Microbiology, 1994 Oct, 140 ( Pt 10), 2657 - 62 The utilization of thiocyanate as a nitrogen source by a heterotrophic bacterium: the degradative pathway involves formation of ammonia and tetrathionate; Stratford J et al.; A Gram-negative soil bacterium (isolate 26B) has been shown to utilize up to 100 mM thiocyanate as a source of nitrogen when supplied with glucose as the source of carbon and energy . During growth of isolate 26B with thiocyanate as the source of nitrogen, no ammonia, nitrate, nitrite, cyanide, cyanate, sulfate, sulfite, sulfide or carbonyl sulfide was detected in the growth medium . Growth of the bacterium on 14C-labelled thiocyanate (1.6 microCi) and glucose, yielded 14C-labelled carbon dioxide (0.9 microCi) . The addition of 2.9 mM thiocyanate to a bacterial suspension in phosphate buffer (50 mM, pH 7.4) resulted in the utilization of 2.1 mM thiocyanate and the production of 2.0 mM ammonia . This activity was inducible and only occurred after growth of the bacterium with thiocyanate as the source of nitrogen . Tetrathionate (0.7 mM) was detected in the medium after the utilization of thiocyanate (2.4 mM) by a suspension of the bacterium in phosphate buffer, and thiosulfate (1.0 mM) was detected as an intermediate . The addition of sulfide or thiosulfate to the bacterial suspension also resulted in the formation of tetrathionate . The utilization of both of these compounds appeared to be constitutive . A pathway for thiocyanate utilization by isolate 26B is proposed which involves the hydrolysis of thiocyanate to produce cyanate and sulfide . The cyanate then undergoes further hydrolysis to form ammonia and carbon dioxide . The sulfide is ultimately oxidized to tetrathionate via a pathway which includes thiosulfate. Plant Cell, 1994 Oct, 6(10), 1375 - 89 A plant plasma membrane proton-ATPase gene is regulated by development and environment and shows signs of a translational regulation; Michelet B et al.; A proton-pumping ATPase is present in the plasma membrane of plant cells where it sustains transport-related functions . This enzyme is encoded by a family of genes that shows signs of both transcriptional and post-transcriptional regulation . The regulation of pma1, one of the Nicotiana plumbaginifolia H+-ATPase genes, was characterized with the help of the beta-glucuronidase (gusA) receptor gene in transgenic plants . pma1 is active in the root epidermis, the stem cortex, and guard cells . This activity depends on developmental and growth conditions . For instance, pma1 activity in guard cells was strongly enhanced when the plant material (young seedlings or mature leaves) was incubated in liquid growth medium . pma1 is also expressed in several tissues of the reproductive organs where active transport is thought to occur but where scarcely any ATPase activity has been identified, namely in the tapetum, the pollen, the transmitting tissue, and the ovules . Several pma genes have a long 5'untranslated region (leader sequence) containing an upstream open reading frame (URF) . Analysis of translational and transcriptional fusions with gusA in transgenic plants suggests that the pma1 leader sequence might activate translation of the main open reading frame, even though the URF is translated by a large majority of the scanning ribosomes . As confirmation, transient expression experiments showed that the pma1 leader causes a fourfold post-transcriptional increase of main open reading frame expression . Deletion of the URF by site-directed mutagenesis stimulated the main open reading frame translation 2.7-fold in an in vitro translational assay . These results are consistent with a regulatory mechanism involving translation reinitiation . Altogether, they suggest a fine, multilevel regulation of H+-ATPase activity in the plant. Gynecol Oncol, 1994 Oct, 55(1), 91 - 5 Effect of nicotine on proliferation of normal, malignant, and human papillomavirus-transformed human cervical cells; Waggoner SE et al.; Nicotine is concentrated in the cervical mucus of smokers relative to serum levels . In this experiment, the effect of nicotine on cellular proliferation of human ectocervical, endocervical, malignant, and human papillomavirus (HPV) 16 DNA-transformed cervical cell lines was studied . Ectocervical and endocervical cell lines were derived from benign hysterectomy specimens and cultured in keratinocyte growth medium . HPV 16 DNA-transformed cell lines were derived through transfection of ectocervical cells with cloned HPV 16 DNA . HPV-transformed lines and malignant cell lines established from three women with newly diagnosed cervical cancer were maintained in E-media with 5% fetal calf serum . Cells (2500-5000) were cultured in 96-well tissue culture plates with varying concentrations of nicotine (100 to 10 mg/ml) and proliferation was assessed 72 hr later with a semiautomated colorimetric assay . Experiments were performed three times and proliferation of nicotine-exposed cells was compared to unexposed cells with one-way analysis of variance . Nicotine, at 100 ng/ml to 10 micrograms/ml, significantly stimulated epithelial cell growth in two ectocervical and three HPV DNA-transformed cell lines (proliferation 118-180% of control, P < .05) . Nicotine at 100 ng/ml to 10 micrograms/ml did not significantly alter proliferation of four endocervical, three malignant, and two other ectocervical cell lines . Toxic effects of nicotine (> 50% inhibition of cellular proliferation) were noted between 100 micrograms/ml and 10 mg/ml and exceed the concentrations of nicotine reported in smoker's cervical mucus . These findings demonstrate that nicotine, in physiologically attainable concentrations, does not impair and occasionally enhances the proliferation of human cervical cells in vitro . The selective mitogenic effect noted among normal ectocervical and HPV-transformed ectocervical cells may relate to epidemiologic studies showing, among smokers, an increased risk of squamous cell carcinoma, and not adenocarcinoma, of the cervix. J Invest Dermatol, 1994 Oct, 103(4), 564 - 8 Normalization of essential-fatty-acid-deficient keratinocytes requires palmitic acid; Marcelo CL et al.; Cultured adult human keratinocytes show accelerated growth rates in medium that is essential fatty acid deficient . The cells also show decreased amounts of the essential fatty acids 18:2, 20:3, and 20:4 and contain increased amounts of the monounsaturated fatty acids 16:1 and 18:1 . These lower levels of polyunsaturated fatty acids were only partially restored by supplementing the medium with 18:2 and 20:4 fatty acid . The addition of the non-essential fatty acid 16:0 (5 microM), along with the essential fatty acids, resulted in the successful normalization of the major fatty acids in the deficient keratinocytes . Normalized cells showed a constant total fatty acid/mg of protein in the phospholipid fraction, as the total cell fatty acid content per cell increased with augmenting fatty acid supplementation . Supplementation of the medium with 16:0 and essential fatty acids decreased the growth and passage potential of the cells . Use of 18:1 in lieu of 18:2 fatty acid yielded essential-fatty-acid-deficient keratinocyte growth values . Likewise the least supplemented medium (5 microM 18:2 + 5 microM 16:0) also gave the accelerated cell growth rates . This study shows that manipulation of the essential fatty acid levels, if accompanied by 5 microM 16:0 in the growth medium, alters the growth properties of adult human primary keratinocytes. Infect Immun, 1994 Oct, 62(10), 4261 - 9 Surface-associated filamentous hemagglutinin induces autoagglutination of Bordetella pertussis; Menozzi FD et al.; Filamentous hemagglutinin (FHA) is a major adhesin produced by Bordetella pertussis, the etiologic agent of whooping cough . FHA has been shown to be surface associated but is also secreted by virulent bacteria . Microscopic observations of lungs of mice infected with B . pertussis showed that the bacteria grow as clusters within the alveolar lumen . When B . pertussis was cultivated in vitro with chemically defined medium, bacteria grew as aggregates, mimicking growth observed in vivo . This aggregation was abolished by the addition of cyclodextrin (CDX) to the growth medium and depended on the production of FHA, because a mutant lacking the FHA structural gene failed to form aggregates in a CDX-free medium . Western blot (immunoblot) analyses revealed that, in the absence of CDX, FHA was attached to the bacterial surface and was not efficiently released into the growth medium . Hydrophobic chromatography of FHA showed that CDX drastically reduced the hydrophobicity of FHA, suggesting a direct binding of CDX to FHA, which was further supported by the partial protection of FHA from trypsin digestion in the presence of CDX . In addition, free FHA can interact in a CDX-inhibitable manner with solid phase-immobilized FHA . It can therefore be postulated that the B . pertussis aggregates are most likely due to direct FHA-FHA interaction. Epidemiol Infect, 1994 Oct, 113(2), 235 - 45 Toxin production, adherence and protein expression by clinical Aeromonas spp . isolates in broth and human pooled ileostomy fluid; Wilcox MH et al.; The physiological behaviour of clinical Aeromonas spp . isolates was compared following culture in a conventional broth and human pooled ileostomy fluid (PIF) . Protein expression was markedly affected by the growth medium, with an overall reduction in whole cell proteins in bacteria grown in ileostomy fluid . In addition, novel outer membrane proteins were produced in PIF but not in broth . The majority of A . hydrophila and A . sobria isolates produced toxin in both broth and PIF, whereas no cytotoxin positive A . caviae were found . Toxin titres were at least two doubling dilutions higher in 40% and 21% of A . hydrophila and A . sobria isolates, respectively, following culture in brain heart infusion broth compared with PIF . Bacterial adherence to Vero and A-549 cells was significantly more common in A . hydrophila (53%) and A . sobria (64%) than in A . caviae (15%) (P < 0.01) . We observed increased adherence by 6 aeromonas strains previously classified as adherence-positive, but not by 6 non-adherers, in PIF compared with brain heart infusion broth . The influence of growth medium on the expression of potential virulence determinants by Aeromonas spp . provides a rationale for the use of human ileostomy fluid in future in vitro studies, in order to simulate the nutrient conditions found in vivo. Biochem Mol Biol Int, 1994 Oct, 34(4), 789 - 99 Properties of yeast cells depleted of the OSCP subunit of mitochondrial ATP synthase by regulated expression of the ATP5 gene; Prescott M et al.; OSCP is a subunit of the FA stalk sector of yeast mitochondrial ATP synthase complex . Cells of a null mutant for OSCP, constructed by disruption of the chromosomal ATP5 gene of Saccharomyces cerevisiae, exhibited a high level of genetic instability (petite formation) . Study of the effects of ablation of OSCP required the development of a progressive depletion strategy . Introduction of a vector bearing an ATP5 gene cassette under GAL1 transcriptional control into null mutant cells gave rise to a stable yeast strain from which OSCP could be depleted in a controlled manner by manipulation of the level of galactose in the growth medium . Cells progressively depleted of OSCP exhibited properties of cellular respiration indicative of a decline in the functional coupling of the catalytic F1 sector to the proton channel F0 sector (normally linked by FA) . Cells depleted of OSCP also exhibited a physical uncoupling of F1 from other subunits of the complex such that other FA subunits and F0 subunit 6 were not recovered in immunoprecipitates of ATP synthase complexes . Thus, OSCP plays a role in the assembly as well as function of the enzyme complex. Protein Expr Purif, 1994 Oct, 5(5), 449 - 57 Cloning and expression in murine erythroleukemia cells: the soluble forms of the type I and type II tumor necrosis factor receptors fused to an immunogenic affinity tag; Newton CR et al.; We have cloned, expressed, and purified the extracellular domains of types I and II human tumor necrosis factor receptors . Both proteins were expressed in and secreted by murine erythroleukemia cells under the control of the human beta-globin promoter placed down-stream from the human globin locus control region . Secretion of both proteins was directed by the respective tumor necrosis factor receptor signal sequence . Each tumor necrosis factor receptor extracellular domain was expressed as a chimeric protein, fused to a carboxy terminal flexible peptide linker and an antigenic affinity tag . Secretion of both proteins into the growth medium in a hollow fiber bioreactor was achieved . A monoclonal antibody generated against the affinity tag allowed the purification of both proteins . These were isolated as biologically active products in that they bound human tumor necrosis factor-alpha in a 125I-radioiodinated ligand binding assay . The two proteins also bound tumor necrosis factor-alpha at approximately equimolar ratios as demonstrated by BIAcore sensorgram analysis. J Biol Chem, 1994 Sep 16, 269(37), 23192 - 6 cDNA cloning and chromosome mapping of human dihydropyrimidine dehydrogenase, an enzyme associated with 5-fluorouracil toxicity and congenital thymine uraciluria; Yokota H et al.; The pig and human dihydropyrimidine dehydrogenase (DPD) cDNAs were cloned and sequenced . The pig enzyme, expressed in Escherichia coli, catalyzed the reduction of uracil, thymine, and 5-fluorouracil with kinetics approximating those published for the enzyme purified from mammalian liver . DPD could be expressed in significant quantities only when uracil was added to the bacterial growth medium . The pig and human enzymes contained 1025 amino acids and calculated M(r) = 111,416 and 111,398, respectively . Conserved domains corresponding to a possible NADPH binding site and FAD binding site were found in the NH2-terminal half of the proteins and two motifs of putative {4Fe-4S} binding sites were found near to the carboxyl terminus of the enzyme . The latter