phosphotransacetylase, which are incapable of synthesizing acetyl phosphate, and mutants in the osmoregulator OmpR divided at a lower rate than did wild-type cells . The mutants did not increase their cell division rate upon the addition of serine, as observed for wild-type cells . These data are consistent with the idea that the previously described effect of serine upon the cell division rate is mediated by acetyl phosphate and phosphorylation of OmpR.

Biol Trace Elem Res, 1998 Jun, 62(3), 135 - 53
Dietary flavonoids interact with trace metals and affect metallothionein level in human intestinal cells; Kuo SM et al.; Flavonoids are natural compounds found in food items of plant origin . The study examined systematically the interaction of structurally diverse dietary flavonoids with trace metal ions and the potential impact of dietary flavonoids on the function of intestinal cells . Spectrum analysis was first performed to determine flavonoid-metal interaction in the buffer . Among the flavonoids tested, genistein, biochanin-A, naringin, and naringenin did not interact with any metal ions tested . Members of the flavonol family, quercetin, rutin, kaempferol, flavanol, and catechin, were found to interact with Cu(II) and Fe(III) . On prolonged exposure, quercetin also interacted with Mn(II) . Quercetin at 1:1 ratio to Cu(II) completely blocked the Cu-dependent color formation from hematoxylin . When quercetin was added to the growth medium of cultured human intestinal cells, Caco-2, the level of metal binding antioxidant protein, metallothionein, decreased . The effect of quercetin on metallothionein was dose- and time-dependent . Genistein and biochanin A, on the contrary, increased the level of metallothionein . The interaction between dietary flavonoids and trace minerals and the effect of flavonoids on metallothionein level imply that flavonoids may affect metal homeostasis and cellular oxidative status in a structure-specific fashion.

Anal Chem, 1998 Jul 1, 70(13), 2704 - 9
Monitoring protein expression in whole bacterial cells with MALDI time-of-flight mass spectrometry; Easterling ML et al.; We report the application of matrix-assisted laser desorption ionization (MALDI) to monitor recombinant protein expression in whole bacteria . This technique is characterized by rapid sample preparation that provides analysis of samples extracted directly from the growth media in less than 10 min . The mass spectrometric method holds several advantages over gel electrophoresis, the conventional method for examining the protein content of cells . Comparisons between the two methods of analysis are presented in terms of increased speed, efficiency, resolution, and mass accuracy . Delayed extraction time-of-flight mass spectrometry identifies posttranslational modifications and other changes in the expected structure which are not recognized by gel electrophoresis . The utility of this method is demonstrated for proteins with molecular masses ranging from 5 to 50 kDa . Low molecular mass proteins (< 10 kDa) can be efficiently analyzed without any treatment of the bacterial broth prior to MALDI sample preparation . The MALDI analysis of higher molecular weight proteins shows enhanced sensitivity when the bacterial solutions are first sonicated.

In Vitro Cell Dev Biol Anim, 1998 Jun, 34(6), 482 - 5
Three-dimensional culture of hepatocytes in a continuously flowing medium; Takeshita K et al.; Rat hepatocytes were maintained on three-dimensional cultures on sponge discs kept in Spinner Baskets (New Brunswick Scientific Co., New Brunswick, NJ, USA) with continuously circulating serum-free hepatocyte growth medium (HGM) containing hepatocyte growth factor (HGF) and epidermal growth factor (EGF) . Hepatocytes were embedded in polyester sponge discs with a collagen gel at the concentration of 5 million cells/ml . Atmospheric gas containing 7% CO2 was directly bubbled into the medium . Agitation by the impeller created a continuous medium-flow through the packed hepatocytes . Comparison between identically prepared perfused and stationery cultures showed that hepatocytes in the perfused cultures maintain higher levels of DNA synthesis . These results demonstrate the value of perfusion systems and also show that hepatocytes can proliferate and maintain differentiation in three-dimensional culture environments.

Am J Trop Med Hyg, 1998 Jun, 58(6), 812 - 5
A single tissue culture system for the propagation of the agents of the human ehrlichioses; Heimer R et al.; Two newly emergent human diseases found in the United States, human monocytotropic ehrlichiosis (HME) and human granulocytotropic ehrlichiosis (HGE), are caused by pathogens of the genus Ehrlichia . The causative agent of HGE can be propagated in HL-60 human promyelocytic leukemia cells . Herein, we report the development of a method to propagate E . chaffeensis, the causative agent of HME, in HL-60 cells, thus providing a common system for the study of both species . The continuous propagation of E . chaffeensis requires the induction of HL-60 differentiation along the monocytic pathway toward phenotypically mature macrophages by the addition of 25-OH vitamin D3 to the growth medium.

J Bacteriol, 1998 Jul, 180(14), 3686 - 91
A periplasmic and extracellular c-type cytochrome of Geobacter sulfurreducens acts as a ferric iron reductase and as an electron carrier to other acceptors or to partner bacteria; Seeliger S et al.; An extracellular electron carrier excreted into the growth medium by cells of Geobacter sulfurreducens was identified as a c-type cytochrome . The cytochrome was found to be distributed in about equal amounts in the membrane fraction, the periplasmic space, and the surrounding medium during all phases of growth with acetate plus fumarate . It was isolated from periplasmic preparations and purified to homogeneity by cation-exchange chromatography, gel filtration, and hydrophobic interaction chromatography . The electrophoretically homogeneous cytochrome had a molecular mass of 9.57 +/- 0.02 kDa and exhibited in its reduced state absorption maxima at wavelengths of 552, 522, and 419 nm . The midpoint redox potential determined by redox titration was -0.167 V . With respect to molecular mass, redox properties, and molecular features, this cytochrome exhibited its highest similarity to the cytochromes c of Desulfovibrio salexigens and Desulfuromonas acetoxidans . The G . sulfurreducens cytochrome c reduced ferrihydrite (Fe(OH)3), Fe(III) nitrilotriacetic acid, Fe(III) citrate, and manganese dioxide at high rates . Elemental sulfur, anthraquinone disulfonate, and humic acids were reduced more slowly . G . sulfurreducens reduced the cytochrome with acetate as an electron donor and oxidized it with fumarate . Wolinella succinogenes was able to reduce externally provided cytochrome c of G . sulfurreducens with molecular hydrogen or formate as an electron donor and oxidized it with fumarate or nitrate as an electron acceptor . A coculture could be established in which G . sulfurreducens reduced the cytochrome with acetate, and the reduced cytochrome was reoxidized by W . succinogenes in the presence of nitrate . We conclude that this cytochrome can act as iron(III) reductase for electron transfer to insoluble iron hydroxides or to sulfur, manganese dioxide, or other oxidized compounds, and it can transfer electrons to partner bacteria.

J Biotechnol, 1998 Apr 15, 61(2), 95 - 108
Secretion in Escherichia coli and phage-display of recombinant insulin-like growth factor binding protein-2; Lucic MR et al.; Insulin-like growth factors (IGFs) promote cell growth and differentiation . Their actions are regulated by six different, but related, binding proteins (IGFBPs) . To investigate the molecular interactions between IGFs and IGFBPs, an Escherichia coli based production method and a phage display system has been developed . The cDNA for bovine IGFBP-2 was inserted between regions coding for the pelB signal sequence and geneIII product, g3p, of bacteriophage fd in a phagemid vector to generate pGF14 . The coding sequences of IGFBP-2 and g3p were separated by an amber stop codon and a flexible linker containing the cleavage recognition site for H64A subtilisin . Using this system in BL21, a non-supE strain lacking ompT, most product, approximately 4 mg 1(-1) of IGFBP-2, was obtained in the growth medium . The bacterially derived IGFBP-2 had a correct N-terminal sequence, molecular mass on SDS-PAGE and the same affinity for IGF-1 and IGF-II as IGFBP-2 from mammalian cells . In a supE strain of E . coli, IGFBP-2 was produced as an IGF-binding fusion to g3p . Procedures for display and approximately 10000 fold enrichment of IGFBP-2 bearing phage using adsorption to IGF-II coated microtitre plates were developed . Thus IGFBP-2 can be secreted in E . coli and displayed on filamentous phage . These can be selectively enriched by binding to immobilised IGF-II.

J Vasc Surg, 1998 Jun, 27(6), 1128 - 40
The differential effect of contrast agents on endothelial cell and smooth muscle cell growth in vitro; Sawmiller CJ et al.; PURPOSE: This study was designed to evaluate the effects of ionic and nonionic contrast agents on endothelial cell (EC) and smooth muscle cell (SMC) proliferation, and to determine the role of osmolality as the etiology of these effects . METHODS: Cultured bovine aorta EC and SMC were exposed to ionic (iothalamate meglumine) or nonionic (ioversol or iopamidol) contrast, or varying osmolar solutions of mannitol, for periods of 1, 3, 5, 10, or 20 minutes . Cells were then incubated in growth media at 37 degrees C and proliferation and structure were assessed 1, 3, 5, and 7 days later . RESULTS: Both EC and SMC showed decreased proliferation after brief exposure to both ionic and nonionic contrast . Proliferation was markedly decreased at 24 hours after exposure, and began to recover by day 3 after exposure . EC showed a significant decrease up to 7 days after exposure to ionic contrast (p < 0.03), whereas SMC showed a significant decrease up to 7 days after exposure to nonionic contrast (p < 0.001) . The decrease in proliferation was directly dependent on the length of exposure to the contrast and the concentration of the contrast . EC proliferation decreased in proportion to increasing osmolality of the test solution (p < 0.05) . SMC proliferation did not show a decrease proportional to osmolality . No change was observed in cell viability as assessed by LDH activity studies . After contrast exposure, bare areas with no cells present were noted in the previously confluent EC and SMC culture wells . Cell structure was altered immediately after exposure to contrast, with normal structure recovered by 24 hours after exposure . CONCLUSION: This study demonstrates that brief exposure to contrast agents injures EC and SMC, altering their structure and decreasing proliferation for up to 7 days in vitro . This response is both dose and time dependent . EC are more severely affected by ionic contrast, and SMC are more severely affected by nonionic contrast . EC injury appears to be mediated by the osmolar effect of the contrast, but the effects of contrast on SMC seem to be due to a different mechanism.

Am J Respir Cell Mol Biol, 1998 Jul, 19(1), 55 - 62
Enhanced expression of GM-CSF in differentiating eosinophils of atopic and atopic asthmatic subjects; Gauvreau GM et al.; Higher numbers of eosinophil/basophil colony-forming units (Eo/B CFU) are observed in blood of atopic individuals, and can be enhanced in atopic asthmatics by allergen-inhalation challenge . It is known that mature basophils and eosinophils synthesize cytokines relevant to allergic inflammation . To investigate the potential role of growth factors in allergic disease we examined the expression of the hemopoietic cytokines, granulocyte-macrophage colony-stimulating factor (GM-CSF) and interleukin (IL)-5, in differentiating Eo/B colony cells from normal and atopic individuals, and from atopic asthmatics before and after allergen-inhalation challenge . Peripheral blood was collected from two normal and 12 atopic individuals, and also from 25 atopic asthmatics before and 24 h after allergen challenge . Nonadherent mononuclear cells were isolated and grown in semisolid growth medium . Eo/B colonies were selected and cytospins were prepared for immunocytochemical analysis of colony cells . Eo/B colonies, especially carbol chromotrope 2R+ cells, selected at Days 10, 14, and 18 from atopic donors contained messenger RNA for GM-CSF by combined in situ reverse transcription-polymerase chain reaction and cytochemistry, and demonstrated time-dependent expression of GM-CSF by immunocytochemistry (P = 0.007) . Atopic individuals demonstrated a higher percentage of cells expressing GM-CSF than did normal subjects under all growth conditions when examined at Day 14 (P = 0 . 04) . Atopic asthmatics challenged with inhaled allergen who demonstrated a dual airway response, an increase in the number of blood eosinophils (P = 0.0001), and an increase in the number of Eo/B CFU (P = 0.02) also demonstrated a significant increase in the percentage of colony cells expressing immunostainable GM-CSF (P = 0 . 0009), but only a variable effect on those expressing IL-5, 24 h after allergen . These results suggest that GM-CSF expression by differentiating Eo/Bs may provide an additional stimulus in vivo to enhance Eo/B progenitor differentiation in atopic and asthmatic individuals, especially after allergen challenge . The concept of microenvironmental differentiation, where blood progenitor cells may aid in their own differentiation, is supported by these ex vivo findings.

Appl Microbiol Biotechnol, 1998 May, 49(5), 531 - 7
Recombinant bioprocess optimization for heterologous protein production using two-stage, cyclic fed-batch culture; Chang CC et al.; A two-stage, cyclic fed-batch bioprocess was designed, and its performance evaluated to improve rice alpha-amylase productivity by the yeast Yarrowia lipolytica SMY2 (MatA, ade1, ura3, xpr2), ATCC 201847, containing a replicative plasmid coding for a rice alpha-amlyase . Transcription of the recombinant gene is controlled by the XPR2 promoter . The first stage (or growth stage) was operated in the fed-batch mode, and the growth medium, designed to maintain a constant high cell density (i.e., 60 g/l), was fed according to a predetermined and preprogrammed optimal feed rate which, in turn, maintained the specific cell growth rate at an optimal value (i.e., 0.1 h-1) . Typically, when the volume in the first stage reached a preset value, a portion of culture broth (i.e., 55%) was transferred to the second stage (or production stage) . The remaining cells in the growth stage were then fed with fresh growth medium according to the bioprocess control strategy developed, while induction of alpha-amylase expression and its production was taking place in the second stage . The second stage was also operated in the fed-batch mode, and the production medium designed to maintain a constant high cell density and high productivity of heterologous protein was fed at a predetermined and preprogrammed rate, which maintained the specific cell growth rate at an optimal level . The volumetric alpha-amylase productivity achieved (1835 units l-1 h-1) from the two-stage, cyclic fed-batch culture process was twofold higher than that of the fed-batch culture process . The genetic stability of the recombinant strain and the design of optimal media for growth and production stages are also critically important to a successful implementation of the two-stage, cyclic fed-batch process for production of heterologous protein.

J Urol, 1998 Feb, 159(2), 559 - 62
Loss of fimbrial adhesion with the addition of Vaccinum macrocarpon to the growth medium of P-fimbriated Escherichia coli; Ahuja S et al.; PURPOSE: Vaccinium macrocarpon--the American cranberry--irreversibly inhibits the expression of P-fimbriae of E . coli . Further effects on the function and expression of P-fimbriae were studied by growing P-fimbriated E . coli in solid media laced with cranberry juice . METHODS: Cranberry concentrate at pH 7.0 was added to CFA medium to a final concentration of 25% . E . coli strains JR1 and DS17 were plated on this medium with a plain CFA control and incubated at 37C . Cultures were tested for ability to agglutinate P-receptor specific beads . Bacteria were washed in PBS and agglutination retested . Cultures were also replated on plain CFA agar and rechecked for their ability to agglutinate . Transmission electron micrographs were performed on positive control and test bacteria . RESULTS: For E . coli strain JR1, P-fimbrial agglutination was inhibited after the third plating . DS17 was fully inhibited after the second plating . Washing in PBS did not affect agglutination, but replating on CFA agar allowed agglutination to recur . Electron micrographic study of control populations confirmed fimbriae . Fully inhibited bacteria had a 100% reduction in expression of fimbriae . Additionally, inhibited bacteria showed cellular elongation . CONCLUSIONS: Cranberry juice irreversibly inhibits P-fimbriae . Electron micrographic evidence suggests that cranberry juice acts on the cell wall preventing proper attachment of the fimbrial subunits or as a genetic control preventing the expression of normal fimbrial subunits or both.

Appl Environ Microbiol, 1998 Jul 1, 64(7), 2539 - 44
Increase in Endogenous and Exogenous Cyclic AMP Levels Inhibits Sclerotial Development in Sclerotinia sclerotiorum; Rollins JA et al.; Growth and development of a wild-type Sclerotinia sclerotiorum isolate were examined in the presence of various pharmacological compounds to investigate signal transduction pathways that influence the development of sclerotia . Compounds known to increase endogenous cyclic AMP (cAMP) levels in other organisms by inhibiting phosphodiesterase activity (caffeine and 3-isobutyl-1-methyl xanthine) or by activating adenylate cyclase (NaF) reduced or eliminated sclerotial development in S . sclerotiorum . Growth in the presence of 5 mM caffeine correlated with increased levels of endogenous cAMP in mycelia . In addition, incorporation of cAMP into the growth medium decreased or eliminated the production of sclerotia in a concentration-dependent manner and increased the accumulation of oxalic acid . Inhibition of sclerotial development was cAMP specific, as exogenous cyclic GMP, AMP, and ATP did not influence sclerotial development . Transfer of developing cultures to cAMP-containing medium at successive time points demonstrated that cAMP inhibits development prior to or during sclerotial initiation . Together, these results indicate that cAMP plays a role in the early transition between mycelial growth and sclerotial development.

J Biol Chem, 1998 Jul 3, 273(27), 17192 - 8
Porcine spleen deoxyribonuclease II . Covalent structure, cDNA sequence, molecular cloning, and gene expression; Wang CC et al.; Porcine spleen DNase II, a lysosomal acid hydrolase, is a noncovalently linked alpha.beta heterodimer (Liao, T.-H . (1985) J . Biol . Chem . 260, 10708-10713) . The alpha subunit, after disulfide cleavage, yields two chains, alpha1 and alpha2 . The complete amino acid sequences of the alpha1, beta, and alpha2 chains were elucidated by protein sequencing, and the pairings of one interchain disulfide between alpha1 and alpha2 and of three intrachain disulfides in alpha2 were assigned . Six carbohydrate attachment sites, two in beta and four in alpha2, were detected by sugar analyses . The cDNA of DNase II was amplified using primers synthesized on the basis of the amino acid sequences determined . The amplified fragments shown to be a cDNA sequence of 1,292 bases . This cDNA sequence has an open reading frame encoding a 364-amino acid polypeptide containing a putative transmembrane peptide at the NH2-end, two small connecting peptides in the middle, and a peptide at the COOH terminus . These are evidently removed to form mature DNase II . Thus, all three chains in the sequence alpha1, beta, and alpha2 are coded by the same cDNA . When Chinese hamster ovary cells were transfected with a cloned plasmid with an inserted cDNA fragment encoding the entire reading frame, the expressed protein was released into the growth medium as an active form of DNase II.

Biochem Biophys Res Commun, 1998 Jun 18, 247(2), 379 - 82
p202 prevents apoptosis in murine AKR-2B fibroblasts; Koul D et al.; p202 is an interferon (IFN)-inducible, primarily nuclear, phosphoprotein (52-kDa) whose overexpression in transfected cells inhibits colony formation . p202 binds to the retinoblastoma tumor suppressor protein and two other members of the pocket family proteins (p107 and p130) . Moreover, overexpression of p202 in transfected cells inhibits the transcriptional activity of E2Fs (E2F-1/DP-1 and E2F-4/DP-1), p53, AP-1 c-Fos and c-Jun, NF-kappaB p50 and p65 . Here we demonstrate that inhibition of endogenous p202 production in murine AKR-2B fibroblasts did not result in an increase in cell proliferation . Instead, these cells exhibited increased susceptibility to apoptosis in response to decrease in serum concentrations in the growth medium . These observations are consistent with the notion that normal levels of p202 may be needed for the regulation of cell proliferation .

Microbiology, 1998 Jun, 144 ( Pt 6), 1575 - 81
Inducible chitinolytic system of Aspergillus fumigatus; Escott GM et al.; Incubation of Aspergillus fumigatus NCPF 2140 in growth medium containing 1% chitin as sole carbon source led to induction of specific extracellular chitinolytic activity of 1.5 mumol GlcNAc released min-1 (mg protein)-1 . The effect was repressed by the inclusion of GlcNAc in the medium, indicating regulation by a negative feedback mechanism . Extracellular chitinase activity was inhibited by allosamidin (IC50 0.12 microM) . Multiple chitinolytic enzymes were detected on zymograms of extracellular preparations; levels of individual enzymes induced were dependent upon whether cells were incubated with purified colloidal chitin or a crude preparation of crystalline chitin . A major, inducible, 45 kDa chitinase was purified using ammonium sulphate precipitation, chitin affinity chromatography and a novel procedure involving the electroelution of the enzyme from a substrate gel containing glycol chitin . The enzyme is a glycoprotein with endochitinase activity.

Microbiology, 1998 Jun, 144 ( Pt 6), 1557 - 63
Pleiotropic effects of potassium deficiency in a heterocystous, nitrogen-fixing cyanobacterium, Anabaena torulosa; Alahari A et al.; Omission of potassium from the growth medium caused multiple metabolic impairments and resulted in cessation of growth of the filamentous, heterocystous, nitrogen-fixing cyanobacterium Anabaena torulosa, during both diazotrophic and nitrogen-supplemented growth . Prominent defects observed during potassium deprivation were: (i) the loss of photosynthetic pigments, (ii) impairment of photosynthetic functions, (iii) reduced synthesis of dinitrogenase reductase (Fe-protein), (iv) inhibition of nitrogenase activity, and (v) specific qualitative modifications of protein synthesis leading to the repression of twelve polypeptides and synthesis and accumulation of nine novel polypeptides . The observed metabolic defects were reversible, and growth arrested under prolonged potassium deficiency was fully restored upon re-addition of potassium . Such pleiotropic effects of potassium deficiency demonstrate that apart from its well-known requirement for pH and turgor homeostasis, K+ plays other vital specific roles in cyanobacterial growth and metabolism.

Int J Cancer, 1998 Jul 3, 77(1), 94 - 100
ras mutation and platinum resistance in human ovarian carcinomas in vitro; Holford J et al.; A panel of 16 human ovarian carcinoma cell lines comprising cisplatin naive as well as those with acquired cisplatin resistance was studied to determine if there was a relationship between ras status and cisplatin sensitivity . From the ras expression studies alongside data produced by direct DNA sequencing, there was very little to suggest that ras overexpression or mutation plays a role in the cisplatin sensitivity of the panel of human ovarian carcinoma cell lines tested . A weak correlation (r2 = 0.53) was found between total Ras protein levels and resistance to cisplatin . No relationship was found between Kirsten-Ras protein levels and cisplatin sensitivity (r2 = 0.0) . Only one ras mutation (codon 13, Kirsten exon 1, glycine --> aspartate in the HX62 cell line) was observed in the cisplatin naive cell lines from the panel which comprised both cisplatin sensitive and resistant models . Of interest, however, was that the HX62 cell line was the most resistant to cisplatin . No ras mutations were found in those cell lines which had repeatedly been exposed, and acquired resistance, to cisplatin . The A2780 and CH1 human ovarian carcinoma cell lines were transfected with activated, mutant Harvey-ras and, as a result, were shown to display elevated MAP kinase phosphorylation in low serum concentration growth medium . No changes in cisplatin sensitivity were found following transfection with activated Harvey-ras in these 2 human ovarian carcinoma tumor cell models which, importantly, differed greatly in their expression of Bcl-2 . Therefore, when conducted under similar conditions to previously published studies, very little evidence was found to support Harvey-ras activation as a factor which can either sensitize or confer resistance to cisplatin in human ovarian carcinoma cell lines.

Exp Cell Res, 1998 Jun 15, 241(2), 445 - 57
Phenobarbital suppresses growth and accelerates restoration of differentiation markers of primary culture rat hepatocytes in the chemically defined hepatocyte growth medium containing hepatocyte growth factor and epidermal growth factor; Miyazaki M et al.; Phenobarbital (PB), a liver-tumor promoter, at a concentration of 3 mM dramatically inhibited the growth of adult rat hepatocytes in the chemically defined medium, HGM, with added hepatocyte growth factor (HGF) and epidermal growth factor (EGF) . In concurrence with these findings, PB down-regulated expression of the HGF receptor (c-met) and suppressed production of the autocrine growth factor transforming growth factor-alpha (TGF-alpha) . Furthermore, PB down-regulated expression of transcription factors associated with proliferation such as AP1 and NF-kappaB . In the presence of PB, hepatocytes remained morphologically differentiated and restoration of the expression of mature hepatocyte markers, such as albumin and cytochrome P450s (1A, 2B1/2, and 2E1), was accelerated after an initial phase of growth . Additionally, PB strongly suppressed expression of the mRNA for alpha-fetoprotein, a protein primarily expressed by fetal liver, and the accelerative effect of PB on restoration of mature hepatocyte markers showed a correlation with the up-regulation of the hepatocyte-enriched transcription factors HNF3 and HNF4 . When the effects of PB on various extracellular matrix proteins were examined, the data indicated that PB specifically suppressed laminin and fibronectin production by hepatocytes, suggesting an important role for these proteins in growing hepatocyte cultures .

Biochemistry, 1998 Jun 16, 37(24), 8754 - 63
Overproduction and characterization of a dimeric non-zinc glyoxalase I from Escherichia coli: evidence for optimal activation by nickel ions; Clugston SL et al.; The ubiquitous glyoxalase system converts toxic alpha-keto aldehydes into their corresponding nontoxic 2-hydroxycarboxylic acids, utilizing glutathione (GSH) as a cofactor . The first enzyme in this system, glyoxalase I (GlxI), catalyzes the isomerization of the hemithioacetal formed nonenzymatically between GSH and cytotoxic alpha-keto aldehydes . To study the Escherichia coli GlxI enzyme, the DNA encoding this protein, gloA, was isolated and incorporated into the plasmid pTTQ18 . Nucleotide sequencing of the gloA gene predicted a polypeptide of 135 amino acids and Mr of 14 919 . The gloA gene has been overexpressed in E . coli and shown to encode for GlxI . An effective two-step purification protocol was developed, yielding 150-200 mg of homogeneous protein per liter of culture . Electrospray mass spectrometry confirmed the monomeric weight of the purified protein, while gel filtration analysis indicated GlxI to be a homodimer of 30 kDa . Zinc, the natural metal ion found in the Homo sapiens and Saccharomyces cerevisiae GlxI, had no effect on the activity of E . coli GlxI . In contrast, the addition of NiCl2 to the growth medium or to purified E . coli apo-GlxI greatly enhanced the enzymatic activity . Inductively coupled plasma and atomic absorption analyses indicated binding of only one nickel ion per dimeric enzyme, suggesting only one functional active site in this homodimeric enzyme . In addition, the apoprotein regained maximal activity with one molar equivalence of nickel chloride, indicative of tight metal binding . The effects of pH on the kinetics of the nickel-activated enzyme were also studied . This is the first example of a non-zinc activated GlxI whose maximal activation is seen with Ni2+.

Biochemistry, 1998 Jun 16, 37(24), 8714 - 23
BglF, the sensor of the bgl system and the beta-glucosides permease of Escherichia coli: evidence for dimerization and intersubunit phosphotransfer; Chen Q et al.; The Escherichia coli BglF protein, also designated EIIbgl, is an enzyme II of the phosphoenolpyruvate-dependent phosphotransferase system (PTS) that catalyzes transport and phosphorylation of beta-glucosides . In addition, BglF has the ability, unusual for an EII, to regulate the activity of a transcriptional regulator, BglG, by phosphorylating and dephosphorylating it according to beta-glucoside availability . Together, BglF and BglG constitute a novel sensory system . The membrane-bound sensor, BglF, has two phosphorylation sites: site 1 accepts a phosphoryl group from HPr and delivers it to site 2; site 2 delivers the phosphoryl group either to beta-glucosides or to BglG . Here, we provide several lines of evidence for the dimerization of BglF and for the occurrence of productive intersubunit phosphotransfer within the BglF dimers . (1) Two inactive BglF mutant proteins, one lacking phosphorylation site 1 and the other lacking site 2, complement one another to allow beta-glucoside utilization by bglF strains . (2) The pairs of mutant proteins complement one another in regulating BglG activity as a transcriptional antiterminator in vivo . (3) Only when they are present in the same membrane preparation do the mutant protein pairs efficiently transfer the phosphoryl group from HPr to beta-glucosides and to BglG in vitro . (4) Gentle extraction of cellular proteins followed by SDS-PAGE reveals the existence of BglF homodimers . A portion of the phosphorylated form of BglF can also be extracted from the membrane as a dimer . Dimerization is mediated by the membrane-bound IICbgl domain, as indicated by the dimerization of IICbgl by itself and of BglF derivatives that contain this domain . Since dimers persist in the presence of a reducing agent, they are apparently not held together by disulfide bonds . Rather, BglF dimerization might involve hydrophobic interactions between residues in the membrane-spanning domain . In addition, we show that BglF dimerization is not modulated by beta-glucosides and is therefore not part of the mechanism that diverts the phosphoryl group away from BglG to the transported sugar upon addition of beta-glucosides to the growth medium.

