Purification of prostaglandin E2-9-oxoreductase from human decidua vera

Prostaglandin E2-9- oxoreductase (PGE2-9-OR), the enzyme which converts prostaglandin E2 (PGE2) to prostaglandin F2 alpha (PGF2 alpha), has been detected in human decidua vera. A 105-fold purification was achieved when the centrifuged homogenate was fractionated sequentially by DEAE-Trisacryl, hydroxyapatite-agarose gel, ultrogel AcA 44 and Matrex gel blue A gel chromatographies. The following kinetic constants for PGE2-9-OR have been obtained. The equilibrium constant with respect to PGE2 is 83 microM, the Michaelis constant, Km, for PGE2 is 80 microM, for NADPH 1.6 microM. The maximal velocity for the forward reaction is V1 = .203 pmol/min. The enzyme was inhibited by progesterone, oestradiol-17 beta, cortisol and pharmaceutical drugs. An activating effect could be demonstrated with Ca2+ and oxytocin. The occurrence of PGE2-9-OR in the decidua vera suggests that this enzyme may be responsible for the transformation of PGE2 to PGF2 alpha in these tissues. This may be an important mechanism for the initiation and maintenance of uterine contractions.     

Electron Microscopy of Pseudomonas φ6 Bacteriophage

phi6 bacteriophage from Pseudomonas phaseolicola has a pleomorphic shape due to an outer layer of lipid. This layer was removed with organic solvents or sodium dodecyl sulfate revealing a 50-nm cubic particle. The virus was found only in the central portion of the cell and not near the cell wall.     

Exposure to caffeine and suppression of DNA replication combine to stabilize the proteins and RNA required for premature mitotic events

Caffeine had been shown to induce mitotic events in Syrian hamster fibroblast (BHK) cells that were arrested during DNA replication (Schlegel and Pardee, Science 232:1264-1266, 1986). Inhibition of protein synthesis blocked these caffeine-induced events, while inhibition of RNA synthesis showed little effect. We now report that the protein(s) that are required for inducing mitosis in these cells were synthesized shortly after caffeine addition, the activity was very labile in the absence of caffeine, and the activity was lost through an ATP-dependent mechanism. Caffeine dramatically increased the stability of these putative proteins while having no effect on overall protein degradation. Experiments with an inhibitor of RNA synthesis indicated that mitosis-related RNA had accumulated during the suppression of DNA replication, and this RNA was unstable when replication was allowed to resume. These results suggest that the stability of RNA needed for mitosis is regulated by the DNA replicative state of the cell and that caffeine selectively stabilizes the protein product(s) of this RNA. Conditions can therefore be selected that permit mitotic factors to accumulate in cells at inappropriate times in the cell cycle. Two-dimensional gel electrophoresis has demonstrated several protein changes resulting from caffeine treatment; their relevance to mitosis-inducing activity remains to be determined.     

Phylogenetic relationships of Blepharisma americanum and Colpoda inflata within the phylum ciliophora inferred from complete small subunit rRNA gene sequences

The complete small subunit rRNA gene sequences of the heterotrich Blepharisma americanum and the colpodid Colpoda inflata were determined to be 1719 and 1786 nucleotides respectively. The phylogeny produced by comparisons with other ciliates indicated that C. inflata is allied more closely with the nassophoreans and oligohymenophoreans than the spirotrichs. This is consistent with the placement of the colpodids in the Class Copodea. Blepharisma americanum was not grouped with the hypotrichs but instead was placed as the earliest branching ciliate. The distinct separation of B. americanum supports the elevation to class status given the heterotrichs based on morphological characters.     

Prostaglandin-E2 9-ketoreductase from human uterine decidua vera

Prostaglandin-E2 9-ketoreductase, the enzyme which catalyzes the reaction from prostaglandin E2 (PGE2) to prostaglandin F2 alpha (PGF2 alpha), has been purified 232-fold from human uterine decidua vera. The molecular mass of the enzyme, as estimated by fast protein liquid chromatography, was 29 kDa. Sodium dodecyl sulfate disc gel electrophoresis of the denatured enzyme revealed a molecular mass of 31 kDa. These data suggest that the enzyme consists of a single polypeptide chain. The rate equation of the enzyme reaction for two substrates was used for the determination of five kinetic constants. The equilibrium constant with respect to PGE2 was 83 microM, the Michaelis constant, Km, for PGE2 was 93 microM. For NADPH, the equilibrium constant was 1.0 microM and Km was 1.6 microM. The maximal velocity for the forward reaction was V1 = 217 pmol/min. The inhibition constants for the analgesic agents indomethacin and fentiazac were Ki = 850 microM and Ki = 450 microM and for the steroid progesterone Ki = 1.5 mM, respectively. Prostaglandin-E2 9-ketoreductase might be responsible for the control of the PGE2/PGF2 alpha ratio in human decidua vera. The enzyme, therefore, might be an important factor in the cascade of events leading to uterine contractions and parturition.     