corresponds to the labile COOH-terminal fragment previously shown to contain the iron sulfur centers . A sequence encompassing a peptide corresponding to the uracil binding site was found between the NADPH/FAD-containing NH2-terminal portion of the protein and the iron-sulfur binding sites near to the COOH terminus . Thus, the DPD appears to be derived from at least three distinct domains . The DPYD gene was localized to the centromeric region of human chromosome 1 between 1p22 and q21. Biochem Biophys Res Commun, 1994 Sep 15, 203(2), 857 - 65 Thrombospondin (TSP) and transforming growth factor beta 1 (TGF-beta) promote human A549 lung carcinoma cell plasminogen activator inhibitor type 1 (PAI-1) production and stimulate tumor cell attachment in vitro; Albo D et al.; A growing body of evidence has recently implicated TSP and TGF-beta in the process of malignancy, such as tumor cell proliferation, tumor angiogenesis, and metastasis . The purpose of the present study was to evaluate potential mechanisms of TSP and TGF-beta in tumor cell attachment and invasion . Our results indicate that both TSP and TGF-beta promoted tumor cell attachment and spreading in the presence of plasminogen . The mechanism for these effects appeared to be due, in part, to the capacity of TSP and TGF-beta to induce tumor cell production of (PAI-1) . PAI-1, which is a natural inhibitor of tumor-cell associated urokinase-type plasminogen activator (uPA) activity, inhibited activation of plasminogen to plasmin in the growth media, thereby preventing plasmin-induced detachment of cells . The TSP-promoted production of PAI-1 could be inhibited not only by anti-TSP antibodies but also by a neutralizing antibody against TGF-beta . These results suggest that TSP by a mechanism involving TGF-beta can promote cell adhesion through stimulation of tumor cell secretion of PAI-1 . These data provide evidence that TSP not only has the capacity of functioning as a matrix protein to directly promote cell-substratum adhesion but that TSP can also stimulate cell adhesion and spreading by modulating cell surface protease expression through stimulation of tumor-associated production of PAI-1. FEMS Microbiol Lett, 1994 Sep 15, 122(1-2), 97 - 101 Variable expression of O-antigen and the role of lipopolysaccharide as an adhesin in Aeromonas sobria; Francki KT et al.; On initial isolation of Aeromonas sobria 3767 from a diarrhoeal stool specimen, two colony types were obtained: opaque (3767O) and translucent (3767T) . Strain 3767O consistently produced lipopolysaccharide (LPS) core and O-antigen side chain, detectable by SDS-PAGE and by Western blotting with an O-antigen-specific monoclonal antibody . Strain 3767T produced LPS core but the amount of O-antigen was dependent on factors including growth medium and bacterial growth phase . Strain 3767T exhibited significantly lower levels of adhesion to HEp-2 cells than 3767O and this correlated with the level of LPS expression, with the greatest reduction (61%) at stationary phase when no LPS was detectable . The results implicate LPS as an adhesin for A . sobria 3767. J Biol Chem, 1994 Sep 9, 269(36), 22719 - 25 Reversal of signal-mediated cellular retention by subunit assembly of human acetylcholinesterase; Velan B et al.; The interrelationship between signal-mediated endoplasmic reticulum retention and control of subunit assembly in secreted complex proteins was examined in recombinant 293 cells expressing human acetylcholinesterase (HuAChE) . This was achieved by analyzing the mutual effects of co-residing retention and dimerization signals on enzyme secretion by transfected cells . The function of putative signals within the COOH-terminal tetrapeptide CSDL of HuAChE was examined by site-directed mutagenesis . The CSDL tetrapeptide carries the free cysteine (Cys-580) involved in subunit assembly, yet it fails to function as a KDEL-type retention signal . This was demonstrated by mutations that increase similarity to the canonical retention signal (substitution of CSDL by KSDL) or those that deviate from it (substitution to CSAL) . Cells expressing both types of mutants exhibited cell-associated HuAChE levels identical to that of wild type enzyme . Appendage of an engineered KDEL retention signal to a dimerization-impaired HuA-ChE subunit (the C580A mutant) resulted in intracellular retention of large amounts of fully active enzyme not prone to proteolytic degradation . On the other hand, attachment of KDEL to a native, dimerization-competent HuAChE polypeptide did not lead to intracellular retention and allowed efficient secretion of enzyme to the cell growth medium . Yet, appendage of KDEL to the native HuAChE led to some retardation in the transport of enzyme molecules through the Golgi apparatus, as manifested by increase in cellular population of endo H-resistant dimers, when compared with wild type enzyme . Taken together, these results indicate (alpha) that sub-unit dimerization mediated by the COOH-terminal cysteine of HuAChE can reverse the signal-mediated retention by masking recognition of KDEL by its cognate receptor and (b) that the native sequences of the acetylcholinesterase subunit polypeptide do not appear to function as a coupled retention/dimerization signal in the control of secretion of assembled enzyme molecules. J Cell Biol, 1994 Sep, 126(6), 1433 - 44 Ponticulin is the major high affinity link between the plasma membrane and the cortical actin network in Dictyostelium; Hitt AL et al.; Interactions between the plasma membrane and underlying actin-based cortex have been implicated in membrane organization and stability, the control of cell shape, and various motile processes . To ascertain the function of high affinity actin-membrane associations, we have disrupted by homologous recombination the gene encoding ponticulin, the major high affinity actin-membrane link in Dictyostelium discoideum amoebae . Cells lacking detectable amounts of ponticulin message and protein also are deficient in high affinity actin-membrane binding by several criteria . First, only 10-13% as much endogenous actin cosediments through sucrose and crude plasma membranes from ponticulin-minus cells, as compared with membranes from the parental strain . Second, purified plasma membranes exhibit little or no binding or nucleation of exogenous actin in vitro . Finally, only 10-30% as much endogenous actin partitions with plasma membranes from ponticulin-minus cells after these cells are mechanically unroofed with polylysine-coated coverslips . The loss of the cell's major actin-binding membrane protein appears to be surprisingly benign under laboratory conditions . Ponticulin-minus cells grow normally in axenic culture and pinocytose FITC-dextran at the same rate as do parental cells . The rate of phagocytosis of particles by ponticulin-minus cells in growth media also is unaffected . By contrast, after initiation of development, cells lacking ponticulin aggregate faster than the parental cells . Subsequent morphogenesis proceeds asynchronously, but viable spores can form . These results indicate that ponticulin is not required for cellular translocation, but apparently plays a role in cell patterning during development. Invest Ophthalmol Vis Sci, 1994 Sep, 35(10), 3582 - 8 Localization of NaK ATPase on cultured human retinal pigment epithelium; Hu JG et al.; PURPOSE . To localize NaK ATPase sites on cultured human retinal pigment epithelium (RPE) . METHODS . Cultured human RPE from fetal, 2-year-old, and 21-year-old donors was grown to confluence in microporous culture wells for 4 months to 2 years, mounted in a small-volume Ussing chamber, and perfused with growth medium . Ouabain (10(-5)-M) was applied to the basal and apical sides of the RPE . Changes in transepithelial resistance (Rt), transepithelial potential (TEP), and apical and basal membrane potentials were measured . RESULTS . Application of ouabain to the basal side of RPE produced a small sustained increase in TEP after 6 minutes and, simultaneously, small depolarizations of both apical and basal membranes . During the continued presence of ouabain on the basal side, application of ouabain to the apical side produced a significantly larger TEP decrease and greater depolarization of both membranes . Significant changes in Rt were not observed . CONCLUSIONS . These results indicate that NaK ATPase sites are present on both the apical and basolateral membranes of cultured human RPE . The greater effect of ouabain when applied to the apical side suggests that functional NaK ATPase sites are more abundant on the apical membrane. Fertil Steril, 1994 Sep, 62(3), 555 - 8 Cumulus mass maintains embryo quality; Saito H et al.; OBJECTIVE: To determine if co-culture of early stage embryos with their own cumulus mass improves embryo quality . DESIGN: Before insemination, cumulus masses along with cumulus matrices were separated from the oocytes surrounded by a few remaining cumulus cells . Fourteen hours after insemination, fertilized oocytes were each placed onto the cumulus cells, and matrix and co-culture commenced . The embryos were observed every 24 hours . Fifty-three oocytes were treated in co-culture (C) and 59 oocytes were treated in routine culture (U) . RESULTS: Thirty-four (C) and 43 (U) oocytes were fertilized and placed on growth media for further culture . Twenty-four hours after culture, 10 embryos (29%) in C and 12 (28%) in U were good quality, and 4 embryos (12%) in C and 7 (16%) in U were of poor quality . Seventy-two hours after culture, 10 (29%) in C and 8 (18%) in U were of good quality, and 3 (9%) in C and 13 (30%) were of poor quality . The percentage of good quality embryos in the co-culture group was significantly higher than in the control group after 72 hours . Conversely, the percentage of poor quality embryos in the co-culture group was significantly lower than that in the control group after 72 hours . CONCLUSION: Co-culture maintains embryo quality over prolonged culture times . This facilitates the development of good quality embryos for ET. J Dermatol, 1994 Sep, 21(9), 633 - 8 Effects of etretinate on keratinocyte proliferation and secretion of interleukin-1 alpha (IL-1 alpha) and IL-8; Zhang JZ et al.; Etretinate has proven to be effective in the treatment of psoriasis . Since abnormal proliferation and cytokine secretion are well-known features of psoriatic epidermis, we studied the in vitro effects of etretinate on these two processes using human keratinocytes . Etretinate promoted proliferation of normal human keratinocytes (NHKs) grown in keratinocyte growth medium (KGM) but not in growth factor-deficient keratinocyte basic medium (KBM) . Moreover, etretinate partly overcame growth inhibition by PMA . Etretinate was shown to have an effect on either IL-1 alpha or IL-8 secretion in unstimulated NHKs . In HSC-1, a human squamous cell carcinoma cell line cultured in 20% FCS/DMEM, inhibited IL-1 alpha secretion and enhanced IL-8 secretion . These results indicate that the effects of etretinate on keratinocyte proliferation and cytokine secretion may depend on cell type and culture conditions . Stimulation of NHKs with PMA significantly enhanced IL-1 alpha and IL-8 secretion, and these effects were inhibited by etretinate . However, etretinate failed to inhibit rTNF alpha-induced IL-8 secretion, suggesting that etretinate regulation of NHK cytokine secretion may also depend on the stimulus . As treatment of keratinocytes or epidermis with PMA can induce psoriasis-like changes, so might the experimental "anti-PMA" activity of etretinate be related to its therapeutic benefit in the treatment of psoriasis. Appl Environ Microbiol, 1994 Sep, 60(9), 3466 - 9 Biodegradation of 2-nitrotoluene by Pseudomonas sp . strain JS42; Haigler BE et al.; A strain of Pseudomonas sp . was isolated from nitrobenzene-contaminated soil and groundwater on 2-nitrotoluene as the sole source of carbon, energy, and nitrogen . Bacterial cells growing on 2-nitrotoluene released nitrite into the growth medium . The isolate also grew on 3-methylcatechol, 4-methylcatechol, and catechol . 2-Nitrotoluene, 3-methylcatechol, and catechol stimulated oxygen consumption by intact cells regardless of the growth substrate . Crude extracts from the isolate contained catechol 2,3-dioxygenase and 2-hydroxy-6-oxohepta-2,4-dienoate hydrolase activity . The results suggest that 2-nitrotoluene is subject to initial attack by a dioxygenase enzyme that forms 3-methylcatechol with concomitant release of nitrite . The 3-methylcatechol is subsequently degraded via the meta ring fission pathway. Mol Microbiol, 1994 Sep, 13(6), 1045 - 55 Nitrite and nitrate regulation at the promoters of two Escherichia coli operons encoding nitrite reductase: identification of common target heptamers for both NarP- and NarL-dependent regulation; Tyson KL et al.; Expression from both the Escherichia coli nir and nrf promoters is dependent on anaerobic induction by FNR but is further regulated by NarL and NarP in response to the presence of nitrite and nitrate in the growth medium . The nir promoter is activated by NarL in response to nitrate and nitrite and activated by NarP in response to nitrate but not nitrite . The effects of point mutations suggest that NarL and NarP both bind to the same target, which is a pair of heptamer sequences organized as an inverted repeat, centred 69 1/2 bp upstream of the transcript startpoint . The nrf promoter can be activated by either NarP or NarL in response to nitrite but is repressed by NarL in response to nitrate . Mutational analysis of the nrf promoter has been exploited to corroborate the location of the -10 hexamer and the FNR-binding site, and to find the sites essential for nitrite-dependent activation and nitrate-dependent repression . Optimal activation by NarP or NarL in response to nitrite requires an inverted pair of heptamer sequences, similar to that found at the nir promoter, but centred 74 1/2 bp upstream from the transcript start . NarL-dependent repression by nitrate is due to two heptamer sequences that flank the FNR-binding sequence . We conclude that NarL and NarP bind to the same heptamer sequences, but that the affinities for the two factors vary from site to site. Mol Biochem Parasitol, 1994 Sep, 67(1), 41 - 7 Characterization of enzymatically active Onchocerca volvulus Cu/Zn superoxide dismutase expressed in Escherichia coli; Henkle-Duhrsen K et al.; The Onchocerca volvulus superoxide dismutase was expressed in Escherichia coli, using a protocol designed to produce the native enzyme rather than a fusion protein . The recombinant O . volvulus superoxide dismutase (rOVSOD) was found in the cytosol of the disrupted bacteria and represented > 10% of the total bacterial protein . The enzyme was purified to homogeneity using DEAE-Sepharose chromatography, followed by phenyl-Sepharose chromatography . The rOVSOD was enzymatically active which was demonstrated by its reactivity with O2.