Growth media

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 sequence element, UASICL1 (5' CATTCATCCG 3'), necessary and sufficient for conferring carbon source-dependent regulation on a heterologous reporter gene . Similar sequence motifs were also found in the upstream regions of coregulated genes involved in gluconeogenesis . This carbon source-responsive element (CSRE) interacts with a protein factor, designated Ang1 (activator of nonfermentative growth), detectable only in extracts derived from derepressed cells . Gene activation mediated by the CSRE requires the positively acting derepression genes CAT1 (= SNF1 and CCR1) and CAT3 (= SNF4) . In the respective mutants, Ang1-CSRE interaction was no longer observed under repressing or derepressing conditions . Since binding of Ang1 factor to the CSRE could be competed for by an upstream sequence derived from the fructose-1,6-bisphosphatase gene FBP1, we propose that the CSRE functions as a UAS element common to genes of the gluconeogenic pathway.

J Virol, 1994 Jun, 68(6), 3491 - 8
Epstein-Barr virus nuclear antigen EBNA3C/6 expression maintains the level of latent membrane protein 1 in G1-arrested cells; Allday MJ et al.; The Epstein-Barr virus in the Burkitt lymphoma-derived cell line Raji has a deletion in the EBNA3C gene . When Raji cells are allowed to grow to high density and most of the cells become growth arrested in the G1 phase of the cell cycle, the level of detectable latent membrane protein 1 (LMP1) is substantially reduced . After dilution of the cells with fresh growth medium, within 8 h, there is a large increase in LMP1 mRNA, and by 12 h, LMP1 is expressed to a high level (H . Boos, M . Stoehr, M . Sauter, and N . Mueller-Lantzch, J . Gen . Virol . 71:1811-1815, 1990) . Here we show that in Raji cells which constitutively express a transfected EBNA3C gene, the down-regulation of LMP1 in growth-arrested cells does not take place . Furthermore, we show that in wild-type Raji cells, low-level LMP1 expression occurs when most of the cells are arrested at a point(s) early in G1 (or G0) when the product of the retinoblastoma gene, pRb, is hypophosphorylated . The dramatic synthesis of LMP1 coincides with the progression of these cells to late G1 when pRb becomes hyperphosphorylated . Thus, in Raji cells, the LMP1 gene is apparently regulated in a cell cycle- or proliferation-dependent manner, but when EBNA3C is present, sustained LMP1 expression occurs as it does in a lymphoblastoid cell line . EBNA3C appears to either relieve the apparent repression of LMP1 in cells progressing through early G1 or possibly alter the stage at which the cells growth arrest to one where they are permissive for LMP1 expression.

Exp Cell Res, 1994 Jun, 212(2), 329 - 37
Effect of lovastatin on the cytotoxicity of ricin, modeccin, Pseudomonas toxin, and diphtheria toxin in brefeldin A-sensitive and -resistant cell lines; Oda T et al.; Lovastatin, a specific inhibitor of 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase, inhibits the biosynthesis of cholesterol and the prenylation of proteins . In this study, we have found that lovastatin inhibited the cytotoxicities of modeccin, ricin, Pseudomonas toxin, and diphtheria toxin in Vero and a brefeldin A (BFA)-resistant mutant of Vero cells (BER-40) to different extents . Among these toxins tested, the cytotoxicity of modeccin was most strongly inhibited by lovastatin in a dose-dependent manner . The protective effect of lovastatin was completely reversed by the addition of mevalonic acid, while the addition of cholesterol had no effect on the cytotoxicity of modeccin in lovastatin-treated cells . These results suggest that prenylated proteins are involved in the intoxication process of modeccin . The addition of cycloheximide to the growth medium also reversed the protective effect of lovastatin, suggesting a requirement of de novo protein synthesis for the protection by lovastatin against toxins . In contrast to Vero and BER-40 cells, no significant effect of lovastatin was observed in naturally BFA-resistant cell lines, PtK1 and MDCK cells, even though similar morphological changes and disassembly of the actin microfilaments were induced by lovastatin in these cell lines as observed in Vero and BER-40 cells . Lovastatin did not affect the binding and internalization of ricin and modeccin in Vero and BER-40 cells . Our results suggest that prenylated cellular proteins are involved in intracellular trafficking or processing of protein toxins, especially modeccin . In PtK1 and MDCK cells, such intracellular vesicle trafficking of protein toxins may be regulated by lovastatin-resistant mechanisms.

In Vitro Cell Dev Biol Anim, 1994 Jun, 30A(6), 356 - 65
Serum-free culture and characterization of renal epithelial cells isolated from human fetal kidneys of varying gestational age; Hazen-Martin DJ et al.; Cell cultures were initiated from seven human fetal kidneys that varied in gestational age from 90 days to newborn . The growth medium utilized was a 1:1 mixture of Dulbecco's modified Eagle's and Ham's F12 supplemented with selenium (5 ng/ml), insulin (5 micrograms/ml), transferrin (5 micrograms/ml), hydrocortisone (36 ng/ml), triiodothyronine (4 pg/ml), and epidermal growth factor (10 ng/ml) . For all the kidney isolates, initial cell attachment occurred within 12 h through multicell spheroids, and by 24 h a rapidly growing population of cells was obtained . Confluency was reached within 3 to 6 days . A combination of light microscopy, immunohistochemistry, and ultrastructural evaluation was utilized to characterize the resulting cultures as epithelial and homogeneous within each isolate and among the isolates . That is, regardless of gestational age of the fetal kidney used as starting material, an identical or highly similar population of cells was obtained . By light microscopy, the cultures were noted to form very few domes, the number being an indication of transport activity . However, ultrastructural examination revealed that the cells were noted to form domes composed of only a few cells or "micro-domes" that would not be visible by light microscopy . Within the micro-domes as well as other areas of the monolayer an apparent absence of tight junctions was noted by routine transmission electron microscopy . However, by freeze fracture analysis cells were shown to possess sealing strands, the structural component of tight junctions . It is postulated that the tight junctions of fetal epithelial cells are structurally altered as compared to tight junctions in adult renal epithelial cell cultures.

Curr Genet, 1994 Jun, 25(6), 475 - 9
Control of the expression of the ADE2 gene of the yeast Saccharomyces cerevisiae; Gedvilaite A et al.; The ADE2 gene encodes AIR-carboxylase which catalyzes the sixth step of the purine biosynthetic pathway in Saccharomyces cerevisiae . We have analyzed the effect of deletions in the promoter region of this gene on the expression of the enzyme using a fusion of the ADE2 gene promoter to the bacterial lacZ gene . Adenine added to the growth medium repressed the expression of the fusion at the level of mRNA . The ADE2-lacZ fusion expression can be slightly activated in response to amino-acid starvation, but only in Gcn4+ strains and in an adenine-supplemented medium . In the absence of adenine in the medium ADE2 gene expression is derepressed, and neither starvation for histidine nor a gcd1 general control regulatory mutation leads to additional derepression . Our experiments indicate that the ADE2 gene of the purine biosynthetic pathway is under both specific adenine control and the general amino-acid control system . The cis-acting promoter elements mediating both modes of regulation overlap each other and are located around the proximal TGACTC sequence.

Appl Environ Microbiol, 1994 Jun, 60(6), 1978 - 83
4-Ethylphenol metabolism by Aspergillus fumigatus; Jones KH et al.; Aspergillus fumigatus ATCC 28282 was found to be capable of growth on 4-ethylphenol as its sole carbon and energy source . A pathway for the metabolism of this compound has been proposed . The initial step involves hydroxylation of the methylene group of 4-ethylphenol to form 1-(4'-hydroxyphenyl)ethanol, followed by oxidation to 4-hydroxyacetophenone . The hydroxylase was NADPH and oxygen dependent, which is a characteristic of a monooxygenase type of enzyme . The 1-(4'-hydroxyphenyl)ethanol isolated from growth medium was a racemic mixture of R-(+) and S-(-) enantiomers . 4-Hydroxyacetophenone undergoes an NADPH-dependent Baeyer-Villiger type of oxygenation to give 4-hydroxyphenyl acetate, which is hydrolyzed to form hydroquinone (1,4-dihydroxybenzene) . Hydroxylation of hydroquinone by an NADPH-dependent enzyme produces 1,2,4-trihydroxybenzene, the ring fission substrate, which is cleaved by ortho fission to form maleylacetate . The pathway was elucidated by various kinds of investigations . Analysis of culture medium sampled during growth on 4-ethylphenol revealed the transient appearance of 1-(4'-hydroxyphenyl)ethanol, 4-hydroxyacetophenone, and hydroquinone . Cells grown on 4-ethylphenol were able to oxidize all of these compounds immediately, whereas oxidation by succinate-grown cells showed a lag period . Extracts prepared from cells grown on 4-ethylphenol contained enzyme activities for all of the proposed steps . Apart from a low level of esterase activity towards 4-hydroxyphenyl acetate, extracts prepared from cells grown on succinate did not contain any of these enzyme activities.

C R Acad Sci III, 1994 Jun, 317(6), 535 - 41
On the relation between phosphate uptake and growth of the cyanobacterium Anacystis nidulans; Falkner G et al.; The energy dependent phosphate uptake by the cyanobacterium Anacystis nidulans has been analysed in terms of a "linear energy converter" that describes the interrelationship between phosphate incorporation into the polyphosphate pool and the photosynthetic proton flux at the thylakoid membrane . It is assumed that both processes are coupled in such a way that uptake proceeds with optimal efficiency at the prevailing phosphate concentration in the growth medium . On the basis of this model two important parameters of the uptake system can be calculated: first, a conductivity coefficient that reflects the activity of the uptake system and second, a minimal threshold value of external phosphate at which uptake ceases . The theoretical data are shown to fit the experimentally observed uptake behaviour of cyanobacteria when the same population is subjected to a transition from growth on low to high phosphate concentrations, mimicking a situation that frequently leads in natural waters to an algal bloom.

Steroids, 1994 Jun, 59(6), 357 - 61
Isolation and identification of testosterone and androstenedione in the fungus Cochliobolus lunatus; Kastelic-Suhadolc T et al.; The cytosol of the filamentous fungus Cochliobolus lunatus was found to contain binding proteins for testosterone and androst-4-ene-3,17-dione . Both of these steroids were also found to be good exogenous substrates for the constitutive 17 beta-hydroxysteroid dehydrogenase, also found in this fungus . We were looking for the possible endogenous substrate . The procedures for isolation and identification of the endogenous steroid molecules in the fungus C . lunatus are described in this paper . The lipids were extracted from the cells and from the growth medium and purified by column and thin-layer chromatography . Analysis of the steroids, isolated from the cells of C . lunatus by gas chromatography and combination of gas chromatography-mass spectrometry, revealed the presence of testosterone and androst-4-ene-3,17-dione . Their structures were confirmed by comparison with the standards on the basis of chromatographic behavior and mass spectra . No such structures were found in the growth medium . Endogenous synthesis of androgens in this fungus was independently confirmed by the detection of radioactively labeled testosterone when the growing cells of C . lunatus were labeled with radioactive precursor molecule {5-3H} mevalonate.

Biotechnology (N Y), 1994 Jun, 12(6), 614 - 8
Production and secretion of recombinant proteins in Dictyostelium discoideum; Dittrich W et al.; We have expressed useful amounts of three recombinant proteins in a new eukaryotic host/vector system . The cellular slime mold Dictyostelium discoideum efficiently secreted two recombinant products, a soluble form of the normally cell surface associated D . discoideum glycoprotein (PsA) and the heterologous protein glutathione-S-transferase (GST) from Schistosoma japonicum, while the enzyme beta-glucuronidase (GUS) from Escherichia coli was cell associated . Up to 20mg/l of recombinant PsA and 1mg/l of GST were obtained after purification from a standard, peptone based growth medium . The secretion signal peptide was correctly cleaved from the recombinant GST- and PsA-proteins and the expression of recombinant PsA was shown to be stable for at least one hundred generations in the absence of selection.

In Vitro Cell Dev Biol Anim, 1994 Jun, 30A(6), 394 - 9
Isolation and characterization of vasoformative endothelial cells from the rat aorta; Nicosia RF et al.; We describe here a modified nonenzymatic method for the isolation of rat aortic endothelial cells with vasoformative properties . Aortic rings placed on plastic or gelatin-coated surfaces generated outgrowths primarily composed of endothelial cells . Prompt removal of aortic explants after endothelial migration minimized fibroblast contamination . However, fibroblasts, because of their high proliferative rate tended to overgrow the endothelial cells even when present in small numbers . This potential pitfall was avoided by weeding out fibroblasts with the rounded tip of a bent glass pipette . Primary endothelial colonies free of fibroblasts were segregated in cloning rings, trypsin-treated, and transferred to gelatin-coated dishes . Endothelial cells were cultured in MCDB 131 growth medium containing 10% fetal bovine serum, endothelial cell growth supplement, and heparin . Using this technique, pure endothelial cell strains were obtained from single aortic rings . Confluent endothelial cells formed a contact-inhibited monolayer with typical cobblestone pattern . The endothelial cells were positive for Factor VIII-related antigen, took up DiI-Ac-LDL, and bound the Griffonia Simplicifolia-isolectin-B4 . Endothelial cells cultured on collagen gel formed a polarized monolayer, produced basement membrane, displayed Weibel-Palade bodies and caveolae, and were connected by tight junctions . In addition, they reorganized into a network of microvascular cords and tubes when overlaid with a second layer of collagen and formed microvascular sprouts in response to fibroblast-conditioned medium . This isolation procedure yields stable strains of vasoformative endothelial cells, which can be used to study aortic endothelium-related angiogenesis and its mechanisms.

J Biol Chem, 1994 May 20, 269(20), 14723 - 9
1,25-Dihydroxyvitamin D3 potentiates the keratinocyte response to calcium; Su MJ et al.; Extracellular calcium (Cao) stimulates the differentiation of keratinocytes; 1,25 dihydroxyvitamin D3 1,25(OH)2D3) does likewise . Since 1,25(OH)2D3 regulates calcium flux in other cells, we hypothesized that 1,25(OH)2D)3 acted through and promoted the effects of calcium on keratinocyte differentiation . To test this hypothesis, we evaluated the effects of calcium and 1,25(OH)2D3 alone and in combination on the mRNA and protein levels of involucrin and transglutaminase in neonatal human keratinocytes . Cao alone increased these mRNA levels in a dose-dependent fashion (0.03 to 1.2 mM) over a 24-h period . This increase in mRNA levels was associated with a stimulation by calcium of involucrin and transglutaminase gene transcription . However, by 72 h, the mRNA levels of involucrin and transglutaminase decreased . At 0.03 mM Cao, 1,25(OH)2D3 showed a dose-dependent stimulation of involucrin and transglutaminase mRNA for up to 48 h and potentiated the initial (4-h) stimulation by Cao of involucrin and transglutaminase mRNA . As for calcium alone, this increase in mRNA was associated with an increase in transcription of the involucrin and transglutaminase genes . However, by 24 h of exposure to both calcium and 1,25(OH)2D3, a dose-dependent fall in mRNA levels was seen . The mRNA levels of involucrin and transglutaminase were stable for 24 h when neonatal human keratinocytes were grown in serum-free keratinocyte growth medium containing 0.03 or 1.2 mM Cao alone . However, the mRNAs of both genes underwent rapid degradation when neonatal human keratinocytes were treated with 1,25(OH)2D3, especially in high Cao . 1,25(OH)2D3 and Cao increased the protein levels of involucrin and transglutaminase activity in a synergistic fashion throughout the 48-h time course . These data support the hypothesis that 1,25(OH)2D3 promotes calcium-induced differentiation at the level of both gene expression and mRNA stability.

Cancer Res, 1994 May 15, 54(10), 2531 - 5
Interleukin-2-secreting mouse fibroblasts transfected with genomic DNA from murine melanoma cells prolong the survival of mice with melanoma; Kim TS et al.; A retrovirus was used to introduce a provirus (pZipNeoSVIL-2) containing the gene for interleukin-2 (IL-2) along with a neor gene (confers resistance to G418) into LM cells, a mouse cell line expressing defined major histocompatibility complex class I antigens (H-2k) . After initial selection in growth medium containing G418, IL-2 secretion was confirmed, and the cells were then cotransfected with genomic DNA from B16F1 or B16F10 melanoma cells, along with DNA from a plasmid (pHyg) that confers resistance to hygromycin . After a second round of selection in growth medium containing sufficient quantities of hygromycin to kill 100% of nontransfected cells but without further modification, the unfractionated populations of transfected cells were tested for their immunotherapeutic properties in C57BL/6 mice (H-2b) with established B16 melanomas (H-2b) . Animals with melanomas treated with either of the transfected cell populations survived significantly (P < 0.01) longer than untreated mice or mice treated with irradiated (5000 rads) B16F1 melanoma cells . The animals also survived longer (P < 0.05) than mice with melanoma treated with IL-2-secreting LM cells transfected with genomic DNA from MOPC-315 cells, a nonimmunologically cross-reactive murine tumor . As determined by the capacity of monoclonal antibodies to T-cell subsets to inhibit the antimelanoma response in a 51Cr release assay, the antimelanoma immunity in mice immunized with cells transfected with genomic DNA from either B16F1 or B16F10 cells was mediated primarily by Lyt-2.2+ T-cells . These data raise the possibility that a generic, live cell tumor vaccine can be developed that can be modified to provide specificity for the neoplasms of individual patients.

Biochim Biophys Acta, 1994 May 11, 1191(2), 331 - 42
Similar regulatory mechanisms despite differences in membrane lipid composition in Acholeplasma laidlawii strains A-EF22 and B-PG9 . A multivariate data analysis; Wieslander A et al.; Mycoplasmas are small, cell wall-deficient bacteria . The metabolic regulation of the lipid composition in the membrane of the species Acholeplasma laidlawii, strains A-EF22 and B-JU, is governed mainly by the balance between the potential formation of lamellar and nonlamellar phase structures . However, the regulatory features have not been consistently observed in the B-PG9 strain . A comparison has been performed between the membrane lipid composition for strains A-EF22 and B-PG9, simultaneously changing eight experimental conditions known to affect the regulation and packing properties of the A-EF22 lipids . Multiple regression and partial least-square discriminant analyses of many variables showed: (i) quantitative differences in membrane lipid and protein composition, and in membrane protein molecular masses of the two strains; (ii) different molar fractions of the major polar lipids monoglucosyldiacylglycerol (nonlamellar) and diglucosyldiacylglycerol (lamellar), which were caused by differences in lipid acyl chain length and unsaturation inherent in the strains and by the type of growth medium used; and (iii) similar regulatory mechanisms for changes in the lipid composition under most conditions, responding to the experimentally varied bilayer and nonbilayer properties of the lipid matrix . These regulatory principles are probably valid in other bacteria as well.

Mol Gen Genet, 1994 May 10, 243(3), 277 - 85
Nitrogen limitation induces expression of the avirulence gene avr9 in the tomato pathogen Cladosporium fulvum; Van den Ackerveken GF et al.; The avirulence gene avr9 of the fungal tomato pathogen Cladosporium fulvum encodes a race-specific peptide elicitor that induces the hypersensitive response in tomato plants carrying the complementary resistance gene Cf9 . The avr9 gene is not expressed under optimal growth conditions in vitro, but is highly expressed when the fungus grows inside the tomato leaf . In this paper we present evidence for the induction of avr9 gene expression in C . fulvum grown in vitro under conditions of nitrogen limitation . Only growth medium with very low amounts of nitrogen (nitrate, ammonium, glutamate or glutamine) induced the expression of avr9 . Limitation of other macronutrients or the addition of plant factors did not induce the expression of avr9 . The induced expression of avr9 is possibly mediated by a positive-acting nitrogen regulatory protein, homologous to the Neurospora crassa NIT2 protein, which induces the expression of many genes under conditions of nitrogen limitation . The avr9 promoter contains several putative NIT2 binding sites . The expression of avr9 during the infection process was explored cytologically using transformants of C . fulvum carrying an avr9 promoter-beta-glucuronidase reporter gene fusion . The possibility that expression of avr9 in C . fulvum growing in planta is caused by nitrogen limitation in the apoplast of the tomato leaf is discussed.

Gene, 1994 May 3, 142(1), 97 - 102
Sequence analysis and transcription of the apxI operon (hemolysin I) from Actinobacillus pleuropneumoniae; Frey J et al.; The DNA sequence of the entire apxI operon from Actinobacillus pleuropneumoniae serotype 1 reference strain 4074 has been determined . This 8292-bp fragment of the chromosomal DNA contains four open reading frames (ORFs) of the strongly hemolytic ApxI toxin . These ORFs correspond to the genes apxIC, apxIA, apxIB and apxID, encoding the activator, the structural toxin protein and the two secretion proteins, respectively . Each of the four ORFs is preceded by a consensus sequence for a putative ribosome-binding site (RBS) . The region upstream from apxIC contains several sites that could act as promoters . The transcription start point (tsp) of the apxI operon in A . pleuropneumoniae has been determined by primer extension analysis and was found to be located 133-bp upstream from the translation start codon . The tsp is preceded by sequences matching the -10 and -35 consensus sequence of promoters from Escherichia coli . This is the first promoter identified in A . pleuropneumoniae . The same tsp was used when the expression of apxI was induced by a high concentration of free Ca2+ in the growth medium, as well as when the expression of apxI was not induced by growing the cells in medium depleted of free Ca2+ ions . However, the signal strength of the primer extension was approximately tenfold stronger in Ca(2+)-grown cells . The leader sequence of the transcript is unusually long and very A+U rich (75% A+U).

J Clin Invest, 1994 May, 93(5), 1981 - 6
Antiproliferative effect of retinoid compounds on Kaposi's sarcoma cells; Corbeil J et al.; A panel of retinoid compounds (tretinoin, isotretinoin, acitretin, and RO13-1470) were tested for inhibitory activity against Kaposi's sarcoma cell (KSC) cultures in vitro . Tretinoin was found to be the most effective retinoid tested, inhibiting the growth of KSC in vitro while having no effect on the expression of interleukin-6 and basic fibroblast growth factor, two important cytokines involved in KSC growth . Tretinoin also did not appear to downregulate the expression of receptors for these two cytokines . At low concentrations (10(-9) M), acitretin and tretinoin selectively inhibited growth of early passage KSC . At higher concentrations (10(-6)-10(-5) M), retinoid treatment induced a pattern of DNA degradation and morphological changes in KSC characteristic of apoptosis (programmed cell death) . The inhibitory activity of tretinoin on KSC growth was decreased if human serum (but not fetal calf serum) was present in the growth medium, and partially restored by removal of serum lipids . These data suggest that retinoids possess potential as therapeutic agents in Kaposi's sarcoma.

Can J Microbiol, 1994 May, 40(5), 393 - 6
Production of 2-aminobutyrate by Megasphaera elsdenii; Furtado AF et al.; Production of the amino acid 2-aminobutyrate was studied in four strains of Megasphaera elsdenii grown on a lactate-based growth medium containing Bacto-casamino acids and yeast extract . Supplementation with threonine increased the production of 2-aminobutyrate in three of the four strains, but no substantial increase in production was noted with serine, methionine, or aspartate, all of which are potential sources for the precursor of 2-aminobutyrate, 2-oxobutyrate . L-Cycloserine, an inhibitor of alanine transaminases, decreased both alanine and 2-aminobutyrate production, suggesting that 2-aminobutyrate synthesis may share the same metabolic pathway as alanine synthesis or that 2-oxobutyrate can act as a substrate for alanine transaminases . Decreases in the production of 2-aminobutyrate were associated with a reduction in the catabolism of branched-chain amino acids in two of the four strains.

In Vitro Cell Dev Biol Anim, 1994 May, 30A(5), 312 - 20
Establishment of human parotid pleomorphic adenoma cells in culture: morphological and biochemical characterization; Prasad KN et al.; This study reports the establishment of alpha-amylase-producing human parotid pleomorphic adenoma cell lines (2HP and 2HP1) which have been maintained in culture for over 1 yr . The procedures required preparation of cellular clumps from tumor tissue and plating them on plasma clot or precoated dishes . During the initial phase of growth they required modified MCDB-153 medium without serum . When cells showed signs of degeneration they were changed to MCDB-153 medium containing first 2% and then 10% heat inactivated fetal bovine serum . Although cells grew well in MCDB-153 containing 10% serum, the epithelial cell morphology was not distinct . Therefore, the growth and morphology of cells grown in MCDB-10% serum were compared with those in RPMI growth medium containing 10% fetal bovine serum and F12 containing 10% agammaglobulin newborn bovine serum . Although the growth of cells was a little slower in F12 medium than those in MCDB and RPMI, the epithelial cell morphology was maintained better than in other growth media . The cells of 2HP and 2HP1 produce low levels of alpha-amylase and relatively high levels of alpha-amylase mRNAs of 1176 and 702 bp and contain neurofilament-160, a neuronal-specific marker . The cells of 2HP1 are tumorigenic when tested in athymic mice, but the cells of 2HP are not . The establishment of amylase-producing human parotid adenoma cell lines of different characteristics in culture provides a new opportunity to study the mechanisms of differentiation and transformation, and regulation of alpha-amylase in these cells.

Antimicrob Agents Chemother, 1994 May, 38(5), 1004 - 7
Fenpropimorph affects uptake of uracil and cytosine in Saccharomyces cerevisiae; Crowley JH et al.; Fenpropimorph was shown to inhibit the accumulation of the pyrimidine bases uracil and cytosine from the growth media in Saccharomyces cerevisiae . Uracil prototrophs of S . cerevisiae were more resistant to the growth-inhibitory effects of fenpropimorph than were uracil auxotrophs . High concentrations of uracil rescued fenpropimorph-treated uracil auxotrophs, and cytosine, which is accumulated by a separate mechanism, could also support growth of treated uracil auxotrophs . Fenpropimorph caused a significant decrease in the uptake of radiolabeled uracil, which was not due to accumulation of ergosta-8,14-dienol (ignosterol) in the treated cultures . Radiolabeled cytosine uptake was unaffected by drug treatment in a wild-type strain but was inhibited in a sterol mutant, in which ergosterol was absent from the cell . The role of fenpropimorph in causing membrane dysfunction through a mechanism other than altered sterol metabolism is discussed.

Somat Cell Mol Genet, 1994 May, 20(3), 183 - 94
Somatic cell genetic and biochemical characterization of cell lines resulting from human genomic DNA transfections of Chinese hamster ovary cell mutants defective in sterol-dependent activation of sterol synthesis and LDL receptor expression; Hasan MT et al.; We have isolated several non-leaky mutant Chinese hamster ovary (CHO) cell clones (M4, M19, and M21) requiring cholesterol and unsaturated fatty acid for growth . These mutants belong to the same complementation group as the mutant M1 cells previously reported from this laboratory . M19 cells reverted to lipid prototrophy at very low frequency and were chosen as recipients to perform DNA-mediated gene-transfer experiments using total human genomic DNAs . Biochemical characterization of these transfectant clones indicated that, unlike their parental M19 cells, they were able to exhibit activation of cholesterol biosynthesis and LDL receptor expression in response to sterol removal from the growth medium . RNA blotting analysis indicated that these transfectants were able to increase HMG-CoA synthase gene transcripts in response to sterol removal . From the genomic DNAs of a representative secondary transfectant cells, we cloned a unique human DNA fragment (designated as h lambda 2) and showed that h lambda 2 closely linked with the presumptive human M1 gene.

J Cell Sci, 1994 May, 107 ( Pt 5), 1169 - 79
Serum deprivation induces apoptotic cell death in a subset of Balb/c 3T3 fibroblasts; Kulkarni GV et al.; Little is known about the regulation of apoptosis in fibroblasts although several model systems including serum deprivation and treatment with staurosporine or topoisomerase inhibitors have been used to induce apoptosis in vitro . To validate a reproducible in vitro model for the study of apoptosis in fibroblasts, we cultured density-inhibited monolayer cultures of Balb/c 3T3 fibroblasts in Dulbecco's modified essential medium plus 15% fetal calf serum and then withdrew serum . Time-lapse video microscopy demonstrated that within minutes of serum withdrawal, cells lost substrate attachment and floated to the top of the liquid growth medium . There was a time-dependent increase in the number of non-adherent cells . Some of these cells regained attachment and spread momentarily, but they eventually rounded up and lost attachment permanently . In contrast to serum-containing cultures in which similar morphological changes were followed by mitosis, in serum-free cultures repeated attempts at mitosis were followed by permanent attachment loss and presumably cell death . To assess whether all the non-adherent cells were in fact dead, the percentages of cells that continued to proliferate upon return to serum-supplemented conditions was computed . After various periods of serum starvation a decreasing proportion (approx . 75% at 30 minutes; < 2% at 24 hours) of the non-adherent cells could be rescued by addition of serum . Transmission electron microscopy of cells 3 hours after serum withdrawal showed that the majority (approximately 60%) of non-adherent cells exhibited marked intranuclear chromatin condensation but maintained integrity of cell and nuclear membranes and cell organelles, morphological changes consistent with those of apoptotic cell death . Scanning electron microscopy of cultures 3 hours following serum withdrawal showed rounded cells with marked surface blebbing . Fluorescence and confocal microscopy revealed increased intensity of nuclear staining with DAPI while actin filaments became indistinct or collapsed around the nucleus . After cycloheximide treatment to inhibit protein synthesis, there was no reduction of apoptosis . Gel electrophoresis of DNA from both control and 3 hour-serum-deprived cells showed intact DNA with no oligonucleosomal length fragmentation . After serum withdrawal, intracellular calcium was reduced by about 32% over 5 minutes as measured by fura2 ratio fluorimetry in single cells . Serum-starved cells showed a time-dependent shrinkage in mean cell diameter compared to trypsinized, adherent control cells (at 0 hours, mean diameter = 18.0 microns--viable; at 4 hours, mean diameter = 15.5 microns--apoptotic) . Flow cytometric analysis showed increased propidium iodide staining and reduced fluorescein diacetate uptake over 3 hours, changes that were contemporaneous with the reduction of cell diameter.(ABSTRACT TRUNCATED AT 400 WORDS)

Enferm Infecc Microbiol Clin, 1994 May, 12(5), 241 - 5
{Utility of the direct culture examination (Ziehl-Neelsen) using the Bactec system for the presumptive identification of Mycobacterium tuberculosis complex, Mycobacterium avium complex, Mycobacterium xenopi, and Mycobacterium kansasii}; Moreno C et al.; BACKGROUND: The usefulness of the microscopic examination of Bactec 12B and 13A growth medium as a method for the possible identification of M . tuberculosis complex, M . avium complex, M . xenopi, and M . kansasii was performed out to guide the selection of different genetic identification probes and, in the case of M . xenopi, the selection of the temperature of subcultures incubation . METHODS: Upon detection of an index of growth greater than 100 in Bactec tubes, staining was performed by the Ziehl-Neelsen technique . On the basis of the morphology observed, the possible identification was performed by genetic probes . Subcultures were used for definitive identification . RESULTS: Three hundred forty-four positive samples were studied by radiometric technique . A total of 190 strains were identified as M . tuberculosis, 88 strains as M . avium-intracellulare (MAI), 33 strains as M . xenopi, 14 strains as M . kansasii and 19 strains were identified as: M . gordonae (10), unpigmented rapid growth microbacteria (7), and M . simiae (2) . Sensitivity, specificity, positive predictive value, and negative predictive value were 97.9%, 95.4%, 96.4%, and 97.3%, respectively for M . tuberculosis complex, 84.0%, 99.2%, 97.3% 94.7% for M . avium complex; 63.6%, 98.3%, 80.7%, 96.2% for M . xenopi; 35.7%, 98.1%, 45.5% 97.2% for M . kansasii . CONCLUSIONS: The morphology of M . tuberculosis complex examined in the radiometric system in useful to differentiate this species from other microbacteria (MOTT), allowing the selection of specific probe used . Within the MOTT, M . avium complex also has morphological characteristics which are useful for its differentiation, the morphology usually described for the remaining species was frequently not observed.

