Reaction of selenomethionine with o-benzoquinone

A new reaction of selenomethionine with o-benzoquinone is described. This reaction can be used to distinguish between methionine and selenomethionine.     

Maintenance of the Adult Rat Superior Cervical Ganglion In Vitro: Comparison of Organ and Explant Culture Systems

Abstract: Concentrations of selected intermediates of energy metabolism whole rat superior cervical ganglia maintained in vitro by an organ culture technique were compared with values measured in small slices of this maintained under essentially the same conditions. Rates of incorporation [³H]leucine into trichloroacetic acid-precipitable material in whole ganglia mained constant for at least 48 h: however, the oxidation-reduction state tissue as indexed by (NAD):(NADH) ratios calculated from measured amounts of lactate and pyruvate decreased more than 50% within ³h in vitro . Ganglion explants prepared by cutting the tissue into ³⁰⁰-pm transverse sections played (NAD):(NADH) ratios that were about three times greater than noted in whole ganglia maintained in vitro for the same period of time. explants contained significantly higher concentrations of pyruvate and α-ketoglutarate than whole ganglia maintained in culture. Maintenance of vorable metabolic state may support the extensive growth of neurites seen explant cultures of superior cervical ganglia. Outgrowth of processes containing catecholamines could be detected readily in explant cultures of ganglia adult rats; however, this was somewhat slower and less consistent than growth observed in explants from neonatal rats. Outgrowth of neurites adult ganglia was minimal without the addition of Nerve Growth Factor.     

The effect of tunicamycin on development of the mammalian embryonic pancreas

The role of cell-surface glycoproteins in histogenesis of the embryonic rat pancreas was investigated by studying the effect of tunicamycin (TM) on in vitro development. TM has been shown to block glycosylation of asparagine residues in glycoproteins by inhibiting formation of dolichol oligosaccharide intermediates. Exposure of Day 15 pancreatic rudiments to 1.0 μg TM/ml for 15 or 24 hr inhibited [³H]mannose, [³H]glucosamine, and [³H]fucose incorporation by 95, 85, and 90%, respectively, while [³H]leucine incorporation was reduced by 35%. Similar results were obtained with Day 17 rudiments. These trends were confirmed using sodium dodecyl sulfate-polyacrylamide slab gel electrophoresis and fluorography. Inhibition of [³H]monosaccharide incorporation correlated with reduced binding of RCA I-ferritin conjugates to the cell surface and both effects of TM were reversed by reculturing rudiments in medium lacking the antibiotic. Morphologically, TM treatment resulted in a delay in pancreatic histogenesis and this delay correlated with an inhibition of the normal increase in specific activity of amylase, an acinar cell secretory protein. These effects were not mimicked by treatment with cycloheximide at a concentration which inhibited [³H]leucine incorporation to the same degree observed with TM. The percentage of delayed rudiments decreased as reculturing in the absence of TM was extended.     

DNA and protein content of mouse sperm: Implications regarding sperm chromatin structure

A variety of biochemical and histochemical techniques have been used to compare the composition of chromatin in sperm nuclei isolated from the epididymides of five mouse strains. The DNA content was determined by phosphorus analysis, deoxyribose analysis, absorption spectroscopy at 260 nm, and cytomorphometry following gallocyanine chrome alum staining. All four methods indicate that the mouse sperm nucleus contains approx. 3.3 pg DNA and that the DNA content does not vary significantly among the strains tested. Three different techniques, quantitative amino acid analysis, absorption spectroscopy at 230 nm, and sperm head density analysis in cesium chloride, were used to determine the protein content. Sperm nuclei from each strain of mouse were found to have a protein to DNA ratio of 0.9 and a chromatin protein content of 3 pg/nucleus. Comparisons of the basic proteins by disc gel electrophoresis demonstrate that the sperm nuclei contain only protamine and lack significant levels of somatic histones or transition proteins. The sperm from each strain contained both mouse protamine variants and the relative distribution of the two proteins did not appear to differ among strains. Using this information, we have been able to draw certain conclusions regarding DNA-protamine interactions and the mode of DNA packaging in the sperm nucleus. The most important of these is that the DNA in the mouse sperm nucleus cannot be packaged in nucleosomes. The protamines in sperm chromatin do not function as structural proteins, providing a subunit core around which the DNA is wrapped, but appear to completely neutralize the phosphodiester backbone of the DNA molecule, thereby minimizing the repulsion between neighboring segments of DNA and allowing it to be condensed into a biochemically inactive particle of genetic information.     

Differential effects of ouabain on human cell-mediated cytotoxicity. I. Inhibition of mitogen-induced cellular cytotoxicity and antibody-dependent cellular cytotoxicity against chicken red cell targets.

The effects of ouabain, a known inhibitor of lymphoproliferation, were studied in relation to the cytotoxic effector function of human peripheral blood mononuclear leukocytes (MNL) against chicken red blood cell (CRC) targets. MNL effectors lysed ⁵¹Cr-labeled CRC targets in the presence of PHA (mitogen-induced cellular cytotoxicity—MICC) or rabbit anti-CRC antibody (antibody-dependent cellular cytotoxicity—ADCC) in the absence of ouabain. The addition of ouabain to the cytotoxic reaction caused profound diminution of MICC with greater than 90% suppression of killing at ouabain concentrations of 5 × 10^?4M; ADCC was much more resistant to the effects of ouabain with only 60 to 70% inhibition of killing at similar ouabain concentrations (P < 0.01). Similar ouabain inhibition of MICC occurred whether the effector cell populations were unseparated MNL, depleted of monocytes, enriched for T cells, or depleted of T cells, suggesting a generalized activity by ouabain against all effector cells active in MICC. Ouabain inhibition of MICC could be overcome by increasing PHA concentrations, indicating that ouabain inhibition was not due to irreversible toxic effects on effector cells. Increasing the concentration of anti-CRC antibody resulted in increased killing in this ADCC system and, paradoxically, ADCC cultures with the highest antibody concentrations were more completely inhibited by ouabain. This enhanced inhibitory effect of ouabain on ADCC cultures with the highest antibody concentrations was not observed when the effector cell population was first depleted of phagocytic cells, suggesting a preferential inhibitory action by ouabain against monocyte effectors in ADCC. Thus, the differential inhibitory effects of ouabain on MICC and ADCC against CRC targets may be in part explained by the differing ouabain sensitivities of the various effector cell subpopulations involved in these cell-mediated cytotoxic events.     

