A role of heme side-chains of human hemoglobin in its function revealed by circular dichroism and resonance Raman spectroscopy

Structural changes of heme side-chains of human adult hemoglobin (Hb A) upon ligand (O₂ or CO) dissociation have been studied by circular dichroism (CD) and resonance Raman (RR) spectroscopies. We point out the occurrence of appreciable deformation of heme side-chains like vinyl and propionate groups prior to the out-of-plane displacement of heme iron. Referring to the recent fine resolved crystal structure of Hb A, the deformations of heme side-chains take place only in the β subunits. However, these changes are not observed in the isolated β chain (β₄ homotetramer) and, therefore, are associated with the α–β inter-subunit interactions. For the communications between α and β subunits in Hb A regarding signals of ligand dissociation, possible routes are proposed on the basis of the time-resolved absorption, CD, MCD (magnetic CD), and RR spectroscopies. Our finding of the movements of heme side-chains would serve as one of the clues to solve the cooperative O₂ binding mechanism of Hb A.     

Appressorium-localized NADPH oxidase B is essential for aggressiveness and pathogenicity in the host-specific,toxin-producing fungus Alternaria alternata Japanese pear pathotype

Black spot disease, Alternaria alternata Japanese pear pathotype, produces the host-specific toxin AK-toxin, an important pathogenicity factor. Previously, we have found that hydrogen peroxide is produced in the hyphal cell wall at the plant–pathogen interaction site, suggesting that the fungal reactive oxygen species (ROS) generation machinery is important for pathogenicity. In this study, we identified two NADPH oxidase (NoxA and NoxB) genes and produced nox disruption mutants. ΔnoxA and ΔnoxB disruption mutants showed increased hyphal branching and spore production per unit area. Surprisingly, only the ΔnoxB disruption mutant compromised disease symptoms. A fluorescent protein reporter assay revealed that only NoxB localized at the appressoria during pear leaf infection. In contrast, both NoxA and NoxB were highly expressed on the cellulose membrane, and these Nox proteins were also localized at the appressoria. In the ΔnoxB disruption mutant, we could not detect any necrotic lesions caused by AK-toxin. Moreover, the ΔnoxB disruption mutant did not induce papilla formation on pear leaves. Ultrastructural analysis revealed that the ΔnoxB disruption mutant also did not penetrate the cuticle layer. Moreover, ROS generation was not essential for penetration, suggesting that NoxB may have an unknown function in penetration. Taken together, our results suggest that NoxB is essential for aggressiveness and basal pathogenicity in A. alternata.     

Characteristics of Asian black bears stripping bark from coniferous trees

Stripping of conifer tree bark by Asian black bears (Ursus thibetanus) has been observed in parts of Japan. To identify and characterize the bears exhibiting this behavior, we performed a genetic analysis using DNA extracted from the hairs left on damaged trees. We analyzed 219 samples of bear hair collected from damaged trees at 33 sites and 64 tissue samples from captured bears as controls by using ten microsatellite DNA loci, ca. 706 bp of the mitochondrial DNA d-loop region, and the amelogenin locus. Sixteen bears were identified; some of them had damaged trees at more than one site. bark-stripping and the captured bears. Spatial autocorrelation analysis for increasing distance class revealed a significantly positive genetic correlation coefficient within 40 km among the bark-stripping bears (P < 0.05). Relatedness among the bark-stripping bears was higher than among the captured bears when the distance between bears was within 25 km. We concluded that bark-stripping behavior is associated with relatedness.     

Soluble Epoxide Hydrolase as an Anti-inflammatory Target of the Thrombolytic Stroke Drug SMTP-7

Although ischemic stroke is a major cause of death and disability worldwide, only a small fraction of patients benefit from the current thrombolytic therapy due to a risk of cerebral hemorrhage caused by inflammation. Thus, the development of a new strategy to combat inflammation during thrombolysis is an urgent demand. The small molecule thrombolytic SMTP-7 effectively treats ischemic stroke in several animal models with reducing cerebral hemorrhage. Here we revealed that SMTP-7 targeted soluble epoxide hydrolase (sEH) to suppress inflammation. SMTP-7 inhibited both of the two sEH enzyme activities: epoxide hydrolase (which inactivates anti-inflammatory epoxy-fatty acids) and lipid phosphate phosphatase. SMTP-7 suppressed epoxy-fatty acid hydrolysis in HepG2 cells in culture, implicating the sEH inhibition in the anti-inflammatory mechanism. The sEH inhibition by SMTP-7 was independent of its thrombolytic activity. The simultaneous targeting of thrombolysis and sEH by a single molecule is a promising strategy to revolutionize the current stroke therapy.     

