Retention and Degradation of N-Glycoproteins in the Rough Endoplasmic Reticulum

Recent studies have shown that newly synthesized proteins and glycoproteins are submitted to a quality control mechanism in the rough endoplasmic reticulum (ER). In this report we present two models: One model will illustrate a transient retention in rough ER leading to a further degradation of glycoproteins in the cytosol, (soluble alkaline phosphatase expressed in Man-P-Dol deficient CHO cells lines). The second model will illustrate a strict retention of glycoproteins in rough ER without degradation nor recycling through the Golgi (E1, E2 glycoproteins of Hepatitis C virus in stably transfected UHCV-11.4 cells and in infected Hep G2 cells).In both cases, oligomannoside structures are markers of these phenomena, either as free soluble released oligomannosides in the case of degradation, or as N-linked oligomannosides for strict retention in rough ER.     

拟南芥AtJ3基因的克隆和分析

克隆并分析了拟南芥（Ａｒａｂｉｄｏｐｓｉｓｔｈａｌｉａｎａ（Ｌ．）Ｈｅｙｎｈ．）ＡｔＪ３的ｃＤＮＡ核苷酸序列,并证明其翻译产物与Ｅ．ｃｏｌｉ的ＤｎａＪ蛋白高度同源。ＡｔＪ３蛋白分子中具有全部这类蛋白的典型特征包括Ｊ结构域（ｄｏｍａｉｎ）、Ｇ或ＧＦ结构域、富含半胱氨酸的锌指结构域,Ｃ末端的ＣＡＱＱ是一个蛋白法尼基化信号。用ＡｔＪ３的ｃＤＮＡ序列末端非翻译区作探针,从拟南芥基因组文库中分离获得ＡｔＪ３基因。基因序列分析表明该基因是由被５个内含子分隔的６个外显子组成。根据Ｓｏｕｔｈｅｒｎ杂交分析,ＡｔＪ３基因为单拷贝基因。Ｎｏｒｔｈｅｒｎ分析结果表明,ＡｔＪ３在子叶、叶、根、花及长角果中都表达。３５℃的热激能增加叶中ＡｔＪ３的ｍＲＮＡ表达     

The Ku70 ?1310C/G promoter polymorphism is associated with breast cancer susceptibility in Chinese Han population

Ku70 plays an important role in the DSBR (DNA double-strand breaks repair) and maintenance of genomic integrity. Genetic variations within human Ku70 have been demonstrated to be associated with increased risk of several types of cancers. In this hospital-based case–control study, we aimed to investigate whether a single nucleotide polymorphism (SNP) in the promoter region (rs2267437) of Ku70 gene is associated with susceptibility to breast cancer in Chinese Han population. A total of 293 patients with breast cancer and 301 age-matched healthy controls were enrolled in this study. The Ku70 −1310C/G polymorphism was determined by polymerase chain reaction-restriction fragment length polymorphism (PCR–RFLP) analysis. A significant difference in genotype distribution and allele frequency was observed between patients and controls. The CG or GG carries were at higher risk of breast cancer compared with the CC homozygotes (OR = 1.43, 95% CI = 1.02–2.00, P = 0.038 and OR = 3.53, 95% CI = 1.60–7.80, P = 0.002, respectively). Further stratification analysis revealed that G allele was associated with an increased risk of breast cancer among premenopausal women (OR = 1.68, 95% CI = 1.21–2.33, P = 0.002), but not in postmenopausal women (OR = 1.33, 5% CI = 0.85–2.10, P = 0.216). Our study suggests that the Ku70 −1310C/G promoter polymorphism may be a susceptibility factor for breast cancer in Chinese Han population.     

Detection of quantitative trait loci for growth- and fatness-related traits in a large-scale White Duroc × Erhualian intercross pig population

Growth and fatness are economically important traits in pigs. In this study, a genome scan was performed to detect quantitative trait loci (QTL) for 14 growth and fatness traits related to body weight, backfat thickness and fat weight in a large-scale White Duroc × Erhualian F(2) intercross. A total of 76 genome-wide significant QTL were mapped to 16 chromosomes. The most significant QTL was found on pig chromosome (SSC) 7 for fatness with unexpectedly small confidence intervals of ～2 cM, providing an excellent starting point to identify causal variants. Common QTL for both fatness and growth traits were found on SSC4, 5, 7 and 8, and shared QTL for fat deposition were detected on SSC1, 2 and X. Time-series analysis of QTL for body weight at six growth stages revealed the continuously significant effects of the QTL on SSC4 at the fattening period and the temporal-specific expression of the QTL on SSC7 at the foetus and fattening stages. For fatness traits, Chinese Erhualian alleles were associated with increased fat deposition except that at the major QTL on SSC7. For growth traits, most of White Duroc alleles enhanced growth rates except for those at three significant QTL on SSC6, 7 and 9. The results confirmed many previously reported QTL and also detected novel QTL, revealing the complexity of the genetic basis of growth and fatness in pigs.     

