A Novel Prolyl Hydroxylase Inhibitor Protects Against Cell Death After Hypoxia

Hypoxia-inducible factor 1 (HIF-1) is regulated by the oxygen-dependent hydroxylation of proline residues by prolyl hydroxylases (PHDs). We recently developed a novel PHD inhibitor, TM6008, that suppresses the activity of PHDs, inducing continuous HIF-1α activation. In this study, we investigated how TM6008 affects cell survival after hypoxic conditions capable of inducing HIF-1α expression and how TM6008 regulates PHDs and genes downstream of HIF-1α. After SHSY-5Y cells had been subjected to hypoxia, TM6008 was added to the cell culture medium under normoxic conditions. Apoptotic cell death was significantly augmented just after the hypoxic conditions, compared with cell death under normoxic conditions. Notably, when TM6008 was added to the media after the cells had been subjected to hypoxia, the expression level of HIF-1α increased and the number of cell deaths decreased, compared with the results for cells cultured in media without TM6008 after hypoxia, during the 7-day incubation period under normoxic conditions. Moreover, the protein expression levels of heme oxygenase 1, erythropoietin, and glucose transporter-3, which were genes downstream of HIF-1α, were elevated in media to which TM6008 had been added, compared with media without TM6008, during the 7-day incubation period under normoxic conditions. However, the protein expression levels of PHD2 and p53 which suppressed cell proliferation were suppressed in the media to which TM6008 had been added. Thus, TM6008, which suppresses the protein expressions of PHD2 and p53, might play an important role in cell survival after hypoxic conditions, with possible applications as a new compound for treatment after ischemic stroke.     

Molecular cloning and biochemical characterization of two novel Neu3 sialidases, neu3a and neu3b, from medaka (Oryzias latipes)

Mammalian Neu3 sialidases are involved in various biological processes, such as cell death and differentiation, through desialylation of gangliosides. The enzymatic profile of Neu3 seems to be highly conserved from birds to mammals. In fish, the functional properties of Neu3 sialidase are not clearly understood, with the partial exception of the zebrafish form. To cast further light on the molecular evolution of Neu3 sialidase, we identified the encoding genes in the medaka Oryzias latipes and investigated the properties of the enzyme. PCR amplification using medaka brain cDNA allowed identification of two novel medaka Neu3 genes, neu3a and neu3b. The YRIP, VGPG motif and Asp-Box, characteristic of consensus motifs of sialidases, were well conserved in the both medaka Neu3 sialidases. When each gene was transfected into HEK293 to allow cell lysates for the use of enzymatic characterization, two Neu3 sialidases showed strict substrate specificity toward gangliosides, similar to mammalian Neu3. The optimal pH values were at pH 4.2 and pH 4.0, respectively, and neu3b in particular showed a broad optimum. Immunofluorescence assays indicated neu3a localization at plasma membranes, while neu3b was found in cytosol. The tissue distribution of two genes was then investigated by estimation of mRNA expression and sialidase activity, both being dominantly expressed in the brain. In neu3a gene-transfected neuroblastoma cells, the enzyme was found to positively regulate retinoic acid-induced differentiation with the elongation of axon length. On the other hand, neu3b did not affect neurite formation. These results and phylogenetic analysis suggested that the medaka neu3a is an evolutionally conserved sialidase with regard to enzymatic properties, whereas neu3b is likely to have originally evolved in medaka.     

Substrate‐shielding and hydrolytic reaction in hydrolases

In general, transferases undergo large structural changes and sequester substrate molecules, to shield them from water. By contrast, hydrolases exhibit only small structural changes, and expose substrate molecules to water. However, some hydrolases deeply bury their substrates within the proteins. To clarify the relationship between substrate‐shielding and enzymatic functions, we investigated 70 representative hydrolase structures, and examined the relative accessible surface areas of their substrates. As compared to the hydrolases employing the single displacement reaction, the hydrolases employing the double displacement reaction bury the substrate within the proteins. The exo hydrolases display significantly more substrate‐shielding from water than the endo hydrolases. It suggests that the substrate‐shielding is related to the chemical reaction mechanism of the hydrolases and the substrate specificity. Proteins 2013; © 2012 Wiley Periodicals, Inc.     

