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.     

Decreased in vivo protein and phospholipid methylation after in vivo elevation of brain S-adenosyl-homocysteine

The administration of adenosine together with homocysteine resulted in a dose-related elevation of cerebral S-adenosyl-L-homocysteine without concomitant perturbation of S-adenosyl-L-methionine levels. The adenosine + homocysteine treatment also decreased the incorporation of labile and stable methyl groups into brain proteins. Brain [³H]-phosphatidyl N,N-dimethylethanolamine and [³H]-phosphatidylcholine were also significantly decreased while [³H]-phosphatidyl-N-monomethylethanolamine remained unchanged. The data indicate that elevated brain S-adenosylhomocysteine can markedly and selectively inhibit the $\text{in vivo}$ methylation of brain proteins and phospholipids.     

The effects of n-alcohols on sarcoplasmic reticulum vesicles

Short, mild treatments of sarcoplasmic reticulum vesicles with aqueous $\text{n-}\text{alcohols}$ from methanol to $\text{n-}\text{heptanol}$ caused an inhibition of calcium uptake and an enhancement of ATPase activity. The $\text{n-}\text{alcohol}$ treatments increased both calcium-dependent (extra) ATPase activity and calcium-independent (basic) ATPase activity of vesicles. The apparent initial reaction rate of ATPase of $\text{n-}\text{alcohol-treated}$ vesicles was about twice that of control vesicles. With increasing number ($\text{n}$) of carbon atoms of the $\text{n-}\text{alcohols}$, the maximum increment of ATPase activity increased, and both the alcohol concentration ($\text{N}{}_{\mathrm{<mtext>Ca</mtext>}}$) required to inhibit calcium uptake by 50% and the alcohol concentration ($\text{N}{}_{\mathrm{<mtext>ATPase</mtext>}}$) required to enhance ATPase activity by 50% of the maximum increment of ATPase activity decreased as follows.

$\text{N}{}_{\mathrm{<mtext>Ca</mtext>}}\text{=23.5·10}{}^{\mathrm{?0.593n}}\text{M}$

$\text{N}{}_{\mathrm{ATPase}}\text{=35.5·10}{}^{\mathrm{?0.593n}}\text{M}$

The ratio, $\text{N}{}_{\mathrm{<mtext>ATPase</mtext>}}$ to $\text{N}{}_{\mathrm{<mtext>Ca</mtext>}}$, was constant for all $\text{n}$ values. The apparent free energy of binding of the methylene groups of $\text{n-}\text{alcohols}$ to sarcoplasmic reticulum vesicles was evaluated (?796 cal/mole) and compared with data from the partition of $\text{n-}\text{alcohols}$ in octanol and water (?670 cal/mole). The effects of $\text{n-}\text{alcohols}$ on membrane vesicles are discussed on the basis of these data.     

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.     

RNA synthesis in aphids, Lachnus tropicalis

Aphid cells contain unique RNAs with molecular weights of 1.2 × 10⁶ and 0.6 × 10⁶. These RNAs are larger in amount in the nucleus and labeled rapidly when the insect is injected with [³H] uridine. Seemingly, the 28S and 18S rRNAs are completed at the cost of these RNAs.     

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.     

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.     

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 $\text{A}$.     

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.     

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.     

Targeting of the antiviral protein from Phytolaccaamericana with an antibody

The antiviral protein (PAP) of $\text{Phytolacca}\text{americana}$ was conjugated with the Fab' fragment of IgG from a rabbit antiserum against murine leukemia L1210 cells via a disulfide bond employing $\text{N}$-succinimidyl 3-(2-pyridyldithio)-propionate (SPDP) as the coupling agent. The conjugate showed a potent $\text{in}\text{vitro}$ cytotoxicity against L1210 cells which was competitively blocked by F(ab′)₂ directed against L1210 cells. PAP itself did not exhibit the cytotoxicity at the concentration corresponding to the PAP content in the conjugate concurrently tested.     

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.     

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.     

Invitro effect of d-lysergic acid diethylamide on immunoglobulin synthesis

Rabbit anti-fluorescyl antibody producing lymphoid cells incubated $\text{in}$$\text{vitro}$ with LSD do not secrete the 7S form of immunoglobulin. The low molecular weight extracellular labeled material shows no measurable anti-fluorescyl antibody activity. Results indicate that during a short incubation period LSD interferes with tryptophan incorporation into antibody protein.     

Studies on the red oxidase (cytochrome o) of Azotobacter vinelandii

In an attempt to isolate and to study the electron transport system of $\text{Azotobacter vinelandii}$, we have isolated and purified a membrane-bound cytochrome $\text{o}$. The cytochrome $\text{o}$, purified as a detergent (Triton X-100) and hemoprotein complex, contained 1.6 nmoles heme per mg of protein. Cold-temperature spectrum showed that no other cytochrome was associated with the purified preparation, and electrophoresis revealed that only one type of hemoprotein was obtained. The purified cytochrome $\text{o}$ reacted with both carbon monoxide and cyanide readily. Only in the reduced form did it combine with carbon monoxide, whereas the oxidized form reacted with cyanide. An “oxygenated” form of the cytochrome $\text{o}$ was demonstrated to be spectrally distinguishable from both the oxidized and the reduced forms.     

mRNA-dependent synthesis of rat apolipoprotein E in vitro: Cotranslational processing and identification of an endoglycosidase H-sensitive glycopeptide intermediate

Total polyadenylated enriched mRNA was prepared from rat liver by guanidine-HCl extraction and oligo(dT)-cellulose chromatography. It was translated $\text{in vitro}$ in an mRNA-dependent wheat germ system and rabbit reticulocyte lysate system, using radiolabeled leucine or methionine as amino acid precursor. A product, designated preapoE, was specifically precipitated by a rabbit anti-rat apoE serum and accounted for 1.5% of the total radioactive peptides. It migrated as a single band of radioactivity on SDS gels with an apparent molecular weight similar to that of mature plasma apoE. Inclusion of dog pancreatic microsomal membranes in the translation reaction resulted in a slightly smaller product (by 500 daltons). It also converted the preapoE from an endoglycosidase H-resistant to an enzyme-sensitive species. This suggests that processing of preapoE takes place by the cotranslational removal of a signal peptide and core glycosylation of the mature protein.