Vascular wall energetics in arterioles during nitric oxide-dependent and -independent vasodilation.

The objective of this study was to evaluate whether the nitric oxide (NO) released from vascular endothelial cells would decrease vessel wall oxygen consumption by decreasing the energy expenditure of mechanical work by vascular smooth muscle. The oxygen consumption rate of arteriolar walls in rat cremaster muscle was determined in vivo during NO-dependent and -independent vasodilation on the basis of the intra- and perivascular oxygen tension (Po2) measured by phosphorescence quenching laser microscopy. NO-dependent vasodilation was induced by increased NO production due to increased blood flow, whereas NO-independent vasodilation was induced by topical administration of papaverine. The energy efficiency of vessel walls was evaluated by the variable ratio of circumferential wall stress (amount of mechanical work) to vessel wall oxygen consumption rate (energy cost) in the arteriole between normal and vasodilated conditions. NO-dependent and -independent dilation increased arteriolar diameters by 13 and 17%, respectively, relative to the values under normal condition. Vessel wall oxygen consumption decreased significantly during both NO-dependent and -independent vasodilation compared with that under normal condition. However, vessel wall oxygen consumption during NO-independent vasodilation was significantly lower than that during NO-dependent vasodilation. On the other hand, there was no significant difference between the energy efficiency of vessel walls during NO-dependent and -independent vasodilation, suggesting the decrease in vessel wall oxygen consumption produced by NO to be related to reduced mechanical work of vascular smooth muscle.     

Neighboring base damage induced by permanganate oxidation of 8-oxoguanine in DNA.

We found that single-stranded DNA oligomers containing a 7, 8-dihydro-8-oxoguanine (8-oxo-G) residue have high reactivity toward KMnO4; the oxidation of 8-oxo-G induces damage to the neighboring nucleotide residues. This paper describes the novel reaction in detail, including experiments that demonstrate the mechanism involved in the induction of DNA damage. The results using DNAs of various base compositions indicated that damaged G, T and C (but not A) sites caused strand scissions after hot piperidine treatment and that the damage around the 8-oxo-G occurred at G sites in both single and double strands with high frequency. The latter substrates were less sensitive to damage. Further, kinetic studies of the KMnO4reaction of single-stranded oligomers suggested that thereactivity of the DNA bases at the site 5'-adjacent to the 8-oxo-G was in the order G >A >T, C. This preference correlates with the electron donating abilities of the bases. In addition, we found that the DNA damage at the G site, which was connected with the 8-oxo-G by a long abasic chain, was inhibited in the above order by the addition of dG, dA or dC. On the other hand, the damage reactions proceeded even after the addition of scavengers for active oxygen species. This study suggests the involvement of a redox process in the unique DNA damage initiated by the oxidation of the 8-oxo-G.     

The mutations induced by oxidatively damaged nucleotides, 5-formyl-dUTP and 5-hydroxy-dCTP,in Escherichia coli.

The mutational properties of 5-formyl-2'-deoxyuridine 5'-triphosphate (5-CHO-dUTP) and 5-hydroxy-2'-deoxycytidine 5'-triphosphate (5-OH-dCTP), the major oxidatively damaged pyrimidine nucleotides derived from dTTP and dCTP, respectively, were analyzed by an in vivo assay. 5-CHO-dUTP and 5-OH-dCTP were directly incorporated into Escherichia coli , and their mutagenicities were evaluated by the chromosomal lacI forward mutation assay. The mutation frequencies increased, depending on the dose of these damaged nucleotides, indicating that these nucleotides were incorporated into E.coli and acted as mutagens in vivo . The mutagenicities of 5-CHO-dUTP and 5-OH-dCTP were comparable to that of 8-hydroxy-2'-deoxyguanosine 5'-triphosphate, a major form of dGTP oxidative damage. 5-CHO-dUTP induced G.C to A.T, A.T to G.C and G.C to T.A mutations, and 5-OH-dCTP elicited G.C to A.T, A.T to C.G and G.C to T.A mutations.     

Genomic organization and mutational analysis of HERG, a gene responsible for familial long QT syndrome

Familial long QT syndrome (LQTS) is characterized by prolonged ventricular repolarization. Clinical symptoms include recurrent syncopal attacks, and sudden death may occur as a result of ventricular tachyarrhythmias. Three genes responsible for this syndrome (KVLQT1, HERG, and SCN5A) have been identified so far, and mutations have been reported on the basis of partially characterized genomic organization. To optimize the search for HERG mutations, we have determined the genomic structure of HERG and investigated mutations in LQTS families. Human genomic clones containing the HERG gene were isolated from a human genomic library by using reverse-transcribed polymerase chain reaction (RT-PCR) products from this gene as probes. We determined exon/intron boundaries and flanking intronic sequences by using primers synthesized on the basis of the HERG cDNA sequence available in the DNA database. HERG was shown to consist of 15 exons spanning approximately 19 kb on chromosome 7q35. Subsequently, we synthesized oligonucleotide primers to cover the entire coding region and searched for mutations in 36 Japanese LQTS families. When genomic DNA from each proband was examined by the PCR/single-strand conformation polymorphism technique followed by direct DNA sequencing, five novel mutations were detected. Each mutation was present in affected relatives of the respective proband. This work should increase the efficiency of screening mutations associated with HERG. Received: 4 November 1997 / Accepted: 5 January 1998     

