Molecular cloning and characterization of novel splicing variants of human decay-accelerating factor

Decay-accelerating factor (DAF) is one of the complement regulatory proteins. Two isoforms of DAF have been identified in humans. In this study, we isolated novel cDNAs encoding five isoforms of DAF from the human lung, which were generated by insertion of new exonic sequences. RT-PCR revealed that all isoforms were expressed in almost all tissues tested, although the expression patterns and levels differed among the tissues. Transfection of isoform vDAF1, 2, and 3 cDNAs into CHO cells showed that these molecules are soluble forms secreted after glycosylation. Isoform vDAF4 and vDAF5 cDNAs included a part of and the entire intron 7 sequence, respectively, and the transfection of vDAF4 cDNA produced a large, glycosylated, membrane-bound form. These results suggest that more than seven isoforms of human DAF are involved in the regulation of complement activation under physiological conditions through their specific structures and localization.     

A novel NADP+-dependent L-1-amino-2-propanol dehydrogenase from Rhodococcus erythropolis MAK154: a promising enzyme for the production of double chiral aminoalcohols

AIM: A novel NADP(+)-dependent L-1-amino-2-propanol dehydrogenase was isolated from Rhodococcus erythropolis MAK154, and characterized. METHODS AND RESULTS: The enzyme was inducibly produced on cultivation with aminoalcohols such as 1-amino-2-propanol, 1-amino-2-butanol and 2-aminocyclohexanol. The enzyme catalyses the NADP(+)-dependent oxidation of several aminoalcohols, and also the NADPH-dependent asymmetric reduction of an aminoketone compound to a double chiral aminoalcohol, d-pseudoephedrine. Amino acid sequence analysis showed that the enzyme might belong to the short-chain dehydrogenase/reductase family. CONCLUSIONS: NADP(+)-dependent L-1-amino-2-propanol dehydrogenase isolated from R. erythropolis MAK154 reversibly catalysed dehydrogenation of aminoalcohols, and exhibited a unique sterospecifity for the reduction reaction. SIGNIFICANCE AND IMPACT OF THE STUDY: The enzyme is a promising catalyst for the production of double chiral compound, d-pseudoephedrine, from prochiral substrate.     

No association between complement factor H gene polymorphism and exudative age-related macular degeneration in Japanese

Age-related macular degeneration (ARMD) is the leading cause of blindness in the elderly population not only Western but also Asian industrial countries. In Caucasian, a polymorphism of the complement factor H gene (CFH), the C allele of rs1061170 (Y402H), was established as the first strong genetic factor for excursively exudative type of ARMD. In this study, we performed an extensive sequencing of the 22 exons in the CFH gene by recruiting 146 exudative ARMD patients and 105 normal controls of Japanese origin and identified 61 polymorphisms. We found that the frequency of the C allele of rs1061170 (Y402H) is much lower (0.04) in Japanese controls than in Caucasians (0.45). No case disease susceptibility to exudative ARMD was noted for rs1061170 (Y402H) (χ ² = 3.19, P _corr = 0.423), or other 12 single nucleotide polymorphisms (SNPs) whose frequency is greater than 0.05. When haplotypes were inferred for 13 SNPs (these 12 SNPs with a frequency greater than 0.05 and rs1061170), three haplotypes whose pattern was similar to those in Caucasians were identified but with substantial difference in frequency. Again we failed to identify genetic association between Japanese exudative ARMD and any of the haplotypes including the J1 haplotype which was shown to be susceptible to ARMD in Caucasians (χ ² = 3.92, P _corr = 0.157). CFH does not appear to be a primary hereditary contributor to ARMD in Japanese. The absence of CFH contribution to ARMD in Japanese may correlate with the findings in ethnic differences of ARMD phenotypes.Electronic Supplementary Material Supplementary material is available for this article at and is accessible for authorized users.This work was accomplished by equal contribution of two groups organized by the last two authors.     

Large scale international replication and meta-analysis study confirms association of the 15q14 locus with myopia. The CREAM consortium

Myopia is a complex genetic disorder and a common cause of visual impairment among working age adults. Genome-wide association studies have identified susceptibility loci on chromosomes 15q14 and 15q25 in Caucasian populations of European ancestry. Here, we present a confirmation and meta-analysis study in which we assessed whether these two loci are also associated with myopia in other populations. The study population comprised 31 cohorts from the Consortium of Refractive Error and Myopia (CREAM) representing 4 different continents with 55,177 individuals; 42,845 Caucasians and 12,332 Asians. We performed a meta-analysis of 14 single nucleotide polymorphisms (SNPs) on 15q14 and 5 SNPs on 15q25 using linear regression analysis with spherical equivalent as a quantitative outcome, adjusted for age and sex. We calculated the odds ratio (OR) of myopia versus hyperopia for carriers of the top-SNP alleles using a fixed effects meta-analysis. At locus 15q14, all SNPs were significantly replicated, with the lowest P value 3.87?×?10(-12) for SNP rs634990 in Caucasians, and 9.65?×?10(-4) for rs8032019 in Asians. The overall meta-analysis provided P value 9.20?×?10(-23) for the top SNP rs634990. The risk of myopia versus hyperopia was OR 1.88 (95?% CI 1.64, 2.16, P?相似文献   

TRIM50 Protein Regulates Vesicular Trafficking for Acid Secretion in Gastric Parietal Cells

Of the TRIM/RBCC family proteins taking part in a variety of cellular processes, TRIM50 is a stomach-specific member with no defined biological function. Our biochemical data demonstrated that TRIM50 is specifically expressed in gastric parietal cells and is predominantly localized in the tubulovesicular and canalicular membranes. In cultured cells ectopically expressing GFP-TRIM50, confocal microscopic imaging revealed dynamic movement of TRIM50-associated vesicles in a phosphoinositide 3-kinase-dependent manner. A protein overlay assay detected preferential binding of the PRY-SPRY domain from the TRIM50 C-terminal region to phosphatidylinositol species, suggesting that TRIM50 is involved in vesicular dynamics by sensing the phosphorylated state of phosphoinositol lipids. Trim50 knock-out mice retained normal histology in the gastric mucosa but exhibited impaired secretion of gastric acid. In response to histamine, Trim50 knock-out parietal cells generated deranged canaliculi, swollen microvilli lacking actin filaments, and excess multilamellar membrane complexes. Therefore, TRIM50 seems to play an essential role in tubulovesicular dynamics, promoting the formation of sophisticated canaliculi and microvilli during acid secretion in parietal cells.     

