The role of the lipid matrix in the biosynthesis of dolichyl-linked oligosaccharides

The enzymes in the dolichol pathway are membrane-proteins that utilize a combination of hydrophilic and extremely hydrophobic substrates. The enzymes in this pathway that have been purified and characterized to any extent have either been shown to be stabilized by mixed phospholipid/detergent micelles, or else require a lipid matrix for catalytic activity. Further understanding of the mechanisms of these essential enzymes may require developing methods for the reconstitution of the glycosyltransferases and their hydrophobic substrates in appropriate lipid matrices. Abbreviations: CHO, Chinese hamster ovary; Dol, dolichol; DAG, diacylglycerol; DOPC, dioleolylphosphatidylcholine; DOPE, dioleolyphosphatidylethanolamine; ER, endoplasmic reticulum; PC, phosphatidylcholine; PE, phosphatidylethanolamine; PG, phosphatidylglycerol; PI, phosphatidylinositol This revised version was published online in November 2006 with corrections to the Cover Date.     

A method for the harvest,culture, and characterization of human adult atrial myocardial cells: Correlation with age of donor

Summary Myocardial cell culture methods are now well established for animal and fetal human tissue. We present here a method for harvesting and culturing adult human atrial myocardiocytes. Cells are obtained from fresh atrial tissue normally discarded after being removed to cannulate the right atrium during open heart surgery. The atrial tissue is minced and then digested using collagenase. The single cell suspension is initially cultured in serum-containing growth medium, then transferred to defined medium, selective for myocardial cell growth. The cells are characterized by immunoperoxidase stains and transmission electron microscopy. The cultured cells stain positive for myoglobin, whereas control cultured fibroblasts and endothelial cells do not. Electron microscopy shows the presence of numerous myofibrils, Z-bodies, pleomorphic mitochondria, and secretory granules. The chronological age of the donor was an important factor in culturing the adult tissue, the younger tissue correlated with a higher success rate. This method provides a means for in vitro study of human adult myocardial cells and provides guidelines for appropriate atrial tissue to use.     

Comparison of the heavy chains of physiologically different myosins by isoelectric focusing

The technique of isoelectric focusing in polyacrylamide gels was used to determine whether differences could be distinguished between the heavy chains of myosin prepared from physiologically different muscles of chicken. The results of focusing a mixture of fast, slow, embryonic skeletal and cardiac myosin indicated that two different heavy chains only were resolved. In fast, slow and embryonic myosin these were present in approximately equal amounts but the chain with the more acidic isoelectric point was present in greater quantity in cardiac myosin.     

Generation of aspartate aminotransferase multiple forms by deamidation.

The development of aspartate aminotransferase subforms in vitro was followed by densitometry after thin-film isoelectric focusing. At the same time ammonia production was measured. Each reaction can be expressed in terms of a first-order process in which 2 mol of glutamine or asparagine/mol of dimer are deamidated with a half time of 22 days. The more negatively charged subforms developed in vitro were almost fully active. Another process occurred leading to inactivation by coenzyme modification, and this was independent of deamidation. Although the enzyme formed absorbed maximally at 340nm, it was different from the naturally occurring inactive enzyme that absorbs at this wavelength.     

Substrate specificity of the erythrocyte Ca2+-ATPase

In the absence of Mg²⁺, the observed activity of the erythrocyte plasma membrane Ca²⁺-ATPase is due to the hydrolysis of CaATP at a low rate. In the presence of Mg²⁺, the activity of the enzyme is much higher, but it is inhibited by high levels of free Mg²⁺. This inhibition appears to be due to competition of Mg²⁺ and Ca²⁺ for a site on the enzyme, rather than for ATP.     

Early-replication DNA patterns in a derivative chromosome in a syrian hamster with only 42 chromosomes

From crosses within a 2n = 43 line of Syrian hamsters (Mesocricetus auratus) lacking one derivative (der 11) of an 11;20 reciprocal translocation we have obtained homozygotes with only 42 chromosomes These animals are homozygous deficient (nullisomic) for the centromere and short arm of chromosome 11 and for the bulk of the long arm of chromosome 20. — During cytogenetic studies, we investigated the frequency patterns of early-replicating bands in the surviving derivative (der 20) at two cytologically defined sub-phases of S using short-term fibroblast cultures. These patterns were compared with those observed in the component, untranslocated arms in normal 2n = 44 cells at the same two sub-phases. — Very close agreement was found, indicating that neither the nullisomy, nor the new arm combination has interfered detectably with the pattern or programme of early band replication.