An arginyl residue in rice UDP-arabinopyranose mutase is required for catalytic activity and autoglycosylation

Plants use UDP-arabinofuranose (UDP-Araf) to donate Araf residues in the biosynthesis of Araf-containing complex carbohydrates. UDP-Araf itself is formed from UDP-arabinopyranose (UDP-Arap) by UDP-arabinopyranose mutase (UAM). However, the mechanism by which this enzyme catalyzes the interconversion of UDP-Arap and UDP-Araf has not been determined. To gain insight into this reaction, functionally recombinant rUAMs were reacted with UDP-Glc or UDP-Araf. The glycosylated recombinant UAMs were fragmented with trypsin, and the glycopeptides formed were then identified and sequenced by LC-MS/MS. The results of these experiments, together with site-directed mutagenesis studies, suggest that in functional UAMs an arginyl residue is reversibly glycosylated with a single glycosyl residue, and that this residue is required for mutase activity. We also provide evidence that a DXD motif is required for catalytic activity.     

Highly Pathogenic H5N1 Avian Influenza Virus Induces Extracellular Ca2+ Influx,Leading to Apoptosis in Avian Cells

In this study, we show that the highly pathogenic H5N1 avian influenza virus (AIV) (A/crow/Kyoto/53/04 and A/chicken/Egypt/CL6/07) induced apoptosis in duck embryonic fibroblasts (DEF). In contrast, apoptosis was reduced among cells infected with low-pathogenic AIVs (A/duck/HK/342/78 [H5N2], A/duck/HK/820/80 [H5N3], A/wigeon/Osaka/1/01 [H7N7], and A/turkey/Wisconsin/1/66 [H9N2]). Thus, we investigated the molecular mechanisms of apoptosis induced by H5N1-AIV infection. Caspase-dependent and -independent pathways contributed to the cytopathic effects. We further showed that, in the induction of apoptosis, the hemagglutinin of H5N1-AIV played a major role and its cleavage sequence was not critical. We also observed outer membrane permeabilization and loss of the transmembrane potential of the mitochondria of infected DEF, indicating that mitochondrial dysfunction was caused by the H5N1-AIV infection. We then analyzed Ca²⁺ dynamics in the infected cells and demonstrated an increase in the concentration of Ca²⁺ in the cytosol ([Ca²⁺]_i) and mitochondria ([Ca²⁺]_m) after H5N1-AIV infection. Regardless, gene expression important for regulating Ca²⁺ efflux from the endoplasmic reticulum did not significantly change after H5N1-AIV infection. These results suggest that extracellular Ca²⁺ may enter H5N1-AIV-infected cells. Indeed, EGTA, which chelates extracellular free Ca²⁺, significantly reduced the [Ca²⁺]_i, [Ca²⁺]_m, and apoptosis induced by H5N1-AIV infection. In conclusion, we identified a novel mechanism for influenza A virus-mediated cell death, which involved the acceleration of extracellular Ca²⁺ influx, leading to mitochondrial dysfunction and apoptosis. These findings may be useful for understanding the pathogenesis of H5N1-AIV in avian species as well as the impact of Ca²⁺ homeostasis on influenza A virus infection.Avian influenza viruses (AIVs) are classified as highly or low-pathogenic AIVs (HPAIVs or LPAIVs, respectively) based on their pathogenicity in chickens (1). HPAIVs cause systemic infections and high mortality in chickens (28), whereas poultry are asymptomatic or develop mild respiratory problems and/or intestinal illness after LPAIV infection (49). Hemagglutinin (HA) cleavability is a critical determinant of AIV pathogenicity in avian species (61). Other determinants, such as nonstructural (NS) protein and neuraminidase (NA) protein, reportedly regulate the virulence of AIVs (9, 29, 44). However, waterfowl, known as the natural host for AIVs, do not usually have any symptoms during an HPAIV infection (21), whereas they show neurologic symptoms and death after infection with some of the recently emerged HPAIVs, such as the Asian H5N1 virus (11, 46, 62). Thus, the entire mechanism responsible for the pathogenicity of the AIVs is not yet known. Unknown cellular and viral factors probably underlie the pathogenesis of HPAIVs in avian species, especially waterfowl.The alveolar epithelial cells (66) or vascular endothelial cells (32) of human patients and chickens infected by H5N1-AIV show apoptosis. Other reports suggest that apoptosis of these cells is essential for the development of acute lung injury in mice and acute respiratory distress syndrome in humans (39), which is often observed in H5N1-AIV-infected patients. Therefore, it is necessary to evaluate whether apoptosis is critical for the pathogenesis of H5N1-AIV in vivo and to understand the molecular mechanisms of the apoptotic cell death induced by H5N1-AIV infection.Ca²⁺ is a key regulator of cell survival, and the breakdown of Ca²⁺ homeostasis, due to sustained elevations in Ca²⁺ inside cells, triggers programmed cell death involving apoptosis (24). Indeed, disruption of Ca²⁺ homeostasis plays a key role in apoptosis during the pathogenic process of several types of viral infections, including those with human immunodeficiency virus (HIV), hepatitis C virus, and human T-cell leukemia virus type 1 (3, 4, 31, 57). In addition, the HIV gp120 envelope protein induces neuronal cell death through Ca²⁺ dysregulation, even in the absence of viral particles (25).In this study, we used duck embryonic fibroblasts (DEF) to elucidate the molecular mechanisms of the apoptotic cell death induced by H5N1-AIV. We show here that H5N1-AIV infection triggered extracellular Ca²⁺ influx and that this alteration in the concentration of Ca²⁺ inside the cells subsequently induced mitochondrial dysfunction and led to apoptotic cell death. In addition, we demonstrate that H5N1-HA was a critical viral factor for inducing apoptosis.     