Biotechnology (N Y), 1995 May, 13(5), 498 - 503
A system for production of commercial quantities of human lactoferrin: a broad spectrum natural antibiotic; Ward PP et al.; We previously reported the production of limited quantities of biologically active recombinant human lactoferrin in the filamentous fungus Aspergillus oryzae . In the present study, we report a modification of this production system combined with a classical strain improvement program that has enabled production of levels of recombinant human lactoferrin in excess of 2 g/l . The protein was expressed in Aspergillus awamori as a glucoamylase fusion polypeptide which was secreted into the growth medium and processed to mature human lactoferrin by an endogenous KEX-2 peptidase . The recombinant protein retains full biological activity in terms of its ability to bind iron and human enterocyte receptors . Furthermore, the recombinant protein functions as a potent broad spectrum antimicrobial protein.

J Biol Chem, 1998 Jun 19, 273(25), 15621 - 7
Cationic liposomes coated with polyethylene glycol as carriers for oligonucleotides; Meyer O et al.; Modification of liposome surface with polyethylene glycol was used to improve oligodeoxyribonucleotide (ODN) loading, stability of the resulting complexes, and specificity of cellular delivery of ODN by cationic liposomes . Liposomes composed of a cationic lipid (DOTAP, DOGS, DDAB), a neutral lipid (DOPE), and a phospholipid derivative of polyethylene glycol (PEG-PE) formed a complex with 18-mer phosphorothioate up to ODN/lipid molar ratio of 0.25 . The complexes showed intact vesicular structures similar to original liposomes and their size (100-130 nm) was unchanged after several weeks of storage, whereas complexes lacking PEG-PE showed progressive aggregation and/or precipitation . After exposure to human plasma, PEG-modified cationic liposomes retained over 60% of the originally bound ODN . PEG-coated complexes resulted in 4-13-fold enhancement of the ODN uptake by human breast cancer cells in serum-supplemented growth medium, relative to free ODN . Complexes containing conjugated anti-HER2 F(ab') fragments at the distal termini of PEG chains efficiently delivered ODN primarily into the cytoplasm and nuclei of HER2 overexpressing cancer cells and greatly enhanced the biological activity of antisense ODN . The development of PEG-modified cationic liposomes may lead to improved ODN potency in vivo.

J Biol Chem, 1998 May 15, 273(20), 12032 - 40
Cytochrome b558/566 from the archaeon Sulfolobus acidocaldarius . A novel highly glycosylated, membrane-bound b-type hemoprotein; Hettmann T et al.; In this study we re-examined the inducible cytochrome b558/566 from the archaeon Sulfolobus acidocaldarius (DSM 639), formerly thought to be a component of a terminal oxidase (Becker, M., and Schafer, G . (1991) FEBS Lett . 291, 331-335) . An improved purification method increased the yield of the protein and allowed more detailed investigations . Its molecular mass and heme content have been found to be 64,210 Da and 1 mol of heme/mol of protein, respectively . It is only detectable in cells grown at low oxygen tensions . The composition of the growth medium also exerts significant influence on the cytochrome b558/566 content of S . acidocaldarius membranes . The cytochrome exhibits an extremely high redox potential of +400 mV and shows no CO reactivity; a ligation other than a His/His-coordination of axial ligands appears likely . It turned out to be highly glycosylated (more than 20% of its molecular mass are sugar residues) and is probably exposed to the outer surface of the plasma membrane . The sugar moiety consists of several O-glycosidically linked mannoses and at least one N-glycosidically linked hexasaccharide comprising two glucoses, two mannoses, and two N-acetyl-glucosamines . The gene of the cytochrome (cbsA) has been sequenced, revealing an interesting predicted secondary structure with two putative alpha-helical membrane anchors flanking the majority of a mainly beta-pleated sheet structure containing unusually high amounts of serine and threonine . A second gene (cbsB) was found to be cotranscribed . The latter displays extreme hydrophobicity and is thought to form a functional unit with cytochrome b558/566 in vivo, although it did not copurify with the latter . Sequence comparisons show no similarity to any entry in data banks indicating that this cytochrome is indeed a novel kind of b-type hemoprotein . A cytochrome c analogous function in the pseudoperiplasmic space of S . acidocaldarius is discussed.

Exp Eye Res, 1998 May, 66(5), 521 - 9
Apoptosis and morphologic changes in drug-treated trabecular meshwork cells in vitro; Sibayan SA et al.; Using an in vitro culture system, we investigated whether bovine trabecular meshwork cells undergo apoptosis (programmed cell death) following exposure to anti-glaucoma medications (timolol, pilocarpine and epinephrine) and known inducers of apoptosis (5-fluorouracil, mitomycin-C and dexamethasone) . Third to fifth passage bovine trabecular meshwork cells were grown to confluence and incubated for 1-12 days in growth media with timolol (1-1000 microM), pilocarpine (15-15,000 microM), epinephrine (5-5000 microM), 5-fluorouracil (10-100 micrograms ml-1), mitomycin-C (0.01-100 micrograms ml-1) and dexamethasone (0.01-100 microM) . The cultures were evaluated for apoptosis by phase-contrast microscopy, transmission electron microscopy and in situ apoptosis labeling . 5-Fluorouracil (10-100 micrograms ml-1), mitomycin-C (0.1-100 micrograms ml-1) and epinephrine (500-5000 microM) induced apoptosis in a dose and time-dependent manner . Timolol, pilocarpine, and dexamethasone-treated specimens did not show evidence of apoptosis at any of the concentrations tested . Trabecular meshwork cells incubated in timolol (100-1000 microM) developed cytoplasmic granules, and specimens treated with pilocarpine (15,000 microM) developed cytoplasmic vacuoles . These granules and vacuoles have the appearance of secondary lysosomes . Dexamethasone-treated cells developed an increased number of mitochondria . This study suggests that the trabecular meshwork may undergo apoptosis following exposure to 5-fluorouracil, mitomycin-C and epinephrine . Timolol, pilocarpine and dexamethasone did not induce apoptosis . However, these drugs can incite characteristic morphologic changes in cultured trabecular meshwork cells.

Free Radic Biol Med, 1998 May, 24(7-8), 1193 - 201
Mechanism of regulation of 8-hydroxyguanine endonuclease by oxidative stress: roles of FNR, ArcA, and Fur; Lee HS et al.; We found previously that 8-hydroxyguanine (oh8Gua) endonuclease in E . coli is induced in response to oxidative stress in a fashion similar to the oxidative response of the Mn-superoxide dismutase (MnSOD) . In this study, attempts were made to identify the genes involved in the co-regulation of E . coli endonuclease and MnSOD (sodA) . oh8Gua nuclease is induced by molecular oxygen and a superoxide radical generator (paraquat) but not by H2O2, suggesting that the regulation of this endonuclease is dependent on SoxRS but independent of OxyR . This enzyme was induced by paraquat in all of the soxRS mutant strains used (soxR-, soxS- and soxRc), whereas glucose-6-phosphate dehydrogenase (a member of the soxRS regulon) showed the expected responses; therefore, this possibility was excluded . The presence of metal chelators in the growth medium caused the induction of this enzyme, and this induction was suppressed by the addition of Fe++ . Consistent with this finding, this enzyme was expressed under anaerobiosis in all of the mutant strains of fnr in particular, as well as fur, arcA, and combinations thereof . These findings suggest that the oxidative regulation of oh8Gua endonuclease is under control of fnr, fur, and arcA, where fnr plays a predominant role . The multiple involvement of regulatory genes as well as co-regulation with antioxidant enzyme will enhance the efficiency of cellular growth and survival in the aerobic environment.

J Biol Chem, 1998 May 8, 273(19), 11638 - 42
Regulation of phosphatidylglycerophosphate synthase levels in Saccharomyces cerevisiae; Shen H et al.; The PGS1 gene of Saccharomyces cerevisiae encodes phosphatidylglycerophosphate (PG-P) synthase . PG-P synthase activity is regulated by factors affecting mitochondrial development and through cross-pathway control by inositol . The molecular mechanism of this regulation was examined by using a reporter gene under control of the PGS1 gene promoter (PPGS1-lacZ) . Gene expression subject to carbon source regulation was monitored both at steady-state level and during the switch between different carbon sources . Cells grown in a non-fermentable carbon source had beta-galactosidase levels 3-fold higher than those grown in glucose . A shift from glucose to lactate rapidly raised the level of gene expression, whereas a shift back to glucose had the opposite effect . In either a pgs1 null mutant or a rho mutant grown in glucose, PPGS1-lacZ expression was 30-50% of the level in wild type cells . Addition of inositol to the growth medium resulted in a 2-3-fold reduction in gene expression in wild type cells . In ino2 and ino4 mutants, gene expression was greatly reduced and was not subject to inositol regulation consistent with inositol repression being dependent on the INO2 and INO4 regulatory genes . PPGS1-lacZ expression was elevated in a cds1 null mutant in the presence or absence of inositol, indicating that the capacity to synthesize CDP-diacylglycerol affects gene expression . Lack of cardiolipin synthesis (cls1 null mutant) had no effect on reporter gene expression.

Biotechnol Prog, 1998 Mar-Apr, 14(2), 275 - 8
Factors influencing parathion degradation by recombinant Escherichia coli with surface-expressed organophosphorus hydrolase; Kaneva I et al.; A unique approach for organophosphorus pesticides detoxification was developed previously by anchoring organophosphorus hydrolase (OPH) onto the surface of Escherichia coli with a tightly regulated tac promoter . The resulting recombinant cells degraded parathion very effectively without the diffusional limitation observed in cells expressing OPH intracellularly . However, the precise conditions for surface targeting or pesticide degradation were not fully understood . In this paper, several factors influencing parathion degradation were investigated . Production of active OPH onto the cell surface was highly host-specific; a high rate of parathion degradation was observed from strains JM105 and XL1-Blue, which regulated production of the OPH fusion very tightly . However, in the absence of ampicillin selection, plasmids were only favorably maintained in strain XL1-Blue . OPH activity was highly dependent on growth conditions . Optimal OPH activity was observed when cells were grown in Luria-Bertani (LB)-buffered medium at 37 degrees C . OPH activity was further improved by supplementing the growth medium with cobalt chloride, which favors the formation of the metal active center . The timing of cobalt addition also influenced parathion degradation . Maximum OPH activity was obtained by adding cobalt to induced cultures during the late stationary phase . The resulting cultures grown under the optimized conditions had an eight-fold increase in parathion degradation.

Atherosclerosis, 1998 Apr, 137(2), 277 - 89
Fibroblast growth factor-2-toxin induced cytotoxicity: differential sensitivity of co-cultured vascular smooth muscle cells and endothelial cells; Lin PH et al.; Recombinant FGF-2-SAP is a mitotoxin consisting of the plant-derived ribosome-inactivating toxin saporin (SAP) fused to basic fibroblast growth factor (FGF-2) . FGF-2-SAP targets and kills cells bearing upregulated FGF receptors . In vivo, FGF-2-SAP inhibits smooth muscle cell hyperplasia in models of restenosis . The present study examined the potential for a differential effect of FGF-2-SAP on canine vascular endothelial cells (EC) and smooth muscle cells (SMC) separately as well as in a novel co-culture model . Canine vascular SMC and EC cultures were established separately and made quiescent once cells reached 80% confluence . Following the release from growth arrest, both cell types were treated with FGF-2-SAP, or FGF-2, or SAP alone for 48 h . {3H}TdR incorporation was used to determine the growth response of SMC and EC . The co-culture system was created by plating canine vascular SMC and EC on either side of a microporous 13 microm thick polyester membrane insert . Both cell types were grown to 80% confluence and independently made quiescent . Following the release from growth arrest, cells were treated with FGF-2-SAP, or FGF-2, or SAP alone . Negative and positive control groups were untreated wells containing phosphate buffered saline and complete growth media, respectively . After 48 h, both {3H}TdR incorporation and total DNA content, by fluorometric measurement, were quantitated in SMC and EC independently . FGF-2-SAP showed a concentration-dependent cytotoxicity in both canine SMC and EC but cytotoxicity for EC required substantially higher concentrations . In co-cultured SMC, FGF-2-SAP significantly decreased both {3H}TdR uptake and total DNA content at 0.5, 5, 50, and 500 ng/ml (0.01-10 nM) compared to positive controls . In co-cultured EC, FGF-2-SAP decreased {3H}TdR uptake at 50 and 500 ng/ml and total DNA content at 500 ng/ml compared to positive controls . Neither SAP alone nor FGF-2 alone showed a significant effect on {3H}TdR uptake or DNA content of either SMC or EC . In this unique co-culture model, which better replicates the relationship between SMC and EC in vivo, we demonstrated a dose-response range of FGF-2-SAP at which both the proliferation and total cell number of SMC, but not EC, is significantly reduced . These data suggest that FGF-2-SAP may have therapeutic utility in inhibiting myointimal hyperplasia in the absence of a deleterious effect on regenerating endothelium following vascular reconstructions.

Neurotoxicology, 1998 Jun, 19(3), 413 - 20
Diminished blocking effect of acute lead exposure on high-threshold voltage-gated calcium currents in PC12 cells chronically exposed to the heavy metal; Hegg CC et al.; Rat pheochromocytoma (PC12) cells were grown in 0, 10, 25, or 50 microM lead-containing growth media for up to twelve weeks . High-threshold whole-cell calcium currents from these PC12 cells were recorded in lead-free recording media (control), then in 1 microM lead-containing recording media (acute challenge), and finally again in lead-free recording media (wash) . The acute lead challenge decreased calcium currents in all treatment groups (including 0 microM lead) . However, this blocking effect of acute lead application diminished with prolonged chronic exposure to 25 and 50 microM lead . Although the acute lead challenge mainly caused a decrease in calcium currents, in some chronically exposed PC12 cells increased calcium currents were recorded during the application of 1 microM lead acetate . In other chronically exposed PC12 cells, the acute lead challenge caused the peak of the current-voltage curve to shift from +10 mV to 0 mV . The number of cells exhibiting either an increase in calcium current or a shift in the current-voltage relationship following acute lead challenge increased with prolonged chronic exposure to the heavy metal . The time-dependent increase in calcium influx may be responsible for at least one manifestation of lead neurotoxicity.

Radiat Environ Biophys, 1998 Apr, 37(1), 63 - 7
Effects of melanin on high- and low- linear energy transfer (LET) radiation response of human epithelial cells; Grossi GF et al.; The search for effective radioprotectors is of major concern in the medical, military, environmental, and space sciences . Conventional radioprotectors are generally effective only during a single irradiation and display their radioprotective properties only at high, toxic concentrations . In addition, they reduce somatic radiation effects but are poorly efficient in protecting from hereditary stochastic radiation effects . In this respect, the pigment melanin merits attention . Experiments referring to potential melanin effects on the ionising radiation response have been carried out with different biological systems, both in vivo and in vitro . In this paper, we present results on the response to high- and low-linear energy transfer (LET) radiation of a human mammary epithelial cell line, H184B5 F5-1 M/10, supplemented by melanin . The incorporation of auto-oxidative (L-dopa) melanin was linear for concentrations from 3 to 10 micrograms/ml in the growth medium . Concentrations of up to 250 micrograms/ml did not significantly impair the cells proliferative ability . No significant protective effect of melanin on the survival of cultured cells after exposure to alpha-particles (130 keV/micron) or x-rays was observed.

J Bacteriol, 1998 Jun, 180(11), 3013 - 6
Analysis of cis-acting elements required for bfpA expression in enteropathogenic Escherichia coli; Bustamante VH et al.; bfpA expression in enteropathogenic Escherichia coli is regulated by growth medium, temperature, and ammonium concentration and requires the BfpT protein (also called PerA), a member of the AraC family of transcriptional activators . Site-directed and PCR random mutagenesis, as well as deletion analysis of the bfpA upstream regulatory region, supported assignment of the promoter elements and demonstrated that the cis-acting elements that mediate BfpT-dependent regulation of bfpA are located between positions -85 and -46 . Interestingly, this region shares 73% identity with a 40-bp-long AT-rich tract located upstream of the bfpT gene, which is essential for bfpT autoregulation.

J Bacteriol, 1998 Jun, 180(11), 2992 - 4
Determining the optimal thymidine concentration for growing Thy- Escherichia coli strains; Molina F et al.; Changes of thymidine concentration in the growth medium affect the chromosome replication time of Thy- strains without at the same time causing a detectable difference in the growth rate (R . H . Pritchard and A . Zaritsky, Nature 226:126-131, 1970) . Consequently, the optimal thymidine concentration cannot be determined by ascertaining which concentration produces the highest growth rate . Here we present a method for determining the optimal thymidine concentration of any Thy- Escherichia coli strain . Using this method, we found that the E . coli "wild-type" strain MG1655 has a partial Thy- phenotype.

Rev Med Chil, 1997 Oct, 125(10), 1157 - 64
{Antibiotic effect of wild Streptomyces strains isolated from Chilean soils}; Garcia-Quintana H et al.; BACKGROUND: The soils of the southern part of Chile, that are isolated, cold, humid, poorly oxygenated and with a low acidity, could contain new strains of antimicrobial producing Streptomyces . AIM: To demonstrate that the soil of the Southern region of Chile contains Streptomyces strains with antimicrobial activity towards pathogenic bacteria and fungi . MATERIAL AND METHODS: Two hundred fifty eight soil and sediment samples were collected from 148 places in Southern regions of Chile . They were cultured in Kuster-Williams growth media and the presence of Streptomyces was confirmed by microscopic examination and biochemical characterization . The antimicrobial activity against reference microorganisms of each wild strain was tested using the disk method . Among active Streptomyces strains, 38 with the higher activity were selected and tested against 142 clinical microorganisms . RESULTS: Seventy seven percent of soils were positive and 542 wild strains of Streptomyces were isolated; of these, 266 had antimicrobial activity . Fifty three percent of isolates had activity against S aureus 43% against B subtilis and 0.7% against E coli . Most Streptomyces were active against more than one organism . When there was activity against single organisms, these were mostly eucariotic, such as C albicans and T mentagrophytes . Among clinical microorganisms, 29% of S aureus strains were inhibited, while P aeruginosa, Alternaria sp, P vulgaris and Y enterocolitica strains were not inhibited . The most frequent Streptomyces morphotypes were those showing pigmented colonies with flexuous and spiral shaped chains of arthrospores . CONCLUSIONS: Soils of the Southern region of Chile allow the growth of abundant native strains of Streptomyces with a promising antimicrobial activity.

J Biol Chem, 1998 Apr 24, 273(17), 10313 - 6
Growth in iron-enriched medium partially compensates Escherichia coli for the lack of manganese and iron superoxide dismutase; Benov L et al.; Enrichment of the growth medium with iron partially relieves the phenotypic deficits imposed on Escherichia coli by lack of both manganese and iron superoxide dismutases . Thus iron supplementation increased the aerobic growth rate, decreased the leakage of sulfite, and diminished sensitivity toward paraquat . Iron supplementation increased the activities of several {4Fe-4S}-containing dehydratases, and this was seen even in the presence of 50 microg/ml of rifampicin, an amount which completely inhibited growth . Assessing the O-2 scavenging activity by means of lucigenin luminescence indicated that the iron-enriched sodAsodB cells had gained some means of eliminating O-2, which was not detectable as superoxide dismutase activity in cell extracts . It is noteworthy that iron-enriched cells were not more sensitive toward the lethality of H2O2 despite having the usual amount of catalase activity . This indicates that iron taken into the cells from the medium is not available for Fenton chemistry, but is available for reconstitution of iron-sulfur clusters . We suppose that oxidation of the {4Fe-4S} clusters of dehydratases by O-2 and their subsequent reductive reconstitution provides a mechanism for scavenging O-2 and that speeding this reductive reconstitution by iron enrichment both spared other targets from O-2 attack and maintained adequate levels of these enzymes to meet the metabolic needs of the cells.

Infect Immun, 1998 Jun, 66(6), 2845 - 53
Expression of two members of the pMGA gene family of Mycoplasma gallisepticum oscillates and is influenced by pMGA-specific antibodies; Markham PF et al.; Certain monoclonal antibodies and polyclonal antisera directed to pMGA, the major protein of Mycoplasma gallisepticum, were tested for the ability to influence the surface phenotype of the cell population which resulted from their inclusion in growth medium . The polyclonal antiserum and one monoclonal antibody (MAb 66) resulted in an alteration of surface phenotype; specifically, populations of cells grown either on plates or in broth cultures which contained these reagents ceased the expression of pMGA and instead expressed an antigenically unrelated new polypeptide (p82) . Upon the removal of antibody, the progeny of these cells regained pMGA expression and produced antigenically sectored colonies . The basis of this switch between pMGA+ and pMGA- states was shown to be transcriptional . The p82 polypeptide, the expression of which resulted from growth of cells in antibodies, was another member of the pMGA gene family and was located just downstream from the pMGA gene normally expressed by the M . gallisepticum cells used . Collectively the results of this work suggest that this organism has evolved an unusual means of altering the antigenic composition of its surface in response to antibodies or to other environmental cues.

EMBO J, 1998 Apr 15, 17(8), 2235 - 45
Cell wall integrity modulates RHO1 activity via the exchange factor ROM2; Bickle M et al.; The essential phosphatidylinositol kinase homologue TOR2 of Saccharomyces cerevisiae controls the actin cytoskeleton by activating a GTPase switch consisting of RHO1 (GTPase), ROM2 (GEF) and SAC7 (GAP) . We have identified two mutations, rot1-1 and rot2-1, that suppress the loss of TOR2 and are synthetic-lethal . The wild-type ROT1 and ROT2 genes and a multicopy suppressor, BIG1, were isolated by their ability to rescue the rot1-1 rot2-1 double mutant . ROT2 encodes glucosidase II, and ROT1 and BIG1 encode novel proteins . We present evidence that cell wall defects activate RHO1 . First, rot1, rot2, big1, cwh41, gas1 and fks1 mutations all confer cell wall defects and suppress tor2(ts) . Second, destabilizing the cell wall by supplementing the growth medium with 0.005% SDS also suppresses a tor2(ts) mutation . Third, disturbing the cell wall with SDS or a rot1, rot2, big1, cwh41, gas1 or fks1 mutation increases GDP/GTP exchange activity toward RHO1 . These results suggest that cell wall defects suppress a tor2 mutation by activating RHO1 independently of TOR2, thereby inducing TOR2-independent polarization of the actin cytoskeleton and cell wall synthesis . Activation of RHO1, a subunit of the cell wall synthesis enzyme glucan synthase, by a cell wall alteration would ensure that cell wall synthesis occurs only when and where needed . The mechanism of RHO1 activation by a cell wall alteration is via the exchange factor ROM2 and could be analogous to signalling by integrin receptors in mammalian cells.

Genes Chromosomes Cancer, 1998 Jun, 22(2), 145 - 51
Cytogenetic analysis shows that carcinosarcomas of the breast are of monoclonal origin; Teixeira MR et al.; Carcinosarcoma of the breast is a rare biphasic neoplasm composed of a carcinomatous component contiguous or admixed with a pleomorphic spindle cell component . The issues of the histogenesis and clonal composition of carcinosarcomas have long been debated . We present the first cytogenetic characterization of mammary carcinosarcomas by analysis of eight tumor samples from two patients with this disease . In the first case, the same karyotypically complex clone, as well as evidence of clonal evolution, was found in samples from three separate areas of the primary tumor . The analysis of one intramammary and one axillary lymph node metastasis from the same patient, both showing only the sarcomatous tumor component, also revealed the common complex stemline and one of the two sidelines found in the primary tumor . The carcinosarcoma of the second patient contained six complex but karyotypically related clones unevenly distributed among the three samples examined . From this case, cells belonging to the carcinomatous and sarcomatous tumor components were separated by differential sedimentation and culturing in specific growth media . Analysis of both fractions showed largely the same karyotype, although one of the subclones was restricted to the epithelial component . Our findings indicate that the epithelial and mesenchymal components of mammary carcinosarcomas are both part of the neoplastic parenchyma and that they have evolved from a single common stem cell, in agreement with the hypothesis that the tumors are of monoclonal origin.

Mol Cell Biol, 1998 May, 18(5), 2514 - 23
Dynamic regulation of copper uptake and detoxification genes in Saccharomyces cerevisiae; Pena MM et al.; The essential yet toxic nature of copper demands tight regulation of the copper homeostatic machinery to ensure that sufficient copper is present in the cell to drive essential biochemical processes yet prevent the accumulation to toxic levels . In Saccharomyces cerevisiae, the nutritional copper sensor Mac1p regulates the copper-dependent expression of the high affinity Cu(I) uptake genes CTR1, CTR3, and FRE1, while the toxic copper sensor Ace1p regulates the transcriptional activation of the detoxification genes CUP1, CRS5, and SOD1 in response to copper . In this study, we characterized the tandem regulation of the copper uptake and detoxification pathways in response to the chronic presence of elevated concentrations of copper ions in the growth medium . Upon addition of CuSO4, mRNA levels of CTR3 were rapidly reduced to eightfold the original basal level whereas the Ace1p-mediated transcriptional activation of CUP1 was rapid and potent but transient . CUP1 expression driven by an Ace1p DNA binding domain-herpes simplex virus VP16 transactivation domain fusion was also transient, demonstrating that this mode of regulation occurs via modulation of the Ace1p copper-activated DNA binding domain . In vivo dimethyl sulfate footprinting analysis of the CUP1 promoter demonstrated transient occupation of the metal response elements by Ace1p which paralleled CUP1 mRNA expression . Analysis of a Mac1p mutant, refractile for copper-dependent repression of the Cu(I) transport genes, showed an aberrant pattern of CUP1 expression and copper sensitivity . These studies (i) demonstrate that the nutritional and toxic copper metalloregulatory transcription factors Mac1p and Ace1p must sense and respond to copper ions in a dynamic fashion to appropriately regulate copper ion homeostasis and (ii) establish the requirement for a wild-type Mac1p for survival in the presence of toxic copper levels.

Exp Cell Res, 1998 May 1, 240(2), 340 - 8
The signals for starvation response are transduced through elevated {Ca2+}i in Dictyostelium cells; Tanaka Y et al.; The mechanism by which cells recognize starvation to allow subsequent cellular development was analyzed using Dictyostelium discoideum, with special emphasis on Ca2+ as a crucial signal transducer in intra- and intercellular communications . As was expected, the cytosolic Ca2+ concentration ({Ca2+}i) in aequorin-expressing cells (RHI76 derived from D . discoideum Ax-3) was temporarily increased, when 3-5 microM thapsigargin (Tg), a specific inhibitor of the Ca(2+)-ATPase, was added into the cells incubated in semistarvation medium (SS-medium: 1 vol of growth medium plus 7 vol either of 20 mM Na2/K-phosphate buffer (pH 6.2) or of Bonner's salt solution (BSS)) . Essentially the same result was obtained by the application of 5 microM nigericin (Ng), an acid ionophore to cells under the semistarved condition . Here it is of interest to note that in the SS-medium Tg and Ng are capable of enhancing cell differentiation as exemplified well by the earlier acquisition of chemotactic response to cAMP, possibly inducing the starvation response through the {Ca2+}i increase . From Western blot analysis of phosphotyrosine (pTyr)-containing proteins using anti-pTyr antibody, it was found that the pTyr-phosphorylation levels of 97-, 80-, and 45-kDa proteins increase specifically in response to starvation . Interestingly, Tg and Ng induced such a change of the 80-kDa protein in the cells incubated in the SS-medium . Taken together these results strongly suggest that the temporal increase of {Ca2+}i may be a matter of importance for signal transduction coupled with starvation response.

Mol Cell Biol, 1998 Jun, 18(6), 2514 - 23
Dynamic regulation of copper uptake and detoxification genes in saccharomyces cerevisiae
Pena MMO, Koch KA, Thiele DJ.
The essential yet toxic nature of copper demands tight regulation of the copper homeostatic machinery to ensure that sufficient copper is present in the cell to drive essential biochemical processes yet prevent the accumulation to toxic levels . In Saccharomyces cerevisiae, the nutritional copper sensor Mac1p regulates the copper-dependent expression of the high affinity Cu(I) uptake genes CTR1, CTR3, and FRE1, while the toxic copper sensor Ace1p regulates the transcriptional activation of the detoxification genes CUP1, CRS5, and SOD1 in response to copper . In this study, we characterized the tandem regulation of the copper uptake and detoxification pathways in response to the chronic presence of elevated concentrations of copper ions in the growth medium . Upon addition of CuSO4, mRNA levels of CTR3 were rapidly reduced to eightfold the original basal level whereas the Ace1p-mediated transcriptional activation of CUP1 was rapid and potent but transient . CUP1 expression driven by an Ace1p DNA binding domain-herpes simplex virus VP16 transactivation domain fusion was also transient, demonstrating that this mode of regulation occurs via modulation of the Ace1p copper-activated DNA binding domain . In vivo dimethyl sulfate footprinting analysis of the CUP1 promoter demonstrated transient occupation of the metal response elements by Ace1p which paralleled CUP1 mRNA expression . Analysis of a Mac1p mutant, refractile for copper-dependent repression of the Cu(I) transport genes, showed an aberrant pattern of CUP1 expression and copper sensitivity . These studies (i) demonstrate that the nutritional and toxic copper metalloregulatory transcription factors Mac1p and Ace1p must sense and respond to copper ions in a dynamic fashion to appropriately regulate copper ion homeostasis and (ii) establish the requirement for a wild-type Mac1p for survival in the presence of toxic copper levels.

