Redox regulation of RyR-mediated Ca2+ release in muscle and neurons

Changes in the redox state of the intracellular ryanodine receptor/Ca2+ release channels of skeletal and cardiac muscle or brain cortex neurons affect their activity. In particular, agents that oxidize or alkylate free SH residues of the channel protein strongly enhance Ca(2+)-induced Ca2+ release, whereas reducing agents have the opposite effects. We will discuss here how modifications of highly reactive cysteine residues by endogenous redox agents or cellular redox state influence RyR channel activation by Ca2+ and ATP or inhibition by Mg2+. Possible physiological and pathological implications of these results on cellular Ca2+ signaling will be addressed as well.     

Folding and insertion of the outer membrane protein OmpA is assisted by the chaperone Skp and by lipopolysaccharide

We have studied the folding pathway of a beta-barrel membrane protein using outer membrane protein A (OmpA) of Escherichia coli as an example. The deletion of the gene of periplasmic Skp impairs the assembly of outer membrane proteins of bacteria. We investigated how Skp facilitates the insertion and folding of completely unfolded OmpA into phospholipid membranes and which are the biochemical and biophysical requirements of a possible Skp-assisted folding pathway. In refolding experiments, Skp alone was not sufficient to facilitate membrane insertion and folding of OmpA. In addition, lipopolysaccharide (LPS) was required. OmpA remained unfolded when bound to Skp and LPS in solution. From this complex, OmpA folded spontaneously into lipid bilayers as determined by electrophoretic mobility measurements, fluorescence spectroscopy, and circular dichroism spectroscopy. The folding of OmpA into lipid bilayers was inhibited when one of the periplasmic components, either Skp or LPS, was absent. Membrane insertion and folding of OmpA was most efficient at specific molar ratios of OmpA, Skp, and LPS. Unfolded OmpA in complex with Skp and LPS folded faster into phospholipid bilayers than urea-unfolded OmpA. Together, these results describe a first assisted folding pathway of an integral membrane protein on the example of OmpA.     

Functional conservation of subfamilies of putative UDP-N-acetylgalactosamine:polypeptide N-acetylgalactosaminyltransferases in Drosophila, Caenorhabditis elegans, and mammals. One subfamily composed of l(2)35Aa is essential in Drosophila

The completed fruit fly genome was found to contain up to 15 putative UDP-N-acetyl-alpha-d-galactosamine:polypeptide N-acetylgalactosaminyltransferase (GalNAc-transferase) genes. Phylogenetic analysis of the putative catalytic domains of the large GalNAc-transferase enzyme families of Drosophila melanogaster (13 available), Caenorhabditis elegans (9 genes), and mammals (12 genes) indicated that distinct subfamilies of orthologous genes are conserved in each species. In support of this hypothesis, we provide evidence that distinctive functional properties of Drosophila and human GalNAc-transferase isoforms were exhibited by evolutionarily conserved members of two subfamilies (dGalNAc-T1 (l(2)35Aa) and GalNAc-T11; dGalNAc-T2 (CG6394) and GalNAc-T7). dGalNAc-T1 and novel human GalNAc-T11 were shown to encode functional GalNAc-transferases with the same polypeptide acceptor substrate specificity, and dGalNAc-T2 was shown to encode a GalNAc-transferase with similar GalNAc glycopeptide substrate specificity as GalNAc-T7. Previous data suggested that the putative GalNAc-transferase encoded by l(2)35Aa had a lethal phenotype (Flores, C., and Engels, W. (1999) Proc. Natl. Acad. Sci. U. S. A. 96, 2964-2969), and this was substantiated by sequencing of three lethal alleles l(2)35Aa(HG8), l(2)35Aa(SF12), and l(2)35Aa(SF32). The finding that subfamilies of GalNAc-transferases with distinct catalytic functions are evolutionarily conserved stresses that GalNAc-transferase isoforms may serve unique biological functions rather than providing functional redundancy, and this is further supported by the lethal phenotype of l(2)35Aa.     

The EGF receptor provides an essential survival signal for SOS-dependent skin tumor development

The EGF receptor (EGFR) is required for skin development and is implicated in epithelial tumor formation. Transgenic mice expressing a dominant form of Son of Sevenless (SOS-F) in basal keratinocytes develop skin papillomas with 100% penetrance. However, tumor formation is inhibited in a hypomorphic (wa2) and null EGFR background. Similarly, EGFR-deficient fibroblasts are resistant to transformation by SOS-F and rasV12, however, tumorigenicity is restored by expression of the anti-apoptotic bcl-2 gene. The K5-SOS-F papillomas and primary keratinocytesfrom wa2 mice display increased apoptosis, reduced Akt phosphorylation and grafting experiments imply a cell-autonomous requirement for EGFR in keratinocytes. Therefore, EGFR functions as a survival factor in oncogenic transformation and provides a valuable target for therapeutic intervention in a broader range of tumors than anticipated.     

