Targeting eEF1A by a Legionella pneumophila effector leads to inhibition of protein synthesis and induction of host stress response

The Legionella pneumophila Dot/Icm type IV secretion system is essential for the biogenesis of a phagosome that supports bacterial multiplication, most likely via the functions of its protein substrates. Recent studies indicate that fundamental cellular processes, such as vesicle trafficking, stress response, autophagy and cell death, are modulated by these effectors. However, how each translocated protein contributes to the modulation of these pathways is largely unknown. In a screen to search substrates of the Dot/Icm transporter that can cause host cell death, we identified a gene whose product is lethal to yeast and mammalian cells. We demonstrate that this protein, called SidI, is a substrate of the Dot/Icm type IV protein transporter that targets the host protein translation process. Our results indicate that SidI specifically interacts with eEF1A and eEF1Bγ, two components of the eukaryotic protein translation elongation machinery and such interactions leads to inhibition of host protein synthesis. Furthermore, we have isolated two SidI substitution mutants that retain the target binding activity but have lost toxicity to eukaryotic cells, suggesting potential biochemical effect of SidI on eEF1A and eEF1Bγ. We also show that infection by L. pneumophila leads to eEF1A‐mediated activation of the heat shock regulatory protein HSF1 in a virulence‐dependent manner and deletion of sidI affects such activation. Moreover, similar response occurred in cells transiently transfected to express SidI. Thus, inhibition of host protein synthesis by specific effectors contributes to the induction of stress response in L. pneumophila‐infected cells.     

Malnutrition impairs alveolar fluid clearance in rat lungs

Inadequate nutrition complicates the clinical course of critically ill patients, and many of these patients develop pulmonary edema. However, little is known about the effect of malnutrition on the mechanisms that resolve alveolar edema. Therefore, we studied the mechanisms responsible for the decrease in alveolar fluid clearance in rats exposed to malnutrition. Rats were allowed access to water, but not to food, for 120 h. Then, the left and right lungs were isolated for the measurement of lung water volume and alveolar fluid clearance, respectively. The rate of alveolar fluid clearance was measured by the progressive increase in the concentration of Evans blue dye that was instilled into the distal air spaces with an isosmolar 5% albumin solution over 1 h. Malnutrition decreased alveolar fluid clearance by 38% compared with controls. Amiloride (10(-3) M) abolished alveolar fluid clearance in malnourished rats. Either refeeding for 120 h following nutritional deprivation for 120 h or an oral supply of sodium glutamate during nutritional deprivation for 120 h restored alveolar fluid clearance to 91 and 86% of normal, respectively. Dibutyryl-cGMP, a cyclic nucleotide-gated cation channel agonist, increased alveolar fluid clearance in malnourished rats supplied with sodium glutamate. Terbutaline, a beta(2)-adrenergic agonist, increased alveolar fluid clearance in rats under all conditions (control, malnutrition, refeeding, and glutamate-treated). These results indicate that malnutrition impairs primarily amiloride-insensitive and dibutyryl-cGMP-sensitive alveolar fluid clearance, but this effect is partially reversible by refeeding, treatment with sodium glutamate, or beta-adrenergic agonist therapy.     

Molecular Cloning and Functional Analysis of a Na<Superscript>+</Superscript>/H<Superscript>+</Superscript> Antiporter Gene <Emphasis Type="Italic">ThNHX1</Emphasis> from a Halophytic Plant <Emphasis Type="Italic">Thellungiella halophila</Emphasis>

Na⁺/H⁺ exchanger catalyzes the countertransport of Na⁺ and H⁺ across membranes. Using the rapid amplification of cDNA ends method, a Na⁺/H⁺ antiporter gene (ThNHX1) was isolated from a halophytic plant, salt cress (Thellungiella halophila). The deduced amino acid sequence contained 545 amino acid residues with a conserved amiloride-binding domain (⁸⁷LFFIYLLPPI⁹⁶) and shared more than 94% identity with that of AtNHX1 from Arabidopsis thaliana. The ThNHX1 mRNA level was upregulated by salt and other stresses (abscisic acid, polyethylene glycol, and high temperature). This gene partially complemented the Na⁺/Li⁺-sensitive phenotype of a yeast mutant that was deficient in the endosomal–vacuolar Na⁺/H⁺ antiporter ScNHX1. Overexpression of ThNHX1 in Arabidopsis increased salt tolerance of transgenic plants compared with the wild-type plants. In addition, the silencing of ThNHX1 gene in T. halophila caused the transgenic plants to be more salt and osmotic sensitive than wild-type plant. Together, these results suggest that ThNHX1 may function as a tonoplast Na⁺/H⁺ antiporter and play an important role in salt tolerance of T. halophila. Chunxia Wu, Xiuhua Gao, and Xiangqiang Kong contributed equally to this work.     