- produced either by the xanthine-xanthine oxidase system or by stimulated eosinophils . The specific activity was determined to be 4668 U mg-1 . This activity could be blocked by rabbit antiserum raised against the rOVSOD . The maximal activity was obtained upon supplementation of the bacterial growth media and enzyme buffer with copper and zinc ions . Activity characteristics in the presence of inhibitors was also characteristic of a Cu/Zn superoxide dismutase . The rOVSOD has an apparent subunit molecular mass of 16,000 in SDS-PAGE . The active enzyme behaves as a dimer of 32 kDa as determined by gel filtration. Biomaterials, 1994 Sep, 15(11), 950 - 2 Cultural technique for in vitro modelling of prolonged bioadhesion; Needleman IG et al.; This paper describes the development of a culture system for bioadhesion testing of gels over prolonged periods . Hamster cheek pouch mucosa was placed on collagen gels or steel mesh supports and submerged in growth medium for periods up to 7 d . Mucosal integrity and morphology were well maintained on mesh supports, but necrosis quickly occurred on collagen gels . Pilot studies with Orabase adhering to the tissue showed no detrimental effects, with the adhesive remaining in place for 4-6 h . It is concluded that the system shows promise for the investigation of prolonged bioadhesion. Am J Reprod Immunol, 1994 Sep, 32(2), 101 - 7 H5D12 monoclonal antibody identifies a 24-kD immunosuppressor factor produced by human chorio carcinoma cells; Bose R et al.; PROBLEM: The purpose of this study was to identify and characterize embryo-associated immunosuppressor factor (EASF) secreted by chorio carcinoma cells with the help of EASF-specific monoclonal antibody H5D12 (raised against EASF purified from embryo growth media of in vitro fertilized human ova) . METHOD: Paraffin-embedded slides of human chorio carcinoma as well as control cell lines were prepared, and immunohistochemistry was done by the avidin-biotin-peroxidase technique . EASF was affinity purified using H5D12-Sepharose 4B from culture media of cell lines and analyzed for immunosuppressive activity (by Concanavalin-A-induced lymphocyte proliferation assay) and molecular weight identity (by metabolic-labelling studies with 35S-methionine followed by immunoprecipitation and SDS-PAGE) . RESULTS: H5D12 showed intense immunostaining of BeWo chorio carcinoma cells . Biosynthetic labeling studies identifies this factor as a 24-kD molecule, and EASF bioassay indicates that this factor possesses immunosuppressive activity . No such immunosuppressive activity or similar molecules were identified when control cell lines were analyzed . CONCLUSIONS: Monoclonal antibody H5D12 recognizes a 24-kD factor with immunosuppressive activity that is secreted by chorio carcinoma cells, which suggests that this is a unique factor and may be one of the key regulators of reproductive functions. In Vitro Cell Dev Biol Anim, 1994 Sep, 30A(9), 596 - 603 Establishment and characterization of immortalized clonal cell lines from fetal rat mesencephalic tissue; Prasad KN et al.; This investigation reports for the first time the establishment of immortalized clones of dopamine-producing nerve cells in culture . Freshly prepared single-cell suspensions from fetal (12-day-old) rat mesencephalic tissue were transfected with plasmid vectors, pSV3neo and pSV5neo, using an electroporation technique . Cells were plated in tissue culture dishes which were precoated with a special substrate and contained modified MCDB-153 growth medium with 10% heat inactivated fetal bovine serum . The immortalized cells were selected by placing the transfected cells in a selection medium (modified MCDB-153 containing 400 micrograms/ml geneticin) . The survivors showed the presence of T-antigens and were non-tumorigenic . Two cell lines, 1RB3 derived from cells transfected with pSV3neo, and 2RB5 derived from cells transfected with pSV5neo revealed only 1 to 2% tyrosine hydroxylase (TH)-positive cells . Repeated single-cell cloning of these cell lines by a standard technique failed to increase the number of TH-positive cells in any clones . Using three cycles of growth, alternating between hormone-supplemented, serum-free medium and serum-containing medium produced a cell line (1RB3A) that was very rich in TH-positive cells . The recloning of 1RB3A yielded clones some of which contained over 95% TH-positive cells . These cells produced homovanillic acid, a metabolite of dopamine, and may be useful not only for neural transplant but also for basic neurobiological studies. Indian J Exp Biol, 1994 Sep, 32(9), 637 - 42 Differential modification of radiation damage in 5-bromo-2-deoxy-uridine sensitized human glioma cells and PHA-stimulated peripheral leukocytes by 2-deoxy-D-glucose; Kalia VK et al.; Effects of 5-bromo-2-deoxy-uridine (BrdU) and 2-deoxy-D-glucose (2-DG) on 60-Co-gamma-ray induced damage were studied in monolayer cultures of glioma (BMG-1) cells, and PHA-stimulated peripheral leukocytes from normal donors . Micronuclei formation was used as an index of cytogenetic damage . BrdU and 2-DG treatments did not induce micronuclei formation in unirradiated cultures . Presence of BrdU (0.8 microM) for more than one cell cycle (24 hr) significantly increased gamma-ray (1-4 Gy) induced micronuclei formation in exponentially growing BMG-1 cells . Incubation of irradiated cells under sub-optimal growth conditions (DMEM with 1% serum) for 3 hr, instead of growth medium, significantly decreased micronuclei formation . Post-irradiation presence of 2-DG (5 mM; 3 hr, in DMEM + 1% serum) significantly increased radiation damage . In BrdU sensitized cells also, 2-DG significantly increased radiation damage further . In PHA-stimulated leukocytes from normal donors, 2-DG (5mM, equimolar with glucose; for 2 hr) did not increase gamma-ray (2-Gy, 42 hr after PHA-stimulation) induced micronuclei formation . Pre-irradiation presence of BrdU (1.6 microM) significantly increased micronuclei . On the contrary, 2-DG treatment reduced radiation induced micronuclei formation in BrdU sensitized leukocyte cultures . These results suggest that (i) radiation induced lesions leading to micronuclei formation in proliferating tumour cells, are, at least, partly repairable; (ii) combination of 2-DG could reduce BrdU doses required for radiosensitization of proliferating tumour cells; and (iii) 2-DG could differentially increase radiation damage in BrdU sensitized proliferating tumour cells, while reducing manifestation of damage in normal proliferating cells. Br J Pharmacol, 1994 Sep, 113(1), 151 - 8 Infection by HIV-1 blocked by binding of dextrin 2-sulphate to the cell surface of activated human peripheral blood mononuclear cells and cultured T-cells; Shaunak S et al.; 1 . Structural analogues of a sulphated polysaccharide, dextrin sulphate, were synthesized and tested for their ability to block infection by HIV-1 . Using the T-cell lines, C8166 and HPB-ALL, and the laboratory adapted strains of HIV-1.MN, HIV-1.IIIb and HIV-1.RF, dextrin 2-sulphate (D2S) combined the best combination of high anti-HIV-1 activity (95% inhibitory concentration (IC95) = 230 nM) and low anticoagulant activity . It also blocked infection of activated peripheral blood mononuclear (PBMN) cells by five primary viral isolates at an IC95 of 230-3700 nM depending upon the primary viral isolate tested . 2 . In saturation binding studies, {3H}-D2S bound to a cell surface protein on HPB-ALL cells in a specific and saturable manner with a Kd of 82 +/- 14 nM and a Bmax of 4.8 +/- 0.3 pmol/10(6) cells . It bound to other human T-cell lines in a similar manner . 3 . There was very little binding of {3H}-D2S to freshly isolated PBMN cells (Bmax 0.18 +/- 0.03 pmol/10(6) cells) and these cells could not be infected by HIV-1 . Culture of PBMN cells in lymphocyte growth medium (LGM) containing IL-2 did not significantly change the Bmax of {3H}-D2S . In contrast, PBMN cells which had been cultured with phytohaemagglutinin (PHA; 5 micrograms ml-1) for 72 h had a Bmax of {3H}-D2S binding of 7.2 +/- 0.1 pmol/10(6) cells and these cells could be infected by HIV-1 . Removal of the PHA and further culture of the PBMN cells in LGM containing IL-2 resulted in a fall in the Bmax to 2.0 +/- 0.1 pmol/10(6) cells.(ABSTRACT TRUNCATED AT 250 WORDS) Biochem Biophys Res Commun, 1994 Aug 30, 203(1), 281 - 8 Alpha-interferon induces depletion of intracellular iron content and upregulation of functional transferrin receptors on human epidermoid cancer KB cells; Caraglia M et al.; We have demonstrated that interferon-alpha (IFN alpha) upregulates the epidermal growth factor receptor (EGF-R) on human epidermoid carcinoma cells . Here we report that IFN alpha induces growth inhibition and upregulation of transferrin receptor (TRF-R) on epidermoid cancer KB cells . IFN alpha does not alter TRF-R affinity for its ligand and induces a two-fold increase of TRF binding sites . IFN alpha does not modify receptor internalization and cycling . Intracellular iron levels are known to regulate TRF-R expression: we have, therefore, evaluated whether changes in the iron content could be determined by IFN alpha . Iron levels are transiently increased after addition of fresh growth medium in untreated controls but not in KB cells exposed for 48 h to IFN alpha . Iron depletion is however completely reversed 24 h later when maximal TRF-R upregulation occurs in IFN alpha-treated cells . We suggest that IFN alpha-induced iron depletion elicits a homeostatic cellular response through upregulation of TRF-R. Brain Res, 1994 Aug 29, 655(1-2), 222 - 32 Growth conditions differentially modulate the vulnerability of developing cerebellar granule cells to excitatory amino acids; Resink A et al.; The survival of immature nerve cells in a cerebellar culture, predominantly excitatory granule cells, is known to be promoted by chronic exposure to high K+ (> 20 mM) or glutamate (Glu) receptor agonists . These treatments are believed to mimic the in vivo effect of the incoming glutamatergic afferents, the mossy fibres . Here we report that with maturation the cells become vulnerable to excitatory amino acids (EAAs) and that the characteristics of EAA sensitivity are dependent on the environmental influences being either "trophic" (25 mM K+ or 140 microM NMDA, K25 or K10 + NMDA) or "non-trophic" (10 mM K+, K10) . Toxicity was assayed routinely at 9 days in vitro (DIV) after 24 h exposure to EAAs . Under all the tested conditions, the effect of Glu was mediated exclusively through NMDA receptors . However, the efficacy and potency of Glu were high in K25- and K10 + NMDA-grown cells compared with K10-grown cells . Growth conditions had the same influence on NMDA as on Glu-induced toxicity, but with the following special features: (1) in comparison with K25 cells, the potency of NMDA was significantly lower in K10 + NMDA cells . The K10 + NMDA cultures behaved as if they were completely insensitive to the NMDA which is present in their growth medium . (2) The K10-grown cells were not vulnerable to NMDA, unless the cell membrane was depolarised by shifting the cells into K25 medium . The efficacy of NMDA became then similar to that in K25 cultures, although the potency was about 7-fold less . Thus NMDA receptors can be activated by the depolarisation of K10 cells, implying the operation of Mg2+ blockade of the channel at normal resting membrane potential . Although non-NMDA receptors did not seem to be involved in Glu toxicity, cells were vulnerable to kainate, which killed significantly more cells than Glu (about 80% vs 70%) . This was partly due to the resistance of GABAergic interneurons present in the cultures to Glu- or NMDA-induced toxicity . In contrast to the effects of Glu or NMDA, KA vulnerability was lower in cells grown in K25 or K40 than K10 medium (rank order K10 > K25 > K40) . Under our experimental conditions, cultured cells were resistant to AMPA, quisqualate and the selective metabotropic Glu receptor agonist 1S,3R-ACPD . Collectively, the observations indicated that EAA sensitivity of cultured cerebellar interneurons is significantly and differentially influenced by environmental factors, believed to mimic in vivo trophic influences on these cells. J Biol Chem, 1994 Aug 19, 269(33), 21072 - 7 The folding of bovine pancreatic trypsin inhibitor in the Escherichia coli periplasm; Ostermeier M et al.; PreOmpA-bovine pancreatic trypsin inhibitor (BPTI) (Goldenberg, D . P . (1988) Biochemistry 27, 2481-2489) was expressed in Escherichia coli, and the folding pathway of the mature protein in the periplasmic space was analyzed by pulse-chase experiments . Folding intermediates were trapped with iodoacetamide, immunoprecipitated with antisera specific for either the reduced or the native protein, and resolved by electrophoresis . In vivo, native BPTI formed with a half-life of 7 min which is 3-fold faster than the optimal in vitro folding rate in growth media supplemented with low molecular weight disulfides . The measured in vivo half-life includes the time required for translocation and processing by leader peptidase and therefore represents the lower limit for the actual folding rate in the cell . In addition to the native species, two-disulfide intermediates accumulated in the cell at appreciable levels and did not chase to the native species for at least 20 min . We found that the folding of