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.     

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.     

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.     

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 $\text{A}$.     

Prostaglandin levels in preovulatory follicles from rabbit ovaries perfused invitro

Prostaglandin (PG) levels in follicular fluid from preovulatory follicles of rabbit ovaries perfused $\text{in}$$\text{vitro}$ were measured in order to compare PG changes in this model system with those that occur $\text{in}$$\text{vivo}$ and in isolated, LH-treated follicles $\text{in}$$\text{barvitro}$. One ovary from each rabbit was perfused without further treatment (control). The other ovary was exposed to LH (0.1 or 1 ug/ml) beginning 1 hour (h) after initiation of perfusion. Samples of perfusion medium were taken at frequent intervals for measurement of PGE, PGF, progesterone and estradiol 17β. The perfusions were terminated when the first ovulation occurred or appeared imminent as judged by changes in the size and shape of the follicles. Follicular fluid was then rapidly aspirated from all large follicles on both ovaries for PGE and PGF measurement.Ovulations occurred only in the LH-treated ovaries. Progesterone and estradiol levels were significantly elevated in the perfusion medium within 1 h of LH treatment in comparison to controls. PG levels in perfusion medium from the control and LH-treated ovaries were not different throughout perfusion and increased in both groups. In contrast, PG levels measured in follicular fluid from LH-treated ovaries were 4- to 5-fold greater than in fluid from control ovaries. It is concluded that ovulation induced by LH in this experimental model is accompanied by an increase in follicular PG levels similar to that seen in other $\text{in}$$\text{vivo}$ and $\text{in}$$\text{vitro}$ models. This difference in follicular PG levels between the LH-treated and control ovaries is, however, not reflected in the perfusion medium.     

Anestrus in cycling beef heifers experimentally inoculated with Anaplasmamarginale

Normal estrous cycles were established for twenty beef heifers. Ten heifers were inoculated with blood from a known $\text{Anaplasma}$$\text{marginale}$ carrier. The inoculated heifers experienced anemia, anorexia, and positive $\text{Anaplasma}$$\text{marginale}$ complement-fixing antibody titers, and $\text{A}$. $\text{marginale}$ was observed on the erythrocytes while uninoculated control heifers remained normal. Further observation following inoculation revealed that five of ten inoculated heifers experienced anestrus while controls continued to cycle normally. Anestrus coincided with clinical signs of acute anaplasmosis. Normal estrus patterns returned following treatment and recovery. This study provides evidence that acute anaplasmosis in beef heifers may cause anestrus and therefore lead to reproductive inefficiency.     

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.     

Denovo synthesis of prolactin by human decidua

Relatively large amounts of immunoreactive prolactin were measured in homogenates of human decidual tissue obtained immediately after delivery of normal term pregnancies. In order to study the release and possible synthesis of prolactin by this tissue, explants of decidua were incubated for 24 hours at 37°C in oxygenated Gey's buffer containing 20% fetal calf serum. When cycloheximide was added to the medium in concentrations sufficient to prevent $\text{in}$$\text{vitro}$ protein synthesis, 85–90% of the prolactin present in the tissue was released into the medium during the first 3 hours of incubation. No additional prolactin accumulated in either the medium or the tissue during the remainder of the incubation period. In the absence of cycloheximide, the prolactin concentration in the medium increased progressively during incubation, so that after 24 hours the total amount of hormone present in the tissue and medium was significantly greater than that in the tissue and medium prior to incubation (37.6 ± 9.6 ng/ml at 0 time vs 82.2 ± 7.7 ng/ml at 24 hours). When ³H-1-leucine (100 u Ci) was supplied during incubation, radioactive proteins were detected in the medium at 24 hr, 14–20% of which were specifically precipitated by antiserum to human pituitary prolactin. When aliquots of this medium were chromatographed on Sephadex G-100, 80–95% of the ³H-proteins precipitated by antiserum to pituitary prolactin eluted in the same position as did purified, iodinated pituitary prolactin. These data indicate that a species of prolactin which is identical to pituitary prolactin by the criteria of immunoprecipitation and gel chromatography is synthesized by human decidual tissue $\text{in}$$\text{vitro}$.     

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 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.     

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.     

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.     

An attempt to isolate Brucellaabortus from uterine flushings of brucellosis-reactor donor cattle

Two experiments were conducted to test for the recovery of brucella organisms from uterine flushings and harvested embryos of sero-positive embryo donor females. In Experiment I, 16 sero-positive cows were superovulated with FSH treatments and artificially inseminated at 12, 24 and 36 hours following the onset of estrus with brucella-free semen. At 48 hours after the onset of estrus, one half the potential donor females were administered an intrauterine inoculation of 3.3 to 4.6 × 10⁴$\text{Brucella}$$\text{abortus}$ (strain 2308) organisms while the remainder received a control inoculation. In Experiment II, the same 16 cows were similarly administered superovulatory treatments and inseminated following estrus. The uterine inoculation was increased to 1.5 to 2.5 × 10⁸ organisms administered 48 hours following estrus. Samples of recovered flushing medium and homogenized embryo residues were placed into a validated $\text{in}$$\text{vitro}$ culture system to detect the presence of brucella bacteria. Uterine flushings and embryos recovered from 31 females exhibiting estrus following FSH treatments were free from either field strain or the inoculated $\text{B.}$$\text{abortus}$ (strain 2308) contamination. The flushings obtained from a single female, which did not respond with estrus following FSH treatment but was inoculated at appointment, did contain $\text{B.}$$\text{abortus}$ which was identified as the inoculated strain 2308 and not field strain organisms. These results indicate that brucella contamination of flushing media and harvested embryos will not likely be incurred when collecting embryos from sero-positive donor females. These findings offer further encouragement for the use of embryo transplantation as a method to produce brucella-free offspring from infected cows.     