Biochem Biophys Res Commun, 1998 Apr 28, 245(3), 804 - 9
Aquaporin-1: an osmoinducible water channel in cultured mIMCD-3 cells; Jenq W et al.; The expressions of aquaporin-1 (AQP-1) in cultured mIMCD-3 cells were studied . There was no detectable AQP-1 in cells grown in serum-containing growth medium (SM, 297 +/- 2 mOsm/kg . H2O) . When SM was supplemented with NaCl (406 +/- 2 mOsm/kg . H2O), cellular AQP-1 was induced . A further increase in medium osmolarity with NaCl (493 +/- 3 mOsm/kg . H2O) had conferred cells an 2.5 to 3-fold increase of AQP-1 expression over those grown in the 406 +/- 2 mOsm/kg . H2O medium . Moreover, AQP-1 was found to be translocated from cytosol to membrane . In addition, exposing the mIMCD-3 cells to vasopressin (AVP, 10(-8) M) and/or NaCl-supplemented serum-free media (496 +/- 3 mOsm/kg . H2O) for 6h did not render them to produce AQP-1 . However, AQP-1 was induced after 24h of incubation, with an 1.5-fold additive effect by AVP . Our RT-PCR data had confirmed the NaCl inducibility and AVP synergism in AQP-1 expression at both mRNA and protein levels . This suggests a new role for cellular AQP-1 and AVP in overcoming osmotic stress in an in vitro system.

Biochem Pharmacol, 1998 Mar 15, 55(6), 873 - 82
Agonist-induced desensitization of A2B adenosine receptors; Peters DM et al.; Agonist-induced desensitization has been described for the A1, A2A, and A3 adenosine receptor subtypes of the G protein-coupled receptor superfamily . Desensitization of the fourth adenosine receptor subtype, the A2B adenosine receptor (A(2B)R), has not been studied extensively . We sought to determine whether the A(2B)R is subject to agonist-induced desensitization . COS 7 cells, which exhibit endogenous expression of the A(2B)R, and transfected CHO cells, which stably express a modified rat A(2B)R bearing a 5' FLAG epitope tag, were studied . Cyclic AMP (cAMP) responsiveness to an acute challenge was measured after pretreating (desensitizing) cells with the adenosine receptor agonist 5'-N-ethylcarboxamidoadenosine (NECA) . Incubation with NECA resulted in hyporesponsiveness to acute agonist challenge in both COS 7 and transfected CHO cells . Desensitized cells exhibited restoration of cAMP responses after recovery for 24 hr in growth medium . Choleratoxin-induced cAMP responses were preserved in desensitized cells, and high concentrations of NECA were unable to overcome the desensitization . Membrane levels of the epitope-tagged A(2B)R were assessed by western blot in transiently transfected COS 7 cells . The expression of epitope-tagged A(2B)Rs was not different between control and desensitized cells . In northern blot analysis, levels of endogenous A(2B)R mRNA were similar in control and desensitized COS 7 cells . We conclude that the A(2B)R is subject to agonist-induced desensitization with preserved expression of A(2B)R mRNA and protein . Uncoupling of the A2B adenosine receptor from the G protein complex may contribute to the mechanism of desensitization.

Biochemistry, 1998 Apr 21, 37(16), 5344 - 8
Rational design of novel antimicrobials: blocking purine salvage in a parasitic protozoan; Somoza JR et al.; All parasitic protozoa obtain purine nucleotides solely by salvaging purine bases and/or nucleosides from their host . This observation suggests that inhibiting purine salvage may be a good way of killing these organisms . To explore this idea, we attempted to block the purine salvage pathway of the parasitic protozoan Tritrichomonas foetus . T . foetus is a good organism to study because its purine salvage depends primarily on a single enzyme, hypoxanthine-guanine-xanthine phosphoribosyltransferase (HGXPRTase), and could provide a good model for rational drug design through specific enzyme inhibition . Guided by the crystal structure of T . foetus HGXPRTase, we used structure-based drug design to identify several non-purine compounds that inhibited this enzyme without any detectable effect on human HGPRTase . One of these compounds, 4-{N-(3, 4-dichlorophenyl)carbamoyl}phthalic anhydride (referred to as TF1), was selected for further characterization . TF1 was shown to be a competitive inhibitor of T . foetus HGXPRTase with respect to both guanine (in the forward reaction; Ki = 13 microM) and GMP (in the reverse reaction; Ki = 10 microM), but showed no effect on the homologous human enzyme at concentrations of up to 1 mM . TF1 inhibited the in vitro growth of T . foetus with an EC50 of approximately 40 microM . This inhibitory effect was associated with a decrease in the incorporation of exogenous guanine into nucleic acids, and could be reversed by supplementing the growth medium with excess exogenous hypoxanthine or guanine . Thus, rationally targeting an essential enzyme in a parasitic organism has yielded specific enzyme inhibitors capable of suppressing that parasite's growth.

Arch Androl, 1998 May-Jun, 40(3), 175 - 80
Significance of antisperm antibodies in female serum in a gamete intrafallopian transfer program; Vandendael A et al.; The influence of antisperm antibodies in the female serum on fertilization and pregnancy rate in patients undergoing GIFT was assessed . A study group of 52 couples (69 cycles) with significant levels of antisperm antibodies in the female serum were compared to a control group of 749 couples (1185 cycles) . Maternal serum or donor serum was used as growth medium supplement . The TAT test was performed for the detection of antisperm antibodies in the serum . Antisperm-antibody presence in female serum was associated with similar fertilization and pregnancy rates in a GIFT program compared to the control group . The type of serum used as growth medium supplement did not affect statistically the fertilization or pregnancy rate.

Actas Urol Esp, 1998 Jan, 22(1), 11 - 6
{Histomorphometry study of the effect of an inhibitor of 5-alpha reductase on the coating of human prostate explants in tissue culture}; Lopez Munoz A et al.; Histomorphometric study of hyperplastic human prostate explants coating grown "in vitro" to evaluate the response to a 5-alpha-reductase inhibitor (finasteride) . Explants were grown for four weeks in three groups, one in growth medium alone (RPM1-1640), one supplemented with testosterone and one supplemented with testosterone plus finasteride . Explants underwent histological studies, quantifying the surface and thickness of the coating epithelium . Study of the values obtained from the different samples show significant differences (p < 0.001) for the coated surface and its thickness between groups supplemented with testosterone and testosterone + finasteride, and so it can be inferred that this compound (finasteride) would blockade basal cells proliferation and their migration on the explant surface.

FEMS Microbiol Lett, 1998 Mar 15, 160(2), 225 - 9
Osmolarity modulates the expression of the Hha protein from Escherichia coli; Mourino M et al.; The effect of the osmolarity of the culture medium on the expression of the hha gene of Escherichia coli was investigated . When cells were grown in LB medium, expression reached a maximum in the exponential phase of growth and decreased in the stationary phase . Increasing the osmolarity of the LB medium had no significant effect on the expression of the hha gene, but depletion of NaCl led to a significant decrease in expression . Expression of the hha gene is thus sensitive to the osmolarity of the growth medium . High levels of expression of the hha gene when cells are grown at medium to high osmolarity are consistent with the finding that the Hha protein appears to play its main modulatory role when cells grow under these conditions.

Am J Physiol, 1998 Apr, 274(4 Pt 1), C866 - 74
Taurine synthesis and cysteine metabolism in cultured rat astrocytes: effects of hyperosmotic exposure; Beetsch JW et al.; We investigated mechanisms controlling taurine synthesis in cultured rat cerebral astrocytes . The mean +/- SE rate of taurine synthesis from extracellular cysteine was 21.2 +/- 2.0 pmol.mg protein-1.min-1, whereas taurine degradation was < 1.3% of this rate . Eliminating cellular glutathione and inhibiting glutathione biosynthesis increased taurine synthesis from extracellular cysteine by 39% . In cell homogenates, cysteine dioxygenase (CDO) and cysteine-sulfinate decarboxylase activities were 2.4 +/- 0.2 and 8.3 +/- 2.8 nmol.mg protein-1.min-1, respectively . CDO activity was strongly dependent on cysteine concentration over physiological and pathophysiological ranges of intracellular cysteine concentration . Growth in hyperosmotic medium caused a greater increase in culture medium taurine content than that measured from cells in isosmotic growth medium . Hyperosmotic treatment transiently increased the rate of cysteine accumulation and cellular cysteine and glutathione contents but had no effect on the synthesis rate of taurine from extracellular cysteine . Thus cysteine is accumulated and then metabolized to taurine through CDO, whose activity depends on the intracellular cysteine concentration and appears to be rate limiting for taurine synthesis . Hyperosmotic exposure increases net taurine production yet has no effect on taurine synthesis from exogenously applied cysteine . Availability of substrate from intracellular pools must contribute to maintenance of high intracellular taurine during hyperosmotic exposure.

J Clin Microbiol, 1998 May, 36(5), 1232 - 5
Growth and survival of Helicobacter pylori in defined medium and susceptibility to Brij 78; Albertson N et al.; The gastrointestinal pathogen Helicobacter pylori requires supplementation with either fetal calf serum (FCS), bovine serum albumin (BSA), or (2,6-dimethyl)-beta-cyclodextrin (CD) for growth in a complex or defined medium . Because the availability of medium in which all components were chemically defined would facilitate metabolic studies of H . pylori, growth of the type strain, ATCC 43504, was compared in a defined medium with different growth additives . The dependency of H . pylori growth on FCS or BSA in a defined medium could partially be replaced by dependency on CD and cholesterol when the last two components were both added to the defined medium . Growth and cell yield were not affected by the addition of glucose, but the culture viability (numbers of CFU per milliliter was extended . Because therapeutic antifoams are used to relieve gastrointestinal symptoms we studied whether the unique susceptibility of H . pylori to the emulsifier polyoxyethylene-20-stearylether (Brij 78) was growth dependent or medium specific . The bactericidal activity exerted in buffer at pH 5 was independent of the preculture medium, and a 5-h exposure of the bacteria to 1.28 to 2.56 microg of Brij 78 per ml reduced the numbers of viable bacteria by >5 log10 . The MICs (0.16 to 0.32 microg/ml) were lower than the corresponding minimal bactericidal concentrations in different growth media and were affected by FCS or BSA . In conclusion, CD plus cholesterol promotes the growth of H . pylori in a serum-free defined medium in which glucose enhances cell viability . The antibacterial activity exerted by Brij 78 is neither growth dependent nor medium specific.

Pract Periodontics Aesthet Dent, 1997 Aug, 9(6), 683 - 90; quiz 692
Viral infections of the oral mucosa in children: a clinical review; Fenton SJ et al.; The oral cavity is a microcosm of the world around us, exposed to a variety of microorganisms present in the local environment . Some of these microorganisms establish a permanent presence in the oral tissues, which serve as a suitable growth medium . These locations include soft and hard tissue, areas of high and low oxygen content, flowing secretions and dryness, and flat or grooved surfaces . Most of the normal oral flora does not cause disease; some even provide a protective benefit . However, occasionally one or more groups become pathologic, producing a disease that may have serious consequences for the host . Many of the pathologic microorganisms are viruses, and children are particularly prone to such infections, since their immune systems are still in the development stage . The learning objective of this article is to review the viral infections of the oral mucosa in children, including varicella, herpes zoster, mononucleosis, and herpangina.

Oral Microbiol Immunol, 1997 Dec, 12(6), 345 - 9
Cytotoxic effects of short-chain carboxylic acids on human gingival epithelial cells; Zhang J et al.; Previous studies showed that foods that are retained on the dentition can accumulate high levels of short-chain carboxylic acids (acetic, formic, lactic and propionic) . Since gingival epithelium is the first periodontal tissue to be challenged by oral factors, a study was undertaken to determine whether short-chain carboxylic acids can affect epithelial cells in vitro . Immortalized human oral epithelial cells were grown in supplemented keratinocyte growth medium at 37 degrees C, and the effects of short-chain carboxylic acids were determined with tetrazolium-based and trypan blue exclusion assays . Low concentrations of short-chain carboxylic acids inhibited the growth of human oral epithelial cells, while higher concentrations led to cell death . The effects of short-chain carboxylic acids on the cells were dose-dependent and varied among the individual acids (propionate > formate > lactate > acetate) . Growth inhibition was partly reversible and growth resumed after removal of the acids . However, the time needed for recovery of the cells increased with short-chain carboxylic acids concentration, consistent with progressively greater damage to the cells at higher short-chain carboxylic acids concentrations . The observed effects of short-chain carboxylic acids on gingival cells in vitro supported our hypothesis that short-chain carboxylic acids can damage the integrity of gingival epithelium in situ.

J Biol Chem, 1998 Apr 3, 273(14), 7957 - 66
The particulate methane monooxygenase from methylococcus capsulatus (Bath) is a novel copper-containing three-subunit enzyme . Isolation and characterization; Nguyen HH et al.; The particulate methane monooxygenase (pMMO) is known to be very difficult to study mainly due to its unusual activity instability in vitro . By cultivating Methylococcus capsulatus (Bath) under methane stress conditions and high copper levels in the growth medium, membranes highly enriched in the pMMO with exceptionally stable activity can be isolated from these cells . Purified and active pMMO can be subsequently obtained from these membrane preparations using protocols in which an excess of reductants and anaerobic conditions were maintained during membrane solubilization by dodecyl beta-D-maltoside and purification by chromatography . The pMMO was found to be the major constituent in these membranes, constituting 60-80% of total membrane proteins . The dominant species of the pMMO was found to consist of three subunits, alpha, beta, and gamma, with an apparent molecular mass of 45, 26, and 23 kDa, respectively . A second species of the pMMO, a proteolytically processed version of the enzyme, was found to be composed of three subunits, alpha', beta, and gamma, with an apparent molecular mass of 35, 26, and 23 kDa, respectively . The alpha and alpha' subunits from these two forms of the pMMO contain identical N-terminal sequences . The gamma subunit, however, exhibits variation in its N-terminal sequence . The pMMO is a copper-containing protein only and shows a requirement for Cu(I) ions . Approximately 12-15 Cu ions per 94-kDa monomeric unit were observed . The pMMO is sensitive to dioxygen tension . On the basis of dioxygen sensitivity, three kinetically distinct forms of the enzyme can be distinguished . A slow but air-stable form, which is converted into a "pulsed" state upon direct exposure to atmospheric oxygen pressure, is considered as type I pMMO . This form was the subject of our pMMO isolation effort . Other forms (types II and III) are deactivated to various extents upon exposure to atmospheric dioxygen pressure . Under inactivating conditions, these unstable forms release protons to the buffer (approximately 10 H+/94-kDa monomeric unit) and eventually become completely inactive.

Anal Biochem, 1998 May 1, 258(2), 216 - 22
Stable transfection of mammalian cells by syringe-mediated mechanical loading of DNA; Waldman AS et al.; We show that mammalian cells can be stably transfected by a mechanical loading procedure in which cells are forced through a small opening in the presence of DNA . A suspension of cells and plasmid DNA in growth medium was passed up and down through a 30-gauge needle attached to a 1-ml syringe . Cells were immediately plated at appropriate densities for subsequent selection for stable expression of a marker gene . Two rodent cell lines, Chinese hamster ovary and mouse Ltk- cells, were successfully transfected with an efficiency of about one transfectant per 5 x 10(4) cells . The human HeLa cell line was transfected with a somewhat lower efficiency . Pluronic F-68, a detergent believed to aid in healing of membrane injuries, had no beneficial effect when present during the loading procedure . Successful transfection was accomplished using three different genes as selectable markers . Southern blotting analysis revealed that transfectants contained one or very few copies of the introduced DNA construct integrated into the genome . Several transfectants were demonstrated to remain stable for more than 20 generations of growth in the absence of selection . This procedure is fast, economical, and of general utility.

Cytobios, 1997, 91(364), 45 - 52
In vitro characteristics of myogenic satellite cells derived from the pectoralis major and biceps femoris muscles of the chicken; McFarland DC et al.; Satellite cells were isolated from the pectoralis major (PM) and biceps femoris (BF) muscles of 5-week-old broiler chickens to compare growth and differentiation characteristics in vitro . BF cells proliferated at greater rates in the growth medium and were more responsive to the mitogenic effects of chicken serum than PM cells at all levels tested (p < or = 0.05) . When low serum-containing medium was administered, the levels of creatine kinase, a marker of differentiation, increased at greater rates in PM cultures than in BF cultures (p < or = 0.05) . Administration of increasing levels of fibroblast growth factor in serum-free medium resulted in similar responsiveness of the two lines to this mitogen . No differences were detected in rates of protein synthesis or degradation in myotube cultures from the two muscle sources . The results suggest that satellite cells derived from PM and BF muscles of the chicken have different responsiveness to serum mitogens.

Matrix Biol, 1998 Mar, 16(9), 541 - 61
Aggrecan and link protein affect cell adhesion to culture plates and to type II collagen; Yang BB et al.; Cartilage is a hypocellular tissue in which a balance of matrix molecules, especially aggrecan and link protein, play a critical role in maintaining structural integrity . To study the role of aggrecan and link protein in mediating cell activities, we have stably expressed them in NIH/3T3 fibroblasts and observed the effect on cell-substratum interactions . Overexpression of either protein destabilized the cell-substratum interaction . However, when both were co-expressed, the interaction between cell and substratum was less impaired . Similar results were obtained on type II collagen-coated plates . The addition of exogenous gene products into fibroblast cell lines and chondrocyte culture had the same effect as expression of the genes . The addition of exogenous hyaluronan to the growth medium or treatment of cells with hyaluronidase also decreased cell adhesion, indicating that hyaluronan also plays a role in the cell-substratum adhesion . The presence of aggrecan seems to increase the amount of link protein on the cell surface . Chondrocytes expressing high concentrations of aggrecan and link protein were maintained within a matrix network and were able to survive in suspended culture . Imbalances in aggrecan or link protein concentrations, or degradation of hyaluronan, disrupted the network and caused the chondrocytes to aggregate or adhere to the plates.

Mutagenesis, 1998 Mar, 13(2), 145 - 9
Micronucleus induction in V79 cells after direct exposure to whole cigarette smoke; Massey E et al.; Previous investigations on the effects of cigarette smoke on cultured cells have used mainly smoke condensate dissolved in culture medium . A system has been designed which allows direct exposure of cells to fresh cigarette smoke, without an intervening layer of growth medium between the cells and the smoke . Preliminary results have been obtained which demonstrate the viability of the system . V79 cells were cultured on porous membranes (Transwell; Costar) . During smoke exposure only the lower surface of each Transwell is supplied with culture medium from the bottom of the culture chambers . In this way the cells had direct contact with the atmosphere at the upper surface and could be exposed directly to the test compound . The constructed exposure system consists of a smoke generator and an exposure unit containing six Transwells, the latter contained in an incubator . Cigarette smoke was generated using a standard 2 s, 35 ml puff once per min . The puff is diluted with conditioned air from the incubator and injected into the exposure unit . Following exposure of the cells to air only for 3 h there was no effect upon V79 cell viability . However, after exposure to smoke containing between 88 and 224 mg/m3 particulate matter, an inhibition of cell proliferation and induction of micronuclei was measured . When a Cambridge filter pad was placed between the cigarette and the cell exposure system to remove particulate matter cell proliferation was also reduced and an increased frequency of micronuclei above the control value was measured.

Indian J Exp Biol, 1997 Nov, 35(11), 1175 - 81
In vitro propagation of Theileria annulata infected schizonts in different media supplemented with heterologous sera; Nichani AK et al.; Efficacy of medium RPMI-1640 (supplied by Gibco USA, Centron and Hi-media) supplemented with horse, donkey, sheep and goat sera was evaluated for in vitro propagation of Theileria annulata (Hisar) infected bovine mononuclear cells . The results were compared with the growth rate in RPMI-1640 supplemented with foetal bovine serum (Gibco) . RPMI-1640 (Gibco) proved to be the best medium for in vitro cultivation of the parasite infected cells . Foetal bovine serum could be easily, safely and reliably substituted with goat and sheep sera in the growth medium . Horse and donkey sera also gave comparable growth of T . annulata infected cells in vitro . Successful use of heterologous sera greatly helped in reducing the cost of in vitro cultivation of T . annulata schizonts . These findings have important implications on mass production of an attenuated cell culture vaccine for the control of bovine tropical theileriosis.

Antimicrob Agents Chemother, 1998 Apr, 42(4), 795 - 800
Accumulation of norfloxacin by Mycobacterium aurum and Mycobacterium smegmatis; Williams KJ et al.; The modified fluorescence method was used to determine the accumulation of norfloxacin by Mycobacterium aurum A+ and Mycobacterium smegmatis mc(2)155 . By using an exogenous norfloxacin concentration of 10 microg/ml, a steady-state concentration (SSC) of 160 to 180 ng of norfloxacin/mg of cells was obtained for M . aurum, and an SSC of 120 to 140 ng of norfloxacin/mg of cells obtained for M . smegmatis . For both species of mycobacteria, the SSC was achieved within 5 min . The silicon oil method was investigated and gave higher SSCs than the modified fluorescence method . Further studies on the mechanism of norfloxacin accumulation by M . aurum were performed . An increase in the pH of the wash buffer from 7.0 to 9.0 did not significantly affect the final SSC obtained . Accumulation was nonsaturated over a norfloxacin concentration range of 0 to 100 microg/ml, and the proton motive force inhibitor 2,4-dinitrophenol (1 and 2 mM), whether it was added before or after norfloxacin was added, had no effect on the final SSC obtained . 2,4-Dinitrophenol also had no effect on norfloxacin accumulation by M . smegmatis . Furthermore, norfloxacin accumulation by M . aurum was unaffected by the presence of either Tween 80 or subinhibitory concentrations of ethambutol in the growth medium . Therefore, it is proposed that norfloxacin accumulation by mycobacteria occurs by simple, energy-independent diffusion.

Cell Mol Biol (Noisy-le-grand), 1998 Feb, 44(1), 231 - 8
The in situ infrared microspectroscopy of bacterial colonies on agar plates; Lang PL et al.; The specular reflectance infrared spectra of thirty species of bacteria were obtained in situ, without their removal from the agar growth media, using the infrared microscope . Compared to transmittance spectra obtained from dehydrated films, the transformed reflectance spectra show significant band shifts which are greater for gram positive bacteria . The most notable shift occurs in the v{a}(PO2-) band which shifts 8 cm(-1) higher upon removal of water . In addition, some measure of the differentiation potential of our in situ reflectance spectra was gathered by using principal components regression as a method of discrimination . We assigned a +1 for a gram positive property and a -1 to a gram negative property for each different bacteria species . The prediction results from a cross validation show that all the gram positive bacteria retain positive values for Gram stain predictions while all the gram negative species retain their negative values, suggesting a simple marker for differentiation . Overall, using reflectance spectra from colonies directly on the agar requires no sample preparation and provides slightly better differentiating spectral information than the transmittance spectra of dehydrated films.

Biochemistry, 1998 Mar 31, 37(13), 4502 - 9
Identification of histidine 105 in the beta1 subunit of soluble guanylate cyclase as the heme proximal ligand; Zhao Y et al.; Soluble guanylate cyclase isolated from bovine and rat lung is a heterodimeric hemoprotein composed of alpha1 and beta1 subunits . The heme binding region has been localized to residues 1-385 of the beta1 subunit {beta1(1-385)}, while the catalytic site(s) have been localized to the C-terminal region of sGC . There are four conserved histidine residues in the heme binding region of sGC . H220 and H346 are conserved among all known sGC subunits (alpha and beta), while H105 and H134 are conserved only in the beta subunits (beta1 and beta2) . Site-directed mutagenesis was used to individually change each of the conserved histidines in sGC beta1(1-385) to alanine or glycine, and the resulting mutants were expressed in E . coli . All of the mutants except for H105A and H105G had heme bound as isolated . Imidazole (Im) was able to rescue heme binding to H105G when added to the growth medium and purification buffers . The heme in H105G isolated in the presence of imidazole {H105G(Im)} was ferric and a mixture of 5-coordinate, high-spin and 6-coordinate, low-spin complexes . After reduction, the ferrous heme in H105G(Im) was 5-coordinate, high-spin as indicated by resonance Raman spectroscopy . When imidazole in H105G(Im) was exchanged with N-methylimidazole (MeIm), the Fe-N(Im/MeIm) stretching frequency was shifted from 221 to 212 cm-1 . A shift of this magnitude is expected when the ligand is directly coordinated to the heme iron . All of the data are consistent with the conclusion that H105 in the beta1 subunit is the heme proximal ligand.

Arch Microbiol, 1998 Mar, 169(3), 211 - 9
Characterization of the alternative sigma-factors SigD and SigE in Synechococcus sp . strain PCC 7002 . SigE is implicated in transcription of post-exponential-phase-specific genes; Gruber TM et al.; The sigD and sigE genes, which encode two alternative sigma-factors from the unicellular marine cyanobacterium Synechococcus sp . PCC 7002, were cloned and characterized . Strains in which the sigD and sigE genes were insertionally inactivated were viable under standard laboratory conditions, indicating that SigD and SigE are group 2 sigma-factors . When stationary-phase cells were diluted into fresh growth medium, it was observed that the sigE mutant strain required longer times to re-establish exponential growth than the wild-type strain . By monitoring the growth rates in such dilution experiments, it was observed that the lag times for the mutant strain became progressively longer as the original cultures progressed towards stationary phase . Transcripts for the sigE gene initially increased and subsequently decreased as cells grew further into stationary phase . It was determined that a functional SigE protein is required for the expression of the starvation-induced protein DpsA/PexB . The results suggest that SigE is involved in the transcription of genes specifically expressed in the post-exponential phase.

Genes Genet Syst, 1997 Dec, 72(6), 323 - 34
The phosphatase system in Saccharomyces cerevisiae; Oshima Y; The yeast Saccharomyces cerevisiae has at least six species of acid and alkaline phosphatases with different cellular localizations, as well as inorganic phosphate (Pi) transporters . Most of the genes encoding these enzymes are coordinately repressed and derepressed depending on the Pi concentration in the growth medium . The Pi signals are conveyed to these genes through a regulatory circuit consisting of a set of positive and negative regulatory proteins . This phosphatase system is interested as one of the best systems for studying gene regulation in S . cerevisiae due to the simplicity of phenotype determination in genetic analysis . With this methodological advantage, considerable amounts of genetic and molecular evidence in phosphatase regulation have been accumulated in the past twenty-five years . This article summarizes the current progress of research into this subject.

J Bacteriol, 1998 Apr, 180(7), 1624 - 31
Characterization of dnaC2 and dnaC28 mutants by flow cytometry; Withers HL et al.; Escherichia coli strains containing thermosensitive dnaC alleles were studied by flow cytometry . Strains containing either the dnaC2 or dnaC28 allele were shifted between different temperatures, and DNA content distributions were gathered . Inhibition of initiation of chromosome replication at nonpermissive temperature, as well as reinitiation of replication at permissive temperature, were found to be affected by a number of parameters . These included the choice of permissive and nonpermissive temperatures, the length of the time of incubation at the nonpermissive temperature, the growth medium, the type of temperature shift used for reinitiation of replication (transient or nontransient), the genetic background of the host cell, and the cell concentration . Reinitiation of replication required neither transcription nor translation, whereas the elongation stage of replication was dependent upon ongoing protein synthesis in the mutants . Efficient use of dnaC mutants for cell cycle studies is discussed.

Biochim Biophys Acta, 1998 Feb 25, 1363(2), 125 - 33
Two NADH:ubiquinone oxidoreductases of Azotobacter vinelandii and their role in the respiratory protection; Bertsova YV et al.; Initial steps of the Azotobacter vinelandii respiratory chain have been studied on the inside-out subcellular vesicles . Two NADH:ubiquinone oxidoreductases were revealed: (i) proton-motive, capsaicin-sensitive and oxidizing dNADH as well as NADH enzyme and (ii) enzyme non-coupled to the energy conservation, capsaicin-resistant and oxidizing only NADH . The level of the oxidoreductases strongly depends upon {O2} and {NH3} in the growth medium . Increase in {O2} results in lowering of the coupled-enzyme level and in rise of the non-coupled one . Exclusion of NH3 from the growth medium increases the level of the non-coupled enzyme whereas that of the coupled enzyme remains constant . The O2-linked control of NADH:ubiquinone oxidoreductases requires CydR, a Fnr-like regulatory protein . Summarizing the above observations with those made in this group on the terminal steps of the A . vinelandii respiratory chains, one can assume that the respiratory protection of nitrogenase could be carried out by co-operation of the non-coupled NADH:ubiquinone oxidoreductase and the "partially coupled" quinoloxidase of the bd-type . Efficiency of this chain seems to be five-fold lower than that of the usual proton-motive chain (the coupled NADH:ubiquinone oxidoreductase, the Q-cycle and cytochrome oxidase of the o-type) which is also present in A . vinelandii and operates at low {O2} .

