Ion pathways in transverse tubules. Quantification of receptors in membranes isolated from frog and rabbit skeletal muscle

The presence of four cation pathways in membrane vesicles isolated from transverse tubules of frog and rabbit skeletal muscle was studied by measuring binding of specific blockers. Transverse tubules purified from frog muscle have a maximal binding capacity for [3H]nitrendipine (a marker for voltage-dependent calcium channels) of 130 pmol/mg of protein; this binding is strongly dependent on temperature and, at 37 degrees C, on the presence of diltiazem. Receptors for [3H]ethylenediamine tetrodotoxin (a marker for voltage-dependent sodium channels) and for 125I-labeled alpha-bungarotoxin (a marker for acetylcholine-mediated channels) showed maximal binding values of about 5 pmol/mg. The number of sodium-pumping sites in the isolated tubule vesicles, inferred from [3H]ouabain binding, was 215 pmol/mg. The high purity of this preparation makes feasible the use of these values as a criterion to judge the degree of purity of isolated preparations, and it allows investigation of transverse tubule contamination in other muscle membrane fractions.     

Effects of partial hepatectomy on various blood and hepatic parameters in the rainbow trout (Salmo gairdnerii, Rich.)

After 36% of hepatic mass removal , rainbow trout recovered its initial liver weight in 20-30 days, i.e., with a regeneration rate clearly lower than in mammals. During early regeneration process hematocrit index and hemoglobin content were slightly decreased, but both parameters rapidly reached their normal values. The evolution of both glycaemia and hepatic glycogen content supported the idea of the existence of a late regeneration wave, which, in this case, could begin at about the 20th post-operative day.     

Isolation of peroxisomes from frozen human liver samples.

This paper shows the successful isolation of peroxisomes from human liver samples that were kept frozen at -70 degrees C. Purification of these peroxisomes was obtained by a combination of two subcellular fractionation techniques: differential centrifugation and isopycnic fractionation in Nycodenz density gradients. Peroxisome integrity was evaluated by latency measurements and by ultrastructural observation. The procedure described here may be useful for the isolation of other subcellular organelles from frozen human samples.     

Effect of stress,adrenalectomy and changes in glutathione metabolism on rat kidney metallothionein content: comparison with liver metallothionein

Eighteen hours of immobilization stress, accompanied by food and water deprivation, increased liver metallothionein (MT) but decreased kidney MT levels. Food and water deprivation alone had a significant effect only on liver MT levels. In contrast, stress and food and water deprivation increased both liver and kidney lipid peroxidation levels, indicating that the relationship between MT and lipid peroxidation levels (an index of free radical production) is unclear. Adrenalectomy increased both liver and kidney MT levels in basal conditions, whereas the administration of corticosterone in the drinking water completely reversed the effect of adrenalectomy, indicating an inhibitory role of glucocorticoids on MT regulation in both tissues. Changes in glutathione (GSH) metabolism produced significant effects on kidney MT levels. Thus, the administration of buthionine sulfoximine, an inhibitor of GSH synthesis, decreased kidney GSH and increased kidney MT content, suggesting that increased cysteine pools because of decreased GSH synthesis might increase kidney MT levels through an undetermined mechanism as it appears to be the case in the liver. However, attempts to increase kidney MT levels by the administration of cysteine or GSH were unsuccesful, in contrast to what is known for the liver. The present results suggest that there are similarities but also substantial differences between liver and kidney MT regulation in these experimental conditions.     

Molecular analysis of a microaerobically induced operon required for hydrogenase synthesis in Rhizobium leguminosarum biovar viciae

The nucleotide sequence (6138 bp) of a microaerobically inducible region (hupV/VI) from the Rhizobium leguminosarum bv. viciae hydrogenase gene cluster has been determined. Six genes, arranged as a single operon, were identified, and designated hypA, B, F, C, D and E based on the sequence similarities of all of them, except hypF, to genes from the hydrogenase pleiotropic operon (hyp) from Escherichia coli. The gene products from hypBFCDE were identified by in vivo expression analysis in E. coli, and their molecular sizes were consistent with those predicted from the nucleotide sequence. Transposon Tn5 insertions into hypB, hypF, hypD and hypE resulted in R. leguminosarum mutants that lacked any hydrogenase activity in symbiosis with peas, but still were able to synthesize the polypeptide for the hydrogenase large subunit. The gene products HypA, HypB, HypF and HypD contained CX₂C motifs characteristic of metal-binding proteins. In addition, HypB bore a long histidine-rich stretch of amino acids near the N-terminus, suggesting a possible role in nickel binding for this protein. The gene product HypF, which was translationally coupled to HypB, presented two cysteine motifs (CX₂CX₈₁CX₂C) with a capacity to form zinc finger-like structures in the N-terminal third of the protein. A role in nickel metabolism in relation to hydrogenase synthesis is postulated for proteins HypB and HypF.     

Solution structure of the potassium channel inhibitor agitoxin 2: caliper for probing channel geometry.