Biochim Biophys Acta, 1994 Apr 28, 1221(3), 279 - 85
Characterization of a COS cell line deficient in polyamine transport; Hyvonen T et al.; In the present study, we describe the isolation and characterization of a COS cell line deficient in polyamine uptake that may provide an important tool for the molecular cloning of polyamine transporter(s) . The cells were selected by isolation for resistance against the cytotoxic agent, methylglyoxal bis(guanylhydrazone) (MGBG), which is entering the cells using the same transport system as the polyamines . The isolated cell line was capable of growing in the presence of 100 microM MGBG, which totally inhibited the growth of the wild-type cells . The transport of putrescine and spermidine was markedly decreased in the COS-MGBGr cells . The decrease in putrescine transport was mainly a result of a 14-fold decrease in Vmax, whereas the reduced spermidine uptake was due to a 3-4-fold decrease in Vmax as well as 12-fold increase in Km, indicating the existence of at least two separate transport systems . No major difference in polyamine content was seen between the parental and the COS-MGBGr cells when grown without MGBG . In the presence of MGBG, both cell lines exhibited an increase in putrescine content . Treatment with MGBG also resulted in a decrease in spermidine and spermine contents in the wild-type cells . In the COS-MGBGr cells, on the other hand, there were no statistically significant effects on the spermidine and spermine contents by MGBG treatment . In the wild-type cells, depletion of polyamines, e.g., by treatment with the ornithine decarboxylase inhibitor 2-difluoromethylornithine (DFMO), stimulated the uptake of polyamines (3-7-fold), whereas in the COS-MGBGr cells the effect of DFMO treatment on polyamine transport was only minor . In contrast to the growth-medium of the wild-type cells, large amounts of polyamines accumulated in the medium of the COS-MGBGr cells, presumably indicating that COS cells normally excrete polyamines and then salvage them using the polyamine transport system.

Brain Res, 1994 Apr 18, 643(1-2), 108 - 14
Cytotoxicity of the cocaine metabolite benzoylecgonine; Lin Y et al.; The NG108-15 and C6 cell lines were used in the present study as neuronal and glial models, respectively, to examine the cytotoxicity of the major metabolite of cocaine, benzoylecgonine (BE) . Exposure of both cell types to varying concentrations of BE resulted in a loss of cells from the growth surface . Analysis of the unattached cells after such exposure, using a variety of techniques, revealed that these cells were not viable . Therefore, this effect could not be ascribed to BE interfering with cell-substratum interactions . The early events in BE cytotoxicity were examined by observing cells cultivated on the stage of an inverted microscope, using differential interference contrast (Nomarski) optics . Upon exposure of either cell type to 10 microM BE a retraction of cellular processes could be observed within 30 min . Within 6 h cell death was apparent . Similar analyses using 50 microM BE in the growth medium resulted in similar results, except that process retraction could be observed as early as 15 min after exposure . These results demonstrate that the major metabolite of cocaine, benzoylecgonine, is cytotoxic to in vitro models of neuronal and glial cells.

Biochem J, 1994 Apr 15, 299 ( Pt 2), 467 - 72
Cell proliferation status, cytokine action and protein tyrosine phosphorylation modulate leukotriene biosynthesis in a basophil leukaemia and a mastocytoma cell line; Hagmann W; Mast cells, mastocytoma cells and basophil leukaemia cells are well-established producers of leukotrienes when grown and stimulated appropriately . I report that the cells' ability to produce leukotrienes is dependent on the cells' proliferative status or their provision with growth factors . Proliferating MC/9 and subconfluent RBL2H3 cells respond maximally to stimulation by 1 microM ionomycin with the production of 56 and 32 pmol of cysteinyl-leukotrienes/10(6) cells respectively . In contrast, confluent RBL2H3 or growth-arrested MC/9 cells lose their ability to generate leukotrienes in response to ionomycin treatment . This rapid down-regulation of leukotriene synthesis is also observed when proliferating RBL2H3 cells are transferred to growth-factor-free medium, wherein cellular leukotriene-synthesis capacity has an apparent half-lifetime of 60 min . Transfer back into growth medium results in the regeneration of leukotriene synthesis capacity within 6 h . In growth-arrested MC/9 cells, leukotriene production ability can at least partially be restored by priming the cells with interleukin 3, but not with interleukin 4 . In RBL2H3 cells, pretreatment with protein tyrosine kinase inhibitors such as genistein (5 min, 37 microM), herbimycin A (6 h, 3 microM) or tyrphostin 25 (16 h, 100 microM) completely inhibits leukotriene generation, whereas okadaic acid (15 min, 0.5 microM) has no effect . Under these conditions, both genistein and herbimycin A strongly impair ionomycin-induced protein tyrosine phosphorylation . Our study indicates that leukotriene generation in these tumour cells is tightly regulated by their proliferation status and supply with growth factors, and cell stimulation towards leukotriene synthesis appears to involve protein tyrosine kinase activity.

FEBS Lett, 1994 Apr 11, 342(3), 319 - 24
Structural differences in chlorosomes from Chloroflexus aurantiacus grown under different conditions support the BChl c-binding function of the 5.7 kDa polypeptide; Lehmann RP et al.; Structurally different chlorosomes were isolated from the green photosynthetic bacterium Chloroflexus aurantiacus grown under different conditions . They were analysed with respect to variable pigment-protein stoichiometries in view of the presumed BChl c-binding function of the 5.7 kDa chlorosome polypeptide . Under high-light conditions on substrate-limited growth medium the pigment-protein ratio of isolated chlorosomes was several times lower than under low-light conditions on complex medium . Proteolytic degradation of the 5.7 kDa polypeptide in high-light chlorosomes led to a 60% decrease of the absorbance at 740 nm . The CD spectrum of high-light chlorosomes exhibited a sixfold lower relative intensity at 740 nm (delta A/A740) than low-light chlorosomes, but it showed a fivefold increase in intensity upon degradation of the 5.7 kDa polypeptide compared to a twofold increase in low-light chlorosomes . It seems probable that BChl c in the chlorosomes is present as oligomers bound to the 5.7 kDa polypeptide . Our data suggest further that compared to low-light chlorosomes smaller oligomers or single BChl c molecules are bound to the 5.7 kDa polypeptide in high-light chlorosomes resulting in lower rotational strength.

J Biol Chem, 1994 Apr 8, 269(14), 10485 - 91
Response of human fibroblasts to hypertonic stress . Cell shrinkage is counteracted by an enhanced active transport of neutral amino acids; Dall'Asta V et al.; Regulatory volume increase (RVI) has been studied in cultured human fibroblasts (CHF) incubated in a complete hypertonic growth medium (400 mosmol/kg) . After the initial cell shrinkage induced by hypertonic treatment, cells recover their volume almost completely within 3 h . This RVI response is associated with a marked increase of the cell content of free amino acids . The cell content of potassium increases only slightly . Chromatographic analysis of the intracellular amino acid pool shows that the RVI-associated increase in cell amino acids is mainly a result of changes in the L-glutamine content . The intracellular accumulation of the analog 2-methylaminoisobutyric acid, a specific substrate of transport system A, is increased in CHF undergoing RVI . Hypertonic treatment causes an immediate and sustained cell hyperpolarization, as demonstrated by changes in the trans-membrane distribution ratio of L-arginine and in the fluorescence of the potential-sensitive dye bis-1,3-diethylthiobarbiturate-trimethineoxonol . Because of cell hyperpolarization, at the end of RVI the trans-membrane gradient of the sodium electrochemical potential is higher than that of the control . The increase in the extracellular potassium concentration ({K+}out = 40 mM) abolishes the hyperpolarization induced by hypertonic treatment and delays volume recovery . Cycloheximide suppresses RVI at a high but not at physiologic {K+}out . It is proposed that CHF counteract hypertonic shrinkage through an enhanced accumulation of substrates of transport system A sustained, initially, by an increase in the energy available for transport and, subsequently, also by the synthesis of new site A carriers.

Comp Biochem Physiol Biochem Mol Biol, 1994 Apr, 107(4), 525 - 31
Lipid biosynthesis by axenic strains of Blastocystis hominis; Keenan TW et al.; Axenic strains of Blastocystis hominis incorporated 32P, added to the medium as orthophosphate, into a number of phospholipids, including sphingomyelin, cardiolipin, phosphatidic acid, the phosphoglycerides of choline, ethanolamine, serine, and inositol and some other minor phospholipids . Radioactive palmitate and glycerol provided in the growth medium introduced radiolabel into diacylglycerols, triacylglycerols, and all major phosphoglycerides found in the organism . Palmitate is a major fatty acid of cholesterol esters in B . hominis, but radioactive palmitate did not enter the cholesterol ester pool . Radioactive acetate was not incorporated into any lipids . Cholesterol and cholesterol esters of the organism were not labeled when cells were grown in the presence of radioactive glucose, mevalonic acid, or mevalonolactone . Radioactive cholesterol added to the medium became stably associated with B . hominis cells, but none of the radioactive cholesterol entered the cholesterol ester pool . Cholesterol-{3H}-palmitate added to the medium became stably associated with the organism, and most of the radioactivity associated with the cells remained in the cholesterol ester fraction on extended incubation . These results show that this parasitic protozoan has the capacity to synthesize most cellular lipids de novo, but suggest that it acquires free cholesterol and intact cholesterol esters directly from growth medium.

Radiat Res, 1994 Apr, 138(1), 34 - 43
Evidence that the product of the xrs gene is predominantly involved in the repair of a subset of radiation-induced interphase chromosome breaks rejoining with fast kinetics; Okayasu R et al.; We classified interphase chromosome breaks into alpha and beta forms to study the requirement for the xrs gene product in the repair of each of these forms of damage . The alpha form of damage comprises radiation-induced interphase chromosome breaks whose rejoining is slow and sensitive to treatment with beta-arabinofuranosyladenine (beta-araA), whereas the beta form of damage comprises interphase chromosome breaks whose rejoining is fast and sensitive to treatment in hypertonic medium . Interphase chromosome breaks of the alpha form are visualized in plateau-phase cells by premature chromosome condensation (PCC) carried out in the absence of any treatment during the condensation period . More interphase chromosome breaks of the alpha form can be uncovered by treatment with beta-araA during the period of PCC . Interphase chromosome breaks of the beta form are not visualized in experiments using standard PCC protocols but can be uncovered by treatment in hypertonic growth medium during the period allowed for PCC . In the present report, we show that the yield of interphase chromosome breaks of the alpha form is similar in CHO and xrs-5 cells and demonstrate that xrs-5 cells rejoin this type of interphase chromosome breaks with an efficiency similar to that observed in repair-proficient CHO cells . Furthermore, we provide evidence supporting the notion that xrs-5 cells are deficient in the rejoining of the beta form of interphase chromosome breaks . These results strongly suggest that the product of the xrs gene is required predominantly in the repair of the beta form of interphase chromosome damage and emphasize the need for discrimination between different forms of interphase chromosome breaks in irradiated cells.

Blood, 1994 Apr 1, 83(7), 1883 - 91
Induction of differentiation of promyelocytic NB4 cells by retinoic acid is associated with rapid increase in urokinase activity subsequently downregulated by production of inhibitors; Tapiovaara H et al.; 13-trans retinoic acid (13-trans RA) is an effective inducer of differentiation of acute promyelocytic (APL) cells both in vivo and in vitro . It is used in the induction of remission of patients with APL . We found, by using the promyelocytic NB4 cell line established from a patient with APL, that the induction of differentiation with RA was accompanied by modulation of the plasminogen activation system . The expression of urokinase (uPA) activity was rapidly increased in the growth medium and at the surface of cells treated with RA . The high uPA activity was counteracted both in the growth medium and at the cell surface by an increased plasminogen activator inhibitor (PAI) production and reduction of uPA synthesis . The expression of uPA receptor and PAI-2 were stimulated and persisted at 48 hours from RA addition . The simultaneous induction of CD11b suggests that differentiation results in increased production of both . APL patients often encounter episodes of disseminated intravascular coagulation that are associated with secondary fibrinolytic events . Our results suggest that downregulation of uPA activity results in the decrease of plasmin on the surface of the differentiated cells, which may reduce the occurrence of fibrinolytic episodes of patients with APL.

Immunomethods, 1994 Apr, 4(2), 139 - 47
Lymphocyte activation-31P magnetic resonance studies of energy metabolism and phospholipid pathways; Kaplan O et al.; 31P NMR spectra of perfused lymphocytes embedded in alginate capsules and activated by interleukin-2 (IL-2) are remarkably different from those of control lymphocytes . The main differences are the appearance and gradual increase of phosphodiester signals, glycerophosphocholine and glycerophosphoethanolamine . These metabolic changes also occur following perfusion with phorbol ester and after incubation with phytohemagglutinin (PHA) and are not dependent on a special growth medium . Nifedipine, a calcium channel blocking drug, inhibits the effects of PHA, but not of IL-2 . There are no NMR spectral differences between peripheral lymphocytes, stimulated for 3 weeks, and tumor-infiltrating lymphocytes . Thus, sustained accelerated turnover of phosphatidylcholine (PC) and phosphatidylethanolamine is an inherent feature of the activation process . 31P NMR spectra of lymphocytes are characterized by a low phosphocholine signal . Perfusion studies with high concentrations of choline and the use of dapsone, an inhibitor of phosphocholine cytidyltransferase, indicate that choline kinase plays a key role in regulating PC synthesis in human lymphocytes.

Pigment Cell Res, 1994 Apr, 7(2), 116 - 22
Proliferation and propagation of human melanocytes in vitro are affected by donor age and anatomical site; Abdel-Malek ZA et al.; We report the effects of two factors, donor age and anatomical site, on the proliferation and melanization of human melanocytes (MC) derived from (1) neonatal foreskins, (2) adult foreskins, or (3) adult breast or arm skin . Two different growth media have been used for this purpose . Medium I supports the long-term proliferation of neonatal MC, and medium II supports the growth of both neonatal and adult MC . We found that neonatal foreskin MC proliferated equally well in both media for more than 12 passages . However, adult MC behaved differently in the two growth media . In very early passages (passages 1-5), all three types of MC proliferated well and reached comparable final cell densities in medium I, but adult foreskin MC proliferated at a higher rate than neonatal or adult non-foreskin MC in medium II . The non-foreskin adult MC had the least proliferative rate in medium II . Unlike neonatal MC, both adult foreskin and non-foreskin MC underwent a drastic reduction in their proliferative rate after only a few (9-10) passages in culture . While the three types of MC differed in their proliferative rates, they responded similarly to melanogenic stimulation by cholera toxin (CT) and isobutyl methylxanthine (IBMX) . These results demonstrate that the proliferation of human MC in standard culture media is affected by the donor age and the anatomical site from which these cells are derived . We conclude that human MC are heterogeneous, and caution must be used in extrapolating the results of studies on MC derived from different anatomical sites or age groups.

J Virol Methods, 1994 Apr, 47(1-2), 27 - 35
Purification of infectious pancreatic necrosis virus by anion exchange chromatography increases the specific infectivity; Carlsson A et al.; An improved method for the isolation and purification of infectious pancreatic necrosis virus (IPNV) is described . Virions released into the clarified growth medium are adsorbed to an anion exchange resin of diethylaminoethyl cellulose at pH 8.1 . IPNV together with the likewise released and accumulated excess pool of the precursor to the major capsid protein, ICP62, are eluted at a salt concentration between 100 and 125 mM NaCl . The bovine serum albumin content of the growth medium supplement also elutes close to this position . Upon one step of combined sucrose- and CsCl-gradient centrifugation the recovered viruses display lower levels of aggregation, higher specific nucleic acid contents and an approximately 350% higher specific infectivity as compared with pools of viruses processed in parallel and isolated according to the established method relying on precipitation with poly(ethylene glycol).

Microbiology, 1994 Apr, 140 ( Pt 4), 703 - 15
Effects of oxygen limitation on sugar metabolism in yeasts: a continuous-culture study of the Kluyver effect; Weusthuis RA et al.; Growth and metabolite formation were studied in oxygen-limited chemostat cultures of Saccharomyces cerevisiae CBS 8066 and Candida utilis CBS 621 growing on glucose or maltose at a dilution rate of 0.1 h-1 . With either glucose or maltose S . cerevisiae could be grown under dual limitation of oxygen and sugar . Respiration and alcoholic fermentation occurred simultaneously and the catabolite fluxes through these processes were dependent on the magnitude of the oxygen feed . C . utilis could also be grown under dual limitation of glucose and oxygen . However, at very low oxygen feed rates (i.e . below 4 mmol l-1 h-1) growth was limited by oxygen only, as indicated by the high residual glucose concentration in the culture . In contrast to S . cerevisiae, C . utilis could not be grown anaerobically at a dilution rate of 0.1 h-1 . With C . utilis absence of oxygen resulted in wash-out, despite the presence of ergosterol and Tween-80 in the growth medium . The behaviour of C . utilis with respect to maltose utilization in oxygen-limited cultures was remarkable: alcoholic fermentation did not occur and the amount of maltose metabolized was dependent on the oxygen supply . Oxygen-limited cultures of C . utilis growing on maltose always contained high residual sugar concentrations . These observations throw new light on the so-called Kluyver effect . Apparently, maltose is a non-fermentable sugar for C . utilis CBS 621, despite the fact that it can serve as a substrate for growth of this facultatively fermentative yeast . This is not due to the absence of key enzymes of alcoholic fermentation . Pyruvate decarboxylase and alcohol dehydrogenase were present at high levels in maltose-utilizing cells of C . utilis grown under oxygen limitation . It is concluded that the Kluyver effect, in C . utilis growing on maltose, results from a regulatory mechanism that prevents the sugar from being fermented . Oxygen is not a key factor in this phenomenon since under oxygen limitation alcoholic fermentation of maltose was not triggered.

Indian J Med Res, 1994 Apr, 99, 171 - 2
A simple medium without blood modified for successful isolation & cultivation of LD bodies; Bhattacharya J et al.; A simple growth medium for primary isolation and subsequent cultivation of Leishmania donovani promastigotes without using whole blood is described . This medium is modified from Aljeboori's biphasic medium (used originally only for cultivation), containing only beef extract, peptone, sodium chloride, bactoagar and foetal calf serum (FCS) . We have modified the medium by adding glucose and ascertaining the pH in the solid phase and by drastically reducing (91%) FCS in the liquid phase . The medium helps in isolation of L . donovani promastigotes from kala-azar patients, in addition to luxurious growth of parasites . The medium is simple, reliable, reproducible and convenient, with minimal interference in using the parasitic cells for immunological, molecular and isoenzyme studies.

Exp Eye Res, 1994 Apr, 58(4), 439 - 51
Modulation of human fibroblast activity by selected angiogenesis inhibitors; Wong J et al.; Tenon's fibroblast (TF) bleb encapsulation is a common cause of filtering surgery failure . Various pharmacologic agents have been used in an attempt to inhibit this response . The effects of three angiogenesis inhibitors were studied . AGM-1470, a fumagillin analog and hydrocortisone 21-phosphate (HC21P) complexed with heparin or with the heparin analog beta-cyclodextrin tetradecasulfate (BCD-TDS) in modulating human tenon's fibroblasts in cell culture . It has been clinically demonstrated that well vascularized blebs are associated with a poorer prognosis . In addition to reducing bleb neovascularization, angiogenesis inhibitors may have an additional therapeutic role in decreasing fibroblast activity . Drug effects were assessed by studying cell growth rates by cell Coulter counting and rate of wound closure . TF's were grown in DMEM with 10% FBS and 1% PCN-strep with fungizone . Angiogenesis inhibitors were added to growth medium in varying concentrations: AGM-1470 alone, HC21P complexed with heparin and HC21P complexed with BCD-TDS . Controls were grown in control medium alone, medium with added ethanol, or with added BCD-TDS, heparin or HC21P . We found a dose related inhibition of cell growth with all of the angiogenesis inhibitor combinations, which was not seen in the control groups . Tenon's fibroblast proliferation was significantly inhibited by AGM-1470 as seen in the four higher concentrations (P < 0.05) with insignificant inhibition at the lowest concentration of AGM-1470 (P = 0.38) . The heparin:HC21P and heparin:BCD-TDS combinations demonstrated significant inhibition at all concentrations (P < 0.05) . Wound closure was significantly inhibited by all the added agents, except AGM-1470 and the controls . The rate of wound closure was significantly reduced by the highest concentrations of the heparin:HC21P and heparin:BCD-TDS combinations (P < 0.05), although it was not significantly affected by lower concentrations (P > 0.05) . Rank order of potencies of these agents for inhibition of TF proliferation was AGM-1470, BCD-TDS:HC21P, heparin:HC21P, HC21P, while the rank order of potencies for these agents for inhibition of wound closure was HC21P, heparin:HC21P, BCD-TDS:HC21P, AGM-1470 . These selected angiogenesis inhibitors appear to have marked inhibitory effects on TF proliferation and migration, which may have a potential clinical role in modulating wound healing associated with glaucoma filtering surgery.

J Biol Chem, 1994 Mar 25, 269(12), 9011 - 8
Cytosolic Ca2+ transients are not required for platelet-derived growth factor to induce cell cycle progression of vascular smooth muscle cells in primary culture . Actions of tyrosine kinase; Kobayashi S et al.; We investigated interrelations among changes in cytosolic Ca2+ concentrations ({Ca2+}i), tyrosine phosphorylation, and the cell cycles of rat aorta smooth muscle cells in primary culture, as stimulated with platelet-derived growth factor (PDGF) . Changes in {Ca2+}i were monitored using the microfluorometry of Fura-2 . The phase of the cell cycle and the extent of tyrosine phosphorylation were examined by immunocytochemical analysis of monoclonal antibodies against cell cycle-specific nuclear antigens and against phosphotyrosine, respectively . Prior to the application of PDGF, the cell cycle was synchronized in the G0 phase by serum deprivation for 24 h . In the presence of extracellular Ca2+, PDGF induced an initial transient (first component) and a subsequent lower steady-state (second component) elevation of {Ca2+}i . NiCl2 and the removal of extracellular Ca2+ inhibited the second, but not the first, component . The first component was inhibited by pretreatment with ryanodine . These results are compatible with the notion that the first and second components may be mediated mainly through the release of intracellular Ca2+ and the influx of extracellular Ca2+, respectively . After pretreatment with ryanodine and in the presence of NiCl2, PDGF also stimulated the entry of G0 cells into G1 phase, but there were no {Ca2+}i transients . Genistein, a tyrosine kinase blocker, inhibited tyrosine phosphorylation induced by PDGF and blocked the first, but not the second, component of {Ca2+}i elevation induced by PDGF . However, genistein did not inhibit the release of intracellular Ca2+ induced by angiotensin II or by caffeine . Genistein prevented G0 cells from entering the G1 phase, as induced by PDGF, but this was not the case when serum was reapplied to the growth medium . Similar results were obtained with another tyrosine kinase blocker, tyrphostin . These data suggest that in vascular smooth muscle cells: 1) an increase in {Ca2+}i is not required for competent (G0 to G1) cell proliferation induced by PDGF; and 2) tyrosine kinase plays an important role in the release of intracellular Ca2+ and in cell proliferation, as induced by PDGF.

Int J Cancer, 1994 Mar 1, 56(5), 766 - 71
Effect of dibutyryl cyclic AMP on morphologic features and marker production of human cervical argyrophil small-cell carcinoma cell line; Shimizu H et al.; The effect of dibutyryl cyclic AMP (dB-cAMP) on the morphologic features and marker production of a human cervical argyrophil small-cell carcinoma (ASCC) cell line was examined . Following 1-5 days' exposure to 5 mM dB-cAMP, morphologic differentiation as defined by the expression of cytoplasmic processes (stellate cells) was observed . The number of stellate cells depended on the dose of dB-cAMP and incubation time . Shortly after removal of dB-cAMP from the culture medium, the treated cells returned to their original spherical shape . dB-cAMP caused a reduction in the growth rate of cells which recovered after removal of the agent . The morphological changes appeared not to be the result of growth inhibition by dB-cAMP, because the cells maintained in a serum-free medium did not show any change in shape . Electron microscopic study revealed the development of intracytoplasmic microtubules, microfilaments, and an increase in the number of neurosecretory granules in the treated cells . The levels of neuron-specific enolase, serotonin and gastrin in treated cells were significantly higher than those in untreated controls . These findings indicate that a reversible differentiation of cultured ASCC cells into neuroendocrine cells occurs in a growth medium containing dB-cAMP.

J Anim Sci, 1994 Mar, 72(3), 537 - 45
Effect of the addition of live yeast (Saccharomyces cerevisiae) on growth and carcass quality of steers fed high-forage or high-grain diets and on feed digestibility and in situ degradability; Mir Z et al.; The effects of adding yeast culture (Saccharomyces cerevisiae, 5 x 10(9) live organisms/g of growth medium) at 10 g/steer daily to three diets consisting of 75% alfalfa silage and 25% barley, 96% corn silage and 4.0% soybean meal, or 75% dry-rolled barley and 25% alfalfa hay on performance of growing and finishing steers and carcass characteristics, feed digestibility, and degradability in the rumen were determined . In separate trials over 2 yr, the performance of steers fed the three diets with no supplementary live yeast (control; C) or with live yeast (Y) was determined using 72 Hereford steer calves in eight pens each year in a randomized complete block design . The diets were fed sequentially and the steers were randomized before diets were switched . Each year at the end of the high-grain feeding segment of the trial, the steers were slaughtered when ultrasound backfat was such that the carcass would grade A1 or A2 (Canadian grades) . The digestibility of feed DM, CP, and NDF of the three diets was determined sequentially using six and eight mature, ruminally cannulated steers in 2 yr, respectively . The degradation characteristics of the three diets in the rumen were determined using the nylon bag technique in two steers for each additive treatment . In the 1st yr, the feed efficiency of the corn silage diet was lower (P < .05) for C than for Y steers (5.9 vs 6.8) . Differences (P < .05) were not observed between C and Y steers for any of the performance or carcass characteristics.(ABSTRACT TRUNCATED AT 250 WORDS)

J Bacteriol, 1994 Mar, 176(6), 1695 - 701
Effects of nitrate respiration on expression of the Arc-controlled operons encoding succinate dehydrogenase and flavin-linked L-lactate dehydrogenase; Iuchi S et al.; Expression of sdhCDAB (encoding succinate dehydrogenase) and lctD (encoding the flavin-linked L-lactate dehydrogenase) is elevated aerobically and repressed anaerobically in Escherichia coli . The repression is initiated by autophosphorylation of the sensor protein ArcB, followed by phosphoryl group transfer to the regulator ArcA . ArcA-P, a global transcriptional regulator, then prevents sdh and lct expression . The stimulus for ArcB is not O2 deficiency per se . In vitro experiments showed that ArcB phosphorylation is enhanced by pyruvate, D-lactate, acetate, and NADH, the concentrations of which are likely to increase with the lack of an effective exogenous electron sink . In addition to their aerobic function, the two primary dehydrogenases also have roles in anaerobic nitrate respiration . Results presented here indicate that the increase of sdh and lct expression by nitrate depended on its chemical reduction, which in turn diminished the ArcA-P pool . Unexpectedly, a mutation in the fnr gene (encoding a global regulator involved in anaerobic metabolism) also alleviated the anaerobic repressions . Mutations in arcB or arcA were epistatic over that of fnr . Moreover, since this relief was counteracted by pyruvate in the growth medium, Fnr appears to affect formation of stimuli for ArcB . It is possible that Fnr also indirectly affects some of the other members of the arcA modulon, e.g., cyoABCDE (encoding the cytochrome o complex), cydAB (encoding the cytochrome d complex), and sodA (encoding the manganese-dependent superoxide dismutase).

Endocrinology, 1994 Mar, 134(3), 1297 - 304
The effect of pertussis toxin on the growth of vascular smooth muscle cells stimulated by serum or platelet-derived growth factor; Zhang LM et al.; The involvement of a Gi- or G(o)-related G-protein as a regulator of the growth of guinea pig thoracic aorta smooth muscle (TASM) cells was studied by investigating the effects of pertussis toxin (PTX) on the growth of these cells . PTX treatment decreased the growth rate of TASM cells by 70-100% . This effect was apparent within 24 h after exposure to the toxin and persisted for at least 10 days after starting the treatment . The effect of the toxin appeared to be the result of the inactivation of a G-protein because 1) TASM cell membranes contained a 40-kilodalton substrate for the toxin in in vitro assays that was absent in membranes prepared from cells pretreated with toxin; and 2) the effect required both the enzymatic component (A-protomer) of the toxin that inactivates Gi/G(o)-related G-proteins and its B-oligomer necessary for binding and internalization of the A-protomer . The effect of the toxin was not due to an increased level of intracellular cAMP brought about by inactivation of a G-protein that normally inhibits adenylyl cyclase activity . Further, the toxin did not merely make some unknown mitogen rate limiting, because neither increasing concentrations of serum in the growth medium nor supplementation with platelet-derived growth factor could overcome its inhibition of TASM cell growth . Instead, some unknown process regulated by a PTX-sensitive G-protein appears to be required for the normal growth of these cells.

Blood, 1994 Mar 1, 83(5), 1216 - 25
Abnormal lymphokine production: a novel feature of the genetic disease Fanconi anemia . II . In vitro and in vivo spontaneous overproduction of tumor necrosis factor alpha; Rosselli F et al.; We have previously shown an unbalanced cytokine production in Fanconi anemia (FA) cells, ie, an underproduction of interleukin 6 (IL-6) during growth . Among a number of cytokines analyzed, the only other anomalies detected concern tumor necrosis factor alpha (TNF alpha) . In comparison to normal cells, this cytokine is overproduced by FA lymphoblasts from the four genetic complementation groups . Indeed, up to an eight-fold increase in TNF alpha is observed in the growth medium of FA cells . Moreover, addition of anti-TNF alpha antibodies partially corrects the FA hypersensitivity to treatment by mitomycin C (MMC) . Treatment of FA cells with IL-6, which partially restored an almost normal sensitivity to MMC of FA cells also reduces the TNF alpha overproduction in FA lymphoblasts . No anomalies at the molecular level (Southern and Northern blot analyses) are detected for the TNF alpha gene and its mRNA . We have investigated the in vivo situation by assaying TNF alpha levels in the serum from FA homozygotes and obligate heterozygotes . In contrast to normal healthy donors or to aplastic anemia patients in whom serum TNF alpha is present only in trace amounts, all 36 FA patients and 21 FA parents monitored show a significantly (P < .001) higher level of serum TNF alpha activity . Consequently, abnormal TNF alpha production seems to be associated with the FA genetic background.