Ital J Anat Embryol, 1997 Oct-Dec, 102(4), 9 - 119
Investigations into mechanisms modulating proliferation, differentiation, and apoptosis in cultured liver, adrenal, skin, and bone cells; Dal Pra I et al.; The intricate modulatory roles played by manifold hormones, growth factors, cytokines, extracellular calcium concentrations, intracellular second messengers, protein kinases, and nuclear poly(ADP-ribose) polymerase in proliferative, differentiative, and apoptotic processes have been the subject of investigations that were carried out by means of in vitro either primary or secondary/tertiary cultures of differentiated epithelial (hepatocytes, keratinocytes, and adrenocytes) and connective tissue cells (osteoblasts and fibroblasts) obtained from man and/or other mammalians . In most cases, an ad hoc model system, in which cells were floated on the top of the growth medium and, hence, could enjoy nearly normal respiratory exchanges, was used . Such a system increased cell viability and the ability of parenchymal epithelial cells to respond to extremely low concentrations of growth factors, hormones, and pharmaco-toxicological agents in a way conceivably very close to their behaviour in vivo.

Biotechniques, 1998 Mar, 24(3), 438 - 44
Bacterial growth medium that significantly increases the yield of recombinant plasmid; Duttweiler HM et al.; Isolation of plasmid DNA from Escherichia coli is a daily activity in many molecular biology laboratories . A number of protocols and media recipes have been reported in an effort to make this process more efficient . Here we describe a growth medium that supports much higher E . coli cell densities and, concomitantly, a much higher yield of plasmid than previously reported for small-scale applications . On a unit volume basis, E . coli cultures grown in this medium, termed H15, produce up to 30-fold more recombinant plasmid than in conventional rich media, paralleling the increase in cell density . This phenomenon is independent of E . coli host strain, DNA insert size and plasmid copy number . H15 medium is also very economical; as much as 6 mg of plasmid can be harvested per dollar of medium.

Arch Microbiol, 1998 Feb, 169(2), 98 - 105
Complex I of Rhodobacter capsulatus and its role in reverted electron transport; Herter SM et al.; The activities of NAD+-photoreduction and NADH/decyl-ubiquinone reductase in membrane preparations of Rhodobacter capsulatus changed to the same extent under different conditions . These results indicated that NADH:ubiquinone oxidoreductase (complex I) catalyzes the electron transport in the downhill direction (respiratory chain) and in the uphill direction (reverted electron flow) . This conclusion was confirmed by the characterization of a complex-I-deficient mutant of R . capsulatus . The mutant was not able to reduce NAD+ in the light . Since this mutant was not able to grow photoautotrophically, we concluded that complex I is the enzyme that catalyzes the reverted electron flow to NAD+ to provide reduction equivalents for CO2 fixation . Complex I is not essential for the reverted electron flow to nitrogenase since the mutant grew under nitrogen-fixing conditions . As shown by immunological means, NuoE, a subunit of complex I from R . capsulatus having an extended C-terminus, was modified depending on the nitrogen source present in the growth medium . When the organism used N2 instead of NH4+, a smaller NuoE polypeptide was synthesized . The complex-I-deficient mutant was not able to modify NuoE . The function of the modification is discussed.

J Pharm Sci, 1998 Feb, 87(2), 256 - 8
Effect of altered serum lipid concentrations on the IC50 of halofantrine against Plasmodium falciparum; Humberstone AJ et al.; Halofantrine (Hf) is a highly lipophilic antimalarial which significantly associates with triglyceride (TG) rich plasma lipoproteins, and this is likely manifest as a decrease in the free fraction of drug . This study assessed the effect of using growth media containing 10% serum containing different concentrations of TG (i.e . TG-rich plasma lipoproteins) on the IC50 of Hf determined using continuous in vitro culture of Plasmodium falciparum . Serum was collected from a human subject in either a fasted state or at various times after ingestion of a fatty meal . There was a linear and statistically significant 2.5-fold increase in the IC50 of Hf across a 6-fold range of increasing TG concentrations, with the increased IC50 values being ascribed to a decreased free fraction of Hf in the growth media due to sequestration by TG-rich lipoproteins . Chloroquine diphosphate, which is hydrophilic and not significantly bound by TG-rich lipoproteins, was used as a control and its IC50 values were independent of TG concentrations . These data indicate that consideration should be given to the adoption of standard conditions for the collection of serum with respect to pre- or postprandial states, and that subject- and disease-related factors which alter plasma lipoprotein profiles should be considered when interpreting the IC50 profile of Hf (and possibly other lipophilic antimalarials) . Furthermore, although food is known to affect the pharmacokinetics of Hf, these data suggest that altered plasma lipoprotein profiles could also influence its pharmacodynamic profile.

J Bacteriol, 1998 Mar, 180(6), 1466 - 72
Physiological and genetic analyses leading to identification of a biochemical role for the moeA (molybdate metabolism) gene product in Escherichia coli; Hasona A et al.; A unique class of chlorate-resistant mutants of Escherichia coli which produced formate hydrogenlyase and nitrate reductase activities only when grown in medium with limiting amounts of sulfur compounds was isolated . These mutants failed to produce the two molybdoenzyme activities when cultured in rich medium or glucose-minimal medium . The mutations in these mutants were localized in the moeA gene . Mutant strains with polar mutations in moeA which are also moeB did not produce active molybdoenzymes in any of the media tested . moeA mutants with a second mutation in either cysDNCJI or cysH gene lost the ability to produce active molybdoenzyme even when grown in medium limiting in sulfur compounds . The CysDNCJIH proteins along with CysG catalyze the conversion of sulfate to sulfide . Addition of sulfide to the growth medium of moeA cys double mutants suppressed the MoeA- phenotype . These results suggest that in the absence of MoeA protein, the sulfide produced by the sulfate activation/reduction pathway combines with molybdate in the production of activated molybdenum . Since hydrogen sulfide is known to interact with molybdate in the production of thiomolybdate, it is possible that the MoeA-catalyzed activated molybdenum is a form of thiomolybdenum species which is used in the synthesis of molybdenum cofactor from Mo-free molybdopterin.

FEMS Microbiol Lett, 1997 Nov 15, 156(2), 265 - 9
An agarose-in-plug bridge method to study chemotaxis in the Archaeon Halobacterium salinarum; Yu HS et al.; A simple agarose-in-plug bridge method was developed to study chemotaxis in the Archaeon Halobacterium salinarum . Preheated liquid agarose solution with chemoeffectors is pipetted in the middle of a microscope slide bridge, constructed by placing two plastic strips 16 mm apart . A coverslip is immediately placed over the agarose . The solidified agarose plug is completely encircled with the halobacterial cell suspension . Within a certain time concentrated halobacteria were seen as a ring at the edge of the agarose plug containing attractant amino acids and the control growth medium . Chemotaxis mutant Pho60 cells do not accumulate either around the attractants or around the growth medium . The kinetics of the ring formation can be readily videotaped or photographed using either phase-contrast or dark-field microscopy.

Chem Res Toxicol, 1998 Feb, 11(2), 119 - 29
Permeability, cytotoxicity, and genotoxicity of chromium (V) and chromium (VI) complexes in V79 Chinese hamster lung cells; Dillon CT et al.; The genotoxicity of Cr(V) complexes in mammalian cells (V79 Chinese hamster lung cells) has been studied for the first time using the in vitro micronucleus assay . Two complexes were investigated, {CrO(ehba)2}-, which undergoes ligand-exchange and disproportionation reactions in the cell growth medium, and {CrO(mampa)}-, which is chemically inert in the medium for the duration of the exposure period . Results of in vitro micronucleus assays show that both complexes are genotoxic and exhibit similar potencies to that of {Cr2O7}2- . The permeabilities of the Cr(V) complexes were also investigated for the first time using particle-induced X-ray emission (PIXE) analysis of individual cells . The Cr uptake increased in the order: {Cr(phen)2-(H2O)2}3+ < {CrO(ehba)2}- < {CrO(mampa)}- < {Cr2O7}2- . Clonal assays showed that Cr(VI) exhibits an expectedly higher cytotoxicity than the Cr(V) complexes . While the genotoxicities of the Cr(V) and Cr(VI) complexes increase according to their permeabilities, the genotoxicities of the Cr(V) complexes are equal to, if not greater than, that of Cr(VI) in terms of the amount of Cr entering the cell . This supports other evidence that Cr(V), produced as a metabolic intermediate from the intracellular reduction of Cr(VI), may be important in Cr-induced cancers.

Appl Environ Microbiol, 1998 Mar, 64(3), 1115 - 22
Isolation of copper biochelates from Methylosinus trichosporium OB3b and soluble methane monooxygenase mutants; Tellez CM et al.; Methylosinus trichosporium OB3b produces an extracellular copper-binding ligand (CBL) with high affinity for copper . Wild-type cells and mutants that express soluble methane monooxygenase (sMMO) in the presence and absence of copper (sMMOc) were used to obtain cell exudates that were separated and analyzed by size exclusion high-performance liquid chromatography . A single chromatographic peak, when present, contained most of the aqueous-phase Cu(II) present in the culture medium . In mutant cultures that were unable to acquire copper, extracellular CBL accumulated to high levels both in the presence and in the absence of copper . Conversely, in wild-type cultures containing 5 microM Cu(II), extracellular CBL was maintained at a low, steady level during exponential growth, after which the external ligand was rapidly consumed . When Cu(II) was omitted from the growth medium, the wild-type organism produced the CBL at a rate that was proportional to cell density . After copper was added to this previously Cu-deprived culture, the CBL and copper concentrations in the medium decreased at approximately the same rate . Apparently, the extracellular CBL was produced throughout the period of cell growth, in the presence and absence of Cu(II), by both the mutant and wild-type cultures and was reinternalized or otherwise utilized by the wild-type cultures when it was bound to copper . CBL produced by the mutant strain facilitated copper uptake by wild-type cells, indicating that the extracellular CBLs produced by the mutant and wild-type organisms are functionally indistinguishable . CBL from the wild-type strain did not promote copper uptake by the mutant . The molecular weight of the CBL was estimated to be 500, and its association constant with copper was 1.4 x 10(16) M-1 . CBL exhibited a preference for copper, even in the presence of 20-fold higher concentrations of nickel . External complexation may play a role in normal copper acquisition by M . trichosporium OB3b . The sMMOc phenotype is probably related to the mutant's inability to take up CBL-complexed copper, not to a defective CBL structure.

J Clin Invest, 1998 Feb 1, 101(3), 578 - 83
Reduced extracellular pH increases endothelin-1 secretion by human renal microvascular endothelial cells; Wesson DE et al.; Because dietary acid increases renal secretion of endothelin-1 (ET-1) which is synthesized by renal microvascular endothelium, we examined if reduced extracellular pH increases ET-1 secretion by cultured human renal microvascular endothelial cells (RMVECs) . Confluent cells were exposed to serum-free media for 24 h, then incubated in either control, acid, or alkaline serum-free media for 12 h . Standard growth media pH was 7.2 after equilibration with 5% CO2 at 37 degrees C and was made the pH of control media . Acid and alkaline media pH were 7.0 and 7.4, respectively . Added Hepes and Tris maintained all assigned pHs . Media ET-1 measured by RIA after column extraction was higher for RMVECs exposed to acid compared with control media (170.0+/-17.1 vs . 64.6+/-9.6 pM, P < 0.004) but those exposed to alkaline media (56.6+/-25.1 pM, P = NS vs . control) were not . Human aortic endothelial cells exposed to control, acid, and alkaline media had similar ET-1 (166.6+/-18.1, 139.3+/-18.5, and 205.9+/-25.3 pM, P = NS) . The data show acid-stimulated ET-1 secretion by RMVECs but not aortic endothelial cells, demonstrating a new environmental factor that influences ET-1 secretion by renal microvascular endothelium and thereby possibly modulates endothelin-dependent processes in vivo.

Biochem Biophys Res Commun, 1998 Feb 24, 243(3), 852 - 7
Ceramide-induced apoptosis is mediated by caspase activation independently from retinoblastoma protein post-translational modification; Spinedi A et al.; Recent evidence suggests that untimely retinoblastoma protein (RB) dephosphorylation and/or proteolytic degradation might provide key events down-stream cysteine protease (caspase) activation in apoptosis induction . We have dealt with this issue by studying apoptosis induced by N-hexanoylsphingosine (C6-Cer) in CHP-100 human neuroepithelioma cells, maintained in complete growth medium . We report that C6-Cer-induced apoptosis occurred predominantly in G1/S phases of the cycle and was associated with RB dephosphorylation, in the setting of negligible Bcl-2 expression . Apoptosis was also associated with poly(ADP-ribose) polymerase (PARP) cleavage, thus indicating activation of CPP32/Yama/apopain (caspase-3); however, while the tripeptide caspase inhibitor Z-Val-Ala-DL-Asp-fluoromethylketone was able to prevent both C6-Cer-induced PARP cleavage and apoptosis, it was ineffective in preventing RB dephosphorylation . Moreover proteolytic RB cleavage occurred only to a marginal extent after C6-Cer treatment . These results indicate that apoptosis induced by ceramide in CHP-100 cells is caspase-mediated, but RB post-translational modification does not provide a key step, downstream caspase activation, in apoptosis execution.

J Mol Cell Cardiol, 1998 Jan, 30(1), 157 - 166
Regulation of cardiac Kv1.5 K+ channel expression by cardiac fibroblasts and mechanical load in cultured newborn rat ventricular myocytes; Guo W et al.; Of the six voltage-gated K+ channel alpha subunits detected in rat heart, the Kv1.5 channel is abundantly expressed, and its gene transcription and protein expression are reduced during cardiac remodeling . Since cardiac fibroblasts and mechanical load have been known to play important roles in myocardial hypertrophy, we studied the regulation of Kv1.5 K+ channel protein expression by these factors in cultured newborn rat ventricular myocytes, using immunofluorescent cytochemistry and Western blot analysis . Ventricular cells were isolated from 1-day-old Wistar rats and cultured for a period of 5 days . The effect of cardiac fibroblasts was examined by co-culturing myocytes with fibroblasts or incubating pure myocytes in fibroblast-conditioned growth medium (FCGM) for 72 h . In addition, a 48-h cyclic stretch at 0.5 Hz with 20% elongation in length was applied to pure myocyte cultures to mimic mechanical load . With a polyclonal antibody against rat Kv1.5 K+ channel protein, single cultured myocytes showed a weak and uniform antibody labeling . Co-culturing with fibroblasts or incubating pure myocytes in FCGM both induced a significant increase in myocyte size implying cell hypertrophy, but neither allowed normal expression of the Kv1.5 K+ channel as indicated by almost negative anti-Kv1.5 labeling . Western blots of cell proteins prepared from ventricular myocyte cultures revealed a single protein band at 75 kD recognized by the anti-Kv1.5 antibody and a 45% decrease in Kv1.5 immunoreactive protein level in the FCGM-treated preparations . Application of 1 microM losartan, an angiotensin II type I receptor blocker, significantly attenuated the FCGM-induced myocyte hypertrophy and reduction of Kv1.5 K+ channel expression . On the other hand, although no cell hypertrophy was stimulated by mechanical stretch, intense punctate antibody labeling with a 48% increase in Kv1.5 protein level was observed in the stretched myocytes . These results suggest that the protein expression of cardiac Kv1.5 K+ channel is differentially regulated by cardiac fibroblasts and mechanical load . Some soluble factors produced from cardiac fibroblasts contribute to the depressed Kv1.5 K+ channel expression in myocardial hypertrophy . This channel regulation may be mediated by angiotensin II type I receptor.

Biol Trace Elem Res, 1998 Jan, 61(1), 1 - 8
Differences in the cellular zinc content and 5'-nucleotidase activity of normal and acrodermatitis enteropathica (AE) fibroblasts; Grider A et al.; The acrodermatitis enteropathica (AE) mutation affects zinc (Zn) metabolism in human fibroblasts . We hypothesize that the mutation affects the cell Zn content, which subsequently affects the activity of various zinc-dependent enzymes, such as 5'-nucleotidase . Therefore, normal and AE fibroblasts were grown in normal medium containing physiological levels of Zn (16 micromol/L) for approximately 24 h . The medium was replaced by normal medium (16 micromol/L Zn), Zn-depleted medium (1.5 micromol/L Zn), or Zn-supplemented medium (200 micromol/L Zn) for another 24 h . Regardless of the Zn concentration of the growth medium, the AE fibroblasts contained significantly less Zn than normal fibroblasts grown in comparable medium . Nevertheless, growth of the fibroblasts in 200 micromol/L Zn medium significantly increased the cell Zn content fourfold of both normal and AE fibroblasts . The activity of 5'-nucleotidase in the AE fibroblasts grown in 16 micromol/L Zn or 1.5 micromol/L Zn medium was also significantly lower than in normal fibroblasts . Changing the growth medium from 16 micromol/L Zn to 1.5 micromol/L Zn medium did not affect the activity of the enzyme in either genotype . Cells grown in 200 micromol/L Zn medium exhibited threefold greater 5'-nucleotidase activity in AE fibroblasts, but had no affect on enzyme activity in normal cells . In summary, altering the cell Zn content of normal fibroblasts did not result in a significant change in their 5'-nucleotidase activity . However, AE fibroblasts grown in 200 micromol/L Zn medium exhibited recovery of their 5'-nucleotidase activity to normal levels . These results support the hypothesis that the AE mutation affects the cellular Zn content . The lower cell Zn content subsequently affects the activity of 5'-nucleotidase.

J Bacteriol, 1998 Mar, 180(5), 1030 - 6
Importance of RpoS and Dps in survival of exposure of both exponential- and stationary-phase Escherichia coli cells to the electrophile N-ethylmaleimide; Ferguson GP et al.; The mechanisms by which Escherichia coli cells survive exposure to the toxic electrophile N-ethylmaleimide (NEM) have been investigated . Stationary-phase E . coli cells were more resistant to NEM than exponential-phase cells . The KefB and KefC systems were found to play an important role in protecting both exponential- and stationary-phase cells against NEM . Additionally, RpoS and the DNA-binding protein Dps aided the survival of both exponential- and stationary-phase cells against NEM . Double mutants lacking both RpoS and Dps and triple mutants deficient in KefB and KefC and either RpoS or Dps had an increased sensitivity to NEM in both exponential- and stationary-phase cells compared to mutants missing only one of these protective mechanisms . Stationary- and exponential-phase cells of a quadruple mutant lacking all four protective systems displayed even greater sensitivity to NEM . These results indicated that protection by the KefB and KefC systems, RpoS and Dps can each occur independently of the other systems . Alterations in the level of RpoS in exponentially growing cells correlated with the degree of NEM sensitivity . Decreasing the level of RpoS by enriching the growth medium enhanced sensitivity to NEM, whereas a mutant lacking the ClpP protease accumulated RpoS and gained high levels of resistance to NEM . A slower-growing E . coli strain was also found to accumulate RpoS and had enhanced resistance to NEM . These data emphasize the multiplicity of pathways involved in protecting E . coli cells against NEM.

Mol Gen Genet, 1998 Jan, 257(2), 238 - 48
Characterisation of Saccharomyces cerevisiae ARO8 and ARO9 genes encoding aromatic aminotransferases I and II reveals a new aminotransferase subfamily; Iraqui I et al.; The ARO8 and ARO9 genes of Saccharomyces cerevisiae were isolated by complementation of the phenylalanine/tyrosine auxotrophy of an aro8 and aro9 double-mutant strain that is defective in aromatic aminotransferase I (aro8) and II (aro9) . The genes were sequenced, and deletion mutants were constructed and analysed . The expression of ARO8 and ARO9 was studied . The deduced amino acid sequences of Aro8p and Aro9p suggest that the former is a 500-residue, 56168-Da polypeptide and the latter a 513-residue, 58516-Da polypeptide . They correspond, respectively, to Ygl202p and Yhr137p, two putative proteins of unknown function revealed by systematic sequencing of the yeast genome . We show that aromatic aminotransferases I and II are homologous proteins, members of aminotransferase subgroup I, and, together with three other proteins, they constitute within the subgroup a new subfamily of enzymes specialised for aromatic amino acid and alpha-aminoadipate transamination . ARO8 expression is subject to the general control of amino acid biosynthesis . ARO9 expression is induced when aromatic amino acids are present in the growth medium and also in aro8 mutants grown on minimal ammonia medium . An autonomously replicating sequence (ARS) element is located between the ARO8 gene and YGL201c which encodes a protein of the minichromosome maintenance family.

Eur J Biochem, 1998 Feb 1, 251(3), 716 - 23
The Saccharomyces cerevisiae LYS7 gene is involved in oxidative stress protection; Gamonet F et al.; Saccharomyces cerevisiae Lys7p was proposed to be the enzyme catalyzing the dehydratation of homocitrate to cis-homoaconitate, the second step of the lysine biosynthetic pathway . In this communication we provide evidence that Lys7p is involved in oxidative stress protection . Cells deleted for the LYS7 gene displayed, in addition to lysine auxotrophy, methionine auxotrophy, sensitivity to superoxide generating drugs and light irradiation, and diminution of calcineurin activity . The SOD1 gene encoding the Cu/Zn-superoxide dismutase was expressed in strains lacking Lys7p, and although Sodlp was produced in normal amounts no detectable enzyme activity was found . In contrast, the mitochondrial Mn-superoxide dismutase activity did not seem to be impaired . lys7 cells exhibited a normal uptake of Cu from growth medium . The Cu/Zn-superoxide dismutase activity was restored by addition of Cu (but not by addition of other metallic cations) to the growth medium or to cellular extracts, suggesting a lack of Cu2+ at the active site . These results render it necessary to reconsider the role of the Lys7p . Its involvement in Cu metabolism and oxidative-stress protection, and the possibility of a human equivalent in amyotrophic lateral sclerosis are discussed.

Cell Transplant, 1998 Jan-Feb, 7(1), 71 - 82
Tissue engineered skeletal muscle: preparation of highly dense, highly oriented hybrid muscular tissues; Okano T et al.; We prepared highly dense, highly oriented hybrid muscular tissues that are composed of C2C12 cells (skeletal muscle myoblast cell line) and type I collagen . A cold mixture of C2C12 cells suspended in DMEM and type I collagen solution was poured into capillary tube molds of two different sizes (inner diameters; 0.90 and 0.53 mm, respectively) . One end of each mold was sealed . Upon centrifugation (1000 rpm, 5 min) and subsequent thermal gelation, a rod-shaped gel was obtained . It was cultured in an agarose gel-coated dish for 7 days (first for 3 days in a growth medium and then for 4 days in a differentiation medium), during which time it shrank to become a highly dense tissue . Small-diameter rod-shaped, highly dense cellular assemblages with multinucleated myotubes were formed and only few necrotic cells at the core of the tissue were observed . On the other hand, a ring-shaped tissue prepared using a specially devised agarose gel mold was subjected to cyclic stretching at 60 rpm, resulting in the formation of a highly dense, highly oriented hybrid muscular tissue in which both densely accumulated cells and collagen fiber bundles tended to be aligned in the direction of stretching . The hybrid muscular tissues that were prepared using via sequential procedures of a centrifugal cell packing method and a mechanical stress-loading method became closer to native muscular tissues in terms of cell density and orientation.

Invest Ophthalmol Vis Sci, 1998 Feb, 39(2), 449 - 54
Antimetabolite-induced apoptosis in Tenon's capsule fibroblasts; Crowston JG et al.; PURPOSE: To determine whether treatment with mitomycin-c and 5-fluorouracil induces apoptotic death in cultured subconjunctival fibroblasts . METHODS: Cultured human subconjunctival Tenon's capsule fibroblasts were exposed to 5-minute applications of mitomycin-C (up to 1 mg/ml) or 5-fluorouracil (up to 50 mg/ml) or phosphate-buffered saline solution (PBS) . Fibroblast apoptosis was determined by cell morphology, apoptosis-specific protein expression, and DNA fragmentation by TdT-mediated dUTP nick-end labeling (TUNEL) . In addition, apoptosis was quantified by direct cell counts based on morphology or lactate dehydrogenase release . RESULTS: Morphologic changes characteristic of apoptosis included nuclear and cytoplasmic condensation and occasional nuclear fragmentation while the plasma membrane remained intact . Apoptosis-specific protein expression and DNA fragmentation was observed in fibroblasts 48 hours after mitomycin-C treatment but not in control PBS-treated fibroblasts . The amount of apoptosis induced was dose dependent and partially inhibited by the addition of fetal calf serum to growth medium immediately after treatment . CONCLUSIONS: Mitomycin-C and high-dose 5-fluorouracil induce apoptosis in cultured Tenon's fibroblasts . Mitomycin-C-induced apoptosis is inhibited by fetal calf serum, indicating that exogenous factors influence the susceptibility of a fibroblast population to apoptosis . The induction and regulation of fibroblast apoptosis provides a novel target for the potential regulation of scarring.

Mund Kiefer Gesichtschir, 1997 Feb, 1(1), 26 - 30
{Vital long-term preservation of human gingiva in perfusion culture}; Lehmann P et al.; Perfusion culture offers the advantage of keeping gingiva alive for a long time as an stable explant according to cell biological parameters . To investigate the suitability of cultured human gingival explants for transplantations the biopsies were put into a newly developed perfusion chamber and cultured for at least 21 days . Gingiva explants were derived from healthy donors undergoing surgical removal of molar teeth . The tissue pieces were cultured without prior proteolytic desintegration or subculture . Immediately after excision a morphological and immunohistochemical analysis of the tissue was carried out and the distribution pattern of cytokeratin and vimentin was examined . Gingival explants cultured for 7, 14 and 21 days in serum-free keratinocyte growth medium in perfusion culture were analyzed in the same way . The morphology of the cultured explant (21 days) was well preserved from stratum basale up to stratum corneum . As proved by immunohistochemical incubation with antibodies to CK 5/6, CK 14 and CK 19, a tissue-specific cytokeratin (CK) expression pattern was maintained during the whole perfusion period . After 7 days of culture vimentin was synthesized in the fibroblast layer and was found in small quantities in each layer of the epithelium . In contrast to conventional cultures, where dissociation of the tissue and a subculture interruption is usually needed for long-term culture, this is not necessary for perfusion cultured tissue . The use of perfusion-cultured gingival explants as autogenous transplants is investigated herein.

Biochem J, 1998 Jan 15, 329 ( Pt 2), 265 - 73
Transfection of L6 myoblasts with adipocyte fatty acid-binding protein cDNA does not affect fatty acid uptake but disturbs lipid metabolism and fusion; Prinsen CF et al.; We studied the involvement of fatty acid-binding protein (FABP) in growth, differentiation and fatty acid metabolism of muscle cells by lipofection of rat L6 myoblasts with rat heart (H) FABP cDNA or with rat adipocyte (A) FABP cDNA in a eukaryotic expression vector which contained a puromycin acetyltransferase cassette . Stable transfectants showed integration into the genome for all constructs and type-specific overexpression at the mRNA and protein level for the clones with H-FABP and A-FABP cDNA constructs . The rate of proliferation of myoblasts transfected with rat A-FABP cDNA was 2-fold higher compared with all other transfected cells . In addition, these myoblasts showed disturbed fusion and differentiation, as assessed by morphological examination and creatine kinase activity . Uptake rates of palmitate were equal for all clone types, in spite of different FABP content and composition . Palmitate oxidation over a 3 h period was similar in all clones from growth medium . After being cultured in differentiation medium, mock- and H-FABP-cDNA-transfected cells showed a lower fatty acid-oxidation rate, in contrast with A-FABP-cDNA-transfected clones . The ratio of {14C}palmitic acid incorporation into phosphatidylcholine and phosphatidylethanolamine of A-FABP-cDNA-transfected clones changed in the opposite direction in differentiation medium from that of mock- and H-FABP-cDNA-transfected clones . In conclusion, transfection of L6 myoblasts with A-FABP cDNA does not affect H-FABP content and fatty acid uptake, but changes fatty acid metabolism . The latter changes may be related to the observed fusion defect.

