Identification of a new subpopulation of triad junctions isolated from skeletal muscle; Morphological correlations with intact muscle

Summary It has been previously recognized that a number of protocols may cause breakage of the triad junction and separation of the constituent organelles of skeletal muscle. We now describe a fraction of triad junctions which is refractory to the known protocols for disruption. Triads were passed through a French press and the dissociated organelles were separated on a sucrose density gradient, which was assayed for PN200-110, ouabain and ryanodine binding. Ryanodine binding showed a single peak at the density of heavy terminal cisternae. On the other hand, the PN200-110 and ouabain, which are external membrane ligands, bound in two peaks: one at the free transverse tubule region and the other at the light terminal cisternae. Similarly, a two peak pattern of PN200-110 and ouabain binding was observed when triad junctions were broken by the Ca²⁺-dependent protease, calpain, which selectively hydrolyzes the junctional foot protein. The light terminal cisternae vesicles were subjected to three different procedures of junctional breakage: French press, hypertonic salt treatment, and protease digestion using calpain or trypsin. The treated membranes were then centrifuged on density gradients. Only extensive trypsin digestion caused a partial shift of ouabain activity into the free transverse tubule region. These observations suggest that the triads are a composite mixture of breakage susceptible, weak, and breakage resistant, strong, triads. Scatchard analysis of PN200-110 suggests that the transverse tubules of strong triads contain a relatively high number of dihydropyridine receptors compared to those of weak triads. Thin section electron microscopic images of the strong triads comparable to those of intact muscle are presented.     

Inhibitors of sterol synthesis. Chemical synthesis, structure, and biological activities of (25R)-3 beta,26-dihydroxy-5 alpha-cholest-8(14)-en-15-one, a metabolite of 3 beta-hydroxy-5 alpha-cholest-8(14)-en-15-one

3 beta-Hydroxy-5 alpha-cholest-8(14)-en-15-one (I) is a potent inhibitor of sterol synthesis with significant hypocholesterolemic activity. (25R)-3 beta,26-Dihydroxy-5 alpha-cholest-8(14)-en-15-one (II) has been shown to be a major metabolite of I after incubation with rat liver mitochondria. Described herein is the chemical synthesis of II from diosgenin. As part of this synthesis, improved conditions are described for the conversion of diosgenin to (25R)-26-hydroxycholesterol. Benzoylation of the latter compound gave (25R)-cholest-5-ene-3 beta,26-diol 3 beta,26-dibenzoate which, upon allylic bromination followed by dehydrobromination, gave (25R)-cholesta-5,7-diene-3 beta,26-diol 3 beta,26-dibenzoate. Hydrogenation-isomerization of the delta 5.7-3 beta,26-dibenzoate to (25R)-5 alpha-cholest-8(14)-ene-3 beta,26-diol 3 beta,26-bis(cyclohexanecarboxylate) followed by controlled oxidation with CrO3-dimethylpyrazole gave (25R)-3 beta,26-dihydroxy-5 alpha-cholest-8(14)-en-15-one 3 beta,26-bis(cyclohexanecarboxylate). Acid hydrolysis of the delta 8(14)-15-ketosteryl diester gave II. 13C NMR assignments are given for all synthetic intermediates and several major reaction byproducts. The structure of II was unequivocally established by X-ray crystal analysis. II was found to be highly active in the suppression of the levels of 3-hydroxy-3-methylglutaryl coenzyme A reductase in cultured mammalian cells and to inhibit oleoyl coenzyme A-dependent esterification of cholesterol in jejunal microsomes.     

