Sudden cardiac death in diabetes mellitus: risk factors in the Rochester diabetic neuropathy study.

OBJECTIVES: To determine risk factors for sudden cardiac death and the role of diabetic autonomic neuropathy (DAN) in the Rochester diabetic neuropathy study (RDNS). METHODS: Associations between diabetic and cardiovascular complications, including DAN, and the risk of sudden cardiac death were studied among 462 diabetic patients (151 type 1) enrolled in the RDNS. Medical records, death certificates, and necropsy reports were assessed for causes of sudden cardiac death. RESULTS: 21 cases of sudden cardiac death were identified over 15 years of follow up. In bivariate analysis of risk covariates, the following were significant: ECG 1 (evolving and previous myocardial infarctions): hazard ratio (HR) = 4.4 (95% confidence interval (CI), 1.6 to 12.1), p = 0.004; ECG 2 (bundle branch block or pacing): HR = 8.6 (2.9 to 25.4), p<0.001; ECG 1 or ECG 2: HR = 4.2 (1.3 to 13.4), p = 0.014; and nephropathy stage: HR = 2.1 (1.3 to 3.4), p = 0.002. Adjusting for ECG 1 or ECG 2, autonomic scores, QTc interval, high density lipoprotein (HDL) cholesterol, 24 hour microalbuminuria, and 24 hour total proteinuria were significant. However, adjusting for nephropathy, none of the autonomic indices, QTc interval, HDL cholesterol, microalbuminuria, or total proteinuria was significant. At necropsy, all patients with sudden cardiac death had coronary artery or myocardial disease. CONCLUSIONS: Sudden cardiac death was correlated with atherosclerotic heart disease and nephropathy, and to a lesser degree with DAN and HDL cholesterol. Although DAN is associated with sudden cardiac death, it is unlikely to be its primary cause.

Normal and dystrophic rat retinal pigment epithelia display different sensitivities to plant lectins.

Previous studies showed that cultured retinal pigment epithelial (RPE) cells from the Royal College of Surgeons (RCS) rat incorporate 50% less fucose into a number of cell-surface glycoproteins compared with controls. The cause of reduced fucose incorporation may be a generalized defect in glycoprotein processing in the RCS rat RPE. This hypothesis has been further explored by comparing the relative sensitivities of normal and dystrophic rat RPE to the toxicity of plant lectins of various specificities. Freshly isolated RPE cells from normal and dystrophic rats were cultured in the presence of increasing concentrations of lectin. For each lectin, the concentration at which less than 10% of the cells survived was determined. These tests showed that the dystrophic RPE cells were more sensitive to lectins that bound to core sugar moieties such as mannose and N-acetylglucosamine; normal RPE cells were more sensitive to lectins which bound to the more terminally located sugars, galactose and N-acetylgalactosamine. Overall, the results suggest that decreased incorporation of fucose into the RCS RPE may be due to failure of the dystrophic RPE to add either N-acetylgalactosamine or galactose (to which fucose is subsequently added) to oligosaccharide structures.

Partial characterization of fucosylated cell surface glycoproteins of cultured RPE.

The major high molecular weight, fucose containing, cell surface glycoproteins of cultured rat retinal pigment epithelial (RPE) cells were partially characterized. One dimensional peptide mapping by the Cleveland method showed that the polypeptide chains of these proteins were not highly related in structure. Incorporation of 3H-mannose into these glycoproteins was equivalent for normal and dystrophic (RCS rdy-p+) RPE. Furthermore, treatment of the glycoproteins from either normal or dystrophic RPE with Endo-beta-N-acetylglucosaminidase H (Endo H) did not cause a shift in their Mr's, as determined by SDS PAGE. These results suggest that the high Mr glycoproteins do not contain a large quantity of unprocessed, mannose containing core type N-linked oligosaccharides in either normal or dystrophic RPE. Digestion of the 3H-fucose labeled glycoproteins with Peptide N-glycosidase F (PNGase F) demonstrated that at least 90% of the 3H-fucose incorporated into these glycoproteins is in N-linked oligosaccharides. Endo-beta-N-acetylglucosaminidase F (Endo F) treatment showed that at least 75-80% of the 3H-fucose is located in more terminal positions (distal to the fucose that is found in alpha 1,6 linkage to the asparagine-linked N-acetylglucosamine residue) in N-linked carbohydrate. Overall, these results support the hypothesis that if the dystrophic RPE possesses a defect in glycoprotein processing, then this defect affects terminal processing of oligosaccharides and addition of terminally located fucose residues. A homologous group of high Mr, fucosylated glycoproteins was found in plasma membranes from cultured monkey RPE, suggesting atht they may be common to other species.