FEMS Immunol Med Microbiol, 1994 Mar, 8(3), 259 - 69
Tracheobronchial washings from seven vertebrate species as growth media for the four species of Bordetella; Porter JF et al.; The four species of Bordetella differed in their ability to grow at 37 degrees C in membrane-filtered tracheobronchial washings (TBW) from seven vertebrate species, including their natural hosts . From washed inocula of approximately 2 x 10(3) colony-forming units per ml (cfu ml-1), Bordetella bronchiseptica and B . avium grew much better than the other two bordetellae and yielded stationary-phase cultures containing 10(8)-10(9) cfu ml-1 in most of the TBW samples . These counts were only moderately lower than those attained in CL medium which contains about a 450-times higher concentration of amino acids . B . bronchiseptica and B . avium also grew to a limited extent in phosphate-buffered saline without nutrient supplements . B . parapertussis grew in TBW from man, sheep, rabbit, mouse and chicken, but not in TBW from a dog and a horse or in PBS . B . pertussis grew well in CL medium, but not in PBS or in any of 13 samples of TBW from the seven vertebrate species, which included three samples of lung lavage fluid from human patients . Analysis of the TBW samples for known Bordetella nutrients revealed concentrations of amino acids and nicotinic acid averaging 0.35 mM and 0.56 microgram ml-1 respectively.

Southeast Asian J Trop Med Public Health, 1994 Mar, 25(1), 96 - 101
Isolation of HIV-1 from Filipino commercial sex workers (CSWs); Torres CA et al.; Peripheral blood mononuclear cells (PBMC) from 36 HIV-1 antibody positive Filipino female commercial sex workers (CSWs) were co-cultivated at a 1:1 ratio with phytohemagglutinin-P activated PBMC from healthy, HIV-1 antibody negative donors . After 3-18 (mean 7.2) days of incubation at 37 degrees C in 5% CO2, 29 cultures showed evidence of replication of HIV-1: increasing concentrations of p24 antigen in the growth medium and the appearance of multinucleated giant cells . Although the length of incubation required for the appearance of cytopathogenic effect for each particular isolate was essentially the same when either 6 microwell plates were seeded with 3.0 x 10(6) cells/well or 24 well plates were seeded with 1.5 x 10(6) cells/well, the 24 well format was more sensitive . The ability to isolate HIV-1 from PBMC did not appear to be associated with the progression of disease or the presence or absence of any specific clinical findings . However, if the PBMC were from individuals with a concomitant p24 antigenemia, the incubation time required for isolation was significantly shorter (mean 3.8 days) . The absolute CD4+ lymphocyte count was also slightly reduced in the culture positive, p24 antigenemic patients (range 302-813 cells/mm3, mean 502 cells/mm3) compared to the culture positive, p24 serum negative cases (range 311-1,511 cells/mm3, mean 830 cells/mm3) . The p24 serum negative cases with CD4+ counts of < 500 cells/mm3 had positive PBMC cultures by 6 days of incubation.(ABSTRACT TRUNCATED AT 250 WORDS)

Biodegradation, 1994 Mar, 5(1), 47 - 54
Physiological properties and substrate specificity of a pentachlorophenol-degrading Pseudomonas species; Resnick SM et al.; A bacterial strain capable of utilizing pentachlorophenol (PCP) as sole source of carbon and energy for growth was isolated from enrichment cultures containing 100 mg/l PCP in a mineral salts medium inoculated with contaminated soil from a lumber treatment waste site . The isolate, designated strain SR3, was identified as a species of Pseudomonas by virtue of its physiological and biochemical characteristics . Mineralization of PCP by Pseudomonas sp . strain SR3 was demonstrated by loss of detectable PCP from growth medium, stoichiometry of chloride release (5 equivalents of chloride per mole of PCP), and formation of biomass consistent with the concentration of PCP mineralized . PCP-induced cells of strain SR3 showed elevated rates of oxygen consumption in the presence of PCP, and with different chlorinated phenols, with complete degradation of 2,3,5,6-, 2,3,6-, 2,4,6-, 2,4-, and 2,6-chloro-substituted phenols . Concentrations of PCP up to 175 mg/liter supported growth of this organism, but maximal rates of PCP removal were observed at a PCP concentration of 100 mg/liter . Based on its degradative properties, Pseudomonas sp . strain SR3 appears to have utility in bioremediation of soil and water contaminated with PCP.

Cancer Lett, 1994 Feb 28, 77(1), 57 - 63
Release of intracellular calcium and stimulation of cell growth by ATP and histamine in human ovarian cancer cells (SKOV-3); Batra S et al.; The effects of ATP and histamine on cell proliferation and intracellular calcium concentrations ({Ca2+}i) were examined in the human ovarian cancer cell line SKOV-3 . Micromolar concentrations of ATP induced a biphasic increase in {Ca2+}i representing a rapid rise to a peak level followed by a smaller but more sustained phase . When influx of extracellular calcium was blocked by calcium chelation to EGTA, the ATP-stimulated rise in {Ca2+}i was rapid and only monophasic . Histamine, in contrast to ATP, caused only a monophasic rise in {Ca2+}i both in the presence and absence of external calcium . The histaminergic H1 receptor antagonist pyrilamine, but not the H2 receptor antagonist cimetidine, totally blocked rises in {Ca2+}i caused by histamine . Fetal calf serum (FCS) induced a slow and monophasic increase in {Ca2+}i in these cells, distinctly different from rises in {Ca2+}i caused by ATP and histamine . Inclusion of low micromolar concentrations of ATP in the growth medium stimulated proliferation of these cells, while higher concentrations (100 microM-1 mM) significantly decreased cellular proliferation . Histamine, in micromolar concentrations, also stimulated cell proliferation . From these results it was concluded that the release of intracellularly bound Ca2+ following receptor stimulation is sufficient to induce cellular proliferation in SKOV-3 cells.

J Biol Chem, 1994 Feb 18, 269(7), 5036 - 42
Photoautotrophic growth of the cyanobacterium Synechocystis sp . PCC 6803 in the absence of cytochrome c553 and plastocyanin; Zhang L et al.; In the cyanobacterium Synechocystis sp . PCC 6803, photosynthetic electron transport from the cytochrome bf complex to photosystem I can be mediated by cytochrome c553 or plastocyanin . The concentration of copper in the growth medium determines which protein is synthesized . To investigate the role of cytochrome c553 in photosynthetic and respiratory electron transport, we cloned the petJ gene encoding cytochrome c553 from Synechocystis 6803 and determined its nucleotide sequence . The 360-base pair open reading frame encodes an 85-amino acid mature protein (predicted molecular mass = 8,742 Da) with a 35-amino acid presequence . Two mutants were constructed, one in which the petJ gene for cytochrome c553 was deleted and another in which the petE gene for plastocyanin was insertionally inactivated . The cytochrome c553 deletion mutant (M109) grew photoautotrophically, even in the absence of copper that prevented the synthesis of plastocyanin, as did the plastocyanin-deficient mutant (M114) grown in the presence of copper that prevented the synthesis of cytochrome c553 . The M109 strain exhibited photosynthetic electron transport rates similar to those of wild-type cells when grown under conditions that prevented the synthesis of plastocyanin . Moreover, in M109 cells grown without copper, cytochrome f was completely photooxidized in less than 10 ms by photosystem I . These observations show that electrons can be transferred from the cytochrome bf complex to photosystem I in the absence of both cytochrome c553 and plastocyanin . Additionally, the M109 cells exhibited dark respiration rates comparable with those of wild-type cells, indicating that cytochrome c553 is not obligately required for respiratory electron transport in Synechocystis 6803.

J Bacteriol, 1994 Feb, 176(3), 665 - 72
A new gene required for cellulose production and a gene encoding cellulolytic activity in Acetobacter xylinum are colocalized with the bcs operon; Standal R et al.; Recently, it was shown that a cellulose-negative mutant (Cel1) of Acetobacter xylinum ATCC 23769 carried an insertion of an indigenous transposable element (IS1031A) about 500 bp upstream of the bcs operon, required for cellulose synthesis . Here we show that Cel1 can be complemented by wild-type DNA covering the insertion point . Nucleotide sequencing of this region revealed the presence of two open reading frames, ORF1 and ORF2 . ORF2, which is disrupted by the IS1031A insertion in Cel1, potentially encodes the complementing function . ORF1 encodes a protein (CMCax) with significant homology to previously described endoglucanases . A cloned DNA fragment containing ORF1 expressed a carboxymethyl cellulose-hydrolyzing activity in Escherichia coli . In A . xylinum, CMCax is secreted into the culture growth medium . The CMCax mature protein consists of 322 amino acids and has a molecular mass of 35.6 kDa.

Exp Cell Res, 1994 Feb, 210(2), 287 - 97
Localization of protein kinase C isozymes in cardiac myocytes; Disatnik MH et al.; Activation of protein kinase C (PKC) isozymes is associated with their translocation from the cell-soluble fraction to the cell-particulate fraction, presumably near their protein substrates . Therefore, identifying the subcellular localization of each activated PKC isozyme may help to elucidate its role in cardiac functions . In the present work, we have determined the subcellular localization of six PKC isozymes (alpha, beta I, beta II, delta, epsilon, and zeta) in nonstimulated cardiac myocytes and in myocytes stimulated by norepinephrine (2 microM) or phorbol 12-myristate 13-acetate (100 nM) . Activated PKC isozymes were localized in various subcellular compartments such as inside the nucleus and on myofibrils . The presence of serum in the growth medium also caused a redistribution of PKC isozymes in the cells distinct from that obtained with cells cultured in defined medium . We suggest that isozyme-specific localization may determine phosphorylation of different protein substrates present at these respective translocation sites and the resulting PKC-mediated cellular responses.

Br J Cancer, 1994 Feb, 69(2), 264 - 73
Conserved cis-elements bind a protein complex that regulates Drosophila ras2/rop bidirectional expression; Lightfoot K et al.; The Drosophila ras2 promoter region exhibits bidirectional activity, as has been demonstrated for the human c-Ha-ras1 and the mouse c-Ki-ras . Here we address a unique case of ras regulation, as Drosophila ras2 provides the only example to date in which the flanking gene (rop) and its product have been isolated . A linking mechanism of control suggests a mutual interaction between the two gene products . Our studies indicate that the Drosophila ras2 promoter region shares with the human c-Ha-ras1 promoter a CACCC box and an AP-1-like sequence . A 14 bp promoter fragment which holds a CACCC element is demonstrated to interact with a specific transcription factor (factor B) . This CACCC promoter element represents a stretch of imperfect palindrome . We present evidence that this factor can form a complex with another specific DNA-binding protein (factor A) . The binding sites (A + B) for these protein factors are essential for 95% expression of both genes flanking the promoter (ras2 and rop) . Region A consists of four overlapping consensus sequences: a TATA-like element, a DSE-like motif (the core sequence of the serum response element), a DRE octamer, which has been shown to play a role in cell proliferation, and a 5 bp direct repeat representing the GATA consensus sequence . Factor A has a very weak affinity to the full promoter region, but when complexed with factor B binding efficiency is enhanced . We also show that alterations of DNA-protein binding specificities can be achieved by supplementing the growth media with different sera.

Mol Microbiol, 1994 Feb, 11(4), 777 - 85
An ABC-transporter from Streptomyces longisporoflavus confers resistance to the polyether-ionophore antibiotic tetronasin; Linton KJ et al.; Streptomyces longisporoflavus produces the polyketide-polyether antibiotic, tetronasin, which acts as an ionophore and depolarizes the membrane of bacteria sensitive to the drug . A genomic library of S . longisporoflavus DNA was cloned in Streptomyces lividans and screened to identify tetronasin-resistance determinants . The inclusion of 0.2M NaCl in the growth medium with tetronasin markedly improved the sensitivity of the screen . Two different resistance determinants, designated tnrB (ptetR51) and tnrA (ptetR11) respectively, were identified . The determinant tnrB (ptetR51) but not tnrA (ptetR11), also conferred resistance to tetronasin when cloned into Streptomyces albus . The tnrB determinant was further localized, by subcloning, to a 2.8 kb KpnI fragment . DNA sequence analysis of this insert revealed one incomplete and two complete open reading frames (ORFs 1, 2 and 3) . The deduced sequence of the gene product of ORF2 (TnrB2) revealed significant similarity to the ATP-binding domains of the ABC (ATP binding cassette) superfamily of transport-related proteins . The adjacent gene, ORF3, is translationally coupled to ORF2 and would encode a hydrophobic protein (TnrB3) with six transmembrane helices which probably constitutes the integral membrane component of the transporter . The mechanism of tetronasin resistance mediated by tnrB is probably an ATP-dependent efflux system.

Anticancer Drugs, 1994 Feb, 5(1), 99 - 104
Effects of 5-fluorouracil on collagen synthesis in the developing palate of hamster; Ben-Khaial GS et al.; A study was undertaken to examine the effect of 5-fluorouracil (5-Fu) on collagen synthesis in the developing secondary palate . Pregnant hamsters were given 81 mg/kg 5-FU intramuscularly or 1 ml saline on day 11 of gestation . Control and treated embryonic palates, dissected from hamsters between days 11 and 13 of gestation, were incubated in a growth medium supplemented with {14C}proline . The rate of collagen synthesis, total protein and collagen isotypes were determined . The data showed that in control hamster palate the collagen synthesis peaked between days 12:00 (12 day: 0 h) and 12:04 of gestation, which is the period of shelf reorientation . In 5-FU-exposed hamster palates, the rate of collagen synthesis was lower than controls until day 12:04 of gestation followed by a rise on day 12:12 of gestation . In 5-FU-treated embryos palatal shelf reorientation took place between days 12:16 and 13:00 of gestation . Electrophoresis showed that only type I collagen was synthesized during palate development in both the control and 5-FU-treated hamster embryos . It was suggested that collagen synthesis may play a critical role in shelf reorientation in hamster since (i) new collagen was synthesized in both the control and 5-FU-treated hamster embryos prior to and during the period of normal reorientation, and (ii) in 5-FU-treated hamster embryos, a recovery in collagen synthesis precedes reorientation . Inhibition of collagen synthesis during abnormal development is only a step in the cascade of events of 5-FU-induced effects on protein synthesis.

Endocr Res, 1994 Feb, 20(1), 21 - 37
The presence of a human chorionic gonadotropin-like protein and its binding site in Saccharomyces cerevisiae; Caticha O et al.; In this study, we characterize from Saccharomyces cerevisiae: 1) a protein that has immunological similarities to human chorionic gonadotropin (hCG), and 2) a binding site for this hCG-like protein which also binds hCG and human luteinizing hormone (hLH) . Saccharomyces cerevisiae chorionic gonadotropin-like protein (ScCGLP) was purified in several steps . This protein when analyzed by SDS-PAGE, under nondenaturing conditions, produced two bands, one at 110-kDa, and another at 57.5-kDa . Under denaturing conditions, only the 57.5-kDa band appeared . This 57.5-kDa band also reacted in a Western blot, using a polyclonal antibody directed against hCG . Purified ScCGLP reacted in the following hCG immunoassays: 1) polyclonal rabbit anti-hCG equilibrium assay, 2) carboxyl-tail hCG equilibrium assay, 3) two equilibrium assays using monoclonal antibodies, and 4) free alpha-chain subunit equilibrium assay using a monoclonal antibody . Characterization of hCG/hLH binding sites in Saccharomyces cerevisiae was performed, and the ability of the ScCGLP to displace I125-hCG was also shown . Human CG and hLH were able to compete for I125-hCG binding to Saccharomyces cerevisiae blastospores (Kds of approximately 10(-8) M), while ScCGLP competed with higher affinity (Kd = 9.41 x 10(-10) M) . The hCG-like immunoactivity was also present in Saccharomyces growth media, as well as in all brands of commercial beer studied.

Mol Carcinog, 1994 Feb, 9(2), 76 - 86
Combined effects of human papillomavirus-18 and N-methyl-N'-nitro-N-nitrosoguanidine on the transformation of normal human oral keratinocytes; Shin KH et al.; We immortalized oral keratinocytes by transfecting them with recombinant human papillomavirus (HPV) type 18 DNA and established three cell lines . These lines were morphologically different from their normal counterpart, contained integrated entire HPV-18 DNA, and expressed the viral E6/E7 genes . The cells contained less p53 protein and more c-myc mRNA than normal cells . However, they proliferated only in keratinocyte growth medium (KGM) containing low calcium and were not tumorigenic in nude mice . To test the hypothesis that tumors result from the combined effect of a "high-risk" HPV and chemical carcinogens in the human oral cavity, we exposed the immortalized cells to the chemical carcinogen N-methyl-N'-nitro-N-nitrosoguanidine . Three chemically transformed cell colonies were isolated . These cells (a) proliferated well in both KGM and Dulbecco's modified minimum essential medium containing physiological levels of calcium; (b) were capable of proliferating in nude mice; (c) contained intact, integrated HPV-18 sequences; (d) transcribed substantially more HPV-18 E6/E7, transforming growth factor-alpha, and c-myc than the immortalized counterpart; and (e) contained, like the immortalized counterpart, less wild-type p53 protein and DCC message . These data indicate that human oral keratinocytes can be transformed by sequential exposure of normal keratinocytes to a "high-risk" HPV and chemical carcinogens.

Toxicol Appl Pharmacol, 1994 Feb, 124(2), 212 - 20
Transgenerational, ultrastructural analysis on the antioxidative effects of tocopherol on early gametogenesis in Caenorhabditis elegans grown in 100% oxygen; Goldstein P et al.; The random, free-radical-mediated oxidations of biological molecules result in membrane degradation leading to cellular deterioration (B . Halliwell, Free Radical Res . Commun . 9, 1-32, 1990) . External oxygen, prooxidants, and internally produced oxygen free radicals (oxyradicals), interact and alter the nature of biomembranes . Antioxidants, e.g., tocopherol (Vitamin E), inhibit such oxidative damage of free radicals . In the present study, the nematode Caenorhabditis elegans was grown under hyperoxia (100% oxygen) with or without the addition of Vitamin E to the growth media . The nematodes were viable under such conditions for at least eight generations, although fecundity gradually decreased through successive generations, presumably due to genetic load . Vitamin E was also shown to have a protective effect against paraquat, which is a strong, intracellular, oxidizing agent . Ultrastructural observations of early meiosis showed that the formation of synaptonemal complexes was compromised and that the telomeres failed to attach to the nuclear envelope . Those nematodes grown in 100% oxygen with 200 micrograms/ml Vitamin E had normal meiotic structures and normal fecundity . Thus, the presence of enhanced levels of intracellular Vitamin E resulted in protection against oxidative stress during gametogenesis.

J Cell Physiol, 1994 Feb, 158(2), 299 - 308
Invadopodia promote proteolysis of a wide variety of extracellular matrix proteins; Kelly T et al.; Chicken embryo fibroblasts (CEF) transformed by Rous sarcoma virus invade the extracellular matrix (ECM) using plasma membrane protrusions, termed invadopodia, that contact and dissolve the matrix . Normal cells neither form invadopodia nor degrade the ECM . Here we show that cells expressing invadopodia degrade and enter into a fibronectin-rich matrix produced by normal fibroblasts . Within 6 h after seeding onto the matrix, the invasive cells create an area devoid of matrix fibrils surrounding the cell body . Proteolysis mediates this matrix clearing because sevenfold more radiolabeled matrix is released into the growth media by the transformed cells relative to the normal cells . In addition to this assembled matrix, transformed cells were grown on thin layers of purified ECM proteins, revealing that invadopodia can degrade fibronectin, collagen type I, collagen type IV, and laminin . A 160 kDa protease that is extracted from transformed cells by Triton X-114 partitions into the detergent phase and is prominent in ventral plasma membranes that contact the ECM suggesting that it is a membrane associated protease.

Biochem Mol Biol Int, 1994 Feb, 32(2), 251 - 8
Cell density-dependent regulation of ATP levels during the growth cycle of cultured Chinese hamster ovary cells; Fiorani M et al.; Chinese hamster ovary (CHO) cells show a cell density-dependent modification of ATP levels during the growth cycle . Cells were seeded at a density of 500,000 cells/75 cm2 flask in 10 ml of growth medium and at various time intervals, samples were taken and assayed for cell number, for adenine (ATP, ADP, AMP) and pyridine (NAD+, NADP+) nucleotide levels and for the activity of some glycolytic enzymes . Glucose consumption was also evaluated . Experimental results indicated that the rate of cell growth was exponential for up to the 4th day of culture after which the cell number remained pratically unchanged up to the 9th day . Under these experimental conditions we found that, whereas the intracellular levels of NAD+ and some glycolytic enzymes were not significantly affected, a drop in ATP content was apparent after 48 hr of culture . The decline in ATP levels progressively increased, reaching a maximum after 4 days of culture, and then remained unchanged . In order to evaluate whether this effect on ATP was determined by a reduced availability of nutritional factors or was really a function of cell density, we also performed experiments similar to those reported above, with the exception that the cells were grown in 40 ml of culture medium . Under these experimental conditions, the exponential growth was longer (in comparison with the cell growth in 10 ml of medium) and a plateau was reached after 6 days of culture.(ABSTRACT TRUNCATED AT 250 WORDS)

Exp Hematol, 1994 Feb, 22(2), 208 - 14
Identification of cytidine deaminase as inhibitor of granulocyte-macrophage colony formation; Boyum A et al.; Mature human blood granulocytes produce regulatory factors that inhibit colony formation by human and murine granulocyte-macrophage colony-forming cells (GM-CFC) . The inhibition of GM-CFC by granulocyte extract (GRE) was strongly enhanced by the addition of thymidine (3 to 6 x 10(-5) M for human cells) and by the presence of fetal calf serum (FCS) in the growth medium . Deoxycytidine and deoxyuridine produced effects similar to those of thymidine, but at higher concentrations (2 to 4 x 10(-4) M) . It was further observed that GRE prevented the antiproliferative effects of cytosine arabinoside (Ara-C) and azadeoxycytidine, suggesting that GRE contained cytidine deaminase (CDD) activity, since CDD is known to abolish the effects of these nucleoside analogs . Accordingly, the GRE was tested for and shown to contain an enzymatic activity that converted deoxycytidine to deoxyuridine, confirming the presence of CDD activity in GRE . The GM-CFC inhibition factor was found to copurify with CDD activity during three succeeding chromatographic separations, indicating that CDD was indeed the inhibiting factor itself . This conclusion was further substantiated by gel filtration experiments demonstrating a molecular weight (MW) of approximately 50 kd, which corresponds to the MW previously published for CDD activity . Furthermore, addition of tetrahydrouridine (THU), a known specific inhibitor of CDD, abolished the suppressive effect of GRE on GM-CFC, which independently confirmed the identification of CDD as an inhibitor of GM-CFC . The growth-regulating property of CDD could be explained by depletion of deoxycytidine nucleotides necessary for DNA synthesis or by a direct effect of CDD binding to specific receptors on progenitor cells.

Neurosci Lett, 1994 Jan 31, 166(2), 178 - 82
Oligodendrocyte-type-2 astrocyte (O-2A) progenitors increase the survival of rat mesencephalic, dopaminergic neurons from death induced by serum deprivation; Takeshima T et al.; When a primary culture of E16 rat striatal cells was grown in a serum-free medium, treatment with basic fibroblast growth factor (bFGF, 10 ng/ml) caused the generation of the progenitor cell for oligodendrocytes and type-2 astrocytes (O-2A) . Immunostaining tests confirmed that > 90% of the cells were positive for A2B5, and < 5% positive for glial fibrillary acidic protein (GFAP) . When E14, mesencephalic, dopaminergic neurons were co-cultured with established O-2A progenitor cells in a serum-free growth medium, the survival of tyrosine hydroxylase-positive (TH+) neurons increased 23-fold and 668-fold at the 5th and 10th days, respectively, compared with control cultures plated on poly-D-lysine . Conditioned medium from the O-2A progenitor cultures also decreased the death of TH+ neurons . The mitotic inhibitor, cytosine arabinoside (1.0 microM), did not block the protective effect of the O-2A progenitor cells . O-2A progenitor cells produce a potent, soluble factor, that mediates the increased survival of dopaminergic neurons in vitro.

Blood, 1994 Jan 15, 83(2), 580 - 6
Elimination of the O-linked glycosylation site at Thr 104 results in the generation of a soluble human-transferrin receptor; Rutledge EA et al.; The transferrin receptor (TfR) is the plasma membrane protein responsible for the binding and internalization of the major iron-transport protein, transferrin . The function of the single O-linked oligosaccharide near the transmembrane domain of the TfR at amino acid Thr 104 is unknown . To elucidate the effect of the O-linked carbohydrate on TfR function, the oligosaccharide was eliminated by replacing Thr 104 with Asp and the mutated cDNA was expressed in a cell line lacking endogenous TfR . Elimination of the oligosaccharide at Thr 104 results in a form of the receptor that is susceptible to cleavage . A 78-kD soluble TfR that can bind transferrin is released into the growth medium . The intact mutant TfR is not grossly altered in its structure and does not differ significantly from the wild-type human receptor in many respects: (1) It shows the same distribution between the plasma membrane and intracellular compartments; (2) the binding constant for transferrin is similar to that of the wild-type TfR; and (3) it is not rapidly degraded . Protein-sequence analysis of the soluble form indicates that the sequence begins at amino acid 101 of the intact receptor . This is the same cleavage site reported for a soluble form of normal receptor found in human serum . Substitution of Gly, Glu, or Met at position 104 also results in increased cleavage of the TfR and suggests that elimination of the O-linked carbohydrate at position 104 enhances the susceptibility of TfR to cleavage and may mimic a naturally occurring process previously described as being related to erythropoiesis.

FEMS Microbiol Lett, 1994 Jan 15, 115(2-3), 163 - 7
Alternatives to arginine as energy sources for the non-fermentative Mycoplasma gallinarum; Taylor RR et al.; In contrast to previously studied non-fermentative arginine-hydrolysing (F-/A+) Mycoplasma species, M . gallinarum cells suspended in a salts solution oxidised ethanol and L-lactic, pyruvic and 2-oxobutyric acids . The organic acids were additionally shown effectively to replace arginine as energy sources in growth media . However, their presence did not inhibit arginine hydrolysis, nor did arginine inhibit organic acid catabolism . The ability to oxidise organic acids is a potentially useful diagnostic character enabling sub-division of the F-/A+ Mycoplasma species . M . gallinarum also differed from previously studied F-/A+ mycoplasmas in possessing relatively high NADH oxidase activity and producing H2O2 as only a minor product of NADH oxidation.

Biochim Biophys Acta, 1994 Jan 13, 1220(2), 139 - 45
Regulatory volume decrease of cultured human fibroblasts involves changes in intracellular amino-acid pool; Dall'Asta V et al.; Regulatory volume decrease (RVD) has been studied in cultured human fibroblasts incubated in a complete growth medium at low osmolality (215 mosmolal) . After the initial swelling induced by hypotonic treatment, cells recover their volume almost completely within about 60 min . This RVD is associated with comparable losses of cell potassium and amino acids . After an initial increase, cell content of sodium is kept at values close to control . Chromatographic analysis of intracellular amino-acid pool has shown that RVD-associated decrease in cell amino acids is due for the most part to changes in the intracellular concentration of L-glutamine . RVD-exerting cells undergo a rapid and marked depolarization that is maintained after cell volume recovery . This change in membrane potential has been detected with measurements of both the transmembrane distribution ratios of L-arginine and of fluorescence of potential-sensitive dye bis-oxonol . Due to depolarization, the trans-membrane gradient of sodium electrochemical potential is lowered . It is proposed that cell depolarization concurs to keep the intracellular concentration of amino acids low by inhibiting sodium-coupled uptake through system A.

Int J Cancer, 1994 Jan 2, 56(1), 106 - 12
Protoporphyrin biosynthesis in melanoma B16 cells stimulated by 5-aminolevulinic acid and chemical inducers: characterization of photodynamic inactivation; Schoenfeld N et al.; The stimulation of protoporphyrin (PP) biosynthesis in B16 melanoma cells in order to facilitate photodynamic cell killing was studied . Biosynthesis and accumulation of PP in the melanoma cells was increased from 8 to 15 pmol/mg protein by the use of dimethyl-sulfoxide (DMSO), a differentiation-inducer . Treatment of the cells with the porphyrogenic agent allylisopropyl-acetamide (AIA) stimulated an additional PP increase . The most remarkable enhancement of intracellular PP was achieved by the supplementation of 5-aminolevulinic acid (5-ALA) to the growth medium following the addition of DMSO and AIA during the induction phase . The intracellular concentration of PP exceeded 21,950 pmol/mg protein following combined stimulation by DMSO/AIA and 5-ALA . The porphyrins produced in the incubated cells, in serum-depleted medium, consisted of 95% PP; 88% of it was recovered from the cells and only 7% was excreted into the medium . Photosensitization of the B16 melanoma cells containing high PP concentrations was effective even at low light doses . Potassium (K) efflux was the first measurable sign of cell damage determined by X-ray microanalysis (XRMA) following fast liquid-nitrogen fixation . During a 1 min interval, 70% of cellular K was lost . After 5 min illumination, complete cell destruction was detected by scanning electron microscopy (SEM) and XRMA . The photodamaged cells showed influx of Na, Cl and Ca ions accompanying the immediate K losses . Ultrastructural cell damage was manifested by disintegration of the outer membrane . Total cell death of B16 melanoma cells was achieved by chemical induction of endogenous PP and photosensitization.

J Neurosci, 1994 Jan, 14(1), 75 - 87
Death of developing septal cholinergic neurons following NGF withdrawal in vitro: protection by protein synthesis inhibition; Svendsen CN et al.; Fetal septal neurons were grown in vitro under glass coverslips . This sandwich culture method significantly increased general neuronal survival, reduced glial proliferation, and permitted the removal of serum from the growth medium after 5 d in vitro . Thereafter, a simple, and completely defined, medium was used, and the effects of NGF, NGF withdrawal, and protein synthesis inhibition were examined on septal cholinergic neurons . NGF added to septal cultures at the time of plating resulted in a threefold increase in the number of cholinergic neurons seen at 14 d in vitro but had no effect on the survival of non-cholinergic cells . Cholinergic neurons identified by staining for AChE, ChAT, and p75NGFR could be maintained in serum-free, NGF-supplemented medium for over 40 d . When NGF was removed and NGF antibodies added to 14-d-old cultures, less than 30% of cholinergic neurons survived a further 4 d, but when NGF was similarly withdrawn from 35-d-old cultures, over 75% of cholinergic neurons survived . Reapplication of NGF after 3 but not after 12 or more hours of NGF withdrawal from 14-d-old cultures prevented the death of most cholinergic neurons . When NGF was withdrawn from 14-d-old cultures in the presence of the protein synthesis inhibitor cycloheximide, over 75% of the cholinergic neurons survived . These findings suggest that septal cholinergic neurons are dependent on NGF for survival only during a critical period of development and that growth factor-regulated developmental cell death may occur in CNS neurons by activation of programmed cell death requiring protein synthesis.