Evaluation of Fucosylated Haptoglobin and Mac-2 Binding Protein as Serum Biomarkers to Estimate Liver Fibrosis in Patients with Chronic Hepatitis C

Fucosylated haptoglobin (Fuc-Hpt) and Mac-2 binding protein (Mac-2 bp) are identified as cancer biomarkers, based on the results from a glyco-proteomic analysis. Recently, we reported that these glyco-biomarkers were associated with liver fibrosis and/or ballooning hepatocytes in patients with nonalcoholic fatty liver disease (NAFLD). We evaluated the ability of these glycoproteins to estimate liver fibrosis in 317 patients with chronic hepatitis C. We measured the serum Fuc-Hpt and Mac-2 bp levels using a lectin-antibody ELISA and ELISA, respectively. The serum levels of both Fuc-Hpt and Mac-2 bp increased with the progression of liver fibrosis. The multivariate analysis revealed that Mac-2 bp was an independent factor associated with moderate liver fibrosis (F ≥ 2). In contrast, Fuc-Hpt was an independent factor associated with advanced liver fibrosis (F ≥ 3). In terms of evaluating liver fibrosis, the serum levels of these glycomarkers were correlated with well-known liver fibrosis indexes, such as the aspartate aminotransferase to platelet ratio index (APRI) and Fibrosis-4 (FIB4) index. An assay that combined the APRI or FIB4 index and the Fuc-Hpt or Mac-2 bp levels increased the AUC value for diagnosing hepatic fibrosis. Interestingly, the cumulative incidence of hepatocellular carcinoma (HCC) was significantly higher in the patients with elevated serum levels of Fuc-Hpt and Mac-2 bp. In conclusion, both Fuc-Hpt and Mac-2 bp could be useful glyco-biomarkers of liver fibrosis and predictors of HCC in patients with chronic hepatitis C.     

Cloning and expression of the human 5-HT1B-type receptor gene.

We report the cloning and expression of a novel 5-HT receptor gene from human genomic DNA. This clone, HGCR1, contains an apparently intronless open reading frame of 390 amino acids with the seven hydrophobic regions, typical of G-protein coupled receptors. The deduced amino acid sequence of HGCR1 is 39%, 55% and 87% identical to that for the human 5-HT1A, the human 5-HT1D and the rat 5-HT1B receptor, respectively. [3H]5-HT binding to transfected COS-7 cell membranes yields a pharmacological profile similar to that of 5-HT1B receptor. Thus these findings indicate the presence of 5-HT1B-type receptor in the human.     

Pancreastatin: characterization of biological activity

Pancreastatin (PST) (1-49) was first isolated from the porcine pancreas and can inhibit glucose-induced insulin release. PST (33-49), a PST C-terminal fragment, can also inhibit insulin release. The purpose of this study was to determine the shortest C-terminal biologically active fragment of PST, in terms of inhibition of insulin release from the isolated perfused rat pancreas. Porcine PST (1-49) and C-terminal fragments, PST (33-49), PST (35-49), PST (37-49) and PST (39-49) were synthesized by solid-phase methodology. PST (1-49), PST (33-49) and PST (35-49), at 10 nM, significantly (p less than 0.05) inhibited insulin release from isolated perfused rat pancreas: the first phase was inhibited by 15.6 +/- 2.4, 24.4 +/- 6.5 and 12.5 +/- 1.9% and the second phase, 18.9 +/- 2.7, 25.7 +/- 4.8 and 20.1 +/- 1.9% by PST (1-49), PST (33-49) and PST (35-49), respectively. PST (35-49) shows a dose-dependent inhibition of insulin release. PST (37-49) and PST (39-49) were, however, inactive. Our results indicate that the shortest C-terminal biologically active fragment is PST (35-49). These data further indicate that the C-terminal portion of PST is primarily responsible for the biological activity of PST.     

Phosphorylation of 4EBP by oral leucine administration was suppressed in the skeletal muscle of PGC-1α knockout mice

Leucine is known to increase mTOR-mediated phosphorylation of 4EBP. In this study, leucine was administered to skeletal muscle-PGC-1α knockout mice. We observed attenuated 4EBP phosphorylation in the skeletal muscle, but not in the liver, of the PGC-1α knockout mice. These data suggest that skeletal muscle-PGC-1α is important for leucine-mediated mTOR activation and protein biosynthesis.     

Plasmalogen Augmentation Reverses Striatal Dopamine Loss in MPTP Mice

Plasmalogens are a class of glycerophospholipids shown to play critical roles in membrane structure and function. Decreased plasmalogens are reported in the brain and blood of Parkinson’s disease (PD) patients. The present study investigated the hypothesis that augmenting plasmalogens could protect striatal dopamine neurons that degenerate in response to 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) treatment in mice, a PD model. First, in a pre-treatment experiment male mice were treated for 10 days with the docosahexaenoic acid (DHA)-plasmalogen precursor PPI-1011 (10, 50 and 200 mg/kg). On day 5 mice received MPTP and were killed on day 11. Next, in a post-treatment study, male mice were treated with MPTP and then received daily for 5 days PPI-1011 (5, 10 and 50 mg/kg). MPTP treatment reduced serum plasmalogen levels, striatal contents of dopamine (DA) and its metabolites, serotonin, DA transporter (DAT) and vesicular monoamine transporter 2 (VMAT2). Pre-treatment with PPI-1011 (10 and 50 mg/kg) prevented all MPTP-induced effects. Positive correlations were measured between striatal DA contents and serum plasmalogen levels as well as striatal DAT and VMAT2 specific binding. Post-treatment with PPI-1011 prevented all MPTP-induced effects at 50 mg/kg but not at lower doses. Positive correlations were measured between striatal DA contents and serum plasmalogen levels as well as striatal DAT and VMAT2 specific binding in the post-treatment experiment. PPI-1011 treatment (10 days at 5, 10 and 50 mg/kg) of intact mice left unchanged striatal biogenic amine contents. These data demonstrate that treatment with a plasmalogen precursor is capable of protecting striatal dopamine markers in an animal model of PD.