Quantitative and Qualitative Involvement of P3N-PIPO in Overcoming Recessive Resistance against Clover Yellow Vein Virus in Pea Carrying the cyv1 Gene

In pea carrying cyv1, a recessive gene for resistance to Clover yellow vein virus (ClYVV), ClYVV isolate Cl-no30 was restricted to the initially infected cells, whereas isolate 90-1 Br2 overcame this resistance. We mapped the region responsible for breaking of cyv1-mediated resistance by examining infection of cyv1 pea with chimeric viruses constructed from parts of Cl-no30 and 90-1 Br2. The breaking of resistance was attributed to the P3 cistron, which is known to produce two proteins: P3, from the main open reading frame (ORF), and P3N-PIPO, which has the N-terminal part of P3 fused to amino acids encoded by a small open reading frame (ORF) called PIPO in the +2 reading frame. We introduced point mutations that were synonymous with respect to the P3 protein but nonsynonymous with respect to the P3N-PIPO protein, and vice versa, into the chimeric viruses. Infection of plants with these mutant viruses revealed that both P3 and P3N-PIPO were involved in overcoming cyv1-mediated resistance. Moreover, P3N-PIPO quantitatively affected the virulence of Cl-no30 in cyv1 pea. Additional expression in trans of the P3N-PIPO derived from Cl-no30, using White clover mosaic virus as a vector, enabled Cl-no30 to move to systemic leaves in cyv1 pea. Susceptible pea plants infected with chimeric ClYVV possessing the P3 cistron of 90-1 Br2, and which were therefore virulent toward cyv1 pea, accumulated more P3N-PIPO than did those infected with Cl-no30, suggesting that the higher level of P3N-PIPO in infected cells contributed to the breaking of resistance by 90-1 Br2. This is the first report showing that P3N-PIPO is a virulence determinant in plants resistant to a potyvirus.     

Technical note: Quantification of neurocranial shape variation using the shortest paths connecting pairs of anatomical landmarks

Three‐dimensional geometric morphometric techniques have been widely used in quantitative comparisons of craniofacial morphology in humans and nonhuman primates. However, few anatomical landmarks can actually be defined on the neurocranium. In this study, an alternative method is proposed for defining semi‐landmarks on neurocranial surfaces for use in detailed analysis of cranial shape. Specifically, midsagittal, nuchal, and temporal lines were approximated using Bezier curves and equally spaced points along each of the curves were defined as semi‐landmarks. The shortest paths connecting pairs of anatomical landmarks as well as semi‐landmarks were then calculated in order to represent the surface morphology between landmarks using equally spaced points along the paths. To evaluate the efficacy of this method, the previously outlined technique was used in morphological analysis of sexual dimorphism in modern Japanese crania. The study sample comprised 22 specimens that were used to generate 110 anatomical semi‐landmarks, which were used in geometric morphometric analysis. Although variations due to sexual dimorphism in human crania are very small, differences could be identified using the proposed landmark placement, which demonstrated the efficacy of the proposed method. Am J Phys Anthropol 151:658–666, 2013. © 2013 Wiley Periodicals, Inc.     

A paradoxical method for NAD(+)/NADH accumulation on an electrode surface using a hydrophobic ionic liquid

In this communication, we describe a novel and facile method for the immobilization of NAD(+)/NADH on an electrode surface using a hydrophobic ionic liquid, 1-butyl-3-methylimidazolium bis(trifluoromethanesulfonyl)imide ([C4mim][Tf(2)N]). By taking advantage of the insolubility of NAD(+)/NADH in hydrophobic ionic liquids, it is expected that NAD(+)/NADH can be retained on the electrode's surface. Alcohol dehydrogenase (ADH) and NAD(+)/NADH were immobilized with a gelatin hydrogel on an electrode that was modified with an electropolymerized ruthenium complex containing 5-amino-1,10-phenanthroline (pAPRu) as a mediator for NADH oxidation. The (ADH, NAD(+))/pAPRu-immobilized electrode exhibited the electrocatalytic oxidation of ethanol in [C4mim][Tf(2)N]. The obtained catalytic current in [C4mim][Tf(2)N] was comparable to that in buffer solution containing NAD(+). It was confirmed by UV-vis spectroscopy that NAD(+) did not dissolve in the [C4mim][Tf(2)N] and was retained on the electrode's surface. Furthermore, we succeeded in constructing an ethanol/O(2) biofuel cell comprised of an (ADH, NAD(+))/pAPRu anode and a bilirubin oxidase cathode using [C4mim][Tf(2)N] as an electrolyte.     

Biochemical Studies on “Bakanae” Fungus. Part 55 Synthesis of Gibberellins

In order to clarify the structure of ring A of gibberellins, thirteen lactones of cyclohexan series, of which eight were new, were prepared to examine their infrared spectra. So far; the experiment is concerned, γ-lactones show the characteristic absorption band in the rang 1775~1782 cm^?1 in dioxane, while 5-ones in the range 1730/~1762 cm^?1. Since the absorptio band due to lactone carbonyl of gibberellins occurs at the range 1777~1786 cm^?1 in dioxane, the lactone ring of gibberellins seems to be γ.