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Several thermophilic hydrogenotrophic methanogens naturally aggregate in their habitats in association with hydrogen-producing bacteria for efficient transfer of the methane fermentation intermediates to produce methane. However, physiology of aggregation and the identity of aggregation-specific genes remain to be elucidated. Here, we isolated and characterized a hydrogen and formate-utilizing M ethanothermobacter sp. CaT2 that is capable of self-aggregation and utilizing formate. CaT2 produced methane from propionate oxidation in association with a syntrophic propionate-oxidizing bacterium faster than other methanogens, including M ethanothermobacter thermautotrophicus Δ H and M ethanothermobacter thermautotrophicus Z-245. CaT2 also aggregated throughout the culture period and was coated with polysaccharides, which was not found on the Δ H and Z-245 cells. Sugar content (particularly of rhamnose and mannose) was also higher in the CaT2 cells than the Δ H and Z-245 cells. Comparative genomic analysis of CaT2 indicated that four candidate genes, all of which encode glycosyltransferase, were involved in aggregation of CaT2. Transcriptional analysis showed that one glycosyltransferase gene was expressed at relatively high levels under normal growth conditions. The polysaccharide layer on the CaT2 cell surface, which is probably assembled by these glycosyltransferases, may be involved in cell aggregation. [ABSTRACT FROM AUTHOR]

Plants belonging to the Brassicaceae family exhibit species-specific profiles of glucosinolates ( GSLs), a class of defence compounds against pathogens and insects. GSLs also exhibit various human health-promoting properties. Among them, glucoraphanin (aliphatic 4-methylsulphinylbutyl GSL) has attracted the most attention because it hydrolyses to form a potent anticancer compound. Increased interest in developing commercial varieties of Brassicaceae crops with desirable GSL profiles has led to attempts to identify genes that are potentially valuable for controlling GSL biosynthesis. However, little attention has been focused on genes of kale ( Brassica oleracea var. acephala). In this study, we established full-length kale c DNA libraries containing 59 904 clones, which were used to generate an expressed sequence tag ( EST) data set with 119 204 entries. The EST data set clarified genes related to the GSL biosynthesis pathway in kale. We specifically focused on BoMYB29, a homolog of Arabidopsis MYB29/PMG2/HAG3, not only to characterize its function but also to demonstrate its usability as a biological resource. BoMYB29 overexpression in wild-type Arabidopsis enhanced the expression of aliphatic GSL biosynthetic genes and the accumulation of aliphatic GSLs. When expressed in the myb28myb29 mutant, which exhibited no detectable aliphatic GSLs, BoMYB29 restored the expression of biosynthetic genes and aliphatic GSL accumulation. Interestingly, the ratio of methylsulphinyl GSL content, including glucoraphanin, to that of methylthio GSLs was greatly increased, indicating the suitability of BoMYB29 as a regulator for increasing methylsulphinyl GSL content. Our results indicate that these biological resources can facilitate further identification of genes useful for modifications of GSL profiles and accumulation in kale. [ABSTRACT FROM AUTHOR]

Abstract: Kufor-Rakeb syndrome (KRS) was originally described as an autosomal recessive form of early-onset parkinsonism with pyramidal degeneration and dementia. ATP13A2 was identified as the causative gene in KRS. ATP13A2 encodes the ATP13A2 protein, which is a lysosomal type5 P-type ATPase, and ATP13A2 mutations are linked to autosomal recessive familial parkinsonism. Here, we report that normal ATP13A2 localizes in the lysosome, whereas disease-associated variants remain in the endoplasmic reticulum. Cathepsin D activity was decreased in ATP13A2-knockdown cells that displayed lysosome-like bodies characterized by fingerprint-like structures. Furthermore, an atp13a2 mutation in medaka fish resulted in dopaminergic neuronal death, decreased cathepsin D activity, and fingerprint-like structures in the brain. Based on these results, lysosome abnormality is very likely to be the primary cause of KRS/PARK9. [Copyright &y& Elsevier]

