Metabarcoding data of mitochondrial cytochrome c oxidase subunit 1 gene from bulk community of aquatic organisms collected from Nara Prefecture, Japan.

Riverine metabarcoding data were obtained from the Takamigawa River, a tributary of the Kinokawa River, in Nara Prefecture (Central Honshu, Japan). We extracted DNA from bulk community samples of aquatic organisms, most of which could not be morphologically identified at species level due to their small body size (0.12 - 2 mm length). A partial coding region of the mitochondrial cytochrome c oxidase subunit 1 gene (cox1) was amplified using PCR, and the amplicon was subjected to high-throughput parallel sequencing (Illumina MiSeq). The 313 bp paired-end sequence reads were classified into operational taxonomic units (OTUs), their species boundaries were delineated using the Generalised Mixed Yule Coalescent (GMYC) method, and taxonomic names of the GMYC species were assigned using basic local alignment search tool (BLAST) against International DNA Databases (INSD: GenBank, ENA, and DDBJ).

Pairwise sequence comparison data of the DNA barcodes of aquatic insects.

This study compared the DNA sequences of cytochrome c oxidase subunit I (COI) and histone H3 of Ephemeroptera, Odonata, Plecoptera, and Trichoptera in a pairwise manner, and calculated the sequence similarities based on uncorrected P-distance (number of identical sites in both sequences per total number of the sites compared). Datasets of annotated sequences, the source organisms of which are identified at the species level in taxonomy, were retrieved from INSD (GenBank/ ENA/ DDBJ) as of the end of May 2020. Similarity scores of the pairwise comparison were sorted by the combinations of taxonomic groups; intraspecific variations, intrageneric-interspecific divergences, intrafamily-intergeneric divergences, and intraorder-interfamily divergences for Ephemeroptera, Odonata, Plecoptera, and Trichoptera. Similarity scores at the cumulative relative frequency points (1%, 5%, 10%, and median) may be used as the threshold to differentiate between the taxonomic groups based on sequence match. This is often done in the characterization of morphologically-unidentified specimens using barcode sequences, in the metabarcoding analysis of the local fauna, and environmental DNA analysis.

Evolutionary Modification of Pereopods in Phronimid Amphipods (Crustacea: Amphipoda: Hyperiidea: Phronimidae) Reflects Host Differences.

Phronimid amphipods are oceanic crustaceans associated with gelatinous zooplankters. Their host organisms belong mainly to two taxonomic groups: tunicates (salps or pyrosomes; subphylum Tunicata) and siphonophores (Cnidaria). After these amphipods devour the inner tissues of their hosts, they display the unique behavior of modifying their hosts into hollow barrel-shaped shelters, which are then utilized as neonatal nurseries by the females. Although previous studies have revealed the host specificity of these amphipods, it has not been inferred which types of hosts ancestral phronimids could have originally used. Moreover, morphological changes associated with host switching have not yet been studied. To deduce the evolutionary patterns of host switching, we investigated the phylogenetic relationships of phronimid species by using two genes: (1) cytochrome c oxidase subunit I (COI) and (2) 18S ribosomal RNA (18S). In addition, a morphometric analysis was conducted in order to better understand the morphological relationships between phronimids and their host organisms. Our phylogenetic analysis suggests that the ancestral host animals of phronimids could have been tunicates and that the host organisms have independently switched from tunicates to siphonophores at least twice in the family Phronimidae. Our morphometric analysis revealed that phronimids using siphonophores as hosts have a relatively shorter pereopod 5 compared to those using tunicates. The shortening of pereopod 5 seems to be an adaptation to the narrower internal space of siphonophore barrels compared to those of tunicates.

A Novel and Highly Potent Urotensin II Receptor Antagonist Inhibits Urotensin II-Induced Pressure Response in Mice.

