Current limitations of SNP data from the public domain for studies of complex disorders: a test for ten candidate genes for obesity and osteoporosis

Background

Public SNP databases are frequently used to choose SNPs for candidate genes in the association and linkage studies of complex disorders. However, their utility for such studies of diseases with ethnic-dependent background has never been evaluated.

Results

To estimate the accuracy and completeness of SNP public databases, we analyzed the allele frequencies of 41 SNPs in 10 candidate genes for obesity and/or osteoporosis in a large American-Caucasian sample (1,873 individuals from 405 nuclear families) by PCR-invader assay. We compared our results with those from the databases and other published studies. Of the 41 SNPs, 8 were monomorphic in our sample. Twelve were reported for the first time for Caucasians and the other 29 SNPs in our sample essentially confirmed the respective allele frequencies for Caucasians in the databases and previous studies. The comparison of our data with other ethnic groups showed significant differentiation between the three major world ethnic groups at some SNPs (Caucasians and Africans differed at 3 of the 18 shared SNPs, and Caucasians and Asians differed at 13 of the 22 shared SNPs). This genetic differentiation may have an important implication for studying the well-known ethnic differences in the prevalence of obesity and osteoporosis, and complex disorders in general.

Conclusion

A comparative analysis of the SNP data of the candidate genes obtained in the present study, as well as those retrieved from the public domain, suggests that the databases may currently have serious limitations for studying complex disorders with an ethnic-dependent background due to the incomplete and uneven representation of the candidate SNPs in the databases for the major ethnic groups. This conclusion attests to the imperative necessity of large-scale and accurate characterization of these SNPs in different ethnic groups.     

Platelet interactions in thrombosis

Patho/physiological platelet aggregate (thrombus) formation is initiated by engagement of platelet surface receptors, glycoprotein (GP)Ib-IX-V and GPVI that bind von Willebrand factor or collagen. Although beneficial in response to vascular injury by preventing blood loss (haemostasis), platelet aggregation in a sclerotic coronary artery or other diseased blood vessel (thrombosis) can cause thrombotic diseases like heart attack and stroke. At the molecular level, ligand interactions with GPIb-IX-V or GPVI trigger signalling responses, including elevation of cytosolic Ca2+, dissociation of calmodulin from their cytoplasmic domains, cytoskeletal actin-filament rearrangements, activation of src-family kinases or PI 3-kinase, and 'inside-out' activation of the integrin, alphaIIbbeta3 (GPIIb-llla), that binds von Willebrand factor or fibrinogen and mediates platelet aggregation. Furthermore, emerging evidence supports a topographical co-association of these receptors of the leucine-rich repeat family (GPIb-IX-V) and immunoglobulin superfamily (GPVI) in an adhesive cluster or 'adhesosome'. This arrangement may underlie common mechanisms of initiating thrombus formation in haemostasis or thrombotic disease.     

Double-stranded DNA bacteriophage prohead protease is homologous to herpesvirus protease

Double-stranded DNA bacteriophages and herpesviruses assemble their heads in a similar fashion; a pre-formed precursor called a prohead or procapsid undergoes a conformational transition to give rise to a mature head or capsid. A virus-encoded prohead or procapsid protease is often required in this maturation process. Through computational analysis, we infer homology between bacteriophage prohead proteases (MEROPS families U9 and U35) and herpesvirus protease (MEROPS family S21), and unify them into a procapsid protease superfamily. We also extend this superfamily to include an uncharacterized cluster of orthologs (COG3566) and many other phage or bacteria-encoded hypothetical proteins. On the basis of this homology and the herpesvirus protease structure and catalytic mechanism, we predict that bacteriophage prohead proteases adopt the herpesvirus protease fold and exploit a conserved Ser and His residue pair in catalysis. Our study provides further support for the proposed evolutionary link between dsDNA bacteriophages and herpesviruses.     

Design, synthesis, and biological evaluation of novel 7-azaindolyl-heteroaryl-maleimides as potent and selective glycogen synthase kinase-3beta (GSK-3beta) inhibitors

Two approaches were developed to synthesize the novel 7-azaindolyl-heteroarylmaleimides. The first approach was based upon the palladium-catalyzed Suzuki cross-coupling or Stille cross-coupling of 2-chloro-maleimide 5 with various arylboronic acids or arylstannanes. The second approach was based upon the condensation of ethyl 7-azaindolyl-3-glyoxylate 12 with various acetamides. The hydroxypropyl-substituted 7-azaindolylmaleimide template was first used to screen different heteroaryls attached to the maleimide. Replacement of hydroxypropyl with different chain lengths and different functional groups were studied next. Many compounds synthesized were demonstrated to have high potency at GSK-3beta, good GS activity in HEK293 cells and good to excellent metabolic stability in human liver microsomes. Three representative compounds (21, 33, and 34) were demonstrated to have good selectivity against a panel of 80 kinase assays. Among them, compound 33 exhibited very weak inhibitions at the other 79 kinase assays, and behaved as a highly selective GSK-3beta inhibitor.     

