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.     

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}$.     

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.     

In vivo radiation-induced thymine residue release from E. coli DNA

Cells of $\text{E. coli}$ C thy^?321 are examined for thymine residue release from DNA following gamma-irradiation from 5 to 15 krad. Experimental conditions are designed to inhibit enzyme activity that might promote base residue release. Enzyme action is restricted in order to assess the physicochemical action of radiation on cellular DNA, and to this end irradiation is done under O₂, N₂, and N₂O saturating conditions. Both thymine and thymidine release from bacterial DNA are detected and quantitated, and three oxygen effects are noted in comparing yields of these products. No difference in effect is observed between N₂ and N₂O gassing conditions, suggesting that the hydroxyl radical has little effect on thymine or thymidine release from irradiated DNA $\text{in vivo}$.     

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.     

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.     

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).     

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.     

Rapid invivo metabolism of Leukotriene C3 in the monkey, Macacairus

[5,6,8,9,11,12-³H₆] Leukotriene C₃ (5 μCi) was injected through a catheter into the right atrium of an anesthetized male monkey. Blood samples were drawn from the aorta via a second catheter. The concentration of tritium in blood decreased from 100 nCi/ml after 5 sec to 1 nCi/ml 15 min after injection, suggesting that leukotriene C₃ was rapidly eliminated from the circulation. Chromatographic analyses of radioactive material in blood collected before recirculation had occurred (15 sec after injection) demonstrated that 40% of the radioactive material had been converted into two less polar metabolites. These products had the same chromatographic properties as leukotrienes D₃ and E₃, respectively. The results indicate that leukotriene C₃ is rapidly transformed by monkey lung $\text{in}\text{vivo}$. Two minutes after injection, the component corresponding to leukotriene E₃ was the predominating metabolite in blood.     

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.     

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.     

A new test of peripheral insulin sensitivity invivo using artificial beta cell

A new $\text{in}$$\text{vivo}$ test of insulin sensitivity is described. It utilizes closed-loop insulin delivery device (GCIIS, Biostator^®) capable of infusing glucose and insulin according to preselected algorithms. In euglycemic patients, insulin was infused by GCIIS to maintain euglycemia in the face of challenges with gradually increasing doses of intravenously administered glucose. Under the described experimental conditions, the endogenous insulin release was minimized as evidenced by serum C-peptide levels of less than 2 ng/ml, and thus the peripheral disposal of glucose should have depended entirely on the exogenous insulin. The amount of the insulin infused was considered to be a measure of peripheral insulin sensitivity. The test was applied to normal and non diabetic obese individuals, and to diabetics, both insulin dependent and independent. Significant insulin resistance was demonstrated in the obese and diabetic patients. In two obese females, the test was repeated after a prolonged period of starvation, and showed marked increase in insulin sensitivity. In two poorly controlled insulin dependent diabetics, marked increase in insulin sensitivity was also observed, here following a prolonged period of euglycemia (48 hours). It is concluded that the GCIIS controlled insulin sensitivity test is a simple, reliable test of peripheral insulin sensitivity, most convenient for clinical and experimental studies $\text{in}$$\text{vivo}$     

Actions of a nicotinic agonist,DMPP, on intestinal ion transport invitro

High-dose carbachol (10^?3 M) has previously been shown to cause NaCl absorption in short-circuited rabbit ileum. The mechanism of this effect may be norepinephrine release induced by carbachol activation of presynaptic nicotinic receptors on adrenergic neurons. Norepinephrine then interacts with postsynaptic α-adrenergic receptors on intestinal mucosal cells to stimulate neutral NaCl absorption and inhibit electrogenic bicarbonate secretion. The present paper examines the $\text{in vitro}$ intestinal ion transport effects of DMPP an agent which is more specific than carbachol on nicotinic cholinergic receptors. DMPP (10^?5 M) caused a transient increase followed by prolonged depression of the short-circuit current, increased NaCl absorption and increased tissue conductance. This effect was antagonized by hexamethonium and phentolamine. It is concluded that nicotinic cholinergic agents stimulate norepinephrine release from adrenergic nerves and effect intestinal ion transport just as norepinephrine does.     

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.     

Tubulin-like protein from Aspergillus nidulans

[³⁵S] labeled extracts of the fungus $\text{Aspergillus nidulans}$ were copolymerized with purified porcine brain tubulin. The [³⁵S] $\text{A. nidulans}$ protein which copurified with porcine microtubules was found to be similar to [³H] chick tubulin when the two were coelectrophoresed on several polyacrylamide gel electrophoresis systems. These results strongly suggest the presence in $\text{A. nidulans}$ of a tubulin-like protein.     

The ionophore X537A (Lasolocid) transiently increases acetylcholine release from rat brain invitro

The effect of X537A on acetylcholine (ACh) release was examined $\text{in vitro}$ in superfused slices of rat cerebrum and striatum. The ionophore (30 μM) induced a transient release of ACh which was not dependent on calcium in the medium. Also in contrast to K⁺-stimulated release, X537A-induced release was not sustained by 10^?5M choline in the superfusion medium and not inhibited by 5 × 10^?4M pentobarbital. The ionophore did not transport ACh or choline from an aqueous to an organic phase. Both K⁺ and X537A inhibited 1 μM (³H) choline uptake into striatal synaptosomes but this effect of X537A was more extensive and less reversible than that caused by K⁺. X537A did not inhibit choline acetyltransferase 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.     

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.     

Specific in vivo binding of 77Br-p-bromospiroperidol in rat brain: A potential tool for gamma ray imaging

The binding of the gamma labeled neuroleptic, ⁷⁷Br-$\text{p}$-bromosprioperidol, in the rat brain was examined $\text{in vivo}$. This binding parallels the binding of ³H-spiroperidol, in that binding is especially high in dopaminergically innervated areas, is saturable, and is displaced by high doses of unlabeled spiroperidol (1–5). Thus, ⁷⁷Br-$\text{p}$-bromospiroperidol is a suitable ligand for use in gamma ray imaging techniques for $\text{in vivo}$ monitoring of receptor binding.