Hiroshi Teraoka

Endothelial Nitric Oxide Synthase Gene Is Positively Associated With Essential Hypertension

Essential hypertension has a genetic basis. Accumulating evidence, including findings of elevation of arterial blood pressure in mice lacking the endothelial nitric oxide synthase (eNOS) gene, strongly suggests that alteration in NO metabolism is implicated in hypertension. There are, however, no reports indicating that polymorphism in the eNOS gene is associated with essential hypertension. We have identified a missense variant, Glu298Asp, in exon 7 of the eNOS gene and demonstrated that it is associated with both coronary spastic angina and myocardial infarction. To explore the genetic involvement of the eNOS gene in essential hypertension, we examined the possible association between essential hypertension and several polymorphisms including the Glu298Asp variant, variable number tandem repeats in intron 4 (eNOS4b/4a), and two polymorphisms in introns 18 and 23. We performed a large-scale study of genetic association using two independent populations from Kyoto (n=458; 240 normotensive versus 218 hypertensive subjects) and Kumamoto (n=421; 223 normotensive versus 187 hypertensive subjects), Japan. In both groups, a new coding variant, Glu298Asp, showed a strong association with essential hypertension (Kyoto: odds ratio, 2.3 [95% confidence interval, 1.4 to 3.9]; Kumamoto: odds ratio, 2.4 [95% confidence interval, 1.4 to 4.0]). The allele frequencies of 298Asp in hypertensive subjects were significantly higher than those in normotensive subjects in both groups (Kyoto: 0.103 versus 0.050, P<0.0017; Kumamoto: 0.120 versus 0.058, P<0.0013, respectively). No such disequilibrium between genotypes was significantly associated with any other polymorphisms we examined; the Glu298Asp variant was also not linked to any other polymorphisms. In conclusion, the Glu298Asp missense variant was significantly associated with essential hypertension, which suggests that it is a genetic susceptibility factor for essential hypertension.

Group II phospholipase A2 mRNA synthesis is stimulated by two distinct mechanisms in rat vascular smooth muscle cells.

Two potent inflammatory mediators, interleukin 1 (IL‐1) and tumor necrosis factor (TNF) as well as lipopolysaccharide (LPS) increased group II phospholipase A2 (PLA2) mRNA levels, which resulted in enhanced secretion of the PLA2 enzyme from rat smooth muscle cells. cAMP‐elevating agents also stimulated the release of PLA2 and increased the mRNA, but IL‐1, TNF and LPS did not affect cAMP levels. Furthermore, the effects of TNF and cAMP‐elevating agents were not additive but synergistic. Therefore, we concluded that the level of rat group II PLA2 mRNA is controlled at least by two distinct mechanisms, one involves cAMP and the other is mediated by TNF, IL‐1 and LPS. This study also suggests important roles of group II PLA2 in pathogenesis of vascular inflammation.

Presence of pancreatic-type phospholipase A2 mRNA in rat gastric mucosa and lung.

The content of mRNA for a pancreatic-type phospholipase A2 present in rat gastric mucosa was much greater than that in pancreas. In lung the mRNA for this pancreatic-type phospholipase A2 was also detected, but less than in pancreas. Nucleotide sequence analysis showed that these pancreatic-type phospholipase A2 cDNAs derived from rat gastric mucosa and lung were completely identical to that from rat pancreas (Ohara et al. (1986) J. Biochem. 99, 733-739). This demonstrates that the pancreatic-type phospholipase A2 present in gastric mucosa and lung does not originate from pancreas.

Molecular cloning and sequence analysis of cDNA encoding rat adrenal cytochrome P-45011β

A cDNA clone encoding cytochrome P‐45011β of rat adrenal has been cloned and sequenced using a bovine P‐45011β cDNA insert (pcP‐450(11β)‐2; (1987) J. Biochem. 102, 559–568) as a probe. The nucleotide sequence contains an open reading frame sufficient to encode the entire amino acid sequence of a P‐45011β precursor protein consisting of 499 amino acids including an extension peptide of 24 amino acids at the NH2‐terminus. The cDNA contains 1247 nucleotides at the 3′‐noncoding region including 51 nucleotides of poly A, but lacks the 5′‐noncoding region. The deduced amino acid sequence shows 61% similarity to that of bovine P‐45011β. Putative binding sites for heme and steroid are highly conserved among steroidogenic P‐450s of known structure.

cDNA cloning and sequence determination of rat membrane-associated phospholipase A2

Based on the partial amino acid sequences of membrane-associated phospholipase A2 (PLA2M), belonging to group II, purified from rat spleen, the cDNA encoding PLA2M was cloned by a new cloning strategy utilizing enzymatic cDNA amplification. At the N-terminus of the coded 146 residues, which were deduced from the cDNA sequence, the putative signal peptide was found despite the tight adherence of this enzyme to the membrane. The sequence of rat PLA2M exhibits 75% homology with that of human group II PLA2 in the protein-coding region. The result of RNA blot analysis showed that rat ileal mucosa contains the largest amount of the PLA2 transcript among the tissues examined.