Mol Cell Biol, 1994 Jan, 14(1), 104 - 15
Isolation and characterization of a Saccharomyces cerevisiae peptide transport gene; Perry JR et al.; We have cloned and characterized a Saccharomyces cerevisiae peptide transport gene (PTR2) isolated from a genomic DNA library by directly selecting for functional complementation of a peptide transport-deficient mutant . Deletion and frameshift mutageneses were used to localize the complementing activity to a 3.1-kbp region on the transforming plasmid . DNA sequencing of the complementing region identified an open reading frame spanning 1,803 bp . The deduced amino acid sequence predicts a hydrophobic peptide consisting of 601 amino acids, having a molecular mass of 68.1 kDa, composed in part of 12 hydrophobic segments, and sharing significant similarities with a nitrate transport protein encoded by the CHL1 gene of Arabidopsis thaliana . Northern (RNA) hybridization experiments demonstrated a single transcript that was 1.8 kb in length and that was transiently induced by the addition of L-leucine to the growth medium . The PTR2 gene was localized to the right arm of chromosome XI by contour-clamped homogeneous electric field gel chromosome blotting and by hybridization to known chromosome XI lambda phage clones of S . cerevisiae DNA . PTR2 was tightly linked to the UBI2 gene, with the coding sequences being separated by a 466-bp region and oriented so that the genes were transcribed convergently . A chromosomal disruption of the PTR2 gene in a haploid strain was not lethal under standard growth conditions . The cloning of PTR2 represents the first example of the molecular genetic characterization of a eucaryotic peptide transport gene.

Life Sci, 1994, 54(23), 1785 - 91
Effects of cell density and conditioned medium on alpha 2A adrenergic receptor density and messenger RNA abundance in HT-29 cells; Sakaue M et al.; We have examined the relationship between cell density and alpha 2A adrenergic receptor number and mRNA in HT-29 cells . alpha 2A receptors increased with increasing cell density from 75 +/- 25 to 400 +/- 73 fmol/mg protein; alpha 2A receptor mRNA also increased about 4 fold with no change in abundance of beta-actin mRNA . We prepared serum-free conditioned medium (SFCM) from confluent dishes of HT-29 cells and applied this SFCM to cells seeded at low density . A 4.7-fold increase in receptor number was observed in HT-29 cells cultured in SFCM for 3 days and alpha 2A receptor mRNA level increased about 3-fold after 24 h . Dialysis of SFCM against fresh growth medium did not abolish these effects . These results suggest that a substance secreted from HT-29 cells regulates expression of alpha 2A receptors.

Actas Urol Esp, 1994 Jan, 18(1), 29 - 33; discussion 34
{Potentiation of cytotoxicity of electromagnetic shock waves by suramin, "in vitro" study}; Rosell D et al.; The objective of this experimental study is to assess the inhibition of tumoral cells growth induced by electromagnetic shockwaves at different energy levels in PC-3, the human prostate adenocarcinoma cell line . Also, an assessment is made of the inhibition of cell growth caused by adding Suramin to the growth medium and the enhancement of cytotoxicity when associated to that produced by electromagnetic shockwaves . Cells viability is determined by life staining exclusion methods and nucleoside incorporation . Cytotoxic action of electromagnetical shockwaves in the PC-3 cell line is dose dependent (p < 0001) . Suramin significantly inhibits cell viability (p < 0001) . The association of both therapeutical approaches enhances significantly their individual cytotoxicities (p < 0001).

ASAIO J, 1994 Jan-Mar, 40(1), 20 - 3
Peritoneal dialysis solution pH and Ca2+ concentration regulate peritoneal macrophage and mesothelial cell activation; Carozzi S et al.; We evaluated the in vitro effects of pH and Ca2+ concentration of peritoneal dialysis solution (PDS) on (1) the release of interleukin-1 (IL-1), tumor necrosis factor alpha (TNF-alpha), interleukin-6 (IL-6), and interleukin-8 (IL-8) from peritoneal macrophages (PM0) and peritoneal mesothelial cells (PMC); (2) the release of IL-6 and IL-8 by PMC; and (3) the PM0 and PMC intracellular Ca2+ concentration . Aliquots of 5 x 10(6) PM0 and PMC were incubated (2 hr, 37 degrees C) in 1 ml of physiologic growth medium (RPMI 1640) and in 1 ml of four different PDS (1.36 g/dl glucose): (1) type A PDS (pH 5.5, Ca2+ 1.75 mM), (2) type B PDS (pH 5.5, Ca2+ 1.25 mM), (3) type C PDS (bicarbonate buffered pH 7.4, Ca2+ 1.75 mmol/L, and (4) type D PDS (bicarbonate buffered pH 7.4, Ca2+ 1.25 mM); each was stimulated with S . epidermidis . Results showed that type A PDS samples induced an average 60% increase in PM0 and PMC cytoplasmic levels and in cytokine release, whereas with type B PDS samples there was a 90% decrease . Type C PDS samples did not modify the PM0 and PMC IL-6 and IL-8 production, whereas a 3-fold rise in the production of IL-1 and TNF-alpha by PM0 was seen; this was associated with an increase in PM0 and PMC Ca2+ cytoplasmic levels . When type D PDS samples were incubated, however, there was an average 40% decrease in PM0 and PMC cytoplasmic Ca2+ levels and in cytokine release.(ABSTRACT TRUNCATED AT 250 WORDS)

Int J Hyperthermia, 1994 Jan-Feb, 10(1), 79 - 88
Effects of heat and amino acid supplementation on the uptake of arginine and its incorporation into proteins in Escherichia coli; Eshraghi N et al.; Hyperthermic treatment reduces protein synthesis and modifies amino acid transport in Escherichia coli . The present study examined the role of nutrient availability on these processes . Cultures of E . coli in log phase were aliquoted into growth medium with or without complete amino acid supplementation and exposed to 37, 44, or 48 degrees C for 10 min . Amino acid supplementation increased radiolabelled arginine uptake at 48 degrees C when compared with unsupplemented cells . Exposure to 48 degrees C also reduced protein synthesis in both groups by at least 50% as reflected by labelled arginine incorporation . Two-dimensional gel electrophoresis indicated that this heat-related decrement in synthesis was most apparent in basic proteins . Total density analysis of the fluorographs demonstrated reductions in basic proteins of 15% at 44 degrees C and 89% at 48 degrees C, while acidic proteins only showed an 80% reduction at 48 degrees C . Amino acid supplementation appears to raise the baseline, but not to modify the final results of hyperthermia-induced inhibition of protein synthesis . The sensitivity of basic protein synthesis seems to be a key event in this process.

Life Sci, 1994, 54(13), 863 - 75
Growth of S49 wild type cells in 3 nM epinephrine increases cyclic AMP phosphodiesterase activity; Richardson MD et al.; In this communication we report that intact cell measurements of cAMP decay have shown an increase averaging over 80% in the cAMP decay constant (kdy) in intact S49 WT cells following a 24 hour growth in 3 nM epinephrine (24 hr/3 nM epinephrine) . A comparable percentage increase was seen in cell free PDE activities from cells similarly treated . The enhanced PDE activity and kdy were detectable after 6 hours of incubation with 3 nM epinephrine, and were maximal by 24 hours of incubation . Lysate PDE activity returned to control levels within 24 hours after the cells were transferred to epinephrine-free growth medium . The increased PDE activity present in S49 WT cells after 24 hr/3 nM epinephrine was the result of increased concentrations of the enzymes already present or the expression of very similar enzymes, rather than the expression of enzymes with markedly different characteristics . Firstly, the apparent Km values for the PDE's in lysates from control and 24 hr/3 nM epinephrine cells were both in the region of 1 microM . Secondly, PDE activities in lysates from 24 hr/control and 24 hr/3 nM epinephrine S49 WT cells showed similar sensitivities to PDE inhibitors . There was support for the hypothesis that the increase in PDE activity in 24 hr/3 nM epinephrine S49 WT cells is dependent on the presence of cAPK . That is, although 24 hr/3nM epinephrine caused a distinct homologous desensitization of adenylate cyclase in S49 kin cells, neither kdy nor lysate PDE activity were affected . Additionally, 24 hours incubation of S49 WT cells with 3 microM dibutyryl cAMP resulted in increased PDE activity . Finally, the stimulatory effect of 24 hr/3 nM epinephrine on PDE activity was inhibited in the presence of cycloheximide, suggesting the involvement of protein synthesis in the process . This study shows that prolonged treatment with very low concentrations of epinephrine results in an increase in PDE activity which had a significant effect on cAMP accumulation . The increases were quantified by intact cell and cell free measurements, with kinetic data which were consistent with the hypothesis that the increased PDE activity in 24 hr/3 nM epinephrine cells reflected an increase PDE synthesis which was dependent on activation of the cAPK.

J Eukaryot Microbiol, 1994 Jan-Feb, 41(1), 38 - 46
Modulation of biological functions of Naegleria fowleri amoebae by growth medium; Marciano-Cabral F et al.; Two strains of Naegleria fowleri amoebae were studied when the amoebae were maintained in the same growth medium or in two different media . A weakly pathogenic strain of N . fowleri, LEE, and a highly pathogenic strain, LEEmpC1, were compared for growth properties, the presence or absence of surface structures termed food cups, cytopathogenicity, cellular locomotion, susceptibility to complement-mediated lysis and immunological relatedness by western immunoblot analysis when grown in Nelson medium or in Cline medium . The two different strains of N . fowleri, LEE and LEEmpC1, were more similar in protein profiles and functional activity when both strains were grown in the same nutritional medium . Differences in growth, proteins synthesized, cytopathogenicity, susceptibility to complement lysis and rate of locomotion were noted when the same strain was grown in different media . Naegleria fowleri grown in Cline medium demonstrated an increased rate of growth, an increase in its rate of locomotion, an increased resistance to complement lysis, and destroyed target nerve cells by contact-dependent lysis . In contrast, the same strain of amoeba grown in Nelson medium showed slower growth, destroyed target cells by trogocytosis, and was less resistant to complement-mediated lysis.

Lasers Surg Med, 1994, 14(4), 347 - 54
Effect of low level diode laser irradiation of human oral mucosa fibroblasts in vitro; Loevschall H et al.; The effects of low level laser (LLL) irradiation on the proliferation of human buccal fibroblasts were studied . A standardized LLL set-up was developed (812 nm, 4.5 +/- 0.5 mW/cm2) . Cultures in petridishes were divided into eight groups (1 group served as control) . On day 6 after seeding, routine growth medium was replaced with PBS for 1/2 hour . At the beginning of this period, LLL irradiation was performed for 0, 1, 3, 10, 32, 100, 316, or 1,000 seconds, respectively--corresponding to the radiant exposures 0, 4.5, 13.5, 45, 144, 450, 1,422, 4,500 mJ/cm2 . Subsequently the cells received 3H-dT in fresh medium for 16 hours DNA-incorporation . Scintillations from tritium and total protein concentration per culture dish were determined . The individual 3H-cpm/protein-concentration ratios were calculated in % of control . Three experiments were performed (N = 151) . Following LLL exposure the 3H-cpm/protein ratio was increased with maximum cpm/protein ratio (132.5% +/- 10.6% SEM) in the group receiving 450 mJ/cm2 (P < 0.03 nonparametric Kruskal Wallis one-way ANOVA-test) . This study demonstrated an increased incorporation on tritiated thymidine in cultured human oral fibroblasts following LLL exposure and suggests that LLL irradiation can induce increased DNA synthesis.

Tumour Biol, 1994, 15(3), 153 - 9
Effect of medium exchange rate on colony growth of the human tumor cell line MDA-231 cloned within the perfused capillary cloning system; Weisser H et al.; The conventional human tumor stem cell assay is limited by the lack of flexibility for drug scheduling with single agents and its inability to test drug combinations . Recently, we described the cloning of tumor cell lines within porous glass capillary tubes which allow free exchange of substances . The present study describes the influence of various perfusion modalities on the colony growth of the human tumor cell line MDA-231 cloned within the perfused capillary cloning system (PCCS) . Colony growth of tumor cells within the PCCS is strongly dependent on perfusion tube volume, flow rate and duration of perfusion with growth medium . Best colony growth was achieved using a perfusion tube volume of 12 ml resulting in a cloning efficiency of 36.3% . Continuous perfusion with fresh medium did not improve the cloning efficiency; in fact, colony growth was hampered compared to colony growth within unperfused porous capillaries . However, cloning efficiency was acceptable when continuous perfusion was started at day 6 (26.4%) instead of day 0 (17.2%), or when a short perfusion with high volume (12 ml/h) was discontinued after 1 h at day 0 . In contrast to the conventional capillary cloning system the PCCS has the potential for investigating secretion and kinetics of tumor-specific factors and the effect of growth-stimulating or growth-inhibiting drugs.

Anticancer Res, 1994 Jan-Feb, 14(1A), 37 - 43
Morphological and biochemical features of a medroxyprogesterone acetate (MPA)-resistant MCF-7 breast cancer cell line; Gibelli N et al.; An MCF-7 human breast cancer line variant (MCF-7/MPA), resistant to medroxyprogesterone-acetate (MPA), was obtained by continuous exposure in vitro to the drug . MCF-7/MPA cells were grown in the presence of 12.5 x 10(-6) M MPA and were selected by increasing the concentration of the drug in the growth medium in a stepwise manner from 0.025 x 10(-6) M up to 12.5 x 10(-6) M . Comparative studies of cellular morphology, cytosolic steroid receptor content and P-Glycoprotein expression were performed on both MCF-7 parental line and MCF-7/MPA variant . MCF-7/MPA cells, when compared to the parental line, exhibit a different morphology in terms of membrane alterations, reduced content of cytosolic progesterone receptor, increased expression of P-glycoprotein along with reduction of Doxorubicin (Dx) activity on the growth of MCF-7/MPA resistant cells.

Microbiol Immunol, 1994, 38(11), 897 - 900
Growth medium containing cyclodextrin and low concentration of horse serum for cultivation of Helicobacter pylori; Morshed MG et al.; The growth of Helicobacter pylori, a Gram-negative microaerophilic bacterium, is often difficult and requires complex media with the supplementation of 5% to 10% blood or blood derivatives . We have found that Brucella broth supplemented with 1% heated horse serum and 0.1% beta-cyclodextrin supports the good growth of H . pylori . The degree of growth and production of urease and vacuolating cytotoxin in this medium were equal to those in the medium supplemented with 5% horse serum . This medium was found to be suitable for both the routine laboratory culture and primary isolation of H . pylori from biopsy samples.

Biochimie, 1994, 76(9), 880 - 6
Regulation and function of non-AUG-initiated proto-oncogenes; Hann SR; A small, yet growing, number of cellular eukaryotic mRNAs encoding important regulatory proteins, such as c-myc and other proto-oncogenes, initiate translation from a non-AUG codon, usually in addition to initiating at a downstream AUG . The efficiency of non-AUG initiation on these natural cellular mRNAs varies considerably and appears to be governed by several features, including the codon sequence, the context surrounding the codon and the secondary structure of the transcript . In addition to factors which control the overall efficiency of c-myc non-AUG initiation, the relative efficiency of the upstream non-AUG initiation compared with the AUG initiation changes during the growth of cells . As lymphoid and fibroblast cells approach high densities in culture there is a sustained 5-10-fold induction in the synthesis of the non-AUG-initiated c-Myc 1 protein to levels comparable to or greater than the AUG-initiated c-Myc 2 protein . This increased efficiency of c-myc non-AUG initiation, due to methionine depletion of the growth medium, suggests that the scanning preinitiation complex can be regulated to enhance the recognition of a suboptimal non-AUG codon . The significance of non-AUG initiation for the growth-regulatory genes is illustrated by the different localizations of the int-2, bFGF and hck non-AUG-initiated proteins, the disruption of the c-myc and lyl-1 non-AUG initiation in tumor-derived cell lines, and the distinct biological function of the non-AUG-initiated forms of bFGF.

J Cancer Res Clin Oncol, 1994, 120(12), 695 - 9
Growth inhibition of human gastrointestinal cancer cells by cyclosporin A; Piontek M et al.; We have studied the ability of cyclosporin A (CsA) to inhibit the growth of human AGS gastric and HT29 colon carcinoma cells in vitro . Using continuous drug exposure in growth assays of cultured tumour cells we found that CsA produced a dose-dependent growth inhibition in gastric and colon cancer cells with a half-maximal effect at 5 microM and 6 microM CsA respectively . The growth inhibition of CsA was reversible in AGS cells, when the tumour cells were incubated in normal growth medium following CsA treatment . Trypan blue dye exclusion in AGS cells indicated a cytostatic rather than a cytotoxic effect in the concentration range used . Coincubation of CsA-treated cells with 10-400 U/ml interleukin-2 (IL-2) could not abrogate this growth inhibition, suggesting an IL-2 independent mechanism of action . Flow-cytometric analysis did not reveal a phase arrest of the gastric cancer cells within the cell cycle . We conclude from our experiments that CsA cytostatically and reversibly inhibits the growth of human gastric cancer cells in a dose-dependent manner . In contrast to its mechanism of action in lymphocytes, this direct antiproliferative effect of CsA seems not to be mediated by an IL-2-dependent pathway or a cell-cycle-phase arrest of the tumour cells.

Acta Neurochir (Wien), 1994, 131(3-4), 282 - 8
Involvement of protein kinase C in growth regulation of human meningioma cells; Todo T et al.; In order to investigate the possible role of protein kinase C (PKC)-mediated signal pathways in growth regulation of meningiomas, we examined the effect of two PKC-activating phorbol esters, 12-O-tetradecanoyl-13-phorbol acetate (TPA) and phorbol 12, 13-dibutyrate (PDBu), and PKC inhibitor, staurosporine, on cell proliferation using low-passage human meningioma cells in culture . TPA (0.1 to 100 ng/ml) caused a dose-dependent stimulation of cell proliferation in six of eight meningioma cultures . At optimal concentrations of TPA, the cell growth ranged from 113% to 251% versus control . In contrast, PDBu (0.1 to 100 ng/ml) caused a significant inhibition of cell proliferation in three of five meningioma cultures . At optimal concentrations of PDBu, the cell growth ranged from 52% to 79% of control . Staurosporine exhibited a stimulation of cell proliferation (135% to 178%) in three of four meningioma cultures studied at a concentration of 10(-10) to 10(-9)M, although a tendency of growth inhibition was observed at a lower concentration . A time course of DNA synthesis in response to TPA, assessed by {3H} thymidine incorporation studies, revealed a time- and dose-dependent stimulation and/or inhibition which further depended on the serum concentration of the growth medium used . The overall results indicate that PKC-mediated signal pathways are closely involved in growth regulation of human meningioma cells . The results further suggest that the signalling processes consist of complex mechanisms which await to be elucidated.

Acta Biomed Ateneo Parmense, 1994, 65(3-4), 147 - 55
Haemmaccel, a key for lung preservation?
Spaggiari L, Alfieri R, Cattelani L, Bobbio A, Rusca M, Urbani S, Foletti G, Tecchio T, Carbognani P, Petronini P, et al.
Several and different solutions have been used for lung preservation but, at present, fluids and solutions are quite alike . In the last years extracellular type solutions have been progressively tested in experimental researches and have shown a better protection vs intracellular one . In this research we have studied the effect of a low-potassium solution normally used as plasma expander (Haemmaccel, HM) on isolated foetal human fibroblasts (WI-38) . HM has been compared with Belzer solution (UWS) after 16 hrs incubation at 10 degrees C and low-potassium solutions with dextran 2% and 5% after 6 hrs and 16 hrs incubation at 10 degrees C . Wi-38 cells have been seeded at the density of 9x10(4)/cm2 onto plastic well plates . Cellular viability was measuring using the rate of protein synthesis through the incorporation of 3H leucine in growth medium during a 1 hr incubation at 37 degrees C . The results were expressed as nmol 3 H leu/mg of proteins/minute and presented as means +/- SD; the comparison has been performed by the one way variance analysis test . After 16 hrs incubation at 10 degrees C HM preserves WI-38 cells significantly better than UWS . Comparing HM with LPD 2% and 5% a significant difference both at 6 hrs and at 16 hrs was observed . Our preliminary in vitro results confirm that low-potassium solutions are less toxic on isolated lung cells than intracellular one . Polygelin contained in HM could be considered a suitable colloidal substance that determine a better preservation if compared with Low-Potassium Dextran solutions.

Symp Soc Exp Biol, 1994, 48, 141 - 53
The plant vacuolar Na+/H+ antiport; Barkla BJ et al.; Salt stress imposes severe limitations on plant growth, however, the extent of growth reduction depends upon the soil salinity level and the plant species . One of the mechanisms employed by salt tolerant plants is the effective vacuolar compartmentalization of sodium . The sequestration of sodium into the vacuole occurs by the operation of a Na+/H+ antiport located at the tonoplast . Evidence for a plant vacuolar Na+/H+ antiport has been demonstrated in tissues, intact vacuoles and isolated tonoplast vesicles . In sugar beet cell suspensions, the activity of the vacuolar Na+/H+ antiport increased with increasing NaCl concentrations in the growth medium . This increased activity was correlated with the increased synthesis of a 170 kDa tonoplast polypeptide . In vivo labelling of tonoplast proteins showed the enhanced synthesis of the 170 kDa polypeptide not only upon exposure of the cells to salt, but also when the cells were grown in the presence of amiloride . Exposure of the cells to amiloride also resulted in increased vacuolar Na+/H+ antiport activity . Polyclonal antibodies raised against the 170 kDa polypeptide almost completely inhibited the antiport activity, suggesting the association of this protein with the plant vacuolar Na+/H+ antiport . Antibodies against the Na+/H+ antiport-associated polypeptide were used to screen a Beta lambda ZAP expression library . A partial clone of 1.65 kb was sequenced and found to encode a polypeptide with a putative transmembrane domain and a large hydrophilic C terminus . This clone showed no homology to any previously cloned gene at either the nucleic acid or the amino acid level.

J Basic Microbiol, 1994, 34(1), 3 - 10
Polysaccharide production in Pseudomonas cepacia; Allison DG et al.; The effects of glucose, osmolarity, temperature and mode of growth on exopolysaccharide production in Pseudomonas cepacia was studied in batch culture using a chemically defined growth medium . Polymer production was maximal under conditions of a 2% (w/v) glucose supplement, 0.4 M NaCl and an incubation temperature of 35 degrees C . In addition, polysaccharide composition and molecular weight varied with mode of growth . On agar culture there was a decrease in pyruvate and rhamnose content yet an increase in the amount of acetate compared to the polymer isolated from broth culture equivalents . The clinical implications of these results are discussed in relation to the potential pathogenicity of P . cepacia in cystic fibrosis patients.

J Invest Dermatol, 1994 Jan, 102(1), 84 - 8
Differential inhibition of cutaneous T-cell-mediated reactions and epidermal cell proliferation by cyclosporin A, FK-506, and rapamycin; Duncan JI; Although cyclosporin A is a highly effective treatment for several skin disorders, particularly psoriasis, its use in dermatology appears limited due to drug-induced hypertension and nephrotoxicity . Newer, similar-acting anti-T-cell agents such as FK-506 and rapamycin may be more effective; therefore a comparison was made with cyclosporin A to assess their inhibitory action on T-cell responses and keratinocyte proliferation . Using a guinea-pig model of delayed-type hypersensitivity to dinitrofluorobenzene (DNFB), drugs were given systemically (25 mg/kg cyclosporin A, rapamycin; 2.5 mg/kg FK-506) and topically (0.02% and 2%) at the time of DNFB challenge or several hours after and were assessed with respect to erythema and the numbers of infiltrating T lymphocytes entering skin-challenge sites . FK-506, at all concentrations, significantly inhibited both T-cell infiltration and skin reddening when used by both routes . Rapamycin displayed no inhibitory effect, whereas cyclosporin A only suppressed the erythema response when given systemically . The inhibition of normal human keratinocyte growth by the drugs was assessed using a protein dye-binding assay . After 2 weeks, FK-506 had no effect, whereas cyclosporin A and rapamycin both inhibited keratinocyte growth in a dose-dependent fashion and almost equivalently in serum-containing and serum-free keratinocyte growth medium . The findings showed that in vivo only FK-506 suppressed T-cell involvement in sensitized animals . In contrast, it failed to have any effect on keratinocyte growth, whereas rapamycin was more potent than cyclosporin A in inhibiting their proliferation . The future benefit of these drugs in dermatology may ultimately lie in their combined use.

FEBS Lett, 1993 Dec 20, 336(1), 75 - 8
Cytochrome d induction in Escherichia coli growing under unfavorable conditions; Bogachev AV et al.; Growth of E . coli in the presence of the protonophorous uncoupler pentachlorophenol is shown to strongly enhance levels of cytochrome d, a putative Na(+)-motive oxidase . This effect was found to be arrested by chloramphenicol and stimulated by high Na+ concentration in the growth medium . The induction of cytochrome d takes place in a mutant deficient in the F0F1 ATP-synthase but does not occur in mutants deficient in either of two different components of the Arc system . Similar relationships were revealed when pentachlorophenol was replaced by ferricyanide and phenazine methosulfate, agents oxidizing the respiratory chain . Induction of cytochrome d is also shown to occur in riboflavin-deficient mutants growing in the presence of such low riboflavin concentrations as to be insufficient to maintain a high respiration rate . It is suggested (i) that it is delta mu H+ decrease rather than reduction of the respiratory chain that is the signal for the induction of cytochrome d, and (ii) the Arc system is involved in this type of metabolic regulation.

J Clin Microbiol, 1993 Dec, 31(12), 3200 - 3
Factors affecting detection of Brucella melitensis by BACTEC NR730, a nonradiometric system for hemocultures; Gamazo C et al.; The detection of Brucella bacteremia by subculture does not always correlate with a positive signal in the BACTEC NR730 nonradiometric system (Becton Dickinson Diagnostic Instrument Systems, Sparks, Md.) . The effect of the inoculum size, pH, sodium polyanetholesulfonate, carbon sources (i-erythritol, sodium pyruvate, monosodium glutamate, D-glucose, and L-alanine), and urea in the release of CO2 was evaluated by using the reference strain Brucella melitensis 16M . In standard NR6 vials with or without blood, inocula 5 to 10 times larger (at least 265 CFU per vial) than those usually found in the blood of patients with brucellosis were necessary to produce a positive growth value (GV) in 4 days or less, and similar results were obtained with vials supplemented with the substrates listed above . GVs were consistently lower in vials with sodium polyanetholesulfonate than in vials without this agent . Vials with no blood inoculated with 265 CFU per vial showed turbidity 1 day before GVs became positive, proving that the major limiting detection factor was the low level of release of CO2 and not an inadequate growth medium . In NR6 vials buffered to pH 6.2, GVs became positive faster and were higher than those in standard vials . NR6 vials at pH 6.2 with 0.3% sodium pyruvate yielded a positive GV in the first day of bacterial turbidity.

J Steroid Biochem Mol Biol, 1993 Dec, 46(6), 681 - 6
Localization of the approximately 12 kDa M(r) discrepancy in gel migration of the mouse glucocorticoid receptor to the major phosphorylated cyanogen bromide fragment in the transactivating domain; Hutchison KA et al.; The intact wild-type mouse glucocorticoid receptor has a theoretical molecular weight of approximately 96 kDa based on amino acid sequence, but on SDS-polyacrylamide gel electrophoresis it migrates as a protein of approximately 98 kDa . It is not known where the unusual primary structure or covalent modification responsible for this anomalous migration is located within the amino acid chain . In the course of examining the pattern of fragmentation of 32P-labeled glucocorticoid receptors from Chinese hamster ovary (CHO) cells containing amplified mouse receptor cDNA, we have found a localized region in the amino-terminal half of the receptor that accounts for this anomalous behavior . Cyanogen bromide treatment of the intact receptor produces a 23.4 kDa (theoretical) fragment consisting of residues 108-324 and containing all of the identified phosphorylated serines within the receptor . We find that the only large resolvable 32P-labeled receptor fragment produced after complete cyanogen bromide cleavage of intact receptors migrates with an apparent molecular weight of approximately 35 kDa . Because the apparent difference between the theoretical and the experimentally observed molecular weights of this cyanogen bromide fragment is essentially the same as the difference between the theoretical and experimental molecular weights of the intact mouse glucocorticoid receptor, we propose that some feature lying within this fragment accounts for slower migration . Although the existence of an additional phosphorylation site lying within the 15 kDa tryptic receptor fragment containing the DNA-binding domain has been contested, we also demonstrate that this fragment of the mouse glucocorticoid receptor is phosphorylated in vivo upon incubation of CHO cells in growth medium containing {32P}orthophosphate.

Neurology, 1993 Dec, 43(12), 2581 - 6
Cytosol protein kinase C downregulation in fibroblasts from Alzheimer's disease patients; Govoni S et al.; We attempted to determine whether changes in protein kinase C (PKC) activity in Alzheimer's disease (AD) brains are also present in cultured skin fibroblasts from living patients . Biopsies collected from shoulder skin were transferred to culture plates with an appropriate growth medium, and histone-directed PKC activity as well as phorbol ester binding were individually determined in soluble and particulate fractions prepared from AD and non-AD fibroblast cell lines . Binding experiments indicated that PKC was unevenly distributed between cytosol (78%) and particulate (22%) . The Bmax values for phorbol ester binding in soluble and particulate fractions were similar in AD and non-AD patients . Kd values in the cytosol were 94% higher in AD patients, indicating lower affinity of the enzyme for the ligand . Accordingly, the soluble PKC activity was 30% lower in AD patients . The data suggest that the changes in PKC phosphorylating activity represent a diffuse cellular defect in AD and are not confined to the brain . The alterations of the enzyme may participate in the disregulation in processing of beta-amyloid precursor protein in AD.

J Dermatol Sci, 1993 Dec, 6(3), 214 - 8
Testosterone metabolism by cultured human beard outer root sheath cells in comparison with epidermal keratinocytes; Sonoda T et al.; In order to elucidate the mechanism of action of androgen in human hair follicles, we studied testosterone metabolism by cultured beard outer root sheath cells in comparison with that of epidermal keratinocytes . When cells were incubated as a monolayer in serum-free keratinocyte growth medium with physiological concentrations (25 nM) of {3H}testosterone, the major metabolite was androstenedione in either type of cells . Small amounts of androstanedione, dihydrotestosterone and androsterone were also formed . The ratio of apparent 5 alpha-reductase to 17 beta-hydroxy-steroid dehydrogenase activities ranged from 0.11 to 0.81 and did not differ between these two kinds of cells . After these cells were cultured in the presence of 10% fetal calf serum for 10 days, the activities of both metabolic pathways markedly increased without significant changes of the ratio of activities of these two enzymes . Thus, testosterone was rather converted to weak androgens even in beard outer root sheath cells under the experimental conditions.

Biochem Med Metab Biol, 1993 Dec, 50(3), 346 - 57
Copper effects on metal regulatory factors of cultured human fibroblasts; Hahn SH et al.; The effects of copper on metal regulatory factors were studied in cultured human fibroblasts using mobility shift assays and protein blotting . A mobility shift was apparent on polyacrylamide gels when fibroblast whole cell extracts were reacted with a 32P-labeled 14-base-pair MREa sequence of the hMT IIA gene . Binding was specific, since the shifted band was not competed away by MREa analogs containing two base changes . Known transcription factors SP1, AP1, AP2, and OCT1 did not compete with the fibroblast metal regulatory element binding protein (MREBP) for binding to the MREa probe . When fibroblasts were grown in 200 or 800 microM copper, binding of the MREBP was enhanced . Protein blotting experiments were performed by electrophoresing cell extracts, blotting onto nitrocellulose, and probing with the 32P-labeled MREa 14-mer . A 112-kDa band, apparent in HeLa cell nuclear extract as well as fibroblast whole cell extract, was not influenced by the presence of copper in the growing fibroblasts . A 34-kDa protein, also present in both HeLa cells and fibroblasts, appeared to correspond with the protein causing the mobility shift . Copper added to the fibroblast growth media enhanced binding of the 34-kDa protein to MREa using the blotting procedure . We conclude that the fibroblast 34-kDa MREBP is a positive regulatory factor influenced by copper . Human fibroblasts can be employed to further study metal regulatory factors whose binding to metal regulatory elements is influenced by copper and other heavy metals.

