Interconversion of thymine and thymidine in a thymine requiring strain of Bacillus subtilis

In a $\text{B. subtilis}$ Thy^? strain, thymidine is rapidly converted into thymine and, at the steady state, the pool size of thymidine is very small as compared to that of thymine. Consequently when such strain is used for pulse incorporation experiments with labelled thymidine paradoxical results are obtained. A quantitative estimation of the rate of DNA synthesis can only be obtained by thymine pulses or by cumulative incorporation experiments. We also pr$\text{e}$ sent evidence that, during a short pulse, thymidine is mainly utilized for replicative DNA synthesis.     

OKY-1581: A selective inhibitor of thromboxane synthesis invivo and invitro

OKY-1581 is an effective inhibitor of thromboxane synthesis $\text{in}$$\text{vivo}$ and $\text{in}$$\text{vitro}$. The generation of thromboxane B₂ (TxB₂), prostaglandin E (PGE) and prostaglandin F (PGF) was measured following clotting and during platelet aggregation induced by collagen. The presence of OKY 1581 either $\text{in}$$\text{vivo}$ or $\text{in}$$\text{vitro}$ caused a reduction in TxB₂ generation during clotting and platelet aggregation with a concomitant increase in PGE and PGF. The effect could be observed two hours after oral or subcutaneous administration of 5 to 100 mg per rabbit and lasted for 24 to 48 hours. The reduction in TxB₂ was not accompanied by an inhibition of clotting or platelet aggregation. OKY-1581 appears to be a suitable agent for studying the role of TxB₂ in atherosclerosis.     

Iron-sulfur clusters and cysteine distribution in a ferredoxin from Azotobactervinelandii

In 80% dimethyl sulfoxide/H₂O, $\text{Azotobacter}$ ferredoxin FeS clusters can be extruded with benzene thiol. The extruded clusters have an absorption spectra maximum at 458 nm which is characteristic of 4Fe4S centers. The amino terminal sequence of the $\text{Azotobacter}$ ferredoxin has 7 of the 8 Cys residues at residue numbers 8, 11, 16, 20, 24, 39 and 42. Except for Cys 24, all of these residues can be correlated to homologous Cys residues in other bacterial ferredoxins. Although two thirds of the first 45 residues are identical to or conservative replacements for the first 43 residues of other bacterial ferredoxins, the insertion of Cys-24 indicates a major change in the environment of one of the two 4Fe4S clusters.     

The argECBH-bearing EcoRI segment of the Escherichia coli chromosome

The transducing phage λd$\text{arg}\text{14}$, carrying a portion of the $\text{E. coli}$ chromosome including $\text{argECBH}$, is derived from the heat-inducible, lysis-defective strain λy199, which has the $\text{b}\text{519}$ and $\text{b}\text{515}$ deletions. Cleavage of λy199 DNA by $\text{Eco}$RI endonuclease, followed by agarose slab gel electrophoresis, results in bands corresponding to the known C, D, E, and F segments of λ, and a segment A′ (A plus B minus $\text{b}\text{519}$ minus $\text{b}\text{515}$, the cleavage site between A and B being eliminated). Cleavage of λd$\text{arg}\text{14}$ DNA by $\text{Eco}$RI yields the expected D, E, and F segments of λ and four other segments, termed 14-1 through 14-4, whose length is 17.5, 6.2, 3.0, and 2.0 kilobases, respectively, as determined by electron microscopy and corroborated by electrophoretic mobility. Heteroduplex analysis shows that the $\text{E. coli argECBH}$ cluster is on the 14-1 segment.     

Syntheses of E- and Z-pseudodiethylstilbestrol and Z-1-hydroxypseudodiethylstilbestrol

The synthesis and characterization of $\text{E}$- and $\text{Z}$-3,4-bis(4-hydroxyphenyl)-2-hexene ($\text{E}$- and $\text{Z}$-pseudo-DES) and of $\text{Z}$-3,4-bis(4-hydroxyphenyl)-2-hexen-1-ol ($\text{Z}$-1-hydroxypseudo-DES) are described. These compounds are useful as probes in the study of hormone action.     