Roberts syndrome and SC phocomelia ( RBS/ SC) are genetic autosomal recessive syndromes caused by establishment of cohesion 1 homolog 2 ( ESCO 2) mutation. RBS/ SC appear to have a variety of clinical features, even with the same mutation of the ESCO2 gene. Here, we established and genetically characterized a medaka model of RBS/ SC by reverse genetics. The RBS/ SC model was screened from a mutant medaka library produced by the Targeting Induced Local Lesions in Genomes method. The medaka mutant carrying the homozygous mutation at R80 S in the conserved region of ESCO2 exhibited clinical variety (i.e. developmental arrest with craniofacial and chromosomal abnormalities and embryonic lethality) as characterized in RBS/ SC. Moreover, widespread apoptosis and downregulation of some gene expression, including notch1a, were detected in the R80 S mutant. The R80S mutant is the animal model for RBS/ SC and a valuable resource that provides the opportunity to extend knowledge of ESCO2. Downregulation of some gene expression in the R80 S mutant is an important clue explaining non-correlation between genotype and phenotype in RBS/ SC. [ABSTRACT FROM AUTHOR]

Opalin is a transmembrane protein detected specifically in mammalian oligodendrocytes. Opalin homologs are found only in mammals and not in the genome sequences of other animal classes. We first determined the nucleotide sequences of Opalin orthologs and their flanking regions derived from four prosimians, a group of primitive primates. A global comparison revealed that an evolutionarily conserved region exists in the first intron of Opalin. When the conserved domain was assayed for its enhancer activity in transgenic mice, oligodendrocyte-directed expression was observed. In an oligodendroglial cell line, Oli-neu, the conserved domain showed oligodendrocyte-directed expression. The conserved domain is composed of eight subdomains, some of which contain binding sites for Myt1 and cAMP-response element binding protein (CREB). Deletion analysis and cotransfection experiments revealed that the subdomains have critical roles in Opalin gene expression. Over-expression of Myt1, treatment of the cell with leukemia inhibitory factor (LIF), and cAMP analog (CREB activator) enhanced the expression of endogenous Opalin in Oli-neu cells and activated the oligodendrocyte enhancer. These results suggest that LIF, cAMP signaling cascades and Myt1 play significant roles in the differentiation of oligodendrocytes through their action on the Opalin oligodendrocyte enhancer. [ABSTRACT FROM AUTHOR]

Peroxisomes play an important role in β-oxidation of fatty acids. All peroxisomal matrix proteins are synthesized in the cytosol and post-translationally sorted to the organelle. Two distinct peroxisomal signal targeting sequences (PTSs), the C-terminal PTS1 and the N-terminal PTS2, have been defined. Import of precursor PTS2 proteins into the peroxisomes is accompanied by a proteolytic removal of the N-terminal targeting sequence. Although the PTS1 signal is preserved upon translocation, many PTS1 proteins undergo a highly selective and limited cleavage. Here, we demonstrate that Tysnd1, a previously uncharacterized protein, is responsible both for the removal of the leader peptide from PTS2 proteins and for the specific processing of PTS1 proteins. All of the identified Tysnd1 substrates catalyze peroxisomal β-oxidation. Tysnd1 itself undergoes processing through the removal of the presumably inhibitory N-terminal fragment. Tysnd1 expression is induced by the proliferator-activated receptor α agonist bezafibrate, along with the increase in its substrates. A model is proposed where the Tysnd1-mediated processing of the peroxisomal enzymes promotes their assembly into a supramolecular complex to enhance the rate of β-oxidation. [ABSTRACT FROM AUTHOR]

Background: Recent studies have demonstrated a significant association between mutations in genes involved in the GHR/IGF1 signaling pathway and extension of the lifespan of model organisms. Exon 3 insertion or deletion is one common polymorphism in the growth hormone receptor ( GHR) of humans. The exon 3 deletion allele is reported to have stronger signaling in the GH/GHR pathway, which may correlate to short lifespan. Methods: We investigated the common polymorphic variation in 119 Japanese semisuper centenarians (SSC; older than 105) compared with 104 healthy younger controls via the polymorphism-based polymerase chain reaction method. Results: The frequency of exon 3 deletion variation of GHR in SSC was found to be higher than controls, although this was not significant statistically. Also, the single nucleotide polymorphism genotype frequency and allele frequency exhibited no differences between SSC and controls. Conclusions: These results show that SSC in Japan do not tend to have the allele of GHR, which has a lower signaling capacity. [ABSTRACT FROM AUTHOR]