3H-fucose incorporation into RPE cell surface proteins of normal and dystrophic rats.

Glycoproteins were metabolically labeled with 3H-fucose in cultured RPE cells from RCS rdy-p+ and Long Evans rats. 3H-labeled glycoproteins associated with a plasma membrane-enriched subcellular fraction were separated by two-dimensional gel electrophoresis. Relative incorporation of 3H-fucose into high molecular weight cell surface glycoproteins (Mr of 128,000-183,000) was measured by quantitative autoradiography and densitometry. The results of these experiments show that 3H-fucose incorporation into four glycoproteins (Mr of 183,000, 175,000, 164,000 and 156,000) was reduced by 30-50% in the dystrophic RPE as compared to the normal cells. This reduction was not due to an absence of the protein core of glycoproteins on the dystrophic RPE cell surface; when RPE cells were labeled with 3H-leucine prior to analysis, no reduction in label was found in the dystrophic RPE as compared to normal. Therefore, the results of this study suggest that the RCS rat RPE processes the oligosaccharide portion of some cell surface glycoproteins differently than normal rat RPE.

Surface modification of retinal pigment epithelial cells: effects on phagocytosis and glycoprotein composition.

Proteases have been used as a tool to investigate the role of cell-surface molecules of cultured retinal pigment epithelial cells (RPE) in the phagocytosis of rod outer segments (ROS). Proteolytic digestion of RPE cells by pronase, thermolysin and Staphylococcus aureus V8 protease (V8 protease) inhibited the phagocytosis of ROS without affecting the viability of the RPE cells. A particular feature of RPE cell proteolysis was that those macromolecules responsible for ROS ingestion were susceptible, while those macromolecules that mediated ROS binding were resistant to cleavage by all three proteases. By taking advantage of this phenomenon, ROS were used as affinity particles to obtain a plasma membrane-enriched fraction of RPE cells before and after proteolytic digestion. All three proteases partially or completely removed several glycoproteins from the cell surfaces. Removal of these glycoproteins was correlated with a loss in phagocytic ability by RPE cells. Two high-molecular-weight (MW) glycoproteins of MWs 160,000 and 214,000 were consistently removed by all proteases tested. Protease-treated RPE cells restored their phagocytic capabilities and normal glycoprotein composition within 24 hr after proteolytic treatment. These data suggest that glycoproteins located on the surfaces of RPE cells may be involved in mediating the phagocytosis of ROS by these cells.

Membrane receptors for retinol-binding protein in cultured human retinal pigment epithelium.

The retinal pigment epithelium (RPE) is responsible for transport of retinol from the choroidal circulation to the photoreceptors. In the intact eye, this process is mediated by membrane receptors for plasma retinol binding protein (RBP) distributed basolaterally on the RPE cells. We have shown that cultured human RPE expresses this receptor. A binding curve exhibiting saturation was generated by incubating enzymatically detached epithelial sheets with increasing concentrations of 125I-labelled RBP. 125I-RBP binding experiments also show that the receptor is expressed at a high level in first passage subcultures, suggesting de novo synthesis, and that basally oriented receptors predominate over those associated with the apical surface, reflecting the polarization characteristic of RPE in vivo. Cultured RPE can internalize 3H-retinol carried by RBP, resulting in synthesis of labelled retinyl palmitate. Production of labelled retinyl ester is competitively inhibited when incubations include an excess of holo-RBP containing non-radioactive retinol. These results indicate that RBP not only binds to the receptor specifically, but also that this interaction is functional, effecting uptake of retinol by the RPE cells. The expression of this property of differentiated RPE favors the use of cultured RPE as a model system for studying vitamin A transport and metabolism.

