Cardiolipin biosynthesis and remodeling enzymes are altered during development of heart failure

Cardiolipin (CL) is responsible for modulation of activities of various enzymes involved in oxidative phosphorylation. Although energy production decreases in heart failure (HF), regulation of cardiolipin during HF development is unknown. Enzymes involved in cardiac cardiolipin synthesis and remodeling were studied in spontaneously hypertensive HF (SHHF) rats, explanted hearts from human HF patients, and nonfailing Sprague Dawley (SD) rats. The biosynthetic enzymes cytidinediphosphatediacylglycerol synthetase (CDS), phosphatidylglycerolphosphate synthase (PGPS) and cardiolipin synthase (CLS) were investigated. Mitochondrial CDS activity and CDS-1 mRNA increased in HF whereas CDS-2 mRNA in SHHF and humans, not in SD rats, decreased. PGPS activity, but not mRNA, increased in SHHF. CLS activity and mRNA decreased in SHHF, but mRNA was not significantly altered in humans. Cardiolipin remodeling enzymes, monolysocardiolipin acyltransferase (MLCL AT) and tafazzin, showed variable changes during HF. MLCL AT activity increased in SHHF. Tafazzin mRNA decreased in SHHF and human HF, but not in SD rats. The gene expression of acyl-CoA: lysocardiolipin acyltransferase-1, an endoplasmic reticulum MLCL AT, remained unaltered in SHHF rats. The results provide mechanisms whereby both cardiolipin biosynthesis and remodeling are altered during HF. Increases in CDS-1, PGPS, and MLCL AT suggest compensatory mechanisms during the development of HF. Human and SD data imply that similar trends may occur in human HF, but not during nonpathological aging, consistent with previous cardiolipin studies.     

Alcohol dehydrogenase isozymes in the mouse: Genetic regulation, allelic variation among inbred strains and sex differences of liver and kidney A2 isozyme activity

Genetic analysis of a proposed cis-acting temporal locus ( Adh-3t ), which regulates alcohol dehydrogenase C₂ (ADH-C₂) acitivity in mouse epididymis extracts, among F₁ (ddN × BALB/c) × ddN male backcross progeny provided evidence for genetic distinctness between the structural ( Adh-3 ) and temporal ( Adh-3t ) loci on chromosome 3. Genetic analysis also confirmed the close, linkage of Adh-1 (encoding liver and kidney ADH-A₂) and Adh-3 (encoding stomach ADH-C₂) to within 0.3 centimorgans on the mouse genome. Evidence is presented for a proposed closely linked cis-acting temporal locus (designated Adh-1t ) for the A₂ isozyme (encoded by Adh-1 ) controlling the activity of this enzyme in mouse kidney extracts, but having no apparent affect on liver and intestine ADH-A₂ activities. An extensive survey of the distribution of Adh-1, Adh-3 and Adh-3t alleles among 65 strains of mice is reported — with the exception of two Japanese strains (ddN and KF), linkage disequilibrium between Adh-3 and Adh-3t was observed. Sex differences in mouse liver and kidney ADH-A₂ activities were observed, with male/female ratios of approximately 0.6 and 3 respectively for these tissue extracts.     

Photoaffinity labeling of GDP-fucose:nLcOse4Cer alpha 1----3-fucosyltransferase from human small cell lung carcinoma NCI-H69 cells with the GDP-fucose analog GDP-hexanolaminyl-4-azidosalicylic acid

An iodinatable photoactive analog of GDP-fucose, GDP-hexanolaminyl-4-azidosalicylic acid, has been prepared and applied to studies of the previously described alpha 1----3-fucosyltransferase from NCI-H69 cells (Holmes, E. H., Ostrander, G. K., and Hakomori, S. (1985) J. Biol. Chem. 260, 7619-7627). The NCI-H69 cell alpha 1----3-fucosyltransferase was obtained from a 0.2% Triton X-100-solubilized enzyme fraction after affinity purification on a GDP-hexanolamine-Sepharose column and gel filtration through a fast protein liquid chromatography Superose 12 column. Increasing concentrations of the photoaffinity reagent were found to result in loss of up to 35% of the original enzyme activity at under 100 microM final concentrations. The inactivation was photolysis dependent and could be prevented by the addition of GDP-fucose prior to photolysis. The photoprobe behaved as a competitive inhibitor with respect to GDP-fucose with a Ki of 23 microM, identical to that of GDP. Photoincorporation of 125I-labeled GDP-hexanolaminyl-4-azidosalicylic acid into the enzyme fraction labeled a slow migrating protein band in a native polyacrylamide gel which corresponded to enzyme activity. Inclusion of GDP-fucose prevented photolabeling of this band. Sodium dodecyl sulfate gel electrophoresis of the photolabeled, GDP-fucose-protected band yielded a 125I-labeled protein band that migrated at Mr 45,000, most probably corresponding to an alpha 1----3-fucosyltransferase protein subunit. These studies suggest photoaffinity labeling using nucleotide affinity ligands linked to photoactivatable, heterobifunctional cross-linking reagents may be generally applicable to photoaffinity labeling glycosyltransferase enzyme proteins.     

Candidate mechanical stimuli for hypertrophy during volume overload.