Arch Environ Contam Toxicol, 1998 Jan, 34(1), 6 - 11
Liquid-gas partitioning of the gasoline oxygenate methyl tert-butyl ether (MTBE) under laboratory conditions and its effect on growth of selected algae; Rousch JM et al.; The partitioning of the widely used gasoline additive methyl tert-butyl ether (MTBE) between liquid growth media and gaseous phase was measured daily under laboratory conditions to determine how closely dissolved MTBE concentrations matched nominal concentrations . Total (gaseous and dissolved) MTBE averaged across 6 days for 29.6, 503.2, and 1005.7 mg L-1 MTBE treatments were 89.9, 90.3, and 73.0% of nominal, respectively, and mean dissolved MTBE in these same treatments were 74.6, 73.8, and 69.6% of total MTBE, respectively . This suggests that dissolved MTBE concentrations can vary substantially from nominal . The effect of MTBE on the growth of selected algae was also evaluated under laboratory conditions . Three unicellular algae, Selenastrum capricornutum (Chlorophyta), Navicula pelliculosa (Bacillariophyta), and Synechococcus leopoliensis (= Anacystic nidulans, Cyanophyta = Cyanobacteria), representative of three taxonomic groups, were used as test organisms . Toxicity tests were acute and increase in cell number was used as an indicator of growth . Algal species were exposed by injection of MTBE into sealed vessels containing defined liquid growth media . The growth of N . pelliculosa and S . leopoliensis was negatively affected at nominal 2400 mg L-1 MTBE, whereas the growth of S . capricornutum was negatively affected at nominal 4800 mg L-1 MTBE and positively affected at nominal 600 mg L-1 MTBE . The differential sensitivity of the growth of these representative species suggests that MTBE may alter algal community composition in the natural environment.

Hum Mol Genet, 1998 Jan, 7(1), 85 - 90
Biosynthesis and intracellular targeting of the CLN3 protein defective in Batten disease; Jarvela I et al.; Batten disease (juvenile-onset neuronal ceroid lipofuscinosis, JNCL), the most common neurodegenerative disorder of childhood, is caused by mutations in a recently identified gene ( CLN3 ) localized to chromosome 16p11.2-12.1 . To elucidate the biosynthesis and localization of the CLN3 protein, we expressed CLN3 cDNA in COS-1 and HeLa cell lines . In vitro translation, immunoprecipitation and Western blotting analyses detected an approximately 43 kDa polypeptide . Pulse-chase experiments indicated that the CLN3 protein is synthesized as an N -glycosylated single-chain polypeptide, which was not detected in growth medium . Confocal immunofluorescence microscopy revealed that the CLN3 protein is localized to the lysosomal compartment . These results provide evidence that Batten disease can be classified as a member of lysosomal diseases.

Pharmazie, 1998 Jan, 53(1), 51 - 7
Effects of growth factors on the proliferation of human keratinocytes and fibroblasts in vitro; Kim DS et al.; Growth/differentiation factor-5 (GDF-5) is a new member of the transforming growth factor-beta (TGF-beta) superfamily of multifunctional peptide growth factors that appear to mediate many key events in cell growth and development . The effects of GDF-5 and other growth factors (epidermal growth factor, EGF; TGF-beta 1) on the proliferation of human keratinocytes and fibroblasts compared with desoximetasone and calcipotriol have been investigated . The proliferation rate was determined by a hemocytometer, MTT assay and the incorporation of {3H}-thymidine . Moreover, cell cycle analyses were performed and the influence on interleukin-1 alpha (IL-1 alpha) production in keratinocytes was measured by enzyme-linked immunosorbent assay (ELISA) because of its pronounced proinflammatory effect . In keratinocytes, GDF-5 stimulated cell proliferation to a minor extent . The drug already proved to be effective at very low concentrations (0.1 ng/ml) . Growth stimulatory effects with EGF have been observed only in keratinocyte basal medium (KBM), but not in keratinocyte growth medium (KGM) . TGF-beta 1 markedly inhibited the proliferation of keratinocytes at concentrations > 1 ng/ml . Calcipotriol and desoximetasone also showed a dose-dependent cell growth inhibition in epidermal cell cultures . IL-1 alpha synthesis was greatly suppressed by calcipotriol 10(-8)-10(-6) M . EGF at 10 ng/ml, in contrast, strongly stimulated IL-1 alpha production . Neither GDF-5 nor TGF-beta 1 had a significant effect on IL-1 alpha production in keratinocyte monolayer cultures . In fibroblasts, GDF-5 induced very weak antiproliferative effects . Calcipotriol and desoximetasone also inhibited cell growth in fibroblast cultures whereas proliferation and DNA synthesis were strongly stimulated by 1 ng/ml EGF . There was, however, a contradiction between TGF-beta 1 results on fibroblasts . Whereas TGF-beta 1 increased proliferation in cell number determination and in the thymidine incorporation assay, MTT assays showed slight antiproliferative effects . Due to these controversial results, in addition cell cycle analysis was employed . TGF-beta 1 led to an increased S phase, which indicates a stimulation of cell division . The different results obtained with the MTT test suggest that TGF-beta 1 may stimulate cell division of fibroblasts not only by increasing the S phase, but also by shortening the G1 phase of the cell cycle.

Indian J Exp Biol, 1997 Aug, 35(8), 866 - 70
Effect of combined nitrogen on the expression of nitrate reductase and nitrite reductase in Azorhizobium caulinodans; Raju KS et al.; In aerobically grown Azorhizobium caulinodans strain IRBG 46, in vivo expression of nitrate reductase (NR) and nitrite reductase (NiR) requires the presence of either nitrate or nitrite . On the contrary mere microaerobic conditions are sufficient for the expression of NR and NiR, however, addition of nitrate to the growth medium enhanced the activities of the enzymes . Optimum concentration of nitrate for maximum expression of NR and NiR activities was different in aerobic and microaerobic conditions . Nitrite was released into the medium both in aerobic and microaerobic conditions beyond a particular concentration of nitrate in the medium . Dissimilatory nitrate reduction was affected to a lesser extent by ammonium compared to assimilatory nitrate reduction.

J Mol Biol, 1998 Jan 16, 275(2), 197 - 209
DNA protein-interactions at the Saccharomyces cerevisiae 35 S rRNA promoter and in its surrounding region; Vogelauer M et al.; This study represents a detailed analysis of the structural context of the RNA polymerase I promoter of Saccharomyces cerevisiae . We determined the presence of regularly spaced nucleosomes in the non-transcribed spacer (NTS) and found that five of them have well defined positions . We show that this nucleosome positioning is restricted to the region between the 35 S and 5 S rRNA promoters, beyond which a more delocalized chromatin structure is evident . A more refined analysis detects the DNA-protein interactions on the RNA polymerase I promoter at nucleotide resolution and provides the first in vivo footprints, attributable to factors like REB1, CF, UAF and an additional protection that seems to be sensitive to the topological context . Moreover, when this analysis is extended to different growth media (YPD versus YNB), some of these protections show a growth condition dependent behaviour.

Dev Biol, 1998 Jan 1, 193(1), 100 - 13
A study of the potential of the embryonic rat telencephalon to generate oligodendrocytes; Birling MC et al.; A major question in neural development is whether each part of the telencephalon has an equal potential to generate each cell type . In this study, we address this question specifically in regard to the generation of oligodendrocytes . We cultured precursor cells from two different regions of the rat embryonic telencephalon--the ganglionic eminence that the anlage of the cerebral cortex--from different stages of development, and labeled the cells with a retroviral vector to follow their fate . We discovered that multipotential precursor cells from E13 ganglionic eminence have several orders of magnitude higher capacity to generate oligodendrocyte than the equivalent cells from E13 cerebral cortex . This failure of cortical precursor cells to generate oligodendrocytes at early development stages (E12-E13) could not be reversed by growth factors, permissive growth media, or a permissive striatal cell environment . A combination of striatal contact and plus specific growth factors, however, did induce the production of oligodendrocytes . We conclude that telencephalic precursor cells do not have the potential to generate oligodendrocytes, but that this potential is significantly greater in striatal than cortical multipotential precursor cells.

J Infect Dis, 1998 Feb, 177(2), 508 - 11
The neutral cysteine proteinase of Entamoeba histolytica degrades IgG and prevents its binding; Tran VQ et al.; Patients infected with Entamoeba histolytica generate specific IgG that does not prevent invasive amebiasis or recurrent infection . Studies investigated whether the effectiveness of the human humoral response was limited by cleavage of IgG by the extracellular neutral cysteine proteinase of E . histolytica trophozoites, one of the first amebic products to interact directly with components of host defenses . Purified proteinase cleaved polyclonal human and monoclonal murine IgG in a dose-dependent manner . Peptide sequencing of the major cleavage fragment(s), which contained the protein A binding site, suggested that cleavage occurred near the hinge region . Intact trophozoites also cleave IgG in both growth media and serum-free media . Cleaved monoclonal antibody to a 29-kDa surface antigen of E . histolytica bound to trophozoites 83.5% +/- 6.7% less than did uncleaved antibody . These results suggest that cleavage of IgG by the extracellular cysteine proteinase may limit the effectiveness of the host humoral response.

Ukr Biokhim Zh, 1997 Mar-Apr, 69(2), 126 - 30
Human leukemia CEM C-1 cells possess a high affinity binding site for farnesol; Iazlovitskaia EM et al.; Binding of 15-carbon isoprenoid farnesol in human acute leukemia CEM C-1 cells has been studied by addition of radio-labeled isoprenoid to cell growth medium . Significant time-dependent accumulation of the cell-associated radioactivity was detected at 37 degrees C . When experiments were carried out at 4 degrees C, about 10 times decrease in cell labeling was observed . In contrast, binding of farnesol by the liposomes prepared from cellular lipids was independent of the temperature . When binding experiments were performed in the presence of the excess of unlabeled farnesol, a saturable specific component was separated from the total binding . Analysis of the specific binding revealed about 335,000 binding sites per cell with Kd of 5.9 x 10(-8) M . The data suggest that CEM C-1 cells may possess receptors capable of binding farnesol.

Phytochemistry, 1998 Feb, 47(4), 605 - 12
Action of proteolysis-resistant systemin analogues in wound signalling; Schaller A; In cultured cells of Lycopersicon peruvianum, the oligopeptide systemin which mediates systemic signalling in the tomato wound response is rapidly inactivated by proteolytic cleavage of the bond carboxy-terminal to Lys14 . A systemin derivative in which this peptide bond had been modified by N-methylation was resistant to proteolytic inactivation . Systemin elicits a rapid, transient alkalinization of the growth medium in L . peruvianum cells . Consistent with its metabolic stability, the response elicited by the N-methylated peptide was found to be more sustained than that caused by systemin . In differentiated tomato plants, the stabilized peptide was found to be 3 times more active than systemin with respect to the induction of proteinase inhibitors I and II . This result indicates the possible physiological significance of the observed proteolytic degradation for systemin inactivation in planta . The activity of a protease capable of processing systemin carboxy-terminal of Lys14 was detected in tomato plasma membranes and may be responsible for the inactivation process . Two further peptides, N-methylated at the bonds carboxy-terminal of Gln3 and Arg10 had proteinase inhibitor inducing activities lower by a factor of 8 and 80, respectively, as compared to systemin . Correspondingly, the alkalinization response elicited by these two peptides in cultured cells was found to be more transient than the systemin response . The correlation between the duration of the alkalinization response and the proteinase inhibitor inducing activities of systemin analogues may be indicative of a casual relationship between ion fluxes and defense gene induction.

Am J Physiol, 1998 Jan, 274(1 Pt 2), F104 - 10
Mechanism and regulation of riboflavin uptake by human renal proximal tubule epithelial cell line HK-2; Kumar CK et al.; Riboflavin (RF), a water-soluble vitamin, is essential for normal cellular functions, growth, and development . Normal RF body homeostasis depends on intestinal absorption and recovery of the filtered vitamin in renal tubules . The mechanism and cellular regulation of the RF renal reabsorption process, especially in the human situation, are poorly understood . The aim of this study was therefore to address these issues, using a recently established human normal renal epithelial cell line, HK-2, as a model . Uptake of RF by HK-2 cells was found to be 1) linear with time for 5 min of incubation and occurring with minimal metabolic alterations, 2) temperature dependent, 3) Na+ independent, 4) saturable as a function of concentration {apparent Michaelis constant (K(m)) of 0.67 +/- 0.21 microM and maximal velocity (Vmax) of 10.05 +/- 0.87 pmol.mg protein-1.3 min-1}, 5) inhibited by structural analogs and anion transport inhibitors, and 6) energy dependent . Protein kinase C-, protein kinase A-, and protein tyrosine kinase-mediated pathways were found to have no role in regulating RF uptake . On the other hand, a Ca2+/calmodulin-mediated pathway appeared to play a role in the regulation of RF uptake by HK-2 cells via an effect on the Vmax, as well as on the apparent K(m) of the RF uptake process . The uptake process of RF was also found to be adaptively regulated by the level of the substrate in the growth medium, with the effect being mediated through changes in the apparent K(m) and the Vmax of the uptake process . These results demonstrate that RF uptake by the human-derived renal epithelial cell line HK-2 is via a carrier-mediated system that is temperature and energy dependent and appears to be under the regulation of a Ca2+/calmodulin-mediated pathways and substrate level in the growth medium.

Br J Dermatol, 1997 Nov, 137(5), 721 - 7
Glutathione efflux associated with a low gamma-glutamyl transpeptidase activity in human melanoma cells; Benathan M; The cellular concentration of reduced glutathione (GSH) modulates the sensitivity of human melanoma cells to alkylating drugs in vitro . To investigate whether the membrane-associated enzyme gamma-glutamyl transpeptidase (gamma-GTP) involved in GSH breakdown was expressed in melanoma cells, the enzymatic activity of gamma-GTP as well as the secretion of GSH were measured in human melanoma cells from four different cell lines (Me8, JUSO, GLL19, Swift) . All the cells showed low gamma-GTP activities (0-1 mU/mg protein) and released GSH in culture supernatants at significant rates . After incubation for 24 h in growth medium containing 0.1 mmol/L cystine, the levels of GSH in supernatants ranged from 56 to 111 nmol GSH/mg protein . The GSH metabolism of melanoma cells was also evaluated by measuring the levels of the melanogenesis intermediate 5-S-cysteinyldopa under different experimental conditions . The results of these experiments suggest that melanoma cells have a low ability to metabolize the tripeptide GSH, which appears to be responsible for GSH secretion and accumulation in culture supernatants.

Zentralbl Hyg Umweltmed, 1997 Apr, 199(6), 513 - 26
{Fungi and bacteria on air filters from heating, ventilation and air-conditioning systems: a method for determination of fungi and bacteria on air filters}; Moritz M et al.; A method was developed for the determination of microorganism concentrations on air filters of HVAC systems, and the influence of different test parameters on the microbiological results was examined by considering various used air filters from several such systems . Microorganisms are detected by shaking air filter samples in fluid, where their concentration is then determined as surface cultures . Since varying the shaking time (30, 60, 90 min) had no influence on the quantitative microorganism determination, a shaking time of 60 minutes was chosen for detection of bacteria, yeasts and moulds . Incubation of blood agar plates either for 4 days at 20 degrees C +/- 2 degrees C or for 2 days at 36 degrees C +/- 1 degree C yielded identical concentrations of bacteria and yeasts . Since results obtained with malt extract agar and Czapek-Dox agar are comparable, one of the two culture media is sufficient for the quantitative determination of moulds on air filters . For statistical evaluation, the inoculation of three parallel agar plates per growth medium was found to be adequate, and the arithmetic mean and the median proved to be equivalent . Investigation on the detection rate showed that, on the average, the method developed demonstrated 80% of the microorganisms detectable on an air filter sample . Thus a simple method is available for quantitative determination of microorganisms on air filters.

Mol Cell Biol, 1998 Feb, 18(2), 926 - 35
Trinucleotide insertions, deletions, and point mutations in glucose transporters confer K+ uptake in Saccharomyces cerevisiae; Liang H et al.; Deletion of TRK1 and TRK2 abolishes high-affinity K+ uptake in Saccharomyces cerevisiae, resulting in the inability to grow on typical synthetic growth medium unless it is supplemented with very high concentrations of potassium . Selection for spontaneous suppressors that restored growth of trk1delta trk2delta cells on K+-limiting medium led to the isolation of cells with unusual gain-of-function mutations in the glucose transporter genes HXT1 and HXT3 and the glucose/galactose transporter gene GAL2 . 86Rb uptake assays demonstrated that the suppressor mutations conferred increased uptake of the ion . In addition to K+, the mutant hexose transporters also conferred permeation of other cations, including Na+ . Because the selection strategy required such gain of function, mutations that disrupted transporter maturation or localization to the plasma membrane were avoided . Thus, the importance of specific sites in glucose transport could be independently assessed by testing for the ability of the mutant transporter to restore glucose-dependent growth to cells containing null alleles of all of the known functional glucose transporter genes . Twelve sites, most of which are conserved among eukaryotic hexose transporters, were revealed to be essential for glucose transport . Four of these have previously been shown to be essential for glucose transport by animal or plant transporters . Eight represented sites not previously known to be crucial for glucose uptake . Each suppressor mutant harbored a single mutation that altered an amino acid(s) within or immediately adjacent to a putative transmembrane domain of the transporter . Seven of 38 independent suppressor mutations consisted of in-frame insertions or deletions . The nature of the insertions and deletions revealed a striking DNA template dependency: each insertion generated a trinucleotide repeat, and each deletion involved the removal of a repeated nucleotide sequence.

Mol Microbiol, 1997 Oct, 26(1), 121 - 32
A new Escherichia coli gene, dsbG, encodes a periplasmic protein involved in disulphide bond formation, required for recycling DsbA/DsbB and DsbC redox proteins; Andersen CL et al.; We have identified and functionally characterized a new Escherichia coli gene, dsbG, whose product is involved in disulphide bond formation in the periplasm . The dsbG gene was cloned from a multicopy plasmid library lacking the dsbB redox protein-encoding gene . Multicopy dsbG-carrying clones were selected, since they allowed E . coli to grow at lethal concentrations of dithiothreitol . In a complementary genetic approach, point mutations were independently obtained and mapped to the dsbG gene . Such mutations led simultaneously to a dithiothreitol-sensitive phenotype and an increased sigmaE-dependent heat shock response, which reflects the presence of misfolded proteins in the extracytoplasm . In agreement with these observations, dsbG mutants were shown to accumulate reduced forms of a variety of disulphide bond-containing proteins in the periplasm . This DsbG defect could be rescued by addition to the growth medium of either oxidized dithiothreitol or cystine, or by overexpression of the dsbA or dsbB genes . DsbG is synthesized as a precursor form of 27.5 kDa and processed to a 25.7kDa mature species located in the periplasm . DsbG was overproduced, purified to homogeneity and shown to have redox properties of thiol-disulphide oxidoreductases in vitro . Replacement of the first Cys residue of the predicted active site, Phe-(Xaa)4-Cys-Pro-Tyr-Cys by Ala, completely inactivated DsbG protein function . Taken together, all our results demonstrate that DsbG acts in vivo as an efficient thiol-disulphide oxidase . In addition, dsbG is the first member of the dsb family for which null mutations are conditionally lethal and can be propagated only if supplemented with oxidants in the growth medium . We propose that the main role of DsbG is to maintain the proper redox balance between the DsbA/DsbB and DsbC systems.

Microb Drug Resist, 1997 Winter, 3(4), 359 - 63
Culture in the presence of sugars increases activity of multi-drug efflux transporter on Haloferax volcanii; Miyauchi S et al.; We found that when a growth medium contained glucose, wild-type cells of Haloferax volcanii were able to grow even in the presence of doxorubicin (DOX), an anti-cancer reagent, whereas they usually cannot grow in its presence . The reason was that cells grown in the presence of glucose (glucose-grown cells) showed high multi-drug efflux activity even though the growth medium contained no DOX or substrates of the transporter . This transporter was ATP-driven and the elevation of efflux activity was not due to an increase in intracellular ATP contents . The activity was increased not only by glucose but also by sugars that could be metabolized.

Biochim Biophys Acta, 1998 Jan 5, 1389(1), 43 - 9
Occurrence of monoacyl-diglucosyl-diacyl-glycerol and monoacyl-bis-glycerophosphoryl-diglucosyl-diacyl-glycerol in membranes of Acholeplasma laidlawii strain B-PG9; Andersson AS et al.; It is shown by thin-layer and high-performance liquid chromatography that the two membrane lipids monoacyl-diglucosyl-diacyl-glycerol (MADGlcDAG) and monoacyl-bis-glycerophosphoryl-diglucosyl-diacyl-glycerol are synthesized by Acholeplasma laidlawii strain B-PG9 when the cells are grown in two different growth media . The two lipids are also synthesized by A . laidlawii strain A-EF22 and their chemical structures have been determined previously by NMR spectroscopy . Since a reversed hexagonal phase is the only liquid-crystalline phase formed by MADGlcDAG, it is concluded that A . laidlawii strain B-PG9, in resemblance to strain A-EF22, synthesizes three membrane lipids that are able to form reversed nonlamellar phases . A comparison of the membrane lipids from the two strains shows that there is essentially one lipid from each strain that differs . However, both these lipids have common physico-chemical properties, namely the ability to form reversed nonlamellar phases . Finally, it is also shown that novel lipids may be synthesized by A . laidlawii through long-time adaptation to altered growth conditions.

J Dermatol Sci, 1997 Nov, 16(1), 52 - 8
Phenotype and proliferation characteristics of cultured spindle-shaped cells obtained from normal human skin and lesions of dermatofibroma, Kaposi's sarcoma, and dermatofibrosarcoma protuberans: a comparison with fibroblast and endothelial cells of the dermis; Bonish BK et al.; Normal human dermis contains mesenchymal cells that are generally referred to as fibroblasts . However the relationships between fibroblasts and endothelial cells with respect to the types of spindle-shaped cells that are present in cultures obtained from tumor bearing-skin is unclear . To explore the potential heterogeneity amongst dermal-derived cells that grow in culture with a spindle-shaped morphology, we compared the immunophenotype and growth characteristics of several types of cells . Besides dermal fibroblasts and microvascular endothelial cells derived from normal adult skin, we also studied large vessel-derived endothelial cells, and spindle-shaped cells derived from three different tumor-bearing dermal-based neoplasms . Kaposi's sarcoma (KS), dermatofibroma (DF), and dermatofibrosarcoma protuberans (DFSP) . A broad panel of eight different antibodies were used to immunophenotype the multi-passaged cultured cells . Spindle-shaped cells from all three neoplasms could be distinguished from the normal skin derived fibroblasts by their constitutive expression of factor XIIIa, and the gamma-interferon induced expression of VCAM-1 . All seven types of cultured cells stained positive for s-actin and proline-4-hydroxylase, and none of the cells expressed CD34 . Both large and small-vessel derived endothelial cells expressed factor VIII, ELAM-1, and VCAM-1 . Using two different types of growth media, significant differences were also observed amongst these cultured cell types . Spindle-shaped cells from DFSP did not grow in DMEM containing 10% fetal bovine serum (DMEM-FBS); but they proliferated in KS cell growth medium (KSGM) . Spindle-shaped cells from DF grew best in KSGM, but not in DMEM-FBS . KS tumor cells grew well in KSGM, but not in DMEM-FBS . Fibroblasts proliferated in DMEM-FBS, but failed to grow in KSGM; and even when pre-treated with conditioned medium from a transformed KS cell line (i.e . SLK cells), no fibroblast proliferation could be induced in KSGM . These results indicate that KS cell line (i.e . SLK cells), no fibroblast proliferation could be induced in KSGM . These results indicate that even though dermal-derived cells can have an identical spindle-shape by light microscopy, significant heterogeneity can be defined amongst such cells from normal and tumor-bearing human skin . Having established culture conditions to propagate these different cell types and phenotypic criteria to distinguish them from one another, will provide new research opportunities to explore the function and ontogeny of the diverse mesenchymal cells that take on a spindle-shaped morphology in culture.

J Biol Chem, 1997 Dec 19, 272(51), 32150 - 7
Mutations in a bacterial mechanosensitive channel change the cellular response to osmotic stress; Blount P et al.; MscL is a channel found in bacterial plasma membranes that opens a large pore in response to mechanical stress . Here we demonstrate that some mutations within this channel protein (K31D and K31E) evoke a cellular phenotype in which the growth rate is severely depressed . Increasing the osmolarity of the growth medium partially rescues this "slowed growth" phenotype and decreases an abnormal cytosolic potassium loss observed in cells expressing the mutants . In addition, upon sudden decrease in osmolarity (osmotic downshock) more cytoplasmic potassium is released from cells expressing the mutants than cells expressing wild-type MscL . After osmotic downshock, all cells remained viable; hence, the differences in potassium efflux observed are not due to cell lysis but instead appear to be an exaggeration of the normal response to this sudden change in environmental osmolarity . Patch clamp studies in native bacterial membranes substantiate the hypothesis that these mutant channels are more sensitive to mechanical stresses, especially at voltages approaching those estimated for bacterial membrane potentials . These data are consistent with a crucial role for MscL in the adaptation to large osmotic downshock and suggest that if the normally tight regulation of MscL gating is disrupted, cell growth can be severely inhibited.

Am J Physiol, 1997 Dec, 273(6 Pt 1), C1859 - 67
Phosphorylation of occludin correlates with occludin localization and function at the tight junction; Wong V; Multiple forms of occludin were found in Madin-Darby canine kidney (MDCK) cells . In the absence of cell-to-cell contacts achieved by incubating cells in low-calcium growth medium, a cluster of lower-molecular-weight (LMW) occludin bands (approximately 65,000-68,000) was present in both MDCK I and II cells . On formation of tight junctions, achieved by changing the low-calcium growth medium to normal-calcium growth medium, a cluster of higher-molecular-weight (HMW) bands (approximately 72,000-75,000 for MDCK I cells and approximately 70,000-73,000 for MDCK II cells) was also expressed . The HMW occludin bands could be eliminated by phosphatase treatment . Therefore, the HMW forms of occludin appeared to be the hyperphosphorylated product of the LMW forms . These HMW forms were Triton X-100 insoluble, which correlated with their localization at the tight junctions . Furthermore, depletion of tight junction-localized occludin by an occludin extracellular domian peptide (20) correlated with a decrease in the HMW forms of occludin . In conclusion, phosphorylation of occludin may be a mechanism by which occludin localization and function are regulated.

J Immunol Methods, 1997 Oct 27, 208(2), 151 - 8
Optimization of the sulforhodamine B colorimetric assay; Papazisis KT et al.; Sulforhodamine B (SRB) protein staining has been widely used for cell proliferation and chemosensitivity testing, substituting for tetrazolium-based assays . However, the cell fixation step in the original assay is subject to error . We tested whether aspiration of medium with an automatic microplate multiwash device prior to fixation improves the method for adherent cells . A panel of adherent cell lines was used . Signal-to-noise ratios were significantly increased in the new assay . Coefficients of variation (CV) between replicate wells were significantly lower especially at lower cell densities . The linearity of the method improved, with absolute linearity over the whole range of cell densities . The aspiration procedure dislodged only negligible numbers of cells . Cytotoxicity testing using the cytotoxic agent paclitaxel showed no IC50 (50% inhibitory concentration) differences between the new and original methods but a better CV was associated with the optimized protocol . We conclude that aspiration of the growth medium prior to fixing comprises a safe and reliable practice which improves CV, linearity and the signal-to-noise ratio of the SRB assay.

Prostate, 1998 Jan 1, 34(1), 10 - 22
Development and characterization of a mouse prostate adenocarcinoma cell line: ductal formation determined by extracellular matrix; Jorcyk CL et al.; BACKGROUND: Tumor vaccines show promise as a new approach for treating cancer . We have developed a murine prostate cancer cell line which can be used to study growth factor and extracellular matrix regulation of prostate differentiation and will be useful for generating tumor vaccines using the C3(1)/TAG transgenic model of prostate cancer . METHODS: Pr-14 cells were established in defined growth media (GM) and grown in GM, GM + 2% fetal bovine serum (FBS) or DMEM + 10% FBS on plastic, collagen, or Matrigel . Immunofluorescence and Western blot analyses were performed using antibodies to cytokeratin, vimentin, SV40 large T-antigen, and androgen receptor (AR) . RESULTS: Pr-14 cells are cytokeratin-positive, vimentin-negative, and express SV40 large T-antigen . These cells are tumorigenic when injected into athymic nude mice and appear to be androgen-independent . Pr-14 cell lines are nontumorigenic when injected into syngeneic FVB/N mice, but form tumors in transgenic TAG-expressing FVB/N mice . Cell growth and morphology are dependent on media composition which determines whether ductal or acinar structures form when grown on Matrigel . CONCLUSIONS: The mouse prostate adenocarcinoma cell line, Pr-14, undergoes alterations in the state of differentiation dependent upon serum concentration when grown on Matrigel . The Pr-14 cell line is a useful reagent to study prostate cell/extracellular matrix interactions, and for immunotherapy and cancer vaccine studies in C3(1)/TAG transgenic mice.