The effect of processing of Chlorellavulgaris: K-5 on in vitro and in vivo digestibility in rats

Two experiments were done to clarify whether or not cell rupture is necessary to improve the digestibility of major components of Chlorella vulgaris: K-5. Chlorella was treated with or without high pressure homogenization (1 × 10⁸ N/m² at less than −20°C) after a heating process (100-120°C). Chlorella (air-dry matter) contained 934 g dry matter and 244 g essential amino acids (total)/kg. Chemical composition was hardly altered irrespective of the treatment. In the first experiment, pepsin digestibility of chlorella protein was determined in vitro. The cell rupture by high pressure homogenization caused a small but significant improvement in pepsin digestibility of chlorella protein compared with the control. In the second experiment, total tract apparent digestibilities of chlorella were determined in the rat. Digestibility of chlorella protein was significantly enhanced by high pressure homogenization, but the difference (88.6% vs. 87.4%, P < 0.01) due to treatment was small and similar to that observed in the in vitro experiment. These results suggested that Chlorella strain vulgaris: K-5 may be an efficient protein source even without cell rupture.     

Optimal branching structure of the vascular tree

Optimality in branching structure of the vascular tree was studied. Analysis on its physiological roles as the duct system for blood supply to the capillaries predicted that the vascular tree should be constructed with minimum volume under restriction of determinant pressure, flow and location at the origin and the terminals. Mathematical derivations of this conditional extremum problem yielded some equations expressing the relations between the radii of the branches and their branching angles, which provided numerical solutions for branching points of bi- and poli-terminal minimum volume trees. Comparison of the peritoneal vascular tree in a dog with the minimum volume one computed under the same restrictive conditions showed good agreement in their branching structure.     

A mutant of Escherichia coli blocked in peptide elongation: altered elongation factor Ts

Among our transfer RNA-dependent growth mutants, one, HAK88, was found that carries an altered elongation factor Ts. The activity of mutant EFTs to bind GDP to EFTu, or to form the ternary complex (aminoacyl-tRNA-EFTu-GTP) is thermolabile. The effect of magnesium on the formation of EFTu-GDP from the EFTu-EFTs complex of HAK8 shows that a four to fivefold increase of the duplex formation occurs when the magnesium concentration is increased from 10^?6m to 10^?2m at 0 °C and at 41 °C. However, at higher temperatures, formation of the binary EFTu-GDP from the EFTu-EFTs complex of HAK88 is depressed, even at 10^?3m to 10^?2m-magnesium. The binding of GDP to the wild-type or mutant EFTu-EFTs complex at 0 °C and 42 °C indicates that the formation of EFTu-GDP is inhibited at 42 °C only when mutant complex is used for the assay. Binding of GTP to complete bacteriophage Qβ replicase (which is known to contain EFTs) formed in phage-infected HAK88 is also inhibited at 42 °C.     

Enhancement of hypocotyl elongation by LOV KELCH PROTEIN2 production is mediated by auxin and phytochrome-interacting factors in <Emphasis Type="Italic">Arabidopsis thaliana</Emphasis>

Key message

Auxin and two phytochrome-interacting factors, PHYTOCHROME-INTERACTING FACTOR4 (PIF4) and PIF5, play crucial roles in the enhancement of hypocotyl elongation in transgenic Arabidopsis thaliana plants that overproduce LOV KELCH PROTEIN2 (LKP2).

Abstract

LOV KELCH PROTEIN2 (LKP2) is a positive regulator of hypocotyl elongation under white light in Arabidopsis thaliana. In this study, using microarray analysis, we compared the gene expression profiles of hypocotyls of wild-type Arabidopsis (Columbia accession), a transgenic line that produces green fluorescent protein (GFP), and two lines that produce GFP-tagged LKP2 (GFP-LKP2). We found that, in GFP-LKP2 hypocotyls, 775 genes were up-regulated, including 36 auxin-responsive genes, such as 27 SMALL AUXIN UP RNA (SAUR) and 6 AUXIN/INDOLE-3-ACETIC ACID (AUX/IAA) genes, and 21 genes involved in responses to red or far-red light, including PHYTOCHROME-INTERACTING FACTOR4 (PIF4) and PIF5; and 725 genes were down-regulated, including 15 flavonoid biosynthesis genes. Hypocotyls of GFP-LKP2 seedlings, but not cotyledons or roots, contained a higher level of indole-3-acetic acid (IAA) than those of control seedlings. Auxin inhibitors reduced the enhancement of hypocotyl elongation in GFP-LKP2 seedlings by inhibiting the increase in cortical cell number and elongation of the epidermal and cortical cells. The enhancement of hypocotyl elongation was completely suppressed in progeny of the crosses between GFP-LKP2 lines and dominant gain-of-function auxin-resistant mutants (axr2-1 and axr3-1) or loss-of-function mutants pif4, pif5, and pif4 pif5. Our results suggest that the enhancement of hypocotyl elongation in GFP-LKP2 seedlings is due to the elevated level of IAA and to the up-regulated expression of PIF4 and PIF5 in hypocotyls.