RNA diversity has profound effects on the translation of neuronal nitric oxide synthase.

A comprehensive analysis of the structure of neuronal nitric oxide synthase (nNOS; EC 1.14.13.39) mRNA species revealed NOS1 to be the most structurally diverse human gene described to date in terms of promoter usage. Nine unique exon 1 variants are variously used for transcript initiation in diverse tissues, and each is expressed from a unique 5'-flanking region. The dependence on unique genomic regions to control transcription initiation in a cell-specific fashion burdens the transcripts with complex 5'-mRNA leader sequences. Elaborate splicing patterns that involve alternatively spliced leader exons and exon skipping have been superimposed on this diversity. Highly structured nNOS mRNA 5'-untranslated regions, which have profound effects on translation both in vitro and in cells, contain cis RNA elements that modulate translational efficiency in response to changes in cellular phenotype.

Cloning and characterization of murine endothelial constitutive nitric oxide synthase.

Complementary DNA clones encoding mouse endothelial constitutive nitric oxide synthase (ecNOS) were isolated by plaque hybridization from a murine fetal cardiac lambda ZAP II cDNA expression library using a full-length human ecNOS cDNA as the hybridization probe. DNA sequence analysis indicates a 1202 amino acid protein showing significant sequence identity with human as well as bovine ecNOS.

Structural organization of the human neuronal nitric oxide synthase gene (NOS1).

Neuronal nitric oxide (NO) synthase, localized to human chromosome 12, uniquely participates in diverse biologic processes; neurotransmission, the regulation of body fluid homeostasis, neuroendocrine physiology, control of smooth muscle motility, sexual function, and myocyte/myoblast biology, among others. Restriction enzyme mapping, subcloning, and DNA sequence analysis of bacteriophage- and yeast artificial chromosome-derived human genomic DNA indicated that the mRNA for neuronal NO synthase is dispersed over a minimum of 160 kilobases of human genomic DNA. Analysis of intron-exon splice junctions predicted that the open reading frame is encoded by 28 exons, with translation initiation and termination in exon 2 and exon 29, respectively. Determination of transcription initiation sites in brain poly(A) RNA with primer extension analysis and RNase protection revealed a major start site 28 nucleotides downstream from a TATA box. Sequence inspection of 5'-flanking regions revealed potential cis-acting DNA elements: AP-2, TEF-1/MCBF, CREB/ATF/c-Fos, NRF-1, Ets, NF-1, and NF-kappa B-like sequences. Diversity appears to represent a major theme apparent upon analysis of human neuronal NO synthase mRNA transcripts. A microsatellite of the dinucleotide variety was detected within the 3'-untranslated region of exon 29. Multiple alleles were evident in normal individuals indicating the existence of allelic mRNA sequence variation. Characterization of variant human neuronal NO synthase cDNAs indicated the existence of casette exon 9/10 and exon 10 deletions as examples of structural mRNA diversity due to alternative splicing. The latter deletion of a 175-nucleotide exon introduces a frame-shift and premature stop codon indicating the potential existence of a novel NH2 terminus protein. In summary, analysis of the human neuronal NO synthase locus reveals a complex genomic organization and mRNA diversity that is both allelic and structural.

Determination of antimicrobial susceptibilities of Canadian isolates of Haemophilus influenzae and characterization of their beta-lactamases. Canadian Haemophilus Study Group.

Susceptibility testing of 1,688 Haemophilus influenzae isolates found 484 ampicillin-resistant strains; 474 strains (28.4%) were beta-lactamase positive, and 5 strains (0.4%) were non-beta-lactamase producers. Restriction enzyme digestion of the beta-lactamase amplicon determined that, of 157 strains, 11 (7.0%) contained ROB-1 beta-lactamase and 146 (93.0%) contained a TEM-type beta-lactamase.

Cytomegalovirus-associated neonatal hepatitis.

Fifty-five patients with cytomegalovirus (CMV)-associated neonatal hepatitis (NH) were followed for 12 to 90 months. Six patients (10.9%) died from either a fulminant course or a chronic liver disease. Among the remaining 49 patients, whose liver function was completely recovered, there were eight with retardation of developmental or growth status, and two with hearing impairment. Overall, 20.4% of the survivors suffered from a long-term impact. The unfavorable outcome was related to several clinical and pathological parameters. These included persistence of clay-colored stool, presence of splenomegaly, ascites or anemia, high peak total and direct bilirubin, low nadir albumin levels, diffuse giant cell transformation and cirrhosis of the liver. The seropositivity of CMV infection did not significantly correlate with the outcome.

Retinoblastoma, chromosome abnormalities and oncogene expression.

Recurrent chromosomal abnormalities in retinoblastomas involve numbers 13, 1, and 6, as well as homogeneously staining regions (HSR) and double minutes (DMS). Evidence suggesting that chromosome 13 contains a gene responsible for tumorigenesis has already been presented. We postulate that the genetic changes resulting from abnormalities of chromosomes 1 and 6 and the HSR/DMS provide a selective growth advantage to cells in which they occur. Support for this hypothesis, as it relates to the HSR/DMS and oncogene amplification, is discussed.

Dual contribution of embryonic ventral blood island and dorsal lateral plate mesoderm during ontogeny of hemopoietic cells in Xenopus laevis.

The early embryonic development of hemopoietic cells in Xenopus laevis was examined. Either dorsal lateral plate (DLP) or ventral blood island (VBI) mesoderm was reciprocally transplanted between cytogenetically distinct (2N or 3N) stage 14 to 19 (neural fold) embryos. F-DNA content of circulating erythrocytes was assayed at stages 40, 41, 43, 45, and 49. The F-DNA content of cells in the thymus and mesonephros was assayed at stage 49. F-DNA values were used to distinguish between donor or host origin of hemopoietic cells in individual animals. The results demonstrated that DLP mesoderm gave rise to a population of stem cells that colonized the thymus and mesonephros, but not the blood. VBI mesoderm gave rise to a population of stem cells that colonized the blood and thymus, but not the mesonephros. These experiments show that there are two stem cell compartments in the amphibian embryo, separated in both space and time.

The catecholamine mechanisms of prostaglandin E1-induced hypothermia in rats.

Intraperitoneal administration of prostaglandin E1 (PGE1) produced a hypothermia in rats at room temperatue (22 degrees C). The hypothermia in response to PGE1 was due to cutaneous vasodilatation and decreased metabolic heat production. Depletion of brain 5-hydroxytryptamine (with 5,6-dihydroxytryptamine and p-chlorophenylalanine) did not alter the PGE1-induced hypothermia. However, depletion of bran catecholamines (with 6-hydroxydopamine) and blockade of central catecholaminergic receptors (with phentolamine and propranolol) both greatly reduced the PGE1-induced hypothermia. The data indicate that PGE1 lowers body temperature in rats by acting on the central catecholaminergic systems.