Bioanalytical application of surface‐ and tip‐enhanced Raman spectroscopy

Due to its fingerprint specificity and trace‐level sensitivity, surface‐enhanced Raman spectroscopy (SERS) is an attractive tool in bioanalytics. This review reflects the research in this highly interesting topic of the last 3–4 years. The detection of the SERS signature of biomolecules up to microorganisms and cells is introduced. Labeling using modified nanoparticles (SERS tags) is also introduced. In order to establish biomedical applications, SERS analysis is performed in complex matrices such as body fluids. Furthermore, the SERS technique is combined with other methods such as microfluidic devices for online monitoring and scanning probe microscopy (i.e. tip‐enhanced Raman spectroscopy, TERS) to investigate nanoscaled features. The present review illustrates the broad application fields of SERS and TERS in bioanalytics and shows the great potential of these methods for biomedical diagnostics.     

Genome-Wide Association Studies for Growth and Meat Production Traits in Sheep

Background

Growth and meat production traits are significant economic traits in sheep. The aim of the study is to identify candidate genes affecting growth and meat production traits at genome level with high throughput single nucleotide polymorphisms (SNP) genotyping technologies.

Methodology and Results

Using Illumina OvineSNP50 BeadChip, we performed a GWA study in 329 purebred sheep for 11 growth and meat production traits (birth weight, weaning weight, 6-month weight, eye muscle area, fat thickness, pre-weaning gain, post-weaning gain, daily weight gain, height at withers, chest girth, and shin circumference). After quality control, 319 sheep and 48,198 SNPs were analyzed by TASSEL program in a mixed linear model (MLM). 36 significant SNPs were identified for 7 traits, and 10 of them reached genome-wise significance level for post-weaning gain. Gene annotation was implemented with the latest sheep genome Ovis_aries_v3.1 (released October 2012). More than one-third SNPs (14 out of 36) were located within ovine genes, others were located close to ovine genes (878bp-398,165bp apart). The strongest new finding is 5 genes were thought to be the most crucial candidate genes associated with post-weaning gain: s58995.1 was located within the ovine genes MEF2B and RFXANK, OAR3_84073899.1, OAR3_115712045.1 and OAR9_91721507.1 were located within CAMKMT, TRHDE, and RIPK2 respectively. GRM1, POL, MBD5, UBR2, RPL7 and SMC2 were thought to be the important candidate genes affecting post-weaning gain too. Additionally, 25 genes at chromosome-wise significance level were also forecasted to be the promising genes that influencing sheep growth and meat production traits.

Conclusions

The results will contribute to the similar studies and facilitate the potential utilization of genes involved in growth and meat production traits in sheep in future.     

Coastal Synechococcus metagenome reveals major roles for horizontal gene transfer and plasmids in population diversity

The extent to which cultured strains represent the genetic diversity of a population of microorganisms is poorly understood. Because they do not require culturing, metagenomic approaches have the potential to reveal the genetic diversity of the microbes actually present in an environment. From coastal California seawater, a complex and diverse environment, the marine cyanobacteria of the genus Synechococcus were enriched by flow cytometry-based sorting and the population metagenome was analysed with 454 sequencing technology. The sequence data were compared with model Synechococcus genomes, including those of two coastal strains, one isolated from the same and one from a very similar environment. The natural population metagenome had high sequence identity to most genes from the coastal model strains but diverged greatly from these genomes in multiple regions of atypical trinucleotide content that encoded diverse functions. These results can be explained by extensive horizontal gene transfer presumably with large differences in horizontally transferred genetic material between different strains. Some assembled contigs showed the presence of novel open reading frames not found in the model genomes, but these could not yet be unambiguously assigned to a Synechococcus clade. At least three distinct mobile DNA elements (plasmids) not found in model strain genomes were detected in the assembled contigs, suggesting for the first time their likely importance in marine cyanobacterial populations and possible role in horizontal gene transfer.     

