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 (Sabour