Lipids, 1993 Dec, 28(12), 1069 - 74
Effects of trans fatty acids on lipid accumulation in 3T3-L1 cells; Panigrahi K et al.; Previous work had shown that dietary trans fatty acids (tFA) resulted in decreased fat deposition in adipose tissue . This study was conducted to see if tFA influence lipid accumulation in Swiss mouse fibroblast 3T3-L1 cells, which are widely used as an adipocyte model . Cells were cultured in the presence of experimental or control growth media supplemented with fatty acids complexed to bovine serum albumin . Fatty acid compositions of experimental and control growth media were similar except that the octadecenoates in the control growth media were cis fatty acids, whereas those in the experimental media contained both cis and trans fatty acids . Cell-conditioned media and cellular lipids at the preadipocyte and differentiating adipocyte stages were analyzed . At both stages of development, less fat accumulated in cells cultured in the presence of tFA, due primarily to a decrease in the nonpolar lipid content of cells exposed to tFA, and linoleate to arachidonate ratios were higher in cells supplemented with tFA . Calculations comparing sums of saturated and monounsaturated fatty acids in cells at the differentiating adipocyte stage suggested that tFA may have replaced monounsaturated fatty acids in the nonpolar lipid fraction and saturated fatty acids in the polar lipid fraction . The results of these studies are in good agreement with the in vivo effects of tFA seen in previous work with mouse adipose tissue . It was concluded that the 3T3-L1 in vitro model is an appropriate system for further studies of tFA and lipid metabolism in adipose tissue.

Pigment Cell Res, 1993 Dec, 6(6), 423 - 31
Dual appearances of pigment cells from in vitro cultured embryonic cells of Japanese flounder: an implication for a differentiation-associated clock; Seikai T et al.; The cells dissociated from developing embryos of Japanese flounder (Paralichthys olivaceus) are cultured in vitro to examine the developmental fate of their pigment cells in relation to establishment of bilaterally asymmetric integumental coloration in vivo . When neurula embryos are dissociated using trypsin-EDTA in Dulbecco's modified Ca(2+)-, Mg(2+)-free phosphate buffered saline and then cultured in vitro using L-15-based fetal calf serum-supplemented growth medium at 20 degrees C, numerous pigment cells appear twice in the same culture with an interval of approximately 1 month even under similar culture conditions . The first group of pigment cells, which is relatively larger in cell size (about 70 microns wide) and lower in cell density, emerges within 12 hr after plating, whereas the second, which is far smaller in cell size (about 30 microns) and overwhelmingly higher in cell density than the first, does so about 1 month after plating . The timing of their appearances in vitro is in good accordance, respectively, with that observed for the larvae under normal development in vivo; the first group appears at the period corresponding to hatching, whereas the second at the period corresponding to the completion of metamorphosis . Light microscopic examinations disclose that each group of pigment cells is composed of black melanophores and reflecting leucophores, and that the population density of melanophores and leucophores in the first group at the climax of appearance is approximated as 1:4 . Typical xanthophores that are distributed in the skin of the larvae of this species are scarcely observed in culture in vitro.(ABSTRACT TRUNCATED AT 250 WORDS)

Mol Gen Genet, 1993 Dec, 241(5-6), 542 - 53
The GEF1 gene of Saccharomyces cerevisiae encodes an integral membrane protein; mutations in which have effects on respiration and iron-limited growth; Greene JR et al.; We have isolated a new class of respiration-defective, i.e petite, mutants of the yeast Saccharomyces cerevisiae . Mutations in the GEF1 gene cause cells to grow slowly on rich media containing carbon sources utilized by respiration . This phenotype is suppressed by adding high concentrations of iron to the growth medium . Gef1- mutants also fail to grow on a fermentable carbon source, glucose, when iron is reduced to low concentrations in the medium, suggesting that the GEF1 gene is required for efficient metabolism of iron during growth on fermentable as well as respired carbon sources . However, activity of the iron uptake system appears to be unaffected in gef1- mutants . Fe(II) transporter activity and regulation is normal in gef1- mutants . Fe(III) reductase induction during iron-limited growth is disrupted, but this appears to be a secondary effect of growth rate alterations . The wild-type GEF1 gene was cloned and sequenced; it encodes a protein of 779 amino acids, 13 possible transmembrane domains, and significant similarity to chloride channel proteins from fish and mammals, suggesting that GEF1 encodes an integral membrane protein . A gef1- deletion mutation generated in vitro and introduced into wild-type haploid strains by gene transplacement was not lethal . Oxygen consumption by intact gef1- cells and by mitochondrial fractions isolated from gef1- mutants was reduced 25-50% relative to wild type, indicating that mitochondrial function is defective in these mutants . We suggest that GEF1 encodes a transport protein that is involved in intracellular iron metabolism.

J Biol Chem, 1993 Nov 25, 268(33), 24547 - 50
In vivo control of redox potential during protein folding catalyzed by bacterial protein disulfide-isomerase (DsbA); Wunderlich M et al.; The formation of disulfide bonds in Escherichia coli is catalyzed by periplasmic protein disulfide-isomerase (DsbA) . When the alpha-amylase/trypsin inhibitor from Ragi, a protein containing five intramolecular disulfide bridges, is secreted into the periplasm of E . coli, large amounts of misfolded inhibitor with incomplete or incorrect disulfides are accumulated . Folding of the inhibitor in the periplasm is not improved when DsbA is coexpressed and cosecreted . However, an up to 14-fold increase in correctly folded inhibitor is observed by co-expression of DsbA in conjugation with the addition of reduced glutathione to the growth medium . This peptide acts as a disulfide-shuffling reagent and can pass the outer membrane of E . coli . Since the influence of DsbA on the folding yield of the inhibitor is reduced in the presence of oxidized glutathione, the in vivo function of DsbA appears to be dependent on the ratio between oxidizing and reducing thiol equivalents in the periplasm . The high stability of thiol reagents against air oxidation during growth of E . coli allows the investigation of oxidative protein folding in vivo under controlled, thiol-dependent redox conditions.

Science, 1993 Nov 19, 262(5137), 1277 - 80
Sensing structural intermediates in bacterial flagellar assembly by export of a negative regulator; Hughes KT et al.; The ability of a regulatory protein to sense the integrity of the bacterial flagellar structure was investigated . In response to a defective hook-basal body complex, the anti-sigma 28 FlgM protein inhibits flagellin transcription . In cells with a functional hook-basal body complex, the flagellin genes are transcribed normally and the FlgM protein is expelled into the growth medium . In strains with a defective hook-basal body structure, FlgM is absent from the media . The presence of flagellin protein in the media is substantially reduced in strains carrying a FlgM-LacZ protein fusion, suggesting that the fusion is blocking the flagellar export apparatus . These results suggest that the FlgM protein assesses the integrity of the flagellar hook-basal body complex by itself being a substrate for export by the flagellar-specific export apparatus.

Anticancer Res, 1993 Nov-Dec, 13(6A), 1939 - 43
Effect of albumin on antitumor activity of diarylsulfonylureas; Schultz RM et al.; Several diarylsulfonylureas (DSU), including Sulofenur (LY186641) and LY181984, have been described that exhibit wide spectrum and high therapeutic activity against murine solid tumors and human tumor xenografts . The mechanism for antitumor activity is poorly understood . Moreover, in vitro cytotoxic activity in serum-containing medium is not predictive of in vivo antitumor activity for DSU . Since DSU are extensively bound to serum albumin (> 99%), we sought to determine the effect of albumin on tumor cytotoxicity . We adapted human CCRF-CEM leukemia and GC3 colon carcinoma cells for growth in UltraCHO serum- and albumin-free medium . In comparisons between normal growth medium (RPMI-1640 with 10% fetal bovine serum) and UltraCHO medium, the unbound fraction of drug correlated better with cytotoxic activity than did the total drug . Tumor cytotoxicity by DSU required > 24 h and was markedly enhanced in UltraCHO medium . For example, LY181984 and Sulofenur had IC50 values of 7.4 and 12.1 micrograms/ml against CCRF-CEM in normal growth medium and 0.6 and 0.2 microgram/ml in UltraCHO . Moreover, DSU with the lowest IC-50s in albumin-free medium displayed the most potent in vivo antitumor activity in the 6C3HED lymphosarcoma . A Sulofenur-resistant CCRF-CEM cell line was developed by culturing the cells for > 20 passages in UltraCHO medium containing LY186641 at 2 micrograms/ml (10X IC-50) . This line showed approximately 18-fold resistance to LY186641, but did not show cross-resistance to vinblastine, actinomycin D, or doxorubicin . The albumin-free conditions may be useful for further mechanistic studies on the antitumor action by DSU . Further studies are underway to determine whether DSU structural requirements for cytotoxicity an albumin binding are intrinsically linked.

Radiat Res, 1993 Nov, 136(2), 262 - 70
Ionizing radiation induces two forms of interphase chromosome breaks in Chinese hamster ovary cells that rejoin with different kinetics and show different sensitivity to treatment in hypertonic medium or beta-araA; Okayasu R et al.; We have shown previously that incubation of irradiated plateau-phase CHO cells in hypertonic growth medium during the period normally allowed for chromosome condensation, in the premature chromosome condensation (PCC) assay, uncovers a form of interphase chromosome breaks that rejoin with fast kinetics (t1/2 = 1.5 min) . Here, we report that incubation with beta-arabinofuranosyladenine (beta-araA), an inhibitor of DNA, chromosome, and cellular repair processes, during the same period uncovers a different form of interphase chromosome breaks that rejoin with slower kinetics (t1/2 = longer than 15-20 min) . The yield of interphase chromosome breaks increased from 2.0 breaks/cell/Gy in untreated control cells to 3.6 breaks/cell/Gy in cells exposed to 1 mM beta-araA, and was the same as that observed in cells treated in hypertonic medium (500 mM NaCl) . Simultaneous exposure to beta-araA and hypertonic medium increased the yield of interphase chromosome breaks further to 5.3 breaks/cell/Gy . This increase was consistent with an additive effect of each treatment on the overall yield of breaks, and suggested that hypertonic medium and beta-araA affect distinct and independent subsets of radiation-induced interphase chromosome breaks . We tested further the notion of independence by measuring rejoining of interphase chromosome breaks sensitive to hypertonic treatment in the presence of 1 mM beta-araA, and vice versa, rejoining of interphase chromosome breaks sensitive to beta-araA during and after treatment in hypertonic medium (500 mM NaCl, 20 min); under both sets of conditions each treatment caused maximal expression of prematurely condensed chromosome breaks responding sensitively to it when given immediately after irradiation . There was no change in the rejoining kinetics of interphase chromosome breaks sensitive to hypertonic treatment in the presence of beta-araA, and no change in the rejoining kinetics of interphase chromosome breaks sensitive to beta-arA in cells treated in hypertonic medium . These results are consistent with the hypothesis that exposure to ionizing radiation leads to the induction of two forms of prematurely condensed chromosome breaks that can be distinguished from each other on the basis of their repair kinetics and their differential sensitivity to treatment with beta-araA or hypertonic medium . In direct analogy to a classification proposed previously for potentially lethal damage (PLD) based on a similar set of experiments, we introduce the terms alpha form and beta form of interphase chromosome breaks for the slow, beta-araA-sensitive, and the fast, hypertonic treatment-sensitive form, respectively . We also propose that there is a correlation between alpha and beta form of interphase chromosome breaks and alpha and beta form of PLD, and present evidence suggesting that fast and slowly repairing DNA double-strand breaks underlie fast and slowly repairing interphase chromosome breaks.(ABSTRACT TRUNCATED AT 400 WORDS)

Mol Gen Genet, 1993 Nov, 241(3-4), 447 - 56
The vacuolar H(+)-ATPase of Saccharomyces cerevisiae is required for efficient copper detoxification, mitochondrial function, and iron metabolism; Eide DJ et al.; Mutations in the GEF2 gene of the yeast Saccharomyces cerevisiae have pleiotropic effects . The gef2 mutants display a petite phenotype . These cells grow slowly on several different carbon sources utilized exclusively or primarily by respiration . This phenotype is suppressed by adding large amounts of iron to the growth medium . A defect in mitochondrial function may be the cause of the petite phenotype: the rate of oxygen consumption by intact gef2 cells and by mitochondrial fractions isolated from gef2 mutants was reduced 60%-75% relative to wild type . Cytochrome levels were unaffected in gef2 mutants, indicating that heme accumulation is not significantly altered in these strains . The gef2 mutants were also more sensitive than wild type to growth inhibition by several divalent cations including Cu . We found that the cup5 mutation, causing Cu sensitivity, is allelic to gef2 mutations . The GEF2 gene was isolated, sequenced, and found to be identical to VMA3, the gene encoding the vacuolar H(+)-ATPase proteolipid subunit . These genetic and biochemical analyses demonstrate that the vacuolar H(+)-ATPase plays a previously unknown role in Cu detoxification, mitochondrial function, and iron metabolism.

J Bacteriol, 1993 Nov, 175(21), 7105 - 8
Adaptation of Escherichia coli to the uncoupler of oxidative phosphorylation 2,4-dinitrophenol; Gage DJ et al.; Escherichia coli was found to adapt to the uncoupler of oxidative phosphorylation 2,4-dinitrophenol . The rates of synthesis of 53 proteins were increased following exposure to 2,4-dinitrophenol . Adaptation was accelerated when the cofactor pyrroloquinoline quinone was provided in the growth medium.

Invest Ophthalmol Vis Sci, 1993 Nov, 34(12), 3396 - 401
High glucose inhibits retinal capillary pericyte contractility in vitro; Gillies MC et al.; PURPOSE . To study the effect of high glucose concentrations on pericyte contractility . METHODS . Bovine retinal capillary pericytes were cultured on silicone rubber sheets, which could be seen to wrinkle when a cell contracted . Cells were grown in glucose, or mannitol, in concentrations ranging from 5 to 40 mMol . Pericyte contractility was expressed as the percentage of cells wrinkling the silicone substratum . Observations were made fortnightly for 8 weeks . RESULTS . Cells grown in glucose exhibited a dose-dependent inhibition of contractility that was significantly greater than that seen with cells grown in mannitol, which were affected to a lesser extent . After returning to normoglycemic conditions for a further 4 weeks, the contractility of cells grown in lower glucose concentrations recovered partially, but cells grown in 40 mMol glucose did not recover at all . Pericyte proliferation was also impaired by the high-glucose growth medium . CONCLUSIONS . Pericyte contractility is inhibited by high glucose concentrations . This is consistent with the hypothesis that increased retinal blood flow may be a factor in the early pathogenesis of diabetic retinopathy.

Eur J Biochem, 1993 Nov 1, 217(3), 1019 - 26
Characterization of the KdpD protein, the sensor kinase of the K(+)-translocating Kdp system of Escherichia coli; Voelkner P et al.; KdpD and KdpE, proteins that control expression of the kdpFABC operon, are members of the class of sensor kinase/response regulator proteins . Using polyclonal antibodies raised against the KdpD protein, we have been able to identify and to localize the chromosome-encoded KdpD protein in the cytoplasmic membrane of Escherichia coli . Furthermore, it has been possible to detect differences in the expression of the KdpD protein according to the K+ concentration in the growth medium . The phosphorylation capacity of the plasmid-encoded KdpD protein and the phospho-transfer to KdpE was investigated . We found that both reactions were strictly dependent on the ionic conditions of the assay medium . Based on optimized conditions, we were able to detect phosphorylation of the chromosome-encoded KdpD protein . Furthermore, replacement of the conserved histidine (His673), the predicted phosphorylation site in KdpD, by glutamine revealed that phosphorylation of KdpD was no longer possible.

In Vitro Cell Dev Biol Anim, 1993 Nov, 29A(11), 823 - 30
Human microvessel endothelial cells: isolation, culture and characterization; Hewett PW et al.; Over recent years, interest in endothelial cell biology has increased dramatically with our ability to grow and study endothelial cells in vitro . While large veins and arteries remain a quick and convenient source of endothelial cells, the great morphological, biochemical and functional heterogeneity that endothelial cells express has necessitated the development of techniques to isolate microvessel endothelial cells from different tissues to create more realistic in vitro models . The majority of isolation procedures employ selective methods to enrich microvessel endothelial cells from tissue homogenates directly, or after a period in culture . These include sieving/filtration, manual weeding, isopycnic centrifugation, selective growth media, and the use of flow cytometry or magnetic beads coupled with specific endothelial cell markers . The establishment of pure endothelial cell populations is important for studying their biochemistry and physiology and there are many morphological, immunological and biochemical criteria which can be used to characterize human endothelial cells . These range from classical markers such as von Willebrand Factor and angiotensin-converting enzyme to novel markers like platelet endothelial cell adhesion molecule-1 (CD31) and the expression of E-selectin on cytokine-activated endothelial cells.

Mol Microbiol, 1993 Nov, 10(4), 849 - 58
Branched Escherichia coli cells; Akerlund T et al.; We report that the normally rod-shaped bacterium Escherichia coli can form branched cells . These were found in strains in which chromosome replication or nucleoid segregation was disturbed, e.g . in minB mutants, intR1 strains, and in strains exhibiting stable DNA replication . Often chromosome DNA was found to be located in the branch point of the cells . The branching frequency was dependent upon the growth medium: in rich medium no branched cells were found, whereas in minimal medium containing acetate and casamino acids the frequency of branched cells was increased . The genetic background of the strains also affected the tendency to branch . Furthermore, electron microscopy of thin-sectioned branched cells revealed additional membrane-like structures, which were not observed in wild-type cells . Finally, the branched cells are compared with bacteria that normally branch, and probable causes for branching in E . coli are discussed.

Mol Microbiol, 1993 Nov, 10(4), 749 - 57
Identification of a gene required for the oxygen-regulated formation of the photosynthetic apparatus of Rhodobacter capsulatus; Pollich M et al.; The pigment-binding proteins of Rhodobacter capsulatus are encoded by the polycistronic puf and puc operons . Both operons show higher expression under low oxygen tension than under high oxygen tension in the wild-type strain . The Tn5 mutant strain AH2 shows only low levels of puf and puc mRNA under high and low oxygen tension, indicating that it lacks a gene product required for stimulation of puf and puc gene expression under low oxygen tension . The formation of wild-type levels of photosynthetic complexes and normal oxygen regulation could be restored by the expression in trans of a 1.7 kb fragment of the R . capsulatus wild-type chromosome or by addition of 10 micrograms l-1 vitamin B12 to the growth medium . An open reading frame of 798 nucleotides containing the Tn5 insertion was identified on the 1.7 kb fragment . This open reading frame shows no homology to known genes and has a remarkably high GC content of 76%.

J Bacteriol, 1993 Nov, 175(21), 7056 - 65
Cloning, expression in Escherichia coli, and characterization of cellulolytic enzymes of Azoarcus sp., a root-invading diazotroph; Reinhold-Hurek B et al.; We screened members of a new genus of grass-associated diazotrophs (Azoarcus spp.) for the presence of cellulolytic enzymes . Out of five Azoarcus strains representing different species, only in the endorhizosphere isolate BH72, which is also capable of invading grass roots, was significant endoglucanase activity, in addition to beta-glucosidase and cellobiohydrolase activity, present . Reducing sugars were readily released from medium-viscosity carboxymethylcellulose (CMC), but neither CMC, cellulose filter strips, Avicel, cellobiose, nor D-glucose served as the sole carbon source for growth of Azoarcus spp . Clones from a plasmid library of strain BH72 expressed all three enzymes in Escherichia coli, apparently not from their own promoter . According to restriction endonuclease mapping and subclone analysis, beta-glucosidase and cellobiohydrolase activities were localized on a single 2.6-kb fragment not physically linked to a 1.45-kb fragment from which endoglucanase (egl) was expressed . Two isoenzymes of endoglucanase probably resulting from proteolytic cleavage had pI values of 6.4 and 6.1 and an apparent molecular mass of approximately 36 kDa . Cellobiohydrolase and beta-glucosidase activity were conferred by one enzyme 41 kDa in size with a pI of 5.4, which we classified as an unspecific exoglycanase (exg) according to substrate utilization and specificity mapping; hydrolysis of various oligomeric substrates differentiated it from endoglucanase, which degraded substituted soluble cellulose derivatives but not microcrystalline cellulose . Both enzymes were not excreted but were associated with the surface of Azoarcus cells . Both activities were only slightly influenced by the presence of CMC or D-glucose in the growth medium but were enhanced by ethanol . egl was located on a large transcript approximately 15 kb in size, which was detectable only in cells grown under microaerobic conditions on N2 . Surface-bound exo- and endoglucanases with some unusual regulatory features, detected in this study in a strain which is unable to metabolize cellulose or sugars, might assist Azoarcus sp . strain BH72 in infection of grass roots.

Biol Trace Elem Res, 1993 Nov-Dec, 39(2-3), 229 - 43
Glucocorticoid and polyamine involvement in zinc uptake by COMMA-1D mammary epithelial cells; Allen JC et al.; The objective was to determine if a mammary cell line shows glucocorticoid stimulation of Zn uptake, and to determine whether polyamines mediate this stimulation . 65Zn uptake by COMMA-1D mouse mammary epithelial cells over a 24-h period increased significantly in cells administered 10(-7) or 10(-6) M hydrocortisone . Incorporation of 65Zn over a 1-h period was not hydrocortisone-responsive, suggesting that these incubation times represent uptake into different pools . The rate of entry into the cells over a 15-min period was significantly increased by supplementing cells with hydrocortisone with or without prolactin . Initially, cells grown in lactogenic hormone-supplemented media (10(-6) M hydrocortisone + 5 micrograms/mL ovine prolactin) had up to 65% greater 65Zn uptake over 24 h than cells in nonsupplemented growth media . 65Zn uptake from hormone media with the spermidine synthesis inhibitor methylglyoxal-bis-(guanylhydrazone) (MGBG, 10(-5)M) added was less than from growth media . Exogenous spermidine (10(-6)-10(-3)M) added to the MGBG + hormone media increased 65Zn uptake . Difluoromethylornithine (DFMO), an inhibitor of spermidine synthesis that blocks ornithine decarboxylase, caused a slight dose-dependent decrease in 65Zn uptake over the range 10(-6)-5 x 10(-3)M (p < 0.002) and tended to decrease 65Zn-uptake in lactogenic hormone-stimulated cells with 8 h of incubation, but not at other times . These data show that Zn uptake in mammary epithelial cells can be hormonally mediated by glucocorticoids and suggest that polyamines may be intracellular mediators of this effect.

J Chromatogr, 1993 Oct 29, 620(2), 243 - 9
Identification of mycotoxins in keratomycosis-derived Fusarium isolates by gas chromatography-mass spectrometry; Raza SK et al.; The production of mycotoxins from Fusarium species has been demonstrated in isolates cultured from patients suffering from keratomycosis . The method employed a combination of thin-layer chromatography directly performed on gel plugs taken from the growth medium, cartridge column chromatography, silylation and gas chromatography on a non-polar stationary phase capillary column linked to mass spectrometry . The sensitivities of detection obtained for a signal-to-noise ratio of 33:1, were 200 pg for single stage GC-MS and 20 pg using tandem GC-MS-MS . Two mycotoxins, diacetoxyscirpenol and T-2 toxin were identified in three cultures.

Cancer Res, 1993 Oct 15, 53(20), 4811 - 6
HPV-16, tobacco-specific N-nitrosamine, and N-methyl-N'-nitro-N-nitrosoguanidine in oral carcinogenesis; Kim MS et al.; We previously immortalized human oral keratinocytes by transfection with recombinant human papillomavirus type 16 (HPV-16) DNA and established two cell lines . These transfected cells were morphologically different from the normal counterpart, contained intact HPV-16 DNA in an integrated form, and expressed numerous viral genes . These cells contained lower levels of wild-type p53 protein and higher levels of c-myc mRNAs compared to normal cells . However, they proliferated only in keratinocyte growth medium containing a low level of calcium and were not tumorigenic in nude mice . A HPV-16-immortalized cell line was exposed to either 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone or N-methyl-N'-nitro-N-nitrosoguanidine . Four chemically transformed cell colonies were isolated . These cells proliferated well in Dulbecco's minimum essential medium containing a physiological level of calcium . They contained, similar to the immortalized counterpart, integrated HPV-16 sequences and lower levels of both wild-type p53 protein and DCC messages compared to normal cells . Among the chemically transformed cells, two colonies obtained from 4-(methyl-nitrosamino)-1-(3-pyridyl)-1-butanone exposure demonstrated an enhanced proliferation capacity in nude mice and transcribed a substantially higher amount of HPV-16 E6/E7, epidermal growth factor receptors, and c-myc genes compared with the immortalized counterpart . These experiments indicate that malignant transformation of oral keratinocytes can be caused by a sequential combined effect of "high risk" HPV and tobacco-related carcinogens.

Mol Cell Biol, 1993 Oct, 13(10), 6241 - 52
Conditional transformation of cells and rapid activation of the mitogen-activated protein kinase cascade by an estradiol-dependent human raf-1 protein kinase; Samuels ML et al.; We report a strategy for regulating the activity of a cytoplasmic signaling molecule, the protein kinase encoded by raf-1 . Retroviruses encoding a gene fusion between an oncogenic form of human p74raf-1 and the hormone-binding domain of the human estrogen receptor (hrafER) were constructed . The fusion protein was nontransforming in the absence of estradiol but could be reversibly activated by the addition or removal of estradiol from the growth media . Activation of hrafER was accompanied in C7 3T3 cells by the rapid, protein synthesis-independent activation of both mitogen-activated protein (MAP) kinase kinase and p42/p44 MAP kinase and by phosphorylation of the resident p74raf-1 protein as demonstrated by decreased electrophoretic mobility . The phosphorylation of p74raf-1 had no effect on the kinase activity of the protein, indicating that mobility shift is an unreliable indicator of p74raf-1 enzymatic activity . Removal of estradiol from the growth media led to a rapid inactivation of the MAP kinase cascade . These results demonstrate that Raf-1 can activate the MAP kinase cascade in vivo, independent of other "upstream" signaling components . Parallel experiments performed with rat1a cells conditionally transformed by hrafER demonstrated activation of MAP kinase kinase in response to estradiol but no subsequent activation of p42/p44 MAP kinases or phosphorylation of p74raf-1 . This result suggests that in rat1a cells, p42/p44 MAP kinase activation is not required for Raf-1-mediated oncogenic transformation . Estradiol-dependent activation of p42/p44 MAP kinases and phosphorylation of p74raf-1 was, however, observed in rat1a cells expressing hrafER when the cells were pretreated with okadaic acid . This result suggests that the level of protein phosphatase activity may play a crucial role in the regulation of the MAP kinase cascade . Our results provide the first example of a cytosolic signal transducer being harnessed by fusion to the hormone-binding domain of the estrogen receptor . This conditional system not only will aid the elucidation of the function of Raf-1 but also may be more broadly useful for the construction of conditional forms of other kinases and signaling molecules.

J Bacteriol, 1993 Oct, 175(20), 6652 - 8
Fusion of Spiroplasma floricola cells with small unilamellar vesicles is dependent on the age of the culture; Salman M et al.; Small unilamellar vesicles were labeled with the fluorescent probe octadecylrhodamine B chloride and mixed with intact Spiroplasma floricola cells . The increase in fluorescence observed was interpreted as a result of the dilution of the probe in the unlabeled S . floricola membranes because of lipid mixing upon fusion . The progression of S . floricola cultures to the stationary phase of growth was accompanied by a sharp decrease in the ability of the cells to fuse with small unilamellar vesicles . Low fusogenic activity was also detected in cells from cultures that were aged in a growth medium maintained at pH 7.5 throughout the growth cycle . Chemical analysis of the cell membrane preparations isolated from cells harvested at the various phases of growth revealed that the phospholipid content and composition and the cholesterol/phospholipid molar ratio were changed very little upon aging of the cultures . Likewise, no changes in the fatty acid composition of membrane lipids were detected, with palmitic and oleic acids predominating throughout the cycle . Nonetheless, upon aging of S . floricola cultures, a pronounced increase in the levels of both cholesteryl esters, incorporated from the growth medium, and organic peroxides was observed . A decrease in both fluorescence anisotropy of diphenylhexatriene and merocyanine 540 binding to membranes of aged cells was also detected . The possible influence of these changes on the fusogenic activity of the cells is discussed.

J Bacteriol, 1993 Oct, 175(19), 6118 - 25
Suppression of recJ mutations of Escherichia coli by mutations in translation initiation factor IF3; Haggerty TJ et al.; We have isolated genetic suppressors of mutations in the recJ gene of Escherichia coli in a locus we term srjA . These srjA mutations cause partial to complete alleviation of the recombination and UV repair defects conferred by recJ153 and recJ154 mutations in a recBC sbcA genetic background . The srjA gene was mapped to 37.5 min on the E . coli chromosome . This chromosomal region from the srjA5 strain was cloned into a plasmid vector and was shown to confer recJ suppression in a dominant fashion . Mutational analysis of this plasmid mapped srjA to the infC gene encoding translation initiation factor 3 (IF3) . Sequence analysis revealed that all three srjA alleles cause amino acid substitutions of IF3 . Suppression of recJ was shown to be allele specific: recJ153 and recJ154 mutations were suppressible, but recJ77 and the insertion allele recJ284::Tn10 were not . In addition, growth medium-conditional lethality was observed for strains carrying srjA mutations with the nonsuppressible recJ alleles . When introduced into recJ+ strains, srjA mutations conferred hyperrecombinational and hyper-UVr phenotypes . An interesting implication of these genetic properties of srjA suppression is that IF3 may regulate the expression of recJ and perhaps other recombination genes and hence may regulate the recombinational capacity of the cell.

EMBO J, 1993 Oct, 12(10), 3947 - 56
pH regulation is a major determinant in expression of a fungal penicillin biosynthetic gene; Espeso EA et al.; Transcription of the ipnA gene encoding isopenicillin N synthetase, an enzyme of secondary metabolism, is under the control of the pH regulatory system in the fungus Aspergillus nidulans . External alkaline pH or mutations in pacC, the wide domain regulatory gene which mediates pH regulation, override carbon regulation of ipnA transcript levels, resulting in elevation of the levels of this message in sucrose broth . Strains carrying these mutations, which mimic growth at alkaline pH, produce higher levels of penicillins when grown in sucrose broth compared with the wild type strain grown under carbon derepressing conditions . ipnA transcription is regulated by carbon (C) source, but extreme mutations in creA (the regulatory gene mediating carbon catabolite repression) only slightly increase repressed transcript levels . Precise deletion of the only in vitro CreA binding site present in a region of the ipnA promoter involved in carbon regulation has no effect on ipnA expression . The levels of ipnA transcript in broths with acetate or glycerol as principal C sources are inconsistent with direct or indirect creA-mediated transcriptional control of the gene . We conclude that a second, creA-independent mechanism of carbon repression controls expression of this gene . All derepressing C sources tested result in alkalinization of the growth media . In contrast, all repressing C sources result in external acidification . Neither acidic external pH nor pal mutations, mimicking the effects of growth at acid pH, prevent carbon derepression, providing strong support for independent regulatory mechanisms, one mediating carbon regulation (via thus far unidentified genes) and another mediating pH regulation (through the pacC-encoded transcriptional regulator) . External pH measurements suggest that these two independent forms of regulation normally act in concert . We propose that external alkalinity represents a physiological signal which triggers penicillin biosynthesis.

