hsBAFF regulates proliferation and response in cultured CD4(+) T lymphocytes by upregulation of intracellular free Ca(2+) homeostasis

B cell activating factor belonging to the TNF family (BAFF, also called BLyS, TALL-1, THANK, or zTNF4) is an important survival factor for B cells, and is able to regulate T-cell activation. Recently, we have demonstrated that treatment of mice with human soluble BAFF (hsBAFF) causes a significant increase of percentages of splenic CD4(+) T lymphocytes dose-dependently, but the CD8(+) T lymphocyte percentages maintained unchanged. Here, we show that hsBAFF significantly enhanced CD4(+) T lymphocyte response of cultured mouse splenic cells, and hsBAFF induced the proliferation and IL-2/IFN-γ secretion of purified CD4(+) T lymphocytes suboptimally stimulated through anti-CD3. Of importance, we observed that IL-2 or IFN-γ cytokine has additive effect on the proliferation and activity of hsBAFF-stimulated CD4(+) T lymphocytes. Using Flow cytometry with fluorescent probe, Fluo-3/AM, we found that hsBAFF elicited [Ca(2+)](i) elevation contributing to CD4(+) T cell proliferation. This is evidenced by our finding that pretreatment with BAPTA/AM, an intracellular Ca(2+) chelator, significantly attenuated the proliferation of hsBAFF-stimulated CD4(+) T lymphocytes. Subsequently, we revealed that hsBAFF-stimulated CD4(+) T cell proliferation was markedly suppressed after pretreatment with EGTA, an extracellular Ca(2+) chelator, or with 2-APB, an inhibitor of Ca(2+) influx through CRAC channels, respectively, suggesting that extracellular Ca(2+) influx due to hsBAFF is closely associated with [Ca(2+)](i) elevation contributing to CD4(+) T cell proliferation. In addition, we noticed that hsBAFF-treated cells conferred partial resistance to decrease of cellular viability induced by thapsigargin (Tg), an endoplasmic reticulum (ER) Ca(2+)-ATPase inhibitor. Taken together, our data indicate that hsBAFF may promote CD4(+) T cell proliferation and response by upregulation of [Ca(2+)](i) homeostasis.     

模拟胃酸条件下的幽门螺杆菌CAT特异性抗体活性研究

幽门螺杆菌是常见的感染性病原菌,人类多种疾病发生与此菌感染有关。预防和治疗菌体感染及引发的相关疾病仍是现代医学面临的课题。实验利用原核表达的幽门螺杆菌过氧化氢酶(1~380 aa)免疫家兔,获得效价为1∶6 000的特异性抗血清,经硫酸铵沉淀法得到初步纯化的抗体。在体外模拟胃酸环境下(pH3.4)将抗体进行水解。SDS-PAGE结果显示,抗体的重链能被水解。水解后的抗体产物经ELISA方法检测,仍然具有与抗原特异性结合的能力。实验结论证实,在体外环境下特异性幽门螺杆菌抗体保护作用不会被胃蛋白酶的水解而破坏,提示口服特异性抗体预防和治疗幽门螺杆菌感染可能是一条可行的途径。     

亚麻外源基因遗传转化研究进展

亚麻作为重要的经济作物,其遗传转化研究对亚麻新品种的选育工作具有重要意义.分析亚麻遗传转化方法、提高转化效率及转化组织筛选等方面,重点从抗除草剂基因、抗病基因、抗重金属逆性基因、改善亚麻纤维质量基因,以及改善亚麻油质量基因方面综述亚麻遗传转化外源基因的研究和应用进展,并讨论亚麻遗传转化过程中存在的问题和解决策略.     

猪的骨骼肌生长发育研究进展

瘦肉率对生猪产业来说是一个极其重要的经济指标,而这一指标完全取决于骨骼肌的生长发育。因此,猪骨骼肌生长发育机理的研究是十分必要的。然而,在早期由于各种因素的限制,猪骨骼肌单个基因的研究一直进展缓慢;相反,以小鼠为模型,其骨骼肌单基因的功能研究却取得了较大进展。在这一时期,影响肌决定和肌分化的基因,如MRFs家族和MEF2家族相继被发现,这些基因在猪的肌肉发育中也发挥着同样的作用。然而,这些结果并不能很好地揭示骨骼肌发育过程中复杂的基因间互作关系。随着近年来芯片和测序技术的不断发展,更多人试图从整个转录谱的水平来阐述猪肌肉发育的分子机理,并且也取得了较大的进展。为了对猪骨骼肌生长发育有一个更为清晰的认识,该文将以目前猪骨骼肌生长发育研究结果为基础,同时结合模式动物小鼠骨骼肌单基因的研究成果,对猪的骨骼肌生长发育分子调控机理进行详细的阐述。     

Identification and analysis of the gene cluster involved in biosynthesis of paenibactin, a catecholate siderophore produced by Paenibacillus elgii B69

Bacteria belonging to the genus Paenibacillus are recognized as rich sources of bioactive natural products. To date, there are few characterized siderophores from this genus. Here, through genome analysis, we identified a non-ribosomal peptide biosynthetic gene cluster (pae) responsible for siderophore assembly in Paenibacillus elgii B69. The 12.8 kb gene cluster comprises six open reading frames encoding proteins similar to the components of the bacillibactin biosynthetic machinery and bacillibactin esterase. To examine the product of the pae gene cluster, we cultured P. elgii B69 in iron-deficient medium for siderophore expression. A novel siderophore structurally similar to bacillibactin, designated paenibactin, was purified and characterized. Its structure was determined as a cyclic trimeric lactone of 2,3-dihydroxybenzoyl-alanine-threonine. The involvement of the pae gene cluster in paenibactin biosynthesis was confirmed by the biochemical assay of adenylation domain specificity. Furthermore, we demonstrated that the pae gene cluster evolves from an ancestral bacillibactin biosynthetic gene cluster via sequence and phylogenetic analyses. The structural difference between paenibactin and bacillibactin may stem from a mutation-induced change in the adenylation domain specificity. Based on these findings and published models for bacillibactin, we proposed models for paenibactin biosynthesis, ferric-paenibactin uptake and paenibactin-bounded iron release.     

