Isolation and characterization of an anaerobic benzoate-degrading spore-forming sulfate-reducing bacterium, Desulfotomaculum sapomandens sp. nov.

Abstract Spore-forming sulfate-reducing bacteria (SRB) were enriched selectively from various kinds of aerobic soils with fatty acids as the sole carbon and energy source. A Gram-negative motile rod-shaped bacterium, which produced gas vacuoles during sporulation was isolated. It degraded alcohols, aromatic and n-fatty acids (up to C₁₈) except for propionate, completely to CO₂. Sulfate, sulfite, thiosulfate or elemental sulfur served as electron acceptors. Because of its sensitivity to H₂S, the isolate never produced more than 8 mM dissolved sulfide at pH 7.0. G + C-content of the DNA was 48.0 mol %. The isolated strain Pato is described as a new species Desulfotomaculum sapomandens .     

Cloning and characterization of DNA complementary to rat liver UDP-glucuronosyltransferase mRNA

UDP-glucuronosyltransferase (transferase) clones were isolated from a cDNA bank constructed in pBR322 using transferase-enriched mRNA from the livers of phenobarbital-treated rats. The enrichment of mRNA was accomplished by polysome immunoadsorption with antibody to purified mouse liver transferase. This antibody was shown to bind specifically to rat transferase by Ouchterlony double diffusion analysis, immunoadsorption of glucuronidating activities, and selective inhibition of the immunoadsorption of in vitro synthesized enzyme by purified rat liver transferase. The isolated clones were verified to contain DNA complementary to transferase mRNA by hybrid translation-selection. Three classes of transferase cDNAs were characterized by restriction endonuclease mapping, and the largest insert-containing clone of each class was designated pUDPGTr-1, pUDPGTr-2, and pUDPGTr-3. Their insert sizes were approximately 2,400, 2,000, and 2,000 bp, respectively. All three cDNAs hybridized with a 2,300 +/- 150 bp mRNA, and each selected the translation of a 52,000-dalton polypeptide. Immunoadsorption of the 35S-labeled translation product could be competitively inhibited in each case by the addition of purified rat liver transferase. pUDPGTr-1 and pUDPGTr-3 inserts shared extensive sequence homology. This was demonstrated by Southern blot analysis using purified inserts and electron microscopic heteroduplex analysis. Southern blot analysis revealed that these cDNAs hybridized to overlapping genomic fragments. pUDPGTr-2 shared less sequence homology with the other two classes of cDNAs, based on the above criteria. In addition, mRNA corresponding to pUDPGTr-2 was elevated 5-fold by phenobarbital treatment, whereas the other mRNAs levels were unaffected. These studies demonstrate that in rat liver there are a minimum of three distinct transferase mRNAs, two of which may be associated with a common gene or gene family.     

The blood pressure decrease-induced sympathetic discharge following atrial natriuretic factor administration may offset its natriuretic action

Conscious SHR and WKY rats were infused during 7 days with ANF (Arg 101-Tyr 126), 100 ng/hr/rat, by means of miniosmotic pumps and their basal blood pressure (BP), changes in sodium excretion and urinary catecholamines compared with those at the last day of the infusion. The SHR initial BP of 181 +/- 3 mmHg gradually declined to 137 +/- 5 mmHg. No significant change in blood pressure was observed in the ANF-infused WKY group. However, WKY rats exhibited an increased sodium excretion and urinary dopamine/norepinephrine ratio when compared to sham-infused rats. No such differences were observed in SHR. It is suggested that an ANF-induced withdrawal of the renal sympathetic tone permits the manifestation of its natriuretic action in WKY rats. When, however, a BP decrease predominates, as in SHR, this decrease results in a reflex sympathetic discharge with a renal sympathetic activity over-riding the ANF induced natriuresis seen in WKY rats. Secondary sympathetic responses to the ANF-induced BP decrease have to be thus taken into account when a dissociation between the hypotensive and natriuretic action of ANF is observed in vivo.     

Evidence for Plasmid Linkage of Raffinose Utilization and Associated alpha-Galactosidase and Sucrose Hydrolase Activity in Pediococcus pentosaceus

The ability to ferment the trisaccharide raffinose was linked with the presence of plasmid DNA in three strains of Pediococcus pentosaceus. Parental strains showed associated inducible alpha-galactosidase and sucrose hydrolase activities when grown in alpha-galactosides and sucrose, respectively. Derivative strains of PPE1.0, PPE2.0, and PPE5.0, which had lost 30-, 28-, and 23-megadalton plasmids, respectively, had no alpha-galactosidase or sucrose hydrolase activity.     

Establishment of two rabbit mammary epithelial cell lines with distinct oncogenic potential and differentiated phenotype after microinjection of transforming genes.

The goal of this work was to establish an assay for transformation of epithelial cells. Two epithelial cell lines were obtained after microinjecting transforming genes into primary rabbit mammary secretory cells. The cell lines were analyzed for their oncogenic potential and for the maintenance of a differentiated phenotype. A fully transformed cell line, which retained epithelial cell organization, was obtained by coinjecting simian virus 40 DNA and the activated human c-Ha-ras gene. The proliferation rate of these cells was high, with a doubling time of 16 h. Their growth was anchorage independent, and they had lost contact inhibition. The cells were tumorigenic in nude mice, but had no metastatic potential. Both microinjected DNAs were efficiently transcribed and translated, in contrast to the casein genes, which were expressed in primary cells but not in the transformed cell line. An immortalized cell line established after injection with simian virus 40 DNA alone was characterized by a moderate rate of proliferation with a doubling time of approximately 30 h. The growth of these cells was contact inhibited and anchorage dependent. The cells were not tumorigenic in nude mice. The viral DNA was expressed during early passages, as shown by the presence of the large T antigen in cell nuclei, but not at later passages. A high number of lactogenic hormone receptors were found associated with the cell surface. Despite the presence of these receptors, no induction of genes coding for milk proteins was observed after addition of prolactin. These data demonstrate that this assay system can be used to assess the immortalizing and transforming potential of candidate oncogenes in epithelial cells.     

The anthranilate aggregate of Escherichia coli: kinetics of inhibition by tryptophan of phosphoribosyltransferase

The kinetic mechanism of the phosphoribosyltransferase reaction is shown to be rapid equilibrium random bi bi with an enzyme-anthranilate-pyrophosphate abortive complex. We present a rate equation that not only predicts the observed kinetic patterns but also accommodates the fact that feedback inhibition is partial, even though tryptophan (Ki = 0.5 microM) and phosphoribosylpyrophosphate (Km = 50 microM) are competitive. Neither ligand completely abolishes the effect of the other. Instead, the binding of one ligand leads to a mutual elevation in the dissociation constant of the opposing ligand by a factor of two to three. Tryptophan inhibition is noncompetitive with respect to anthranilate (Km = 0.58 microM) and does not diminish the rate of interconversion of ternary complexes. Tryptophan cooperativity, with respect to the inhibition of phosphoribosyltransferase, conforms to the concerted Monod-Wyman-Changeux formulation (kinetic Hill coefficient = 2), whereas tryptophan as an inhibitor of anthranilate synthase more closely conforms to a Koshland model of sequential cooperativity with a kinetic Hill coefficient of 1.4. The aggregate contains only one class of tryptophan sites. Thus the first tryptophan molecule bound to the aggregate maximally inhibits both phosphoribosyltransferase active centers and one of the two anthranilate synthase catalytic sites. The remaining anthranilate synthase subunit thereupon is converted into a form with less (but not zero) affinity for chorismate and a greater affinity for a second molecule of tryptophan.