The influence of dna A and dna C mutations on the initiation of plasmid DNA replication

The dependence of the replication of several plasmids on the chromosome-determined initiation products, $\text{dna}$ A and $\text{dna}$ C, has been studied. The initiation of the replication of $\text{Col}$ E₁ DNA requires the chromosomal $\text{dna}$ A product. In contrast two de-repressed transfer factors ($\text{R 1 drd 16}$ and $\text{Hly}$₁₅₂) seem to determine a corresponding plasmid-specific factor. The $\text{dna}$ C-product is necessary for the ordered initation of all plasmids studied. The addition of low concentrations of chloramphenicol leads to a relaxed replication of $\text{Col}$ E₁ DNA at the restrictive temperature in $\text{dna}$ A-mutants, but not in $\text{dna}$ C-mutants.     

Evidence for phospholipid in plasma membrane penicillinase of Bacilluslicheniformis749C

The plasma membrane-bound penicillinase of $\text{Bacillus}$$\text{licheniformis}$$\text{749}\text{C}$ has been purified. Amino acid analysis showed no significant differences in composition between the enzyme and exopenicillinase. Enzyme purified from cultures containing H₃³³PO₄ or [³H]-glycerol contained ³³P or [³H]-glycerol activity and treatment with 8 M urea, 0.2% sodium dodecyl sulfate at 80° C did not remove the ³H-activity from the enzyme protein. Trypsin readily cleaved the glycerol-containing moiety from the enzyme protein, forming enzyme with molecular weight and heat stability like that of the exoenzyme. Phospholipase D and C also produced enzyme resembling the exo-form.     

Genetic transformation in E.coli: The inhibitory role of the recBC DNase

Genetic transformation of $\text{E.}\text{coli}$ for various chromosomal markers was accomplished by (i) using recipient cells that lack the $\text{recBC}$ DNase but were recombination proficient due to $\text{sbcA}$ or $\text{sbcB}$ mutations and (ii) treating the recipient cells with CaCl₂ at a concentration that facilitates transfection by λ DNA. Cotransformation of three markers ($\text{thr}{}^{\mathrm{+}}\text{ara}{}^{\mathrm{+.}}\text{leu}{}^{\mathrm{+}}$) was found to depend on the molecular weight of the transforming DNA.     

Electron transport-linked nitrous oxide synthesis and reduction by Paracoccusdenitrificans monitored with an electrode

The nitrous oxide reductase activity of $\text{Paracoccus}\text{denitrificans}$ can be conveniently measured using an electrochemical method for determining N₂O. Introduction of this procedure has shown that (i) N₂O reductase activity is reversibly inhibited by oxygen; (ii) antimycin strongly inhibits electron flow to N₂O and that the inhibition is bypassed by tetramethyl-p-phenylenediamine; (iii) ascorbate plus tetramethyl-p-phenylenediamine, presumably by donating electrons to cytochrome c, is an effective reductant for nitrous oxide reductase; (iv) in the presence of the nitrous oxide reductase inhibitor, acetylene, N₂O is promptly produced from nitrite, consistent with the product of nitrite reductase being N₂O.     

Regulation of argF mRNA synthesis,performed in vitro

The specific synthesis of $\text{arg}$F mRNA directed by the $\text{arg}$F gene carried on the specialized transducing bacteriophage $\text{λh80C}{}_1\text{857darg}$F, performed $\text{in vitro}$, is described with the use of an S180 extract from a strain carrying $\text{arg}$R^?. Synthesis of $\text{arg}$F mRNA is biphasic at approximately 7 minutes. The regulation of $\text{arg}$F mRNA synthesis by the specific arginine holorepressor present in an S180 extract prepared from a strain carrying the $\text{arg}$R⁺ allele is described.     