BPTI in E . coli was absolutely dependent on DsbA, a protein which accelerates the formation of disulfide bonds in the periplasm . In a dsbA mutant strain, trace amounts of oxidized BPTI could be detected only in cultures grown under strongly oxidizing conditions . In wild-type cells, the addition of different concentrations of GSH/GSSG or oxidized dithiothreitol did not affect the kinetics of BPTI oxidation by DsbA . Finally, even though the folding of BPTI in vitro decreases by almost 10-fold/unit pH decrease, folding in cells grown at pH 6.0 was only marginally slower than folding in cells grown at neutral pH, despite the fact that the pH of the periplasmic space varies in response to the extracellular fluid. Biochim Biophys Acta, 1994 Aug 11, 1223(1), 23 - 8 Suppression of heat-shock protein synthesis by short-chain fatty acids and alcohols; Munks RJ et al.; We have shown that ethanol, propanol and butanol (at 0.5-2%) and salts of butyric and propionic acids (at 8-40 mM) all cause a major reduction in heat-shock protein (hsp) synthesis when present in the growth medium of Drosophila cultured cells (Kc and SL2) subjected to either increased temperature or chemical stressors . Inhibition of normal protein synthesis in unstressed cells was comparatively slight, and the usual suppression of synthesis of non-heat-shock proteins in stressed cells was unaffected . Maximum suppression of hsp synthesis occurred only if inhibitors were added before initiation of the stress response, an observation that eliminates the possibility that these findings are due to non-specific, toxic effects . Suppression was accompanied by severely reduced levels of both hsp70 mRNA and active heat-shock factor (HSF) . We conclude that the inhibitors act by suppressing the initiation of transcription of heat-shock genes. FEBS Lett, 1994 Aug 8, 349(3), 424 - 8 Na(+)-driven ATP synthesis in Methanobacterium thermoautotrophicum and its differentiation from H(+)-driven ATP synthesis by rhodamine 6G; Smigan P et al.; Rhodamine 6G (3 microM) effectively inhibited delta pH-driven ATP synthesis in Methanobacterium thermoautotrophicum while delta pNA-driven ATP synthesis was not affected by it . Rhodamine 6G inhibited Mg(2+)-stimulated ATPase activity of membrane vesicles prepared from these cells but the ATPase catalytic sector detached from the membrane was insensitive to this inhibitor . Methanogenesis-driven ATP synthesis at pH 6.8 of the cells grown in the presence of 50 mM NaCl was inhibited by rhodamine 6G both in the presence of 5 mM and 50 mM NaCl . On the other hand, the methanogenesis-driven ATP synthesis at pH 8.0 of cells grown in the presence of 50 mM NaCl was slightly inhibited by rhodamine 6G in the presence of 5 mM NaCl and was not inhibited at all in the presence of 50 mM NaCl . The growth experiments have shown that cells of Methanobacterium thermoautotrophicum can grow under alkaline conditions even in the presence of rhodamine 6G and of high NaCl concentration when the growth media were inoculated with the cells which had been grown in the presence of 50 mM NaCl . These results indicate that sodium-motive force-driven ATP synthase in Methanobacterium thermoautotrophicum operates effectively at alkaline conditions and it might be the sole ATP synthesizing system when the proton motive force-supported ATP synthesis is inhibited by rhodamine 6G. J Biol Chem, 1994 Aug 5, 269(31), 19725 - 30 The Saccharomyces cerevisiae PLB1 gene encodes a protein required for lysophospholipase and phospholipase B activity; Lee KS et al.; Several enzymes with lysophospholipase/phospholipase B activity have been described from the budding yeast Saccharomyces cerevisiae . In vitro, these enzymes are capable of hydrolyzing all phospholipids that can be extracted from yeast cells . Two forms of the enzyme have been isolated from plasma membranes and a third from culture supernatants and the periplasmic space, but their biological roles have not been determined . These highly glycosylated enzymes were reported to have very similar catalytic properties but differed with respect to apparent molecular weight . We isolated a gene from S . cerevisiae, encoding a protein predicted to share 45% amino acid sequence identity with phospholipase B from Penicillium notatum . This yeast gene, designated PLB1, was mapped to the left arm of chromosome VIII . No residual lysophospholipase/phospholipase B activity was detected upon assay of extracts or culture supernatants of a plb1 delta mutant . Thus, either the PLB1 gene encodes all of the previously detected isoforms of phospholipase B or its gene product is required for their expression or activation . Deletion of PLB1 did not result in any apparent phenotypic defect, suggesting either that we failed to identify the growth conditions that would betray such a defect or that Plb1p is functionally redundant with another protein, whose activity has gone undetected . A plb1 delta mutant released wild-type levels of the soluble phosphatidylinositol metabolite glycerophosphoinositol into the growth medium but released greatly reduced levels of the corresponding phosphatidylcholine and phosphatidylethanolamine metabolites . These results indicate that PLB1 is principally responsible for the production of the deacylation products of phosphatidylcholine and phosphatidylethanolamine but not phosphatidylinositol. FEMS Microbiol Lett, 1994 Aug 1, 121(1), 35 - 8 Escherichia coli cells with mutations in the gene for adenylate cyclase (cya) exhibit a heat shock response; Lee-Rivera I et al.; Adenylate cyclase mutants of Escherichia coli showed the heat-shock response . The heat-shock response was studied in two different mutants and in different growth media, including rich and minimal media . These results are in disagreement with the proposal that the cya gene regulates the expression of the heat-shock genes. Am J Physiol, 1994 Aug, 267(2 Pt 1), C520 - 8 Regulation of rat ventricular myosin heavy chain expression by serum and contractile activity; Qi M et al.; To quantitatively analyze the effects of serum stimulation and contractile activity and their interaction on cellular growth and cardiac myosin heavy chain (MHC) gene expression, spontaneously contracting neonatal rat ventricular myocytes in primary culture were maintained in serum-free growth medium or growth medium supplemented with fetal bovine serum . Contractile activity in paired cultures was inhibited by addition of the calcium channel blocker verapamil (10 microM) to the culture medium . Both serum stimulation and contractile activity produced myocyte hypertrophy as assessed by increases in total protein, total RNA, protein-to-DNA ratios, and total MHC protein content . MHC isoenzyme analysis indicated that both MHC-alpha and MHC-beta proteins accumulated in response to serum stimulation and/or contractile activity . The increases in MHC-beta protein resulting from serum stimulation and contractile activity occurred in parallel with increases in MHC-beta mRNA . In contrast, MHC-alpha mRNA levels were relatively unaffected by serum stimulation but appeared to decrease in response to contractile activity . The protein kinase inhibitor staurosporine (5 nM) reduced MHC-beta expression in serum-free, contracting cultures and also prevented the serum-induced increase in MHC-beta mRNA observed in both contracting and arrested myocytes . Staurosporine also increased MHC-alpha mRNA levels in serum-free, contracting, and verapamil-arrested myocytes . These data suggest that both humoral and mechanical factors regulate MHC isoenzyme expression and cellular growth in neonatal ventricular myocytes. DNA Cell Biol, 1994 Aug, 13(8), 875 - 82 Expression and export in Escherichia coli of fusion proteins containing carboxy-terminally located honeybee prepromelittin; He M et al.; The aim of this work was to express a eukaryotic pre-protein in Escherichia coli so that it could be obtained intact, without cleavage, by bacterial leader peptidase . To this end, cDNA coding for honeybee prepromelittin was ligated to the 3' end of genes coding for truncated forms of either Protein A or beta-galactosidase (beta-Gal) under the control of inducible promoters, with an oligonucleotide coding for the Factor Xa cleavage site at the junction between the two proteins . The Protein A fusion was expressed in good yield, and about 80% of it formed inclusion bodies . The prepromelittin section of the Protein A fusion caused some export of the intact fusion protein to the growth medium . The prepromelittin beta-Gal fusion was expressed in low yield and became associated with the E . coli cytoplasmic membrane . Its expression was toxic to E . coli . Thus, the synthesis of a full-length eukaryotic pre-protein in E . coli is best achieved when the fusion protein forms inclusion bodies. Br J Cancer, 1994 Aug, 70(2), 204 - 11 Effects of 4-hydroxytamoxifen and a novel pure antioestrogen (ICI 182780) on the clonogenic growth of human breast cancer cells in vitro; DeFriend DJ et al.; We have investigated the effects on breast cancer cell growth of 4-hydroxytamoxifen (4OHT), a conventional antioestrogen with agonist activity, and 7 alpha-{9-(4,4,5,5,5-pentafluoropentylsulphinyl)nonyl}oestra- 1,3,5,(10)- triene-3,17 beta-diol (ICI 182780), a novel, pure antioestrogen, using established human breast cancer cell lines and cancer cells obtained directly from breast cancer patients with malignant pleural effusions who had relapsed on tamoxifen . The effects of the two agents were assessed using the Courtenay-Mills clonogenic assay, which measures the growth of single cancer cells as colonies suspended in soft agar . The standard assay was modified by the use of defined serum- and phenol red-free growth medium . The growth of oestrogen receptor (ER)-positive MCF-7 cells in the assay was oestrogen responsive . Both antioestrogens inhibited the stimulatory effects of 1 nM oestradiol, but ICI 182780 caused significantly greater inhibition than 4OHT at 0.1-1.0 microM concentrations . In the absence of oestradiol, 4OHT but not ICI 182780 caused significant stimulation of colony formation at low (0.01-1.00 nM) concentrations . Neither antioestrogen had any effects on colony formation by the ER-negative Hs578T cell line . Successful colony formation was obtained in primary cultures from six out of eight malignant effusions . Colony formation was significantly stimulated by 0.1 nM oestradiol in four cases and by 10 nM 40HT in two cases . In contrast, ICI 182780 exhibited no intrinsic stimulatory activity and significantly inhibited both oestradiol- and 4OHT-stimulated cell growth . We conclude that the agonist activity of 4OHT and other conventional antioestrogens may cause treatment failure in some patients by stimulating breast cancer cell growth . The new, pure antioestrogen ICI 182780 is a more potent oestrogen antagonist than 4OHT and exhibits no growth-stimulatory activity . This agent may therefore offer therapeutic advantages over conventional antioestrogens in patients with advanced breast cancer and may be effective after conventional agents have failed. J Bacteriol, 1994 Aug, 176(16), 5181 - 3 Regulation of phosphatidylinositol:ceramide phosphoinositol transferase in Saccharomyces cerevisiae; Ko J et al.; Maximal phosphatidylinositol:ceramide phosphoinositol transferase activity was measured in yeast cells harvested during the exponential phase of growth . The addition of inositol to the growth medium resulted in a twofold increase in IPC synthase activity in cells grown in the presence or absence of exogenous choline . Enzyme activity was not regulated in yeast inositol biosynthesis regulatory mutants by the addition of inositol to the growth medium. J Cell Physiol, 1994 Aug, 160(2), 239 - 48 Characterization of rat aortic smooth muscle cells resistant to the antiproliferative activity of heparin following long-term heparin treatment; Barzu T et al.; Vascular smooth muscle cells (SMC) do not represent a homogeneous population (Schwartz et al., 1990, Am . J . Pathol . 136: 1417-1428) . Cellular clones resistant to the antiproliferative activity of heparin were isolated from rat aortic SMC cultures (Pukac et al., 1990, Cell Regul., 1:435-443; San Antonio et al., 1993, Arterioscler . Thromb., 13:748-757) and from explant of human arterial restenotic lesions (Chan et al., 1993, Lancet, 341:341-342) . We have shown in the present study that long-term treatment (growth medium supplemented with 200 micrograms/ml heparin, from the second to the tenth passage) of rat aortic SMC, without cell cloning, resulted in a significant loss of sensitivity to the growth inhibition by heparin and its derivatives . The heparin resistance was stable after growing cells for two passages in heparin-free medium, suggesting the selection of a particular phenotype . We tried to characterize these cells and to determine the causes of the resistance to the growth inhibition by heparin . Heparin-treated SMC (HT-SMC) were smaller than their control culture at the same passage, expressed less alpha-SM actin, and did not overgrow after reaching confluence . As in the heparin-resistant clones (San Antonio et al., 1993, Cell Regul., 1:435-443) expression of alpha-SM actin could be increased in HT-SMC by heparin addition before Western blotting . Heparin resistance was associated with a tenfold decrease in {3H}-heparin binding capacity (Bmax = 1.9 x 10(6) sites per cell) compared to control cultures (Bmax = 1.7 x 10(7) sites per cell), which was irreversible after growing the cells for two additional passages in heparin-free medium . We also investigated protein kinase C (PKC) in HT-SMC in terms of both enzymatic activity and protein expression (evaluated by {3H}-staurosporine and {3H}-phorbol-12,13-dibutyrate binding) . We found that HT-SMC had only half the PKC activity and expression as control SMC . Therefore, long-term treatment of rat aortic SMC with heparin allowed the selection of a less differentiated subpopulation of cells, exhibiting low sensitivity to the growth inhibition by heparin, which could be related to the low capacity of binding heparin and to a lower PKC activity and/or expression. Infect Immun, 1994 Aug, 62(8), 3262 - 9 Acquisition of iron from transferrin and lactoferrin by the protozoan Leishmania chagasi; Wilson ME et al.; Leishmania chagasi, the cause of South American visceral leishmaniasis, requires iron for its growth . However, the extent to which different iron sources can be utilized by the parasite is not known . To address this question, we studied acquisition of iron from lactoferrin and transferrin by the extracellular promastigote form of L . chagasi during growth in vitro . A promastigote growth medium based on minimal essential medium supplemented with iron-depleted serum supported promastigote growth only after the addition of exogenous iron . The addition of 8 microM iron chelated to lactoferrin or hemin resulted in normal promastigote growth . Ferritransferrin also supported promastigote growth, but only after a considerable lag . Promastigotes grown in all three iron sources generated similar amounts of hydroxyl radical upon exposure to hydrogen peroxide, indicating that none of these protected parasites against generation of this toxic radical . Promastigotes were able to take up 59Fe chelated to either transferrin or lactoferrin, although uptake from 59Fe-lactoferrin occurred more rapidly . 