Coronary endothelial dysfunction from ischemia and reperfusion: effect of reactive oxygen metabolite scavengers

Using anesthetized mongrel dogs exposed to 60 min of ligation of the left anterior descending coronary artery followed by 60 min of reperfusion, we examined the effect of superoxide dismutase (SOD) and dimethylthiourea (DMTU) on evidence of endothelial injury in coronary rings studied in vitro. In 13 dogs treated with saline rings from the normal left circumflex coronary artery (LCF) relaxed by 98 +/- 4% when exposed to 10(-5) M acetylcholine whereas rings from the left anterior descending coronary artery (LAD) relaxed by 79 +/- 7% (p less than 0.05). In the same rings maximum relaxation with the ionophore A23187 was 107 +/- 5% versus 87 +/- 8% (p less than 0.05) for the LCF and the LAD, respectively. Comparisons of concentration-response curves through a range of doses of both acetylcholine and A23187 revealed significant differences for both vasodilators between the LCF and the LAD (p less than 0.01 for each). Nine dogs were treated with bovine SOD infused in the left atrium the last 20 min of ligation and throughout reperfusion (140 units/kg/min) and six other dogs were treated with DMTU 500 mg/kg i.v. given the last 30 min of the ligation period. Neither SOD nor DMTU prevented endothelial injury in the LAD. Despite pretreatment with these agents, there were significant reductions in maximum relaxation and in total concentration-response curves in the LAD as compared with the results in rings from the LCF with both acetylcholine and A23187. There were normal responses to nitroprusside in both the LCF and LAD in all three experimental groups.(ABSTRACT TRUNCATED AT 250 WORDS)     

Human progesterone receptor transformation and nuclear down-regulation are independent of phosphorylation

We have studied the phosphorylation of progesterone receptors (PR) in T47Dco human breast cancer cells using a monoclonal antibody directed against human PR called AB-52. This antibody recognizes both the A- (Mr approximately 94,000) and B- (Mr approximately 120,000) hormone binding proteins of PR, and was used to immunoprecipitate phosphorylated receptors isolated from cells incubated in vivo with [32P]orthophosphate. The specific activity, or phosphorylation levels, relative to protein levels was quantified by combined immunoblotting and autoradiography followed by densitometry. We find that immunopurified untransformed hormone-free receptors, which have a characteristic triplet B, singlet A structure, are phosphoproteins with similar levels of phosphate incorporation in all protein bands. If PR are first transformed to the nuclear binding form by treatment of cells with progesterone, and then labeled with [32P]orthophosphate, the receptor proteins are additionally phosphorylated. These chromatin-bound hormone occupied receptors incorporate five to 10 times more labeled phosphate per total receptor protein than do PR from untreated cells during the same [32P]incubation time. The second round of phosphorylation may also account for mobility shifts of transformed A- and B-receptors observed in sodium dodecyl sulfate-polyacrylamide gels. Both untransformed and transformed species of A- and B-receptors are phosphorylated only on serine residues, and neither the extent of phosphorylation, nor the phosphoamino acids, are affected by treatment of the cells with epidermal growth factor or insulin. We previously reported that after hormone binding and transformation of receptors to the tight chromatin binding state, PR undergo processing, or nuclear down-regulation. AB-52 was used to compare PR protein and phosphorylation levels when cells were treated for 0.5-48 h with progesterone or the synthetic progestin R5020. Both agonists lead to hyperphosphorylation of nuclear PR before phosphorylation levels decrease, in parallel with the drop in protein levels as receptors down-regulate. Treatment of cells with RU 486, an antiprogestin, leads to PR transformation as determined by immunoblotting, but subsequent down-regulation does not occur. After transformation, chromatin-bound RU 486-occupied receptors become intensely phosphorylated however, with specific activities 15 times greater than those of untransformed PR. Since these receptors are phosphorylated but not processed, the hormone-induced nuclear phosphorylation of PR is unlikely to be a signal for receptor processing.(ABSTRACT TRUNCATED AT 400 WORDS)     

Comparison of trout hepatocyte culture on different substrates

Summary Comparisons were made of attachment and viability of rainbow trout (Salmo gairdneri) hepatocytes in short-term (2 days), primary culture on plastic, collagen-coated or extracellular matrix (ECM) coated dishes. Hepatocyte isolation routinely yielded cells with good viability (96%). Cells plated on ECM attached with high efficiency (93%) in contrast to cells cultured on plastic or collagen (∼20%). The cells plated on ECM flattened out and formed monolayers, while the cells on plastic and collagen rounded up and formed multi-cell aggregates in suspension. Viability of cells in all substrates remained high over the 2 day culture period. ECM is the first substrate to support trout-hepatocyte attachment in primary culture. Differentiated liver function was maintained in cells cultured on ECM as evidence by the induction of tyrosine aminotransferase by hydrocortisone (200%). This work was supported in part by research grant R809599010 from the U. S. Environmental Protection Agency. Editor's Statement This paper reports improved methods for culture of trout liver-derived cells that make in vitro investigations of fish metabolism, carcinogenesis and chemical toxicity more feasible than previously applied techniques. Recent interest in fish as models for study and indicators of effects of envionmental and food-related toxins make this work timely, poarticularly since many of the compounds of interest are primarily metabolized by hepatocytes or act on liver as a major target. David W. Barnes     

Developmental profiles of three embryo-lethal maize mutants lacking leaf primordia: ptd*-1130, cp*-1418, and bno*-747B