Aureobasidium pullulans metabolism of prostaglandins of the A-type

$\text{Aureobasidium pullulans}$, originally introduced as an inadvertent contaminant in solutions used for evaluating the stability of prostaglandins, proved to lead to the rapid disappearance of the cyclopentenone unit of PGA₂ (as monitored by circular dichroic spectroscopy). The cyclopentenone unit is converted, in various metabolites, to a 9-keto, 9α or 9β-hydroxy group lacking the ring unsaturation. The major EtoAc-soluble 9-hydroxy metabolite (Compound-I) was shown to be 9α, 15α-dihydroxy-2,3,4,5-tetranor-13-$\text{trans}$-prostenoic acid. Similar tetranor 9-hydroxy metabolites with one additional degree of unsaturation, and with a 9β-hydroxy group, also occur but these have not been fully characterized. Only two of the wide range of 9-keto metabolites are fully characterized by mass spectral (MS) data: 9,15-oxo-2,3,4,5-tetranorprostanoic acid and 9,15-oxo-2,3,4,5-tetranor-13-$\text{trans}$-prostenoic acid. The water soluble metabolites have not been characterized further.The fully characterized metabolites together with MS data from mixtures of minor metabolites indicate that $\text{A. pullulans}$ can perform the following transformations: β-oxidation, dehydrogenation at C-15, reduction of the enone carbon-carbon double bonds (both Δ^10,11 and Δ^13,14), reduction of the 9-ketone, and possibly migration of the cyclopentyl double bond (Δ^10,11 → Δ^11,12). $\text{A. pullulans}$ metabolizes 15-epimeric PGA₂ equally readily with the production of similar products. PGA₁ affords less 9-keto metabolites with compound I constituting 33% of the product by HPLC analysis. $\text{A. pullulans}$ displays some enantioselectivity, PGA₂ and 15-epi-PGA₂ are each metabolized more rapidly than their enantiomers. Other prostaglandins appear to be less readily metabolized.     

Effect of removing the zona pellucida on development of hamster and bovine embryos invitro and invivo

Uterine stage embryos collected from the hamster (8-cell) and cow (morula, early blastocyst) were monitored for development $\text{in}$$\text{vitro}$ (embryo culture) and $\text{in}$$\text{vivo}$ (embryo transfer) following premature removal of the zona pellucida.Removal of the zona pellucida did not significantly affect $\text{in}$$\text{vitro}$ development to the blastocyst stage of (1) 8-cell hamster embryos (zonae removed by a combined enzymic-mechanical procedure), (2) bovine morulae (zonae removed by mechanical means only) (3) early bovine blastocysts (zonae removed by the enzymic-mechanical technique).Zona-free hamster embryos formed significantly fewer viable fetuses than did zona-intact embryos. The lower incidence of fetal development observed following transfer of zona-free 8-cell hamster embryos may have resulted in part from the formation of chimeras by fusion of these embryos $\text{in}$$\text{utero}$. Such fusion was observed to occur $\text{in}$$\text{vitro}$ between zona-free embryos placed in close proximity. The proportion of pregnancies resulting from transfer of bovine blastocysts cultured from zona-free morulae was similar to that of zona-intact embryos.In this study we have demonstrated that (1) enzymic and mechanical procedures used to remove zonae pellucidae from uterine-stage hamster and bovine embryos do not adversely affect subsequent development of these embryos $\text{in}$$\text{vitro}$ and $\text{in}$$\text{vivo}$ and (2) zonae pellucidae are not required for normal development of these embryos. These findings have implications for microsurgery of mammalian embryos and for embryo transfer.     

Prostaglandins and in vitro ovarian progestin biosynthesis

Prostaglandin F_2α (PGF_2α) did not alter the $\text{in vitro}$ biosynthesis of progesterone by slices of luteinized rat ovaries when used in concentrations from 1 to 10,000 ng/ml of incubation medium; likewise, PGF_2α did not affect the incorporation of acetate-1-¹⁴C into progestins. PGF_1α, 15-keto PGF_2α, and PGE₁ did not alter the biosynthesis of progesterone by luteinized rat ovaries; PGE₂ inhibited the production of progesterone when used at a concentration of 10 μg/ml, but not at lower doses. PGF_2α in combination with luteinizing hormone (LH) enhanced the metabolism of progesterone to 20α-hydroxypregn-4-en-3-one in luteinized rat ovaries. Interestingly, PGF_2α, at a high concentration of 10 μg/ml, did stimulate progesterone biosynthesis by slices of ovarian tissue from immature rats hormonally primed to simulate “pseudopregnancy,” suggesting a steroidogenic action of prostaglandins on the ovarian follicular or interstitial cell. PGF_2α (10 μg/ml) did not stimulate the $\text{in vitro}$ biosynthesis of progesterone or 20α-hydroxypregn-4-en-3-one by slices of rabbit corpora lutea or rabbit ovarian interstitial tissue. It is concluded that prostaglandins do not stimulate progestin biosynthesis in rat luteal tissue.     