This study was designed to characterize the pharmacological profile of DS37001789, which is a structurally novel piperazine derivative that acts as urotensin II (U-II) receptor antagonist. DS37001789 inhibited [I]-U-II binding to human GPR14, U-II receptor, with an IC50 value 0.9 nM. Its potency was superior to that of ACT-058362, a nonpeptide U-II receptor antagonist whose IC50 was 120 nM. Human U-II-induced vascular contraction was blocked by DS37001789. The dose-response curve of DS37001789 in rats and monkeys did not show species differences, and it shifted to the right without any effects on the maximum vascular response. Moreover, orally administered DS37001789 dose-dependently prevented human U-II-induced blood pressure elevation in mice, and this effect was significant at dose and higher dose (30 and 100 mg/kg), and its potency was superior to that of ACT-058362 (100 mg/kg). These results suggest that DS37001789 is a highly potent U-II receptor antagonist both in vitro and in vivo, with no marked species difference. DS37001789 would be a useful tool to clarify the physiological roles of U-II/GPR14 system. In addition, it can serve as a novel therapeutic agent for diseases in which the U-II/GPR14 system is upregulated, such as hypertension, heart failure, renal dysfunction, and diabetes.

Attenuation of pulmonary fibrosis in type I collagen-targeted reporter mice with ALK-5 inhibitors.

Idiopathic pulmonary fibrosis (IPF) is a progressive and fatal disease, and consequently, effective antifibrotic drugs are strongly desired. Although we have previously reported a validated Col1a1-Luc Tg rat model for fibrosis, there are only a few mouse models that enable the evaluation of fibrosis in a short time period and with high sensitivity. Therefore, we generated a Col1a1-internal ribosome entry site (IRES)-Luc knock-in (KI) mouse in which the IRES-luciferase gene construct was inserted into the 3'-UTR of the type I collagen alpha 1 gene (Col1a1). There was a high correlation between luciferase activity and hydroxyproline content in the KI mice, which is similar to the result that we have previously reported for the Col1a1-Luc Tg rat model. In a bleomycin (BLM)-induced lung fibrosis model, luciferase activity in the lung showed a significant increase 3 days after BLM treatment, while only a slight increase was observed in the hydroxyproline content. An ALK-5 inhibitor-R-268712-was effective in inhibiting the luciferase activity in both the in vivo BLM-induced lung fibrosis model and in vitro primary mouse lung fibroblasts. This suggests that fibroblasts are the major collagen-producing cells in lung fibrosis. In human lung fibroblasts, TGF-ß stimulation induced α-smooth muscle actin as observed by immunostaining, suggesting that myofibroblast transdifferentiation (MTD) plays an important role in lung fibrosis. Together, these results indicated that ALK-5 inhibitors might affect lung fibrosis mainly via the inhibition of MTD. Thus, the Col1a1-IRES-Luc KI mouse might be useful for the evaluation of antifibrotic effects and their underlying mechanisms.

R-268712, an orally active transforming growth factor-ß type I receptor inhibitor, prevents glomerular sclerosis in a Thy1 nephritis model.

R-268712 is a novel and specific inhibitor of activin receptor-like kinase 5 (ALK5), a transforming growth factor ß (TGF-ß) type I receptor. Evaluation of in vitro inhibition indicated that R-268712 is a potent and selective inhibitor of ALK5 with an IC50 of 2.5nM, an approximately 5000-fold more selectivity for ALK5 than p38 mitogen-activated protein kinase (MAPK). Oral administration of R-268712 at doses of 1, 3 and 10mg/kg also inhibited the development of renal fibrosis in a dose-dependent manner in a unilateral ureteral obstruction (UUO) model. Additionally, we evaluated the efficacy of R-268712 in a heminephrectomized anti-Thy1 glomerulonephritis model at doses of 0.3 and 1mg/kg. R-268712 reduced proteinuria and glomerulosclerosis significantly with improvement of renal function. Collectively, these results suggested that R-268712 and other ALK5 inhibitors could suppress glomerulonephritis as well as glomerulosclerosis by an inhibitory mechanism that involves suppression of TGF-ß signaling.

Lead optimization of 5-amino-6-(2,2-dimethyl-5-oxo-4-phenylpiperazin-1-yl)-4-hydroxyhexanamides to reduce a cardiac safety issue: discovery of DS-8108b, an orally active renin inhibitor.

With the aim to address an undesired cardiac issue observed with our related compound in the recently disclosed novel series of renin inhibitors, further chemical modifications of this series were performed. Extensive structure-activity relationships studies as well as in vivo cardiac studies using the electrophysiology rat model led to the discovery of clinical candidate trans-adamantan-1-ol analogue 56 (DS-8108b) as a potent renin inhibitor with reduced potential cardiac risk. Oral administration of single doses of 3 and 10 mg/kg of 56 in cynomolgus monkeys pre-treated with furosemide led to significant reduction of mean arterial blood pressure for more than 12 h.