半胱胺盐酸盐对高温条件下泌乳后期奶牛生产性能的影响

目的:探讨夏季高温、环境温湿指数(THI)高于76条件下半胱胺盐酸盐(Lactonin)对泌乳后期奶牛产奶性能的影响.方法:96头黑白花奶牛,分为Lactonin(3000 U/d)处理(LT,n=49,泌乳中期曾接受Lactonin)和对照组(n=47),再根据高温期以前的产奶量(M)将LT组和对照组分别组分成4个产量组(G,n=12;第4组LT,n=13;对照组,n=11):G1(M＜24 kg/d),G2(24＜M＜28 kg/d),G3(28＜M＜32 kg/d),G4(M＞32kg/d).结果:与对照相比,第一产量组LT奶牛的体温降低(P＜0.05);常乳、标准乳及饲料转化效率提高(P＜0.05)、乳脂率增加(P＜0.05),乳蛋白量倾向增加;体细胞数趋于降低.所有参试LT奶牛平均乳脂率显著增加(P＜0.05),乳蛋白量倾向增加;常乳和标准乳产量趋于提高;血清中胰岛素浓度显著提高(P＜0.01),T3、T4浓度有降低趋势.结论:Lactonin有利于高温季节奶牛正常代谢的维持,改善奶产量和乳品质,提高饲料利用效率.Lactonin对奶牛的积极影响是经过胰岛素、T3、T4等内分泌激素和生长轴介导的.     

Functional difference between <Emphasis Type="Italic">Sinorhizobium meliloti</Emphasis> NifA and <Emphasis Type="Italic">Enterobacter cloacae</Emphasis> NifA

The nifA gene is an important regulatory gene and its product, NifA protein, regulates the expression of many nif genes involved in the nitrogen fixation process. We introduced multiple copies of the constitutively expressed Sinorhizobium meliloti (Sm) or Enterobacter cloacae (Ec) nifA gene into both the nifA mutant strain SmY and the wild-type strain Sm1021. Root nodules produced by SmY containing a constitutively expressed Sm nifA gene were capable of fixing nitrogen, while nodules produced by SmY containing the Ec nifA gene remained unable to fix nitrogen, as is the case for SmY itself. However, transfer of an additional Sm nifA gene into Sm1021 improved the nitrogen-fixing efficiency of root nodules to a greater extent than that observed upon transfer of the Ec nifA gene into Sm1021. Comparative analysis of amino acid sequences between Sm NifA and Ec NifA showed that the N-terminal domain was the least similar, but this domain is indispensable for complementation of the Fix^- phenotype of SmY by Sm NifA. We conclude that more than one domain is involved in determining functional differences between Sm NifA and Ec NifA.     

Molecular phenotype of airway side population cells

Lung epithelial-specific stem cells have been localized to discrete microenvironments throughout the adult conducting airway. Properties of these cells include pollutant resistance, multipotent differentiation, and infrequent proliferation. Goals of the present study were to use Hoechst 33342 efflux, a property of stem cells in other tissues, to purify and further characterize airway stem cells. Hoechst 33342 effluxing lung cells were identified as a verapamil-sensitive side population by flow cytometry. Lung side population cells were further subdivided on the basis of hematopoietic (CD45 positive) or nonhematopoietic (CD45 negative) origin. Nonhematopoietic side population cells were enriched for stem cell antigen-1 reactivity and expressed molecular markers specific to both airway and mesenchymal lineages. Analysis of the molecular phenotype of airway-derived side population cells indicates that they are similar to neuroepithelial body-associated variant Clara cells. Taken together, these data suggest that the nonhematopoietic side population isolated from lung is enriched for previously identified airway stem cells.     

Purification and nucleosome mapping analysis of native yeast plasmid chromatin

There is much evidence indicating the importance in gene regulation of the positions of nucleosomes with respect to DNA sequence. Low resolution chromatin structures have been described for many genes, but there is a dearth of detailed high resolution chromatin structures. In the cases where they are available, high resolution maps have revealed much more complex chromatin structures, with multiple alternative nucleosome positions. The discovery that ATP-dependent chromatin remodelling machines are recruited to genes, with their ability to mobilise nucleosomes on DNA and to alter nucleosomal conformation, emphasises the necessity for obtaining high resolution nucleosome maps, so that the details of these remodelling reactions can be defined in vivo. Here, we describe protocols for purifying plasmid chromatin from cells of the yeast Saccharomyces cerevisiae and for mapping nucleosome positions on the plasmid using the monomer extension mapping method. This method requires purified chromatin, but is capable of mapping relatively long stretches of chromatin in great detail. Typically, it reveals very complex chromatin structures.     

Tumor necrosis factor regulates intestinal epithelial cell migration by receptor-dependent mechanisms

Altered mucosal integrity andincreased cytokine production, including tumor necrosis factor (TNF),are the hallmarks of inflammatory bowel disease (IBD). In this study,we addressed the role of TNF receptors (TNFR) on intestinal epithelialcell migration in an in vitro wound closure model. With mouse TNFR1 orTNFR2 knockout intestinal epithelial cells, gene transfection, andpharmacological inhibitors, we show a concentration-dependentreceptor-mediated regulation of intestinal cell migration by TNF. Aphysiological TNF level (1 ng/ml) enhances migration through TNFR2,whereas a pathological level (100 ng/ml) inhibits wound closure through TNFR1. Increased rate of wound closure by TNFR2 or inhibition by TNFR1cannot be explained by either increased proliferation orapoptosis, respectively. Furthermore, inhibiting Src tyrosine kinase decreases TNF-induced focal adhesion kinase (FAK) tyrosine phosphorylation and cellular migration. We therefore conclude thatTNFR2 activates a novel Src-regulated pathway involving FAK tyrosinephosphorylation that enhances migration of intestinal epithelial cells.