Use of a biotinylated probe and in situ hybridization for light and electron microscopic localization of P 0 mRNA in myelin-forming Schwann cells

Summary A biotinylated P ₀ glycoprotein cDNA was hybridized in situ to aldehyde-fixed vibratome sections and to aldehyde-fixed thin sections of Lowicryl-embedded trigeminal ganglia of 15 day old rats. Alkaline phosphatase and peroxidase detectors were used for light microscopic (LM) studies and peroxidase or colloidal gold were employed for electron microscopic (EM) detection. In both LM and EM sections, probe was found in cytoplasmic areas of myelinforming Schwann cells that were enriched in granular endoplasmic reticulum, demonstrating that these regions contain P ₀ mRNA. Interestingly, P ₀ mRNA tended to cluster in regions close to the developing myelin sheath. Relatively simple methods are here described for EM detection of mRNA with reasonable tissue preservation and high resolution. These methods may be useful for developmental and disease-related studies of specific mRNAs in mammalian tissues.     

Alterations in tissue Mg++, Na+ and spinal cord edema following impact trauma in rats

Alterations in water content and total tissue Na+ and Mg++ of rat spinal cord tissue were followed over time after a 100 g-cm impact injury to the T-9 spinal cord segment. Rats subjected to laminectomy but not trauma served as controls. In the injured segment there was a progressive increase in water content with increased Na+ and decreased Mg++ at 1 hour and 24 hours after trauma. At seven days, water and Na+ content remained elevated, whereas Mg++ levels had returned to preinjury baseline values. Because of its important role in many metabolic and physiological regulatory processes the early decline in Mg++ concentration after trauma may contribute to the development of secondary tissue damage after spinal cord injury.     

A Monte Carlo investigation of homogeneity tests of the odds ratio under various sample size configurations

Epidemiologic data for case-control studies are often summarized into K 2 x 2 tables. Given a fixed number of cases and controls, the degree of sparseness in the data depends on the number of strata, K. The effect of increasing stratification on size and power of seven tests of homogeneity of the odds ratio is studied using Monte Carlo methods. In all the designs considered here, the numbers of cases and controls per stratum are the same. Considering both size and power in non-sparse-data settings, we recommend the Breslow-Day statistic (1980, Statistical Methods in Cancer Research, 1. The Analysis of Case-Control Studies, p. 142; Lyon: International Agency for Research on Cancer) for general use. In sparse-data settings the T4 statistic of Liang and Self (1985, Biometrika 72, 353-358) performs the best when all tables, regardless of sample size, have odds ratios generated from the same distribution. In sparse-data settings characterized by a large table with an odds ratio of 1 and many small tables with odds ratios greater than 1, the T5 statistic of Liang and Self (1985) performs the best. One of the most important results of this study is the generally low power for all homogeneity tests especially when the data are sparse.     

Evolution ofMHC polymorphism: Extensive sharing of polymorphic sequence motifs between human and bovine DRB alleles

The evolution ofMHC polymorphism has been studied by comparing the amino acid and nucleotide sequences of 14 bovine and 32 humanDRB alleles. The comparison revealed an extensive sharing of polymorphic sequence motifs in the two species. Almost identical sets of residues were found at several highly polymorphic amino acid positions in the putative antigen recognition site. Consequently, certain bovine alleles were found to be more similar to certain human alleles than to other bovine alleles. In contrast, the frequencies of silent nucleotide substitutions were found to be much higher in comparisons between species than within species implying that none of the human or bovine DRB alleles originated before the divergence of these distantly related species. The results suggest that the observed similarity inDRB polymorphism is due to convergent evolution and possibly the sharing of short ancestral sequence motifs. However, the relative role of the latter mechanism is difficult to assess due to the biased base composition in the first domain exon of polymorphic class 11 genes. The frequency of silent substitutions betweenDRB alleles was markedly lower in cattle than in man suggesting that theDRB diversity has evolved more rapidly in the former species.     

Effects of sulfhydryl reagents on nitroglycerin-induced relaxation of bovine coronary artery

The mechanism whereby nitroglycerin relaxes vascular smooth muscle remains uncertain. A current hypothesis suggests that nitroglycerin reacts with critical cellular sulfhydryl groups to form an intermediate, which activates guanylate cyclase, resulting in cGMP accumulation and relaxation. This study investigated further the potential involvement of sulfhydryls in nitroglycerin-induced vascular smooth muscle relaxation by evaluating effects of a variety of sulfhydryl alkylating and reducing agents on responses to nitroglycerin and other relaxants in bovine coronary arterial strips submaximally contracted using 30 mM K. Whereas 10(-4) M 5,5'-dithiobis-(2-nitrobenzoic acid), 10(-5) MN-ethylmaleimide, and 10(-4) MN-naphthylmaleimide did not affect nitroglycerin-induced relaxation, 10(-4) MN-ethylmaleimide and 10(-4) M ethacrynic acid significantly inhibited relaxation induced by nitroglycerin. Both ethacrynic acid and N-ethylmaleimide at 10(-4) M also inhibited relaxation induced by sodium nitroprusside. N-ethylmaleimide, but not ethacrynic acid, inhibited relaxation induced by isoproterenol and forskolin. Ethacrynic acid significantly reduced both relaxation and cGMP elevation induced by both 10(-7) M nitroglycerin and 10(-7) M sodium nitroprusside. Ethacrynic acid, but not N-ethylmaleimide, significantly reduced relaxation induced by 8-Br-cGMP. Pretreatment with the sulfhydryl-containing agents N-acetylcysteine, 2-mercaptoethanol, or dithiothreitol, at 10(-3) M did not affect nitroglycerin-induced relaxation in nontolerant arteries. Similarly, N-acetylcysteine and dithiothreitol did not alter the depressed responses to nitroglycerin in arteries in which tolerance to nitroglycerin was induced in vitro. A slight but statistically significant reversal of nitroglycerin-tolerance occurred after treatment of tolerant arteries with 2-mercaptoethanol.(ABSTRACT TRUNCATED AT 250 WORDS)     

Isolation and partial characterization of carbonic anhydrase from erythrocytes of Meleagris gallopavo

Summary A soluble enzyme with carbonic anhydrase activity has been isolated from domestic turkey(Meleagris gallopavo) erythrocytes and purified by chloroformethanol precipitation, ammonium sulfate fractionation and gel filtration on a Sephadex G-75 column. Analytical polyacrylamide gel disc electrophoresis showed one major and two minor bands. The specific activity for the CO₂ hydration reaction was approximately 2000 Wilbur-Anderson units/mg protein at 0°C. The presence of the reducing agents 2-mercaptoethanol or dithioerythritol was required throughout the procedure. Upon removal of the 2-mercaptoethanol by dialysis the activity was lost but could be restored by addition of the reducing agent. The enzymatic activity was inhibited by acetazolamide,p-chloromercuribenzoate ando-iodosobenzoate. Esterase activity was detected withp-nitrophenylacetate as the substrate. The molecular weight of the enzyme was determined as 31,000 by gel filtration and 34,000 ± 2000 with analytical ultracentrifugation. Atomic absorption spectroscopy indicated the presence of zinc in the ratio of one mole of zinc per one mole of enzyme.