Differential effects of ouabain on human cell-mediated cytotoxicity. II. Stimulation of monocyte-mediated, spontaneous cytotoxicity against chicken red cell targets.

We have previously shown that ouabain inhibits mitogen-induced cellular cytotoxicity (MICC) and antibody-dependent cellular cytotoxicity (ADCC) against chicken red cell (CRC) targets. We now report that ouabain increases spontaneous killing of CRC targets in the absence of mitogen or antibody. Spontaneous cytotoxicity by fresh mononuclear leukocytes (MNL) was enhanced by ouabain in a dose-dependent fashion and was maximal at a ouabain concentration of 5 × 10^?5M. Removal of phagocytic cells from the MNL effector cell population abrogated ouabain-induced spontaneous cytotoxicity, suggesting that the effector cell activated by ouabain was a monocyte. Ouabain-induced spontaneous cytotoxicity was relatively inefficient compared to MICC or ADCC and was only demonstrated consistently at effector:target cell ratios higher than those routinely employed for MICC and ADCC. Very low concentrations of ouabain (5 × 10^?9M) also enhanced spontaneous cytotoxicity of MNL precultured for 7 days, when added at either Day 0 or Day 6 of preculture. The cell effecting spontaneous cytotoxicity after 7 days of culture has been previously shown to be a monocyte. Thus, ouabain has opposing effects on cell-mediated cytotoxic functions: it inhibits MICC and ADCC against CRC targets, but stimulates spontaneous, monocyte-mediated cytotoxicity against the same targets.     

Preparation and crystal structure of [NEt4]3[Fe6W2S8(SEt)9]; structural and electrochemical comparisons with its molybdenum analogue

[NEt₄]₃[Fe₆M₂S₈(SEt)₉] (M = Mo or W) compounds are isomorphous and contain molybdenum and tungsten atoms in an essentially identical environment. These complexes undergo an irreversible one-electron oxidation at −0.46 V (Mo) and −0.51 V (W) and two one-electron reductions at −1.56 and −1.76 V (Mo) and −1.52 and −1.84 V (W), in DMSO solution versus (0.1 M). The only distinction between the behavior of these molybdenum and tungsten complexes identified thus far is that, for the former the reductions are reversible whereas for the latter they are irreversible. This difference may be relevant to the low activity found for nitrogenases reconstituted with tungsten in place of molybdenum.     

The action of proteolytic enzymes on chloroplast thylakoid membranes

Envelope- and stroma-free thylakoid membranes of Vicia faba chloroplasts were incubated with trypsin or pronase for several hours. The indigestible residue was analyzed by polyacrylamide gel electrophoresis. Trypsinization resulted in a complete digestion of all proteins with the exception of the pigment-protein complexes as well as a polypeptide not yet characterized. Yet, as compared with untreated material, Complex II was found to have higher electrophoretic mobility. Electron-microscopic studies illustrate that the indigestible residue still has a preserved membrane structure. Disintegration of the thylakoid membranes by sodium dodecyl sulfate followed by trypsinization also resulted in the two complexes while all the other proteins were found to be digested. However, after removal of the lipids the protein moieties of the complexes proved to be easily digestible. From these results it is concluded that pigment-protein interaction may be an important factor in maintaining a conformation rather resistant to perturbants and proteases. In contrast to trypsin, pronase completely digested the polypeptides of the thylakoid membranes including the protein moieties of the pigment-protein complexes leaving an amorphous lipid mass. The results support the assumption that the complexes are necessary to maintain the membrane structure.     

Mechanism of steroid action in inflammation: Inhibition of prostaglandin synthesis and release

Acute arthritis was induced by injection of cell-free extract of group A Streptococci into the knee joints of mature male rats. Slices of control and inflamed synovia were incubated for 30 to 240 minutes and the rate of prostaglandin E (PGE) released into the medium was measured by radioimmunoassay. PGE release from inflamed synovia was 5–8 fold higher than that in normal tissue. Incubation of inflamed synovia with corticosterone acetate, dexamethasone or prednisone (100 μg/ml) for one or four hours reduced PGE release by 33% and 55% respectively. Lower concentrations of corticosterone (10 – 30 μg/ml) were ineffective. Aldosterone and progesterone (100 μg/ml) had no effect on PGE release throughout the incubation period. Chloroquine (10 μg/ml) inhibited PGE release from inflamed synovia by 50%. Indomethacin (1 μg/ml) abolished PGE release by 90%. Corticosterone, dexamethasone and prednisone reduced PGE content of inflamed synovia by approximately 45% during a 4-h incubation period. Aldosterone and progesterone were ineffective, while indomethacin reduced PGE content by 70%. The suppressive action of corticosterone on PGE release was prevented by addition to the medium of arachidonic acid (2 μg/ml). By contrast, the inhibitory action of indomethacin was not affected by provision of exogenous substrate. We suggest that glucocorticosteroids reduce PGE release by limiting the availability of the substrate for prostaglandin biosynthesis, and this may well explain some of their anti-inflammatory properties.