Identification of two genes encoding putative new members of the ECF subfamily of eubacterial RNA polymerase sigma factors in Clostridium acetobutylicum

Two genes from Clostridium acetobutylicum DSM 792 were identified which are predicted to encode new members of the ECF subfamily of eubacterial RNA polymerase sigma factors. The sigX gene has the potential to encode a 184-amino acid protein with a molecular mass of 21,870 Da and with the highest overall similarity to Fecl of Escherichia coli (27 % identical residues). The second gene, which is predicted to encode an alternative sigma factor of the ECF subfamily, is the previously described orf2 gene (Gerischer and Dürre, 1990) located in the adc gene region of C. acetobutylicum. The deduced protein of orf2 has significant similarity to SigX of C. acetobutylicum (22 % identical residues) and shares structural features with other alternative sigma factors. Therefore, it is proposed to rename orf2 as sigY. Analysis of the phylogenetic relationship revealed that SigX from C. acetobutylicum, together with sigmaE from Streptomyces coelicolor and SigX from Bacillus subtilis, form a gram-positive cluster within the ECF subfamily and that SigY from C. acetobutylicum together with UviA from Clostridium perfringens, form a separate cluster located between the gram-positive cluster and the sporulation sigma factor sigmaH from B. subtilis.     

Orthophosphate-pyrophosphate exchange catalyzed by soluble and membrane-bound inorganic pyrophosphatases. Role of H+ gradient

A comparative study of the orthophosphate-pyrophosphate exchange reaction catalyzed by the soluble pyrophosphatase from baker's yeast and by the membrane-bound pyrophosphatase of Rhodospirillum rubrum chromatophores was performed. In both systems the rate of exchange increased when the pH of the medium was raised from 6.0 to 7.8 and when the MgCl2 concentration was raised from 0.1 mM to 20 mM. For the yeast pyrophosphatase the exchange rates measured at different pH values and in the presence of 6.7 to 8.8 mM free Mg2+ superimposed as a single curve when plotted as a function of the concentrations of either HPO4(2-) or MgHPO4. This was not observed with the use of R. rubrum chromatophores. With yeast pyrophosphatase, the Km for Pi was higher than 10 mM and could not be measured when the free Mg2+ concentration in the medium was lower than 0.5 mM. There was a decrease in the Km for Pi when the free Mg2+ concentration was raised to 6.7-8.8 mM or when, in the presence of low free Mg2+, the organic solvents dimethylsulfoxide (20% v/v) or ethyleneglycol (40% v/v) were included in the assay medium. In the presence of 6.7-8.8 mM free Mg2+ the Km for total Pi was 7 mM at pH 7.0 and 12 mM at pH 7.8. For the ionic species HPO4(2-) and MgHPO4, the Km values were 5.8 mM and 4.2 mM respectively. In the presence of 0.24-0.42 mM free Mg2+ and either 20% (v/v) dimethylsulfoxide or 40% (v/v) ethyleneglycol the Km values for total Pi, HPO4(2-) and MgHPO4 were 7.6, 3.5 and 0.5 mM respectively. With R. rubrum chromatophores, the Km for Pi in the presence of 5.5-7.5 mM free Mg2+ was very high and could not be measured. In the presence of 0.24-0.45 mM free Mg2+ the ratio between the velocities of hydrolysis and synthesis of pyrophosphate measured at pH 7.8 with yeast pyrophosphatase and chromatophores of R. rubrum were practically the same. When the free Mg2+ concentration was raised to 5.5-8.8 mM this ratio decreased from 1028 to 540 when the yeast pyrophosphatase was used and from 754 to 46 when chromatophores were used.     

Metabolic syndrome in children and adolescents--risk for sleep-disordered breathing and obstructive sleep-apnoea syndrome?

The clinical relevance of the term "metabolic syndrome", the definition criteria, and predictive power are being disputed. Inclusion of sleep-disordered breathing and sleep apnoea into a definition of metabolic syndrome is also controversial once children and/or adolescents are affected. Nevertheless, along with the increasing prevalence of childhood obesity, the prevalence of the metabolic syndrome in obese children is reported at 30%, irrespective of the definition applied. Moreover, childhood obesity is associated with sleep-disordered breathing. Adipocytokines, cytokines secreted from adipose tissue, are thought to play a major role in the pathophysiology of metabolic syndrome. Leptin was initially suggested as a promising "anti-obesity" hormone. New concepts indicate that in humans leptin and its soluble receptor may be more important in states of energy deficiency rather than a predictor of the metabolic syndrome. Adiponectin, on the other hand, is not only related to obesity and insulin resistance, but appears to be the strongest predictor for metabolic syndrome, even in children. In newborns and infants, both adipocytokines occur in high concentrations, even though this cannot completely explain the increased risk for ensuing metabolic disease later in life. Finally, low-grade systemic inflammation may underlie the clustering of metabolic risk factors. Overall factors from the adipose tissue may constitute not only markers but also mediators of metabolic sequelae of obesity.