The structure of the potassium channel blocker agitoxin 2 was solved by solution NMR methods. The structure consists of a triple-stranded antiparallel beta-sheet and a single helix covering one face of the beta-sheet. The cysteine side chains connecting the beta-sheet and the helix form the core of the molecule. One edge of the beta-sheet and the adjacent face of the helix form the interface with the Shaker K+ channel. The fold of agitoxin is homologous to the previously determined folds of scorpion venom toxins. However, agitoxin 2 differs significantly from the other channel blockers in the specificity of its interactions. This study was thus focused on a precise characterization of the surface residues at the face of the protein interacting with the Shaker K+ channel. The rigid toxin molecule can be used to estimate dimensions of the potassium channel. Surface-exposed residues, Arg24, Lys27, and Arg31 of the beta-sheet, have been identified from mutagenesis studies as functionally important for blocking the Shaker K+ channel. The sequential and spatial locations of Arg24 and Arg31 are not conserved among the homologous toxins. Knowledge on the details of the channel-binding sites of agitoxin 2 formed a basis for site-directed mutagenesis studies of the toxin and the K+ channel sequences. Observed interactions between mutated toxin and channel are being used to elucidate the channel structure and mechanisms of channel-toxin interactions.     

Effect of mexiletine on glucose and oxygen uptake in rat brain, liver and myocardial tissue in vitro (author's transl)

The effect of mexiletine on oxygen and glucose consumption was studied both in homogenate and slices of brain, liver and myocardium of Wistar rats. Oxygen consumption was detected by means of Warburg's manometric techniques, and glucose utilization by the enzymatic method of glucose oxidase. Whilst glucose uptake was not modified in any of the studied preparations, mexiletine promoted a significant increase of oxygen consumption in the homogenized slices, and an inhibition in the intact tissue.     

Inositol 1,4,5-triphosphate phosphatase activity in membranes isolated from amphibian skeletal muscle [corrected

The hydrolysis of [3H]inositol 1,4,5-trisphosphate by a soluble fraction and by isolated transverse tubule and sarcoplasmic reticulum membranes from frog skeletal muscle was studied. Transverse tubule membranes displayed rates of hydrolysis several-fold higher than those of sacroplasmic reticulum and soluble fraction; Km and Vmax were 25.2 microM and 44.1 nmol/mg/min, respectively. Transverse tubule membranes sequentially hydrolyzed inositol trisphosphate to inositol bisphosphate, inositol 1-phosphate and inositol, indicating that these membranes have inositol bis- and monophosphatases in addition to inositol trisphosphatase.     

Oxidant-induced haemoprotein degradation in rat tissue slices: effect of bromotrichloromethane, antioxidants and chelators

Haemoprotein degradation and lipid peroxidation were evaluated in rat liver, kidney and heart slices incubated for 2 h in the presence and absence of bromotrichloromethane, antioxidants and chelators to obtain information about the relationship between oxidants and damage to haemoproteins. Haemoproteins were modified by bromotrichloromethane, and this modification, measured as loss of ferrohaemoproteins, generally was concurrent with lipid peroxidation measured as thiobarbituric acid-reactive substances. These two processes occurred simultaneously as a function of incubation time and oxidant concentration. Inhibition of the two processes by nordihydroguaiaretic acid, butylated hydroxyanisole and Trolox C, and lack of inhibition by mannitol, catalase and superoxide dismutase also were coincident. However, Methylene blue, EDTA, sodium fluoride, 2,4-dinitrophenol, N-ethylmaleimide and o-phenanthroline affected the two processes differently. The results suggested that haemoproteins may compete with other molecules for oxidant radicals, thus serving as protectors of cells against oxidant radicals. Products of haemoprotein degradation such as protein polymers, free amino acids and bilirubin may be indicators of in vivo oxidative stress.     

Oxidant-increased proteolysis in rat liver slices: effect of bromotrichloromethane, antioxidants and effectors of proteolysis

Proteolysis and lipid peroxidation were evaluated in rat liver slices incubated in the presence of the oxidant bromotrichloromethane and effectors of proteolysis. Proteolysis was evaluated by S-amino acids and lipid peroxidation by thiobarbituric acid-reactive substances (TBARS) released into the incubation medium. The increased release of S-amino acids by BrCl3C depended on incubation time and oxidant concentration. S-Amino acid release increased 30% over control value and TBARS increased from 22 to 124 nmol/g liver by incubation for 120 min with 1 mM BrCl3C. Release of S-amino acids and TBARS was decreased when liver slices were treated with nor-dihydroguaiaretic acid (NDG), butylated hydroxyanisole (BHA), Trolox C, or N,N'-diphenyl-1,4-phenylenediamine (DPPD) immediately prior to addition of oxidant, suggesting participation of lipid-soluble free radicals. Oxidant-induced release of S-amino acids but not of TBARS was decreased by mannitol, suggesting participation of hydroxyl radical or a species with similar reactivity; and by superoxide dismutase and catalase, suggesting participation of superoxide and hydrogen peroxide, respectively. The decrease of S-amino acid release by sodium fluoride, sodium arsenate, 2,4-dinitrophenol, chloroquine, leupeptin, phenylmethylsulfonyl fluoride, EDTA and o-phenanthroline was variable, suggesting the presence in liver of several proteases to remove oxidatively-modified proteins.