Guanylate cyclase from Dictyosteliumdiscoideum

Guanylate cyclase from crude homogenates of vegetative $\text{Dictyostelium}$$\text{discoideum}$ has been characterized. It has a pH optimum of 8.0, temperature optimum of 25°C and requires 1 mM dithiothreitol for optimal activity. It strongly prefers Mn⁺⁺ to Mg⁺⁺ as divalent cation, requires Mn⁺⁺ in excess of GTP for detectable activity, and is inhibited by high Mn⁺⁺ concentrations. It has an apparent Km for GTP of approximately 517 μM at 1 mM excess Mn⁺⁺.The specific activity of guanylate cyclase in vegetative homogenates is 50–80 pmoles cGMP formed/min/mg protein. Most of the vegetative activity is found in the supernatant of a 100,000 x g spin (S100). The enzyme is relatively unstable. It loses 40% of its activity after 3 hours storage on ice. Enzyme activity was measured from cells that had been shaken in phosphate buffer for various times. It was found that the specific activity changed little for at least 8 hours. Cyclic AMP at 10^?4 M did not affect the guanylate cyclase activity from crude homogenates of vegetative or 6 hour phosphate-shaken cells.     

Cloning and expression in Streptomyces lividans of a paromomycin phosphotransferase from Streptomyces rimosusforma paromomycinus

The paromomycin producing organism $\text{Streptomyces}$$\text{rimosus}$$\text{forma paromomycinus}$ is resistant to this antibiotic and contains a phosphotransferase which inactivates paromomycin. The gene encoding this enzyme has been inserted in the $\text{Streptomyces}$ vector pIJ702 and then cloned in $\text{Streptomyces}$$\text{lividans}$, selecting for paromomycin-resistance. Three plasmids have been isolated and one of them, pMJ1, contains a 2.2 kb insert with a single HindIII restriction site. Insertion of foreign DNA in this site blocks the expression of the phosphotransferase enzyme indicating that it is within the cloned gene. These findings provide a new dominant selective marker for $\text{Streptomyces}$ cloning vectors with the versatility of insertional inactivation.     

Metabolites of mating pheromone,rhodotorucine A, by a cells of Rhodosporidiumtoruloides

Rhodotorucine $\text{A}$ which induces mating tube formation of $\text{a}$ cells in $\text{Rhodosporidium}\text{toruloides}$ is metabolized rapidly by $\text{a}$ cells. By use of labeled rhodotorucine $\text{A}$, the degradation was found to be proteolytic. Two peptide fragments Tyr-Pro-Glu-Ile-Ser-Trp-Thr-Arg and Asn-Gly-Cys(S-farnesyl) were identified as the metabolites. Proteolysis of the pheromone mainly occurred on the cell surface. Culture filtrate of $\text{a}$ cells at log phase did not metabolize rhodotorucine $\text{A}$.     

Effect of Brucellaabortus infection on spermatogenesis in three Zebu bulls (Bosindicus). A case report

This case report addresses the occurrence of Brucellosis and its effect on the cattle in developing countries. Three Zebu bulls ($\text{Bos}$$\text{indicus}$) are presented and the clinical and pathologic signs are described. Conception rates declined following an abortion storm in one herd and without prior abortions in another herd. Semen collected by electro-ejaculation was found to be azoospermic or with very few spermatozoa. $\text{B. abortus}$ was isolated from seminal vesicles, testes and epididymides. Organs affected and showing microscopic lesions were testes, epididymides and seminal vesicles. The latter were not consistently affected. None of the bulls showed impairment of libido or breeding capacity.     

An endodextranase inhibitor from batch cultures of Streptococcus,mutans

An inhibitor of $\text{Streptococcus}$,$\text{mutans}$ endodextranase was detected in proteins prepared from batch cultures of $\text{S.}$,$\text{mutans}$ strains representing serotypes $\text{a}$ through $\text{g}$. Affinity chromatography of strain 6715-49 proteins, which apparently were free of endodextranase activity, yielded an active endodextranase and, in a separate peak, the endodextranase inhibitor. The presence of the inhibitor in culture fluids accounts for the absence of endodextranase activity in batch-grown cultures of $\text{S.}$,$\text{mutans}$ known to produce this enzyme.     

Disaggregation of elongation factor 1 by extracts of Artemiasalina

Extracts of 40 hr $\text{Artemia}$$\text{salina}$ nauplii can convert a heavy form of elongation factor 1 (EF-1_H) to a light species (EF-1_L). The data indicate that a protease in the extracts is responsible for this reaction, and these findings may explain the observation that extracts from $\text{Artemia}$$\text{salina}$ nauplii have only EF-1_L whereas before hatching of the $\text{Artemia}$$\text{salina}$ embryos EF-1_H is the predominant species (Slobin and M?ller [1975] Nature 258, 452–454).