Two AAA-family peroxin genes, pex-1 and pex-6 of the nematode Caenorhabditis elegans(Department of Sciences for Biospheric Coexistence)

博士学位論文要

Type-II 3-oxoacyl-CoA thiolase of the nematode Caenorhabditis elegans is located in peroxisomes, highly expressed during larval stages and induced by clofibrate

We examined the expression and localization of type-II 3-oxoacyl-CoA thiolase in the nematode Caenorhabditis elegans. Type-II thiolase acts on 3- oxoacyl-CoA esters with a methyl group at the α carbon, whereas conventional thiolases do not. Mammalian type-II thiolase, which is also termed sterol carrier protein x (SCPx) or SCP2/3-oxoacyl-CoA thiolase, is located in the peroxisomes and involved in phytanic acid degradation and most probably in bile acid synthesis. The nematode enzyme lacks the SCP2 domain, which carries the peroxisomal-targeting signal, but produces bile acids in a cell-free system. Northern and Western blot analyses demonstrated that C. elegans expressed type-II thiolase throughout its life cycle, especially during the larval stages, and that the expression was significantly enhanced by the addition of clofibrate at 5 mM or more to the culture medium. Whole-mount in situ hybridization and immunostaining of L4 larvae revealed that the enzyme was mainly expressed in intestinal cells, which are multifunctional like many of the cell types in C. elegans. Subcellular fractionation and indirect immunoelectron microscopy of the nematode detected the enzyme in the matrix of peroxisomes. These results indicate the fundamental homology between mammalian SCPx and the nematode enzyme regardless of whether the SCP2 part is fused, suggesting their common physiological roles

The cDNA sequence and expression of the AAA-family peroxin genes pex-1 and pex-6 from the nematode Caenorhabditis elegans

We cloned cDNAs encoding two peroxins, PEX-1 and PEX-6, of the nematode Caenorhabditis elegans. Peroxins are proteins that play essential roles in peroxisome biogenesis and are encoded by pex genes. Among the peroxins, PEX-1 and PEX-6 constitute the subfamily 2 of AAA (ATPases associated with diverse cellular activities) proteins. Each cDNA agreed well with the respective mRNA in size (3.4 kb for pex-1 and 2.3 kb for pex-6) and did not carry any spliced leader sequence. The pex-1 cDNA was composed of 24 exons, which were encoded by a genomic region containing three open reading frames (ORFs), c11h1.4, c11h1.5, and c11h1.6; the predicted ORF c11h1.5 was encompassed in the 15th intron. Although many exon-intron borders in pex-1 were inconsistent with those predicted for c11h1.4 and c11h1.6, those in pex-6 coincided with those for the ORF f39g3.7. The pex-1 and pex-6 genes encoded proteins with 996 and 720 amino acid residues, respectively. Both pex-1 mRNA and pex-6 mRNA were detectable mainly in intestinal cells throughout the life cycle of C. elegans.The erratam is attached for this article

C-Terminus Glycans with Critical Functional Role in the Maturation of Secretory Glycoproteins

The N-glycans of membrane glycoproteins are mainly exposed to the extracellular space. Human tyrosinase is a transmembrane glycoprotein with six or seven bulky N-glycans exposed towards the lumen of subcellular organelles. The central active site region of human tyrosinase is modeled here within less than 2.5 Å accuracy starting from Streptomyces castaneoglobisporus tyrosinase. The model accounts for the last five C-terminus glycosylation sites of which four are occupied and indicates that these cluster in two pairs - one in close vicinity to the active site and the other on the opposite side. We have analyzed and compared the roles of all tyrosinase N-glycans during tyrosinase processing with a special focus on the proximal to the active site N-glycans, s6:N337 and s7:N371, versus s3:N161 and s4:N230 which decorate the opposite side of the domain. To this end, we have constructed mutants of human tyrosinase in which its seven N-glycosylation sites were deleted. Ablation of the s6:N337 and s7:N371 sites arrests the post-translational productive folding process resulting in terminally misfolded mutants subjected to degradation through the mannosidase driven ERAD pathway. In contrast, single mutants of the other five N-glycans located either opposite to the active site or into the N-terminus Cys1 extension of tyrosinase are temperature-sensitive mutants and recover enzymatic activity at the permissive temperature of 31°C. Sites s3 and s4 display selective calreticulin binding properties. The C-terminus sites s7 and s6 are critical for the endoplasmic reticulum retention and intracellular disposal. Results herein suggest that individual N-glycan location is critical for the stability, regional folding control and secretion of human tyrosinase and explains some tyrosinase gene missense mutations associated with oculocutaneous albinism type I

Standard comparison of local mental health care systems in eight European countries

Aims. There is a need of more quantitative standardised data to compare local Mental Health Systems (MHSs) across international jurisdictions. Problems related to terminological variability and commensurability in the evaluation of services hamper like-with-like comparisons and hinder the development of work in this area. This study was aimed to provide standard assessment and comparison of MHS in selected local areas in Europe, contributing to a better understanding of MHS and related allocation of resources at local level and to lessen the scarcity in standard service comparison in Europe. This study is part of the Seventh Framework programme REFINEMENT (Research on Financing Systems' Effect on the Quality of Mental Health Care in Europe) project. Methods. A total of eight study areas from European countries with different systems of care (Austria, England, Finland, France, Italy, Norway, Romania, Spain) were analysed using a standard open-access classification system (Description and Evaluation of Services for Long Term Care in Europe, DESDE-LTC). All publicly funded services universally accessible to adults (>= 18 years) with a psychiatric disorder were coded. Care availability, diversity and capacity were compared across these eight local MHS. Results. The comparison of MHS revealed more community-oriented delivery systems in the areas of England (Hampshire) and Southern European countries (Verona - Italy and Girona - Spain). Community-oriented systems with a higher proportion of hospital care were identified in Austria (Industrieviertel) and Scandinavian countries (Sor-Trondelag in Norway and Helsinki-Uusimaa in Finland), while Loiret (France) was considered as a predominantly hospital-based system. The MHS in Suceava (Romania) was still in transition to community care. Conclusions. There is a significant variation in care availability and capacity across MHS of local areas in Europe. This information is relevant for understanding the process of implementation of community-oriented mental health care in local areas. Standard comparison of care provision in local areas is important for context analysis and policy planning.Peer reviewe

Strains used in this study. Targeting EDEM protects against ER stress and improves development and survival in C. elegans

S1 Table. Strains used in this study.Peer reviewe

edem response to thapsigargin

S4 Fig. edem response to thapsigargin. (A) Quantitative RT-PCR measurements of edem mRNA levels in WT young animals under non stress (NS) and treatment with 5 μM thapsigargin (TG), (n = 3 independent experiments). (B) Percentage of eggs that developed into L4 larvae after 3 days on 10 μM thapsigargin. Each strain was scored in three independent experiments in triplicates. (C) Quantification of WT (EV) or RNAi-treated day one adults carrying Phsp-4::GFP transgene, treated or not with 5 μM thapsigargin. The worms were kept on RNAi plates for one generation before exposure to ER stress treatments. Values represent mean fluorescence/µm2 x 1000. The red bars indicate the average ±SEM. (D) Analysis of xbp-1 spliced/unspliced ratio from young adults of indicated strains. Total RNA was isolated, reverse transcribed and used for PCR analysis of xbp-1 splicing forms.Peer reviewe