RPE cell surface proteins in normal and dystrophic rats.

Membrane-bound proteins in plasma membrane enriched fractions from cultured rat RPE were analyzed by two-dimensional gel electrophoresis. Membrane proteins were characterized on three increasingly specific levels. Total protein was visualized by silver staining. A maximum of 102 separate proteins were counted in silver-stained gels. Glycoproteins were labeled with 3H-glucosamine or 3H-fucose and detected by autoradiography. Thirty-eight fucose-labeled and 61-71 glucosamine-labeled proteins were identified. All of the fucose-labeled proteins were labeled with glucosamine-derived radioactivity. Proteins exposed at the cell surface were labeled by lactoperoxidase-catalyzed radioiodination prior to preparation of membranes for two-dimensional analysis. Forty separate 125I-labeled surface proteins were resolved by two-dimensional electrophoresis/autoradiography. Comparison with the glycoprotein map showed that a number of these surface labeled proteins were glycoproteins. Two-dimensional maps of total protein, fucose-labeled, and glucosamine-labeled glycoproteins, and 125I-labeled surface proteins of membranes from dystrophic (RCS rdy-p+) and normal (Long Evans or RCS rdy+p+) RPE were compared. No differences in the total protein or surface-labeled proteins were observed. However, the results suggest that a 183K glycoprotein is more heavily glycosylated with glucosamine and fucose in normal RPE membranes as compared to membranes from dystrophic RPE.

Identification of some plasma membrane proteins of cultured rat pigment epithelial cells by labeling with 125I.

Cultured rat retinal pigment epithelial (PE) cells were labeled with 125I by lactoperoxidase (LPO)-catalyzed radioiodination. Examination of 125I-labeled cells by electron microscopic autoradiography suggested that 125I was incorporated into proteins at both the apical and basal surfaces of the PE cells, and into the extracellular matrix (ECM). Analysis of labeled cells by SDS-polyacrylamide gel electrophoresis and autoradiography showed that 125I was incorporated into at least 15 polypeptides. In order to determine which of these labeled proteins were derived from the plasma membrane. 125I-labeled cells were subjected to differential detergent extraction. Triton X-100 (2% v/v), which removed the cell plasma membrane, solubilized only three of the labeled proteins (152 000, 138 000, 123 000 daltons). SDS (0.25% w/v) completely removed cells from the tissue culture dish and solubilized all but four of the labeled proteins (225 000, 173 000, 87 000 and 72 100 daltons). When 125I-labeled PE cells were mechanically disrupted, and the resulting cell fractions separated by sucrose density gradient centrifugation, labeled proteins separated into two subcellular fractions. One fraction was especially enriched in the 152 000, 138 000 and 123 000 dalton labeled proteins, in addition to the plasma membrane marker enzymes Na+, K+-ATPase, and alkaline phosphodiesterase I. The second fraction was enriched in the 225 000, 196 000 and 173 000 dalton labeled proteins, the ECM proteins laminin and fibronectin, and the 43 000 actin band . It is proposed that 125I-labeled proteins in the former cell fraction are truly plasma membrane proteins, while those found in the latter cell fraction are a mixture of 125I-labeled ECM and basal plasma membrane proteins. The 152 000, 138 000 and 123 000 dalton labeled proteins of the plasma membrane fraction are glycoproteins and become firmly anchored to the Triton X-100 insoluble cytoskeleton when labeled cells are treated with concanavalin A.

