Risk factors for early death in non-insulin dependent diabetes and men with known glucose tolerance status.

OBJECTIVE--To identify risk factors for all cause mortality according to glucose tolerance status. DESIGN--Cohort study with an average 15.6 years'' follow up. SETTING--Paris, France. SUBJECTS--7166 working men aged 44-55 in 1968-72 in the Paris prospective study cohort, with non-insulin dependent diabetes or known result of two hour 75 g oral glucose tolerance test. MAIN OUTCOME MEASURES--Risk factors for death from all causes. RESULTS--128 men were known to be diabetic, 180 had diabetes diagnosed, and 697 had impaired glucose tolerance diagnosed. Compared with normoglycaemic men the relative risks of death in these groups were 2.0 (95% confidence interval 1.4 to 3.0), 2.7 (2.0 to 3.6), and 1.6 (1.3 to 2.0) respectively. Obesity, smoking, high blood pressure, and high non-esterified fatty acid concentration were risk factors for death in all subjects and were unaffected by glucose tolerance. The risks for fasting and two hour insulin concentrations and mean corpuscular volume were two times higher in known diabetic men than in men not known to be diabetic. Central obesity was significant only in men not known to be diabetic (1.6 (1.4 to 1.9)). In known diabetic men a two hour glucose concentration higher than 11.1 mmol/l carried a relative risk of death of 3.8 (1.4 to 9.4). CONCLUSIONS--Diabetic men have similar risk factors for early mortality to other men but are at higher risk from hyperinsulinaemia, hyperglycaemia, and high mean corpuscular volume.     

Near ultraviolet light inactivation of dihydroxyacid dehydratase in Escherichia coli

The effects of near ultraviolet (NUV) light on a NUV chromophore-containing oxidant-sensitive enzyme, dihydroxyacid dehydratase (DHAD), were measured in seven strains of Escherichia coli. The strains differed in production of the oxidant-defense enzymes, superoxide dismutases (Fe-SOD and Mn-SOD), and catalases HPI and HPII. With the stress of aerobic growth but without NUV exposure, the strains lacking either Fe or Mn SOD or both SODs had 57%, 25%, and 12%, respectively, of the DHAD-specific activity of the parent (K12) strain. Under the same conditions, the catalase strains that were wild type, overproducing, and deficient had comparable DHAD-specific activities. When aerobic cultures were exposed for 30 min to NUV with a fluence of 216 J/m²/s at 310–400 nm, the percentage decreases in DHAD-specific activities were similar (ranging from 75% to 89%) in strains with none, either, or both SODs missing, and in the catalase-overproducing strain. However, the decreases were only 58% and 52% in the strain with catalase missing and in its parent, respectively. The NUV-induced loss of DHAD enzyme activity was not accompanied by any detectable loss of the DHAD protein as measured by polyclonal antibody to DHAD.     

Knowledge-based simulation of DNA metabolism: prediction of enzyme action

We have developed a knowledge-based simulation of DNA metabolismthat accurately predicts the actions of enzymes on DNA undera large number of environmental conditions. Previous simulationsof enzyme systems rely predominantly on mathematical models.We use a frame-based representation to model enzymes, substratesand conditions. Interactions between these objects are expressedusing production rules and an underlying truth maintenance system.The system performs rapid inference and can explain its reasoning.A graphical interface provides access to all elements of thesimulation, including object representations and explanationgraphs. Predicting enzyme action is the first step in the developmentof a large knowledge base to envision the metabolic pathwaysof DNA replication and repair. Received on February 1, 1990; accepted on October 2, 1990     

Purification and characterization of recombinant human testis angiotensin-converting enzyme expressed in Chinese hamster ovary cells.

Enzymatically active human testis angiotensin-converting enzyme (ACE) was expressed in Chinese hamster ovary (CHO) cells stably transfected with each of three vectors: p omega-ACE contains a full-length testis ACE cDNA under the control of a retroviral promoter; and pLEN-ACEVII and pLEN-ACE6/5, in which full-length and membrane anchor-minus testis ACE cDNAs, respectively, are under the control of the human metallothionein IIA promoter and SV40 enhancer. In every case, active recombinant human testis ACE (hTACE) was secreted in a soluble form into the culture media, up to 2.4 mg/liter in the media of metal-induced, high-producing clones transfected with one of the pLEN vectors. In addition, membrane-bound recombinant enzyme was recovered from detergent extracts of cell pellets of CHO cells transfected with either p omega-ACE or pLEN-ACE-VII. Recombinant converting enzyme was purified to homogeneity by single-step affinity chromatography of conditioned media and detergent-extracted cell pellets in 85 and 70% overall yield, respectively. Purified hTACE from all sources comigrated with the native testis isozyme on sodium dodecyl sulfate-polyacrylamide gel electrophoresis with M(r) approximately 100 kDa. The native and recombinant proteins cross-reacted equally with anti-human kidney ACE antiserum on Western blotting. The catalytic activity of recombinant angiotensin-converting enzyme, in terms of angiotensin I and 2-furanacryloyl-Phe-Gly-Gly hydrolysis, chloride activation, and lisinopril inhibition, was essentially identical to that of the native enzyme. The facile recovery in high yield of fully active hTACE from the media of stably transfected CHO cells provides a suitable system for investigating structure-function relationships in this enzyme.     

Synthesis and expression of genes encoding tuna, pigeon, and horse cytochromes c in the yeast Saccharomyces cerevisiae.

Genes encoding tuna, pigeon, and horse cytochromes c were constructed with synthetic oligodeoxyribonucleotides having preferred codons and portions of the iso-1-cytochrome c-encoding gene from the yeast Saccharomyces cerevisiae. The genes were ligated into an expression vector, which contains the normal 5'- and 3'-untranslated regions of the yeast iso-1-cytochrome c gene, and were integrated in single copy into the chromosome. Yeast strains were also constructed with multiple integrated copies of the pigeon gene. The heterologous and normal mRNA levels of the single-copy strains were equivalent. Although the N-terminal methionines were completely cleaved in the heterospecific proteins, the levels of trimethylation of Lys72 and acetylation of N-terminal glycines ranged from 39-78% and 10-70%, respectively. Horse cytochrome c was produced at a nearly normal level, whereas the pigeon and tuna cytochromes c were produced at approx. 40% of the normal levels. The levels of the cytochromes c and growth of the mutant yeast strains indicated that the heterospecific cytochromes c had approx. 50% specific activity in vivo.