ASIAN: a website for network inference

We constructed a website for inferring a network by applying the graphical Gaussian model, from a large amount of data, including redundant information. The available tools on the website are based on a system, named ASIAN (Automatic System for Inferring A Network), in combination with the two methods in our previous papers, which were designed to analyze gene expression profiles on a genomic scale. One of the remarkable features of the website is its ability to infer a network, concomitant with hierarchical clustering and the following estimation of cluster boundaries. AVAILABILITY: http://eureka.ims.u-tokyo.ac.jp/asian     

Supercritical CO2 as a reaction medium for synthesis of capsaicin analogues by lipase-catalyzed transacylation of capsaicin

Capsaicin analogues having different acyl moiety were synthesized by lipase-catalyzed transacylation of capsaicin with a corresponding acyl donor in supercritical CO₂ as a reaction medium. Transacylation with methyl tetradecanoate using Novozym 435 as a catalyst gave vanillyl tetradecanamide in a 54% yield at 80 °C and 19 MPa over 72 h. Vanillyl (Z)-9-octadecenamide, olvanil, was synthesized from triolein in a 21% yield over 7 d.     

Expression of flap endonuclease-1 during meiosis in a basidiomycete, Coprinus cinereus

In the basidiomycete Coprinus cinereus (C. cinereus), which shows a highly synchronous meiotic cell cycle, the meiotic prophase I cells demonstrate flap endonuclease-1 activity. To investigate its role during meiosis, we isolated a C. cinereus cDNA homolog of flap endonuclease-1 (CcFEN-1), 1377bp in length with the open reading frame (ORF) encoding a predicted molecular mass of 51 kDa. At amino-acid residues Glu276-Pro345, a specific inserted sequence composed of 70 amino acids rich in polar forms was found to exist, without sequence identity to other eukaryotic FEN-1 or the polar amino acid rich sequences found in C. cinereus PCNA and C. cinereus DNA ligase IV, although the lengths and percentages of polar amino acids were similar. Northern hybridization analysis indicated CcFEN-1 to be expressed not only in the pre-meiotic S phase but also in meiotic prophase I. The roles of CcFEN-1 during meiosis are discussed.     

Quantification of neutral cysteine protease bleomycin hydrolase and its localization in rat tissues

A neutral cysteine protease, bleomycin hydrolase (BH), was found to be present in the range 3.7-131.1 ng per mg of rat tissues by enzyme-lined immunosorbent assay (ELISA). Newborn rat skin contained the highest amount of BH, and relatively high levels of BH were detected in the kidney and liver of 6-week-old male rats. The tissue distribution of BH in female rats was similar to that in male rats. Moreover, BH was detected in the extracts of erythrocytes and leukocyte-rich cells as well as in those of rat hemo-lymphocytic lineage cell lines by Western blotting. The BH level was increased at 6 weeks after birth and then slightly decreased. By immunohistochemistry, BH was localized as granular staining in the distal and proximal tubular cells of the kidney, and it was also detected in hepatocytes of the liver, in the red pulpy region of the spleen and in neurons of the brain. An immunoelectron microscopic study showed that BH-immunoreactivity was essentially located in the cytoplasm and at the outer membrane of the rough endoplasmic reticulum of epithelial cells of the kidney, as well as in that of hepatocytes of the liver. These results suggest that BH may play ubiquitous and unique roles in rat tissues.     

Mitochondrial haplogroup A is a genetic risk factor for atherothrombotic cerebral infarction in Japanese females

Mitochondrion-derived reactive oxygen species possibly play an important role in the pathogenesis of atherosclerosis and atherothrombotic cerebral infarction, because mitochondria in vascular endothelial cells are the major site of superoxide production. In the present study, we surveyed mitochondrial haplogroups associated with atherothrombotic cerebral infarction in 1081 Japanese subjects. Twenty-six mitochondrial single nucleotide polymorphisms of 11 major mitochondrial haplogroups (F, B, A, N9a, M7a, M7b, M7c, G1, G2, D4, and D5) were determined by use of 28-plex PCR and fluorescent beads combined with sequence-specific oligonucleotide probes. Multivariate logistic regression analysis with adjustment for conventional risk factors revealed that mitochondrial haplogroup A was associated with atherothrombotic cerebral infarction in female subjects (P< 0.05). However, no significant association was detected for males. Our study shows that haplogroup A confers an increased risk of atherothrombotic cerebral infarction in Japanese females. Validation of our findings will require additional studies with independent subject panels.     

Phospholipid methylation controls Atg32‐mediated mitophagy and Atg8 recycling

Degradation of mitochondria via selective autophagy, termed mitophagy, contributes to mitochondrial quality and quantity control whose defects have been implicated in oxidative phosphorylation deficiency, aberrant cell differentiation, and neurodegeneration. How mitophagy is regulated in response to cellular physiology remains obscure. Here, we show that mitophagy in yeast is linked to the phospholipid biosynthesis pathway for conversion of phosphatidylethanolamine to phosphatidylcholine by the two methyltransferases Cho2 and Opi3. Under mitophagy‐inducing conditions, cells lacking Opi3 exhibit retardation of Cho2 repression that causes an anomalous increase in glutathione levels, leading to suppression of Atg32, a mitochondria‐anchored protein essential for mitophagy. In addition, loss of Opi3 results in accumulation of phosphatidylmonomethylethanolamine (PMME) and, surprisingly, generation of Atg8–PMME, a mitophagy‐incompetent lipid conjugate of the autophagy‐related ubiquitin‐like modifier. Amelioration of Atg32 expression and attenuation of Atg8–PMME conjugation markedly rescue mitophagy in opi3‐null cells. We propose that proper regulation of phospholipid methylation is crucial for Atg32‐mediated mitophagy.     