The Glycoprotein B Disintegrin-Like Domain Binds Beta 1 Integrin To Mediate Cytomegalovirus Entry

Cellular integrins were identified as human cytomegalovirus (HCMV) entry receptors and signaling mediators in both fibroblasts and endothelial cells. The goal of these studies was to determine the mechanism by which HCMV binds to cellular integrins to mediate virus entry. HCMV envelope glycoprotein B (gB) has sequence similarity to the integrin-binding disintegrin-like domain found in the ADAM (a disintegrin and metalloprotease) family of proteins. To test the ability of this region to bind to cellular integrins, we generated a recombinant soluble version of the gB disintegrin-like domain (gB-DLD). The gB-DLD protein bound to human fibroblasts in a specific, dose-dependent and saturable manner that required the expression of an intact β1 integrin ectodomain. Furthermore, a physical association between gB-DLD and β1 integrin was demonstrated through in vitro pull-down assays. The function of this interaction was shown by the ability of cell-bound gB-DLD to efficiently block HCMV entry and the infectivity of multiple in vivo target cells. Additionally, rabbit polyclonal antibodies raised against gB-DLD neutralized HCMV infection. Mimicry of the ADAM family disintegrin-like domain by HCMV gB represents a novel mechanism for integrin engagement by a virus and reveals a unique therapeutic target for HCMV neutralization. The strong conservation of the DLD across beta- and gammaherpesviruses suggests that integrin recognition and utilization may be a more broadly conserved feature throughout the Herpesviridae.Like many other herpesviruses, human cytomegalovirus (HCMV) is an opportunistic pathogen that is able to asymptomatically infect the human population with high incidence throughout the world. Primary infection is followed by a life-long latent phase that may reactivate and cause disease during the immunosuppression experienced by AIDS patients and organ transplant recipients (14, 52). HCMV disease is also a cause of significant morbidity and mortality during primary congenital infections (66). Currently there is no effective HCMV vaccine, and HCMV antiviral therapies, such as ganciclovir, are highly toxic and unsuitable for treating pregnant women in the congenital setting (92).HCMV disease can manifest itself in most organ systems and tissue types. Pathology from HCMV-infected individuals reveals that HCMV can infect most cell types, including fibroblasts, endothelial cells, epithelial cells, smooth muscle cells, stromal cells, monocytes/macrophages, neutrophils, neuronal cells, and hepatocytes (20, 25, 77, 83, 87). The broad intrahost organ and tissue tropism of HCMV is paralleled in vitro with the virus'' ability to bind and fuse with nearly every vertebrate cell type tested (40, 62, 78). However, full productive infection is limited to secondary strains of fibroblasts and endothelial cells. The ability of HCMV to enter such a diverse range of cell types is indicative of multiple cell-specific receptors, broadly expressed receptors, or a complex entry pathway in which a combination of both cell-specific and broadly expressed cellular receptors are utilized.The genes that encode envelope glycoprotein B (gB) and gH are essential (37), play several key roles during virus entry and egress, and are conserved throughout the Herpesviridae (reviewed in reference 80). A soluble form of gB truncated at the transmembrane domain (gBs) binds to permissive cells specifically, blocks virus entry, and is sufficient to trigger signal transduction events that result in the activation of an interferon-responsive pathway that is also activated by HCMV virions (10, 12, 13).HCMV entry requires initial tethering of virions to cell surface heparan sulfate proteoglycans (HSPGs) (22, 80). The HCMV envelope contains at least two separate glycoprotein complexes with affinities for heparan sulfate: gB (22) and the gM/gN complex (48). The gM/gN complex is more abundant than gB within the envelope (88) and binds heparin with higher affinity (49). Thus, the gM/gN complex is thought to be the primary heparin-binding component of the HCMV envelope.Virus-cell tethering via HSPGs is followed by a more stable interaction and subsequent signal transduction cascades. This interaction was proposed to be mediated via cell surface epidermal growth factor receptor (EGFR) (17, 95). These data, however, conflicted with more recent reports that demonstrate EGFR is not explicitly required for infection (21, 42). Platelet-derived growth factor receptor (PDGFR) has also been reported to function as an attachment receptor that functions to activate signaling cascades required for infection (79). The relative contribution of signaling and