59Fe uptake from either 59Fe-transferrin or 59Fe-lactoferrin was inhibited by a 10-fold excess of unlabeled ferrilactoferrin, ferritransferrin, apolactoferrin, apotransferrin, or iron nitrilotriacetate but not ferritin or bovine serum albumin . There was no evidence for a role for parasite-derived siderophores or proteolytic cleavage of ferritransferrin or ferrilactoferrin in the acquisition of iron by promastigotes . Thus, L . chagasi promastigotes can acquire iron from hemin, ferrilactoferrin, or ferritransferrin . This capacity to utilize several iron sources may contribute to the organism's ability to survive in the diverse environments it encounters in the insect and mammalian hosts. Curr Genet, 1994 Aug, 26(2), 184 - 6 Reversion of a long-living, undifferentiated mutant of Podospora anserina by copper; Marbach K et al.; The Podospora anserina nuclear mutant grisea displays an undifferentiated growth phenotype (diminished production of aerial hyphae), is female sterile (lack of perithecia), has a prolonged life-span compared to the wild-type strain, and lacks detectable phenoloxidase (laccase and tyrosinase) activity . Reversion of all of these characteristics to those of the wild-type phenotype was accomplished by supplementing the growth medium with extra amounts of copper salts . These results indicate that the primary defect of the grisea strain is in its copper uptake and/or distribution in the cells. Antimicrob Agents Chemother, 1994 Aug, 38(8), 1838 - 43 Activities of the benzoxazinorifamycin KRM 1648 and ethambutol against Mycobacterium avium complex in vitro and in macrophages; Inderlied CB et al.; KRM 1648 is a 4-aminobenzoxazine derivative of rifamycin S with potent in vitro activity against the Mycobacterium avium complex (MAC); the MIC for 90% of 24 MAC isolates from AIDS patients was 0.25 microgram/ml as determined by a radiometric broth macrodilution assay . KRM 1648 was bactericidal for MAC isolates in Middlebrook 7H9 broth, with a reduction in viability of 1 to 4 orders of magnitude over 72 h . In human macrophages, KRM 1648 also was bactericidal, with a reduction of 3 to 4 orders of magnitude in CFU per ml of macrophage lysate at a concentration of 1 microgram/ml; however, the bactericidal activity varied approximately 10-fold among the three MAC serovars tested . In growth medium, ethambutol potentiated the effect of KRM 1648, but this potentiation was modest when tested against MAC in macrophages and also varied between MAC strains . KRM 1648 has potential as an antimycobacterial agent for MAC disease, perhaps in combination with other agents so that the use of lower dosages of KRM 1648 than are needed with other rifamycins may be possible. J Cell Sci, 1994 Aug, 107 ( Pt 8), 2219 - 28 Cell kinetic characterization of growth arrest in cultured human keratinocytes; van Ruissen F et al.; In this study we have performed a cell kinetic characterization of growth and growth arrest of keratinocytes derived from normal human skin . Proliferative activity of the cell cultures was analysed with a flow cytometric technique, measuring relative DNA content and iododeoxyuridine (IdUrd) incorporation simultaneously . Normal human keratinocytes were grown in keratinocyte growth medium (KGM) and growth arrest was induced by using either keratinocyte basal medium (KBM) or KGM supplemented with TGF-beta 1 . It was found that human keratinocytes grown in KGM plus TGF-beta 1 were growth-arrested within 52 hours . The rate of IdUrd incorporation into DNA decreased by more than 95% after 52 hours and paralleled the decrease of cells in S-phase . Within 52 hours after addition of TGF-beta 1, 79% of the growth-arrested cells were in the G0/G1-phase of the cell cycle, a situation that approaches that of the normal epidermis . Growth arrest of human keratinocytes in KBM showed a similar decrease in the rate of IdUrd incorporation . However, the decrease in IdUrd incorporation was not reflected in a decrease in cells in S-phase, suggesting that the cells were blocked in G0/G1, S or G2/M-phase rather than selectively in the physiological growth arrest state of G0/G1 . Secondly, we investigated the kinetics of the cells when they were restimulated after growth arrest . We found that after termination of the growth arrest in KGM supplemented with TGF-beta 1 the cells require 6 to 8 hours to initiate DNA synthesis, with a continued decrease in the G0/G1 population, suggesting that the cells are recruited as a cohort . After growth arrest induced by KBM, cells also require 6 to 8 hours in KGM to initiate DNA synthesis, but the cells are not recruited as a cohort . We conclude that growth arrest induced by TGF-beta 1 is the preferred system in which to study induction of keratinocyte proliferation, since it induces a state of quiescence that approaches that of normal human epidermis. J Microsc, 1994 Aug, 175 ( Pt 2), 143 - 53 Preparation of cultured airway smooth muscle for study of intracellular element concentrations by X-ray microanalysis: comparison of whole cells with cryosections; Warley A et al.; Methods for growing and preparing smooth muscle cells, isolated from rabbit trachealis, for X-ray microanalysis studies are presented . The cells are grown on Pioloform-covered gold grids supported on Thermanox coverslips . This provides a growth-compatible substrate which is easy to handle and is easily incorporated into routine cell culture studies . The cells are analysed as whole mounts after removal of growth medium by washing, followed by cryofixation and freeze drying . The effects of different washing media (0.3 M sucrose, 0.15 M ammonium acetate and distilled water) on cytoplasmic elemental content are discussed . A method for growing the cells as monolayers and mounting the cryofixed monolayers for cryosectioning is also given . Comparison of elemental concentrations in the cytoplasm of distilled-water washed cells with those of the cytoplasm of cryosectioned cells obtained from the same animal showed good agreement between values obtained from the two preparative procedures . These methods are therefore easily applied to the study of changes in intracellular element concentrations which may be important in understanding the mechanisms of proliferation which lead to increased airway smooth muscle mass in persistent severe asthma. Microbiology, 1994 Aug, 140 ( Pt 8), 2125 - 34 Mechanism of catabolite repression of tryptophanase synthesis in Escherichia coli; Isaacs H Jr et al.; Repression of tryptophanase (tryptophan indole-lyase) by glucose and its non-metabolizable analogue methyl alpha-glucoside has been studied employing a series of isogenic strains of Escherichia coli lacking cyclic AMP phosphodiesterase and altered for two of the proteins of the phosphoenolpyruvate:sugar phosphotransferase system (PTS), Enzyme I and Enzyme IIAGlc . Basal activity of tryptophanase was depressed mildly by inclusion of glucose in the growth medium, but inducible tryptophanase synthesis was subject to strong glucose repression in the parental strain, which exhibited normal PTS enzyme activities . Methyl alpha-glucoside was without effect in this strain . Loss of Enzyme I decreased sensitivity to repression by glucose but enhanced sensitivity to repression by methyl alpha-glucoside . Loss of Enzyme IIAGlc activity largely abolished repression by methyl alpha-glucoside but had a less severe effect on glucose repression . The repressive effects of both sugars were fully reversed by inclusion of cyclic AMP in the growth medium . Tryptophan uptake under the same conditions was inhibited weakly by glucose and more strongly by methyl alpha-glucoside in the parental strain . Inhibition by both sugars was alleviated by partial loss of Enzyme I . Inhibition by methyl alpha-glucoside appeared to be largely due to energy competition and was not responsible for repression of tryptophanase synthesis . Measurement of net production of cyclic AMP as well as intracellular concentrations of cyclic AMP revealed a good correlation with intensity of repression . The results suggest that while basal tryptophanase synthesis is relatively insensitive to catabolite repression, inducible synthesis is subject to strong repression by two distinct mechanisms, one dependent on enzyme IIAGlc of the PTS and the other independent of this protein . Both mechanisms are attributable to depressed rates of cyclic AMP synthesis . No evidence for a cyclic-AMP-independent mechanism of catabolite repression was obtained. Biol Bull, 1994 Aug, 187(1), 1 - 7 Physiological parameters affecting the chemosensory response of Tetrahymena; Koppelhus U et al.; We have investigated the significance of a number of physiological parameters in the preparation of cells for experiments on chemokinesis in Tetrahymena . The study comprises (1) growth state of the cells, (2) composition of the starvation medium, (3) concentration of cells during starvation, (4) oxygen saturation of the starvation medium, (5) temperature during starvation, and (6) starvation period . By controlling the physiological state of the cells, we significantly improved the reproducibility of the results obtained in assays for chemokinesis in Tetrahymena . In short, cells optimal for chemokinesis at an assay temperature of 28 degrees C should be starved from the exponential growth phase in a concentration below 2 x 10(5) cells ml-1 for 10-20 h . The surface-to-volume ratio of the starvation medium--water or Hepes buffer--should be about 5 cm-1 (or more) to ensure more than 95% oxygen saturation of the starvation medium . Maximal chemosensory responses were obtained if the cells were starved at 21 degrees C . The chemokinetic potential of the cells decreased significantly, as did the levels of the ratio of ATP to ADP, if cells were starved at higher temperatures . A tentative correlation between the ATP level in the cells and the chemosensory potential of the cells has been found . We suggest that chemokinesis is a constant quality of Tetrahymena, because we found no sign that prolonged starvation or other changes applied to the cells produced an up-regulation of the chemosensory response . Apparently, starvation is obligatory only to remove the growth medium (which is itself a very potent attractant), thereby making the cells sensitive to the chemoattractants. J Neurosci, 1994 Aug, 14(8), 4769 - 79 Mesencephalic type 1 astrocytes rescue dopaminergic neurons from death induced by serum deprivation; Takeshima T et al.; We have established a primary neuronal culture of the embryonic day 14 rat, ventral mesencephalic region, centered on the A8, A9, and A10 dopaminergic nuclei (approximately 1.0 mm3 of tissue) . At 16 hr after plating on a substrate of poly-D-lysine, in a serum-free or serum-supplemented growth medium, using a microisland culturing method, 95% of the cells stained positive for neuron-specific enolase, 20% for tyrosine hydroxylase, and < 5% for vimentin . When the growth medium was supplemented with 10% fetal calf serum, the percentage of tyrosine hydroxylase-positive neurons increased significantly (p < 0.05) at the 7th and 10th days in culture, compared with the percentage present at 16 hr after plating . When cultured in a serum-free growth medium, the percentage of tyrosine hydroxylase-positive neurons decreased to < 5% and to 0% by the 5th and 7th days, respectively, while the percentage of GABA-IR neurons increased . The addition of serum to the serum-free culture rescued dopaminergic neurons from death induced by serum deprivation . The effect of serum was dependent both on the time of addition after plating, and on the percentage added . When the cells were plated in a serum-free medium, on a confluent, type 1 astrocyte monolayer, prepared from the ventral mesencephalon of the embryonic day 16 rat, the survival of dopaminergic neurons increased significantly (p < 0.01) at DIV5, versus survival after plating on poly-D-lysine . Conditioned medium prepared from the same mesencephalic type 1 astrocyte monolayer also rescued dopaminergic neurons from death . The rescue mediated by the astrocyte monolayer or the conditioned medium was not inhibited by the mitotic inhibitor cytosine arabino furanoside (1.0 microM) . Type 1 astrocyte monolayers and conditioned media prepared from the striatum and cerebral cortex of the embryonic day 16 rat had weaker trophic effects than those mediated by mesencephalic glia . We conclude that serum deprivation causes the selective death of dopaminergic neurons in a primary culture of the rat E14 ventral mesencephalon . Type 1 astrocytes or the conditioned medium from type 1 astrocytes can rescue dopaminergic neurons from death induced by serum deprivation.