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.     

Candidate-gene exclusion in a family with inherited non-syndromic dental disorders

Objectives

Amelogenesis imperfecta, dentinogenesis imperfecta, and dentin dysplasia are the most common non-syndromic dental disorders. In this study, we analysed and localised the gene(s) responsible for inherited non-syndromic dental disorders in a Chinese family.

Methods

This study identified and researched non-syndromic dental disorders in a four-generation Chinese family, including four affected individuals whose clinical phenotype was atypical. Linkage analysis with seven polymorphic markers that localise to six different autochromosomes showed that the family was linked through chromosome 4q. All exons and exon–intron boundaries of dentin sialophosphoprotein (DSPP), enamelin (ENAM), and ameloblastin (AMBN), which are located on chromosome 4q, were sequenced in nine of the family members.

Results

Direct DNA sequence analysis revealed the existence of a G to A transversion in exon 4 (g.13081786G > A, c.727G > A, p.Asp243Asn, based on reference sequences NM_014208.3) of the DSPP gene, and this sequence variation correlated exactly with the presence of the disease.

Conclusion

These results indicate that mutation p.Asp243Asn is a highly probable cause of non-syndromic dental disorder in this Chinese family. The presence of symptom heterogeneity is possible, as the clinical classification system is hampered by the lack of close correlation between the subtype and the molecular defect.     

Affinity Precipitation of Active Rho-GEFs Using a GST-tagged Mutant Rho Protein (GST-RhoA(G17A)) from Epithelial Cell Lysates

Proteins of the Rho family of small GTPases are central regulators of the cytoskeleton, and control a large variety of cellular processes, including cell migration, gene expression, cell cycle progression and cell adhesion ¹. Rho proteins are molecular switches that are active in GTP-bound and inactive in GDP-bound state. Their activation is mediated by a family of Guanine-nucleotide Exchange Factor (GEF) proteins. Rho-GEFs constitute a large family, with overlapping specificities ². Although a lot of progress has been made in identifying the GEFs activated by specific signals, there are still many questions remaining regarding the pathway-specific regulation of these proteins. The number of Rho-GEFs exceeds 70, and each cell expresses more than one GEF protein. In addition, many of these proteins activate not only Rho, but other members of the family, contributing further to the complexity of the regulatory networks. Importantly, exploring how GEFs are regulated requires a method to follow the active pool of individual GEFs in cells activated by different stimuli. Here we provide a step-by-step protocol for a method used to assess and quantify the available active Rho-specific GEFs using an affinity precipitation assay. This assay was developed a few years ago in the Burridge lab ^3,4 and we have used it in kidney tubular cell lines ^5,6,7. The assay takes advantage of a "nucleotide free" mutant RhoA, with a high affinity for active GEFs. The mutation (G17A) renders the protein unable to bind GDP or GTP and this state mimics the intermediate state that is bound to the GEF. A GST-tagged version of this mutant protein is expressed and purified from E. coli, bound to glutathione sepharose beads and used to precipitate active GEFs from lysates of untreated and stimulated cells. As most GEFs are activated via posttranslational modifications or release from inhibitory bindings, their active state is preserved in cell lysates, and they can be detected by this assay⁸. Captured proteins can be probed for known GEFs by detection with specific antibodies using Western blotting, or analyzed by Mass Spectrometry to identify unknown GEFs activated by certain stimuli.     

家庭功能、父母教养方式及夫妻关系对青少年社会适应性的预测

目的:探讨青少年社会适应性状况以及家庭功能、父母教养方式、父母夫妻关系对社会适应性的预测.方法:采用分层随机取样,抽取452名12-18岁的中学生,用中学生社会适应性量表、家庭功能评定量表、简式父母教养方式问卷及Olson婚姻质量问卷中的两个维度进行调查.结果:①夫妻交流(β=0.098,P＜0.05)、情感温暖(β=0.266,P＜0.001)、沟通(β=-0.240,P＜0.001)对青少年的心理优越感有显著的预测力;②情感温暖( β=0.211,P＜0.001)、沟通(β=-0.177,P＜0.05)对青少年的心理能量有显著的预测力;③情感温暖(β＝0.171,P＜0.01)、问题解决(β=-0.125,P＜0.05)、角色(β=-0.133,P＜0.05)对青少年的人际适应性有显著的预测力;④夫妻交流(β=0.130,P＜0.01)、解决冲突的方式(β＝-0.102,P＜0.05)、过度保护(β=-0.172,P＜0.001)、情感温暖(β=0.167,P＜0.01)、问题解决(β=-0.116,P＜0.05)对青少年的心理弹性有显著的预测力.结论:家庭功能对青少年社会适应性的预测力最大.     