Determination of the mRNA start site is the first step in identifying the promoter region, which is of key importance for transcriptional regulation of gene expression. The 'oligo-capping' method enabled us to introduce a sequence tag to the first base of an mRNA by replacing the cap structure of the mRNA. Using cDNA libraries made from oligo-capped mRNAs, we could identify the transcriptional start site of an individual mRNA just by sequencing the 5'-end of the cDNA. The fine mapping of transcriptional start sites was performed for 5880 mRNAs in 276 human genes. Contrary to our expectations, the majority of the genes showed a diverse distribution of transcriptional start sites. They were distributed over 61.7 bp with a standard deviation of 19.5. Our finding may reflect the dynamic nature of transcriptional initiation events of human genes in vivo. [ABSTRACT FROM AUTHOR]

Maintenance of long-term potentiation (LTP) requires de novo gene expression. Here we report the direct isolation, using PCR-differential display, of genes whose expression level was altered after induction of long-lasting LTP in the hippocampus of freely moving awake rats. Differential display using 480 primer combinations revealed 17 cDNA bands that showed a reproducible change in expression level. These cDNAs represented at least 10 different genes (termed RM1-10), all of which showed up-regulation at 75 min after LTP induction and a return to basal expression levels within 24 h. Three of these genes were known only from expressed sequence tags (RM1-3), two were known genes whose up-regulation by LTP has not been described (GADD153/CHOP and ler5), and five were known genes whose up-regulation by LTP has already been reported (MAPK phosphatase, NGFI-A/zif268, vesl-1S/homer-1a, Ag2, and krox-20). We characterized the expression profiles of genes in the two former categories with respect to NMDA receptor dependency, tissue specificity, and developmental regulation using northern blotting and semiquantitative RT-PCR. The up-regulation of all five of these genes was NMDA receptor-dependent and correlated with the persistence of LTP, suggesting that these genes may play functional roles in prolonged LTP maintenance. [ABSTRACT FROM AUTHOR]

The phagocyte NADPH oxidase is activated during phagocytosis to produce superoxide, a precursor of microbicidal oxidants. The formation of the active oxidase complex at the membrane requires translocation of the Rac GTPase and two specialized cytosolic proteins that harbor SH3 domains, p67phox and p47phox. Another SH3-domain-containing protein p40 phox, which is constitutively associated with p67phox in phagocytes, also enters the complex upon cell stimulation. Here we describe how we cloned mouse cDNAs encoding p40 phox and its partner in phagocytes, p67phox. Both p40 phox and p67phox comprise several protein-binding modules that are structurally and functionally well conserved between mouse and human, indicating their nature as adaptor proteins. We have also systematically investigated expression of the gene for p40 phox in comparison with those for p67phox and p47phox. Distributions of the mRNAs for the three proteins among tissues are similar, with the most abundant expression in the spleen. The messages are abundant not only in phagocytic cells, but also in B cell lineage. The p40 phox gene, but not the other two, is expressed in some types of cells such as plasma cells and T lymphocytes. Furthermore, in situ hybridization analysis shows that the p40 phox mRNA is distributed in neuronal cells of mouse brain, providing evidence that one of the genes for the specialized oxidase factors is expressed in neurons. These observations raise the possibility that the adaptor protein p40 phox plays a heretofore unsuspected role via interacting with other proteins in the cells that do not express p67phox or p47phox. [ABSTRACT FROM AUTHOR]

The superoxide-generating NADPH oxidase, dormant in resting phagocytes, is activated during phagocytosis following assembly of the membrane-integrated protein cytochrome b558 and cytosolic factors. Among the latter are the three proteins containing Src homology 3 (SH3) domains, p67phox, p47phox and p40 phox. While the first two factors are indispensable for the activity, p40 phox is tightly associated with p67phox in resting cells and is suggested to have some modulatory role. Here we describe a systematic analysis of the interaction between p40 phox and p67phox using the yeast two-hybrid system and in vitro binding assays with recombinant proteins. Both methods unequivocally showed that the minimum requirements for stable interaction are the C-terminal region of p40 phox and the region between the two SH3 domains of p67phox. This interaction is maintained even in the presence of anionic amphiphiles used for the activation of the NADPH oxidase, raising a possibility that it mediates constitutive association of the two factors in both resting and activated cells. The C-terminal region of p40 phox responsible for the interaction contains a characteristic stretch of amino acids designated as the PC motif, that also exists in other signal-transducing proteins from yeast to human. Intensive site-directed mutagenesis to the motif in p40 phox revealed that it plays a critical role in the binding to p67 phox. Thus the PC motif appears to represent a novel module for protein-protein interaction used in a variety of signaling pathways. [ABSTRACT FROM AUTHOR]