The defective kernel (dek) mutants of maize are altered in both their embryo and endosperm development. Earlier studies have indicated that some of the dek mutants are unable to form shoot apical meristems or leaf primoirda. We have examined three embryo lethal dek mutants of this type, ptd*-1130, cp*-1418, and bno*-747B, to obtain a developmental profile for each. Allelism tests show that these three mutants are not allelic. Embryos were examined in early, mid-, and late kernel development as well as at kernel maturity by dissection and sectioning procedures and also at kernel maturity by scanning electron microscopy. All three mutants lag behind normal embryos in their rate of development. Embryos of ptd*-1130 reached the transition stage by early kernel development and progressed no further but underwent cell enlargement and necrosis during late kernel development. Embryos of cp*-1418 reached an early coleoptilar stage by midkernel development. They subsequently increased in size but did not form any leaf primordia. At kernel maturity, they no longer had a shoot apical meristem but often had a well formed root meristem. They appeared to remain healthy and did not become necrotic. Embryos of bno*747B reached the early coleoptilar stage by early kernel development but progressed no further. By kernel maturity, they had grown into masses of irregularly shaped embryonic tissue that no longer resembled any normal embryo stage but were not necrotic. None of these three mutants responded to attempts to support continued embryo development when cultured, but all three mutants formed callus on N6 and MS media supplemented with 2,4-D. These results indicate that these mutants are all uniformly blocked at specific stages early in embryonic development, have different subsequent developmental fates, and represent three different genes performing unique functions that are essential for embryogenesis.     

Bronchodilatory properties of 2-decarboxy-2-hydroxymethyl prostaglandin E1

We evaluated in a double-blind study the bronchodilatory properties of 2-decarboxy-2-hydroxymethyl prostaglandin E₁ (PGE₁-carbinol), described recently as a nonirritant bronchodilator in animals. Fifteen asthmatic patients received by inhalation single doses of 1, 10, and 30 μg PGE₁-carbinol, 55 μg PGE₂, and placebo (10% ethanol in normal saline, which was also used as diluent for the PGs). Such pulmonary function tests as forced expiratory volume in 1 second, forced vital capacity, and maximal expiratory flow were monitored during 2 hours following inhalation of each compound. 10 and 30 μg PGE₁-carbinol produced significant but short-acting bronchodilation, similar to that caused by 55 μg PGE₂. One-third of the patients reported mild cough and throat irritation during and shortly after inhalation of 30 μg PGE₁-carbinol or 55 μg PGE₂. Placebo and 1 μg PGE₁-carbinol produced minimal side effects, but neither agent caused bronchodilation. In an adjunctive, unblinded trial, the same patients received 400 μg fenoterol. Fenoterol caused greater bronchodilation 15 and 30 minutes after inhalation than did the PGs in the double-blind study.     

Partitioning of variance and estimation of genetic parameters for various bristle number characters ofDrosophila melanogaster

Summary Phenotypic variance for each of several bristle number characters (abdominal, sternopleural, second and third coxal) was partitioned using both hierarchal and dialled designs. Heritabilities and genetic correlations were estimated from parent-offspring regressions and correlations and half-sib correlations.A high proportion of the genetic variance for abdominal bristle number was due to epistatic and sex-linked gene action, but most of the genetic variance for the other characters was additive autosomal.The genetic correlations among sternopleural, and second and third coxal bristle numbers were all high, but that between abdominals and sternopleurals was low, while those between abdominals and either second or third coxals were virtually zero. An appreciable proportion of the covariance between abdominal and sternopleural bristle numbers was non-additive genetic.The diallel method gave more reliable estimates of genetic parameters when non-additive or sex-linked genetic variation was present.

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Zusammenfassung Für eine Anzahl verschiedener Borstenzahl-Charaktere (abdominales, sternopleurales, 2. und 3. coxales Segment) wurde die phänotypische Varianz unter Verwendung hierarchischer und dialleler Versuchsanlagen unterteilt. Anhand von Elter-Nachkommen-Regressionen und-Korrelationen und von Halbgeschwister-Korrelationen wurden Heritabilitäten und genetische Korrelation geschätzt.Ein hoher Anteil der genetischen Varianz für die Zahl abdominaler Borsten wurde durch epistatische Effekte und die Wirkung geschlechtsgekoppelter Gene bedingt. Bei den anderen Charakteren war der größte Anteil der genetischen Varianz additiv autosomal.Die genetische Korrelation zwischen der Zahl der Borsten sternopleural und 2. und 3. Segment coxal war durchweg hoch, zwischen abdominal und sternopleural niedrig und zwischen abdominal und sowohl 2. und 3. coxal praktisch gleich null.Ein bemerkenswerter Anteil der Covarianz zwischen der Zahl abdominaler und sternopleuraler Borsten war nicht-additiv genetisch.Die Diallel-Methode ergab zuverlässigere Schätzungen der genetischen Parameter, wenn nicht-additive oder geschlechtsgebundene genetische Variation vorlag.

     

The fine structure of the hamster seminal vesicle with special reference to pigment formation

Summary This study describes the fine structure of the hamster seminal vesicle. Intact, sexually mature young and aged animals were used to obtain the results presented. The epithelium of the hamster seminal vesicle contains numerous large electron-dense structures, with a single limiting membrane, with many internal granules and, in the aged animals, myelin-like figures. Based on histochemical results presented in this study, and those of others, it is suggested that this material is a lipofuscin pigment. This substance is of special interest inasmuch as in the hamster seminal vesicle epithelium, pigment is a more sensitive indicator of circulating androgen than is, as in the case of the rat seminal vesicle, regression of cell height. It is possible that this pigment takes origin as breakdown products of mitochondria, the endoplasmic reticulum and the Golgi apparatus. The relation of pro-pigment granules and lysosomes is discussed. Results indicate that pigment is formed at sites of increased catabolism in response to a biological insult to the cell.Supported by U.S.P.H.S. grant RG 6583 from the National Institutes of Health, National Institute of General Medical Sciences.