Vet Immunol Immunopathol, 1993 Oct, 38(3-4), 367 - 73
Effect of costimulants on interleukin-2-like activities from goat peripheral blood mononuclear cells; Tomar A et al.; Culture supernatants containing interleukin-2-like activities (CS-IL2) were prepared from goat peripheral blood cells (mononuclear cells 75% and polymorphonuclear cells 25%) . These were stimulated with three costimulants, (tetradecanoyl phorbol acetate, indomethacin and calcium ionophore A23187), either alone or in different combinations, in RPMI-1640 medium (containing 0.5% bovine serum albumin (BSA)) with or without serum . After 18 h of incubation with costimulants, concanavalin A (Con A) was added and the incubation was continued for next 48 h . Higher interleukin-2 (IL-2)-like activities were generated in the culture supernatants prepared in RPMI-1640 growth medium containing 0.5% BSA without serum . Further, IL-2-like activities were much higher in culture supernatants obtained by stimulation with all the three costimulants, as well as Con A, than the two costimulants with Con A or any of the costimulants with Con A.

J Antimicrob Chemother, 1993 Oct, 32(4), 519 - 37
The post-antibiotic effect; MacKenzie FM et al.; Pharmacodynamics are being applied increasingly to the design of antibiotic dosing regimens . One characteristic of pharmacodynamics is the post-antibiotic effect (PAE), the delayed regrowth of bacteria following exposure to an antibiotic . In this review the various laboratory techniques which have been used to determine the PAE are critically evaluated and compared with the standard viable counting method . The potential sources of error associated with these methods are considered and recommendations are made for the optimum testing system; on the basis of current evidence the bioluminescence and impedence techniques appear to be the most suitable in terms of being the least labour-intensive and producing the most reliable results . Whichever technique is used, the properties of the growth medium, including osmolality and pH, the size of the bacterial inoculum, the initial antibiotic concentration, the exposure time to the antibiotic and the method of antibiotic removal should be standardized . Other post-(antibiotic) exposure events and their relationships with the PAE and theories concerning the mechanism (or mechanisms) by which antibiotics produce PAEs are discussed . Finally, consideration is given to the clinical significance of the PAE and how it, in conjunction with other pharmacodynamic parameters, might be used to allow antibiotic dosing regimens to be developed on a more scientific basis.

Mol Biochem Parasitol, 1993 Oct, 61(2), 197 - 205
Regulation of L-proline transport in Leishmania donovani by extracellular pH; Zilberstein D et al.; We have previously shown that Leishmania donovani promastigotes adapted to long-term culture at acidic pH can serve as a model to study parasite development in a lysosomal-like environment . In this study we investigated the effect of growth pH on L . donovani L-proline transport systems . Reducing the pH of the growth medium causes an up to 7-fold decrease in the extent of L-proline transport . Transport resumes after switching the culture from pH 4.5 to pH 7 for 48 h by a protein synthesis-dependent process . The pH optimum for transport changes from 7.5 in promastigotes grown at pH 7 to 5.5 in cells grown at pH 4.5 . In addition, kinetic analysis of L-proline transport showed that Vmax in pH 4.5-grown L . donovani promastigotes is one-tenth that of cells grown at pH 7 (4.5 and 44.7 nmol min-1 (10(8) cells)-1, respectively) . The apparent Km for L-proline in pH 4.5 promastigotes is one-half of the Km in pH 7 cells (0.30 and 0.65 mM, respectively) . In contrast to L-proline transport, D-glucose transport demonstrates a growth pH-independent activity: Km and Vmax as well as optimum pH of transport are similar in promastigotes grown at either pH 7 or pH 4.5 . Taken together, the results indicate that in L . donovani, expression and activity of L-proline transport is regulated by culture pH . The pH-dependent expression of L-proline transporters may be of physiological significance during the promastigote-amastigote transition.

Yeast, 1993 Oct, 9(10), 1075 - 84
Transcriptional regulation by glucose of the yeast PMA1 gene encoding the plasma membrane H(+)-ATPase; Rao R et al.; The yeast plasma membrane H(+)-ATPase generates a membrane electrochemical gradient which is required for the secondary uptake of nutrients . Although the ATPase has previously been shown to be post-translationally regulated in response to the availability of glucose, there has been no evidence to date for transcriptional regulation of the ATPase gene (PMA1) . In this work, we have examined the pool of newly synthesized ATPase that accumulates in secretory vesicles en route to the cell surface in the temperature-sensitive secretory mutant sec6-4, and have observed changes in the level of ATPase polypeptide as a function of the glucose concentration in the growth medium . In parallel, there were rapid and reversible changes in the levels of ATPase mRNA . Finally, when cells were grown on a variety of carbon sources, the amount of ATPase polypeptide was proportional to the specific growth rate, suggesting that PMA1 expression is adjusted according to the metabolic state of the cell . These results complement the findings of Capieaux et al . (Capieaux, E., Vignais, M.-L., Sentenac, A . and Goffeau, A . (1989) . J . Biol . Chem . 264, 7437-7446), who show that the transcriptional factor TUF/RAP1 binds to upstream activating sequences in the PMA1 gene . Taken together, the results suggest a model in which transcriptional regulation of the ATPase gene by glucose is mediated by TUF/RAP1.

Glia, 1993 Oct, 9(2), 157 - 62
Endogenous opioid systems and the growth of oligodendrocyte progenitors: paradoxical increases in oligodendrogenesis as an indirect mechanism of opioid action; Hauser KF et al.; Endogenous opioids inhibit nervous system development by inhibiting the proliferation of certain neuronal and glial progenitors . To determine whether opioids affect the growth of preoligodendrocytes, the effects of the endogenous opioid {Met5}-enkephalin were examined in preoligodendrocytes in primary mixed-glial and preoligodendrocyte-enriched (> 98% pure) cultures . Proliferating preoligodendrocytes in mixed-glial or preoligodendrocyte-enriched cultures were continuously treated for a total of 40 h with either basal growth media (controls), 1 microM {Met5}-enkephalin, 1 microM {Met5}-enkephalin plus the opioid antagonist naloxone (3 microM), or naloxone alone (3 microM), and incubated in {3H}-thymidine (0.2 microCi/ml/4-6 h) after 34-36 h of opioid exposure . Opioid-dependent changes in DNA synthesis were assessed autoradiographically in O4-immunoreactive oligodendrocyte progenitors . Naloxone alone significantly decreased the rate of DNA synthesis and number of O4-immunoreactive preoligodendrocytes in mixed-glial cultures . However, naloxone and/or {Met5}-enkephalin did not affect DNA synthesis or the number of O4-immunoreactive preoligodendrocytes in cultures enriched in preoligodendrocytes . The results suggest that astrocytes, or perhaps another cell type, play a permissive role in opioid-dependent alterations in preoligodendrocyte proliferation . Endogenous opioids affect the genesis of neural cells by both direct and indirect mechanisms.

Eur J Biochem, 1993 Oct 1, 217(1), 89 - 96
Correlation between the inhibition of cell growth by accumulated polyamines and the decrease of magnesium and ATP; He Y et al.; The mechanism of the antiproliferation effect of spermidine and spermine was studied using a cell culture system of mouse FM3A cells . The addition of either 10 mM spermidine or 2 mM spermine to the growth medium containing 0.9 mM Mg2+ greatly inhibited cell growth (more than 90%) . A decrease in the Mg2+ concentration to 50 microM in the growth medium, but without the polyamine addition, did not influence cell growth . However, the concentrations of spermidine and spermine necessary for the inhibition of cell growth when cells were cultured in the presence of 50 microM Mg2+ were much smaller (2 mM spermidine and 0.15 mM spermine) . Nevertheless, the amount of polyamines accumulating in cells which could cause the inhibition of cell growth was almost the same, regardless of the large difference in the added polyamine concentrations . At the early stage of polyamine accumulation, the inhibition of cell growth correlated with the decrease of Mg2+ content, but not with a decrease of the ATP content . The decrease in Mg2+ content correlated well with the inhibition of macromolecular synthesis, especially protein synthesis . Thus, the inhibition of cell growth at the early stage of polyamine accumulation was thought to be due to the inactivation of ribosomes through the replacement of Mg2+ on magnesium-binding sites by polyamines . The decrease in Mg2+ content was mainly caused by the inhibition of Mg2+ transport by polyamines . At the later stage of polyamine accumulation, a decrease in ATP content was also observed . This was followed by swelling of the mitochondria, which may be a symptom of the subsequent cell death.

Eur J Biochem, 1993 Oct 1, 217(1), 479 - 85
Biotin biosynthesis in higher plant cells . Identification of intermediates; Baldet P et al.; Biotin biosynthesis was investigated in lavender cell cultures (Lavandula vera L.) . Two different biological assays and two different HPLC procedures were used to identify all the intermediates involved in biotin biosynthesis . The pathway for biotin biosynthesis could be analyzed starting with {3H}pimelic acid as precursor, leading to labelled biotin and even to labelled biotinylated enzymes . Intermediates known from the bacterial pathway (7-oxo-8-amino-pelargonic acid, 7,8-diamino-pelargonic acid, dethiobiotin) were present in detectable amounts . Pimelic acid activation to pimeloyl-CoA could be observed . In contrast to bacterial cells, an unknown stable labelled intermediate, named compound A, accumulated . This compound coeluted with an authentic sample of 9-mercaptodethiobiotin from HPLC with an anion-exchange column and was as effective as biotin in supporting the growth of the strain bioB105 of Escherichia coli . When 3H-labelled compound A was added to the growth medium of the lavender cells it was incorporated in an acidomycin-sensitive manner into biotin . {3H}Dethiobiotin was incorporated into both compound A and biotin . These results strongly suggest that, in higher plant cells, the reaction catalysed by biotin synthase may proceed in two distinct steps involving mercaptodethiobiotin (9-mercaptodethiobiotin?) as an intermediate.

Biochim Biophys Acta, 1993 Sep 8, 1169(3), 280 - 90
n-6 and n-3 fatty acid accumulation in thp-1 cell phospholipids; Galella G et al.; The human monocytic leukemia cell line THP-1 is depleted of the long-chain n-6, AA, when compared to human monocytes . This reflects the low availability of this FA in the growth medium generally used for cultured cells . The effects of AA, as well as EPA, supplementation of THP-1 cells on the incorporation of these FA in cell PL, especially in PC and PE, was investigated . In addition the incorporation of labeled AA in PL from THP-1 cells was compared to that in human monocytes . Measurements were done through HPLC separation of PL, detected by UV absorption and radioactivity, FA analysis by GC and characterization of PC subclasses by FAB-MS . Marked differences were observed in the incorporation of the two FA in cell PL, particularly two PC subclasses, and in the accumulation in individual PL after supplementation of THP-1 cells . Accumulation of AA and EPA in THP-1 cells appeared to be mutually independent . The incorporation of AA was also quite different in THP-1 from that in monocytes . Thus, characterization of the FA content in lipids of cultured cells is an essential requirement for optimal utilization of these cells.

J Virol, 1993 Sep, 67(9), 5550 - 61
Characterization of a small (25-kilodalton) derivative of the Rous sarcoma virus Gag protein competent for particle release; Weldon RA Jr et al.; Retroviral Gag proteins have the ability to induce budding and particle release from the plasma membrane when expressed in the absence of all of the other virus-encoded components; however, the locations of the functional domains within the Gag protein that are important for this process are poorly understood . It was shown previously that the protease sequence of the Rous sarcoma virus (RSV) Gag protein can be replaced with a foreign polypeptide, iso-1-cytochrome c from a yeast, without disrupting particle assembly (R . A . Weldon, Jr., C . R . Erdie, M . G . Oliver, and J . W . Wills, J . Virol . 64:4169-4179, 1990) . An unexpected product of the chimeric gag gene is a small, Gag-related protein named p25C . This product was of interest because of its high efficiency of packaging into particles . The goal of the experiments described here was to determine the mechanism by which p25C is synthesized and packaged into particles . The results demonstrate that it is not the product of proteolytic processing of the Gag-cytochrome precursor but is derived from an unusual spliced mRNA . cDNA clones of the spliced mRNA were obtained, and each expressed a product of approximately 25 kDa, designated p25M1, which was released into the growth medium in membrane-enclosed particles that were much lighter than authentic retrovirions as measured in sucrose density gradients . DNA sequencing revealed that the clones encode the first 180 of the 701 amino acids of the RSV Gag protein and no residues from iso-1-cytochrome c . This suggested that a domain in the carboxy-terminal half of Gag is important for the packaging of Gag proteins into dense arrays within the particles . In support of this hypothesis, particles of the correct density were obtained when a small segment from the carboxy terminus of the RSV Gag protein (residues 417 to 584) was included on the end of p25.

J Invest Dermatol, 1993 Sep, 101(3), 275 - 9
Prolactin stimulates proliferation of cultured human keratinocytes; Girolomoni G et al.; The effect of the pituitary hormone prolactin on in vitro proliferation of human keratinocytes has been studied . Cell proliferation was determined by {3H} thymidine incorporation and cell enumeration in culture . Physiologic concentrations of prolactin markedly stimulated proliferation of newborn foreskin keratinocytes in serum-free medium . In addition, it was able to replace almost completely the growth-promoting effects of bovine pituitary extract, a commonly added supplement for keratinocyte culture . This activity was also evident in the absence of epidermal growth factor, but required the presence of insulin . Radioligand-binding studies confirmed the expression of specific prolactin binding sites (Kd 8.9 nM; 1350 sites per cell) on freshly procured keratinocyte membranes . These results extend its hormonal influences to include regulation of in vitro proliferation of human keratinocytes, and suggest the possibility of a completely defined growth medium for keratinocytes.

J Bacteriol, 1993 Sep, 175(17), 5655 - 65
Maltose and maltotriose can be formed endogenously in Escherichia coli from glucose and glucose-1-phosphate independently of enzymes of the maltose system; Decker K et al.; The maltose system in Escherichia coli consists of cell envelope-associated proteins and enzymes that catalyze the uptake and utilization of maltose and alpha,1-4-linked maltodextrins . The presence of these sugars in the growth medium induces the maltose system (exogenous induction), even though only maltotriose has been identified in vitro as an inducer (O . Raibaud and E . Richet, J . Bacteriol., 169:3059-3061, 1987) . Induction is dependent on MalT, the positive regulator protein of the system . In the presence of exogenous glucose, the maltose system is normally repressed because of catabolite repression and inducer exclusion brought about by the phosphotransferase-mediated vectorial phosphorylation of glucose . In contrast, the increase of free, unphosphorylated glucose in the cell induces the maltose system . A ptsG ptsM glk mutant which cannot grow on glucose can accumulate {14C}glucose via galactose permeases . In this strain, internal glucose is polymerized to maltose, maltotriose, and maltodextrins in which only the reducing glucose residue is labeled . This polymerization does not require maltose enzymes, since it still occurs in malT mutants . Formation of maltodextrins from external glucose as well as induction of the maltose system is absent in a mutant lacking phosphoglucomutase, and induction by external glucose could be regained by the addition of glucose-1-phosphate entering the cells via a constitutive glucose phosphate transport system . malQ mutants, which lack amylomaltase, are constitutive for the expression of the maltose genes . This constitutive nature is due to the formation of maltose and maltodextrins from the degradation of glycogen.

Dig Dis Sci, 1993 Sep, 38(9), 1697 - 701
Production of chemoattractant by Helicobacter pylori; Reymunde A et al.; Helicobacter pylori is present in the antral region of the stomach in a majority of patients with gastritis type B . The specific mechanism whereby the organism participates in the development of disease remains uncertain . Since the organism is not invasive, we postulate that H . pylori produces a chemoattractant that recruits inflammatory cells to the antral region of the stomach . H . pylori was grown under microaerophilic conditions at 37 degrees C for 72 hr in Brucella broth containing 1% fetal bovine serum . Culture supernates were harvested after removal of organisms by centrifugation and filtration . The putative chemoattractant in culture supernates as well as that which might be present endogenously in the growth medium (negative control) was assayed against human neutrophils (PMN) in modified Boyden blind-well chambers using 3.0-microns membranes . We found that H . pylori supernates are chemotactic and showed up to 130% activity when compared to the positive chemoattractant control (zymosan-activated serum, a source of C5a) . Minimal activity was observed with virgin growth medium . The chemoattractant activity is proportional to the number of colony forming units (CFU) of H . pylori . Preliminary characterization of the activity shows that the chemoattractant is stable in a boiling water bath for 15 min, activity is lost within 1 hr in acid or alkali, and the chemotactic factor has an approximate molecular weight of 8500 daltons . The factor has no amino-sugar and is negative for the lipid A portion of lipopolysaccharide.(ABSTRACT TRUNCATED AT 250 WORDS)

Immunol Lett, 1993 Sep, 38(1), 47 - 54
Production and characterization of monoclonal antibodies to embryo-associated immunosuppressor factor (EASF) produced by human pre-implantation embryo; Bose R et al.; Balb/c mice were immunized with pre-implantation embryo-associated immunosuppressor factor (EASF) (purified from embryo growth media of in vitro fertilized human ova) . Hybridoma clones were produced by fusing their spleen cells with NS1 and P3X653 myeloma cell lines . The presence of specific anti-EASF monoclonal antibodies (mAb) in the hybridoma culture supernatants were tested by enzyme-linked immunosorbent assay . A total of 15 hybridoma clones were selected, and their products were purified and characterized . Each mAb bound specifically to its antigen in a dose-dependent manner . The affinity-purified EASF from embryo growth media demonstrated immunosuppressive activity on Concanavalin A-induced lymphocytes and the presence of 14 kDa, 24 kDa and 37 kDa factors . No such activity or similar molecules were identified when control growth media were analyzed . This clearly demonstrates that these mAb are indeed EASF-specific and are able to recognize biologically active immunosuppressive components in embryo growth media . These mAbs are presently being tested for the development of EASF-specific assay system.

J Biochem (Tokyo), 1993 Sep, 114(3), 329 - 33
An ecto-enzyme from Sulfolobus acidocaldarius strain 7 which catalyzes hydrolysis of inorganic pyrophosphate, ATP, and ADP: purification and characterization; Amano T et al.; Membranes of Sulfolobus acidocaldarius, a thermoacidophilic archaebacterium, show novel enzymatic activities to hydrolyze PPi, ATP, and ADP at an optimal pH of 3, equal to the growth optimum . The activity increased by about 2-fold on addition of PPi and/or Pi to the growth medium, when yeast extract and casamino acids were removed . The enzyme which hydrolyzes PPi at pH 3 was solubilized and purified by successive chromatographies . The final preparation showed a 26 kDa single band on SDS-PAGE, and a molecular mass of 35 kDa on gel permeation chromatography . The Km and Vmax for PPi were 0.16 mM and 33 mumol Pi released/min/mg at 55 degrees C . ATP and ADP were also good substrates . Divalent cations were not essential for activity . Substrate inhibition at more than 5 mM PPi, ATP or ADP was observed . AMP, glucose-6-phosphate, and p-nitrophenyl phosphate were not hydrolyzed at all . The activity was 4-fold stimulated by addition of the lipid fraction extracted from the organism.

NMR Biomed, 1993 Sep-Oct, 6(5), 297 - 301
Growth and magnetic resonance characteristics of human squamous cell carcinoma xenografts implanted with cells suspended in Matrigel; Czerski L et al.; Growth and magnetic resonance characteristics of a human squamous cell carcinoma SQ20B were studied in vivo as xenografts in nu/nu nude mice . Tumor cells injected subcutaneously in the flank using either Matrigel (MTG, an extract of basement membrane proteins) or growth medium (GM) as a vehicle were compared . Much higher tumor growth rates and cell density were observed with Matrigel than with GM implantation . Histology also showed that MTG implanted cells grew as vascularized solid tumors compared to GM tumors which formed cysts . As a result of increased cell density with the improved method, tumors as small as 0.3 cm3 provide high S/N magnetic resonance spectra which yield smaller standard deviations with fewer experiments.

J Gen Microbiol, 1993 Sep, 139 ( Pt 9), 2027 - 31
A new experimental approach to the search for chemical density factors in the regulation of monoculture growth; Degermendzhy AG et al.; In monocultures of micro-organisms, growth is controlled by feedback mechanisms involving chemical factors such as limiting substrates and inhibitory metabolic products . The role of such feedback in the growth regulation of Escherichia coli O-124 was investigated by growing cells in batch culture using a medium containing glucose and mineral salts . In various phases of growth, portions of the native culture were diluted with culture filtrate, so that although cell density decreased, the chemical composition of the growth medium was unaltered . As the diluted cultures grew, variations in growth acceleration were calculated and compared with those of native (undiluted) cultures . Towards the end of the exponential phase and in the growth deceleration phase, the specific feedback level (FBL) was between -20 and -200 (h g l-1)-1 . The feedback components resulting from changes in glucose concentrations were calculated using experimentally determined values of mu max (0.55 +/- 0.05 h-1) and Ks (2.5 +/- 0.7 mg l-1) . Only 0.1-40% of FBL could be accounted for by changes in glucose concentration, indicating the presence of additional growth regulators . The method developed may become a new tool for determination of growth-regulating cell-density factors in microbial cultures.

J Cell Physiol, 1993 Sep, 156(3), 515 - 21
Epidermal growth factor induces terminal differentiation in human epidermoid carcinoma cells; Konger RL et al.; Previous studies have reported that the proliferation of A431 cells, a human squamous cell carcinoma cell line, was stimulated by picomolar epidermal growth factor (EGF) but inhibited by nanomolar EGF . This biphasic dose-response phenomenon is not observed in normal human epithelial cells where nanomolar EGF is usually mitogenic . We have examined the effects of inhibitory and stimulatory concentrations of EGF on the growth and differentiation of A431 cells . In the presence of 100 pM EGF, A431 cells showed a mild increase in growth rate (129% of control) compared to cells grown in the absence of EGF . At 10 nM EGF, growth inhibition to 63% of control was observed . EGF at 10 nM stimulates a twofold increase both in cornified envelope formation and in epidermal transglutaminase activity, suggesting that high concentrations of EGF induce terminal differentiation in A431 cells . Mitogenic concentrations of EGF (100 pM) had no significant effect on these differentiation markers . Chronic exposure of A431 cells to 20 or 50 nM EGF resulted in EGF-resistant A431 variants that are neither growth arrested nor induced to terminally differentiate by 10 nM EGF . Removal of EGF from the growth medium of the EGF-resistant cells resulted in the reversion of these cells back to the wild-type A431 biphasic response pattern within 2 weeks . Our results suggest that A431 cells have the capacity to non-mutatively alter their response pattern to EGF in vitro to maintain themselves in a state of optimum proliferation and away from terminal differentiation.

Mol Microbiol, 1993 Sep, 9(6), 1297 - 303
Activation of potassium channels during metabolite detoxification in Escherichia coli; Ferguson GP et al.; In bacteria the detoxification of compounds as diverse as methylglyoxal and chlorodinitrobenzene proceeds through the formation of a glutathione adduct . In the Gram-negative bacteria, e.g . Escherichia coli, such glutathione adducts activate one, or both, of a pair of potassium efflux systems KefB and KefC . These systems share many of the properties of cation-translocating channels in eukaryotes . The activity of these systems has been found to be present in a range of Gram-negative bacteria, but not in the glutathione-deficient species of Gram-positive organisms . The conservation of the activity of these systems in a diverse range of organisms suggested a physiological role for these systems . Here we demonstrate that in E . coli cells activation of the KefB efflux system is essential for the survival of exposure to methylglyoxal . Methylglyoxal can be added to the growth medium or its synthesis can be stimulated in the cytoplasm . Under both sets of conditions survival is aided by the activity of KefB . Inhibition of KefB activity by the addition of 10 mM potassium to the growth medium stimulates methylglyoxal-induced cell death . This establishes an essential physiological function for the KefB system.

Comp Biochem Physiol C, 1993 Sep, 106(1), 63 - 70
Metabolism of biogenic monoamines in the ciliated protozoan, Tetrahymena pyriformis; Takeda N et al.; 1 . The three-dimensional HPLC system was used to detect the presence and determine the levels of biogenic monoamines, including precursor amino acids and metabolites, simultaneously in an extract of the ciliated protozoan, Tetrahymena pyriformis . 2 . Representative biogenic monoamines, such as dopamine (DA), 5-hydroxytryptamine (5-HT), epinephrine (E) and kynurenine (KYN) were found to be synthesized in Tetrahymena and released into the growth medium . 3 . The following metabolic pathways were suggested to be operative: (L-DOPA)-DA-(Epinine)-E-dihydroxyphenylethyleneglycol (DOPEG)-vanillylmandelic acid (VMA) and tyrosine-4 (TYR-4)-tyramine (TYRA)-hydroxyphenylacetic acid-4 (HPAC-4) in the case of catecholamines, and tryptophan (TRP)-5-HT and TRP-KYN-xanthurenic acid (XA) in the case of indolalkylamines . 4 . As judged from the released metabolites, systems for generation of biogenic monoamines in Tetrahymena seem to be active during the logarithmic phase of its growth.

Mol Microbiol, 1993 Sep, 9(5), 955 - 63
Aeromonas spp . can secrete Escherichia coli alkaline phosphatase into the culture supernatant, and its release requires a functional general secretion pathway; Wong KR et al.; Aerolysin is a channel-forming protein secreted by Aeromonas hydrophila . To determine if regions of aerolysin could direct the secretion of another protein, portions of aerA were fused to phoA, the Escherichia coli alkaline phosphatase gene and cloned into E . coli, Aeromonas salmonicida, and A . hydrophila . We were surprised to find that secretion of the enzyme by both Aeromonas spp . was independent of the aerolysin segments fused to it . The smallest fusion product contained only the signal sequence and two amino acids of aerolysin . The largest had more than 90% of the aerolysin molecule . The fusion proteins were found in the periplasms of E . coli and A . salmonicida grown in LB medium containing glucose, as well as in the shocked cells . Aerolysin itself was secreted by A . salmonicida under these conditions . In contrast, when A . salmonicida containing any of the fused genes was grown in LB medium without glucose, most of the alkaline phosphatase activity was extracellular, whereas beta-lactamase remained in its normal periplasmic location . Similar results were obtained with A . hydrophila . The change in location of the enzyme in A . salmonicida appeared to be related to the pH of the growth medium . A . salmonicida and A . hydrophila also secreted native E . coli alkaline phosphatase, but A . hydrophila strains with mutations in the general secretion pathway were unable to release the enzyme . We conclude that the Aeromonas secretion system can recognize the E . coli enzyme as an extracellular protein and direct it outside the cell.

J Mol Biol, 1993 Aug 20, 232(4), 1017 - 29
UGA codon context which spans three codons . Reversal by ms2i6A37 in tRNA, mutation in rpsD(S4) or streptomycin; Bjornsson A et al.; Mutant UGA codon contexts which previously have been identified at different positions in the lacI part of a fused lacIlacZ gene were characterized with respect to translational readthrough in another genetic surrounding at a constant location . Although readthrough levels are systematically higher in this new location the "tight/leaky" characteristics of these codon contexts are essentially fully determined by the two codons flanking the nonsense codon itself . Analysis of some UGA hybrid contexts shows that the contribution to the codon context character by the codon either at the 5'-side (CCA or AGC) or at the 3'-side (NGU) is independent of the nature of the codon at the other side of UGA if this codon is decoded by trpT(Su9) suppressor tRNA . In a trpT(Su9), miaA double mutant strain, which lacks the ms2i6A37 modification in this tRNA, suppression is decreased at all UGA contexts investigated . However, in one case the contribution to the codon context character by the determinant flanking at one side is negatively affected by the nature of the codon at the other side of UGA . Thus, the character of a nonsense codon context in this case results from both flanking codons acting in a co-operative manner with the tRNA reading the middle UGA codon . This negative context effect is counteracted by a rpsD12 (ribosomal protein S4) mutation or by a sublethal concentration of streptomycin in the growth medium . It is suggested that the ms2i6A37 base in trpT(Su9) suppressor tRNA increases the efficiency of this tRNA by protecting it from ribosomal proofreading which is induced by codon context.

Eur J Biochem, 1993 Aug 15, 216(1), 269 - 79
Both isoforms of protein phosphatase Z are essential for the maintenance of cell size and integrity in Saccharomyces cerevisiae in response to osmotic stress; Hughes V et al.; The sequences of two genes encoding the protein-serine/threonine-phosphatases PPZ1 and PPZ2 from Saccharomyces cerevisiae have been determined . The molecular masses of PPZ1 and PPZ2 are 77.5 and 78.5 kDa, respectively, and each protein consists of two distinct domains . The C-terminal half of each molecule is 93% identical in PPZ1 and PPZ2, and comprises the protein-phosphatase catalytic domain, while the N-terminal halves, which are rich in serine and asparagine (PPZ1) or serine and arginine (PPZ2), are only 43% identical . Both N-termini start with the amino acids Met-Gly-Asn, suggesting that after removal of the initiating methionine, the N-terminal glycine of the mature protein is myristoylated . Disruption of the gene encoding either PPZ1 or PPZ2 leads to an increase in cell size and cell lysis, the latter being more pronounced in cells disrupted in PPZ1 . Haploid cells carrying a double disruption of PPZ1 and PPZ2 genes also show a marked increase in cell size and cell lysis, which can be significantly reduced by the addition of 1 M sorbitol to the growth medium . These results suggest that PPZ1 and PPZ2 play a role in regulating osmotic stability.

Cancer Res, 1993 Aug 15, 53(16), 3670 - 3
Membrane lipid free radicals produced from L1210 murine leukemia cells by photofrin photosensitization: an electron paramagnetic resonance spin trapping study; Buettner GR et al.; We have detected membrane lipid-derived free radicals from neoplastic cells subjected to Photofrin photosensitization . The presence of the prooxidants iron or iron plus ascorbate in the L1210 cell system increased the intensity of the spin-trapped lipid radical electron paramagnetic resonance spectra and correspondingly decreased cell survival . In addition, raising the proportion of unsaturated lipids in the cell membranes by supplementation of the growth medium with docosahexaenoic acid increased lipid radical formation and decreased cell survival when the L1210 cells were subjected to Photofrin and light . These data educe the hypothesis that the extent of radical generation as well as the efficacy of photodynamic therapy can be increased when prooxidant conditions, which enhance free radical processes, are present in conjunction with photosensitizers that target membrane lipids.

Cryobiology, 1993 Aug, 30(4), 353 - 65
Induction of tolerance to freeze-thaw (FT) damage in mammalian cells by pre-FT hypothermia treatment; Glofcheski DJ et al.; Attached asynchronous exponential phase V79 Chinese hamster cells were pretreated by hypothermic cycling in Hepes growth medium by Method 3 (48 h at 25 degrees C + trypsin) or Method 4 (48 h at 25 degrees C + 3 h at 37 degrees C + trypsin) prior to a freeze-thaw (FT) cycle in Hepes growth medium . Pretreatment by Method 3 or 4 increased the FT survival by a factor of 3.4 . This implies that the 3 h at 37 degrees C, after the 25 degrees C exposure, is not necessary to confer resistance to the subsequent FT cycle in the case of V79 cells . However, with RIF-1 mouse cells, the 3 h at 37 degrees C confers increased resistance . The increase in FT survival of V79 cells after the above pretreatments cannot be accounted for by changes in cell cycle age distribution . No heat shock proteins are produced by this pretreatment . Since pretreatment by Method 3 or 4 also makes the cells resistant to hyperthermia, three other pretreatments, making the cells thermotolerant, were tried . None of these pretreatments resulted in a change in FT survival of the cells . Interaction analysis of FT data, when pretreatment by Method 4 is combined with the presence of DMSO during the FT cycle, indicates that the pretreatment and DMSO act synergistically whether exponential or stationary phase cells are used . Furthermore, the pretreatments and L-glutamine also act synergistically . These pretreatments also increase the FT survival of the RIF-1 mouse cell line; again, pretreatment and DMSO act synergistically . Hence, the method is not limited to cells of hibernating mammals.