Cause of subunit heterogeneity in purified rat renal phosphate-dependent glutaminase.

When electrophoresed on polyacrylamide gels in the presence of sodium lauryl sulfate, highly purified rat renal phosphate-dependent glutaminase exhibits subunits which range in molecular weight from 57,000 to 75,000. Peptide mapping of the separated subunits following limited proteolysis in the presence of sodium lauryl sulfate shows that all of the various subunits are related in structure. The glutaminase, immunoprecipitated from Triton X-100-solubilized mitochondria, is composed primarily of subunits which have molecular weights of 83,000. In addition, the series of smaller subunits is generated during storage of the Triton-solubilized glutaminase at 4 degrees C. These results indicate that the heterogeneity of subunit size found in the purified glutaminase results from a noninactivating partial proteolysis of the native form of the enzyme.

Inactivation of rat renal phosphate-dependent glutaminase with 6-diazo-5-oxo-L-norleucine. Evidence for interaction at the glutamine binding site.

Inactivation of rat renal phosphate-dependent glutaminase by 6-diazo-5-oxo-L-norleucine occurs only under conditions where the enzyme is catalytically active. The glutaminase activity and the rate of inactivation by the diazoketone exhibit very similar phosphate concentration-dependent activation profiles. Because of this phosphate dependency, it was not possible to differentiate an apparent protection by glutamine from the strong inhibition of inactivation caused by glutamate. The ability of glutamate to protect the glutaminase against inactivation is reversed by increasing concentrations of phosphate. The observed characteristics of inactivation by 6-diazo-5-oxo-L-norleucine differ considerably from those reported for the inactivation by L-2-amino-4-oxo-5-chloropentanoic acid. In addition, the presence of o-carbamoyl-L-serine was found to stimulate inactivation by 6-diazo-5-oxo-L-norleucine, but to protect the glutaminase against inactivation by the chloroketone. Preinactivation of the glutaminase by the diazoketone only slightly reduced the stoichiometry of binding of [5-14C]chloroketone. These observations suggest that 6-diazo-5-oxo-L-norleucine and L-2-amino-4-oxo-5-chloropentanoic acid interact with different sites on the glutaminase which are specific for binding glutamine and glutamate, respectively.

Covalent interaction of L-2-amino-4-oxo-5-chloropentanoic acid with rat renal phosphate-dependent glutaminase. Evidence for a specific glutamate binding site and of subunit heterogeneity.

Rat renal phosphate-dependent glutaminase is rapidly inactivated by incubating with L-2-amino-4-oxo-5-chloropentanoic scid. Concentrations of phosphate, which increase the glutaminase activity, decrease the rate of inactivation by chloroketone. In addition, inactivation is not blocked by glutamine. Instead, glutamate was shown to specifically reduce the rate of chloroketone inactivation. Upon sodium lauryl sulfate-polyacrylamide gel electrophoresis, the purified glutaminase preparation exhibits at least five protein staining bands which range in molecular weight from 57,000 to 75,000. Studies with 14C-labeled chloroketone indicate that this reagent reacts with each of these peptides. The mean stoichiometry of binding was calculated to be 1.3 mol/mol of enzyme. Therefore, these results indicate that the glutaminase may contain a specific site for binding glutamate and that the purified enzyme consists of a series of related peptides which may have resulted from partial proteolysis.

The structure and properties of histone F2a comprising the heterologous group F2a1 and F2a2 studied by 13C nuclear magnetic resonance.

(13)C n.m.r. spectra have been obtained for aqueous solutions of histones F2a(1) and F2a(2), for the group F2a, for the appropriate amino acid mixturesand for the corresponding hydrolysates. These, when compared with computer simulated spectra give good agreement for secondary structure with that calculated from the known primary structure of the proteins. Evidence based on the spectra obtained at various salt concentrations leads to the conclusion that F2a is not a simple mixture but an interacting heterologous group of histones F2a(1) and F2a(2).