J Biolumin Chemilumin, 1997 May-Jun, 12(3), 165 - 75
Effects of the composition of bacteriological growth media on a chemiluminometric assay of beta-galactosidase in Escherichia coli; Van Poucke SO et al.; The effects of the composition of bacteriological growth media on the light output in a chemiluminometric assay of beta-galactosidase in Escherichia coli using 1,2-dioxetane substrates has been studied . In this assay a basic conflict exists between conditions that promote optimal bacterial growth and those conducive to maximal chemiluminescence . Common medium ingredients such as yeast or beef extract, protein hydrolysates and lactose suppress light emission and/or lead to high backgrounds . Quenching of light emission is probably partly due to light absorption by medium ingredients such as oxgall, and partly to interference with the reaction triggering the chemiluminescent process . Elevated backgrounds are caused by the presence of high concentrations of protein hydrolysates, which interact with the alkali in the accelerator solution . Only two purposely developed media, i.e . ILM and Colicult are shown to reconcile the requirements of growth support with that of optimal luminescent properties.

Protein Expr Purif, 1997 Dec, 11(3), 289 - 96
Divergent effects of chaperone overexpression and ethanol supplementation on inclusion body formation in recombinant Escherichia coli; Thomas JG et al.; The proper folding of aggregation-prone recombinant proteins in Escherichia coli can be facilitated by co-overexpressing specific molecular chaperones or by culturing the cells in the presence of ethanol or other agents that upregulate the synthesis of all heat-shock proteins (hsps) . We have investigated the effect of combining direct chaperone overproduction with ethanol supplementation on the cytoplasmic folding of two aggregation-prone model proteins, preS2-S'-beta-galactosidase and human SPARC . In 25-ml shake flask cultures grown at 30 degrees C, addition of 3% (v/v) ethanol to the growth medium prior to inoculation improved the chaperone-mediated increase in the yields of active preS2-S'-beta-galactosidase 1.5- to 2-fold . When cultures overexpressing the dnaKJ operon were grown in the presence of ethanol, the levels of enzymatic activity were 5-fold higher relative to control cells and preS2-S'-beta-galactosidase aggregation was almost entirely abolished . Combining DnaK-DnaJ overexpression and growth of the cells at temperatures lower than 30 degrees C did not result in a comparable increase in activity . Although the individual effects of ethanol supplementation and dnaKJ overproduction were more limited when the culture volume was raised, a synergistic improvement in preS2-S'-beta-galactosidase activity was observed when the two approaches were used in concert . In contrast, ethanol supplementation promoted the aggregation of human SPARC, a protein exhibiting a chaperone dependency similar to that of preS2-S'-beta-galactosidase . Our results show that ethanol can exert complex and divergent effects on inclusion body formation and that the beneficial effect of the solvent on recombinant protein folding cannot simply be explained by an increase in the intracellular concentration of molecular chaperones.

Invasion Metastasis, 1997, 17(1), 26 - 41
Interrelation of motility, cytoskeletal organization and gap junctional communication with invasiveness of melanocytic cells in vitro; Helige C et al.; Intercellular communication and the active movement of malignant cells into and through host tissue barriers play a critical role during the complex process of tumor invasion . Motile activity, cytoskeletal actin and vinculin organization as well as gap junctional communication of in vivo benign and malignant melanocytes were compared and related to in vitro invasiveness . Normal melanocytes, Melan-a, showed significantly less motile activity, a higher organization of the actin cytoskeleton and more vinculin-containing cell-substratum adhesion plaques than highly metastatic melanoma cells, K1735-M2 . There was no pronounced difference in gap junctional communication under comparable culture conditions . However, cultivation of Melan-a cells in a conventional melanocyte growth medium containing the tumor promoter 12-O-tetradecanoylphorbol-13-acetate (TPA) enhanced intercellular communication . Melanocytes were less invasive than melanoma cells both in the embryonic chick heart model and in the Matrigel invasion assay . The least invasive activity was determined for melanocytes cultivated in TPA-deficient medium indicating that the medium supplement TPA stimulates invasion . The comparison of certain in vitro properties of both melanocytic cell lines revealed a positive correlation of motility with in vitro invasion, whereas an inverse correlation was found for the degree of actin filament organization as well as for the number of vinculin plaques . Gap junctional communication was not directly related to in vitro invasiveness.

J Vasc Surg, 1997 Dec, 26(6), 1002 - 7; discussion 1007-8
Proliferation and extracellular matrix production by human infragenicular smooth muscle cells in response to interleukin-1 beta; Forsyth EA et al.; PURPOSE: Atherosclerotic peripheral vascular disease commonly involves the infragenicular arterial tree . Our study evaluated the effect of interleukin (IL)-1 beta on the proliferation of vascular smooth muscle cells (VSMCs) derived from atherosclerotic infragenicular arteries of human subjects who underwent below-knee amputation, as well as the role of IL-1 beta in VSMCs' production of extracellular matrix components, substances that are important in the transformation of VSMCs from the contractile to the synthetic phenotype . This transformation to the synthetic phenotype is an important step in the formation of the atherosclerotic lesion . METHODS: Cultures were identified as being of smooth muscle origin through staining with the cytoskeletal marker, alpha-smooth muscle actin . Proliferation assays were performed by seeding confluent cultures of passages 4 to 7 into six-well plates at 10,000 cells per well . After serum starvation, samples were incubated with IL-1 beta (1 ng/ml) . Cell number was determined on a daily basis . To study extracellular matrix production, cells were propagated in tissue culture chamber slides in the absence or presence of growth media containing IL-1 beta . After fixation with 100% methanol, each sample was stained with a primary antibody specific for an extracellular matrix component . After staining with the fluorescein-tagged secondary antibody, each sample was examined using immunofluorescent microscopic examination . RESULTS: The results of our proliferation assays showed that IL-1 beta caused a significant increase in the proliferation of VSMCs at 24, 48, 72, and 96 hours (p < or = 0.003 when comparing IL-1 beta-treated samples with control specimens at each time period using unpaired t test) . The number of IL-1 beta-treated cells at 96 hours was double the number present in the control samples (16,033 +/- 238 vs 8102 +/- 824) . When compared with control samples, IL-1 beta was found to affect the production of extracellular matrix proteins by infragenicular VSMCs . IL-1 beta caused an increase in the production of fibronectin, a decrease in the production of laminin, and no change in the production of collagen type IV . CONCLUSIONS: These results suggest that interleukin-1 beta acts as a potent stimulant of the proliferation of human infragenicular VSMCs . IL-1 beta also acts to augment the production of fibronectin by these cells . Fibronectin has been implicated in the phenotypic transformation of VSMCs from the contractile to the synthetic state . Therefore, IL-1 beta may serve as an important regulatory factor in the development of atherosclerosis by stimulating the proliferation of VSMCs and their transformation to the synthetic state, two important steps in the formation of the atherosclerotic lesion.

J Neurosci, 1997 Dec 15, 17(24), 9573 - 82
Nerve growth factor modulates synaptic transmission between sympathetic neurons and cardiac myocytes; Lockhart ST et al.; Regulation of heart rate by the sympathetic nervous system involves the release of norepinephrine (NE) from nerve terminals onto heart tissue, resulting in an elevation in beat rate . Nerve growth factor (NGF) is a neurotrophin produced by the heart that supports the survival and differentiation of sympathetic neurons . Here we report that NGF also functions as a modulator of sympathetic synaptic transmission . We determined the effect of NGF on the strength of synaptic transmission in co-cultures of neonatal rat cardiac myocytes and sympathetic neurons from the superior cervical ganglion (SCG) . Synaptic transmission was assayed functionally, as an increase in the beat rate of a cardiac myocyte during stimulation of a connected neuron . Application of NGF produced a pronounced, reversible enhancement of synaptic strength . We found that TrkA, the receptor tyrosine kinase that mediates many NGF responses, is expressed primarily by neurons in these cultures, suggesting a presynaptic mechanism for the effects of NGF . A presynaptic model is further supported by the finding that NGF did not alter the response of myocytes to application of NE . In addition to the acute modulatory effects of NGF, we found that the concentration of NGF in the growth medium affects the level of synaptic transmission in cultures of sympathetic neurons and cardiac myocytes . These results indicate that in addition to its role as a survival factor, NGF plays both acute and long-term roles in the regulation of developing sympathetic synapses in the cardiac system.

Microbiology, 1997 Dec, 143 ( Pt 12), 3877 - 88
The patB gene of Dictyostelium discoideum encodes a P-type H(+)-ATPase isoform essential for growth and development under acidic conditions; Coukell MB et al.; During growth and early development of Dictyostelium discoideum, the amoebae exhibit transient pH changes in their cytosol (pHi) and external medium which correlate with the extrusion of H+ from the cell by a plasma membrane pump . Moreover, the changes in pHi have been postulated to influence early prestalk/prespore differentiation during development . To learn more about the role of H+ fluxes in Dictyostelium, we cloned and analysed cDNAs of the gene patB, which appears to encode a P-type H(+)-ATPase . The patB ORF encodes a protein (termed PAT2) of 1058 amino acids with a calculated molecular mass of 117,460 Da . When aligned with other P-type ion-transport ATPases, PAT2 showed the greatest amino acid sequence identity with plasma membrane H(+)-ATPases of plants and fungi and considerably lower identity with other monovalent cation pumps and with Ca2+ pumps . Northern and Western analyses revealed that patB is expressed at very low levels in cells growing at neutral pH, but it is up-regulated rapidly and dramatically when the cells are shifted to an acidic medium . Immunofluorescence analysis indicated that PAT2 resides on the plasma membrane . When patB was disrupted by homologous recombination, the cells grew and developed normally at neutral and slightly alkaline pHs but they were unable to grow or develop at pH 5.0, and they slowly died . In growth medium at pH 6.8, patB+ and patB cells exhibited similar levels of vanadate-sensitive ATPase activity . However, when the cells were shifted to pH 5.0, this activity rapidly increased about twofold in the control cells but not in the mutant cells . Despite the lower ATPase activity in patB cells, they showed relatively normal H+ fluxes and only a slight decrease in pHi when incubated in acidic medium . Together, these results suggest that patB encodes an acid-inducible P-type H(+)-ATPase which is indispensable for the survival of Dictyostelium cells in moderately acidic external environments.

Br J Pharmacol, 1997 Dec, 122(7), 1441 - 9
Ruthenium complexes as nitric oxide scavengers: a potential therapeutic approach to nitric oxide-mediated diseases; Fricker SP et al.; 1 . Ruthenium(III) reacts with nitric oxide (NO) to form stable ruthenium(II) mononitrosyls . Several Ru(III) complexes were synthesized and a study made of their ability to bind NO, in vitro and also in several biological systems following expression of the inducible isoform of nitric oxide synthase (iNOS) . Here we report on the properties of two, related polyaminocarboxylate-ruthenium complexes: potassium chloro{hydrogen(ethylenedinitrilo)tetraacetato}ruthenate+ ++ (=JM1226; CAS no.14741-19-6) and aqua{hydrogen(ethylenedinitrilo)tetraacetato}ruthenium (=JM6245; CAS no.15282-93-6) . 2 . Binding of authentic NO by aqueous solutions of JM1226 yielded a product with an infrared (IR) spectrum characteristic of an Ru(II)-NO adduct . A compound with a similar IR spectrum was obtained after reacting JM1226 with S-nitroso-N-acetylpenicillamine (SNAP) . 3 . The effect of JM1226 or JM6245 on nitrite (NO2-) accumulation in cultures of macrophages (RAW 264 line) 18 h after stimulating cells with lipolysaccharide (LPS) and interferon-gamma (IFNgamma) was studied . Activation of RAW264 cells increased NO2- levels in the growth medium from (mean+/-1 s.e.mean) 4.9+/-0.5 microM to 20.9+/-0.4 microM . This was blocked by actinomycin D (10 microM) or cycloheximide (5 microM) . The addition of JM1226 or JM6245 (both 100 microM) to activated RAW264 cells reduced NO2- levels to 7.6+/-0.2 microM and 8.8+/-0.6 microM, respectively . N(G)-methyl-L-arginine (L-NMMA; 250 microM) similarly reduced NO2- levels, to 6.1+/-0.2 microM . 4 . The effect of JM1226 or JM6245 on NO-mediated tumour cell killing by LPS+IFNgamma-activated macrophages (RAW 264) was studied in a co-culture system, using a non-adherent murine mastocytoma (P815) line as the 'target' cell . Addition of JM1226 or JM6245 (both 100 microM) to the culture medium afforded some protection from macrophage-mediated cell killing: target cell viability increased from 54.5+/-3.3% to 93.2+/-7.1% and 80.0+/-4.6%, respectively (n=6) . 5 . Vasodilator responses of isolated, perfused, pre-contracted rat tail arteries elicited by bolus injections (10 microl) of SNAP were attenuated by the addition of JM1226 or JM6245 (10(-4) M) to the perfusate: the ED50 increased from 6.0 microM (Krebs only) to 1.8 mM (Krebs + JM6245) and from 7 microM (Krebs only) to 132 microM (Krebs + JM1226) . Oxyhaemoglobin (5 microM) increased the ED50 value for SNAP from 8 microM to 200 microM . 6 . Male Wistar rats were injected with bacterial LPS (4 mg kg(-1); i.p.) to induce endotoxaemia . JM1226 and JM6245 (both 100 microM) fully reversed the hyporesponsiveness to phenylephrine of tail arteries isolated from animals previously (24 h earlier) injected with LPS . Blood pressure recordings were made in conscious LPS-treated rats using a tail cuff apparatus . A single injection of JM1226 (100 mg kg(-1), i.p.) administered 20 h after LPS (4 mg kg(-1), i.p.) reversed the hypotension associated with endotoxaemia . 7 . The results show that JM1226 and JM6245 are able to scavenge NO in biological systems and suggest a role for these compounds in novel therapeutic strategies aimed at alleviating NO-mediated disease states.

Indian J Exp Biol, 1997 Jul, 35(7), 727 - 34
Comparative in vitro and in vivo evaluation of himachalol in murine invasive aspergillosis; Chowdhry L et al.; Aspergilli are increasingly important infections in immunocompromised patients (ICP) . The available antifungals often cause discrepancies in laboratory determination of MICs and a correlation in therapy . An effort was made to compare in vitro techniques for testing of antifungals, viz . polyenes, imidazoles, 5-fluorocytosine, amorolfine; and screened a phytoproduct- himachalol (a sesquiterpene alcohol) from Cedrus deodara (Roxb.) Loud against A . fumigatus clinical isolates (24) by macrobroth two-fold seal dilution (TFSD), microbroth microtitre (MT) and disc diffusion (DD) techniques using various broth/agar media at varying periods of incubation . The best activity in terms of geometric mean (GM) (GM.MIC < 0.39 microgrmas ml-1) was obtained with SCZ in the broth by both MT or TFSD technique followed by ECZ (GM.MIC 0.39 micrograms ml-1) and ITZ (GM.MIC 0.39-0.8 micrograms ml-1) in RPMI-1640 . Overall RPMI-1640 was found to be the most suitable growth medium for testing of azoles or amorolfine, and YNB for polyene and 5-FC . MT technique was the most sensitive quantitative, reproducible, rapid and economical compared to other techniques . The treatment of Swiss mice with himachalol (200 mg kg-1, po) once a day, for 7 days, provided 60% protection concomitantly with increased MST (15 days) against invasive aspergillosis . A combination of himachalol (200 mgkg-1) plus SCZ (5 mgkg-1) showed better regimen in the therapy evidenced by enhanced survival (80%) of mice significantly (p < 0.001) with prolonged MST (> 15 days) compared to control . The treatments also reduced cfu (mean log10) burden of A . fumigatus from kidney.

Plant J, 1997 Nov, 12(5), 1011 - 20
An Arabidopsis mutant showing reduced feedback inhibition of photosynthesis; Van Oosten JJ et al.; Many plant genes are responsive to sugars but the mechanisms used by plants to sense sugars are unknown . A genetic approach has been used in Arabidopsis to identify genes involved in perception and transduction of sugar signals . For this purpose, an in vivo reporter system was established consisting of the light- and sugar-regulated plastocyanin promoter, fused to the luciferase coding sequence (PC-LUC construct) . At the seedling stage, expression of the PC-LUC gene is repressed by sucrose, and a number of sucrose-uncoupled (sun) mutants were selected in which sucrose is unable to repress the activity of the PC promoter . Three mutants have been characterized in more detail . The sugar analog 2-deoxy-D-glucose (2DG) was used to repress whole plant photosynthesis, PC-LUC gene expression and total ribulose-1,5-bisphosphate activity . It was found that the sun6 mutation makes plants unresponsive to these 2DG-induced effects . Moreover, unlike wild-type plants, sun6 mutants are insensitive to elevated levels of glucose in the growth medium . These findings suggest that the SUN6 gene is active in a hexose-activated signal transduction pathway.

Environ Health Perspect, 1998 Jan, 106(1), 17 - 22
Bay or baylike regions of polycyclic aromatic hydrocarbons were potent inhibitors of Gap junctional intercellular communication; Weis LM et al.; Many polycyclic aromatic hydrocarbons (PAHs) are known carcinogens, and a considerable amount of research has been devoted to predicting the tumor-initiating potential of PAHs based on chemical structure . However, there has been little research into the effects of PAHs on the epigenetic events of tumor promotion and no structural correlation has been made thereof . Gap junctional intercellular communication (GJIC) activity was used in this study as an epigenetic biomarker to determine the structure-activity relationships of twelve different PAHs . The PAHs used were naphthalene, 1-methylnaphthalene, 2-methylnaphthalene, anthracene, 1-methylanthracene, 2-methylanthracene, 9-methylanthracene, 9, 10-dimethylanthracene, phenanthrene, fluorene, 1-methylfluorene, and fluoranthene . Results showed that PAHs containing bay or baylike regions inhibited GJIC more than did the linear PAHs . The nonnaphthalene PAHs were not cytotoxic as determined by a vital dye uptake assay, but the naphthalene compounds were cytotoxic at the higher doses, indicating that the down regulation of GJIC by these naphthalenes could be a consequence of general membrane damage . Inhibition of GJIC by all the inhibitory PAHs was reversed when the cells were refreshed with PAH-free growth medium . Inhibition of GJIC occurred within 0.5-5 min and correlated with the aqueous solubility of the PAHs . The present study revealed that there are structural determinants of epigenetic toxicity as determined by GJIC activity.

Proteins, 1997 Dec, 29(4), 553 - 61
A combinatorial library for the binuclear metal center of bacterial phosphotriesterase; Watkins LM et al.; Phosphotriesterase (PTE) is a zinc metalloenzyme that catalyzes the hydrolysis of an extensive array of organophosphate pesticides and mammalian acetylcholinesterase nerve agents . Although the three-dimensional crystal structure of PTE has been solved (M . M . Benning et al., Biochemistry 34:7973-7978, 1995), the precise functions of the individual amino acid residues that interact directly with the substrate at the active site are largely unknown . To construct mutants of PTE with altered specificities for particular target substrates, a simple methodology for generating a library of mutants at specific sites was developed . In this investigation, four of the six protein ligands to the binuclear metal site (His-55, His-57, His-201, and His-230) were targeted for further characterization and investigation . Using the polymerase chain reaction (PCR) protocols, a library of modified PTE genes was generated by simultaneously creating random combinations of histidine and cysteine codons at these four positions . The 16 possible DNA sequences were isolated and confirmed by dideoxy-DNA sequencing . The 16 mutant proteins were expressed in Escherichia coli and grown with the presence or absence of 1 mM CoCl2, ZnSO4, or CdSO4 in the growth medium . When grown in the presence of CoCl2, the H57C protein cell lysate showed greater activity for the hydrolysis of paraoxon than the wild type PTE cell lysate . H201C and H230C exhibited up to 15% of the wild-type activity, while H55C, a green protein, was inactive under all assay conditions . All other mutants had < 10(-5) of wild-type activity . None of the purified mutants that exhibited catalytic activity had a significantly altered Km for paraoxon.

Microsc Res Tech, 1997 Nov 1, 39(3), 297 - 304
Reevaluation of the effect of lysoyzme on Escherichia coli employing ultrarapid freezing followed by cryoelectronmicroscopy or freeze substitution; Wild P et al.; Lysozyme is able to lyse Gram-positive bacteria acting as muramidase on the peptidoglycan polymer . Gram-negative bacteria in vitro are not lysed by lysozyme . It was assumed that the peptido-glycan is protected by the outer membrane and thus that Gram-negative bacteria are not affected by lysozyme without the aid of other factors such as EDTA or complement which enable lysozyme to penetrate the outer membrane . Accidentally, Pellegrini et al . {(1992) J . Appl . Bacteriol., 72:180-187} found that lysozyme per se is able to kill some Gram-negative bacteria . On the basis of morphological and immunocytochemical findings obtained from chemically fixed bacteria, it was concluded that lysozyme does not lyse Gram-negative bacteria but affects the cytoplasm of for example, Escherichia coli, leading to its disintegration, whilst the membranes do not break down . In an attempt to clarify the action of lysozyme on E . coli, we employed cryotechniques including ultrarapid freezing, cryomicroscopy and freeze substitution, and immunolabeling . Bacteria that were immediately frozen after exposure to lysozyme remained morphologically intact . Individual bacteria plated on agar after exposure to lysozyme were mostly intact when frozen within a few seconds . However, inner and outer membranes of 80% of the bacteria were disrupted, whereas the cytoplasm of only a few bacteria showed signs of disintegration when bacteria were frozen with a delay of only 5 min of plating onto pure agar or agar containing growth medium . After a period of time of 15 min between plating onto agar and freezing, about 97% of the bacteria showed changes of disintegration of various extent . Immunolabeling showed that lysozyme binds to the outer cell membrane and may penetrate the membrane, reaching the periplasmic space and possibly the inner cell membrane . The ultrastructural findings and the results of antibacterial assays suggest that lysozyme is bactericidal for E . coli but is not able to induce disintegration . Disintegration is accomplished by changes of the environment starting at the cell membranes . The mechanism by which lysozyme penetrates the membrane, the way it acts to be bactericidal, and the way disintegration is initiated remain to be clarified.

Am J Physiol, 1997 Nov, 273(5 Pt 1), C1650 - 6
Steroid hormone-dependent expression of blocker-sensitive ENaCs in apical membranes of A6 epithelia; Baxendale-Cox LM et al.; Weak channel blocker-induced noise analysis was used to determine the way in which the steroids aldosterone and corticosterone stimulated apical membrane Na+ entry into the cells of tissue-cultured A6 epithelia . Among groups of tissues grown on a variety of substrates, in a variety of growth media, and with cells at passages 73-112, the steroids stimulated both amiloride-sensitive and amiloride-insensitive Na+ transport as measured by short-circuit currents in chambers perfused with either growth medium or a Ringer solution . From baseline rates of blocker-sensitive short-circuit current between 2 and 7 microA/cm2, transport was stimulated about threefold in all groups of experiments . Single channel currents averaged near 0.3 pA (growth medium) and 0.5 pA (Ringer) and were decreased 6-20% from controls by steroid due to the expected decreases of fractional transcellular resistance . Irrespective of baseline transport rates, the steroids in all groups of tissues stimulated transport by increase of the density of blocker-sensitive epithelial Na+ channels (ENaCs) . Channel open probability was the same in control and stimulated tissues, averaging approximately 0.3 in all groups of tissues . Accordingly, steroid-mediated increases of open channel density responsible for stimulation of Na+ transport are due to increases of the apical membrane pool of functional channels and not their open probability.

J Biol Chem, 1997 Nov 28, 272(48), 30504 - 11
Cytosolic 85-kDa phospholipase A2-mediated release of arachidonic acid is critical for proliferation of vascular smooth muscle cells; Anderson KM et al.; Recent evidence suggests that arachidonic acid (AA) may be involved in regulating cellular proliferation . The predominant mechanism of AA release from cellular phospholipids is via phospholipase A2 (PLA2) hydrolysis . The purpose of this study was to examine the roles of the distinct 14-kDa and 85-kDa PLA2 enzymes in human coronary artery vascular smooth muscle cell (hCAVSMC) proliferation . Cultured hCAVSMCs proliferate in the presence of growth medium with a typical doubling time of 30-40 h, grow at a slower proliferative rate upon reaching confluency (day 8), and eventually undergo contact inhibition of growth (day 10) . Neither Type II 14-kDa PLA2 activity nor mass changed over a 10-day culture period . In contrast, 85-kDa PLA2 protein activity and mRNA decreased as time in culture progressed . This reduction in 85-kDa PLA2 correlated with reductions in DNA synthesis and suggested a possible association between 85-kDa PLA2 and proliferation . To directly evaluate the role of the 85-kDa PLA2 in proliferation we examined the effects of an 85-kDa PLA2 inhibitor (AACOCF3) and 85-kDa PLA2 antisense oligonucleotides on proliferation . Both reagents dose dependently inhibited proliferation, whereas a 14-kDa PLA2 inhibitor (SB203347), a calcium-independent PLA2 inhibitor (HELSS), an 85-kDa sense oligonucleotide, and a nonrelevant scrambled control oligonucleotide had no effect . The mechanism by which 85-kDa PLA2 influences cellular proliferation remains unclear . Inhibition of 85-kDa PLA2 activity produced neither phase-specific cell cycle arrest nor apoptosis (fluorescence-activated cell sorter analysis) . Addition of AA (20 mu M) attenuated the effects of both AACOCF3 and 85-kDa antisense oligonucleotides implicating AA as a key mediator in cellular proliferation . However, although prostaglandin E2 (PGE2) was present in the culture medium, it peaked early (day 3) in culture, and indomethacin had no effect on cellular proliferation indicating that hCAVSMC proliferation was not mediated through PGE2 . These data provide the first direct evidence that PLA2 is involved in control of VSMC proliferation and indicate that 85-kDa PLA2-mediated liberation of AA is critical for cellular proliferation.

Arch Biochem Biophys, 1997 Nov 15, 347(2), 271 - 4
Superoxide imposes leakage of sulfite from Escherichia coli; Benov L et al.; Escherichia coli, which lacks the cytosolic superoxide dismutases, exhibits several nutritional auxotrophies when growing aerobically . The cysteine/methionine requirement, which is one of these, was previously shown to be due to leakage from the cells, and accumulation in the medium, of a metabolic intermediate on the biosynthetic route to these amino acids . The parental strain does not significantly accumulate this compound . It is now shown that treatment with alkaline cyanide releases sulfite from this compound, a property shared by alpha-hydroxy sulfonic acids (carbonyl-bisulfite adducts) . Since E . coli accumulates carbonyl compounds in the growth medium, it appears likely that the sulfitogenic compounds accumulated by the sodA sodB strain are alpha-hydroxy sulfonic acids .

Brain Res, 1997 Oct 24, 772(1-2), 63 - 70
Chronic exposure to inorganic lead increases high-threshold voltage-gated calcium currents in rat pheochromocytoma (PC12) cells; Hegg CC et al.; Rat pheochromocytoma (PC12) cells were exposed to lead acetate (0, 10, 25 and 50 microM) in their growth media for up to 12 weeks . High-threshold voltage-gated calcium currents were recorded each week from nerve growth factor-differentiated PC12 cells using the whole-cell patch-clamp technique . Chronic exposure for 1 month did not modify peak or sustained calcium current amplitudes in lead-treated cells when compared to sister control cultures . Two month exposure to 25 and 50 microM significantly increased peak and sustained calcium current amplitudes, while 10 microM had little effect . During the third month of exposure, peak and sustained calcium current amplitudes remained increased in the cells exposed to 25 and 50 microM lead acetate . By the end of the second month of exposure to 25 and 50 microM lead acetate, the voltage at which maximal current amplitude was attained shifted from + 10 mV to 0 mV . The observed effects of toxicologically relevant lead concentrations on high-threshold calcium currents in chronically exposed mammalian cells provide further support for the notion that at least one cellular target of the heavy metal's neurotoxic action may be the voltage-gated calcium channel.

J Cell Physiol, 1998 Jan, 174(1), 115 - 24
Co-regulation of pituitary tumor cell adhesion and prolactin gene expression by glucocorticoid; Spangler PR et al.; Rat 235-1 pituitary tumor cells are lactotrophs producing high levels of prolactin (PRL) . Dexamethasone (Dex, 100 nM) inhibits PRL gene expression in 235-1 cells by 50%, while simultaneously decreasing cell replication and cell-cell aggregation . To determine the time course of Dex action, we used a quantitative assay for cell-cell interaction, based on the number of single cells present before and after re-aggregation of dispersed cells . 235-1 cells were cultured in growth medium or medium plus 100 nM Dex for 1-4 days before assay . Control cells had 90% re-aggregation on all days of assay . Aggregation of Dex-treated cells decreased to 55% by day 4 . Dex treatment also reduced cell numbers by 40%, but this decrease did not contribute to reduced aggregation . To determine the mechanism of Dex-inhibited cell-cell adhesion, we examined the expression of cadherins and catenins . Cadherin-related mRNAs (P- and N-cadherin probes) were detectable in 235-1 cells, but their levels were unchanged by Dex . A pancadherin antibody was unable to detect classical cadherins in these cells . Both alpha- and beta-catenins were detected by Western blotting and their levels were decreased by Dex . Unlike control aggregates, aggregates of Dex-treated cells were able to inhibit expression of PRL mRNA when added to monolayers of 235-1 cells . These data suggest that Dex influences cadherin function by inhibiting catenin expression and that this has the functional consequence of altering 235-1 cell-cell interactions . Overall the data show that Dex affects important aspects of lactotroph function other than PRL gene expression . These changes may include physical alterations in pituitary cell contacts that further support a change in functional state.

