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.     

Prostaglandin synthesis in rat embryo tissue: The effect of non-steroidal anti-inflammatory drug in vivo and ex vivo

Aspirin and salicylate are well-known but poorly understood teratogens in laboratory animals. Because aspirin inhibits PG synthesis, we systematically examined PG synthesis in rat embryo homogenates, the inhibition of PG synthesis $\text{in vivo}$ and $\text{ex vivo}$ by various non-steroidal anti-inflammatory drugs, and tested the hypothesis that the inhibition of PG synthesis is responsible for aspirin-induced limb defects in rats. We report that embryonic rat homogenates synthesis 6-keto-PGF_1α, PGE, and PGF in large amounts from endogenous substrate, that aspirin and other non-steroidal anti-inflammatory drugs inhibit PG synthesis $\text{in vitro}$ but not necessarily $\text{in vivo}$, and that contrary to our original hypothesis, the inhibition of PG synthesis is likely not responsible for aspirin-induced limb defects in rats.     

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.     

Steganacin: An inhibitor of HeLa cell growth and microtubule assembly invitro

The plant derivative steganacin, an antitumor compound, blocks the replication of HeLa cells in mitosis. Steganacin inhibits microtubule assembly $\text{in}\text{vitro}\text{(ID}{}_{50}\text{= 1.5 μM)}$ and is a competitive inhibitor of colchicine binding to purified tubulin (K_i = 3.1 μM). The structure-activity relationships of steganacin and a series of analogues are reported.     

Photophosphorylation in cell envelope vesicles from Halobacteriumhalobium

We have prepared vesicles from cell envelope membranes of $\text{Halobacterium}\text{halobium}$ strains R₁ and ET-15 which are able to synthesize ATP in response to illumination. This photophosphorylation is inhibited by dicyclohexylcarbodiimide (DCCD) and by phloretin. ATP synthesis in L vesicles from the R₁ strain (which contain bacteriorhodopsin) is inhibited by the protonophore 1799 but not by valinomycin. In M vesicles from the R₁ strain and in ET-15 vesicles (both contain halorhodopsin) photophosphorylation is inhibited by both 1799 and valinomycin. These data are consistent with the idea that light-driven ATP synthesis can be coupled to the electrochemical H⁺ gradient generated by bacteriorhodopsin or by halorhodopsin through the membrane potential component of protonmotive force.     

Detection of nicotinic cholinergic transmission in malapteruruselectricus electrop lax

Biochemical and electrophysiological studies were conducted on the electric organ of the electric fish of the Nile, $\text{Malapterurus}$$\text{electricus}$, in order to determine if transmission was chemically mediated. There was no binding of [³H] acetylcholine, [³H] quinuclidinyl benzilate or [³H]-perhydrohistrionicotoxin; but low acetylcholinesterase activity was observed, as was binding of [¹²⁵I] α-bungarotoxin. The latter binding was detectable at 0.85 ± 0.07 pmol/g tissue, and was totally inhibited by 1 μM α-bungarotoxin or 100 μM d-tubocurarine. A tetrodotoxin-sensitive action potential was measured which was Na⁺- dependent. Depolarization (30–40 mV) was caused by carbamylcholine, and this was blocked by d-tubocurarine or α-bungarotoxin. The data suggest that this electric organ which may be a rich source for electrically excitable channels, is innervated by nicotonic cholinergic motoneurons, but the concentrations of acetylcholine receptors and acetylcholinesterase are very low.     

Uncoupler and anaerobic resistant transport of phosphate in Escherichia coli

Active transport of inorganic phosphate into whole cells of a strain (AB3311) derived from $\text{Escherichia coli}$ K12 was found to be partially resistant to 50 μM carbonyl cyanide $\text{m}$-chlorophenyl hydrazone (CCCP), a powerful uncoupler of oxidative phosphorylation. The presence of 10 mM dithiothreitol (DTT) before the addition of CCCP completely prevented the inhibition of phosphate uptake caused by the uncoupler. The addition of DTT to the CCCP-inhibited system restored phosphate uptake to the control rate even when added 5 min after the phosphate transport assay was started. This uncoupler resistant transport is insensitive to anaerobiosis, or the addition of 10 mM KCN which reduces oxygen consumption to less than 1% that of aerobic controls. Additional studies of transport in a mutant (CBT302) deficient in membranebound Ca²⁺-, Mg²⁺-ATPase activity also demonstrated the retention of appreciable inorganic phosphate uptake under anaerobic conditions.     

