Impaired NO-mediated vasodilation with increased superoxide but robust EDHF function in right ventricular arterial microvessels of pulmonary hypertensive rats

Pulmonary hypertension (PH) causes right ventricular (RV) hypertrophy and, according to the extent of pressure overload, eventual heart failure. We tested the hypothesis that the mechanical stress in PH-RV impairs the vasoreactivity of the RV coronary microvessels of different sizes with increased superoxide levels. Five-week-old male Sprague-Dawley rats were injected with monocrotaline (n=126) to induce PH or with saline as controls (n=114). After 3 wk, coronary arterioles (diameter = 30-100 microm) and small arteries (diameter = 100-200 microm) in the RV were visualized using intravital videomicroscopy. We evaluated ACh-induced vasodilation alone, in the presence of N(omega)-nitro-L-arginine methyl ester (L-NAME), in the presence of tetraethylammonium (TEA) or catalase with or without L-NAME, and in the presence of SOD. The degree of suppression in vasodilation by L-NAME and TEA was used as indexes of the contributions of endothelial nitric oxide (NO) and endothelium-derived hyperpolarizing factor (EDHF), respectively. In PH rats, ACh-induced vasodilation was significantly attenuated in both arterioles and small arteries, especially in arterioles. This decreased vasodilation was largely attributable to reduced NO-mediated vasoreactivity, whereas the EDHF-mediated vasodilation was relatively robust. The suppressive effect on arteriolar vasodilation by catalase was similar to TEA in both groups. Superoxide, as measured by lucigenin chemiluminescence, was significantly elevated in the RV tissues in PH. SOD significantly ameliorated the impairment of ACh-induced vasodilation in PH. Robust EDHF function will play a protective role in preserving coronary microvascular homeostasis in the event of NO dysfunction with increased superoxide levels.     

Novel sequence-responding fluorescent oligoDNA probe bearing a silylated pyrene molecule

A novel fluorescent phosphoramidite derivative of dimethylsilylated pyrene was prepared and incorporated into oligoDNA. The fluorescent oligoDNA exhibited marked fluorescent signal upon binding to the fully matched complementary DNA strand, however, the signal was strongly quenched in the single-stranded form as well as in the duplex having mismatched base pair at the terminus of the duplex-forming region.     

Ultrasound enhances retrovirus-mediated gene transfer

Viral vector systems are efficient for transfection of foreign genes into many tissues. Especially, retrovirus based vectors integrate the transgene into the genome of the target cells, which can sustain long term expression. However, it has been demonstrated that the transduction efficiency using retrovirus is relatively lower than those of other viruses. Ultrasound was recently reported to increase gene expression using plasmid DNA, with or without, a delivery vehicle. However, there are no reports, which show an ultrasound effect to retrovirus-mediated gene transfer efficiency. Retrovirus-mediated gene transfer systems were used for transfection of 293T cells, bovine aortic endothelial cells (BAECs), rat aortic smooth muscle cells (RASMCs), and rat skeletal muscle myoblasts (L6 cells) with beta-galactosidase (beta-Gal) genes. Transduction efficiency and cell viability assay were performed on 293T cells that were exposed to varying durations (5 to 30 seconds) and power levels (1.0 watts/cm(2) to 4.0 watts/cm(2)) of ultrasound after being transduced by a retrovirus. Effects of ultrasound to the retrovirus itself was evaluated by transduction efficiency of 293T cells. After exposure to varying power levels of ultrasound to a retrovirus for 5 seconds, 293T cells were transduced by a retrovirus, and transduction efficiency was evaluated. Below 1.0 watts/cm(2) and 5 seconds exposure, ultrasound showed increased transduction efficiency and no cytotoxicity to 293T cells transduced by a retrovirus. Also, ultrasound showed no toxicity to the virus itself at the same condition. Exposure of 5 seconds at the power of 1.0 watts/cm(2) of an ultrasound resulted in significant increases in retrovirus-mediated gene expression in all four cell types tested in this experiment. Transduction efficiencies by ultrasound were enhanced 6.6-fold, 4.8-fold, 2.3-fold, and 3.2-fold in 293T cells, BAECs, RASMCs, and L6 cells, respectively. Furthermore, beta-Gal activities were also increased by the retrovirus with ultrasound exposure in these cells. Adjunctive ultrasound exposure was associated with enhanced retrovirus-mediated transgene expression in vitro. Ultrasound associated local gene therapy has potential for not only plasmid-DNA-, but also retrovirus-mediated gene transfer.     