Direct binding and regulation of RhoA protein by cyclic GMP-dependent protein kinase Iα.

Vascular smooth muscle cell (VSMC) tone is regulated by the state of myosin light chain (MLC) phosphorylation, which is in turn regulated by the balance between MLC kinase and MLC phosphatase (MLCP) activities. RhoA activates Rho kinase, which phosphorylates the regulatory subunit of MLC phosphatase, thereby inhibiting MLC phosphatase activity and increasing contraction and vascular tone. Nitric oxide is an important mediator of VSMC relaxation and vasodilation, which acts by increasing cyclic GMP (cGMP) levels in VSMC, thereby activating cGMP-dependent protein kinase Iα (PKGIα). PKGI is known to phosphorylate Rho kinase, preventing Rho-mediated inhibition of MLC phosphatase, promoting vasorelaxation, although the molecular mechanisms that mediate this are unclear. Here we identify RhoA as a target of activated PKGIα and show further that PKGIα binds directly to RhoA, inhibiting its activation and translocation. In protein pulldown and immunoprecipitation experiments, binding of RhoA and PKGIα was demonstrated via a direct interaction between the amino terminus of RhoA (residues 1-44), containing the switch I domain of RhoA, and the amino terminus of PKGIα (residues 1-59), which includes a leucine zipper heptad repeat motif. Affinity assays using cGMP-immobilized agarose showed that only activated PKGIα binds RhoA, and a leucine zipper mutant PKGIα was unable to bind RhoA even if activated. Furthermore, a catalytically inactive mutant of PKGIα bound RhoA but did not prevent RhoA activation and translocation. Collectively, these results support that RhoA is a PKGIα target and that direct binding of activated PKGIα to RhoA is central to cGMP-mediated inhibition of the VSMC Rho kinase contractile pathway.

Behavior of a hammerhead ribozyme in aqueous solution at medium to high temperatures.

The "RNA world" hypothesis proposes that--early in the evolution of life--RNA molecules played important roles both in information storage and in enzymatic functions. However, this hypothesis seems to be inconsistent with the concept that life may have emerged under hydrothermal conditions since RNA molecules are considered to be labile under such extreme conditions. Presently, the possibility that the last common ancestor of the present organisms was a hyperthermophilic organism which is important to support the hypothesis of the hydrothermal origin of life has been subject of strong discussions. Consequently, it is of importance to study the behavior of RNA molecules under hydrothermal conditions from the viewpoints of stability, catalytic functions, and storage of genetic information of RNA molecules and determination of the upper limit of temperature where life could have emerged. In the present work, self-cleavage of a natural hammerhead ribozyme was examined at temperatures 10-200 °C. Self-cleavage was investigated in the presence of Mg(2+), which facilitates and accelerates this reaction. Self-cleavage of the hammerhead ribozyme was clearly observed at temperatures up to 60 °C, but at higher temperatures self-cleavage occurs together with hydrolysis and with increasing temperature hydrolysis becomes dominant. The influence of the amount of Mg(2+) on the reaction rate was also investigated. In addition, we discovered that the reaction proceeds in the presence of high concentrations of monovalent cations (Na(+) or K(+)), although very slowly. Furthermore, at high temperatures (above 60 °C), monovalent cations protect the ribozyme against degradation.

Design and optimization of novel (2S,4S,5S)-5-amino-6-(2,2-dimethyl-5-oxo-4-phenylpiperazin-1-yl)-4-hydroxy-2-isopropylhexanamides as renin inhibitors.

Introduction of the 2,2-dimethyl-4-phenylpiperazin-5-one scaffold into the P(3)-P(1) portion of the (2S,4S,5S)-5-amino-6-dialkylamino-4-hydroxy-2-isopropylhexanamide backbone dramatically increased the renin inhibitory activity without using the interaction to the S(3)(sp) pocket. Compound 31 exhibited >10,000-fold selectivity over other human proteases, and 18.5% oral bioavailability in monkey.

Characterization of a novel flooding stress-responsive alcohol dehydrogenase expressed in soybean roots.