The estrogenic activity of DDT: Invivo and invitro induction of a specific estrogen inducible uterine protein by o,p'-DDT

The pesticide o,p'-DDT stimulates the production of a specific uterine protein, the so-called induced protein or IP, normally associated with an estrogenic response of the uterus. $\text{In}$$\text{vivo}$ stimulation of IP production is observed 1 hour after the administration of 250 mg/kg of o,p'-DDT to immature rats. $\text{In}$$\text{vitro}$ stimulation of IP production is observed after a 1 hour incubation of uteri with 100 μM o,p'-DDT. This $\text{in}$$\text{vitro}$ response is blocked by Actinomycin D. In contrast to o,p'-DDT, which binds to the cytoplasmic estrogen receptor and stimulates IP production, p,p'-DDT which does not bind well to the estrogen receptor does not stimulate IP production $\text{in}$$\text{vitro}$. These findings represent the first report of an estrogenic effect of o,p'-DDT in a completely $\text{in}$$\text{vitro}$ system.     

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.     

A soybean lectin having 4-O-Methyl-D-Glucuronic acid specificity

Evidence is presented for the presence of a new lectin activity in soybean seeds [$\text{Glycine}\text{max}$ (L.) Merrill] that has specificity towards the 4-O-methyl-$\text{D}$-glucurono-$\text{L}$-rhamnan exopolysaccharide produced by certain strains of $\text{Rhizobium}\text{japonicum}$. Bacterial agglutination and precipitin reactions revealed the lectin activity in phosphate-buffered saline extracts of seeds of all cultivars tested, including the “lectinless” varieties. Reaction of such extracts with carbohydrate haptens demonstrated that the specificity of the binding was towards 4-$\text{O}$-methyl-$\text{D}$-glucuronic acid, $\text{D}$-glucuronic acid and their methyl glycosides.     

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.     

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).     

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.     

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.     

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 $\text{A}$.     

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.     

An invitro assay system for chitin synthesis in insect tissue

The system described utilizes the abdominal integument of the grasshopper $\text{Melanoplus}$$\text{sanguinipes}$ as the tissue and radioactive glucose, glucosamine or UDP-N-acetyl glucosamine as substrate. A micromethod is described that makes it possible to use as little as 2 μl total volume of incubation. After an initial lag period, incorporation rates become linear for at least 2 hrs. Cell disruption leads to immediate and complete loss of activity.     

The synthesis of 13-cis-dl-erythro-15, 16-dihydroxyprostaglandins

Both pairs of $\text{dl}$-ll-desoxy- and $\text{dl}$-13-$\text{cis}$-$\text{erythro}$-15, 16-dihydroxyprostaglandins have been synthesized via 1,4-conjugate additions of an appropriately functionalized $\text{cis}$-vinyl cuprate to the requisite cyclopentenone. These prostaglandin analogs are considerably less potent than PGE₂ as gastric secretion inhibitors or as bronchodilators.     

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.     

Nitrate reductase from Penicilliumchrysogenum: Kinetic mechanism at sub-optimum pH

The steady-state kinetics of the NADPH + FAD-dependent reduction of nitrate by nitrate reductase from $\text{Penicillium}\text{chrysogenum}$ was studied at pH 6.18. At this sub-optimum pH, V_max was about 83 units × mg protein^?1 compared with 225 units × mg protein^?1 at pH 7.20. All initial velocity reciprocal plot patterns at pH 6.18 as well as the NADP⁺/nitrate product inhibition pattern were intersecting. In contrast, the NADP(H)/nitrate plots at pH 7.20 were parallel (Renosto, F. $\text{et}\text{al.}$ J. Biol. Chem. $\text{256}$, 8616, 1981). A major effect of lowering the assay pH was to change the K_m for FAD from 0.17 μM at pH 7.20 to 4 μM at pH 6.18. The results suggest that nitrate reductase has a steady-state random kinetic mechanism in which k_cat in the forward direction at pH 7.20 (ca. 375 sec^?1) is greater that k_off for the dissociation of one or more substrates. Several observations suggest that k_off for FAD is extremely small at pH 7.20.