Radiat Res, 1993 Aug, 135(2), 160 - 70
Hypertonic treatment during premature chromosome condensation allows visualization of interphase chromosome breaks repaired with fast kinetics in irradiated CHO cells; Iliakis G et al.; A class of interphase chromosome breaks was visualized in irradiated (10 Gy) plateau-phase CHO cells after treatment (2-30 min) in hypertonic (500 mM NaCl) growth medium during the period normally allowed for chromosome condensation, in the premature chromosome condensation (PCC) assay . Rejoining of this class of interphase chromosome breaks was fast (t1/2 = 1.5 min) compared to the rejoining of interphase chromosome breaks normally observed in the absence of hypertonic treatment (t1/2 = 76 min), suggesting that they are formed from a different subset of precursor DNA lesions . A fast (t1/2fast = 12 min) and a slow (t1/2slow = 71 min) component were also observed in the rejoining of radiation-induced (50 Gy) DNA double-strand breaks (DSBs), as measured by pulsed-field gel electrophoresis . We propose that fast-repairing DSBs are the precursor lesions underlying the fast-repairing interphase chromosome breaks observed in these experiments . Slowly repairing DSBs are postulated to be the precursor lesions underlying the slowly repairing interphase chromosome breaks visualized using regular protocols for PCC . The visualization of fast-repairing interphase chromosome breaks achieved in these experiments is assumed to be due to a destabilization of chromatin by the hypertonic medium . This chromatin destabilization may cause either an inhibition of the rejoining of the fast component of DSBs during the period allowed for PCC or a transformation of a defined subset of fast-repairing DSBs into chromosome breaks . The latter hypothesis allows a more consistent interpretation of the available results . Transformation of a defined subset of fast-repairing DSBs to interphase chromosome breaks may be equivalent to damage fixation, and may correspond to the fixation of a form of PLD (beta-PLD) sensitive to treatment in hypertonic medium.

Biochem J, 1993 Aug 1, 293 ( Pt 3), 675 - 82
Biosynthesis of 3,4-didehydroretinol from retinol by human skin keratinocytes in culture; Rollman O et al.; The uptake and metabolism of radiolabelled retinol was studied in cultivated human skin cells . Normal epidermal keratinocytes in primary culture were able to incorporate unbound {11,12-3H}all-trans-retinol from the growth medium and transform it into 3,4-didehydroretinol (dehydroretinol) in a dose- and time-dependent manner . A total of 23% of the radioactive label became cell-associated during a 48-h incubation period when added at 7 nM to differentiated keratinocytes submerged in serum-containing, high-calcium (1.56 mM) culture medium . At that time point, 25-30% of cell-bound radioactive retinol had been converted into dehydroretinol, with no labelled retinal, dehydroretinal, retinoic acid or dehydroretinoic acid being detected in cells or medium . Thus dehydroretinol, which occurs physiologically in mammalian skin tissue in vivo, was identified as the predominant neutral retinol metabolite in cultured keratinocytes using h.p.l.c . and anhydro-derivatization procedures . At least 94% of the product, along with its precursor, was present in the cells in esterified form, with no traces of the compound being secreted into the cell environment . The rate of formation of dehydroretinol from its precursor was significantly lower in keratinocytes grown in serum-free, low-calcium (0.09 mM) culture medium, and in medium pre-incubated with excess unlabelled substrate . Furthermore, the application of 13-cis-retinoic acid (isotretinoin), a therapeutic retinoid drug known to markedly reduce dehydroretinol levels in human skin, blocked the biosynthesis of this metabolite in cultured keratinocytes . The 3,4-dehydrogenation pathway observed in this study could not be shown to operate to any significant extent in cultures of human epidermal melanocytes or dermal fibroblasts, supporting the hypothesis that keratinocytes represent the principal cell type involved in dehydroretinol formation from retinol in human skin.

Biochem Mol Biol Int, 1993 Aug, 30(6), 1143 - 52
Carboxymethylcellulase from Lenzites saepiaria, a brown-rotter; Bhattacharjee B et al.; Three wood-rotting fungi namely, Lenzites saepiaria, Polyporus xeranticus and Trametes gibbosa, were screened as cellulose-degraders from 20 different genera of both brown and white-rotters of Polyporaceae on the basis of their potential to degrade carboxymethylcellulose . The utilization of different carbon sources in the growth medium was studied with these fungi for identification of enzymes involved in saccharification . Carboxymethylcellulase and beta-glucosidase were identified as the two major enzymes involved in this process . Extracellular carboxymethylcellulase from L . saepiaria was purified to homogeneity and the enzyme partially characterized.

Mol Cell Biol, 1993 Aug, 13(8), 5010 - 9
The immunosuppressant FK506 inhibits amino acid import in Saccharomyces cerevisiae; Heitman J et al.; The immunosuppressants cyclosporin A, FK506, and rapamycin inhibit growth of unicellular eukaryotic microorganisms and also block activation of T lymphocytes from multicellular eukaryotes . In vitro, these compounds bind and inhibit two different types of peptidyl-prolyl cis-trans isomerases . Cyclosporin A binds cyclophilins, whereas FK506 and rapamycin bind FK506-binding proteins (FKBPs) . Cyclophilins and FKBPs are ubiquitous, abundant, and targeted to multiple cellular compartments, and they may fold proteins in vivo . Previously, a 12-kDa cytoplasmic FKBP was shown to be only one of at least two FK506-sensitive targets in the yeast Saccharomyces cerevisiae . We find that a second FK506-sensitive target is required for amino acid import . Amino acid-auxotrophic yeast strains (trp1 his4 leu2) are FK506 sensitive, whereas prototrophic strains (TRP1 his4 leu2, trp1 HIS4 leu2, and trp1 his4 LEU2) are FK506 resistant . Amino acids added exogenously to the growth medium mitigate FK506 toxicity . FK506 induces GCN4 expression, which is normally induced by amino acid starvation . FK506 inhibits transport of tryptophan, histidine, and leucine into yeast cells . Lastly, several genes encoding proteins involved in amino acid import or biosynthesis confer FK506 resistance . These findings demonstrate that FK506 inhibits amino acid import in yeast cells, most likely by inhibiting amino acid transporters . Amino acid transporters are integral membrane proteins which import extracellular amino acids and constitute a protein family sharing 30 to 35% identity, including eight invariant prolines . Thus, the second FK506-sensitive target in yeast cells may be a proline isomerase that plays a role in folding amino acid transporters during transit through the secretory pathway.

Cancer Lett, 1993 Jul 30, 71(1-3), 43 - 50
Antiproliferative action of the steroid RU486 in cultured human lymphoma cells; Schuster C et al.; The antiproliferative properties of the synthetic steroid RU486 on the human lymphoma cell line Daudi are described . In suspension cultures, RU486 (10 microM) caused a time dose and cell density dependent reduction of cell proliferation . This effect was reversed within 48 h of withdrawal of RU486 from the growth medium . High concentrations of foetal calf serum can mask the inhibitory effect . In semi-solid cultures RU486 also impaired cell proliferation . Thus RU486 was able to suppress in this tumor cell line the expression of some properties frequently associated with the transformed status of the cells.

Brain Res Dev Brain Res, 1993 Jul 16, 74(1), 83 - 8
Neurite promoting activity of insulin, insulin-like growth factor I and nerve growth factor on spinal motoneurons is astrocyte dependent; Ang LC et al.; Mouse motoneurons were isolated from dissociated E15 mouse spinal cord and grown on polyornithine-coated round coverslips in a growth medium (DMEM/F12) supplemented with progesterone, trans-ferrin, selenium, horse serum and muscle extract . Astrocytes from newborn mouse neopallium were grown on rectangular coverslips . The motoneuron neurite growth was determined at day 8 of culture by counting, using the light microscope, the intersections produced by neurites radiating from the perikaryon placed centrally in a graticule eyepiece of concentric circles . The mean intersections for cultures without addition of astrocytes, insulin, insulin-like growth factor I (IGF-I) or nerve growth factor (NGF) was 12.6 +/- 0.8 . When astrocytes on a separate coverslip were introduced from day 1, there was a small increase in neurite growth (16.3 +/- 0.9) . The neurite growth was further increased significantly with the addition of insulin (27.3 +/- 1.4), IGF-I (31.5 +/- 1.4) or NGF (21.8 +/- 1.1) to cultures with astrocytes . Insulin, IGF-I or NGF in the absence of astrocytes did not greatly increase the neurite growth . We conclude that insulin, IGF-I and NGF promote neurite growth through some interactions with astrocytes.

Gene, 1993 Jul 15, 129(1), 129 - 34
Synthesis and expression of a DNA encoding the Fv domain of an anti-lysozyme monoclonal antibody, HyHEL10, in Streptomyces lividans; Ueda Y et al.; A secretory production system for the Fv domain of a monoclonal antibody (mAb) against hen egg-white lysozyme (HEL) was established in Streptomyces lividans using a chemically synthesized gene . The synthetic DNAs encoding the Fv fragments (VH and VL) of the anti-HEL mAb, HyHEL10, were fused to DNA encoding the signal peptide of Streptomyces subtilisin inhibitor (SPssi) in an SPssi::VH-SPssi::VL dicistronic arrangement . The genes were expressed under the control of the ssi promoter using S . lividans as host . Each Fv fragment was accurately processed and secreted into the growth medium . No inclusion bodies were produced . The Fv fragments were isolated from culture supernatant by a two-step purification (affinity chromatography and gel filtration) with a high yield (approx . 1 microgram/ml) . Purified Fv fragments bound to HEL specifically, and completely inhibited the catalytic activity of HEL at a molar ratio of 1.25 for Fv vs . HEL.

Ugeskr Laeger, 1993 Jul 5, 155(27), 2154 - 5
{Septicemia caused by Malassezia furfur}; Bjerregaard-Andersen H et al.; A case of septicaemia caused by Malassezia furfur in a newborn patient receiving intravenous nutrition is presented . M . furfur, the well known cause of pityriasis versicolor, is a strict lipophilic yeast, with predilection for indwelling catheters conducting lipid solutions . Since the diagnosis of M . furfur is hampered by the slow growth and the requirement of lipid containing growth media, one must keep the organism in mind when signs of septicaemia occur in a patient receiving parenteral nutrition, especially in premature infants.

Antimicrob Agents Chemother, 1993 Jul, 37(7), 1536 - 8
Ethambutol inhibition of glucose metabolism in mycobacteria: a possible target of the drug; Silve G et al.; The addition of D-arabinose, D-galactose, D-glucosamine, or D-mannose to the growth medium of Mycobacterium smegmatis suppressed the inhibitory effects of ethambutol both on acetate labeling of cell wall-linked mycolic acids and on the increase in the delipidated cell dry weight . The addition of D-glucose or D-fructose had no effect . It is proposed that ethambutol inhibits an early step of glucose conversion into the monosaccharides used for the biosynthesis of structurally and biologically important cell wall polysaccharides: arabinogalactan, arabinomannan, and peptidoglycan.

J Med Entomol, 1993 Jul, 30(4), 820 - 3
Egg yolk and bacteria growth medium for Musca domestica (Diptera: Muscidae); Watson DW et al.; Six preparations of egg yolk media inoculated with Escherichia coli (Migula) were evaluated as an in vitro growth system for immature house flies, Musca domestica L . Larval development, based on pupation and eclosion success, was normal on all media supplemented with E . coli, but few larvae survived without bacteria (4%) . Pupation (72%) and eclosion (63%) were significantly higher on egg yolk media without mannitol than on media with mannitol or on blood agar controls.

Curr Genet, 1993 Jul-Aug, 24(1-2), 38 - 44
Cloning and expression of Hormoconis resinae glucoamylase P cDNA in Saccharomyces cerevisiae; Vainio AE et al.; A cDNA coding for glucoamylase P of Hormoconis resinae was cloned using a synthetic oligonucleotide probe coding for a peptide fragment of the purified enzyme and polyclonal anti-glucoamylase antibodies . Nucleotide-sequence analysis revealed an open reading frame of 1848 base pairs coding for a protein of 616 amino-acid residues . Comparison with other fungal glucoamylase amino-acid sequences showed homologies of 37-48% . The glucoamylase cDNA, when introduced into Saccharomyces cerevisiae under the control of the yeast ADC1 promoter, directed the secretion of active glucoamylase P into the growth medium.

J Clin Microbiol, 1993 Jul, 31(7), 1882 - 5
Development and evaluation of a blood-free medium for determining growth curves and optimizing growth of Rochalimaea henselae; Schwartzman WA et al.; Two strains of Rochalimaea henselae were used to optimize a blood-free growth medium . Seven agar bases, four broths, and combinations of eight supplements were evaluated . Acceptable growth was achieved in media containing Fildes solution and hemin, with the best growth demonstrated in brucella broth or on brucella agar with 6 to 8% Fildes solution and 250 micrograms of hemin per ml . R . henselae utilized hemin in concentrations six times that utilized by Rochalimaea quintana . Erythrocyte membrane was necessary to achieve the full growth-promoting effect of rabbit blood.

J Bacteriol, 1993 Jul, 175(14), 4528 - 37
Regulation of kdp operon expression in Escherichia coli: evidence against turgor as signal for transcriptional control; Asha H et al.; Kdp, an inducible high-affinity K+ transporter in Escherichia coli, is encoded by genes of the kdpABC operon, and its expression is regulated by the products of kdpD and kdpE . Loss of cell turgor has been proposed to be the signal which induces kdp expression (L . A . Laimins, D . B . Rhoads, and W . Epstein, Proc . Natl . Acad . Sci . USA 78:464-468, 1981) . We reexamined kdp expression during steady-state growth under a variety of conditions and were able to confirm earlier observations which had indicated that it is primarily affected by the concentration of K+ in the medium and by mutations in genes encoding various K+ transporters in E . coli . Changes in pH of the culture also altered kdp expression . In all of these cases, an increase in {K+} of the medium repressed the operon . Several ionic solutes induced steady-state kdp expression (but to differing extents), whereas nonionic solutes had no effect, indicating that kdp expression is not determined by osmolarity of the growth medium . kdp expression during steady-state growth was shown also to be unaffected by the accumulation of other intracellular compatible solutes such as trehalose or glycine betaine, which would be expected to restore cell turgor during growth in high-osmolarity media . Two mutants that are defective in perception of the signal regulating kdp were isolated, and the mutation in each of them was mapped to the kdpDE regulatory locus . Analysis of kdp expression in one of these mutants provided additional evidence against the turgor regulation model . On the basis of these data, we discuss alternative candidates that might serve as the signal for control of kdp operon transcription.

Biochem J, 1993 Jul 1, 293 ( Pt 1), 115 - 8
The isolation and identification of 6-hydroxycyclohepta-1,4-dione as a novel intermediate in the bacterial degradation of atropine; Bartholomew BA et al.; Growth of Pseudomonas AT3 on the alkaloid atropine as its sole source of carbon and nitrogen is nitrogen-limited and proceeds by degradation of the tropic acid part of the molecule, with the metabolism of the tropine being limited to the point of release of its nitrogen . A nitrogen-free compound accumulated in the growth medium and was isolated and identified as 6-hydroxycyclohepta-1,4-dione . This novel compound is proposed as an intermediate in tropine metabolism . It served as a growth substrate for the organism and was also the substrate for an NAD(+)-linked dehydrogenase present in cell extracts . The enzyme was induced during the tropine phase of diauxic growth on atropine or during growth on tropine alone.

Mol Cell Biol, 1993 Jul, 13(7), 4391 - 9
ADH2 expression is repressed by REG1 independently of mutations that alter the phosphorylation of the yeast transcription factor ADR1; Dombek KM et al.; In Saccharomyces cerevisiae, expression of the ADH2 gene is undetectable during growth on glucose . The transcription factor ADR1 is required to fully activate expression when glucose becomes depleted . Partial activation during growth on glucose occurred in cells carrying a constitutive allele of ADR1 in which the phosphorylatable serine of a cyclic AMP (cAMP)-dependent protein kinase phosphorylation site had been changed to alanine . When glucose was removed from the growth medium, a substantial increase in the level of this constitutive expression was observed for both the ADH2 gene and a reporter construct containing the ADR1 binding site . This suggests that glucose can block ADR1-mediated activation independently of cAMP-dependent phosphorylation at serine 230 . REG1/HEX2/SRN1 was identified as a potential serine 230-independent repressor of ADH2 expression . Yeast strains carrying a deletion of the REG1 gene, reg1-1966, showed a large increase in ADR1-dependent expression of ADH2 during growth on glucose . A smaller increase in ADR1-independent expression was also observed . Additionally, an increase in the level of ADR1 expression and posttranslational modification of the ADR1 protein were observed . When the reg1-1966 allele was combined with various ADR1 constitutive alleles, the level of ADH2 expression was synergistically elevated . This indicates that REG1 can act independently of phosphorylation at serine 230 . Our results suggest that glucose repression in the presence of ADR1 constitutive alleles occurs primarily through a REG1-dependent pathway which appears to affect ADH2 transcription at multiple levels.

Plant Physiol, 1993 Jul, 102(3), 983 - 9
Ammonium inhibition of Arabidopsis root growth can be reversed by potassium and by auxin resistance mutations aux1, axr1, and axr2; Cao Y et al.; A novel effect of ammonium ions on root growth was investigated to understand how environmental signals affect organ development . Ammonium ions (3-12 mM) were found to dramatically inhibit Arabidopsis thaliana seedling root growth in the absence of potassium even if nitrate was present . This inhibition could be reversed by including in the growth medium low levels (20-100 microM) of potassium or alkali ions Rb+ and Cs+ but not alkali ions Na+ and Li+ . The protective effect of low concentrations of potassium is not due to an inhibition of ammonium uptake . Ammonium inhibition is reversible, because root growth was restored in ammonium-treated seedlings if they were subsequently transferred to medium containing potassium . It is known that plant hormones can inhibit root growth . We found that mutants of Arabidopsis resistant to high levels of auxin and other hormones (aux1, axr1, and axr2) are also resistant to the ammonium inhibition and produce roots in the absence of potassium . Thus, the mechanisms that mediate the ammonium inhibition of root development are linked to hormone metabolic or signaling pathways . These findings have important implications for understanding how environmental signals, especially mineral nutrients, affect plant root development.

Cardiovasc Res, 1993 Jul, 27(7), 1229 - 32
Alterations of rabbit aortic smooth muscle cell proliferation in diabetes mellitus; Alipui C et al.; OBJECTIVE: Diabetes mellitus is a known risk factor for atherosclerosis . Because initiation and/or progression of the atherosclerotic process is associated with alterations in vascular smooth muscle cell growth and differentiation, the present studies were conducted to evaluate the effect of diabetes mellitus on the proliferative behaviour of cultured aortic smooth muscle cells . METHODS: Male New Zealand White rabbits were made diabetic with a single intravenous injection of alloxan monohydrate (100 mg.kg-1) in saline . Primary cultures of smooth muscle cells were established from thoracic aortic segments of control and diabetic rabbits and used to develop multiple cell strains . The proliferative capability of secondary cultures was determined by measurements of {3H}-thymidine incorporation into DNA, cell counts, and protein content in control and diabetic cultures . The serum dependence of cellular growth was evaluated by incubation of cultured cells in growth medium supplemented with various fetal calf serum concentrations . RESULTS: Cultures of diabetic origin incorporated thymidine to a greater extent than control cultures . Although the efficiency of cell attachment was not different between control and diabetic cells, diabetic cells had a shorter population doubling time than control cells {41.08(SEM 4.15) h v 58.08(6.79) h} and achieved higher final densities than control cultures . The serum dependence of smooth muscle cell cultures for viability and growth was different between the two groups . CONCLUSIONS: These findings support the hypothesis that diabetes induces changes in vascular smooth muscle cell proliferation which may be associated with the onset or progression of the atherogenic process observed in diabetes.

J Biol Chem, 1993 Jun 15, 268(17), 12427 - 33
Polymorphic regulation of membrane phospholipid composition in Escherichia coli; Rietveld AG et al.; To investigate whether the phase preference of Escherichia coli membrane lipids is regulated by adjustment of the ratio of bilayer to non-bilayer lipids, the lipid biosynthetic mutant AD93 was used . This strain lacks the ability to synthesize phosphatidylethanolamine, a non-bilayer lipid which normally accounts for 70-80% of the phospholipids in the E . coli membrane . The lack of phosphatidylethanolamine is compensated by a large increase in the levels of phosphatidylglycerol and cardiolipin . This strain has an absolute requirement for high concentrations of divalent cations . Since divalent cations are known to induce HII phase formation in cardiolipin model systems, this suggests that cardiolipin in combination with divalent cations replaces phosphatidylethanolamine in the membrane and that it is the non-bilayer forming property of the latter which is important for membrane functioning . The growth of the mutant strain was found to be dependent on the type and concentration of divalent cations present in the growth medium . In media supplemented with MgCl2 and CaCl2, growth was maximal at concentrations of 30-50 mM . In the presence of SrCl2 a growth optimum was observed at 15 mM . The cells did not grow in the presence of BaCl2 or at high concentrations of SrCl2 . Furthermore, this strain was found to adapt the lipid composition of the membrane in reaction to the type of cation present during growth . The phase behavior of dispersions of mutant and parental E . coli derived lipids was studied under a variety of conditions, using 31P NMR . In the presence of the growth-promoting cations at concentrations where there is maximal growth, a bilayer to non-bilayer transition was observed in the lipid dispersions in a narrow temperature range, approximately 10 degrees C above the growth temperature, whereas at concentrations where there is no growth, a bilayer structure was observed at all temperatures tested . This suggests a polymorphic regulation of membrane lipid composition in order to maintain a propensity toward type II structure formation in the membrane.

Eur J Biochem, 1993 Jun 15, 214(3), 935 - 44
The bis(adenosin-N6-yl)alkanes, a family of potential dinucleoside-polyphosphate analogue precursors . Cytotoxicity, adenosine-receptor binding and metabolism; Chen H et al.; A series of bis(adenosin-N6-yl)alkanes, in which two adenosine residues are linked via their N6 positions by alkyl bridges comprising between 2 and 14 methylene units, were synthesized as potential precursors to dinucleoside-polyphosphate analogues . These compounds were moderately cytotoxic to mammalian cells, the toxicity increasing with the length of the alkyl chain . For example, the dose of bis(adenosin-N6-yl)dodecane, A{CH2}12A, leading to 50% inhibition of cell growth (ID50) for BHK fibroblasts, Walker 256 carcinoma cells and S-49 T-lymphoma cells were 90 +/- 8, 100 +/- 5 and 23 +/- 4 microM respectively . A significant amount of A{CH2}12A bound to serum albumin in the growth media; thus the ID50 for S-49 cells grown in serum-free medium was 9 +/- 2 microM . The corresponding bis-cytidine analogues were much less toxic; however the presence of a second adenosine moeity/molecule had little significant effect on cell growth when compared to N6-alkyladenosines . Toxicity to S-49 cells was unaffected by the nucleoside-transporter inhibitor nitrobenzylthioinosine and was even higher (ID50 = 5 +/- 0.5 microM) towards nucleoside-transport-deficient AE-1 cells, showing that the analogues could pass freely through the plasma membrane . Interaction with A1 adenosine receptors was shown by displacement of {3H}N6-R-phenylisopropyladenosine (Kd = 6 nM) from rat adipocyte membranes, with Ki values of 45, 65, 85 and 390 nM for the compounds containing 12, 8, 6 and 4 methylene units, respectively . Affinity for human platelet membrane A2 adenosine receptors was about 100-fold less, however the compounds were weak A2 agonists, producing up to a threefold increase in intracellular cyclic AMP in WI-38/VA-13 cells . Thus, these compounds behave, not surprisingly, as adenosine analogues . In addition, A{CH2}12A was metabolized in vitro and intracellularly by adenosine kinase (Ki = 70 nM) and adenylate kinase to yield a number of phosphorylated derivatives with the potential to act as diadenosine polyphosphate analogues . One of these, the bismonophosphate, was recognized by and inhibited adenylate kinase more effectively than adenosine(5')tetraphospho(5')adenosine (Ap4A, Ki = 3 microM).

J Neurosci Res, 1993 Jun 15, 35(3), 274 - 87
Glial progenitor cells of the nerve fiber layer of the olfactory bulb: effect of astrocyte growth media; Doucette R; There are two morphologically distinct types of glial cells (i.e., ensheathing cells and astrocytes) in the nerve fiber layer (NFL) of the adult mammalian olfactory bulb . Ensheathing cells provide ensheathment for olfactory axons, whereas astrocytes occupy the interfascicular spaces of the olfactory NFL . During embryonic development, however, only one type of glial cell is found in this layer of the olfactory bulb, namely, the ensheathing cell . Even though ensheathing cells take up residence within the CNS, they are actually derived from the olfactory placode . Far less is known about the developmental origin of interfascicular astrocytes, which arise either from the glial progenitor cells that give rise to ensheathing cells or from astrocyte precursor cells that migrate into the NFL from deeper layers of the bulb primordium . In the present study, enriched populations of ensheathing cells were grown in vitro in media known to promote the growth and differentiation of astrocytes to determine whether ensheathing cell progenitors could differentiate into astrocytes . These media failed to induce the appearance of astrocytes in the ensheathing cell cultures . It was concluded that the astrocytes of the NFL most likely arise from progenitor cells that migrate into this layer from deeper parts of the developing bulb.

J Biol Chem, 1993 Jun 15, 268(17), 12818 - 24
Transcriptional regulation by lovastatin and 25-hydroxycholesterol in HepG2 cells and molecular cloning and expression of the cDNA for the human hepatic squalene synthase; Jiang G et al.; Primers, based on the cDNA nucleotide sequences for rat hepatic squalene synthase (EC 2.5.1.21) (McKenzie, T.L., Jiang, G., Straubhaar, J.R., Conrad, D., and Shechter, I . (1992) J . Biol . Chem . 267, 21368-21374), were synthesized and used for the amplification and sequencing of a 1672-base pair (bp) cDNA for the human hepatic squalene synthase (HSS) from human hepatic RNA . An open reading frame of 1251 bp encoding 417 amino acids (M(r) = 48,200) was detected for HSS . We have constructed a pHSS 1286 expression vector by molecular cloning of a 1286-bp cDNA, that includes sequences of the entire coding region for HSS, into pBluescript . Expression in Escherichia coli of a functional, full-length HSS was confirmed by immunoblot analysis and enzymatic activity . Northern blot analyses of poly(A+) RNA obtained from the human hepatoma cell line HepG2 show three distinct size classes of mRNA for HSS . 1.4-, 1.6- and 2.1-kilobase mRNA were observed . The relative abundance is in the order 1.6 > 1.4 > 2.1 and did not change when the cells were grown in the presence of 25-hydroxycholesterol or lovastatin . The ratio between the level of HSS mRNA in cells grown in the absence and presence of 5 micrograms/ml 25-hydroxycholesterol varies between 8- and 16-fold . This lowering of the mRNA level was observed when the cells were grown in 10% of either full serum or lipid-depleted serum . A 2.7- and 4.0-fold increase of HSS mRNA was observed when HepG2 cells were grown in the presence of 5 micrograms/ml lovastatin in lipid-depleted or full serum, respectively . These studies show that HSS exhibit a relatively high level of transcriptional regulation in response to 25-hydroxycholesterol regardless of the presence of cholesterol in the growth media.

J Cell Biol, 1993 Jun, 121(5), 1153 - 63
Accumulation of PDGF B and cell-binding forms of PDGF A in the extracellular matrix; Kelly JL et al.; PDGF is a powerful mitogen initially identified within platelets, but also shown to be produced by a wide variety of cell types . PDGF is encoded on two separate genes . These give rise to three polypeptides, PDGF B and two forms of PDGF A (SA and LA), resulting from alternative splicing of the PDGF A gene primary transcript . We report that in CHO cells transfected with PDGF gene constructs and producing moderate levels of PDGF homodimers, much of the PDGF LA and B produced, but little if any SA, is found in the matrix laid down beneath the cells . Immunoreactive PDGF in cells, and in matrix below expressing cells, was visualized by laser confocal microscopy . Western blotting of protein in matrix extracts, cell extracts, and secreted into the growth medium was used to demonstrate that the range of PDGF A polypeptides seen in the matrix was overlapping with those reported previously to be cell associated in cell types such as NIH3T3 and COS 7 . This suggests that attachment to matrix or cell surface may be alternative fates for these polypeptides, with fate dependent on the characteristics of the producing cells . Immunoreactive PDGF A and B could be partially released by incubation of matrix material with heparin but not with other glycosaminoglycans . Digestion of matrix with chondroitin ABC lyase but not heparitinase or collagenase displaced some PDGF from its attachment sites . The results indicate attachment of PDGF to matrix proteoglycans, at least partly through the glycosaminoglycan moieties, and perhaps to additional components . The significance of matrix deposition for PDGF action is discussed.

J Bacteriol, 1993 Jun, 175(11), 3380 - 7
Inhibition of expression of the tryptophanase operon in Escherichia coli by extrachromosomal copies of the tna leader region; Gish K et al.; Expression of the tryptophanase (tna) operon in Escherichia coli is regulated by catabolite repression and transcription attenuation . Expression is induced by the presence of elevated levels of tryptophan in a growth medium devoid of a catabolite-repressing carbon source . Induction requires the translation of a 24-residue coding region, tnaC, located in the 319-nucleotide transcribed leader region preceding tnaA, the structural gene for tryptophanase . Multicopy plasmids carrying the tnaC leader region were found to inhibit induction of the chromosomal tna operon . Mutational studies established that this inhibition was not due to inhibited transcription initiation, translation initiation, tryptophan transport, or enzyme activity . Rather, multicopy tnaC plasmids inhibited induction by preventing tryptophan-induced transcription antitermination in the leader region of the tna operon . Translation of the single Trp codon in tnaC of the multicopy plasmids was shown to be essential for this inhibition . We hypothesize that translation of the Trp codon of the leader peptide titrates out a trans-acting factor that is essential for tryptophan-induced antitermination in the chromosomal tna operon . We postulate that this factor is an altered form of tRNATrp.