Arrestins and spinophilin competitively regulate Na+,K+-ATPase trafficking through association with a large cytoplasmic loop of the Na+,K+-ATPase

The activity and trafficking of the Na(+),K(+)-ATPase are regulated by several hormones, including dopamine, vasopressin, and adrenergic hormones through the action of G-protein-coupled receptors (GPCRs). Arrestins, GPCR kinases (GRKs), 14-3-3 proteins, and spinophilin interact with GPCRs and modulate the duration and magnitude of receptor signaling. We have found that arrestin 2 and 3, GRK 2 and 3, 14-3-3 epsilon, and spinophilin directly associate with the Na(+),K(+)-ATPase and that the associations with arrestins, GRKs, or 14-3-3 epsilon are blocked in the presence of spinophilin. In COS cells that overexpressed arrestin, the Na(+),K(+)-ATPase was redistributed to intracellular compartments. This effect was not seen in mock-transfected cells or in cells expressing spinophilin. Furthermore, expression of spinophilin appeared to slow, whereas overexpression of beta-arrestins accelerated internalization of the Na(+),K(+)-ATPase endocytosis. We also find that GRKs phosphorylate the Na(+),K(+)-ATPase in vitro on its large cytoplasmic loop. Taken together, it appears that association with arrestins, GRKs, 14-3-3 epsilon, and spinophilin may be important modulators of Na(+),K(+)-ATPase trafficking.     

Molecular cloning and expression of gelatinases (MMP-2 and MMP-9) in the pufferfish Takifugu rubripes

To determine the metabolism location of the extra-cellular matrix proteins in fugu (Takifugu rubripes), we cloned the cDNAs of the fugu gelatinases, matrix metalloproteinase-2 (MMP-2) and MMP-9, and examined their expressions in various adult tissues using a quantitative real-time PCR. The expression profiles of fugu gelatinases were different among tissues. FgMMP-9 mRNA was abundant in tissues that contain blood cells abundantly where fgMMP-2 mRNA was little expressed. We also examined the expression of these genes in fugu embryos during development using a whole mount in situ hybridization. Fugu MMP-2 mRNA was expressed in the pharyngeal area and mesenchyme in embryos at 80 hours post fertilization (hpf). While fugu MMP-9 mRNA was expressed in the vent at 140 hpf and the caudal end of the fin fold at 172 hpf. Although fugu MMP-2 mRNA was expressed in the pectoral fin bud at 120 hpf, fugu MMP-9 mRNA did not appear in this tissue until 10 days post fertilization (dpf). These data show expression profiles differ between the fugu gelatinases and suggest expressions of these genes are controlled at the matrix protein degradation site in fugu embryos during development.     

Expression and distribution of fugu TIMP-2s (fgTIMP-2a and fgTIMP-2b) mRNAs in tissues and embryos

In teleosts, two distinct types of TIMP-2s occur, TIMP-2a and TIMP-2b, but little is known about their locations and quantitative expressions. Here, we examined pufferfish (Takifugu rubripes) TIMP-2a (fgTIMP-2a) and TIMP-2b (fgTIMP-2b) quantities and locations in fugu adult tissues and embryos. To compare the quantitative expression of fgTIMP-2s, we performed a quantitative real-time PCR (qPCR). FgTIMP-2a mRNA was constitutively expressed and significant differences in expression were not observed among adult tissues. Whereas, fgTIMP-2b mRNA was significantly differently expressed in ordinary muscle and gill compared to the expression level in whole blood (P<0.05). Although significant difference was not observed between brain and other tissues, both fgTIMP-2s mRNAs were abundant in the brain. In addition, we examined embryos during development using qPCR. Both fgTIMP-2s mRNAs gradually increased during embryonic development from 48 hpf. However, fgTIMP-2b mRNA was obviously abundant compared to fgTIMP-2a mRNA in embryos. We also examined the specific mRNA distribution in embryos. The fgTIMP-2s mRNAs showed the same distribution during development. Both fgTIMP-2s are expressed in adult fugu tissues and embryos but their expression levels clearly differ, suggesting that there is a predominance of fgTIMP-2b over fgTIMP-2a in vivo.     