眼科手术等候期患者家属对健康教育需求的调查

目的:了解眼科手术等候期患者家属对健康教育的需求。方法:采用自行设计的调查问卷对100名在患者手术过程中等候的家属进行调查。调查内容包括手术等候中希望获得的相关信息及其需求程度,需要的健康教育方式及每种健康教育的需求程度。结果:1).眼科手术等候中,家属强烈需要的信息有手术效果(76%)、术后饮食指导(60.4%)、手术费用(57.3%)、患者目前病情(54.2%)、术后头位指导(51%)。2).最需要的健康教育方式是与医生护士交谈(66%)。结论:对手术等候期的患者家属应根据需求实施科学有效的健康教育,用恰当的方式及时提供家属想要了解的信息,使他们具有良好的应对能力和心理承受能力,在照顾患者的过程中为患者提供有效的支持系统。     

A structural analysis of the catalytic mechanism of methionine sulfoxide reductase A from Neisseria meningitidis

The methionine sulfoxide reductases (Msrs) are thioredoxin-dependent oxidoreductases that catalyse the reduction of the sulfoxide function of the oxidized methionine residues. These enzymes have been shown to regulate the life span of a wide range of microbial and animal species and to play the role of physiological virulence determinant of some bacterial pathogens. Two structurally unrelated classes of Msrs exist, MsrA and MsrB, with opposite stereoselectivity towards the R and S isomers of the sulfoxide function, respectively. Both Msrs share a similar three-step chemical mechanism including (1) the formation of a sulfenic acid intermediate on the catalytic Cys with the concomitant release of the product—methionine, (2) the formation of an intramonomeric disulfide bridge between the catalytic and the regenerating Cys and (3) the reduction of the disulfide bridge by thioredoxin or its homologues. In this study, four structures of the MsrA domain of the PilB protein from Neisseria meningitidis, representative of four catalytic intermediates of the MsrA catalytic cycle, were determined by X-ray crystallography: the free reduced form, the Michaelis-like complex, the sulfenic acid intermediate and the disulfide oxidized forms. They reveal a conserved overall structure up to the formation of the sulfenic acid intermediate, while a large conformational switch is observed in the oxidized form. The results are discussed in relation to those proposed from enzymatic, NMR and theoretical chemistry studies. In particular, the substrate specificity and binding, the catalytic scenario of the reductase step and the relevance and role of the large conformational change observed in the oxidized form are discussed.     

A comparison of BRCT domains involved in nonhomologous end-joining: introducing the solution structure of the BRCT domain of polymerase lambda

Three of the four family X polymerases, DNA polymerase lambda, DNA polymerase mu, and TdT, have been associated with repair of double-strand DNA breaks by nonhomologous end-joining. Their involvement in this DNA repair process requires an N-terminal BRCT domain that mediates interaction with other protein factors required for recognition and binding of broken DNA ends. Here we present the NMR solution structure of the BRCT domain of DNA polymerase lambda, completing the structural portrait for this family of enzymes. Analysis of the overall fold of the polymerase lambda BRCT domain reveals structural similarity to the BRCT domains of polymerase mu and TdT, yet highlights some key sequence and structural differences that may account for important differences in the biological activities of these enzymes and their roles in nonhomologous end-joining. Mutagenesis studies indicate that the conserved Arg57 residue of Pol lambda plays a more critical role for binding to the XRCC4-Ligase IV complex than its structural homolog in Pol mu, Arg43. In contrast, the hydrophobic Leu60 residue of Pol lambda contributes less significantly to binding than the structurally homologous Phe46 residue of Pol mu. A third leucine residue involved in the binding and activity of Pol mu, is nonconservatively replaced by a glutamine in Pol lambda (Gln64) and, based on binding and activity data, is apparently unimportant for Pol lambda interactions with the NHEJ complex. In conclusion, both the structure of the Pol lambda BRCT domain and its mode of interaction with the other components of the NHEJ complex significantly differ from the two previously studied homologs, Pol mu and TdT.     

Role of human RAD51 recombinase in the cycle checkpoint and survival of a cell

The RAD 51 protein, a eukaryotic homologue of Escherichia coli RecA, plays a significant role in both mitotic and meiotic homologous recombination. Here, we demonstrate that short-term silencing of the Rad51 gene by specific small interfering RNA (siRNA) that inhibits cell proliferation and reduces the viability of most cells. Cells with suppressed expression of Rad51 gene have altered cell cycles and accumulate in the S and G₂ phases. Our findings show that the disruption of homologous recombination leads to cell death. However, some cells, e.g., MCF-7 cells, are insensitive to the suppression of Rad51 gene expression.     

Japan’s Hidden Youths: Mainstreaming the Emotionally Distressed in Japan

One of the most talked-about social issues in Japan in recent years has been the problem of the nation's purportedly one million "hidden" youths, known as hikikomori (literally, "the withdrawn"). Most observers agree that the category of hikikomori encompasses a wide range of problems and provocations. The fact that these various dilemmas lead to the shared outcome of shutting oneself away at home is the point of departure here. The article explores the spheres of mental health care, education and family, focusing on the reluctance to highlight underlying psychological dimensions of hikikomori and the desire on the part of schools and families to "mainstream" Japanese children, accommodating as many as possible within standardized public education. Hikikomori can perhaps be seen as a manifestation of Japanese democracy, in which the good society is imagined as cohesive, protective and secure, rather than one in which the individual can freely exercise the right to be different. Schools, families and the sphere of mental health care have focused on producing social inclusion but have discouraged citizens from being labeled as "different" -- even when such a distinction might help them. The dearth of facilities and discourse for caring for the mentally ill or learning disabled is, in many respects, the darker side of Japan's successes. Those who cannot adjust are cared for through the institutions of families, companies and various other spheres that offer spaces to rest and to temporarily "drop out"; however, the expectation is that rest will eventually lead to a re-entry into mainstream society. Often the psychological problem or disability that led to the problem goes unnamed and untreated (hikikomori, psychiatry, special education, youth, family, Japan).