Regulation of protease-activated receptor signaling by post-translational modifications

Protease-activated receptors (PARs) are a unique family of G-protein-coupled receptors (GPCRs) that are irreversibly activated following proteolytic cleavage of their extracellular N-terminus. PARs play critical functions in hemostasis, thrombosis, inflammation, embryonic development, and cancer progression. Because of the irreversible proteolytic nature of PAR activation, signaling by the receptors is tightly regulated. Three distinct processes including desensitization, internalization, and lysosomal degradation, regulate the temporal and spatial aspects of activated PAR signaling. Post-translational modifications play a critical role in regulating each of these processes and here we review the nature of PAR post-translational modifications and their importance in signal regulation. The PARs are activated by numerous proteases, and some can elicit distinct cellular responses, how this biased agonism is determined is unknown. Further study of the function of post-translational modifications of the PARs will lead to a greater understanding of the physiological regulation of baised agonism and how PAR signaling is precisely controlled in different cellular contexts.     

Diet-induced obesity in rats leads to a decrease in sperm motility

Background  

Obesity is rapidly becoming a worldwide epidemic that affects children and adults. Some studies have shown a relationship between obesity and infertility, but until now it remains controversial. Thus, the aim of the present study was to investigate the effect of high-fat diet-induced obesity on male reproductive parameters.     

长爪沙鼠ATPase8,ATPase6,COX3基因的克隆及序列分析

目的对长爪沙鼠线粒体ATPase8,ATPase6,COX3基因全序列进行测定,并对其进行鉴定及进化分析。方法根据长爪沙鼠已知基因序列设计引物,采用PCR产物测序法,对目的片段进行测序鉴定。结合已公布啮齿类动物ATPase8,ATPase6,COX3基因序列,分析其碱基组成、遗传距离、并基于最小进化法和UPGMA法构建系统进化树。结果获得长爪沙鼠线粒体ATPase8,ATPase6,COX3基因全序列,其与家鼠、小家鼠和仓鼠均具有较高的同源性（76～98%）;进化分析结果显示,长爪沙鼠与家鼠、黑家鼠和仓鼠遗传距离较近;碱基G的含量分别为6.9%,10.7%,15.2%,符合mtDNA的特点;A＋T含量分别为68.2%,64.1%,59.2%,明显低于G＋C含量,符合哺乳动物的特点。结论本研究为首次获得长爪沙鼠ATPase8,ATPase6,COX3基因全序列,长爪沙鼠与家鼠、黑家鼠和仓鼠具有较近遗传距离,本研究为长爪沙鼠进化研究、线粒体的结构和功能研究奠定基础。     

PvAMT1;1, a highly selective ammonium transporter that functions as H+/NH4(+) symporter

One of the main forms of nitrogen assimilated by microorganisms and plants is ammonium, despite its toxicity at low millimolar concentrations. Ammonium absorption has been demonstrated to be carried out by highly selective plasma membrane-located transporters of the AMT/MEP/Rh family and characterized by the presence of a well conserved hydrophobic pore through which ammonia is proposed to move. However, uncertainties exist regarding the exact chemical species transported by these membrane proteins, which can be in the form of either hydrophobic ammonia or charged ammonium. Here, we present the characterization of PvAMT1;1 from the common bean and demonstrate that it mediates the high affinity (micromolar), rapidly saturating (1 mM) electrogenic transport of ammonium. Activity of the transporter is enhanced by low extracellular pH, and associated with this acidic pH stimulation are changes in the reversal potential and cytoplasm acidification, indicating that PvAMT1;1 functions as an H(+)/NH(4)(+) symporter. Mutation analysis of a unique histidine present in PvAMT1;1 (H125R) leads to the stimulation of ammonium transport by decreasing the K(m) value by half and by increasing the V(max) 3-fold, without affecting the pH dependence of the symporter. In contrast, mutation of the first conserved histidine within the channel modifies the properties of PvAMT1;1, increasing its K(m) and V(max) values and transforming it into a pH-independent mechanism.     

Z:ZCLA长爪沙鼠微卫星标记的遗传多态性研究

通过对9个微卫星座位的扩增,研究了Z：ZCLA长爪沙鼠的遗传多态性。结果表明,Z：ZCLA长爪沙鼠在其中1个位点上只有一个等位基因,在其它位点上均有2～4个等位基因,平均等位基因数2．6个。平均杂合度0．4684,平均多态信息量0．4166,平均有效等位基因数2．1756。全群基因纯合度从0．1111～0．5555,平均0．3389,提示目前本群遗传多样性水平处于中度多态。     

Sphingosine kinase, phosphatidylinositol 3-kinase, Akt, NF-kappaB, and p300 are required for CCL5 production in Mycobacterium bovis Bacillus Calmette-Guérin (BCG)-infected epithelial cells