Alcohol dehydrogenase (Adh) is the key enzyme in alcohol fermentation. We analyzed Adh expression in order to clarify the role of Adh of soybeans (Glycine max) to flooding stress. Proteome analysis confirmed that expression of Adh is significantly upregulated in 4-day-old soybean seedlings subjected to 2 days of flooding. Southern hybridization analysis and soybean genome database search revealed that soybean has at least 6 Adh genes. The GmAdh2 gene that responded to flooding was isolated from soybean cultivar Enrei. Adh2 expression was markedly increased 6 h after flooding and decreased 24 h after floodwater drainage. In situ hybridization and Western blot indicated that flooding strongly induces Adh2 expression in RNA and protein levels in the root apical meristem. Osmotic, cold, or drought stress did not induce expression of Adh2. These results indicate that Adh2 is a flooding-response specific soybean gene expressed in root tissue.

Lymphokine-activated killer cell therapy combined with high-dose glucocorticoid showed clinical efficacy towards advanced lung carcinoma.

We describe the case of a patient with terminal adenocarcinoma of the lung with no response to lymphokine-activated killer (LAK) cell therapy alone who distinctly responded clinically to a combination of high-dose glucocorticoids (GC) and LAK cell therapy, administered by chance. However, after decreasing the dose of GC because of gastrointestinal bleeding caused by the high-dose treatment, there was no response on restarting GC plus LAK cell therapy. The clinical course of this case strongly suggests that local inflammation in the vicinity of tumors should be adequately suppressed in patients with advanced cancer who receive immunotherapy.

A sensitive short-term evaluation of antifibrotic effects using newly established type I collagen reporter transgenic rats.

Fibrosis is the final common pathway for various tissue lesions that lead to chronic progressive organ failure, and consequently effective antifibrotic drugs are strongly desired. However, there are few animal models in which it is possible to evaluate fibrosis sensitively in a short period of time. We therefore generated two transgenic rats harboring a firefly luciferase reporter gene under the control of the 5'-flanking region of rat α(1)(I) collagen (Col1a1-Luc Tg rats) and α(2)(I) collagen (Col1a2-Luc Tg rats). The luciferase activities of these transgenic rats were highly correlated with the hydroxyproline content in various organs. In unilateral ureteral obstruction (UUO), a well-characterized model of renal fibrosis, the luciferase activity in obstructed kidneys showed a significant increase after even 3 days of UUO, while the hydroxyproline content showed little increase. In addition, the renal hydroxyproline content had a higher correlation with the luciferase activity than α(1)(I) collagen mRNA level for over 2 wk after UUO. Although both an ANG II type 1 receptor blocker (ARB), olmesartan, and a transforming growth factor-ß (TGF-ß) type I receptor kinase (ALK5) inhibitor, SB-431542, inhibited renal luciferase activities in UUO, only SB-431542 inhibited luciferase activity induced by TGF-ß1 in isolated glomeruli. Double immunostaining for luciferase and α-smooth muscle actin (α-SMA) revealed that some α-SMA-positive tubular epithelial cells and tubular interstitial cells produced type I collagen, which would lead to renal fibrosis. Thus collagen reporter transgenic rats would be very useful for the evaluation of antifibrotic effects and analysis of their mechanisms.

A target site for spontaneous insertion of IS10 element in pUC19 DNA located within intrinsically bent DNA.

Residual insertion sequence elements (IS elements) in Escherichia coli strains that are commonly used for DNA cloning are known to cause cloning artifacts by transposing themselves into the recombinant DNA fragments. In such cases, chance insertion of IS elements may occur at integration sites in the cloning targets, which in the case of the IS10 element is a 9-bp consensus sequence. We report here that the integration of IS10-related DNA sequences into the pUC19 cloning vector and its derivative occurred with considerable frequency in E. coli strains JM107 and DH10B, with duplication of a 9-bp segment (TCTAAAGTA). Notably, native polyacrylamide gel electrophoresis revealed that intrinsically bent DNA flanks the insertion site.

Structural characterization of supercoiled DNA containing a minisatellite repeat that has polypurine/polypyrimidine stretch.

The pUC19 derivative pAL79 contains six tandem repeats of a 30-bp unit sequence. Supercoiled pAL79 molecules show aberrant electrophoretic mobility under acidic conditions, and microscopic analysis suggests pAL79 molecules conform to tight plectonemes when deposited on a substrate under acidic conditions. This may mean that a short inserted DNA sequence affects the global topology of plasmid DNA.

Stable minihairpin structures forming at minisatellite DNA isolated from yellow fin sea bream Acanthopagrus latus.