virus-host cell attachment for each of these growth factor receptors remains to be further characterized. The possibility also exists that additional attachment receptors still remain unidentified.Integrins are expressed on the cell surfaces of all vertebrate cells, a characteristic that parallels the promiscuity of HCMV entry. Additionally, β1 integrins are capable of mediating many of the same signal transduction pathways that are triggered during HCMV entry into host cells. Upon binding and fusing with host cell surfaces, HCMV triggers changes in Ca²⁺ homeostasis (36) and the activation of phospholipases C and A2, as well as an increased release of arachidonic acid and its metabolites (2). Additionally, mitogen-activated protein kinase (MAPK) (44, 45), phosphatidylinositol-3-OH kinase (PI3-K) (46), and G proteins are activated (73). Indeed, it was shown that HCMV entry led to an activation of integrin signaling pathways that reorganized the actin cytoskeleton (31) and phosphorylated β1 and β3 integrin cytoplasmic domains (31), focal adhesion kinase (FAK) (31), and Src (94). Integrin antibody blocking studies in combination with HCMV infectivity assays in β1 integrin-null GD25 cells identified α2β1, α6β1, and αVβ3 integrins as HCMV “postattachment” entry receptors (31). Certain integrin signaling events could be triggered by both HCMV and a soluble version of gB and require the expression of β1 integrin, identifying this specific viral ligand in integrin engagement (31).ADAM family members are multifunctional proteins that contain a metalloproteinase domain involved in ectodomain shedding and a disintegrin module of approximately 90 amino acids that confers RGD-independent integrin binding (43, 81, 99). The minimum component of the disintegrin module required for integrin engagement is the 12- to 13-amino-acid disintegrin loop, for which a consensus sequence has been described: RX₆DLXXF (29). The 20-amino-acid stretch encompassing the gB disintegrin-like domain is highly conserved, with greater than 98% amino acid identity among HCMV clinical isolates. Additionally, this domain is present in most gammaherpesviruses and all betaherpesviruses, suggesting that integrin engagement may be a conserved feature for most of the Herpesviridae. Synthetic peptides of the gB disintegrin loop block virus fusion (tegument delivery) but not virus attachment (31). This fact suggests a disintegrin-mediated molecular mechanism of herpesvirus-integrin engagement. Glycoprotein H (gH) has also been identified as an αVβ3 integrin ligand (94). However, gH contains no previously identified integrin recognition motifs, and the αVβ3 integrin heterodimer does not typically engage ADAM family proteins.Herein, we explore the molecular mechanism of integrin engagement by HCMV envelope gB. We provide multiple lines of evidence that demonstrate a physical interaction between the gB disintegrin module with β1 integrin. Furthermore, this interaction has significant consequences to the viral life cycle, since a soluble version of the gB disintegrin module efficiently blocks HCMV infection at a postattachment step during entry into multiple in vivo cell targets. Similarly, polyclonal antibodies directed against the gB disintegrin-like domain neutralize HCMV infectivity. These data identify the molecular mechanism of an HCMV ligand-receptor interaction required for virus-host fusion.     

PI3K/Akt Pathway is Required for Spinal Central Sensitization in Neuropathic Pain

Phosphatidylinositol-3-kinase (PI3K) has been identified in the expression of central sensitization after noxious inflammatory stimuli. However, its contribution in neuropathic pain remains to be determined. Here we address the role of PI3K signaling in central sensitization in a model of neuropathic pain, and propose a novel potential drug target for neuropathic pain. Chronic constriction injury (CCI) rat model was used in the study as the model for neuropathic pain. Western blotting, whole-cell patch clamp, and von Frey assay were performed to study biochemical, electrical, and behavioral changes in CCI rats, respectively. A steroid metabolite of the fungi (wortmannin) was used to block PI3K signaling and its effects on CCI rats were tested. PI3K/Akt signaling increased in the spinal cord L4–L6 sections in the CCI rats. CCI also facilitated miniature excitatory postsynaptic potential of dorsal horn substantia gelatinosa neurons, increased phosphorylation of glutamate receptor subunit GluA1 and synapsin at the synapse, and induced mechanic allodynia. Wortmannin reversed biochemical, electrical, and behavioral changes in CCI rats. This study is the first to show PI3K/Akt signaling is required for spinal central sensitization in the CCI neuropathic pain model.