Eur J Biochem, 1997 Nov 1, 249(3), 684 - 9
Synthesis of soybean agglutinin in bacterial and mammalian cells; Adar R et al.; The cDNA of soybean agglutinin (SBA), a glycoprotein lectin, obtained from the mRNA of soybean seeds at mid-maturation, was cloned in a lambda gt 10 phage and subcloned in a pUC-8 plasmid . Probing with a fragment of the lectin gene {Vodkin, L . O., Rhodes, P . R . & Goldberg, R . B . (1983) Cell 34, 1023-1031} afforded a clone of 1012 nucleotides containing the complete coding region of 858 nucleotides for the precursor to soybean agglutinin . The deduced amino acid sequence contains the 253 residues of the mature lectin and an hydrophobic N-terminal signal peptide of 32 amino acids . Expression in Escherichia coli of the cDNA coding for the precursor to the lectin or for the mature lectin led to the accumulation of large quantities of inclusion bodies, from which mature SBA was isolated in small yield (up to 1 mg/l) . It was identical with the native lectin in the hemagglutinating activity and carbohydrate specificity, N-terminal sequence and oligomeric structure, but, because it was not glycosylated, its subunit mass was lower by 2 kDa . Our findings show that pre-SBA is processed into the mature form in the bacteria, and that, contrary to what has been suggested {Nagai, K . & Yamaguchi, H . (1993) J . Biochem . (Tokyo) 113, 123-125}, glycosylation is not essential for the folding of the lectin, nor for its subunit assembly into a biologically active tetramer . To obtain recombinant SBA in secreted form, the pre-SBA cDNA was subcloned in pTM1 vector and the construct inserted into vaccinia virus . When monkey BS-C-1 cells were infected by the virus, using a double expression protocol, recombinant lectin was secreted into the growth medium, from which it was isolated by immunoaffinity chromatography at a yield similar to that from the bacteria . Except for its lower hemagglutinating activity, the product was indistinguishable from native SBA in all properties tested . It was also susceptible to digestion by endo-beta-N-acetylglucosaminidase H or N-glycanase which caused a decrease of 2 kDa in its subunit mass and gave the same results on lectin blot analysis, indicating that it too is a glycoprotein with a single oligomannose unit.

Endocrinology, 1997 Dec, 138(12), 5434 - 41
Regulation of expression of epidermal growth factor receptors in gonadotropes by epidermal growth factor and estradiol: studies in cycling female rats; Armstrong JL et al.; Changes in expression of epidermal growth factor (EGF) receptors by gonadotropes parallel those of GnRH receptors . Gonadotropes increase their expression of EGF receptors (EGFR) during diestrus to reach a peak on the morning of proestrus . This is followed by a decline in expression to reach a nadir by estrus . We hypothesized that regulatory factors that stimulate changes in GnRH receptors might mediate the same changes in EGFR . To test this hypothesis, pituitary cells were collected from cycling rats and grown overnight in media with or without serum, 100 pM estradiol, or 60 ng/ml activin . On the next day, some of the cultures were further stimulated with 1 nM GnRH (4 h) . The cells were then dual-labeled for EGFR and LHbeta or FSHbeta antigens and analyzed for their content of EGFR and gonadotropins . Neither activin nor estradiol increased percentages of cells with gonadotropin antigens and EGFR . Estradiol decreased percentages of cells with EGFR and LH in proestrous rats and those with EGFR and FSH in diestrous rats . The estradiol-mediated decline in EGFR expression during proestrus is similar to that seen when GnRH receptors are studied . Serum containing media alone increased percentages of LH and FSH cells with EGFR in populations from estrous or metestrous rats . Therefore, further experiments were conducted to learn if serum factors or EGF might be a regulator . Removal of serum from the growth media did not prevent the increase in percentages of LH cells with EGFR over the 18-h growth period . However, removal of serum did prevent the increased percentages of FSH cells with EGFR . Similarly, adding 1:100 anti-EGF to the serum containing media did not affect expression of EGFR by LH cells . However, it did cause a 27% decrease in percentages of FSH cells with EGFR . Finally, when 10 ng/ml EGF was added to metestrous populations in serum-free media there was a 1.4-1.5-fold increase in percentages of LH or FSH cells with EGFR . Collectively, these studies show that EGF receptors are not stimulated in gonadotropes by the same hormones that up-regulate GnRH receptors . Furthermore, EGF itself may be among the factors that up regulate EGFR in gonadotropes . EGF receptors may be down-regulated by estradiol during proestrus, but the effect is limited to LH cells . Finally, EGF's differential effects on LH and FSH cells suggests that it may selectively act on monohormonal gonadotropes . EGF receptors may be a marker for a unique subset of developing gonadotropes.

Microbiology, 1997 Nov, 143 ( Pt 11), 3543 - 53
Characterization of a gene encoding dihydrolipoamide dehydrogenase of the cyanobacterium Synechocystis sp . strain PCC 6803; Engels A et al.; The authors previously reported the isolation and partial characterization of a periplasmically located dihydrolipoamide dehydrogenase (LPD) from the cyanobacterium Synechocystis sp . strain PCC 6803 . In the present work the gene (lpdA; database accession number Z48564) encoding the apoprotein of this LPD in Synechocystis PCC 6803 has been identified, sequenced and analysed . The lpdA gene codes for a protein starting with methionine, which is post-translationally removed . The mature protein contains an N-terminal serine and consists of 473 amino acids with a deduced molecular mass of 51421 Da (including one FAD) . The LPD is an acidic protein with a calculated isoelectric point of 5.17 . Comparison of the amino acid sequence of the Synechocystis LPD with protein sequences in the databases revealed that the enzyme shares identities of 31-35% with all 18 LPDs so far sequenced and published . As a first step in determining the role of this cyanobacterial LPD, attempts were made to generate an LPD-free Synechocystis mutant by insertionally inactivating the lpdA gene with a kanamycin-resistance cassette . However, the selected transformants appeared to be heteroallelic, containing both the intact lpdA gene and the lpdA gene inactivated by the drug-resistance cassette . The heteroallelic mutant studied, which had about 50% of the wild-type LPD activity, caused acidification of the growth medium . Growth over a prolonged time was only possible after an increased buffering of the medium . Since it is reported in the literature that inactivation of the pyruvate dehydrogenase complex (PDC) leads to acidosis, a function of the LPD in a cytoplasmic-membrane-associated PDC is conceivable.

FEBS Lett, 1997 Oct 20, 416(2), 179 - 82
Nitrogen source-dependent expression of a 126 kDa protein in the plasma membrane of the cyanobacterium Synechococcus PCC 7942; Zinovieva M et al.; The expression of a 126 kDa protein in the cytoplasmic membrane of Synechococcus PCC 7942 is shown to be dependent on the nitrogen source . It is absent in ammonium-grown cells and its quantity is inversely related to the concentration of nitrate or nitrite in the growth medium . Addition of ammonium-grown cells to a medium containing nitrate or L-methionine-DL-sulfoximine results in the expression of this protein . It is present in the plasmalemma of the Synechococcus NC3 mutant (nrtC gene deleted) and absent in the NA3 mutant (nrtABCD genes deleted) . These results may suggest involvement of the 126 kDa protein in nitrate transport through Synechococcus cytoplasmic membrane.

J Microsc, 1997 Oct, 188 ( Pt 1), 17 - 23
Specialized scanning ion-conductance microscope for imaging of living cells; Korchev YE et al.; A specialized scanning ion conductance microscope (SICM) for imaging living cells has been developed from a conventional patch-clamp apparatus, which uses a glass micropipette as the sensitive probe . In contrast with other types of scanning probe microscope, the SICM probe has significant advantages for imaging living cells: it is most suitable for imaging samples immersed in water solutions; and since the probe senses ion current and does not need physical contact with the sample during the scan, any preliminary preparation of cells (fixation or adherence to a substrate) is unnecessary . We have successfully imaged murine melanocytes in growth medium . The microscope images the highly convoluted surface structures without damaging or deforming them, and reveals the true, three-dimensional relief of the cells . This instrument has considerable ability to operate, potentially simultaneously, in applications as diverse as real-time microscopy, electrophysiology, micromanipulation and drug delivery.

FEMS Microbiol Lett, 1997 Nov 1, 156(1), 141 - 5
Growth of an Escherichia coli mutant deficient in respiration; Futatsugi L et al.; An Escherichia coli mutant deficient in genes for heme biosynthesis grew in medium of initial pH 8 containing 1% tryptone and glucose under aerobic growth conditions, and its doubling time was approximately 60 min at 37 degrees C . The growth rate was not increased under O2-limiting conditions . When the mutant was grown in medium of initial pH 6, growth stopped at the middle of the exponential growth phase . This could be overcome and the growth yield increased by the addition of 20 mM lysine to the growth medium . Lysine did not prevent the decrease in the medium pH as growth proceeded, making it unlikely that lysine decarboxylation stimulates growth by the alkalinization of the medium . These results indicate that respiration is not obligatory for growth under aerobic conditions, but growth without respiration at low pH requires a large amount of lysine.

Biochem Biophys Res Commun, 1997 Nov 7, 240(1), 104 - 7
EGF inhibits expression of WDNM1 and sulfated glycoprotein-2 genes in mammary epithelial cells; Lee M et al.; We have previously shown that expressions of ferritin heavy chain (FHC), WDNM1, and sulfated glycoprotein-2 (SGP-2) genes are induced at an involution stage of mammary gland . Here we studied the effect of lactogenic hormones and EGF on the expression of involution-induced genes in HC11 mammary epithelial cells . Insulin, dexamethasone, prolactin, and its combinations did not affect expression of the genes . When cells were cultured in growth medium containing EGF, expression of WDNM1 and SGP-2 genes was strongly inhibited in a dose- and time- dependent manner, whereas expression of FHC gene was not influenced by EGF . Results demonstrate that EGF inhibits expression of WDNM1 and SGP-2 genes in mammary epithelial cells.

J Mol Biol, 1997 Oct 17, 273(1), 114 - 21
Dual role for the yeast THI4 gene in thiamine biosynthesis and DNA damage tolerance; Machado CR et al.; The THI4 gene of Saccharomyces cerevisiae encodes an enzyme of the thiamine biosynthetic pathway . The plant homolog thi1, from Arabidopsis thaliana, is also involved in thiamine biosynthesis; but was originally cloned due to its capacity to complement DNA repair deficient phenotypes in Escherichia coli . Here, the behavior of a thi4 disrupted strain was examined for increased sensitivity to treatment with the DNA damaging agents ultraviolet radiation (UV, 254 nm) and methyl methanesulfonate (MMS) . Although the thi4 null mutant showed a similar level of survival as the wild-type strain, a higher frequency of respiratory mutants was induced by the two treatments . A similar phenotype was seen with wild-type strains expressing an antisense THI4 construct . Further analysis of respiratory mutants revealed that these were due to mutations of mitochondrial DNA (mtDNA) rather than nuclear DNA, consisting of rho-petite mutants . Moreover, the frequency of mutations was unaffected by the presence or absence of thiamine in the growth medium, and the defect leading to induction of petites in the thi4 mutant was corrected by expression of the Arabidopsis thi1 gene . Thus, Thi4 and its plant homolog appear to be dual functional proteins with roles in thiamine biosynthesis and mitochondrial DNA damage tolerance.

J Gen Virol, 1997 Nov, 78 ( Pt 11), 2779 - 87
Inhibition of pestivirus infection in cell culture by envelope proteins E(rns) and E2 of classical swine fever virus: E(rns) and E2 interact with different receptors; Hulst MM et al.; Pure preparations of envelope glycoproteins E(rns) and E2 of classical swine fever virus (CSFV) synthesized in insect cells were used to study infection of porcine and bovine cells with the pestiviruses CSFV and bovine viral diarrhoea virus (BVDV) . Almost 100% inhibition of infection of porcine kidney cells with CSFV was produced by 100 microg/ml E(rns) . After removal of the virus no E(rns) was needed in the overlay medium (growth medium) to maintain this level of inhibition . In contrast, 100% inhibition of infection of porcine kidney cells with CSFV by 10 microg/ml E2 was only achieved when E2 was added to the overlay medium . When E2 was omitted, a maximum of 50% inhibition was achieved . This indicated that after the virus and E2 were removed from the cells, infection still occurred, by virus particles which were still bound to the cell surface . Treatment with 100 microg/ml E(rns) released these particles from the cell surface . Furthermore, E(rns) bound irreversibly to the surface of cells susceptible or unsusceptible to pestivirus infection and cell-to-cell spread of CSFV was completely inhibited by E2 but not by E(rns) . These results demonstrated that E(rns) and E2 interacted with different cell surface receptors . Inhibition of BVDV infection of porcine and bovine cells by CSFV E2 suggested that CSFV E2 and BVDV E2 share an identical receptor . BVDV strain 5250 isolated from pigs was efficiently inhibited by CSFV E(rns), whereas several BVDV strains isolated from cattle were not, suggesting that the conformation of E(rns) plays a role in host tropism.

J Biol Chem, 1997 Nov 14, 272(46), 29033 - 8
Identification of the key protein for zinc uptake in Hemophilus influenzae; Lu D et al.; Very little is known about specific mechanisms for zinc accumulation and transport in bacteria . In this study a putative adhesin B in Hemophilus influenzae, the product of gene HI0119, has been identified as a periplasmic zinc-binding protein (PZP1) . A pzp1-deficient mutant has been constructed which is defective for growth under aerobic conditions and grows poorly under anaerobic conditions . The growth defect is specifically rescued by supplementing the growth medium with high concentrations of zinc . Subcellular fractionation was used to localize PZP1 to the periplasmic region in a nontypeable H . influenzae strain and in a transfected recombinant Escherichia coli strain (TApzp1) . Recombinant PZP1, purified from a periplasmic extract of E . coli strain TApzp1, contained approximately two zinc atoms/protein molecule as determined by neutron activation analysis and atomic absorption spectroscopy . The zinc atoms could be removed by incubation with EDTA, and, by further addition of zinc, a total of five zinc atoms/PZP1 could be bound . Direct binding of 65Zn to the recombinant protein by Western blot was demonstrated . Taken together, these results provide direct evidence that PZP1 plays a key role in zinc uptake by H . influenzae.

Glycobiology, 1997 Oct, 7(7), 921 - 7
Acceptor specificity of GDP-Fuc:Gal beta 1-->4GlcNAc-R alpha 3-fucosyltransferase VI (FucT VI) expressed in insect cells as soluble, secreted enzyme; De Vries T et al.; As an extension of previous study (de Vries et al., 1995, J . Biol . Chem., 270, 8712-8722) the acceptor specificity of recombinant FucT VI, expressed in insect cells as soluble enzyme, and purified from the growth medium by affinity chromatography, was analyzed toward a broad panel of oligosaccharide and glycoprotein substrates . It was found that FucT VI effectively utilizes any type-2-chain based structure (Gal beta 1-->4GlcNAc-R) . Neutral as well as sialylated structures are fucosylated with high efficiency . To identify polar groups on acceptors that function in enzyme binding, deoxygenated substrate analogs were tested as acceptors . FucT VI had an absolute requirement for a hydroxyl at C-6 of galactose in addition to the accepting hydroxyl at C-3 . Thus, FucT VI, although different from FucT III, IV, and V in acceptor properties, seems to bind the acceptor in a similar way.

Zentralbl Bakteriol, 1997 Oct, 286(3), 363 - 70
The isolation of Borrelia burgdorferi spirochetes from clinical material in cell line cultures; Tylewska-Wierzbanowska S et al.; It has been found that B . burgdorferi bacteria multiply in mouse fibroblasts . Mouse fibroblast of the L-929 cell line was inoculated with less than 10 up to 10(4) B . burgdorferi cells and incubated for 2-10 days at 35 degrees C in microaerophilic conditions . Within 2 days, visible growth was observed . The bacteria were present in growth medium and on/in mouse fibroblasts as revealed by the indirect immunofluorescence assay . At the same time, development of vacuolized fibroblastic giant cells was observed . Viable spirochetes were also detected in Eagle's medium from a L-929 fibroblast cell line culture, after approximately 2-5 days of incubation with blood, cerebro-spinal and synovial fluids of Lyme borreliosis patients . The bacteria were present in growth medium and on/in endothelial cells as revealed by the indirect immunofluorescence assay . The establishment of B . burgdorferi culture conditions in cell lines gives us a possibility to isolate the etiological agent of Lyme disease from patient blood, cerebrospinal and synovial fluids at different stages of infection . The high sensitivity of this procedure would be helpful in a proper identification of the infection as well as in the control of treatment effectiveness.

Adv Exp Med Biol, 1997, 422, 157 - 66
Regulation of peroxisomal fatty acyl-CoA oxidase in the yeast . Saccharomyces cerevisiae; Small GM et al.; Peroxisomes are specialized organelles found in most eukaryote cells, where their major functions are in cellular respiration and fatty acid oxidation . Proliferation of this organelle, and induction of peroxisomal enzymes, is a phenomenon that occurs in diverse species, and is stimulated by a number of physiological and pharmacological stimuli . A large number of chemically diverse compounds, including hypolipidemic drugs and industrial plasticizers, have been shown to cause peroxisome proliferation and the induction of peroxisomal enzymes in rodents . Chronic exposure to these compounds produces hepatocellular carcinomas, however, the mechanism by which this tumorigenic event occurs is unknown . In the yeast Saccharomyces cerevisiae peroxisomes are induced when a fatty acid such as oleate is supplied as a carbon source in the growth medium . In addition, many peroxisomal enzymes are induced by growth on oleate; these include enzymes of the peroxisomal beta-oxidation cycle . This regulation occurs at the transcription level, and is controlled by specific trans-acting factors . The research in our laboratory has focused on the mechanisms involved in this regulation, and on the identification and characterization of the proteins involved . Our recent results, and current research directions are summarized.

AIDS Res Hum Retroviruses, 1997 Nov 1, 13(16), 1411 - 20
Lymphocyte trafficking and HIV infection of human lymphoid tissue in a rotating wall vessel bioreactor; Margolis LB et al.; The pathogenesis of HIV infection involves a complex interplay between both the infected and noninfected cells of human lymphoid tissue, the release of free viral particles, the de novo infection of cells, and the recirculatory trafficking of peripheral blood lymphocytes . To develop an in vitro model for studying these various aspects of HIV pathogenesis we have utilized blocks of surgically excised human tonsils and a rotating wall vessel (RWV) cell culture system . Here we show that (1) fragments of the surgically excised human lymphoid tissue remain viable and retain their gross cytoarchitecture for at least 3 weeks when cultured in the RWV system; (2) such lymphoid tissue gradually shows a loss of both T and B cells to the surrounding growth medium; however, this cellular migration is reversible as demonstrated by repopulation of the tissue by labeled cells from the growth medium; (3) this cellular migration may be partially or completely inhibited by embedding the blocks of lymphoid tissue in either a collagen or agarose gel matrix; these embedded tissue blocks retain most of the basic elements of a normal lymphoid cytoarchitecture; and (4) both embedded and nonembedded RWV-cultured blocks of human lymphoid tissue are capable of productive infection by HIV-1 of at least three various strains of different tropism and phenotype, as shown by an increase in both p24 antigen levels and free virus in the culture medium, and by the demonstration of HIV-1 RNA-positive cells inside the tissue identified by in situ hybridization . It is therefore reasonable to suggest that gel-embedded and nonembedded blocks of human lymphoid tissue, cocultured with a suspension of tonsillar lymphocytes in an RWV culture system, constitute a useful model for simulating normal lymphocyte recirculatory traffic and provide a new tool for testing the various aspects of HIV pathogenesis.

Biochem J, 1997 Oct 15, 327 ( Pt 2), 341 - 7
The heat-shock transcription factor HSF1 is rapidly activated by either hyper- or hypo-osmotic stress in mammalian cells; Caruccio L et al.; Osmoregulation, the cellular response to environmental changes of osmolarity and ionic strength, is important for the survival of living organisms . We have demonstrated previously that an exposure of mammalian cells to hypo-osmotic stress, either in growth medium (30% growth medium and 70% water) or in binary solution containing sorbitol and water, prominently induced the DNA-binding activity of the heat-shock transcription factor (HSF1) {Huang, Caruccio, Liu and Chen (1995) Biochem . J . 307, 347-352} . Since hyperosmotic and hypo-osmotic stress usually elicit opposite biological responses, we wondered what would be the effect of hyperosmotic stress on HSF activation . In this study we have examined the HSF DNA-binding activity in HeLa cells maintained in the sorbitol/water binary solution over a wide concentration range (0.1-0.9 M) and in Dulbecco's medium supplemented with sorbitol or NaCl . We found that HSF-binding activity could be induced prominently under both hypo-osmotic (0.1-0.25 M) and hyperosmotic conditions (0.50-0.90 M) . In both cases, HSF activation was observed within 5 min after changing the osmotic pressure . The activation was accompanied by both HSF trimerization and nuclear translocation, and appeared to be independent of protein synthesis . The effects of hypo- or hyper-osmotic stress on HSF activation could be reversed once the cells were returned to iso-osmotic conditions (0.30M) with a half-life (t12) of 25 min or less . This rapid turnover of the osmotic-stress-induced HSF-binding activity was inhibited by cycloheximide, a potent inhibitor of protein synthesis . Unlike heat shock, activation of HSF by either hypo- or hyper-osmotic stress did not lead to an accumulation of heat-shock protein 70 (HSP70) mRNA in HeLa cells . We propose that HSF activation during osmotic stress may serve physiological functions independent of the synthesis of heat-shock proteins.

Gene, 1997 Oct 15, 199(1-2), 111 - 21
Identification and characterization of the thiamine transporter gene of Saccharomyces cerevisiae; Singleton CK; A positive selection scheme is described that selects for thiamine transporter clones . The scheme is based on the rescue of lethality, under non-permissive conditions, of Saccharomyces cerevisiae strains that are conditional for thiamine biosynthesis and are defective in thiamine transport . Transport defective strains were generated by selection for resistance to the lethal thiamine analog, pyrithiamine . Pyrithiamine resistance was shown to be a recessive, single gene trait that resulted from the mutation of the thiamine transporter gene, as suggested by previous work . Conditional thiamine biosynthesis was generated by cloning THI4, a thiamine biosynthetic gene, into a URA3 containing plasmid and transforming a strain disrupted in THI4 . Thus, plating on 5-fluoroorotic acid causes the loss of thiamine synthesis ability . The gene for the yeast thiamine transporter, THI7, was cloned using this scheme . The predicted 598 amino acid transporter is a member of the major facilitator superfamily of transporters and thus possesses 12 transmembrane spanning segments with amino and carboxy termini intracellularly located . Several alterations in the coding region were characterized that result in greatly reduced ability to transport thiamine . The level of transporter mRNA was found to be rapidly and dramatically reduced by the addition of thiamine to the growth medium.

Biochem J, 1997 Oct 1, 327 ( Pt 1), 51 - 7
Carbon dioxide and light regulation of promoters controlling the expression of mitochondrial carbonic anhydrase in Chlamydomonas reinhardtii; Villand P et al.; Nuclear genes coding for carbonic anhydrase, a major mitochondrial constituent in Chlamydomonas reinhardtii grown under limited CO2, were characterized . Two genes, ca1 and ca2, were found within 7 kb of genomic DNA, organized 'head to head' in a large inverted repeat . The DNA sequences for the two genes were very similar, even in the promoter regions and in introns, indicating that the repeat is a result of a recent duplication . To study gene regulation, elements from the upstream region of ca1 were fused to the arylsulphatase reporter gene . After transformation, the expression of arylsulphatase was regulated similarly to the endogenous ca1/ca2 genes, even when the promoter was trimmed down to 194 nt . Expression could not be detected when 5% CO2 was bubbled into the growth medium, but was induced within hours after transfer to air . The ca1 promoter was not induced in low light, but at intermediate light levels its activity was dependent on the irradiance . O2 concentration had no effect on the promoter activity, indicating that photorespiratory metabolites are not triggering the response . The availability of cells transformed with a CO2-regulated reporter gene should facilitate further studies on the metabolic adaptations that occur in some green algae in response to the external CO2 level.

Vet Microbiol, 1997 Sep, 57(2-3), 105 - 18
Characterization of a putative receptor protein for bovine viral diarrhea virus; Xue W et al.; In a previous communication, we reported a 50-kDa cell surface protein from Madin-Darby bovine kidney (MDBK) cells as a putative receptor for bovine viral diarrhea virus (BVDV) . The present study delineates further characterization of the receptor protein . Protease treatment of cultured MDBK cells adversely affected the receptor, thus abolishing the binding of anti-D89 (BVDV anti-idiotypes) to the cells . However, pretreatment of the cells with either phospholipases or glycosidases did not significantly change the anti-D89 binding to the cells . Additionally, pretreatment of cell monolayers with proteases decreased BVDV attachment and replication in the cells . These results suggested that the receptor for BVDV is a protein in nature, and glycosylation and phosphorylation may not play a direct role in BVDV attachment to cells . The BVDV receptor gradually regenerated on the cell surface after the protease-treated cells were cultured in normal growth medium . Regeneration of the BVDV receptor to a normal level took about 4 h as indicated by flow cytometric analysis and this process was inhibited in the presence of cycloheximide, a protein synthesis inhibitor . The 50-kDa receptor protein purified by electro-elution inhibited BVDV infection in a plaque reduction assay . It also inhibited anti-D89 binding to cells as analyzed by flow cytometry . These data demonstrated the nature of the 50-kDa protein as a specific receptor for BVDV.

Cancer Res, 1997 Nov 1, 57(21), 4965 - 70
Mechanisms of the regulation of thioredoxin reductase activity in cancer cells by the chemopreventive agent selenium; Gallegos A et al.; Selenium is an essential trace element, the deficiency of which is associated with an increased incidence of some human cancers . Dietary supplementation with selenium has been reported to produce a decrease in the incidence of some cancers in humans . Thioredoxin reductase (TR) is a newly discovered homodimeric selenocysteine (SeCys)-containing protein that catalyzes the NADPH-dependent reduction of the redox protein thioredoxin (Trx) . Trx is overexpressed by a number of human tumors, and experimental studies have shown that Trx contributes to the growth and to the transformed phenotype of some human cancer cells . Thus, TR, by reducing Trx, could play a role in regulating the growth of normal and cancer cells . We have investigated mechanisms by which selenium, in the form of sodium selenite, added to serum-free growth medium regulates TR activity in cancer cell lines . Selenium caused a dose-dependent increase in cellular TR activity . The increase in TR activity produced by 1 microM Se compared to medium with no added selenium was: for MCF-7 breast cancer cells, 37-fold; for HT-29 colon cancer cells, 19-fold; and for A549 lung cancer cells, 8-fold . In contrast, Jurkat and HL-60 leukemia cells showed no increase in TR activity . The half-life of the time course of induction of TR in HT-29 cells after adding selenium was 10 h . The increase in TR activity was accompanied by an increase in TR protein levels up to 3-fold and an increase in the specific activity of the enzyme of 5-32-fold, depending on the cell line . Studies using 75Se showed that the amount of selenium incorporated into TR increased with increasing selenium concentration up to a ratio of 1 selenium per TR monomer . There was an increase in TR mRNA levels of 2-5-fold at 1 microM selenium and an increase in the stability of TR mRNA with a half-life for degradation of 21 h compared to 10 h in the absence of selenium . Trx mRNA and protein levels and Trx mRNA stability were not affected by selenium . The results of the study show that the increase in TR activity caused by selenium is specific and due to several effects, including an increase in the stability of TR mRNA leading to increased TR mRNA levels, an increase in TR protein, but predominantly to an increase in the specific activity of TR associated with increased incorporation of selenium into the enzyme.