The lengths of simple repeat sequences are generally unstable or polymorphic (highly variable with respect to the numbers of tandem repeats). Previously we have isolated a family of minisatellite DNA (GenBank accession AF422186) that appears specifically and abundantly in the genome of yellow fin sea bream Acanthopagrus latus but not in closely-related red sea bream Pagrus major, and found that the numbers of tandem arrays in the homologous loci are polymorphic. This means that the minisatellite sequence has appeared and propagated in A. latus genome after speciation. In order to understand what makes the minisatellite widespread within the A. latus genome and what causes the polymorphic nature of the number of tandem repeats, the structural features of single-stranded polynucleotides were analyzed by electrophoresis, chemical modification, circular dichroism (CD), differential scanning calorimetry (DSC) and electron microscopy. The results suggest that a portion of the repeat unit forms a stable minihairpin structure, and it can cause polymerase pausing within the minisatellite DNA.

DNA secondary structure forming at minisatellite repeat unit sequences.

The lengths of simple repeat sequences are generally unstable or polymorphic; that is, they are highly variable with respect to the numbers of tandem repeats. To determine the structural features that cause such variability, we examined a minisatellite DNA isolated from the yellow fin sea bream Acanthopagrus latus. Electrophoresis, CD spectra, and calorimetric analyses of oligodeoxyribonucleotides comprised of the minisatellite repeat unit suggest a stable secondary structure is formed within the repeat unit.

Mode of evolution of the satellite DNA from the bluegill sunfish Lepomis macrochirus.

Examination of the sequence of a satellite DNA (BG16) isolated from the bluegill sunfish Lepomis macrochirus revealed that the repeat unit contains regions comprised of internal subrepeats. This suggests that the BG16 satellite evolved from a shorter repeat unit into a larger composite repeat, and a stepwise expansion model of the evolution of the BG16 satellite is proposed.

The effect of angiotensin II receptor blockade on an end-stage renal failure model of type 2 diabetes.

The effect of olmesartan medoxomil (OLM), an angiotensin II receptor blocker (ARB), on advanced nephropathy and mortality was evaluated in Zucker Diabetic Fatty (ZDF) rats, a type 2 diabetes model. OLM was administered from 36 weeks of age, when the animals developed advanced proteinuria. OLM effectively suppressed the progression of proteinuria. The ZDF rats started to die at 50 weeks of age, which was accompanied by abrupt increase in blood urea nitrogen, suggesting that the cause of death was renal insufficiency. OLM suppressed increases in blood urea nitrogen and increased the survival rate of the ZDF rats. The histological examination revealed that the renal damage was ameliorated by OLM. The macrophage infiltration and monocyte chemoattractant protein-1 (MCP-1) expression was increased in the glomeruli and tubulointerstitium of the ZDF rat kidneys, and the increase was lessened by OLM. In a separate study, albumin increased MCP-1 release from cultured tubular epithelial cells. These results suggest that protein leakage from the glomeruli stimulates MCP-1 production in tubular cells and that MCP-1 released into the interstitial space induces macrophage infiltration and inflammation. It is conceivable that the beneficial actions of ARB on diabetic nephropathy are, at least in part, due to decrease of proteinuria and the subsequent reduction of inflammatory changes in tubular cells.

Severity of hyperlipidemia does not affect antiatherosclerotic effect of an angiotensin II receptor antagonist in apolipoprotein E-deficient mice.

The purpose of this study was to clarify whether severity of hyperlipidemia affects the antiatherosclerotic effect of angiotensin II receptor blockers (ARBs). The effect of olmesartan medoxomil, an ARB, on atherosclerotic lesion was examined in apolipoprotein E-deficient (ApoEKO) mice fed a normal diet (ND) or a high-fat-supplemented diet (FD) for 25 weeks. ApoEKO mice have high plasma cholesterol levels, which were further increased by feeding of an FD. Both the atherosclerotic lesion area of the aortic luminal surface and the atherosclerotic lesion thickness in the aortic valves were significantly greater in the FD mice than in the ND mice. Olmesartan medoxomil did not affect the plasma cholesterol levels in either the ND or FD ApoEKO mice; however, it reduced effectively both the atherosclerotic lesion surface area and the lesion thickness even in FD ApoEKO mice. It is concluded that the antiatherosclerotic effect of ARBs is not weakened by the high plasma cholesterol level, suggesting the usefulness of ARBs in the treatment of atherosclerosis, even in a situation in which the plasma cholesterol level is not fully controlled.