Hepatitis B Virus (HBV) Surface Antigen Interacts with and Promotes Cyclophilin A Secretion: Possible Link to Pathogenesis of HBV Infection

Cyclophilin A (CypA), predominantly located intracellularly, is a multifunctional protein. We previously reported decreased CypA levels in hepatocytes of transgenic mice expressing hepatitis B virus (HBV) surface antigen (HBsAg). In this study, we found that expression of HBV small surface protein (SHBs) in human hepatoma cell lines specifically triggered CypA secretion, whereas SHBs added extracellularly to culture medium did not. Moreover, CypA secretion was not promoted by the expression of a secretion deficient SHBs mutant, suggesting a close association between secretion of CypA and SHBs. Interaction between CypA and SHBs was observed by using coimmunoprecipitation and glutathione S-transferase pull-down assays. Hydrodynamic injection of the SHBs expression construct into C57BL/6J mice resulted in increased serum CypA levels and ALT/AST levels, as well as the infiltration of inflammatory cells surrounding SHBs-positive hepatocytes. The inflammatory response and serum ALT/AST level were reduced when the chemotactic effect of CypA was inhibited by cyclosporine and anti-CD147 antibody. Furthermore, higher serum CypA levels were detected in chronic hepatitis B patients than in healthy individuals. In HBV patients who had received liver transplantation, serum CypA levels declined dramatically after the loss of HBsAg as a consequence of liver transplantation. Taken together, these results indicate that expression and secretion of SHBs can promote CypA secretion, which may contribute to the pathogenesis of HBV infection.Hepatitis B virus (HBV) infects more than 350 million people worldwide and is a major cause of chronic viral hepatitis and hepatocellular carcinoma (25). Three morphologically distinct forms of viral particles exist in the sera of HBV-infected patients, namely, the 22-nm-diameter spherical particles, tubular particles, and 42-nm-diameter virions (19). Strikingly, the subviral particles (spheres and tubules), containing only viral envelop glycoproteins and host-derived lipids, typically outnumber the virions by a factor of 1,000- to 10,000-fold (6, 11). There are three HBV envelop glycoproteins collectively known as HBV surface antigen (HBsAg), including the large (LHBs), middle (MHBs), and small (SHBs) surface proteins. Among them, SHBs is the most abundant viral envelop protein in virion and subviral particles. Although excess HBsAg subviral particles have been suggested to sequester the neutralizing antibody against HBV and contribute to a state of immune tolerance, thereby enabling the survival of infectious virions and leading to persistent infections (6, 27), the biological and pathological significance of the overproduction of HBsAg subviral particles still remains elusive.HBsAg has been proved extremely effective in inducing protective antibodies (anti-HBs) and thus has been used as the prophylactic vaccine. Thus far, most studies on HBsAg have focused on the development of hepatitis B vaccines (41), identification of HBsAg-interacting membrane proteins as potential host HBV receptors (9, 13), and characterization of the impact of naturally occurring HBsAg mutations on its antigenicity (12, 43). However, specific interactions between HBsAg and host intracellular factors have not been extensively studied.To address this issue, SHBs-secreting cell lines and lineages of HBV transgenic mice persistently expressing HBsAg were used in our previous studies (28, 34, 44). We found that the level of cyclophilin A (CypA) decreased markedly in the livers of HBsAg transgenic mice but increased significantly in their sera (44). CypA is a multifunctional cellular protein. It is the major binding protein for the immunosuppressive drug cyclosporine (Cs) (14) and exhibits peptidyl-prolyl cis-trans isomerase activity (35). Recently, it was found CypA played important roles in regulating inflammatory responses and viral infections. Regarding these newly recognized physiological functions, CypA was speculated to be involved in HBV infection. In the present study, the mechanism and clinical implications of elevated secretion of CypA induced by SHBs were explored in detail, including studies in cell cultures, hydrodynamic injected mouse models, and chronic hepatitis B patients. Our findings indicate that expression and secretion of SHBs can trigger the secretion of CypA, which may induce liver inflammation and contribute to the pathogenesis of HBV infection.     

Distinct Roles for Rho Versus Rac/Cdc42 GTPases Downstream of Vav2 in Regulating Mammary Epithelial Acinar Architecture

Non-malignant mammary epithelial cells (MECs) undergo acinar morphogenesis in three-dimensional Matrigel culture, a trait that is lost upon oncogenic transformation. Rho GTPases are thought to play important roles in regulating epithelial cell-cell junctions, but their contributions to acinar morphogenesis remain unclear. Here we report that the activity of Rho GTPases is down-regulated in non-malignant MECs in three-dimensional culture with particular suppression of Rac1 and Cdc42. Inducible expression of a constitutively active form of Vav2, a Rho GTPase guanine nucleotide exchange factor activated by receptor tyrosine kinases, in three-dimensional MEC culture activated Rac1 and Cdc42; Vav2 induction from early stages of culture impaired acinar morphogenesis, and induction in preformed acini disrupted the pre-established acinar architecture and led to cellular outgrowths. Knockdown studies demonstrated that Rac1 and Cdc42 mediate the constitutively active Vav2 phenotype, whereas in contrast, RhoA knockdown intensified the Vav2-induced disruption of acini, leading to more aggressive cell outgrowth and branching morphogenesis. These results indicate that RhoA plays an antagonistic role to Rac1/Cdc42 in the control of mammary epithelial acinar morphogenesis.     