Analysis of aminophospholipid molecular species by high performance liquid chromatography

A new method is described for the separation of individual molecular species of the aminophospholipids, phosphatidylethanolamine and phosphatidylserine. Trinitrobenzene-sulfonic acid was used to derivatize both aminophospholipids and the derivatives were purified by thin-layer chromatography. A reversed-phase high performance liquid chromatography technique was developed to separate and quantify individual molecular species based upon ultraviolet detection of the attached chromophore. The retention times of the molecular species on the C18 reversed-phase column were longer with increasing carbon chain length and decreasing degree of unsaturation of fatty acyl chain. The overall procedure allowed a quantitative recovery of the aminophospholipid species. The lower limit of detection was about 10 pmol and a linear response was observed in the range of 0.1-10 nmol of phospholipid. Using this method, we were able to separate and quantify trinitrophenyl-phosphatidylethanolamine molecular species of both subclasses (diacyl and alkenyl) from human red blood cells and rat brains. Separation of species was confirmed by gas-liquid chromatographic analysis of the fatty acid content of each peak and by thermospray liquid chromatography-mass spectrometry. This new method provides a convenient and sensitive technique for studies of aminophospholipid molecular species composition. Furthermore, it appears to be a useful tool for the analysis of asymmetric distribution of these species in biological membranes.     

Maturation, fertilization and development of bovine oocytes in vitro using TCM199 and a simple defined medium with co-culture

Bovine oocytes obtained from ovarian follicles (2 to 5 mm in diameter) from slaughtered cattle were cultured in TCM199 with 10% heat-inactivated estrous cow serum (ECS) for 24 to 25 h at 39 degrees C under 5% CO(2) in air. The 10% ECS was selected on the basis of preliminary studies in which in vitro fertilization rates of oocytes with 10, 15 and 20% ECS in the medium were 46, 30 and 31%, respectively (P<0.05). Of 120 oocytes cultured for 24 to 25 h, 63% were classified as being in Metaphase II. The rate of oocytes matured in vitro was 55% (69 125 ), the proportion of penetrated oocytes which contained male and female pronuclei was 94% (65 69 ), and the incidence of polyspermy was very low (0 to 9%). Of 122 oocytes fertilized in vitro and cultured in TCM199 medium with 10% fetal bovine serum for 7 d, 53% were cleaved, but only 2% developed beyond the 16-cell block. However, in simple semi-defined Chatot-Ziomek-Bavister medium co-cultured with bovine oviduct epithelial cells (BOEC), 75% of 138 oocytes cleaved, and 38% of those which cleaved developed into morulae or blastocysts. The results of this study indicate that co-culture with BOEC exerted a pronounced beneficial effect on development of in vitro fertilized bovine oocytes through the 16-cell block. The medium required in the co-culture system was simple and semi-defined.     

The changes of histopathology and serum anti-sparganum IgG in experimental sparganosis of mice

The present study is intended to observe the chronologic changes of experimental sparganosis by histopathological observation and detection of circulating anti-sparganum IgG antibody using ELISA. Each of 25 mice was infected with five spargana, and they were examined after 1, 2, 4, 10 weeks or 6 months from infection. The followings are summarized results. 1. The plerocercoids were detected in the subcutaneous tissue of the trunk, neck or axilla, but a few often extended into the skeletal muscle. The recovery rates were 72% at the first week, 80% at the second week, 95% at the fourth week, 92% at the tenth week and 100% at the sixth month. The larvae grew slowly in both length and weight until 6 months. 2. Histopathologically, most of the larvae were observed alive in the soft tissue or skeletal muscle. Numerous eosinophils, neutrophils, lymphocytes and plasma cells were infiltrated focally around the worms by the second week, but they surrounded the worms to form a layer of inflammatory reaction after 4 weeks of infection. Also histiocytes and fibroblasts began to appear around the inflammatory cells at 4 weeks. After 10 weeks, the worms encircled by a thin fibrous layer were found. After 6 months, the worms were surrounded by either fibrous tissue or active inflammatory cells. The inflammation looked more severe in the tracks left by the worms, rather than around the worms. 3. The level of anti-sparganum IgG antibody in the serum showed an increase by the fourth week, and a rapid and continuous increase was observed thereafter by the tenth week after infection.(ABSTRACT TRUNCATED AT 250 WORDS)     

Structure of a ricin mutant showing rescue of activity by a noncatalytic residue.