A myocyte system that senses and responds to mechanical inputs might be activated by any number of features of the time-varying length or force signals experienced by the myocytes. We therefore characterized left ventricular volume and wall stress signals during early volume overload with high spatial and temporal resolution. Left ventricular pressure and volume were measured in open-chest isoflurane-anesthetized male Sprague-Dawley rats 4 and 7 days after surgical creation of an infrarenal arteriovenous fistula or sham operation. Mean wall stresses were calculated by using a simple thick-walled ellipsoidal model. Consistent with previous reports, this surgical model produced a 66% increase in cardiac output and a 10% increase in left ventricular mass by day 7. A number of features of the time-varying volume signal (maximum, mean, amplitude, rates of rise and fall) were significantly altered during early volume overload, whereas many other proposed hypertrophic stimuli, including peak systolic wall stress and diastolic strain, were not. Treating hemodynamic variables more generally as time-varying signals allowed us to identify a wider range of candidate mechanical stimuli for hypertrophy (including some not previously proposed in the literature) than focusing on standard time points in the cardiac cycle. We conclude that features of the time-varying ventricular volume signal and related local deformations may drive hypertrophy during volume overload and propose that those features of the volume signal that also change during pressure overload might be the most interesting candidates for further exploration.     

In vitro replication of mouse hepatitis virus strain A59.

An in vitro replication system for mouse hepatitis virus (MHV) strain A59 was developed using lysolecithin to produce cell extracts. In extracts of MHV-infected cells, radiolabeled UMP was incorporated at a linear rate for up to 1 h into RNA, which hybridized to MHV-specific cDNA probes and migrated in denaturing formaldehyde-agarose gels to the same position as MHV genomic RNA. The incorporation of [32P]UMP into genome-sized RNA in vitro correlated with the observed increase of [3H]uridine incorporation in MHV-infected cells labeled in vivo. Incorporation of [32P]UMP into genome-sized RNA was inhibited when extracts were incubated with puromycin. The addition to the assay of antiserum to the MHV-A59 nucleocapsid protein N inhibited synthesis of genome-sized RNA by 90% compared with the addition of preimmune serum. In contrast, antiserum to the E1 or E2 glycoproteins did not significantly inhibit RNA replication. In vitro-synthesized RNA banded in cesium chloride gradients as a ribonucleoprotein complex with the characteristic density of MHV nucleocapsids isolated from virions. These experiments suggest that ongoing protein synthesis is necessary for replication of MHV genomic RNA and indicate that the N protein plays an important role in MHV replication.     

Amino-acid sequence of human alpha 2-antiplasmin

The amino-acid sequence of human alpha 2-antiplasmin was determined by Edman degradation of peptides purified from CNBr, tryptic and chymotryptic digests. Of the total sequence of 452 amino acids of mature alpha 2-antiplasmin, as deduced from the cDNA sequence [Holmes et al. (1987) J. Biol. Chem. 262, 1659-1664], 444 residues were identified by amino-acid sequencing. Two differences were found between the peptide and cDNA analyses (Gly instead of Leu at position 10 and Gly instead of Ser at position 369). alpha 2-Antiplasmin contains two disulfide bridges (Cys64-Cys104 and Cys31-Cys113) and four glucosamine-based carbohydrate chains attached to Asn87, Asn256, Asn270 and Asn277. alpha 2-Antiplasmin is homologous with 12 other proteins belonging to the serine protease inhibitor (serpin) superfamily.     

Biosynthesis of fucose containing lacto-series glycolipids in human colonic adenocarcinoma Colo 205 cells

Biosynthesis of fucose containing lacto-series glycolipids has been studied in human colonic adenocarcinoma Colo 205 cells. Transfer of fucose in both alpha 1----3 linkage to type 2 chain acceptors and alpha 1----4 linkage to type 1 chain acceptors was demonstrated with a Triton X-100 solubilized membrane fraction. The enzyme was found to be highly active over a broad pH range between 6.0 and 7.5. Kinetics of the transfer reactions were studied and indicated that the enzyme had an apparent Km for GDPfucose of 53 and 49 microM with acceptors nLc4 and Lc4, respectively. The apparent Km values for acceptors Lc4, nLc4, and IV3NeuAcnLc4 were determined to be 42, 18, and 26 microM, respectively. Transfer of fucose to the type 1 chain acceptor Lc4 alone and in the presence of increasing concentrations of the type 2 chain acceptor IV3NeuAcnLc4 or Gb3 suggested that both type 1 and 2 acceptors were alternate acceptors for a single enzyme. This was further established by the finding that IV3NeuAcnLc4 behaved as a competitive inhibitor of fucose transfer with respect to Lc4. Conditions were defined for preparative scale in vitro synthesis of fucosylated products of nLc6 catalyzed by the Colo 205 cell enzyme. Yields of the monofucosyl derivative of 2.5 mg (46%) and 1 mg (17%) of the difucosyl derivative were obtained from 5 mg of original nLc6. The structures of these biosynthetic products were carefully studied by 1H NMR, +FAB-MS, and methylation analysis. These studies revealed extremely high purity products composed of III3FucnLc6 and III3V3Fuc2nLc6. The significance of the nature of these products and enzymatic properties is discussed.