H102对APP转基因小鼠脑内淀粉样蛋白和淀粉样蛋白前体蛋白表达的影响

目的:研究H102对APP695转基因模型小鼠脑内淀粉样蛋白和淀粉样蛋白前体蛋白表达的影响方法:9月龄转基因小鼠随机分为模型组和药物注射组,正常对照组采用月龄和性别与之相匹配的C57BL/6J小鼠。药物注射组给予侧脑室注射H102,每只每次3μl,连续10d;模型组和正常对照组给予等体积NS。应用免疫组织化学结合刚果红组织学染色,普通光学显微镜观察海马和颞叶皮层蛋白表达的变化。免疫印迹法检测小鼠大脑皮层APP蛋白的表达。结果:Aβ和APP免疫组化染色结果显示对照组海马CA1区神经元胞浆着色呈阴性或弱阳性,模型组较对照组阳性细胞增多,表达增强,胞浆着色明显加深。药物注射组同模型组相比,胞浆着色变淡,表达减弱。刚果红染色观察转基因小鼠模型组和H102注射组大脑颞叶皮层和海马的淀粉样斑块,可见H102注射组淀粉样斑块数较模型组明显减少。正常对照组未见阳性淀粉样斑块。免疫印迹检测显示模型组APP蛋白表达明显增加,给药组与模型组相比具有统计学意义。结论:APP695转基因小鼠大脑CA1区Aβ蛋白和APP蛋白表达增加,H102能够明显抑制该转基因小鼠Aβ蛋白和APP蛋白表达。     

A Simple Genetic Architecture Underlies Morphological Variation in Dogs

Domestic dogs exhibit tremendous phenotypic diversity, including a greater variation in body size than any other terrestrial mammal. Here, we generate a high density map of canine genetic variation by genotyping 915 dogs from 80 domestic dog breeds, 83 wild canids, and 10 outbred African shelter dogs across 60,968 single-nucleotide polymorphisms (SNPs). Coupling this genomic resource with external measurements from breed standards and individuals as well as skeletal measurements from museum specimens, we identify 51 regions of the dog genome associated with phenotypic variation among breeds in 57 traits. The complex traits include average breed body size and external body dimensions and cranial, dental, and long bone shape and size with and without allometric scaling. In contrast to the results from association mapping of quantitative traits in humans and domesticated plants, we find that across dog breeds, a small number of quantitative trait loci (≤3) explain the majority of phenotypic variation for most of the traits we studied. In addition, many genomic regions show signatures of recent selection, with most of the highly differentiated regions being associated with breed-defining traits such as body size, coat characteristics, and ear floppiness. Our results demonstrate the efficacy of mapping multiple traits in the domestic dog using a database of genotyped individuals and highlight the important role human-directed selection has played in altering the genetic architecture of key traits in this important species.     

Successful low dose insemination of flow cytometrically sorted Sika (Cervus nippon) sperm in Wapiti (Cervus elaphus)

The purpose of this study was to determine a practical method in Wapiti (Cervus elaphus) of using predetermined sexed Sika (Cervus nippon) semen. Semen was collected by electro-ejaculation from one stag of proven fertility and transported to the laboratory where it was retained as unsorted (control) or was separated into X- and Y-chromosome-bearing sperm using a modified high-speed cell sorter. Wapiti hinds (n = 81) were inseminated into the uterus by rectum manipulation with 1 × 10⁶ (X1 and Y1 group, respectively) or 2 × 10⁶ (X2 and Y2 group, respectively) of sorted frozen-thawed and 1 × 10⁷ non-sorted frozen-thawed (a commercial dose control) Sika motile sperm 60–66 h after removal of intra-vaginal progesterone-impregnated CIDR devices and administration of 700 IU of PMSG at the time of CIDR removal. The percentage of hinds calving after insemination was similar for X1 (38.5%), X2 (41.7%), Y1 (44.4%), Y2 (38.9%) groups (P > 0.05), but higher for control (75%) treatment (P < 0.05). Ultimately 15 out of the 16 Sika and Wapiti-hybrid calves produced by Wapiti hinds inseminated with Y-sorted sperm were male (93.7%) and 10/10 (100%) Sika and Wapiti-hybrid calves from hinds inseminated with X-sorted sperm were female. The sex ratio of the Sika and Wapiti-hybrid calves born to hinds inseminated with sex-sorted sperm deviated significantly (P < 0.05) from 50% and 50.0% in the control group. All Sika and Wapiti-hybrid calves were born between 237 and 250 d of gestation. Male and female calves in the control group had similar birth weights and weaning weights as calves from hinds inseminated with X- or Y-sorted sperm. In conclusion it can be said that normal Sika and Wapiti-hybrid calves of predicted sex can be produced after artificial insemination of Wapiti does with low numbers of sex-sorted cryopreserved Sika sperm.     

Synthesis and SAR of GlyT1 inhibitors derived from a series of N-((4-(morpholine-4-carbonyl)-1-(propylsulfonyl)piperidin-4-yl)methyl)benzamides

This Letter describes the synthesis and SAR, developed through an iterative analog library approach, of potent and selective non-sarcosine-derived GlyT1 inhibitors.