Ricin A chain is an N-glycosidase which removes a single adenine base from a conservative loop of 28S rRNA, thereby inactivating eukaryotic ribosomes. The mechanism of action has been proposed to include transition-state stabilization of an oxycarbonium ion on the substrate ribose by interaction with Glu 177. Conversion of Glu 177 to Gln reduces activity nearly 200-fold [Ready, M. P., Kim, Y., & Robertus, J. D. (1991) Proteins: Struct., Funct., Genet. 10, 270-278] while conversion to Ala (E177A) reduces activity only 20-fold [Schlossman, D., Withers, D., Welsh, P., Alexander, A., Robertus, J., & Frankel, A. (1989) Mol. Cell. Biol. 9, 5012-5021]. X-ray analysis of the latter mutant protein shows that a residue at the edge of the active site, Glu 208, rotates into the space left vacant by the mutation. Its rearranged carboxylate partially substitutes for that of Glu 177. This is equivalent to the rescue of enzyme activity by a second-site reversion. Kinetic analysis shows the E177A mutation affects kcat and not Km, consistent with the notion that the carboxylate serves in transition-state stabilization.     

A photoactive phosphonamide derivative of GTP for the identification of the GTP-binding domain in beta-tubulin

A GTP photoaffinity probe (125I-APTG) was developed that incorporated an [125I]-N-(4-azidophenyl)-2-amino-3-(4-hydroxy-3-iodophenyl)propionamide group at the gamma-position of GTP through a phosphonamide linkage. A combination of saturation and GTP protection studies (90% protection at 25 microM GTP with an apparent Kd of 5 microM) validated the use of this new probe as a satisfactory GTP mimic. This probe offered the advantage of possessing an 125I radiolabel external to the GTP moiety, in contrast to the previously reported [gamma 32P]-8-N3GTP that possessed an internal 32P radiolabel. This novel feature accommodated the purification of photolabeled peptides using a combination of ion-exclusion, gel filtration, and HPLC techniques. [125I]APTG was used to identify a peptide (beta:65-79) in the exchangeable GTP-binding domain of the beta-subunit of tubulin.     

Nonserine esterases from rat liver cytosol

An effort to identify the major general esterases of rat liver cytosol that are insensitive to the serine esterase inhibitor paraoxon (diethyl 4-nitrophenyl phosphate) has led to the isolation of a dozen enzymes. Four of these are electrophoretically homogeneous. Although purified on the basis of their hydrolytic activity toward 4-nitrophenyl acetate, each of the enzymes has a very broad and overlapping substrate specificity for aromatic esters. Thiol esters serve as substrates but, within the limits of the methods used, amides are not hydrolyzed.     

Regional Reductions of Transketolase in Thiamine-Deficient Rat Brain

Abstract: Thiamine deficiency impairs oxidative metabolism and causes metabolic encephalopathy. An early reduction in transketolase (TK) activity may be an important pathogenic event. To assess the role of TK, we have delineated the regional/cellular distribution of TK protein and mRNA in adult rat brain in pyrithiamine-induced thiamine deficiency. TK activity declined in both vulnerable and spared regions. Immunoblots showed a parallel reduction of TK protein. With a few exceptions, immunocytochemistry indicated an overall decline of TK immunoreactivity and the decrease was not specific to vulnerable areas. In contrast to the pronounced, general decline of TK protein, in situ hybridization revealed a regional decrease of 0–25% of TK mRNA in thiamine deficiency. Northern blots indicated a similar level of TK mRNA in whole brain in thiamine deficiency. These results show that the decline of TK activity results from a proportional decrease of TK protein, and the deficiency may be due to an instability of TK protein or an inhibition of TK mRNA translation. The lack of correlation of the distribution, and the absence of specific alteration, of TK in affected regions suggest that the reduced TK may not be linked directly to selective vulnerability in thiamine deficiency.