No correlation exists between the conjugative transfer of the autotrophic character and that of plasmids in Nocardia opaca strains

A new isolate of Nocardia opaca was obtained by enrichment culture for aerobic lithoautotrophic growth on CO2 and H2. This strain, MR22, is very similar to N. opaca MR11 (formerly 1b) in functioning as a donor for genetic information determining the ability to grow lithoautotrophically (Aut character) in matings with Aut- strains of N. opaca or closely related heterotrophic species. The strain contains a plasmid, pHG33 of about 110 kb. A mutant was isolated from strain MR22 which was plasmid-free, and had lost the Aut character, resistance to 50 microM-thallium salt and susceptibility to the nocardia-specific bacteriophage phi B1. As a recipient of the Aut character, this plasmid-free mutant was as well suited as plasmid-bearing Aut- strains of N. opaca. In matings with the mutant as recipient the frequency of Aut+ transconjugants per donor was 3 X 10(-4) with N. opaca MR11 (pHG31-a, Aut+, Tlr, Strs, phi B1s) and 2 X 10(-3) with N. opaca MR22 (pHG33, Aut+, Tlr, Strs, phi B1r) as donor. Phenotypic characterization of the transconjugants, which had been selected for the Aut marker, revealed that in many cases the Aut marker had been transferred without plasmid transfer. Furthermore, plasmid-free, Aut+ transconjugants functioned as donors for the Aut marker. Both plasmid-free and plasmid-bearing transconjugants transferred the Aut marker to the Aut- strains of N. opaca with a frequency which was one or two orders of magnitude higher than that of the wild-type strains. The plasmids pHG31-a and pHG33 code for thallium resistance (50 microM-thallium acetate). The frequency of thallium-resistant transconjugants was 10(-1) to 10(-2) per donor; all thallium-resistant transconjugants contained the donor plasmid. We conclude that the plasmids pHG31-a of strain MR11 and pHG33 of strain MR22 of N. opaca carry the genetic information for thallium resistance but not the Aut character. As plasmid-free Aut+ strains can function as donors the Aut character is assumed to reside on the chromosome and to function as an independent self-transmissible genetic element.     

Alcaligenes eutrophus CH34 is a facultative chemolithotroph with plasmid-bound resistance to heavy metals.

Alcaligenes eutrophus strain CH34, which was isolated as a bacterium resistant to cobalt, zinc, and cadmium ions, shares with A. eutrophus strain H16 the ability to grow lithoautotrophically on molecular hydrogen, to form a cytoplasmic NAD-reducing and a membrane-bound hydrogenase, and most metabolic attributes; however, it does not grow on fructose. Strain CH34 contains two plasmids, pMOL28 (163 kilobases) specifying nickel, mercury, and cobalt resistance and pMOL30 (238 kilobases) specifying zinc, cadmium, mercury, and cobalt resistance. The plasmids are self-transmissible in homologous matings, but at low frequencies. The transfer frequency was strongly increased with IncP1 plasmids RP4 and pUZ8 as helper plasmids. The phenotypes of the wild type, cured strains, and transconjugants are characterized by the following MICs (Micromolar) in strains with the indicated phenotypes: Nic+, 2.5; Nic-, 0.6; Cob+A, 5.0; Cob+B, 20.0; Cob-, less than 0.07; Zin+, 12.0; Zin-, 0.6; Cad+, 2.5; and Cad-, 0.6. Plasmid-free cells of strain CH34 are still able to grow lithoautotrophically and to form both hydrogenases, indicating that the hydrogenase genes are located on the chromosome, in contrast to the Hox structural genes of strain H16, which are located on the megaplasmid pHG1 (450 kilobases).     

The persistence of micronucleated erythrocytes in the peripheral circulation of normal and splenectomized Fischer 344 rats: implications for cytogenetic screening

Micronucleated erythrocytes are selectively removed from the peripheral circulation of normal rats. Splenectomy prevents this selective removal. In normal rats treated daily for 20 days with 0.2 mg/kg triethylenemelamine (TEM), micronucleated normochromatic (mature) erythrocytes did not accumulate in peripheral blood. In these same animals, the frequencies of micronucleated cells among polychromatic (newly formed) erythrocytes increased from 0.21 to 5.25 per thousand in peripheral blood and from 1.75 to 31.5 per thousand in bone marrow. Since both control and induced frequencies in peripheral blood were approximately 15% of those in bone marrow, the removal appears to be equally efficient for cells containing either spontaneously occurring or clastogen-induced micronuclei. In splenectomized rats treated daily for 11 days with 0.2 mg/kg TEM, the frequency of micronucleated normochromatic erythrocytes (NCEs) in the peripheral blood rose rapidly to 9 times the control value and remained elevated for 50-55 days, indicating a life span approximately equivalent to that of normal erythrocytes. Among splenectomized rats exposed to either 0.15 mg/kg triethylenemelamine, 6.5 mg/kg cyclophosphamide, or 300 mg/kg urethane for periods exceeding the erythrocyte life span, the incidences of micronucleated NCEs in the peripheral blood rose steadily from a control value of 1.0 per thousand to maximum values of 15.0, 12.7 and 8.9 per thousand, respectively. During these extended exposures, the mean frequencies of micronucleated polychromatic erythrocytes (PCEs) in peripheral blood increased from a spontaneous value of 0.9 per thousand to 23.0, 13.0 and 6.6 per thousand, respectively, reflecting the frequencies among PCEs in the bone marrow and approximating the maximum values among NCEs in the peripheral blood. Thus, the frequency of micronucleated erythrocytes in the peripheral blood of splenectomized rats can be used as an index of both acute and cumulative chromosomal damage, while in normal rats the use of peripheral blood for cytogenetic monitoring is restricted by the selective removal of these micronucleated cells.