(ABSTRACT TRUNCATED AT 400 WORDS) Bioorg Med Chem, 1994 Aug, 2(8), 837 - 43 Cloning, overexpression and isolation of the type II FDP aldolase from E . coli for specificity study and synthetic application; Henderson I et al.; A stable overexpression E . coli strain containing the plasmid pKEN 2 for the production of the Zn(2+)-dependent FDP aldolase from E . coli has been developed . Approximately 14,000 U of the enzyme (specific activity = 23.3 U/mg) can be obtained from 4-L of growth medium . The enzyme was isolated, purified to homogeneity and used for the studies of stability, substrate specificity and metal ion replacement and dissociation . Crystals of the enzyme have been obtained for structural analysis . This E . coli strain was deposited with the American Type Culture Collection (ATCC #77472). Cell Struct Funct, 1994 Aug, 19(4), 241 - 52 Selective inhibition of a step of myotube formation with wheat germ agglutinin in a murine myoblast cell line, C2C12; Muroya S et al.; Myoblast cells, C2C12, which is an established cell line from satellite cells of skeletal muscle of C3H mouse, start to fuse and form multinucleated cells (myotubes) and begin to express creatine phosphokinase and myosin, when culture medium is changed from the growth medium to the differentiation medium . Among the 12 lectins that we tested, wheat germ agglutinin apparently suppressed the myotube development judged by phase-contrast microscopy, but did not affect the induction of creatine phosphokinase activity . The addition of N-acetylglucosamine or N,N',N"-triacetylchitotriose, which is a specific ligand for wheat germ agglutinin, to the differentiation medium, recovered this apparently suppressive effect of wheat germ agglutinin on the myotube development . Tachypleus tridentatus (Japanese horseshoe crab) lectin that specifically recognizes N-acetylneuraminic acid, one of the sialic acids, showed no effect on the myotube development . It was suggested that wheat germ agglutinin suppressed the process through recognizing N-acetylglucosamine containing sugar . Surprisingly, even in the presence of wheat germ agglutinin, the ratio of mononucleated cell numbers to the genomic DNA content, which represents the fusion level, decreased after incubation in the differentiation medium, indicating that even when wheat germ agglutinin was present in the medium, cell fusion, which is the initial step of the myotube formation, occurred . Immunostaining with anti-skeletal muscle myosin antiserum confirmed that the myosin expressing cells actually fused and formed multinucleated cells . Their shape, however, was thin compared to that in the absence of wheat germ agglutinin . We propose that the membrane fusion step to form myotubes is composed of two distinct steps in C2C12; one fusion step is to form long and thin myotubes from mononucleated cells and the other one is to develop fat myotubes . Wheat germ agglutinin specifically inhibits the latter fusion step. Brain Res, 1994 Jul 18, 651(1-2), 1 - 6 Differential regulation of phenotypic expression in a pluripotential neuroblastoma cell line; Kentroti S et al.; Our laboratory has recently been involved in investigating factors which influence plasticity of neurotransmitter phenotypic expression both in vivo and in culture . Our previous studies have shown that precursor neuroblasts are pluripotential with respect to neurotransmitter phenotype and respond differentially to microenvironmental signals . In the present study, we examined phenotypic expression in neuroblastoma cells, P2 clone, using the activities of choline acetyltransferase (ChAT) and tyrosine hydroxylase (TH) as neuronal markers for the cholinergic and catecholaminergic phenotypes, respectively . Cells were plated and grown for 4 days in culture, harvested and frozen for assay of ChAT and TH . Basal activity of ChAT was 2.47 +/- 0.22 nmoles Ach formed /h/mg protein and that of TH was 5.23 +/- 0.41 pmoles CO2 formed /h/mg protein in control cultures . When intracellular cAMP levels were increased by addition to the growth medium of 10 micrograms/ml prostaglandin E1 (PGE1; a receptor-mediated enhancer of adenylate cyclase activity) or 200 micrograms/ml RO20-1724 (an inhibitor of cyclic nucleotide phosphodiesterase) the activity of TH was increased 340- and 423-fold, respectively . In marked contrast, the activity of ChAT was not affected by either agent . Double staining immunocytochemical examination demonstrated that both ChAT and TH were colocalized in the same cell . The molecular mechanism whereby catecholaminergic expression exclusively is affected in this cell model is currently under investigation . We conclude that (1) P2 neuroblastoma is a pluripotential cell line, (2) phenotypic expression in a homogenous cell population, such as P2 neuroblastoma, is differentially regulated . Moreover, this cell line is a unique model for studying the molecular mechanisms of phenotypic expression and neuronal plasticity. Proc Natl Acad Sci U S A, 1994 Jul 5, 91(14), 6559 - 63 Reversal of cell determination in yeast meiosis: postcommitment arrest allows return to mitotic growth; Honigberg SM et al.; When yeast from the early stages of meiosis are transferred from sporulation to growth medium, they can reenter the mitotic cell cycle directly . In contrast, cells from later stages of meiosis (after the initiation of the first nuclear division) will complete meiosis and sporulation despite the shift to growth medium, a phenomenon known as "commitment to meiosis." This study reports the surprising finding that when the normal progression of meiosis is arrested, cells from later stages of meiosis can return to growth . Cells were arrested after the first or second meiotic division by three independent means: the spo14 mutation, the spo3-1 mutation, and a high-temperature arrest of wild-type cells . In every case, the arrested cells were able to form buds after transfer to growth medium . These cells, however, experienced a delay upon return to growth relative to uncommitted cells . We propose that the commitment phenomenon results from a transient delay of mitotic growth, which occurs specifically during meiosis, and that commitment does not involve an irreversible inhibition of mitosis as previously thought. J Eukaryot Microbiol, 1994 Jul-Aug, 41(4), 337 - 43 Modulation of complement resistance and virulence of Naegleria fowleri amoebae by alterations in growth media; Toney DM et al.; Highly-pathogenic, mouse-passaged Naegleria fowleri amoebae are complement resistant . The present study evaluates the effect of complement on N . fowleri and the virulence of the amoebae after animal passage and growth in two different axenic media . Pathogenic N . fowleri maintained in "enriched" Cline medium are virulent for mice and resistant to complement lysis . A rapid decline in resistance to complement and virulence for mice is observed when highly-pathogenic N . fowleri are grown in Nelson medium lacking hemin . N . fowleri maintained in Nelson medium can be rendered complement-resistant by shifting the amoebae to growth in Cline medium for 2 h prior to the addition of complement . Cycloheximide treatment of N . fowleri maintained in Nelson medium blocks the transition to a complement-resistant phenotype following a shift in growth medium . Proteins were radiolabeled with {35S} during a shift from Nelson to Cline medium to identify specific polypeptides which may be associated with the functional activities related to virulence and resistance to complement. Appl Environ Microbiol, 1994 Jul, 60(7), 2431 - 7 Effect of biosynthetic manipulation of heme on insolubility of Vitreoscilla hemoglobin in Escherichia coli; Hart RA et al.; Vitreoscilla hemoglobin (VHb) is accumulated at high levels in both soluble and insoluble forms when expressed from its native promoter on a pUC19-derived plasmid in Escherichia coli . Examination by atomic absorption spectroscopy and electron paramagnetic resonance spectroscopy revealed that the insoluble form uniformly lacks the heme prosthetic group (apoVHb) . The purified soluble form contains heme (holoVHb) and is spectroscopically indistinguishable from holoVHb produced by Vitreoscilla cells . This observation suggested that a relationship may exist between the insolubility of apoVHb and biosynthesis of heme . To examine this possibility, a series of experiments were conducted to chemically and genetically manipulate the formation and conversion of 5-aminolevulinic acid (ALA), a key intermediate in heme biosynthesis . Chemical perturbations involved supplementing the growth medium with the intermediate ALA and the competitive inhibitor levulinic acid which freely cross the cell barrier . Genetic manipulations involved amplifying the gene dosage for the enzymes ALA synthase and ALA dehydratase . Results from both levulinic acid and ALA supplementations indicate that the level of soluble holoVHb correlates with the heme level but that the level of insoluble apoVHb does not . The ratio of soluble to insoluble VHb also does not correlate with the level of total VHb accumulated . The effect of amplifying ALA synthase and ALA dehydratase gene dosage is complex and may involve secondary factors . Results indicate that the rate-limiting step of heme biosynthesis in cells overproducing VHb does not lie at ALA synthesis, as it reportedly does in wild-type E . coli (S . Hino and A . Ishida, Enzyme 16:42-49, 1973). Gut, 1994 Jul, 35(7), 879 - 83 Tissue culture of epithelium derived from Barrett's oesophagus; Washington K et al.; Barrett's oesophagus is a preneoplastic condition in which the squamous mucosa of the oesophagus is replaced by columnar epithelium . Epithelial cells of Barrett's oesophagus were isolated from resected oesophagus specimens by two methods not previously applied to the culture of Barrett's oesophagus cells . These techniques included trypsinisation of small fragments of mucosa, followed by plating in tissue culture dishes, and a direct tissue explant technique . A modified MCDB-153 growth medium was used . Primary trypsin technique cultures were plated on uncoated plastic, or plastic coated with type I collagen, type IV collagen, or fibronectin . Growth on type IV collagen and fibronectin plates was slower but produced less contamination from fibroblasts . By 20-40 days most cultures formed confluent monolayers made up of cells with epithelial morphology . The cells were cytokeratin positive, vimentin negative, and contained alcian blue positive vacuoles, confirming their epithelial origin and suggesting their derivation from Barrett's oesophagus . Electron microscopy showed tonofilaments, microvilli, and desmosomes . Cells proliferated through up to eight subcultures before growth slowed and cells showed senescent changes . This study shows that epithelial cells from Barrett's oesophagus can be grown by comparatively simple tissue culture techniques . These methods can provide sufficient material for a variety of molecular biology and biochemical studies of epithelial cells from Barrett's oesophagus. Parasitology, 1994 Jul, 109 ( Pt 1), 23 - 8 Haemoglobin inhibits the development of infective promastigotes and chitinase secretion in Leishmania major cultures; Schlein Y et al.; Haemoglobin or blood in the growth medium of Leishmania major inhibited the formation of infective promastigotes and the secretion of chitinases . Inoculation of mice with stationary-phase parasites from control medium caused infections in 20/29 mice, compared to 3/20 mice injected with parasites grown with 10% rabbit blood, or 1/30 mice that received parasites grown with rabbit haemoglobin . The concentration of peanut lectin (PNA) required to agglutinate promastigotes was used as an index of their infectivity, ranging from a high concentration for infective populations to a low concentration for relatively non-infective populations . Agglutination of 50% of the parasites from control medium or from medium containing rabbit haemoglobin required 4.1 micrograms PNA/ml and 0.1 microgram PNA/ml, respectively . Chitinase activities/10(7) parasites decreased from 4.8 units chitinase and 12.5 units N-acetylglucosaminidase (NAGase) in the control to 2.0 units chitinase and 8.5 units NAGase in cultures containing rabbit haemoglobin . Rabbit, human, bovine and pigeon haemoglobins had various inhibitory effects on the activity of chitinases and not on the virulence, as expressed by PNA agglutination . The relevance of the results to the cycle of Leishmania is discussed. Lab Invest, 1994 Jul, 71(1), 25 - 34 On the analysis of the pathophysiology of Chediak-Higashi syndrome . Defects expressed by cultured melanocytes; Zhao H et al.; BACKGROUND: The Chediak-Higashi syndrome (CHS) is a disorder that affects the synthesis and/or maintenance of storage/secretory granules in various types of cells . Lysosomes of leukocytes and fibroblasts, dense bodies of platelets, azurophilic granules of neutrophils and melanosomes of melanocytes are generally larger in size and irregular in morphology, indicating that a common pathway in storage organellogenesis is affected in patients with CHS . EXPERIMENTAL DESIGN: A pure line of melanocytes has been established using a 2 cm2 shave biopsy from a child with CHS . This 4-week-old male patient had oculocutaneous albinism and expressed neutropenia, impaired platelet function, and no natural killer cell activity . The cultured CHS melanocytes were analyzed for cell biological and biochemical aberrancies . RESULTS: Cultured melanocytes demonstrated some large and/or complexed melanosomes that resembled those observed in melanocytes from ultrastructural sections of the biopsy . Cytoplasmic localization of tyrosinase, tyrosinase-related protein-1 and granulophysin (a 40 kilodalton membrane protein originally identified as a component in dense bodies of platelets) demonstrated a prominent perinuclear accumulation . The basal synthesis of melanin and the activity levels of tyrosine hydroxylase, dihydroxyphenylalanine (DOPA) oxidase, or DOPAchrome tautomerase were comparable to control Caucasian melanocytes in culture . However, melanin synthesis as well as the catalytic activities of tyrosinase were not dramatically upregulated in CHS melanocytes by the addition of isobutyl methylxanthine and cholera toxin in the growth medium when parameters were assayed in cell lysates . In contrast, when assays were performed using live cells, tyrosine hydroxylase