Discovery of 6-benzyloxyquinolines as c-Met selective kinase inhibitors

A novel quinoline derivative that selectively inhibits c-Met kinase was identified. The molecular design is based on a result of the analysis of a PF-2341066 (1)/c-Met cocrystal structure (PDB code: 2wgj). The kinase selectivity of the derivatives is discussed from the view point of the sequence homology of the kinases, the key interactions found in X-ray cocrystal structures, and the structure–activity relationship (SAR) obtained in this work.     

Structural characteristics of green tea catechins for formation of protein carbonyl in human serum albumin

Catechins are polyphenolic antioxidants found in green tea leaves. Recent studies have reported that various polyphenolic compounds, including catechins, cause protein carbonyl formation in proteins via their pro-oxidant actions. In this study, we evaluate the formation of protein carbonyl in human serum albumin (HSA) by tea catechins and investigate the relationship between catechin chemical structure and its pro-oxidant property. To assess the formation of protein carbonyl in HSA, HSA was incubated with four individual catechins under physiological conditions to generate biotin-LC-hydrazide labeled protein carbonyls. Comparison of catechins using Western blotting revealed that the formation of protein carbonyl in HSA was higher for pyrogallol-type catechins than the corresponding catechol-type catechins. In addition, the formation of protein carbonyl was also found to be higher for the catechins having a galloyl group than the corresponding catechins lacking a galloyl group. The importance of the pyrogallol structural motif in the B-ring and the galloyl group was confirmed using methylated catechins and phenolic acids. These results indicate that the most important structural element contributing to the formation of protein carbonyl in HSA by tea catechins is the pyrogallol structural motif in the B-ring, followed by the galloyl group. The oxidation stability and binding affinity of tea catechins with proteins are responsible for the formation of protein carbonyl, and consequently the difference in these properties of each catechin may contribute to the magnitude of their biological activities.     

