Reduced endothelial nitric oxide synthase in lungs of chronically ventilated preterm lambs.

Nitric oxide (NO), produced in lung vascular endothelium and airway epithelium, has an important role in regulating smooth muscle cell growth and tone. Chronic lung disease, a frequent complication of premature birth, is characterized by excess abundance, tone, and reactivity of smooth muscle in the pulmonary circulation and conducting airways, leading to increased lung vascular and airway resistance. Whether these structural and functional changes are associated with diminished pulmonary expression of endothelial nitric oxide synthase (eNOS) protein is unknown. Both quantitative immunoblot analysis and semiquantitative immunohistochemistry showed that there was less eNOS protein in the endothelium of small intrapulmonary arteries and epithelium of small airways of preterm lambs that were mechanically ventilated for 3 wk compared with control lambs born at term. No significant differences were detected for other proteins (inducible NOS, alpha-smooth muscle actin, and pancytokeratin). Lung vascular and respiratory tract resistances were greater in the chronically ventilated preterm lambs compared with control term lambs. These results support the notion that decreased eNOS in the pulmonary circulation and respiratory tract of preterm lambs may contribute to the pathophysiology of chronic lung disease.

Molecular analysis of an esterase-encoding gene from a lipolytic psychrotrophic pseudomonad.

An esterase gene (estA) from a lipolytic psychotroph (Pseudomonas sp. LS107d2), was cloned in Escherichia coli and its nucleotide sequence was determined, revealing an ORF encoding a polypeptide of 389 amino acid residues, with a molecular mass of 42276 Da. Labelling of plasmid-encoded proteins with [35S]methionine, using the maxicell procedure, gave a single polypeptide of molecular mass 42 kDa, consistent with that calculated from the ORF. Colonies of E. coli cells containing estA produced a clear halo when grown on solid media containing tributyrin; no clearance was produced when cells were grown on media containing triolein. Extracts of cells containing estA also hydrolysed water-soluble nitrophenol esters, but were unable to cleave water-insoluble substrates. The preference for water-soluble substrates indicates that the gene product is an esterase.

Cloning and sequencing of cDNA encoding baboon liver alcohol dehydrogenase: evidence for a common ancestral lineage with the human alcohol dehydrogenase beta subunit and for class I ADH gene duplications predating primate radiation.

The baboon has at least five alcohol dehydrogenases (ADH; alcohol:NAD+ oxidoreductase, EC 1.1.1.1) and has distinct liver and kidney class I isozymes. A rat liver class I ADH partial cDNA was used to screen a baboon liver cDNA library. A cDNA clone was isolated and sequenced and found to contain the entire coding region for baboon liver ADH, 12 nucleotides of the 5' noncoding region, and 256 nucleotides of the 3' noncoding region. The amino acid sequence deduced from this cDNA most closely resembles that of human liver ADH beta subunit (ADH-beta): 363 of 374 residues were identical. This suggested that baboon liver class I ADH is of the same ancestral lineage as the human ADH-beta. In contrast to human liver, only a single ADH-beta transcript is observed in baboon liver. A comparison of human and baboon ADH 3' noncoding regions suggests that a single nucleotide change in a polyadenylylation signal consensus sequence may, in part, be responsible for the generation of ADH-beta transcripts with variable-length 3' ends in human liver. A nucleotide substitution rate of 0.5 x 10(-9) substitutions per site per year for primate class I ADH genes was deduced from the data, which suggests that the alpha-beta gamma separation of human ADH genes occurred about 60 million years ago, and that primate class I ADH gene duplications predated primate radiation.