demonstrated dramatic upregulation . Medium conditioned by CHS melanocytes demonstrated phenylthiourea-inhibitable tyrosinase activity . Melanocyte lysates and conditioned medium analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and DOPA staining showed an extra, approximately 100 kilodalton soluble protein band with DOPA positivity and tyrosinase immunoreactivity . In addition to tyrosinase, one of three lysosomal enzymes assayed (beta-glucuronidase) was aberrantly secreted into the medium . CONCLUSIONS: These results demonstrate that melanocytes cultured from CHS express a defect in the structure and/or function of the melanosome and abnormal trafficking of some cellular proteins. J Cell Biol, 1994 Jul, 126(2), 343 - 52 Clathrin heavy chain functions in sorting and secretion of lysosomal enzymes in Dictyostelium discoideum; Ruscetti T et al.; The clathrin heavy chain is a major component of clathrin-coated vesicles that function in selective membrane traffic in eukaryotic cells . We disrupted the clathrin heavy chain gene (chcA) in Dictyostelium discoideum to generate a stable clathrin heavy chain-deficient cell line . Measurement of pinocytosis in the clathrin-minus mutant revealed a four-to five-fold deficiency in the internalization of fluid-phase markers . Once internalized, these markers recycled to the cell surface of mutant cells at wild-type rates . We also explored the involvement of clathrin heavy chain in the trafficking of lysosomal enzymes . Pulse chase analysis revealed that clathrin-minus cells processed most alpha-mannosidase to mature forms, however, approximately 20-25% of the precursor molecules remained uncleaved, were missorted, and were rapidly secreted by the constitutive secretory pathway . The remaining intracellular alpha-mannosidase was successfully targeted to mature lysosomes . Standard secretion assays showed that the rate of secretion of alpha-mannosidase was significantly less in clathrin-minus cells compared to control cells in growth medium . Interestingly, the secretion rates of another lysosomal enzyme, acid phosphatase, were similar in clathrin-minus and wild-type cells . Like wild-type cells, clathrin-minus mutants responded to starvation conditions with increased lysosomal enzyme secretion . Our study of the mutant cells provide in vivo evidence for roles for the clathrin heavy chain in (a) the internalization of fluid from the plasma membrane; (b) sorting of hydrolase precursors from the constitutive secretory pathway to the lysosomal pathway; and (c) secretion of mature hydrolases from lysosomes to the extracellular space. J Cell Physiol, 1994 Jul, 160(1), 107 - 12 Chronic hypoxia impairs the differentiation of 3T3-L1 fibroblast in culture: role of sustained protein kinase C activation; Sahai A et al.; The effect of hypoxia on 3T3-L1 cell differentiation was examined in confluent cultures incubated with differentiation medium (DM) followed by incubation in growth medium (GM) . Control cultures remained in GM throughout the incubation period . Eight days after the incubation, cells were assessed either for changes in morphology by staining with Oil Red O/hematoxylin or harvested to measure protein kinase C activity . Morphological examination of stained cells showed almost complete differentiation of normoxic cells to adipocytes when exposed to DM . By contrast hypoxia caused a dramatic inhibition of differentiation under similar media conditions with only 34 +/- 4% of cells accumulating fat deposits . Cultures sustained in GM under normoxic or hypoxic conditions were devoid of any fat deposits, reflecting an undifferentiated phenotype . Normoxic cells exposed to DM exhibited a significantly lower membrane to cytosolic ratio of protein kinase C in comparison with cells maintained in GM, which is consistent with differentiated and undifferentiated phenotypes, respectively . In comparison with normoxic cells incubated in DM, cells exposed to hypoxia under similar media conditions exhibited a significantly higher membrane to cytosolic ratio of protein kinase C, indicating sustained activation of the enzyme . In addition, cells in differentiation medium exposed to hypoxia in the presence of the protein kinase C inhibitors staurosporine or H7 exhibited a significant increase in the number of fat accumulating cells when compared with hypoxic controls . These studies indicate that chronic hypoxia impairs the differentiation of 3T3-L1 cells to adipocytes in association with the sustained activation of protein kinase C, which appears to play a role in mediating this process. Exp Cell Res, 1994 Jul, 213(1), 121 - 7 Altered regulation of fibronectin gene expression in Werner syndrome fibroblasts; Rasoamanantena P et al.; Fibronectin (FN) production was quantified in the extracellular medium and in extracts of human diploid fibroblasts (HDF) by enzyme-linked immunosorbent assay and by immunoprecipitation of {35S}methionine biosynthetically labeled proteins . FN output was increased in normal late passage (old) HDF and in prematurely senescent Werner syndrome (WS) fibroblasts, compared to normal early passage (young) HDF . Output was maximal when cells were preconfluent, exceeding the level found at confluence and postconfluence . For all cell types the highest proportion of newly synthesized FN was found in the extracellular compartment . Both young and old HDF exposed to regular growth medium containing 15% fetal bovine serum (FBS) secreted twice as much FN as when exposed to serum-free medium, and cognate mRNA levels were commensurate with protein output . WS fibroblasts secreted 1.6-fold more FN than old HDF when both cell types were exposed to medium containing 15% FBS . Immunofluorescent analysis revealed greater association of FN with WS and old HDF than with young HDF . Furthermore, WS cells exposed to 15% FBS contained 3.4-fold more FN mRNA and produced seven times more FN protein compared to WS cells exposed to serum-free medium . Thus, WS fibroblasts secrete more FN than old normal and young normal HDF in the presence of 15% FBS due to augmentation of FN mRNA levels and enhanced efficiency of FN mRNA translation and/or post-translational processing. Radiat Res, 1994 Jul, 139(1), 109 - 14 Misoprostol-induced radioprotection of Syrian hamster embryo cells in utero from cell death and oncogenic transformation; Miller RC et al.; Misoprostol, a PGE1 analog, is an effective radioprotector of murine intestine and hematopoietic and hair cell renewal systems . The radioprotective nature of misoprostol was extended to examine its ability to influence clonogenic cell survival and induction of oncogenic transformation in Syrian hamster embryo cells exposed to X rays in utero and assayed in vitro . Hamsters in their 12th day of pregnancy were injected subcutaneously with misoprostol, and 2 h later the pregnant hamsters were exposed to graded doses of X rays . Immediately after irradiation, hamsters were euthanized and embryonic tissue was explanted into culture dishes containing complete growth medium . After a 2-week incubation period, clonogenic cell survival and morphologically transformed foci were determined . Survival of misoprostol-treated SHE cells was increased and yielded a dose reduction factor of 1.5 compared to SHE cells treated with X rays alone . In contrast, radiation-induced oncogenic transformation of misoprostol-treated cells was reduced by a factor of 20 compared to cells treated with X rays alone . These studies suggest that misoprostol not only protects normal tissues in vivo from acute radiation injury, but also protects cells, to a large extent, from injury leading to transforming events. Proc Soc Exp Biol Med, 1994 Jul, 206(3), 185 - 9 Overexpression of the short form of the growth hormone receptor in 3T3-L1 mouse preadipocytes; Bick T et al.; In rodents, the gene for the growth hormone receptor (GHR) gives rise to two mRNA transcripts encoding two proteins: a larger membrane spanning receptor (GHRL) and a smaller isoform, GHRs that consists of the extracellular domain and a unique hydrophilic carboxyl terminus . We examined the hypothesis that GHRs may contribute to cellular binding of GH and play a role in growth hormone (GH) signaling . Rat cDNA encoding GHRs was ligated into the mammalian expression vector pcDNA-I/neo and stably transfected into mouse 3T3-L1 preadipocytes which have endogenous GH receptors and, when differentiated into adipocytes, have the biochemical machinery to express the various GH effects . Sixteen of 24 neomycin resistant clones secreted at least twice as much GHRs in the growth medium as cells transfected with the vector alone, and in nine of these, GH binding was increased 2- to 4-fold . The amount of GHRL in extracts of these cells was unchanged, indicating that increased binding could not be accounted for by effects on formation or degradation of GHRL . The transfected cDNA for GHRs directs the synthesis of a 50 kDa protein . Cross-linking of {125I}hGH to transfected 3T3-L1 cells indicated a 3.5-fold increase in a 72 kDa GHRs{125I}hGH complex . In the presence of 10% newborn calf serum, incorporation of {35S}methionine into cellular proteins was similar in transfected clones and control cells . Deprivation of serum for 2 hr decreased protein synthesis by approximately 70% in control cells, but in the transfected cells, protein synthesis was reduced only by approximately 50% or 30% in cells exhibiting 2x or 3x increases in GH binding . In all cells, 1 nM IGF-1 restored protein synthesis to the serum replete level . Similarly, although 3H-2-deoxy-D-glucose (2DG) uptake was comparable in all cells after 2 hr of serum deprivation, 18 hr of serum deprivation decreased uptake by approximately 70% in control cells, but only by approximately 30% in cells with increased GH binding . One nanomolar IGF-1 restored 2DG uptake to levels seen after 2 hr or serum deprivation . IGF-1 had no effect after only 2 hr of serum deprivation . Measurement of IGF-1 secreted into the medium, revealed that clones which overexpress GHRs produce greater amounts of IGF-1 than control cells or transfected clones that failed to overexpress GHRs . We conclude that GHRs contributes to GH binding and may therefore be a functional receptor . In addition, overexpression of GHRs in 3T3-L1 cells altered cell function in the absence of GH. Endocrinology, 1994 Jul, 135(1), 53 - 62 Negative feedback regulation of insulin-like growth factor-II gene expression in differentiating myoblasts in vitro; Magri KA et al.; We have previously reported that autocrine secretion of insulin-like growth factor-II (IGF-II) plays a critical role in stimulating spontaneous myogenic differentiation in vitro . Myogenesis and IGF-II gene expression are both negatively controlled by high serum growth medium, and it is likely that serum inhibits terminal differentiation at least in part by blocking autocrine secretion of IGF-II . To investigate this possibility, we assessed the effects of various serum fractions and growth factors on endogenous IGF-II gene expression in rat L6A1 myoblasts . Unexpectedly, we found that IGF-I, IGF-II, and high concentrations of insulin were potent inhibitors of IGF-II gene expression . This is the first example we have seen in which IGFs regulate their own expression by a negative feedback mechanism . Feedback inhibition was not dependent on the stimulation of cell proliferation by IGFs, and differentiated L6A1 myotubes remained sensitive to this action of the IGFs . Results with IGF analogs suggested that the inhibition of IGF-II gene expression by IGFs was mediated by the type I IGF receptor and was strongly suppressed by L6A1-secreted IGF-binding proteins . Human primary myoblasts also exhibited feedback inhibition by the IGFs, whereas the rapidly fusing mouse Sol 8 cell line did not . We conclude that IGF-II gene expression in differentiating L6A1 myoblasts is regulated by a negative feedback mechanism (unusual for the IGFs) that acts primarily through the type I IGF receptor and appears to be inhibited by IGF-binding proteins secreted by L6A1 myoblasts in low serum differentiation medium. Endocrinology, 1994 Jul, 135(1), 321 - 9 Human cytotrophoblast cells cultured in maternal serum progress to a differentiated syncytial phenotype expressing both human chorionic gonadotropin and human placental lactogen; Richards RG et al.; Under appropriate conditions, protease-dispersed cytotrophoblast cells from human term placenta fuse and differentiate into syncytiotrophoblast cells that express hCG . Although human placental lactogen (hPL) is also expressed by the syncytiotrophoblast in vivo, little hPL is expressed by trophoblast cells differentiated in vitro using standard tissue culture medium or keratinocyte growth medium supplemented with fetal bovine serum . In this report, we demonstrate that cytotrophoblast cells fuse and differentiate to a phenotype that expresses large amounts of both hCG and hPL when cultured in medium containing maternal serum . When protease-dispersed cytotrophoblast cells were plated in RPMI-1640 supplemented with 10% second trimester (16-22 week) maternal serum (STMS medium), extensive cell fusion was observed by day 3 in culture . Cell fusion appeared to be essentially complete by day 6, coinciding with the peak of hCG expression and the initiation of hPL expression . The peak of hCG expression occurred between days 4-7 of an experiment; the range for maximal release of hCG was 0.1-3.7 micrograms/24 h.well when cells were plated at a density of 10(6)/well . In contrast, the peak of hPL expression occurred between days 7-11 of an experiment, and the range of maximal hPL release was 0.9-3.5 micrograms/24 h.well . Northern blot analysis indicated that the level of hPL messenger RNA (mRNA) paralleled the release of hormone . However, whereas the hCG beta-subunit mRNA paralleled the release of hCG, the hCG alpha-subunit mRNA did not, suggesting that the two genes are independently regulated . Cells cultured in nonpregnant (male or female) serum-supplemented medium also differentiated to the syncytiotrophoblast phenotype following a temporal pattern almost identical to that observed for cells cultured in