Spatial variation of phosphorus fractions in bottom sediments and the potential contributions to eutrophication in shallow lakes

Spatial variation of phosphorus fractions in bottom sediment, pore water and overlying water in three shallow eutrophic lakes, Nishiura, Kitaura and Sotonasakaura, Japan, and the contributions of the fractional P to mobilization of phosphorus from sediment were examined in this study. The vertical distributions of dissolved inorganic phosphorus (DIP) concentrations in overlying and pore water differed with lake and sampling site. In particular, DIP was high in pore water in the surface layer of the sediment for the middle to downlake areas of Lake Kitaura. DIP release flux calculated from a gradient of the concentrations at the sediment–water interface was high compared with other sites. The distribution of fractional P content in sediments was highly variable. The citrate–dithionite–bicarbonate–non-reactive phosphorus (CDB–NRP) fraction, in particular, differed greatly among the three lakes. According to correlation in the ratios between CDB–NRP and loss on ignition, sediments of these lakes were classified in three clusters. The CDB–NRP fraction was suggested to play a role in DIP release from sediment. The possibility of nitrate concentration playing a role in the control of DIP release was considered.     

Cystathionine γ-Lyase-deficient Mice Require Dietary Cysteine to Protect against Acute Lethal Myopathy and Oxidative Injury

Cysteine is considered a nonessential amino acid in mammals as it is synthesized from methionine via trans-sulfuration. However, premature infants or patients with hepatic failure may require dietary cysteine due to a lack of cystathionine γ-lyase (CTH), a key trans-sulfuration enzyme. Here, we generated CTH-deficient (Cth^−/−) mice as an animal model of cystathioninemia/cystathioninuria. Cth^−/− mice developed normally in general but displayed hypercystathioninemia/hyperhomocysteinemia though not hypermethioninemia. When fed a low cyst(e)ine diet, Cth^−/− mice showed acute skeletal muscle atrophy (myopathy) accompanied by enhanced gene expression of asparagine synthetase and reduced contents of glutathione in livers and skeletal muscles, and intracellular accumulation of LC3 and p62 in skeletal myofibers; they finally died of severe paralysis of the extremities. Cth^−/− hepatocytes required cystine in a culture medium and showed greater sensitivity to oxidative stress. Cth^−/− mice exhibited systemic vulnerability to oxidative injury, which became more prominent when they were fed the low cyst(e)ine diet. These results reveal novel roles of trans-sulfuration previously unrecognized in mice lacking another trans-sulfuration enzyme cystathionine β-synthase (Cbs^−/−). Because Cbs^−/− mice display hyperhomocysteinemia and hypermethioninemia, our results raise questions against the homocysteine-based etiology of CBS deficiency and the current newborn screening for homocysteinemia using Guthrie''s method, which detects hypermethioninemia.     

Deficiency of Chemokine Receptor CCR1 Causes Osteopenia Due to Impaired Functions of Osteoclasts and Osteoblasts

Chemokines are characterized by the homing activity of leukocytes to targeted inflammation sites. Recent research indicates that chemokines play more divergent roles in various phases of pathogenesis as well as immune reactions. The chemokine receptor, CCR1, and its ligands are thought to be involved in inflammatory bone destruction, but their physiological roles in the bone metabolism in vivo have not yet been elucidated. In the present study, we investigated the roles of CCR1 in bone metabolism using CCR1-deficient mice. Ccr1^−/− mice have fewer and thinner trabecular bones and low mineral bone density in cancellous bones. The lack of CCR1 affects the differentiation and function of osteoblasts. Runx2, Atf4, Osteopontin, and Osteonectin were significantly up-regulated in Ccr1^−/− mice despite sustained expression of Osterix and reduced expression of Osteocalcin, suggesting a lower potential for differentiation into mature osteoblasts. In addition, mineralized nodule formation was markedly disrupted in cultured osteoblastic cells isolated from Ccr1^−/− mice. Osteoclastogenesis induced from cultured Ccr1^−/− bone marrow cells yielded fewer and smaller osteoclasts due to the abrogated cell-fusion. Ccr1^−/− osteoclasts exerted no osteolytic activity concomitant with reduced expressions of Rank and its downstream targets, implying that the defective osteoclastogenesis is involved in the bone phenotype in Ccr1^−/− mice. The co-culture of wild-type osteoclast precursors with Ccr1^−/− osteoblasts failed to facilitate osteoclastogenesis. This finding is most likely due to a reduction in Rankl expression. These observations suggest that the axis of CCR1 and its ligands are likely to be involved in cross-talk between osteoclasts and osteoblasts by modulating the RANK-RANKL-mediated interaction.     