CCL5 is a key in limiting mycobacterial infection. Although NF-κB has been implicated, signaling cascades involved in CCL5 production by epithelial cells following infection with Mycobacterium bovis BCG are still not defined. Here we show that using pharmacological inhibition of sphingosine kinase (SPK), striking inhibition of M. bovis BCG-induced CCL5 protein was observed. Phosphatidylinositol 3-kinase (PI3K) and Akt were also important for CCL5 production by epithelial cells infected with M. bovis BCG. Moreover, there was increased activation of PI3K, IKK/αβ and NF-κB in A549 cells infected with M. bovis BCG. Importantly, the PI3K activation was dependent on SPK. Finally, M. bovis BCG increases the recruitment of p300 with NF-κB in A549 cells. Together, these studies are the first to show that M. bovis BCG-induced CCL5 secretion is dependent on the SPK/PI3K/Akt/NF-κB and p300 signaling pathway. The regulatory pathways of M. bovis BCG-induced CCL5 production can potentially be exploited therapeutically.     

用19个生化基因位点研究普通级Z:ZCLA长爪沙鼠的遗传多样性

目的调查Z:ZCLA长爪沙鼠原种群普通级长爪沙鼠的遗传多样性。方法用本实验室自行建立的长爪沙鼠19个生化基因位点的乙酸纤维素膜电泳技术并结合基本群体遗传学指标研究了普通级Z:ZCLA长爪沙鼠100个家系的遗传多态性。结果Z:ZCLA长爪沙鼠在Es-1、Car-2、Hbb、Gpi-1、Cs-1、Ce-2I、dh-l、Mod-1呈单态,在Es-2、Es-3、Es-4、Es-6、Es-8、Es-9、Es-10、pd-1、gm-1、Trf、Akp-1个位点上呈现多态性,等位基因从2~4个不等,平均等位基因数3.0,平均杂合度0.512,平均多态信息量0.455。结论提示目前本群遗传多样性水平处于中度多态。     

Establishing the Yeast Kluyveromyces lactis as an Expression Host for Production of the Saposin-Like Domain of the Aspartic Protease Cirsin

Typical plant aspartic protease zymogens comprise a characteristic and plant-specific insert (PSI). PSI domains can interact with membranes, and a role as a defensive weapon against pathogens has been proposed. However, the potential of PSIs as antimicrobial agents has not been fully investigated and explored yet due to problems in producing sufficient amounts of these domains in bacteria. Here, we report the development of an expression platform for the production of the PSI domain of cirsin in the generally regarded as safe (GRAS) yeast Kluyveromyces lactis. We successfully generated K. lactis transformants expressing and secreting significant amounts of correctly processed and glycosylated PSI, as well as its nonglycosylated mutant. A purification protocol with protein yields of ∼4.0 mg/liter was established for both wild-type and nonglycosylated PSIs, which represents the highest reported yield for a nontagged PSI domain. Subsequent bioactivity assays targeting phytopathogenic fungi indicated that the PSI of cirsin is produced in a biologically active form in K. lactis and provided clear evidence for its antifungal activity. This yeast expression system thereby emerges as a promising production platform for further exploring the biotechnological potential of these plant saposin-like proteins.     

Genome sequencing and comparative analysis of three Chlamydia pecorum strains associated with different pathogenic outcomes

Background

Chlamydia pecorum is the causative agent of a number of acute diseases, but most often causes persistent, subclinical infection in ruminants, swine and birds. In this study, the genome sequences of three C. pecorum strains isolated from the faeces of a sheep with inapparent enteric infection (strain W73), from the synovial fluid of a sheep with polyarthritis (strain P787) and from a cervical swab taken from a cow with metritis (strain PV3056/3) were determined using Illumina/Solexa and Roche 454 genome sequencing.

Results

Gene order and synteny was almost identical between C. pecorum strains and C. psittaci. Differences between C. pecorum and other chlamydiae occurred at a number of loci, including the plasticity zone, which contained a MAC/perforin domain protein, two copies of a >3400 amino acid putative cytotoxin gene and four (PV3056/3) or five (P787 and W73) genes encoding phospholipase D. Chlamydia pecorum contains an almost intact tryptophan biosynthesis operon encoding trpABCDFR and has the ability to sequester kynurenine from its host, however it lacks the genes folA, folKP and folB required for folate metabolism found in other chlamydiae. A total of 15 polymorphic membrane proteins were identified, belonging to six pmp families. Strains possess an intact type III secretion system composed of 18 structural genes and accessory proteins, however a number of putative inc effector proteins widely distributed in chlamydiae are absent from C. pecorum. Two genes encoding the hypothetical protein ORF663 and IncA contain variable numbers of repeat sequences that could be associated with persistence of infection.

Conclusions

Genome sequencing of three C. pecorum strains, originating from animals with different disease manifestations, has identified differences in ORF663 and pseudogene content between strains and has identified genes and metabolic traits that may influence intracellular survival, pathogenicity and evasion of the host immune system.

Electronic supplementary material

The online version of this article (doi:10.1186/1471-2164-15-23) contains supplementary material, which is available to authorized users.