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).     

Purification of the prothoracicotropic hormone from the tobacco hornworm Manducasexta

Standard biochemical procedures were used to purify the prothoracicotropic hormone (PTTH) 4400 fold from whole head extracts of $\text{Mandura}$$\text{sexta}$ fifth instar larvae. Hormonal activity was bioassayed by injection into neck-ligated fourth instar larvae. The hormone was stable to heating at 85°C. Ammonium sulfate and acetone fractionation provided a crude preparation which showed dose-dependent activity in the bioassay. Chromatography on Sephadex G-100, DEAE-Sephadex, and hydroxylapatite gave a preparation with 2.6 $\text{Manduca}$ PTTH units/μg protein (4400-fold purification). Activity was sensitive to proteolytic enzymes. Further purification by preparative electrophoresis gave a preparation which migrated as a single band in two polyacrylamide gel electrophoresis systems. A molecular weight estimate of 25,000 Daltons was obtained for this bands on SDS polyacrylamide gels.     

Invitro evaluation of granulocyte differentiation of diffusion chamber inocula

The semi-soft agar colony assay permits an $\text{in}$$\text{vitro}$ analysis of committed myeloid stem cell (CFU-c) proliferation capacities. In this paper this procedure has been used in combination with prior diffusion chamber culturing to determine the effect of host influences upon this committed stem cell population. This “double-seeding” procedure of first culturing bone marrow cells in diffusion chambers and then re-seeding them in agar furnishes data suggesting a relationship between $\text{in}$$\text{vivo}$ diffusion chamber transitional lymphocytes and $\text{in}$$\text{vitro}$ CFU-c seeding capacities. Diffusion chamber culturing offers a means of monitoring granulopoiesis and selects for enrichment of stem cell numbers. Detection and quantification of diffusion chamber stem cell enrichment is easily assessed by seeding chamber contents into the agar colony assay.     

The occurrence of ethanolamine and galactofuranosyl residues attached to Penicilliumcharlesii cell wall saccharides

$\text{Penicillium}$$\text{charlesii}$ incorporates ³H or ¹⁴C from ³H- or ¹⁴C-labeled ethanolamine into an -alkali soluble, alcohol -insoluble fraction obtained from cell walls. Dansyl ethanolamine was isolated from this alcohol-insoluble fraction following dansylation and hydrolysis. The alcohol-insoluble material was non-dialyzable and contained galactofuranosyl, glucosyl, phosphoryl, amino acyl and variable quantities of uronosyl residues. The lack of detectable quantities of mannosyl residues in this material suggests that the galactofuranosyl-containing cell wall polymer is distinct from the peptidophosphogalactomannan which is obtained from culture filtrates of $\text{P}$. $\text{charlesii}$ (Gander $\text{et}$$\text{al}$., (1974) J. Biol. Chem. $\text{249}$, 2063).     

Escherichiacoli endotoxin absorption from the ewe's uterus and peritoneal cavity

An experiment was conducted to assess the effects of estrogen or parturition on absorption of endotoxin from the ovine uterus. Twelve cycling ewes were assigned to one of four treatment groups (three ewes/group): Group I, no estrogen (NE) + intrauterine infusion of sterile saline (IUS); Group II, NE + intrauterine infusion of 100 mg endotoxin - Lipopolysaccharide W./$\text{E.}$$\text{coli}$ 0127:B8, Difco Laboratories, Detroit, MI (IUE); Group III, 3 days pretreatment with estradiol-17β (50 μg/da, E) + IUS; and Group IV, E + IUE. In addition, the uteri of three early postpartum ewes were infused with 100 mg endotoxin (Group V). Rectal temperatures (RT) and jugular blood samples were obtained at ?40, ?20, 0 (infusion), 20, 40, 60, 80, 100, 120, 180, and 240 minutes. The blood samples were analyzed for total white blood cell counts (WBC) and Limulus Amebocyte Lysate Assays (LAL). There were no alterations in RT, WBC, or LAL observed in Groups I–V. These results indicated that neither prior treatment with estradiol in cycling ewes nor parturition affected absorption of $\text{E.}$$\text{coli}$ endotoxin from the ovine uterus.A second study was conducted to characterize the changes in RT, WBC, and LAL during endotoxemia in cycling ewes. Three ewes received intraperitoneal infusions of 100 mg endotoxin and three ewes received intraperitoneal infusions of sterile saline. Evidence that endotoxin was absorbed from the peritoneal cavity was a decrease (P<0.10) in WBC and positive LAL in endotoxin-infused ewes. WBC and LAL did not change in saline-infused ewes. No changes in RT were observed in either group.     

Effects of experimental Trypanosomavivax on fertility of heifers

Six heifers were used in a series of experiments to study the effects of experimental $\text{Trypanosoma}$$\text{vivax}$ infection on bovine reproduction. Four three-year-old Zebu heifers were intravenously inoculated with T. vivax-strain Y58 — on days 14 and 16 of their estrous cycle and two control heifers in the same phase of estrus were not infected. All the heifers were bred in the research pens with a proven bull. The four infected heifers were bred at the first wave of parasitemia and the onset of pyrexia which characterised the infection. All the heifers were examined rectally 40 days after breeding. The four infected heifers were not pregnant but the two controls were. The infected heifers later became anestrous during the experimental period of more than five months. It is concluded that trypanosomiasis may contribute to high infertility rates in cattle kept in endemic areas.