The Critical Role of Nitric Oxide Signaling,via Protein S-Guanylation and Nitrated Cyclic GMP,in the Antioxidant Adaptive Response

A nitrated guanine nucleotide, 8-nitroguanosine 3′,5′-cyclic monophosphate (8-nitro-cGMP), is formed via nitric oxide (NO) and causes protein S-guanylation. However, intracellular 8-nitro-cGMP levels and mechanisms of formation of 8-nitro-cGMP and S-guanylation are yet to be identified. In this study, we precisely quantified NO-dependent formation of 8-nitro-cGMP in C6 glioma cells via liquid chromatography-tandem mass spectrometry. Treatment of cells with S-nitroso-N-acetylpenicillamine led to a rapid, transient increase in cGMP, after which 8-nitro-cGMP increased linearly up to a peak value comparable with that of cGMP at 24 h and declined thereafter. Markedly high levels (>40 μm) of 8-nitro-cGMP were also evident in C6 cells that had been stimulated to express inducible NO synthase with excessive NO production. The amount of 8-nitro-cGMP generated was comparable with or much higher than that of cGMP, whose production profile slightly preceded 8-nitro-cGMP formation in the activated inducible NO synthase-expressing cells. These unexpectedly large amounts of 8-nitro-cGMP suggest that GTP (a substrate of cGMP biosynthesis), rather than cGMP per se, may undergo guanine nitration. Also, 8-nitro-cGMP caused S-guanylation of KEAP1 in cells, which led to Nrf2 activation and subsequent induction of antioxidant enzymes, including heme oxygenase-1; thus, 8-nitro-cGMP protected cells against cytotoxic effects of hydrogen peroxide. Proteomic analysis for endogenously modified KEAP1 with matrix-assisted laser desorption/ionization time-of-flight-tandem mass spectrometry revealed that 8-nitro-cGMP S-guanylated the Cys⁴³⁴ of KEAP1. The present report is therefore the first substantial corroboration of the biological significance of cellular 8-nitro-cGMP formation and potential roles of 8-nitro-cGMP in the Nrf2-dependent antioxidant response.     

Porphyromonas gingivalis Peptidoglycans Induce Excessive Activation of the Innate Immune System in Silkworm Larvae

Porphyromonas gingivalis, a pathogen that causes inflammation in human periodontal tissue, killed silkworm (Bombyx mori, Lepidoptera) larvae when injected into the blood (hemolymph). Silkworm lethality was not rescued by antibiotic treatment, and heat-killed bacteria were also lethal. Heat-killed bacteria of mutant P. gingivalis strains lacking virulence factors also killed silkworms. Silkworms died after injection of peptidoglycans purified from P. gingivalis (pPG), and pPG toxicity was blocked by treatment with mutanolysin, a peptidoglycan-degrading enzyme. pPG induced silkworm hemolymph melanization at the same dose as that required to kill the animal. pPG injection increased caspase activity in silkworm tissues. pPG-induced silkworm death was delayed by injecting melanization-inhibiting reagents (a serine protease inhibitor and 1-phenyl-2-thiourea), antioxidants (N-acetyl-l-cysteine, glutathione, and catalase), and a caspase inhibitor (Ac-DEVD-CHO). Thus, pPG induces excessive activation of the innate immune response, which leads to the generation of reactive oxygen species and apoptotic cell death in the host tissue.