Misshapen-like kinase 1 (MINK1) is a novel component of striatin-interacting phosphatase and kinase (STRIPAK) and is required for the completion of cytokinesis

Cytokinesis is initiated by constriction of the cleavage furrow and terminated by abscission of the intercellular bridge that connects two separating daughter cells. The complicated processes of cytokinesis are coordinated by phosphorylation and dephosphorylation mediated by protein kinases and phosphatases. Mammalian Misshapen-like kinase 1 (MINK1) is a member of the germinal center kinases and is known to regulate cytoskeletal organization and oncogene-induced cell senescence. To search for novel regulators of cytokinesis, we performed a screen using a library of siRNAs and found that MINK1 was essential for cytokinesis. Time-lapse analysis revealed that MINK1-depleted cells were able to initiate furrowing but that abscission was disrupted. STRN4 (Zinedin) is a regulatory subunit of protein phosphatase 2A (PP2A) and was recently shown to be a component of a novel protein complex called striatin-interacting phosphatase and kinase (STRIPAK). Mass spectrometry analysis showed that MINK1 was a component of STRIPAK and that MINK1 directly interacted with STRN4. Similar to MINK1 depletion, STRN4-knockdown induced multinucleated cells and inhibited the completion of abscission. In addition, STRN4 reduced MINK1 activity in the presence of catalytic and structural subunits of PP2A. Our study identifies a novel regulatory network of protein kinases and phosphatases that regulate the completion of abscission.     

l-Pantoyl lactone dehydrogenase from Rhodococcus erythropolis: genetic analyses and application to the stereospecific oxidation of l-pantoyl lactone

The 1,2-propanediol (1,2-PD) inducible membrane-bound L-pantoyl lactone (L-PL) dehydrogenase (LPLDH) has been isolated from Rhodococcus erythropolis AKU2103 (Kataoka et al. in Eur J Biochem 204:799, 1992). Based on the N-terminal amino acid sequence of LPLDH and the highly conserved amino acid sequence in homology search results, the LPLDH gene (lpldh) was cloned. The gene consists of 1,179 bases and encodes a protein of 392 amino acid residues. The deduced amino acid sequence showed high similarity to the proteins of the FMN-dependent α-hydroxy acid dehydrogenase/oxidase family. The overexpression vector pKLPLDH containing lpldh with its upstream region (1,940 bp) was constructed and introduced into R. erythropolis AKU2103. The recombinant R. erythropolis AKU2103 harboring pKLPLDH showed six times higher LPLDH activity than the wild-type strain. Conversion of L-PL to ketopantoyl lactone was achieved with 92% or 80% conversion yield when the substrate concentration was 0.768 or 1.15 M, respectively. Stereoinversion of L-PL to D-PL was also carried out by using the combination of recombinant R. erythropolis AKU2103 harboring pKLPLDH and ketopantoic acid-reducing Escherichia coli.     

Establishment of consomic strains derived from A/J and SM/J mice for genetic analysis of complex traits

Consomic strains, in which one chromosome is derived from a donor strain and the other chromosomes are derived from the recipient strain, provide a powerful tool for the dissection of complex genetic traits. In this study we established ten consomic strains (A-2^SM, A-6^SM, A-11^SM, A-12^SM, A-13^SM, A-15^SM, A-17^SM, A-18^SM, A-19^SM, A-Y^SM) using the SM/J strain as the donor and the A/J strain as the recipient; these are the parental strains of a set of SMXA recombinant inbred (RI) strains that we had developed previously. We analyzed body weights and blood lipid levels in the consomic and parental strains. The mean values for each trait showed a continuous range of variation in the consomic strains suggesting that they are controlled by multiple genes. We previously identified suggestive QTLs for body weight on chromosome 6 in SMXA RI strains and (SM/J?×?A/J)F₂ mice. The observation that the A-6^SM consomic strain had a significantly lower mean body weight than the A/J strain supports the presence of this QTL on chromosome 6. Similarly, the higher blood triglyceride level in the A-11^SM strain shows the existence of a previously mapped QTL on chromosome 11, and the A-12^SM strain provides evidence of a QTL for blood total cholesterol level on chromosome 12. These consomic strains, along with the previously developed set of SMXA RI strains from A/J and SM/J mice, offer an invaluable and powerful resource for the analysis of complex genetic traits in mice.     

Discovery of novel 5-alkynyl-4-anilinopyrimidines as potent, orally active dual inhibitors of EGFR and Her-2 tyrosine kinases

5-Alkenyl or 5-alkynyl-4-anilinopyrimidines were prepared and evaluated for in vitro inhibition of EGFR/Her-2 kinase activity and the growth of tumor cell lines (BT474 and N87). Several of these compounds inhibited the growth of BT474 and N87 at concentrations below 200nM. Structure-activity relationship studies revealed a critical role for the 5-alkynyl moieties. The representative compound 19 exhibited significant antitumor potency in a mouse xenograft model.