Infect Immun, 1997 Nov, 65(11), 4539 - 47
Response of Chlamydia trachomatis serovar E to iron restriction in vitro and evidence for iron-regulated chlamydial proteins; Raulston JE; Iron is a well-established mediator of virulence in several bacterial pathogens, yet little is known about the role of iron in infectious disease processes caused by obligate intracellular bacterial pathogens . In this study, the effect of iron limitation was examined for the sexually transmitted infectious agent Chlamydia trachomatis in an in vitro model of human genital infection using the intracellular iron-chelating reagent deferoxamine mesylate (Desferal) . Iron restriction caused a significant reduction in infectivity of C . trachomatis elementary bodies (EB) harvested from Desferal-exposed polarized epithelial cells when compared to that of EB harvested from iron-sufficient control cell cultures . Replacement of the Desferal exposure medium with medium containing iron-saturated transferrin restored chlamydial infectivity, whereas replacement with growth medium alone had no effect . The following three prominent morphological features were observed by electron microscopic examination of chlamydia-infected cells exposed to Desferal: (i) inclusions containing chlamydiae greatly delayed in maturation, (ii) substantial blebbing within chlamydial inclusions, and (iii) electron-dense material surrounding inclusions . Protein analyses of highly purified EB by two-dimensional polyacrylamide gel electrophoresis revealed that there were at least 19 candidate iron-repressible proteins in C . trachomatis and at least one protein which was iron inducible . One putative iron-repressible protein was confirmed by Western blot (immunoblot) analysis to be the chlamydial heat shock protein 60 (hsp60) . The enhanced production of this antigen by chlamydiae as a result of iron limitation is of particular importance since there is a well-documented association between chlamydial hsp60 and destructive immunopathological sequelae in infected patients.

Am J Reprod Immunol, 1997 Nov, 38(5), 313 - 9
Suppressive effect on lymphoproliferation in vitro by soluble annexin II released from isolated placental membranes; Aarli A et al.; PROBLEM: Syncytiotrophoblast microvillous plasma membranes (StMPM) are potent suppressors of lymphoproliferation in vitro . We have previously shown that soluble annexin II (AII) is present at higher levels in retroplacental serum (RPS) than in peripheral serum, and that soluble AII has an immunosuppressive effect . The aims of this study were to determine whether AII can be released from StMPM and whether soluble AII from StMPM exerts any immunosuppressive effect . METHOD OF STUDY: Isolated StMPM were incubated in growth medium for 18 hr and supernatants were prepared by ultracentrifugation . Soluble AII was detected by immunoblotting . StMPM, StMPM supernatant, and affinity-purified AII were analysed in a lymphoproliferation assay for immunomodulating activity . RESULTS: AII heavy chain and its p11 light chain were detected both in StMPM supernatant and in RPS after removal of StMPM particles by ultracentrifugation . StMPM, StMPM supernatant, and purified AII suppressed lymphoproliferation in a dose-dependent manner . Absorption of AII from StMPM supernatant reduced the suppressive activity . The suppressive effect of StMPM supernatant and purified AII was completely reversed by heating at 100 degrees C for 30 min or by adding recombinant interleukin-2 at 100 units/ml . Although StMPM and affinity-purified AII suppressed the proliferation of lymphocytes from all donors tested, StMPM supernatant suppressed the proliferation of lymphocytes from 12 of 23 donors . Six of eight female non-suppressed donors were multiparae, whereas five of five female suppressed donors were nulliparae . CONCLUSIONS: Annexin II is released by isolated placental membranes in vitro and is present in RPS, indicating in vivo release of AII at the fetomaternal interface, probably as AII heterotetramer . AII has immunosuppressive activity and may be important in fetal allograft survival.

Lett Appl Microbiol, 1997 Oct, 25(4), 254 - 6
Yeast exoglycoproteins produced under NaCl-stress conditions as efficient cryoprotective agents; Breierova E; Six extracellular yeast glycoproteins were prepared from three yeast species in osmotic equilibrium and unequilibrium environments and used as non-penetrating cryoadditives . The glycoprotein secreted by the strain Dipodascus australiensis into the growth medium containing NaCl (8% w/v) was found to be the most effective cryoadditive . It was possible to use this glycoprotein alone (without penetrating agent DMSO) for the cryoprotection of the yeasts studied.

Lett Appl Microbiol, 1997 Sep, 25(3), 202 - 6
Novel selective and non-selective optical detection of microorganisms; Shelef LA et al.; A new instrument, capable of detecting metabolic changes due to microbiological activity, is described . Optical changes in growth media are monitored in a semi-fluid zone that separates the liquid medium containing the sample . Data demonstrate that common media can be utilized in conjunction with this rapid automated technology . Nutrient broth with the pH dye indicator . bromocresol purple was suitable for total counts . Selective media containing dyes were utilized to assess the presence or absence of specific groups of organisms . Biochemical reactions, such as lysine decarboxylase activity, were identified by the unique generated patterns, and specific enzymatic cleavage reactions with chromogenic substrates, such as 5-bromo-4 chloro-3 indolyl-beta-D-glucuronic acid (X-GLUC), were monitored.

Mol Pathol, 1997 Aug, 50(4), 186 - 93
A two year prospective study to compare culture and polymerase chain reaction amplification for the detection and diagnosis of Lyme borreliosis; Picken MM et al.; AIM: To compare polymerase chain reaction (PCR) amplification of borrelial DNA and culture isolation of spirochaetes for the diagnosis of Lyme borreliosis by direct detection of Borrelia burgdorferi sensu lato in patients with erythema migrans and acrodermatitis chronica atrophicans lesions . METHODS: Skin biopsy specimens from erythema migrans and acrodermatitis chronica atrophicans lesions were subdivided and tested by PCR amplification assay and culture using two artificial growth media, Barbour-Stoenner-Kelly II (BSK II) and modified Kelly-Pettenkofer (MKP) . Five classes of lesions were studied: typical erythema migrans, spontaneously resolved erythema migrans, atypical/partially treated erythema migrans, typical acrodermatitis chronica atrophicans, and atypical/partially treated acrodermatitis chronica atrophicans . RESULTS: For both erythema migrans and acrodermatitis chronica atrophicans lesions, the most sensitive detection method was MKP culture . PCR was less sensitive than MKP culture, but more sensitive than BSK II culture . Results for 758 typical erythema migrans specimens showed positivity rates of 36% for MKP, 25% for PCR, and 24% for BSK II . Differences were statistically significant . The overall positivity rate for all three methods combined was 54%, but few specimens (6%) were positive by all three methods . Examination of multiple erythema migrans lesions from the same patient increased the diagnostic yield . These findings, and similar results for acrodermatitis chronica atrophicans lesions, suggest that the distribution of spirochaetes in skin biopsies is not homogeneous . CONCLUSIONS: Although possessing the potential to provide a rapid diagnosis, PCR is not more sensitive than culture for the direct detection of borrelia . Spirochaetes appear to be unevenly distributed throughout biopsy specimens, suggesting that diagnosis of Lyme borreliosis by direct detection of the causative agent in skin lesions in vulnerable to sample bias.

J Biol Chem, 1997 Oct 31, 272(44), 28149 - 57
Mutations in the CYS4 gene provide evidence for regulation of the yeast vacuolar H+-ATPase by oxidation and reduction in vivo; Oluwatosin YE et al.; The vma41-1 mutant was identified in a genetic screen designed to identify novel genes required for vacuolar H+-ATPase activity in Saccharomyces cerevisiae . The VMA41 gene was cloned and shown to be allelic to the CYS4 gene . The CYS4 gene encodes the first enzyme in cysteine biosynthesis, and in addition to cysteine auxotrophy, cys4 mutants have much lower levels of intracellular glutathione than wild-type cells . cys4 mutants display the pH-dependent growth phenotypes characteristic of vma mutants and are unable to accumulate quinacrine in the vacuole, indicating loss of vacuolar acidification in vivo . The vacuolar proton-translocating ATPases (V-ATPase) is synthesized at normal levels and assembled at the vacuolar membrane in cys4 mutants, but its specific activity is reduced (47% of wild type) and the activity is unstable . Addition of reduced glutathione to the growth medium complements the pH-dependent growth phenotype, partially restores vacuolar acidification, and restores wild type levels of ATPase activity . The CYS4 gene was deleted in a strain in which the catalytic site cysteine residue implicated in oxidative inhibition of the yeast V-ATPase has been mutagenized (Liu, Q., Leng, X.-H., Newman, P., Vasilyeva, E., Kane, P . M., and Forgac, M . (1997) J . Biol . Chem . 272, 11750-11756) . This catalytic site point mutation suppresses the effects of the cys4 mutation . The data indicate that the acidification defect of cys4 mutants arises from inactivation of the vacuolar ATPase in the less reducing cytosol resulting from loss of Cys4p activity and provide the first evidence for the modulation of V-ATPase activity by the redox state of the environment in vivo.

Arch Biochem Biophys, 1997 Nov 1, 347(1), 1 - 8
A structural requirement of zinc for the folding of recombinant link protein; Varelas JB et al.; We have cloned and ligated a full-length bovine link protein (LP) in the pMAL-c2 vector and overexpressed it in fusion with maltose-binding protein (MBP) in Escherichia coli . We have demonstrated dose-dependent binding of MBP/LP to biotinylated hyaluronan in a dot blot assay . A greater percentage of the expressed fusion protein was soluble, monomeric, and undegraded when the growth temperature was lowered, the growth medium was supplemented with zinc, and metal chelators were omitted from the lysis buffers . Similar effects were observed when we tested the effects of lower growth temperature and zinc supplementation on another construct consisting of MBP in fusion with the first proteoglycan tandem repeat of LP . Our results suggest zinc may be necessary for the folding and disulfide bond formation of recombinant LP . In addition, a greater amount of monomeric MBP/LP produced at 27 degrees C with zinc supplementation bound to biotinylated hyaluronic acid-binding region of aggrecan than MBP/LP produced at 27 or 37 degrees C without zinc . This suggests that recombinant LP may have a conformational requirement for zinc necessary for binding to aggrecan . Factor Xa cleavage of MBP/LP expressed in the presence of zinc yielded much more intact LP product than cleavage of MBP/LP expressed without zinc . These data indicate a structural role of zinc that allows MBP/LP to fold in a manner such that it is resistant to proteolytic degradation .

Anal Biochem, 1997 Oct 15, 252(2), 293 - 8
Determination of pheomelanin by measurement of aminohydroxyphenylalanine isomers with high-performance liquid chromatography; Kolb AM et al.; We describe an improved method for the analysis of pheomelanin in biological samples . The method is based on a chemical degradation of the melanin polymer and HPLC analysis of specific degradation products . Hydriodic hydrolysis provides 4-amino-3-hydroxyphenylalanine (AHP) and 3-amino-l-tyrosine (AT) which are detected with an electrochemical detector . We have examined each step of the analysis and the results are presented in this paper . First the samples are hydrolyzed for 16 h . AT and AHP are then isolated from the hydrolysates by ion-exchange chromatography and then separated and quantitated by HPLC and electrochemical detection . The method shows good reproducibility with a total imprecision below 5.6% . The linearity of the method was shown from 0 to 490 ng AT and 0 to 850 ng AHP per sample, using a melanoma cell suspension (27 mg protein/ml) with up to 24-fold dilutions of the original sample . For cultured "normal" human melanocytes a minimal amount of 0.1 mg protein is sufficient for analysis of pheomelanin in the samples . This method provides the opportunity to study the composition of the formed melanin in cell lines, cultured in different growth media .

Mol Gen Genet, 1997 Aug, 255(6), 619 - 27
Constitutive and carbon source-responsive promoter elements are involved in the regulated expression of the Saccharomyces cerevisiae malate synthase gene MLS1; Caspary F et al.; The malate synthase gene, MLS1, of the yeast Saccharomyces cerevisiae is transcriptionally regulated by the carbon source in the growth medium . A MLS1-lacZ fusion gene, expressed at a basal level in the presence of 2% glucose, is derepressed more than 100-fold under conditions of sugar limitation . No evidence for MLS1 induction by oleic acid was found . By deletion analysis of the MLS1 control region, we identified two sites, UAS1 and UAS2, as important for efficient derepression of the gene . Both sites contain sequences that resemble the previously characterized carbon source-responsive element (CSRE) found in the promoter of the isocitrate lyase gene ICL1 . Indeed, UAS1 and UAS2 in the MLS1 upstream region turn out to be functional CSRE sequence variants . This finding allowed us to define a modified version of the CSRE consensus sequence (CCRTYSRNCCG) . Protein binding to UAS1MLS1 was observed with extracts from derepressed but not from repressed cells, and could be competed for by an excess of the unlabelled CSRE (ICL1) sequence . No competition was observed with a mutated CSRE variant . Site-directed mutagenesis of both CSREs in the MLS1 promoter reduced gene activation under derepressing conditions to 20% of the wild-type level . The same decrease was observed with the wild-type MLS1 promoter in a cat8 mutant, lacking an activator of CSRE-dependent transcription . The CSRE/Cat8p-independent activation of MLS1 is mediated by constitutive UAS elements . The pleiotropic transcription factor Abf1p, which binds to the MLS1 upstream region, may contribute to constitutive activation . Thus, in order to ensure the severe glucose repression of MLS1 observed, repressor elements that respond to the carbon source must counteract constitutive activation . In summary, ICL1 and MLS1 share common cis-acting elements, although a distinct mechanism of carbon source control also contributes to MLS1 regulation.

Mol Gen Genet, 1997 Aug, 255(6), 570 - 9
SUR1 (CSG1/BCL21), a gene necessary for growth of Saccharomyces cerevisiae in the presence of high Ca2+ concentrations at 37 degrees C, is required for mannosylation of inositolphosphorylceramide; Beeler TJ et al.; Saccharomyces cerevisiae cells require two genes, CSG1/SUR1 and CSG2, for growth in 50 mM Ca2+, but not 50 mM Sr2+ . CSG2 was previously shown to be required for the mannosylation of inositolphosphorylceramide (IPC) to form mannosylinositolphosphorylceramide (MIPC) . Here we demonstrate that SUR1/CSG1 is both genetically and biochemically related to CSG2 . Like CSG2, SUR1/CSG1 is required for IPC mannosylation . A 93-amino acid stretch of Csg1p shows 29% identity with the alpha-1, 6-mannosyltransferase encoded by OCH1 . The SUR1/CSG1 gene is a dose-dependent suppressor of the Ca(2+)-sensitive phenotype of the csg2 mutant, but overexpression of CSG2 does not suppress the Ca2+ sensitivity of the csg1 mutant . The csg1 and csg2 mutants display normal growth in YPD, indicating that mannosylation of sphingolipids is not essential . Increased osmolarity of the growth medium increases the Ca2+ tolerance of csg1 and csg2 mutant cells, suggesting that altered cell wall synthesis causes Ca(2+)-induced death . Hydroxylation of IPC-C to form IPC-D requires CCC2, a gene encoding an intracellular Cu2+ transporter . Increased expression of CCC2 or increased Cu2+ concentration in the growth medium enhances the Ca2+ tolerance of csg1 mutants, suggesting that accumulation of IPC-C renders csg1 cells Ca2+ sensitive.

Folia Microbiol (Praha), 1997, 42(2), 113 - 6
Standardization in animal cell technology; Stacey GN; Continuous cell lines offer a level of reproducibility, and thus standardization, which cannot normally be achieved using primary cells . However, even with continuous cell lines adoption of correct cell banking and appropriate quality control procedures are critical to the provision of reliable, reproducible and safe cell stocks . These procedures enable establishment of cryopreserved stocks of pure cultures of correct identity and phenotype which are free from adventitious agents . In addition to quality control techniques, culture conditions and growth medium used often require standardization . In particular different sources of serum, growth factors and cell attachment substrates may lead to significant variation in the 'performance' of cell lines . To ensure a high degree of reliability it is essential to obtain cells from authenticated and quality controlled sources . In culture collections, the principles of correct cell banking should be applied with appropriate quality control for which the minimum standard should be confirmation of viability and mycoplasma testing . Such approaches will afford increased confidence in research data and avoid the waste of time and resources which result from the use of cross-contaminated or infected cells.

Biochem Pharmacol, 1997 Sep 1, 54(5), 575 - 82
Thiamine deficiency in cardiac cells in culture; Zangen A et al.; Rat heart cells in culture were found to be a unique model for studying biochemical and pharmacological aspects of thiamine deficiency . When thiamine was excluded from the growth medium, the following effects were observed: (1) Morphological examination did not show any difference between control and thiamine-deprived cells during the first 10 days . However, after 10-11 days spontaneous contractions ceased, accompanied by initiation of cell degeneration; (2) Intensive degeneration and cell death were observed after 14-16 days . (3) Thiamine pyrophosphate (TPP) concentration in thiamine-deprived cells was decreased gradually, with an elimination half-life of 4-5 days . (4) {3H}deoxyglucose uptake by the cells was increased, even after 1 day of thiamine deprivation . (5) ATP level decreased after 8 days and reached 50% of control cells after 10 days . (6) In thiamine-deprived cells, thiamine addition caused a 60% rise in contraction amplitude but contraction rate was not altered significantly . (7) All these effects were reversible if thiamine was supplied before the initiation of the degeneration processes.

Biophys J, 1997 Oct, 73(4), 2183 - 94
AFM review study on pox viruses and living cells; Ohnesorge FM et al.; Single living cells were studied in growth medium by atomic force microscopy at a high--down to one image frame per second--imaging rate over time periods of many hours, stably producing hundreds of consecutive scans with a lateral resolution of approximately 30-40 nm . The cell was held by a micropipette mounted onto the scanner-piezo as shown in Haberle, W., J . K . H . Horber, and G . Binnig . 1991 . Force microscopy on living cells . J . Vac . Sci . Technol . B9:1210-0000 . To initiate specific processes on the cell surface the cells had been infected with pox viruses as reported earlier and, most likely, the liberation of a progeny virion by the still-living cell was observed, hence confirming and supporting earlier results (Haberle, W., J . K . H . Horber, F . Ohnesorge, D . P . E . Smith, and G . Binnig . 1992 . In situ investigations of single living cells infected by viruses . Ultramicroscopy . 42-44:1161-0000; Horber, J . K . H., W . Haberle, F . Ohnesorge, G . Binnig, H . G . Liebich, C . P . Czerny, H . Mahnel, and A . Mayr . 1992 . Investigation of living cells in the nanometer regime with the atomic force microscope . Scanning Microscopy . 6:919-930) . Furthermore, the pox viruses used were characterized separately by AFM in an aqueous environment down to the molecular level . Quasi-ordered structural details were resolved on a scale of a few nm where, however, image distortions and artifacts due to multiple tip effects are probably involved--just as in very high resolution (<15-20 nm) images on the cells . Although in a very preliminary manner, initial studies on the mechanical resonance properties of a single living (noninfected) cell, held by the micropipette, have been performed . In particular, frequency response spectra were recorded that indicate elastic properties and enough stiffness of these cells to make the demonstrated rapid scanning of the imaging tip plausible . Measurements of this kind, especially if they can be proven to be cell-type specific, may perhaps have a large potential for biomedical applications . Images of these living cells were also recorded in the widely known (e.g., Radmacher, M., R . W . Tillmann, and H . E . Gaub . 1993 . Imaging viscoelasticity by force modulation with the atomic force microscope . Biophys . J . 64:735-742) force modulation mode, yet at one low modulation frequency of approximately 2 kHz . (Note: After the cells were attached to the pipette by suction, they first deformed significantly and then reassumed their original spherical shape, which they also acquire when freely suspended in solution, to a great extent with the exception of the portion adjusting to the pipette edge geometry after approximately 0.5-1 h, which occurred in almost the same manner with uninfected cells, and those that had been infected several hours earlier . This seems to be a process which is at least actively supported by the cellular cytoskeleton, rather than a mere osmotic pressure effect induced by electrolyte transport through the membrane . Furthermore, several hours postinfection (p.i.) infected cells developed many optically visible refraction effects, which appeared as small dark spots in the light microscope, that we believed to be the regions in the cell plasma where viruses are assembled; this is known from the literature on electron microscopy on pox-infected cells and referred to there as "virus factories" (e.g., Moss, B . 1986 . Replication of pox viruses . In Fundamental Virology, B . N . Fields and D . M . Knape, editors . Raven Press, New York . 637-655) . Therefore, we assume that the cells stay alive during imaging, in our experience for approximately 30-45 h p.i.).

Invest Ophthalmol Vis Sci, 1997 Sep, 38(10), 1998 - 2007
Single exposures to antiproliferatives: long-term effects on ocular fibroblast wound-healing behavior; Occleston NL et al.; PURPOSE: To determine the long-term effects of single, 5-minute exposures to 5-fluorouracil (5FU) and mitomycin-C (MMC) on Tenon's capsule fibroblast migration, growth factor production, growth factor receptor expression, and extracellular matrix (ECM) production . METHODS: Monolayer cultures and the overlying growth medium of Tenon's capsule fibroblasts exposed to 5FU (0.25 to 25 mg/ml) or MMC (0.001 to 0.1 mg/ml) were harvested up to 48 days after treatment . The expression of growth factors and growth factor receptors, including transforming growth factor beta (TGFbeta), basic fibroblast growth factor (bFGF), and epidermal growth factor (EGF), and ECM molecules (collagen type I, collagen type III, and fibronectin) were quantitated at the mRNA and protein levels . The ability of fibroblasts exposed to 5FU and MMC to migrate to fetal calf serum was also investigated up to 48 days after treatment . RESULTS: Control cultures were found to produce the growth factors TGFbeta and bFGF but not EGF . Exposure to 5FU or MMC resulted in an initial significant increase (P < 0.05) in the production of TGFbeta and bFGF, with levels then decreasing toward those of controls . Cells exposed to 5FU or MMC exhibited an initial significant decrease (P < 0.05) in the number of TGFbeta, bFGF, and EGF growth factor receptors, with subsequent recovery toward control levels by day 48 after treatment . Both 5FU and MMC caused a significant reduction (P < 0.05) in collagen type I and fibronectin production compared to controls throughout the 48-day culture period . The production of collagen type III was initially elevated (P < 0.05) compared to controls after exposure to 5FU or MMC, production then decreasing toward control levels over the remainder of the 48-day culture period . The migration of cells exposed to 5FU or MMC was significantly reduced (P < 0.05) compared to controls up to 48 days after treatment; these cells exhibited a partial recovery of migratory ability throughout this period . CONCLUSIONS: Fibroblasts whose growth was arrested using single, short exposures to 5FU or MMC appear to be capable of performing several crucial aspects of wound healing, including the expression of growth factors and receptors and ECM molecules and the ability to migrate . These findings may help explain why in some patients treated with antiproliferatives, glaucoma filtration surgery fails because of scarring.

Somat Cell Mol Genet, 1997 May, 23(3), 203 - 9
High-efficiency retroviral infection of primary myoblasts; Springer ML et al.; In the past, it has been hard to introduce genes into primary myoblasts without selection, as they have been very difficult to transfect or infect . We describe conditions under which mouse primary skeletal muscle myoblasts can be infected with retroviral vectors at high efficiency . Infection can be greatly increased by minimizing the time during which cells are exposed to virus, adding a minimal centrifugation step, and supplementing the infection cocktail to mimic more closely primary myoblast growth medium . Under these conditions, one round of exposure to virus results in an infection efficiency of up to 80%, whereas 4-5 rounds of infection over a two day period reproducibly yield an infection efficiency of > 99% . These methods greatly enhance the potential for studying genetically engineered primary myoblasts from any mouse strain, transgenic or knockout, and may have useful application to other primary cell types that are refractory to transfection or infection.

Ultrasound Med Biol, 1997, 23(7), 1095 - 105
Mechanism of ultrasound enhanced porphyrin cytotoxicity . Part I: A search for free radical effects; Worthington AE et al.; Intense ultrasound beams may have the potential to treat malignant tumours when combined with sensitizers, often called sonodynamic agents . Some of these agents, e.g., the porphyrins, are currently used for photodynamic therapy . However, the experimental evidence for ultrasound activation of sensitizers is inconsistent . This paper attempts to discover whether they yield of free radicals such as .OH and .H, which are produced by transient cavitation, could explain the killing of Chinese hamster ovary (CHO) cells in vitro with and without sonodynamic agents . CHO cells were irradiated with ultrasound beams in phosphate-buffered saline or in growth medium, and the immediate cell lysis and loss of cell colony forming ability were measured . Under our specific conditions, in which the standing wave patterns were minimized, a general correlation was observed between the transient cavitation, free radical production, and cytotoxicity . However, the yield of free radicals was much too small to explain the cell killing observed . We conclude that cytotoxicity is not linked to attack from free radicals formed outside the cells . In our experiments, immediate cell lysis is closely linked to the transient cavitation, which is known to produce shear forces that disrupt cellular membranes . We hypothesize that the loss of cell colony forming ability is also linked to damage of cellular membranes.

Mol Pharmacol, 1997 Oct, 52(4), 658 - 66
Identification of yeast DNA topoisomerase II mutants resistant to the antitumor drug doxorubicin: implications for the mechanisms of doxorubicin action and cytotoxicity; Patel S et al.; Doxorubicin is a therapeutically useful anticancer drug that exerts multiple biological effects . Its antitumor and cardiotoxic properties have been ascribed to anthracycline-mediated free radical damage to DNA and membranes . Evidence for this idea comes in part from the selection by doxorubicin from stationary phase yeast cells of mutants (petites) deficient in mitochondrial respiration and therefore defective in free radical generation . However, doxorubicin also binds to DNA topoisomerase II, converting the enzyme into a DNA damaging agent through the trapping of a covalent enzyme-DNA complex termed the 'cleavable complex.' We have used yeast to determine whether stabilization of cleavable complexes plays a role in doxorubicin action and cytotoxicity . A plasmid-borne yeast TOP2 gene was mutagenized with hydroxylamine and used to transform drug-permeable yeast strain JN394t2-4, which carries a temperature-sensitive top2-4 mutation in its chromosomal TOP2 gene . Selection in growth medium at the nonpermissive temperature of 35 degrees in the presence of doxorubicin resulted in the isolation of plasmid-borne top2 mutants specifying functional doxorubicin-resistant DNA topoisomerase II . Single-point changes of Gly748 to Glu or Ala642 to Ser in yeast topoisomerase II, which lie in and adjacent to the CAP-like DNA binding domain, respectively, were identified as responsible for resistance to doxorubicin, implicating these regions in drug action . None of the mutants selected in JN394t2-4, which has a rad52 defect in double-strand DNA break repair, was respiration-deficient . We conclude that topoisomerase II is an intracellular target for doxorubicin and that the genetic background and/or cell proliferation status can determine the relative importance of topoisomerase II- versus free radical-killing.

Gastroenterology, 1997 Oct, 113(4), 1198 - 213
Cell dynamics and differentiation of conditionally immortalized human intestinal epithelial cells; Quaroni A et al.; BACKGROUND & AIMS: Intestinal epithelial cell lines capable of differentiating in monolayer culture have been difficult to obtain, prompting the use of novel approaches including immortalization with oncogenes or tumor viruses . The aim of this study was to obtain conditionally immortalized human intestinal epithelial cells (temperature-sensitive fetal human intestinal {tsFHI} cells) and study their growth and differentiation capabilities . METHODS: Intestinal cells from human fetuses were transformed with a temperature-sensitive simian virus 40 large tumor antigen, cloned, and screened for expression of intestinal markers . RESULTS: Establishment of tsFHI cells was crucially dependent on a growth medium based on OptiMEM . At 32 degrees C, the tsFHI cells proliferate and display crypt cell markers . Only a few scattered differentiated cells are observed together with cells displaying the morphological signs of apoptosis . A shift to 39 degrees C results in irreversible growth arrest and acquisition of an enterocyte-like phenotype . Expression of the cyclin-dependent kinase inhibitor p21/WAF1/Cip1 is induced within hours of a shift to 39 degrees C 1-2 days before corresponding increases in brush border enzymes, suggesting a role in the induction rather than in the maintenance of a differentiated phenotype . CONCLUSIONS: The tsFHI cells recapitulate key aspects of intestinal cell dynamics in vitro and seem particularly suited for the study of early events in cell differentiation.

J Biotechnol, 1997 Aug 28, 56(3), 153 - 65
On the safety of Mortierella alpina for the production of food ingredients, such as arachidonic acid; Streekstra H; Mortierella alpina is the most efficient production organism for arachidonic acid (AA) presently known . Since AA is being developed as a food ingredient, and since M . alpina has no history of use for such applications, we have undertaken this safety evaluation . M . alpina is a common soil fungus, to which humans are frequently exposed . The production strains are non-pathogenic and do not form potentially allergenic spores under production conditions . Moreover, there are no reliable reports in the literature connecting the species with disease or allergenic responses . No production of mycotoxins was observed, in line with the absence of literature reports describing such products, and with the results of toxicological tests . On solid growth media the strains showed antibiotic activity against Gram-positive bacteria . In submerged culture, which is used for AA production, no significant antibiotic activity was found . We conclude that M . alpina in general, and the AA production strains CBS 168.95 and CBS 169.95 in particular, should be considered safe for the submerged production of food ingredientsPublication Types:

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