A simple immunoradiometric assay for measuring the entire molecules of adrenomedullin in human plasma

We developed a one-step two-site immunoradiometric assay (IRMA) using two kinds of monoclonal antibodies, which enables us to directly measure the entire molecules of adrenomedullin (AM) (the sum of mature-type AM (abbreviated, m-AM) amidated at the C-terminus and Gly-extended non-amidated AM) in human plasma using a small amount of sample (100 microl) without prior extraction. The detection limit of this assay was 0.5 pmol/l for a 100-microl sample. Intra- and inter-assay precisions were 3.4-7.3% and 5.8-7.6%, respectively. The dilution curves of plasma samples showed good linearity and analytical recovery was 89-118%. The mean total AM in plasma of healthy subjects was 9.00+/-2.13 pmol/l, whereas m-AM was 1.05+/-0.24 pmol/l. This method, together with our previously reported simplified method to specifically measure m-AM (Ohta et al., Clin Chem 1999;45:244-251), allows facile estimation of the plasma concentration of AM-Gly by subtracting m-AM from the total AM measured by the procedure described in this paper. We were able to show that the concentration of total AM in patients with sepsis was markedly higher than that in the healthy controls and that the ratios of m-AM/total AM were significantly different between the controls and patients.

Erythromycin-Resistant Mutant of Escherichia coli with Altered Ribosomal Protein Component

Erythromycin combines with 50S ribosomal subunit of an erythromycin-sensitive Escherichia coli (strain Q13), while ribosomes from an erythromycin-resistant mutant from this strain have little affinity for the antibiotic. A protein component of the 50S subunit of the mutant strain is distinct from that of the parent Q13 strain.

Ribosomes from erythromycin-resistant mutants of Escherichia coli Q13

Mutants from Escherichia coli Q13 were selected for resistance to leucomycin, tylosin or spiramycin. Most of the mutants so selected exhibited cross resistance to all the macrolide antibiotics tested including erythromycin. A few mutants however seem to be less resistant to erythromycin. One mutant, QSP008, was highly resistant to tylosin, leucomycin and spiramycin but relatively sensitive to erythromycin. Another mutant, QSP006, was highly resistant to spiramycin but less resistant to erythromycin, tylosin and leucomycin. This selective resistance of cells to specific antibiotics could be due to the extent of conformational alteration of their ribosomes, which may be demonstrated by the extent of 14C-erythromycin binding to these ribosomes. The ribosomes from QSP008 cells were found to contain an altered 50-8 protein of the 50s ribosomal subunit, while in the ribosomes from QSP006 no such protein change could be detected by the methods used.

Constitutive expression of interleukin-7- mRNA and production of IL-7 by a cloned murine thymic stromal cell line

Mouse interleukin‐7 (IL‐7) cDNA was cloned from mouse thymic stromal cell clone MRL104.8a using a polymerase chain reaction (PCR) technique and expressed in COS‐7 cells. The resulting recombinant interleukin‐7 (rlL‐7) supported the proliferation of mouse antigen‐specific helper T cell (Th) clone 9–16 in the absence of IL‐2 and antigen as well as mouse pre‐B cell line DW34. It was also found that high levels of the mRNA for IL‐7 were constitutively expressed in the MRL104.8a cells, and a potent amount of IL‐7 was produced in its culture supernatant. These results provide the evidence for constitutive expression of IL‐7 mRNA and for production of IL‐7 by thymic stromal cells that have a critical role in intrathymic T cell development. The results are discussed in the context of the functional and molecular relationship between IL‐7 and the previously described cytokines produced by thymic stromal cells.

rig encodes ribosomal protein S15 The primary structure of mammalian ribosomal protein S15

rig, a gene originally isolated from a rat insulinoma cDNA library, codes for a basic 145 amino acid protein [(1986) Diabetes 35, 1178–1180]. Here we show that the immunoreactivity to a monocional antibody against the deduced rig protein and the translation product of rig mRNA comigrated with ribosomal protein S15. The amino acid sequence of ribosomal protein S15 purified from rat liver coincided with that deduced from the nucleotide sequence of rig mRNA, but there were indications that the initiator methionine was removed and the succeeding alanyl residue was monoacetylated. From these results, we conclude that the product of rig is ribosomal protein S15.