Effects of denervation on the contents of cholesterol and membrane systems involved in muscle contraction in rabbit fast-twitch sarcotubular system.

Denervated fast-twitch rabbit muscles were progressively losing their fresh weight and the yield of sarcotubular protein was increasing. The activity of Ca(2+)-ATPase was affected but very slightly, the basal Mg(2+)-ATPase and the Mg(2+)-ATPase/Ca(2+)-ATPase ratio however increased together with a simultaneous depression of the membrane-bound acetylcholinesterase activity. We did not observe any differences in density properties of sarcotubular fractions between control and denervated muscle. However, a relative enrichment in SM and H fraction could be seen after denervation with small changes in the content of the Ca(2+)-pump protein, increased levels of calsequestrin and cholesterol, mostly in the heavy and the SM fraction. After denervation the binding sites for 3H-PN-200-110 did not show any changes in receptor affinity, but the number of putative Ca(2+)-channels increased twice along with a depression of 3H-ouabain binding sites. We suggest that the denervation of fast-twitch muscle leads to the hypertrophy of the junctional sarcoplasmic reticulum and the T-system. Changes in the cholesterol content, in the number of putative Ca(2+)-channels and in Na+, K(+)-ATPase can affect the muscle contraction.     

Structures of the α(1-3)-galactose-containing asparagine-linked glycans of a Lewis lung carcinoma cell subline resistant toAleuria aurantia agglutinin: elucidation by1H-NMR spectroscopy

Four bi-antennary glycan fractions of theN-acetyllactosamine-type, derived from a Lewis lung carcinoma (LL₂) cell subline resistant to theAleuria aurantia agglutinin were studied by 400 MHz¹H-NMR spectroscopy. By this method, their antennae were found to be terminated either by (2-3 or 6)-linkedN-acetylneuraminic acid or (1-3)-linked galactose residues. The primary structure of glycans of these four glycopeptide or derived oligosaccharide-alditols has been determined in full detail.Abbreviations NAc N-acetyl group - NGc N-glycolyl group - GlcNAc N-acetylglucosamine - NeuAc N-acetylneuraminic acid - NeuGc N-glycolylneuraminic acid - Man mannose - Gal galactose - Fuc fucose - Con A concanavalin A - LCA Lens culinaris agglutinin - AAA Aleuria aurantia agglutinin - WGA Wheat germ agglutinin - RCA II Ricinus communis agglutinin II - PBS phosphate buffered saline, 0.01m Na₂HPO₄/0.14m NaCl, pH 7.2 - HPLC high performance liquid chromatography - EMEM Eagle's Minimal Essential Medium - Lec^R lectin resistant - MG -methylglycoside     

Ca2+ calmodulin-dependent protein kinase activity in the ascomycetes Neurospora crassa

DEAE-cellulose column chromatography of Neurospora crassa soluble mycelial extracts leads to the resolution of three major protein kinase activity peaks designated PKI, PKII, and PKIII.PKII activity is stimulated by Ca²⁺ and Neurospora or brain calmodulin. Maximal stimulation was observed at 2 µM-free Ca²⁺ and 1 µg/ml of the modulator. The stimulatory effect of the Ca²⁺-calmodulin complex was blocked by EGTA and by some calmodulin antagonists such as phenothiazine drugs or compound 48/80.PKII phosphorylates different proteins, among which histone II-A at a low concentration and CDPKS, the synthetic peptide specific for Ca²⁺-calmodulin dependent protein kinases, are the best substrates. Some phosphorylation can be detected in the absence of any exogenous acceptor. PKII activity assayed in the presence of histone II-A or in the absence of exogenous phosphate acceptor (autophosphorylation) co-elute in a DEAE-cellulose column at 0.28 M NaCl. As result of the autophosphorylation reaction of the purified enzyme a main phosphorylated component of 70 kDa was resolved by SDS-polyacrylamide gel electrophoresis. It is possible that this component is an active part of this enzyme.     

Ultrastructural visualization of exocytosis in the pig pineal gland

Summary The pinealocytes of the pig contain conspicuous dense bodies, the nature and role of which are not yet fully elucidated. The aim of this study was to demonstrate whether or not these structures are involved in the secretion process. The tannic acid-Ringer incubation (TARI)-method, which allows a clear-cut ultrastructural study of secretory discharge by exocytosis, has been used. The results indicate that pig pinealocytes release the content of the dense bodies with an amorphous inner structure into the extracellular space via exocytosis and that this secretion is quantitatively important. The secreted material is proteinaceous in nature; this indicates that polypeptides are released by the pineal.     

Decrease in insulin-containing secretory granules and mitochondrial gene expression in mouse pancreatic islets maintained in culture following streptozotocin exposure

We have previously described a preferential reduction in the secretory response to nutrient secretagogues in pancreatic mouse islets maintained in culture after in vitro exposure to streptozotocin (SZ). This reduction was associated with an impaired substrate metabolism at the mitochondrial level. To further clarify this issue, mouse pancreatic islets were exposed in vitro to 2.2 mM SZ for 30 min. At 4 h after SZ treatment ultrastructural changes were apparent in the endoplasmic reticulum and Golgi areas of the B-cells. However, 2 and 6 days following SZ exposure the B-cells appeared well preserved, except for a marked decrease in the number of insulin-containing secretory granules. A morphometric analysis of the B-cells 6 days after SZ exposure showed a normal B-cell size and a normal volume fraction of B-cell mitochondria. However, there was a decrease in total islet size and a 13% decrease in the volume fraction of B-cells in the islets. These mouse islets exhibited a decreased content of the mitochondrial DNA-encoded cytochrome b mRNA, as evaluated by dot-blot analysis. As a whole, the data obtained indicate that SZ treatment does not induce a decrease in the number of mitochondria or long-lasting ultrastructural damage to this organelle. However, there is a clear decrease in the cytochrome b mRNA, suggesting that SZ can induce damage to the mitochondrial DNA.