Chemokine PARC Gene (SCYA18) Generated by Fusion of Two MIP-1α/LD78α-like Genes

Two loci in the human genome, chromosomes 4q12–q21 and 17q11.2, contain clusters of CXC and CC chemokine subfamily genes, respectively. Since mice appear to contain fewer chemokine genes than humans, numerous gene duplications might have occurred in each locus of the human genome. Here we describe the genomic organization of the human pulmonary and activation-regulated CC chemokine (PARC), also known as DC-CK1 and AMAC-1. Despite high sequence similarity to a CC chemokine macrophage inflammatory protein-1α (MIP-1α)/LD78α, PARC is chemotactic for lymphocytes and not for monocytes and does not share its receptor with MIP-1α. Analyses of the BAC clones containing the humanPARCgene indicated that the gene is located most closely toMIP-1α(HGMW-approved symbolSCYA3) andMIP-1β(HGMW-approved symbolSCYA4) on chromosome 17q11.2. Dot-plot comparison suggested that thePARCgene had been generated by fusion of twoMIP-1α-like genes with deletion and selective usage of exons. Base changes accumulated before and after the fusion might have adapted the gene to a new function. Since there are variably duplicated copies of theMIP-1αgene calledLD78β(HGMW-approved symbolSCYA3L) in the vicinity of theMIP-1αgene, the locus surrounding theMIP-1αgene seems to be a “hot spring” that continuously produces new family genes. This evidence provides a new model, duplication and fusion, of the molecular basis for diversity within a gene family.     

Use of gene networks for identifying and validating drug targets

We propose a new method for identifying and validating drug targets by using gene networks, which are estimated from cDNA microarray gene expression profile data. We created novel gene disruption and drug response microarray gene expression profile data libraries for the purpose of drug target elucidation. We use two types of microarray gene expression profile data for estimating gene networks and then identifying drug targets. The estimated gene networks play an essential role in understanding drug response data and this information is unattainable from clustering methods, which are the standard for gene expression analysis. In the construction of gene networks, we use the Bayesian network model. We use an actual example from analysis of the Saccharomyces cerevisiae gene expression profile data to express a concrete strategy for the application of gene network information to drug discovery.     

Role of hydroperoxide lyase in white-backed planthopper (Sogatella furcifera Horváth)-induced resistance to bacterial blight in rice, Oryza sativa L.

A pre-infestation of the white-backed planthopper (WBPH), Sogatella furcifera Horváth, conferred resistance to bacterial blight caused by Xanthomonas oryzae pv. oryzae ( Xoo ) in rice ( Oryza sativa  L.) under both laboratory and field conditions. The infestation of another planthopper species, the brown planthopper (BPH) Nilaparvata lugens Stål, did not significantly reduce the incidence of bacterial blight symptoms. A large-scale screening using a rice DNA microarray and quantitative RT-PCR revealed that WBPH infestation caused the upregulation of more defence-related genes than did BPH infestation. Hydroperoxide lyase 2 ( OsHPL2 ), an enzyme for producing C₆ volatiles, was upregulated by WBPH infestation, but not by BPH infestation. One C₆ volatile, ( E )-2-hexenal, accumulated in rice after WBPH infestation, but not after BPH infestation. A direct application of ( E )-2-hexenal to a liquid culture of Xoo inhibited the growth of the bacterium. Furthermore, a vapour treatment of rice plants with ( E )-2-hexenal induced resistance to bacterial blight. OsHPL2 -overexpressing transgenic rice plants exhibited increased resistance to bacterial blight. Based on these data, we conclude that OsHPL2 and its derived ( E )-2-hexenal play some role in WBPH-induced resistance in rice.     

The B2 alternatively spliced isoform of nonmuscle myosin II-B lacks actin-activated MgATPase activity and in vitro motility

We report the initial biochemical characterization of an alternatively spliced isoform of nonmuscle heavy meromyosin (HMM) II-B2 and compare it with HMM II-B0, the nonspliced isoform. HMM II-B2 is the HMM derivative of an alternatively spliced isoform of endogenous nonmuscle myosin (NM) II-B, which has 21-amino acids inserted into loop 2, near the actin-binding region. NM II-B2 is expressed in the Purkinje cells of the cerebellum as well as in other neuronal cells [X. Ma, S. Kawamoto, J. Uribe, R.S. Adelstein, Function of the neuron-specific alternatively spliced isoforms of nonmuscle myosin II-B during mouse brain development, Mol. Biol. Cell 15 (2006) 2138-2149]. In contrast to any of the previously described isoforms of NM II (II-A, II-B0, II-B1, II-C0 and II-C1) or to smooth muscle myosin, the actin-activated MgATPase activity of HMM II-B2 is not significantly increased from a low, basal level by phosphorylation of the 20 kDa myosin light chain (MLC-20). Moreover, although HMM II-B2 can bind to actin in the absence of ATP and is released in its presence, it cannot propel actin in the sliding actin filament assay following MLC-20 phosphorylation. Unlike HMM II-B2, the actin-activated MgATPase activity of a chimeric HMM with the 21-amino acid II-B2 sequence inserted into the homologous location in the heavy chain of HMM II-C is increased following MLC-20 phosphorylation. This indicates that the effect of the II-B2 insert is myosin heavy chain specific.