Nucleotide substitutions in the acetolactate synthase genes of sulfonylurea-resistant biotypes of Monochoria vaginalis (Pontederiaceae)

Some point mutations in acetolactate synthase (ALS) confer resistance to ALS-inhibiting herbicides in weeds. To clarify the evolution of the herbicide resistance of Monochoria vaginalis, a weed in rice fields in Japan, the nucleotide sequences of four genes encoding ALS were surveyed in five sulfonylurea-resistant (SU-R) and five sulfonylurea-susceptible (SU-S) biotypes. In the ALS1 gene, two SU-R biotypes showed nucleotide substitutions changing Pro197 to Ser and Leu, respectively. In a different gene, ALS3, three other SU-R biotypes showed either of the two nonsynonymous nucleotide substitutions seen in ALS1. Only two biotypes geographically located distantly from each other shared the same mutation conferring SU resistance in the same gene. These patterns of nucleotide substitutions indicate that the SU-R phenotype was acquired independently by different biotypes. Nucleotide diversity values of the genes showing SU-R mutations were higher than those of ALS2 lacking any SU-R mutation and of a putative pseudogene, ALS4. This result suggests that the maintenance of nucleotide variability within target genes provides an opportunity for the evolution of SU-R phenotypes by herbicide-driven selection for mutations conferring resistance.     

Analysis of the exon-intron structures of fish, amphibian, bird and mammalian hatching enzyme genes, with special reference to the intron loss evolution of hatching enzyme genes in Teleostei

Using gene cloning and in silico cloning, we analyzed the structures of hatching enzyme gene orthologs of vertebrates. Comparison led to a hypothesis that hatching enzyme genes of Japanese eel conserve an ancestral structure of the genes of fishes, amphibians, birds and mammals. However, the exon-intron structure of the genes was different from species to species in Teleostei: Japanese eel hatching enzyme genes were 9-exon-8-intron genes, and zebrafish genes were 5-exon-4-intron genes. In the present study, we further analyzed the gene structures of fishes belonging to Acanthopterygii. In the species of Teleostei we examined, diversification of hatching enzyme gene into two paralogous genes for HCE (high choriolytic enzyme) and LCE (low choriolytic enzyme) was found only in the acanthopterygian fishes such as medaka Oryzias latipes, Fundulus heteroclitus, Takifugu rubripes and Tetraodon nigroviridis. In addition, the HCE gene had no intron, while the LCE gene consisted of 8 exons and 7 introns. Phylogenetic analysis revealed that HCE and LCE genes were paralogous to each other, and diverged during the evolutionary lineage to Acanthopterygii. Analysis of gene synteny and cluster structure showed that the syntenic genes around the HCE and LCE genes were highly conserved between medaka and Teraodon, but such synteny was not found around the zebrafish hatching enzyme genes. We hypothesize that the zebrafish hatching enzyme genes were translocated from chromosome to chromosome, and lost some of their introns during evolution.     

Substrate specificities of wild and mutated farnesyl diphosphate synthases from Bacillus stearothermophilus with artificial substrates

To determine the substrate specificities of wild and mutated types of farnesyl diphosphate (FPP) synthases from Bacillus stearothermophilus, we examined the reactivities of 8-hydroxygeranyl diphosphate (HOGPP) and 8-methoxygeranyl diphosphate (CH(3)OGPP) as allylic substrate homologs. The wild-type FPP synthase reaction of HOGPP (and CH(3)OGPP) with isopentenyl diphosphate (IPP) gave hydroxyfarnesyl- (and methoxyfarnesyl-) diphosphates that stopped at the first stage of condensation. On the other hand, with mutated type FPP synthase (Y81S), the former gave hydroxygeranylgeranyl diphosphate as the main double-condensation product together with hydroxyfarnesyl diphosphate as a single-condensation product and a small amount of hydroxygeranylfarnesyl diphosphate as a triple-condensation product. Moreover, the latter gave a double-condensation product, methoxygeranylgeranyl diphosphate, as the main product and only a trace of methoxyfarnesyl diphosphate was obtained.