Plast Reconstr Surg, 1993 Jun, 91(7), 1287 - 93
The inhibition of fibroblast-populated collagen lattice contraction by human amniotic fluid: a chronologic examination; Wider TM et al.; The effect of human amniotic fluid on fetal wound healing remains to be fully elucidated and may lead to the isolation of factors that could modulate adult wound healing . This study uses an in vitro model of wound contraction, the fibroblast-populated collagen lattice, to examine the effects of chronologically sampled human amniotic fluid on contraction of lattices composed of either human adult or fetal fibroblasts . This chronology has not been reported previously . Human amniotic fluid was obtained in a sterile fashion via amniocentesis from 120 different women at different time points in gestation, ranging from 13 to 24 weeks . At each time point of gestation, three to five samples were individually examined in duplicate sets . Only fluid from pregnancies deemed normal by amniocentesis was included . Contaminated specimens were discarded . Using Bell's protocol, lattices were constructed of acid-soluble rat tail collagen, growth medium, and either human adult fibroblasts or human fetal fibroblasts . Lattices contained 20% v/v human amniotic fluid . In the control lattices, phosphate-buffered saline replaced amniotic fluid in equal volumes . Area was measured at 24-hour intervals, and all tests were run in duplicate for each specimen . The mean area at each interval was computed for each gestational week examined . Data were analyzed for significance with ANOVA and Dunnett's t test against control.(ABSTRACT TRUNCATED AT 250 WORDS)

Cell Calcium, 1993 Jun, 14(6), 507 - 16
Intracellular calcium signalling after binding of low-density lipoprotein to confluent and nonconfluent cultures of an endothelial cell line, EA.hy 926; Schaefer HI et al.; The effect of lipoproteins on cellular calcium ion concentration ({Ca2+}i) in EA.hy 926 endothelial cells was investigated, particularly with respect to the difference in response on {Ca2+}i between native low-density lipoprotein (LDL) which binds to the apo B/E receptor, and acetylated LDL (AcLDL) which binds to the scavenger receptor . The scavenger receptor recognizes chemically or cell-induced modified LDL . LDL as well as AcLDL caused a transient increase of {Ca2+}i lasting 1-2 min . On a protein basis, LDL was more effective that AcLDL in raising {Ca2+}i . Preincubation of confluent cultures in growth medium with a reduced fetal bovine serum content (2% FBS instead of 10%) increased the potency of LDL to increase {Ca2+}i . The LDL-induced peak {Ca2+}i was dependent on cell density . The effect of AcLDL on {Ca2+}i did not differ between confluent and nonconfluent cultures . Also, preincubation with 2% FBS did not modify the AcLDL-induced calcium response . We conclude that binding of lipoproteins to membrane lipoprotein receptors is responsible for the transient rise of {Ca2+}i although the characteristics of the calcium response are dependent on the receptor involved, i.e . the apo B/E (LDL) receptor or the scavenger receptor . We suggest that Ca2+ acts as a second messenger, and that the LDL-induced calcium response is controlled by the proliferative state of the cells.

J Gen Microbiol, 1993 Jun, 139 ( Pt 6), 1253 - 9
Processing and secretion by Escherichia coli of a recombinant form of the immunogenic protein MPB70 of Mycobacterium bovis; Hewinson RG et al.; The gene encoding an immunodominant secreted antigen, MPB70, of Mycobacterium bovis was cloned into the plasmid vector pBluescript II KS+ along with its native ribosome-binding site . In this construct translation of the protein in Escherichia coli was from the native AUG initiation codon and was directed by the mycobacterial ribosome-binding site . Two different molecular mass forms (26 kDa and 22 kDa) of MPB70 were observed in whole-cell pellets of recombinant E . coli . The difference in size indicates cleavage of the signal peptide of MPB70 by an endopeptidase of E . coli . MPB70 was secreted into the periplasm of recombinant E . coli, where the 22 kDa form of the protein was predominant . The culture filtrate contained only the 22 kDa form of the protein, which was soluble . The passage of MPB70 from the periplasm into the growth medium was found to be due, at least in part, to non-specific leakage of periplasmic proteins across the outer membrane associated with the expression of recombinant MPB70.

Exp Dermatol, 1993 Jun, 2(3), 121 - 4
Mode of release of interleukin-8 from proliferating human epidermal keratinocytes in vitro; Takematsu H et al.; Keratinocytes have been shown to express interleukin-8 (IL-8) mRNA on stimulation with IL-1 and other substances . This has been assumed to account for the large amount of this neutrophil chemotactic cytokine in psoriasis . We found that, without any added agents, commercially available normal human epidermal keratinocytes proliferating in Keratinocyte Growth Medium (KGM) released a chemotactic peptide extracellularly, which was confirmed to be IL-8 . To determine whether most of the IL-8 is secreted extracellularly from proliferating keratinocytes or is mainly stored to be released only on stimulation . We quantified cell-associated and released immunoreactive IL-8 from keratinocytes cultured in KGM for up to 11 days at the peptide level . The keratinocytes proliferated, taking a sigmoid growth curve, to reach a plateau at day 7 . We found that the amounts of immunoreactive IL-8 gradually increased in the culture supernatant with cell growth but its prominent release took place only after the cell growth reached a plateau . The cell-associated IL-8 was much smaller in amount than that noted in the supernatant . These results suggest that IL-8 constitutively produced by keratinocytes was mostly released extracellular but that the production by actively proliferating cells seems to be far less than that by non-proliferating cells that probably occurred in an autocrine fashion under the stimulation of keratinocyte-derived cytokines accumulated in the culture medium . Neutrophil chemotactic activity assayed concomitantly showed a consistent increase during the culture period, indicating that, with their growth, the keratinocytes release substances other than IL-8 that exert an influence on neutrophil chemotactic functions.

Biochim Biophys Acta, 1993 May 14, 1148(1), 161 - 72
In-vitro stability and cytostatic activity of liposomal formulations of 5-fluoro-2'-deoxyuridine and its diacylated derivatives; van Borssum Waalkes M et al.; The water-soluble antineoplastic agent 5-fluoro-2'-deoxyuridine (FUdR) was encapsulated in the water phase of liposomes of different lipid compositions . The retention of this drug upon storage and during contact with plasma was assessed . It was found that, upon refrigeration, diffusion of FUdR across the liposome bilayer was considerably faster when the drug was encapsulated in fluid-type liposomes (egg PC/PS/CHOL) than in solid-type liposomes (DSPC/DPPG/CHOL) . With either composition, leakage of the drug from the liposomes was accelerated upon contact with plasma . To achieve improved liposomal retention of the drug, FUdR was converted to a lipophilic prodrug by esterifying the free hydroxyl groups in the deoxyribose moiety with fatty acids of different chain lengths . Thus FUdR-dipalmitate (C-16) and FUdR-dioctanoate (C-8) were synthesized and incorporated in liposomes . The dipalmitoyl derivative could be incorporated upto 13 mol% in solid-type liposomes but to only 2 mol% in fluid-type liposomes . Freeze-fracture electron microscopy revealed no major differences between control liposomes and those containing the prodrug . FUdR-dipalmitate was found to be firmly associated with the liposomal bilayer in both liposome-types: no exchange of the pro-drug with blood constituents or hydrolysis by serum esterases could be registered when the liposomes were incubated with serum . On the other hand, liposome-incorporated FUdR-dioctanoate was found to be readily extracted from the liposomes by serum components (predominantly albumin) and was found to be degraded rapidly by serum esterase activity . The antitumor activity of FUdR-prodrugs was determined using C26 colon adenocarcinoma cells . This cell line was found to be highly sensitive to FUdR . Liposomal FUdR-dioctanoate inhibited cell growth in the same concentration range as unesterified FUdR . FUdR-dipalmitate, however, was more than two orders of magnitude less potent in inhibiting cell proliferation . Its antiproliferative activity was dependent on the liposome-type used: when incorporated in fluid-type liposomes, antiproliferative activity of FUdR-dipalmitate was several-fold higher than in solid-type liposomes . The difference in antitumor activity between FUdR-dipalmitate and FUdR-dioctanoate and between FUdR-dipalmitate in the fluid- and solid-type liposomes could be explained by differences in the rate of hydrolysis of the prodrugs to FUdR by esterase activity in the tumor cells or in the growth medium.

Appl Environ Microbiol, 1993 May, 59(5), 1642 - 6
Regulated expression of the nor-1 and ver-1 genes associated with aflatoxin biosynthesis; Skory CD et al.; RNA transcript accumulation for the ver-1 and nor-1 genes, which are associated with aflatoxin biosynthesis in the fungus Aspergillus parasiticus, was measured before and during aflatoxin production in liquid shake culture . Transcripts were not detected until near the end of trophophase (growth phase) and could still be observed well into stationary phase during batch fermentation in an aflatoxin-supporting growth medium . Maximum accumulation of both transcripts occurred just prior to the onset of stationary phase . Aflatoxin B1 was first detected approximately 8 h after the appearance of the ver-1 and nor-1 transcripts . In contrast, maximum transcript accumulation for the pyrG gene (encoding orotidine monophosphate decarboxylase), which is involved in primary metabolism, was observed at the onset of trophophase when the ver-1 and nor-1 transcripts could not be detected . Accumulation of the ver-1 and nor-1 transcripts was also studied following a nutritional shift from a non-aflatoxin-supporting medium (peptone mineral salts {PMS}) to a glucose-containing medium (glucose mineral salts {GMS}), which does support aflatoxin biosynthesis . Transcripts from ver-1 and nor-1 could not be detected on PMS medium but did accumulate approximately 4 to 7 h following transfer to GMS medium . Additionally, aflatoxins were not detected in PMS medium but were observed to accumulate within 24 h after the shift from PMS to GMS . These data suggest that aflatoxin biosynthesis is in part regulated by the accumulation of the ver-1 and nor-1 transcripts.

Exp Hematol, 1993 May, 21(5), 675 - 82
Inhibitors of hematopoietic colonies are produced by certain rat fibroblastoid cell lines and are modulated by corticosteroids; Wang H et al.; Both stimulatory (CSA) and inhibitory (INH) factors may contribute to hematopoietic regulation, but little is known about how their physiologic balance is maintained . Previously we have shown that antigen-defined fibroblastoid cells cultured from rat lung (ST3-/ST4+) constitutively produce INH, and those derived from bone marrow (ST3+/ST4-) respond to macrophage cytokines to release both CSA and INH into their conditioned media (CM) . Here we show that this pattern was maintained in cell strains ("ST3" and "ST4") propagated from the primary cultures, and that the presence of CSA was measured in "ST4" CM if the inhibitory > 100 kd fraction was removed . Two subclones of the "ST3" line, called 2A and 9D, were selected for high or low expression of the ST3 antigen, respectively . Both produced CSA, but only 9D produced the > 100 kd inhibitor . In the CM of cells cultured in the presence of hydrocortisone, there was less INH detected but CSA was not changed . From these data, however, we cannot assess how many individual cell products might be contributing to the INH activity . These results demonstrate that the appearance of inhibitory activity in the growth media differs among fibroblast subpopulations, and that it can be modified by natural regulators such as corticosteroids.

Mol Gen Genet, 1993 May, 239(1-2), 273 - 80
Dual regulation by heat and nutrient stress of the yeast HSP150 gene encoding a secretory glycoprotein; Russo P et al.; We have cloned and characterized the HSP150 gene of Saccharomyces cerevisiae, which encodes a glycoprotein (hsp150) that is secreted into the growth medium . Unexpectedly, the HSP150 gene was found to be regulated by heat shock and nitrogen starvation . Shifting the cells from 24 degrees C to 37 degrees C resulted in an abrupt increase in the steady-state level of the HSP150 mRNA, and de novo synthesized hsp150 protein . Returning the cells to 24 degrees C caused a rapid decrease in mRNA and protein synthesis to basal levels . The HSP150 5'-flanking region contains several heat shock element-like sequences (HSE) . To study the function of these sequences, a strain bearing a disrupted copy of the HSP150 gene was transformed with plasmids in which the coding region of HSP150, or a HSP150-lacZ fusion gene, was preceded by 5' deletion derivatives of the HSP150 promoter . Site-directed mutagenesis of one HSE-like element, located between the TATA box and transcription initiation sites, abolished heat activation of transcription . In addition to heat shock, the HSP150 gene is regulated by the availability of nutrients in the growth medium . The HSP150 mRNA level was increased by nitrogen limitation at 24 degrees C, even when under the control of a HSP150 promoter region of 137 bp carrying the mutagenized HSE.

Anat Rec, 1993 May, 236(1), 129 - 35
Cholinergic-nicotinic control of growth and secretion of cultured pulmonary neuroendocrine cells; Nylen ES et al.; Dispersed newborn hamster lung cells were established in vitro in a defined, low-serum growth medium . Neuroendocrine markers (immunohistochemistry for bombesin/gastrin-releasing peptide and calcitonin) revealed a cellular predominance of pulmonary neuroendocrine (PNE) cells . While the supernatant concentration remained stable, the concentration of PNE cell immunoreactive calcitonin (iCT) gradually declined over 4 weeks . Supplementation of the medium with nicotine for 3 weeks prevented this decline in cellular iCT . Concurrently, the number of cells and {3H}thymidine incorporation were significantly increased . The stimulatory effect of chronic nicotine was reversed by the coadministration of the nicotinic antagonist hexamethonium . In another set of experiments, prior multiple transplacental nicotine pretreatments resulted in a significant increase in iCT in the lungs of newborns; when these lungs were subsequently placed in cell culture without nicotine, despite the higher concentration of iCT, there was a drop in iCT similar to that observed in the control culture . In contrast, in vivo, the lung iCT remained significantly elevated at 1 week post-parturition . Cell culture supernatants were analyzed at week 4 for the evoked release of iCT; cholinergic-nicotinic agonists promptly increased the supernatant iCT, which was blocked by nicotinic but not by muscarinic antagonists . We suggest that this in vitro system provides a useful tool to study directly the PNE cell . The acute and chronic effects of nicotine are most likely related to stimulation of cholinergic-nicotinic receptors on iCT-containing PNE cells.

J Periodontal Res, 1993 May, 28(3), 161 - 5
Cell cycle-specific growth inhibitory effect on human gingival fibroblasts of a toxin isolated from the culture medium of Actinobacillus actinomycetemcomitans; Helgeland K et al.; A toxin isolated from the growth medium of Actinobacillus actinomycetemcomitans by ammonium sulfate precipitation was shown to inhibit irreversibly the multiplication of human gingival fibroblasts . DNA histograms from flow cytometric measurements showed that the cells accumulated preferentially in the G2 phase of the cell cycle . Such cells exhibited sheetlike protrusions, and an increased frequency of micronuclei was evident in cells treated with low concentrations of the toxin . Toxin-treated cells were viable for several weeks, as shown by staining with trypan blue and fluorescein diacetate, and the general cell metabolism as measured by oxygen consumption was unimpaired.

J Gen Microbiol, 1993 May, 139 ( Pt 5), 987 - 93
Cloning, sequence analysis and expression in Escherichia coli of a gene encoding an alginate lyase from Pseudomonas sp . OS-ALG-9; Maki H et al.; A gene (aly) encoding alginate lyase (ALY; EC 4.2.2.3) was isolated from a library constructed with the cosmid vector pHC79 and Sau3AI-digested genomic DNA of Pseudomonas sp . OS-ALG-9 . Successive subcloning of the aly-containing cosmid enabled us to locate the gene on a 2.3 kb HpaI fragment . Nucleotide sequencing of this fragment revealed a single open reading frame (ORF) of 1365 bp . The directly determined N-terminal amino acid sequence of the ALY protein purified from Pseudomonas sp . OS-ALG-9 was found in the amino acid sequence deduced from this ORF between nucleotides 282 and 366 . Expression of aly was induced by IPTG in Escherichia coli and leakage of the enzyme into the extracellular milieu was significantly enhanced by addition of glycine to the growth medium . The ALY enzyme had a greater specificity for the homopolymer of mannuronate than for that of guluronate.

J Gen Microbiol, 1993 May, 139 ( Pt 5), 897 - 906
Regulation of carotenoid and bacteriochlorophyll biosynthesis genes and identification of an evolutionarily conserved gene required for bacteriochlorophyll accumulation; Armstrong GA et al.; The temporal expression of ten clustered genes required for carotenoid (crt) and bacteriochlorophyll (bch) biosynthesis was examined during the transition from aerobic respiration to anaerobiosis requisite for the development of the photosynthetic membrane in the bacterium Rhodobacter capsulatus . Accumulation of crtA, crtC, crtD, crtE, crtF, crtK, bchC and bchD mRNAs increased transiently and coordinately, up to 12-fold following removal of oxygen from the growth medium, paralleling increases in mRNAs encoding pigment-binding polypeptides of the photosynthetic apparatus . The crtB and crtI genes, in contrast, were expressed similarly in the presence or absence of oxygen . The regulation patterns of promoters for the crtA and crtI genes and the bchCXYZ operon were characterized using lacZ transcriptional fusion and qualitatively reflected the corresponding mRNA accumulation patterns . We also report that the bchI gene product, encoded by a DNA sequence previously considered to be a portion of crtA, shares 49% sequence identity with the nuclear-encoded Arabidopsis thaliana Cs chloroplast protein required for normal pigmentation in plants.

Biophys J, 1993 May, 64(5), 1602 - 9
Monitoring electropermeabilization in the plasma membrane of adherent mammalian cells; Ghosh PM et al.; When an electrical potential of order one volt is induced across a cell membrane for a fraction of a second, temporary breakdown of ordinary membrane functions may occur . One result of such a breakdown is that molecules normally excluded by the membrane can now enter the cells . This phenomenon, generally referred to as electropermeabilization, is known as electroporation when actual pores form in the membrane . This paper presents a unique approach to the measurement of pore formation and closure in anchored mammalian cells . The cells are cultured on small gold electrodes, and by constantly monitoring the impedance of the electrode with a low-amplitude AC signal, small changes in cell morphology, cell motion, and membrane resistance can be detected . Because the active electrode is small, the application of a few volts across the cell-covered electrode causes pore formation in the cell membrane . In addition, the heat transfer is very efficient, and the cells can be porated in their regular growth medium . By this method, the formation and resealing of pores due to applied electric fields can be followed in real time for anchorage-dependent cells.

In Vitro Cell Dev Biol Anim, 1993 May, 29A(5), 408 - 14
Growth of separated and recombined neonatal rat uterine luminal epithelium and stroma on extracellular matrix: effects of in vivo tamoxifen exposure; Branham WS et al.; We have developed a system for serum-free culture of separated uterine epithelium and stroma from 11-day-old rats recombined on extracellular matrix extracted from Englebreth-Holm-Swarm tumors . Epithelium grew and, after 2 days in culture, developed into luminal epithelial spheres (LES) surrounding a fluid-filled lumen . Individual LES cells maintained epithelial cell characteristics such as basally located nuclei, apical microvilli (oriented toward the lumen), lateral membranes with interdigitations and desmosomes, secretory Golgi complexes, and abundant mitochondria and rough endoplasmic reticulum . Secretory vesicles were ubiquitous throughout the luminal fluid . Addition of 17 beta-estradiol to the growth medium increased the number and longevity of the LES . Prior exposure of uteri to tamoxifen via s.c . injection in vivo on postnatal Days 1 to 5 reduced or completely inhibited formation of LES in vitro . These effects occurred regardless of whether the stromal or epithelial component of the recombinant tissue was exposed to tamoxifen . These data suggest a directive property of neonatal stroma in culture resulting in the formation of highly secretory spherical epithelial structures completely enclosing a lumen . LES formation is responsive to both estrogen (positive response) and antiestrogen (negative response).

Res Microbiol, 1993 May, 144(4), 317 - 26
Proteolytic and oxidoreductase activity of Treponema denticola ATCC 35405 grown in an aerobic and anaerobic gaseous environment; Syed SA et al.; The cells of a human oral spirochete, Treponema denticola ATCC 35405, and of seven clinical isolates of this organism obtained from the subgingival dental plaque of periodontitis patients were studied for their ability to grow in an aerobic and an anaerobic environment, and for their profile of peptidohydrolase and oxidoreductase enzymes . The growth yield of aerobically grown cultures was either comparable to or higher than that of anaerobically grown ones regardless of whether prereduced broth, freshly prepared broth or oxidized broth was used . However, elimination of certain supplements from the growth media resulted in poor growth regardless of the nature of the gaseous environment . The microscopic morphology and motility of the cells were not affected by differences in the gaseous atmosphere . Quantitative studies on several peptidohydrolase activities suggest that anaerobically grown cells displayed higher specific activity especially toward N alpha-L-prolyl-2-naphthylamine, indicating that increased synthesis of proline iminopeptidase enzymes (or enzyme) of the cells was associated with anaerobic growth conditions . The formation of enzymes hydrolysing N alpha-benzoyl-DL-arginyl-2-naphthylamine (and the corresponding p-nitroaniline) was not affected to the same extent . Growth experiments suggest that T . denticola ATCC 35405 is a facultatively anaerobic spirochete instead of an obligate anaerobe as reported in previous literature . The quantitative enzyme studies suggest that the gaseous growth atmosphere of the cells can exert a selective effect on the activity levels of certain peptidolytic enzymes of this organism . Such effects were not observed when the whole cells were studied by means of qualitative or semi-quantitative enzyme tests . The activities of catalase, peroxidase and superoxide dismutase of the cells were low and variable . Because of this, it was not possible to relate these oxidoreductase activities to the composition of the gaseous atmosphere.

Biochem Biophys Res Commun, 1993 Apr 30, 192(2), 714 - 9
p53 gene is up-regulated during skeletal muscle cell differentiation; Halevy O; Differentiation of cells in the mouse myogenic cell line C2, or a primary culture of chicken satellite cells was induced by low serum levels in the growth medium . Endogenous wild-type p53 mRNA was substantially expressed after approximately 5 h of incubation . Induction of p53 mRNA expression was also observed in cells treated with 10(-8) M retinoic acid, after 18 h of incubation . In both cases, the increase in p53 mRNA was transient . c-fos mRNA levels decreased rapidly and were barely detectable after 2 h of exposure to retinoic acid . The down-regulation of c-fos confirms its role in muscle cell differentiation, whereas the up-regulation of wild-type p53 suggests its role during this process.

Biochemistry, 1993 Apr 20, 32(15), 4090 - 7
Formation of DNA-protein cross-links in mammalian cells by levuglandin E2; Murthi KK et al.; Levuglandin E2 (LGE2), a rearrangement product derived from the prostaglandin endoperoxide, PGH2, causes repair-resistant DNA-protein cross-links and cell death (LD50 = 230 nM) in V79 Chinese hamster lung fibroblasts . The half-life for sequestration of LGE2 by covalent binding to cellular nucleophiles is at least an hour for 10 microM LG . This suggests that the in vivo production and distribution of free LGs should be measurable on this time scale . Following removal of the LGE2 and the return of the cultures to normal growth medium, additional DNA-protein cross-links continued to form over the ensuing 6-24 h . The results suggest that LG adducts to DNA or protein are not repaired, but react further at sites on protein or DNA in close proximity to the initial adducts, forming cross-links in a slow phase of the process.

Cancer Lett, 1993 Apr 15, 69(1), 33 - 8
Antileukemic effects of non-metabolizable derivatives of spermidine and spermine; Ask A et al.; To determine whether non-metabolizable derivatives of spermidine and spermine exert anticancer effects, L1210 leukemic mice were treated with 5,8-dimethylspermidine and 5,8-dimethylspermine . Both derivatives cured 5% of the leukemic mice . The increase in median survival time, however, was slight . In combination with alpha-difluoromethylornithine (DFMO), an ornithine decarboxylase inhibitor, only 5,8-dimethylspermine had a favorable effect . Treatment with DFMO is known to increase the uptake of extracellular polyamines and presumably their derivatives, by depleting the intracellular putrescine and spermidine content . However, treatment of L1210 leukemia cells in vitro with DFMO did not affect the uptake of the methyl-substituted polyamines added to the growth medium . 5,8-Dimethylspermidine and 5,8-dimethylspermine repressed the ornithine decarboxylase activity when added to cultures of L1210 leukemia cells . S-Adenosylmethionine decarboxylase activity was only repressed by 5,8-dimethylspermine . This finding may explain the potentiation by this derivative and not by 5,8-dimethylspermidine, of the antileukemic effect of DFMO.

Biochem Biophys Res Commun, 1993 Apr 15, 192(1), 214 - 22
Complex gangliosides modulate the integrin-mediated adhesion in a rat hepatoma cell line; Barletta E et al.; In this study, we investigated whether complex gangliosides influence cell adhesion by modulating the activity of integrin receptors . Our experimental model was represented by CMH5123 cells, a line of neoplastic hepatocytes derived from the minimal deviation Morris hepatoma 5123c of the rat, which adhered to substrata coated with fetal calf serum (FCS) by an integrin-mediated mechanism, being vitronectin the specific serum protein which sustained cell adhesion . We found that ganglioside depletion, obtained by inhibiting complex ganglioside biosynthesis, was accompanied by a reduction of cell adhesiveness to FCS-coated substrata . Integrins appeared to mediate the effect of ganglioside depletion on cell adhesiveness . In fact, sensitivity to the integrin inhibitor GRGDSPC peptide was ten times higher in ganglioside-depleted cells compared to control cells . Moreover, growth of ganglioside-depleted CMH5123 cells in media supplemented with complex gangliosides restored the cell sensitivity to the integrin inhibitor to the same level as that found in control cells . Furthermore, ganglioside depletion of CMH5123 cells decreased the affinity of vitronectin receptors for vitronectin without modifying their number, affinity of vitronectin receptors was re-established in ganglioside-depleted cells by supplementing their growth media with complex gangliosides . In conclusion, these results support the participation of gangliosides to cell adhesion as modulators of integrin receptors.

Protein Expr Purif, 1993 Apr, 4(2), 160 - 3
Overcoming inclusion body formation in a high-level expression system; Moore JT et al.; Attempts at overexpressing T4-phage deoxycytidylate deaminase using the pET3c/BL21(DE3)/pLysS system resulted in this enzyme being part of an inactive inclusion-body complex . However, by employing an enriched growth medium it was found that the deaminase could be induced in a soluble active form to at least 20% of this organism's cellular protein . Insoluble inclusion bodies were obtained with less rich media . This procedure was employed successfully with other highly expressed proteins that formed inclusion bodies . The use of a rich growth medium during the course of protein induction may be a valuable adjunct to limiting inclusion body formation with this as well as other expression systems.

J Bacteriol, 1993 Apr, 175(8), 2304 - 13
5-Aminolevulinic acid availability and control of spectral complex formation in hemA and hemT mutants of Rhodobacter sphaeroides; Neidle EL et al.; In the photosynthetic bacterium Rhodobacter sphaeroides, two genes, hemA and hemT, each encode a distinct 5-aminolevulinic acid (ALA) synthase isozyme (E . L . Neidle and S . Kaplan, J . Bacteriol . 175:2292-2303, 1993) . This enzyme catalyzes the first and rate-limiting step in a branched pathway for tetrapyrrole formation, leading to the biosynthesis of hemes, bacteriochlorophylls, and corrinoids . In an attempt to determine the functions of hemA and hemT, mutant strains were constructed with specific chromosomal disruptions . These chromosomal disruption allowed hemA and hemT to be precisely localized on the larger and smaller of two R . sphaeroides chromosomes, respectively . Mutants carrying a single hemA or hemT disruption grew well without the addition of ALA, whereas a mutant, HemAT1, in which hemA and hemT had both been inactivated required exogenous ALA for growth . The growth rates, ALA synthase enzyme levels, and the amounts of bacteriochlorophyll-containing intracytoplasmic membrane spectral complexes of all strains were compared . Under photosynthetic growth conditions, the levels of bacteriochlorophyll, carotenoids, and B800-850 and B875 light-harvesting complexes were significantly lower in the Hem mutants than in the wild type . In the mutant strains, available bacteriochlorophyll appeared to be preferentially targeted to the B875 light-harvesting complex relative to the B800-850 complex . In strain HemAT1, the amount of B800-850 complex varied with the concentration of ALA added to the growth medium, and under conditions of ALA limitation, no B800-850 complexes could be detected . In the Hem mutants, there were aberrant transcript levels corresponding to the puc and puf operons encoding structural polypeptides of the B800-850 and B875 complexes . These results suggest that hemA and hemT expression is coupled to the genetic control of the R . sphaeroides photosynthetic apparatus.

Endocrinology, 1993 Apr, 132(4), 1621 - 9
Estrogen increases low voltage-activated calcium current density in GH3 anterior pituitary cells; Ritchie AK; The effects of estrogen (17 beta-estradiol) on calcium current density were examined in the GH3 anterior pituitary cell line . The addition of 1 nM estrogen to the growth medium induces a 4- to 5-fold increase in low voltage-activated (LVA) Ca2+ current density without affecting high voltage-activated Ca2+ current density . The increase is significant after 24 h, reaches a maximum level in 3 days, and reverses with a similar time course when the estrogen is withdrawn . The EC50 for estrogen stimulation of LVA Ca2+ current density is about 30 pM . The LVA current induced by estrogen has similar voltage dependence and time course of activation, inactivation, and deactivation as controls . The effect of estrogen requires protein synthesis, since it is blocked by cycloheximide . It is suggested that the estrogen-induced increase in LVA Ca2+ current density may be due to an increase in the number of functional channels in the membrane . This effect could be due to insertion of new channels or synthesis of a protein that converts preexisting silent channels into functional channels.

J Bacteriol, 1993 Apr, 175(7), 1961 - 70
Modulation of the heat shock response by one-carbon metabolism in Escherichia coli; Gage DJ et al.; A genetic screen designed to isolate mutants of Escherichia coli W3110 altered in the ability to induce the heat shock response identified a strain unable to induce the heat shock proteins in a rich, defined medium lacking methionine after exposure to 2,4-dinitrophenol . This strain also grew slowly at 28 degrees C and linearly at 42 degrees C in this medium . The abnormal induction of the heat shock proteins and abnormal growth at both high and low temperatures were reversed when methionine was included in the growth medium . The mutation responsible for these phenotypes mapped to the glyA gene, a biosynthetic gene encoding the enzyme that converts serine and tetrahydrofolate to glycine and 5,10-methylenetetrahydrofolate . This reaction is the major source of glycine and one-carbon units in the cell . Because fixed one-carbon units, in the form of methionine, allowed mutant cells to induce the heat shock response after exposure to 2,4-dinitrophenol, a one-carbon restriction may be responsible for the phenotypes described above.

Glycoconj J, 1993 Apr, 10(2), 152 - 64
Purification and properties of the alpha-3/4-L-fucosyltransferase released into the culture medium during the growth of the human A431 epidermoid carcinoma cell line; Johnson PH et al.; A soluble alpha-3/4-fucosyltransferase secreted into the growth medium of the human A431 epidermoid carcinoma cell line has been purified 700,000 fold by a series of steps involving chromatography on Phenyl Sepharose 4B, CM-Sephadex C-50 and GDP-hexanolamine Sepharose 4B . The untreated spent culture medium transferred almost ten times more fucose to the subterminal N-acetylglucosamine residue in the Type 1 (Gal beta 1-3GlcNAc) disaccharide than to the subterminal sugar in the Type 2 (Gal beta 1-4GlcNAc) disaccharide; the relative activity with these two substrates remained virtually unchanged throughout the purification procedure . At no stage was any alpha-3-fucosyltransferase species acting solely on N-acetylglucosamine residues in Type 2 chains separated from the bulk of the alpha-3/4-fucosyltransferase activity . The purified enzyme preparation showed insignificant activity with glycoprotein substrates having N-linked oligosaccharide chains with terminal Type 2 sequences but transferred fucose to a mucin-type glycoprotein with O-linked oligosaccharide chains with terminal Type 1 structures . Lactose was a poor substrate but the activity of the enzyme was influenced by the presence of substituents on the terminal beta-galactosyl residue and 2'-fucosyllactose was almost as good an acceptor as the Type 1 disaccharide . The properties of the purified enzyme with regard to specificity, divalent cation requirements, pH optimum, and M(r), closely resembled those of the Lewis-blood-group gene associated alpha-3/4-fucosyltransferase isolated from human milk.

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