The heavy form of elongation factor 1 in Artemia salina embryos is functionally analogous to a complex of bacterial factors EF-Tu and EF-Ts

The purified heavy form of elongation factor 1 (EF-1) from cysts of $\text{Artemia salina}$ was found to catalyze the exchange of free GTP with a complex of EF-1_L (EF-1_α) and GDP. Furthermore, after heat treatment of EF-1_H in the presence of GTP, the factor, while inactive by itself, stimulated aminoacyl-tRNA binding to ribosomes as well as polyphenylalanine synthesis when combined with EF-1_α. These functional properties are similar to those reported recently for purified EF-1_β from pig liver [ Nagata,S., Motoyoshi,K., and Iwasaki,K. (1976) Biochem. Biophys. Res. Comm. $\text{71}$, 933–938 ]. We suggest that Artemia EF-1_H consists of a EF-1_α. EF-1_β complex which is functionally analogous to bacterial EF-Tu · EF-Ts.     

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.     

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.     

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.     

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.     

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.     

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.     

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 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.     

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.     

Synthesis of d1-4,5,6-trinor-3,7-inter-m-phenylene-3-oxaprostaglandins including one which inhibits platelet aggregation

The synthesis of $\text{d1}$-4,5,6-trinor-3,7-inter-$\text{m}$-phenylene-3-oxaprostaglandins oxaprostaglandins of the E₁ and F₁α series⁷ from 6-endo-(1-heptenyl)-bicyclo[3:1:0]hexan-3-one (III), is described. Preliminary biological screening data for gerbil colon smooth muscle stimulation, rat blood pressure and substrate specificity toward 15-hydroxyprostaglandin dehydrogenase is presented. Platelet function studies, both $\text{in vitro}$ and $\text{in vivo}$ of $\text{d1}$-4,5,6-trinor-3,7-inter-$\text{m}$-phenylene-3-oxa-PGE₁, methyl ester (VIII) are presented.     

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.     

Metabolism and uptake of L-pipecolic acid by brain and heart

Metabolism and uptake of $\text{L}$-[1-¹⁴C]pipecolate was studied in the rat through tail vein injection at low (30 μg/kg) and high (30 mg/kg) doses. No radioactive compound other than $\text{L}$-pipecolate was detected in the brain or heart samples 0.5 to 60 min after injection. The contents of $\text{L}$-pipecolate in the brain dropped rapidly to reach a plateau value 2 min after injection both in the low and high dose experiments (from 0.06 to 0.05 and from 86 to 55 nmole/g brain, respectively). Similar results were observed for the heart except that the heart had $\text{L}$-pipecolate contents 2–3 fold higher than the brain at every time interval. The influx of $\text{L}$-pipecolate to the brain, as measured by the plasma/brain ratio of its contents, was 3 fold lower than the heart at each sampling point throughout the course of measurement for both dosages. The influx of $\text{L}$-pipecolate from the plasma to the heart reached an equilibrium, i.e., plasma/heart = 1, 60 min after injection for both dosages; the plasma to brain ratio was 3 at 60 min. These results indicate that there is a blood-brain transport barrier for $\text{L}$-pipecolate in the rat, which may account for the differences in neuronal effects of $\text{L}$-pipecolate when administered intracerebrally or intraperitoneally.     

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}$.     

Enzymatic release of nitric oxide from L-alanosine,an antineoplastic antibiotic

L-Alanosine is an antineoplastic drug which is the 3-isonitramino analog of $\text{L}$-aspartic acid. The drug is known to be metabolized to the corresponding 2-oxo acid. Unlike the parent amino acid, the 2-oxo acid is unstable under mild conditions. When the 2-oxo acid is generated $\text{in}\text{vitro}$ by the aerobic action of $\text{L}$-amino acid oxidase on $\text{L}$-alanosine, the reaction mixture contains products capable of diazotizing sulfanilamide and of reducing ferricytochrome $\text{c}$ to ferrocytochrome $\text{c}$. It is thus likely that, as expected from model reactions, the unstable 2-oxo acid derived from $\text{L}$-alanosine decomposes into nitric oxide and other reactive free-radical species. Enzymatically promoted production of highly cytotoxic nitric oxide may pertain to the biological activity of the antibiotic. The reaction should prove extrapolable to the design of other enzyme-activated cytotoxic agents.     

Mycoplasma pneumoniae: A prokaryote which consumes oxygen and generates superoxide but which lacks superoxide dismutase

Superoxide dismutase and catalase were not detected in $\text{M}$. $\text{pneumoniae}$ and several other species of $\text{Mycoplasma}$ some of which consume oxygen and secrete H₂O₂. $\text{M}$. $\text{pneumoniae}$ in suspension formed O₂^? in the presence of NADH and flavins and extracts of $\text{M}$. $\text{pneumoniae}$ formed O₂^? in the presence of either NADH or NADPH. The lack of superoxide dismutase in $\text{M}$. $\text{pneumoniae}$ could not be attributed to superoxide dismutase in the complex medium in which the organisms were grown because organisms grown in medium in which the superoxide dismutase had been inactivated by heat still contained undetectable amounts. Mycoplasmas appear to be an exception to the rule that organisms which consume O₂ synthesize superoxide dismutase.     

Valinomycin inhibited methane synthesis in Methanobacteriumthermoautotrophicum

$\text{Methanobacterium}\text{thermoautotrophicum}$ cells, incubated anaerobically under H₂ in 0.1 M KCl or 0.1 M NaCl, above pH 7.5, are interior acid with respect to the incubation medium. The pH gradient thus established can be discharged by either carbonyl cyanide m-chlorophenylhydrazone or valinomycin at high concentration (17μM). In these cells, which actively synthesize CH₄ from CO₂ and H₂, methanogenesis is strongly inhibited when the pH gradient is discharged.     

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.