STMS . However, the quantity of hormone released was at least 2-fold greater with STMS . Compared to RPMI-1640 or keratinocyte growth medium supplemented with 10% fetal bovine serum, STMS induced equal or greater expression of hCG and substantially greater expression of hPL at the level of both mRNA and protein . The extent of cell fusion, the level of hormone expression, and the regulated patterns of hormone expression suggest that cytotrophoblast cells cultured in maternal serum progress to an advanced stage of trophoblast differentiation. Infect Immun, 1994 Jul, 62(7), 3033 - 7 Degradation of endogenous plasma membrane fibronectin concomitant with Treponema denticola 35405 adhesion to gingival fibroblasts; Ellen RP et al.; Treponema denticola adhesion and degradation of fibronectin (Fn) on human gingival fibroblasts (HGF) were studied by immunofluorescence and enzyme-linked immunosorbent assays . The number of adherent bacteria increased and the amount of immunoreactive Fn decreased as a function of increasing T . denticola concentration . The distribution of cell-bound Fn was punctate in micrographs . Anti-human Fn impaired bacterial adhesion to HGF . Phenylmethylsulfonyl fluoride inhibited Fn degradation but not adhesion . Sonicated extracts and diluted spent growth medium degraded HGF Fn but, unlike intact T . denticola cells, they hardly stimulated F-actin rearrangements. Mol Microbiol, 1994 Jul, 13(2), 369 - 77 A copper-transporting P-type ATPase found in the thylakoid membrane of the cyanobacterium Synechococcus species PCC7942; Kanamaru K et al.; P-type ATPases constitute a large family of cation pumps that play crucial physiological roles in many organisms, including bacteria, plants and mammals . They are postulated to play important roles in a variety of environmental adaptation systems . Recently, we cloned two distinct putative P-type ATPase genes (pacS and pacL) from a photosynthetic cyanobacterium, Synechococcus species PCC7942 . In this study, one of the gene products (named PacS) was found to possess a putative metal-binding motif (Gly-Met-X-Cys-X-X-Cys) in its N-terminal portion . Thus we supposed that this ATPase may function as a metal pump . Indeed, the results of Northern blotting analysis showed that pacS-mRNA specifically increases upon addition of copper or silver to the growth medium . The results of Western blotting analysis confirmed the view that PacS accumulates in copper-treated Synechococcus cells . Thus we concluded that the expression of PacS ATPase is regulated in response to the change in concentration of external metals, namely copper and silver . Consistent with this, an insertional inactivation mutant of pacS exhibited hypersensitivity in terms of growth to these potentially toxic metals . It was also revealed that PacS was mainly located in the thylakoid membrane, in which the photosynthetic reactions take place . This P-type ATPase in the thylakoid membrane is implicated as a copper-transporting system that may be involved in copper-homeostasis crucial to the photosynthetic thylakoid function. Lipids, 1994 Jul, 29(7), 449 - 59 Characterization of lipid hydroperoxides generated by photodynamic treatment of leukemia cells; Bachowski GJ et al.; A new technique, high-performance liquid chromatography with reductive mode electrochemical detection on a mercury drop (HPLC-EC), has been used for analyzing lipid hydroperoxide (LOOH) formation in photooxidatively stressed L1210 leukemia cells . Highly specific and sensitive for peroxides (detection limits < 0.5 pmol for cholesterol hydroperoxides and < 50 pmol for phospholipid hydroperoxides), this approach allows different classes of LOOH to be separated and determined in minimally damaged cells . L1210 cells in serum-containing growth medium were irradiated in the presence of merocyanine 540 (MC540), a lipophilic photosensitizing dye . Lipid extracts from cells exposed to a light fluence of 0.11 J/cm2 (which reduced clonally assessed survival by 30%) showed 12-15 well-defined peaks in HPLC-EC . None of these peaks was observed when cells were irradiated without MC540 or when dye/light-treated samples were reduced with triphenylphosphine prior to analysis . Three peaks of relatively low retention time (< 12 min) were assigned to the following species by virtue of comigration with authentic standards: 3 beta-hydroxy-5 alpha-cholest-6-ene-5-hydroperoxide (5 alpha-OOH), 3 beta-hydroxycholest-4-ene-6 beta-hydroperoxide (6 beta-OOH), and 3 beta-hydroxycholest-5-ene-7 alpha/7 beta-hydroperoxide (7 alpha/7 beta-OOH) . Formation of 5 alpha-OOH and 6 beta-OOH (single oxygen adducts) was confirmed by subjecting {14C}cholesterol-labeled cells to relatively high levels of photooxidation and analyzing extracted lipids by HPLC with radiochemical detection . Material represented in a major peak at 18-22 min on HPLC-EC was isolated in relatively large amounts by semipreparative HPLC and shown to contain phospholipid hydroperoxides (predominantly phosphatidylcholine species, PCOOH) according to the following criteria: (i) decay of 18-22 min peak during Ca2+/phospholipase A2 treatment, with reciprocal appearance of fatty acid hydroperoxides; (ii) reduction of peroxide during treatment with reduced glutathione and phospholipid hydroperoxide glutathione peroxidase, but not glutathione peroxidase; and (iii) comigration with PCOOH standards in thin-layer chromatography . HPLC-EC analysis revealed quantifiable amounts of PCOOH and ChOOH at a light fluence that clonally inactivated < 10% of the cells, which allows for the possibility that photoperoxidative damage plays a causal role in cell killing. J Wildl Dis, 1994 Jul, 30(3), 457 - 9 A new method for the diagnosis of Trichomonas gallinae infection by culture; Cover AJ et al.; Diagnosis of Trichomonas gallinae infection can reliably be made by the demonstration of organisms in material taken from a bird's mouth and crop . This can be examined directly or incubated in a growth medium until organisms are numerous enough to be easily found in aliquots examined with the microscope . We compared two methods of culture diagnosis of Trichomonas gallinae infection in pigeons . A commercially available kit, In-Pouch TF, proved as sensitive for this purpose as in vitro Diamond's medium, but had practical advantages over the usual in vitro system. Neurol Med Chir (Tokyo), 1994 Jul, 34(7), 407 - 11 Pretreatment with interleukin-1 enhances survival of sympathetic ganglionic neuron grafts; Nakao N et al.; The effects of pretreatment with interleukin-1 (IL-1) on neurite growth and cell survival of rat superior cervical ganglion (SCG) explants were examined . Neonate rat SCG explants were cultured with serum-containing growth media . Pretreatment with IL-1 beta (30 U/ml) for 3 hours stimulated neurite outgrowth of the SCG explant, which was inhibited by the addition of anti-nerve growth factor (NGF) antibody to the growth medium . Adult rat autologous SCG with the same pretreatment and non-treated ganglia were transplanted into the lateral ventricle . Histological evaluation of the grafts 2 weeks after transplantation revealed that the pretreated SCG cells survived better . Pretreatment with IL-1 may achieve the NGF-mediated neurotrophic effect in sympathetic ganglionic neurons resulting in enhanced graft survival. FEBS Lett, 1994 Jun 27, 347(2-3), 190 - 4 Na(+)-driven ATP synthesis in Methanobacterium thermoautotrophicum and its differentiation from H(+)-driven ATP synthesis by rhodamine 6G; Smigan P et al.; Rhodamine 6G (3 microM) effectively inhibited delta pH-driven ATP synthesis in Methanobacterium thermoautotrophicum while delta pNa-driven ATP synthesis was not affected by it . Rhodamine 6G inhibited Mg(2+)-stimulated ATPase activity of membrane vesicles prepared from these cells but the ATPase catalytic sector detached from the membrane was insensitive to this inhibitor . Methanogenesis-driven ATP synthesis at pH 6.8 of cells grown in the presence of 50 mM NaCl was inhibited by rhodamine 6G both in the presence of 5 mM and 50 mM NaCl . On the other hand, the methanogenesis-driven ATP synthesis at pH 8.0 of cells grown in the presence of 50 mM NaCl was slightly inhibited by rhodamine 6G in the presence of 5 mM NaCl and was not inhibited at all in the presence of 50 mM NaCl . The growth experiments have shown that cells of Methanobacterium thermoautotrophicum can grow under alkaline conditions even in the presence of rhodamine 6G and of high NaCl concentration when the growth media were inoculated with the cells which had been grown in the presence of 50 mM NaCl . These results indicate that sodium-motive force-driven ATP synthase in Methanobacterium thermoautotrophicum operates effectively in alkaline conditions and it might be the sole ATP synthesizing system when the proton-motive force-supported ATP synthesis is inhibited by rhodamine 6G. Eur J Biochem, 1994 Jun 15, 222(3), 769 - 74 Induction, localization and metal content of hydrogenase in the green alga Chlamydomonas reinhardtii; Happe T et al.; The hydrogenase enzyme occurring in Chlamydomonas reinhardtii is induced by anaerobic adaptation of the cells . In aerobically growing cells, antibodies against the hydrogenase failed to detect either active or inactive enzyme . However, already 10 min after the onset of anaerobic adaptation, the protein could be detected . The maximal amount of enzyme was reached after 2-3 hours anaerobiosis . Addition of nickel or iron to the growth medium did not influence activity . In atomic absorption experiments, a Ni/Fe ratio of about 1:250 was measured . We, therefore, propose the hydrogenase from C . reinhardtii to be of the Fe-only type . Adaptation in the presence of uncouplers of phosphorylation showed this process to be energy-dependent . From protein synthesis inhibition experiments, it is concluded that the protein is synthesized on cytoplasmic ribosomes and, therefore, must be nuclear encoded . After isolation of intact chloroplasts from adapted cells, the active enzyme was shown, by Western-blotting analysis, to be located in the chloroplasts. Arch Biochem Biophys, 1994 Jun, 311(2), 402 - 17 Purification and enzymatic properties of a recombinant fusion protein expressed in Escherichia coli containing the domains of bovine P450 17A and rat NADPH-P450 reductase; Shet MS et al.; A fusion protein containing the heme domain of bovine cytochrome P450 17A and the flavin domains of rat NADPH-cytochrome P450 reductase has been genetically engineered by linking the modified cDNAs for each gene with the codons for serine and threonine . Transformation of Escherichia coli (DH5 alpha) and growth under defined conditions permits expression of 600-700 nmol of membrane-bound fusion protein per liter of growth medium (approximately 4% of cellular protein) . A method has been developed for the solubilization, isolation, and purification to homogeneity of this protein . In the presence of NADPH the purified fusion protein catalyzes the 17 alpha-hydroxylation of progesterone and pregnenolone as well as the conversion of 17 alpha-hydroxypregnenolone to dehydroepiandrosterone . The 17,20-lyase activity is enhanced sixfold by the addition of purified rat liver cytochrome b5 . Further, dehydroepiandrosterone is slowly metabolized to a number of additional more polar metabolites while 17 alpha-hydroxy-progesterone is slowly converted to dihydroxy-progesterone metabolites as well as a small amount of androstenedione in a reaction not influenced by cytochrome b5 . Use of 5 alpha-pregnan steroids as substrates show the importance of the 3 beta-hydroxyl group for cytochrome b5 stimulated 17,20-lyase activity . Studies investigating the factors affecting electron transport between the flavin and heme domains suggest that the protein exists as a tight complex functioning as a self-contained biocatalytic unit. Mol Cell Biol, 1994 Jun, 14(6), 4135 - 44 GPD1, which encodes glycerol-3-phosphate dehydrogenase, is essential for growth under osmotic stress in Saccharomyces cerevisiae, and its expression is regulated by the high-osmolarity glycerol response pathway; Albertyn J et al.; The yeast Saccharomyces cerevisiae responds to osmotic stress, i.e., an increase in osmolarity of the growth medium, by enhanced production and intracellular accumulation of glycerol as a compatible solute . We have cloned a gene encoding the key enzyme of glycerol synthesis, the NADH-dependent cytosolic glycerol-3-phosphate dehydrogenase, and we named it GPD1 . gpd1 delta mutants produced very little glycerol, and they were sensitive to osmotic stress . Thus, glycerol production is indeed essential for the growth of yeast cells during reduced water availability . hog1 delta mutants lacking a protein kinase involved in osmostress-induced signal transduction (the high-osmolarity glycerol response {HOG} pathway) failed to increase glycerol-3-phosphate dehydrogenase activity and mRNA levels when osmotic stress was imposed . Thus, expression of GPD1 is regulated through the HOG pathway . However, there may be Hog1-independent mechanisms mediating osmostress-induced glycerol accumulation, since a hog1 delta strain could still enhance its glycerol content, although less than the wild type . hog1 delta mutants are more sensitive to osmotic stress than isogenic gpd1 delta strains, and gpd1 delta hog1 delta double mutants are even more sensitive than either single mutant . Thus, the HOG pathway most probably has additional targets in the mechanism of adaptation to hypertonic medium. Mol Cell Biol, 1994 Jun, 14(6), 3613 - 22 A carbon source-responsive promoter element necessary for activation of the isocitrate lyase gene ICL1 is common to genes of the gluconeogenic pathway in the yeast Saccharomyces cerevisiae; Scholer A et al.; The expression of yeast genes encoding gluconeogenic enzymes depends strictly on the carbon source available in the growth medium . We have characterized the control region of the isocitrate lyase gene ICL1, which is derepressed more than 200-fold after transfer of cells from fermentative to nonfermentative growth conditions . Deletion analysis of the ICL1 promoter led to the identification of an upstream activating seque