Amino acid changes in P3, and not the overlapping pipo-encoded protein, determine virulence of soybean mosaic virus on functionally immune Rsv1-genotype soybean

A small open reading frame, termed 'pipo', is embedded in the P3 cistron of potyviruses. Currently, knowledge on pipo and its role(s) in the life cycle of potyviruses is limited. The P3 and helper-component proteinase (HC-Pro) cistrons of Soybean mosaic virus (SMV) harbour determinants affecting virulence on functionally immune Rsv1-genotype soybeans. Interestingly, a key virulence determinant of SMV on Rsv1-genotype soybeans (i.e. soybeans containing the Rsv1 resistance gene) that resides at polyprotein codon 947 overlaps both P3 and a pipo-encoded codon. This raises the question of whether PIPO or P3 is the virulence factor. To answer this question, the corresponding pipo of an avirulent and two virulent strains of SMV were studied by comparative genomics, followed by syntheses and analyses of site-directed mutants. Our data demonstrate that the virulence of SMV on Rsv1-genotype soybeans is affected by P3 and not the overlapping pipo-encoded protein.     

Coordinate expression of multiple proteins in plant cells by exploiting endogenous kex2p-like protease activity

Simultaneous expression of multiple proteins in plants finds ample applications. Here, we examined the biotechnological application of native kex2p-like protease activity in plants for coordinate expression of multiple secretory proteins from a single transgene encoding a cleavable polyprotein precursor. We expressed a secretory red fluorescent protein (DsRed) or human cytokine (GMCSF), fused to a downstream green fluorescent protein (GFP) by a linker containing putative recognition sites of the kex2p-like protease in tobacco cells and referred to them as RKG and GKG cells, respectively. Our analyses showed that GFP is cleaved off the fusion proteins and secreted into the media by both RKG and GKG cells. The cleaved GFP product displayed the expected fluorescence characteristics. Using GFP immunoprecipitation and fluorescence analysis, the cleaved DsRed product in the RKG cells was found to be functional as well. However, DsRed was not detected in the RKG culture medium, possibly due to its tetramer formation. Cleaved and biologically active GMCSF could also be detected in GKG cell extracts, but secreted GMCSF was found to be only at a low level, likely because of instability of GMCSF protein in the medium. Processing of polyprotein precursors was observed to be similarly effective in tobacco leaf, stem and root tissues. Importantly, we also demonstrated that, via agroinfiltration, polyprotein precursors can be efficiently processed in plant species other than tobacco. Collectively, our results demonstrate the utility of native kex2p-like protease activity for the expression of multiple secretory proteins in plant cells using cleavable polyprotein precursors containing kex2p linker(s).     

Metabolically engineered soybean seed with enhanced threonine levels: biochemical characterization and seed‐specific expression of lysine‐insensitive variants of aspartate kinases from the enteric bacterium Xenorhabdus bovienii

Threonine (Thr) is one of a few limiting essential amino acids (EAAs) in the animal feed industry, and its level in feed rations can impact production of important meat sources, such as swine and poultry. Threonine as well as EAAs lysine (Lys) and methionine (Met) are all synthesized via the aspartate family pathway. Here, we report a successful strategy to produce high free threonine soybean seed via identification of a feedback‐resistant aspartate kinase (AK) enzyme that can be over‐expressed in developing soybean seed. Towards this goal, we have purified and biochemically characterized AK from the enteric bacterium Xenorhabdus bovienii (Xb). Site‐directed mutagenesis of XbAK identified two key regulatory residues Glu‐257 and Thr‐359 involved in lysine inhibition. Three feedback‐resistant alleles, XbAK_T359I, XbAK_E257K and XbAK_E257K/T359I, have been generated. This study is the first to kinetically characterize the XbAK enzyme and provide biochemical and transgenic evidence that Glu‐257 near the catalytic site is a critical residue for the allosteric regulation of AK. Furthermore, seed‐specific expression of the feedback‐resistant XbAK_T359I or XbAK_E257K allele results in increases of free Thr levels of up to 100‐fold in R₁ soybean seed when compared to wild‐type. Expression of feedback‐sensitive wild‐type AK did not substantially impact seed Thr content. In addition to high Thr, transgenic seed also showed substantial increases in other major free amino acid (FAA) levels, resulting in an up to 3.5‐fold increase in the total FAA content. The transgenic seed was normal in appearance and germinated well under greenhouse conditions.     

MTML-msBayes: Approximate Bayesian comparative phylogeographic inference from multiple taxa and multiple loci with rate heterogeneity

Background  

MTML-msBayes uses hierarchical approximate Bayesian computation (HABC) under a coalescent model to infer temporal patterns of divergence and gene flow across codistributed taxon-pairs. Under a model of multiple codistributed taxa that diverge into taxon-pairs with subsequent gene flow or isolation, one can estimate hyper-parameters that quantify the mean and variability in divergence times or test models of migration and isolation. The software uses multi-locus DNA sequence data collected from multiple taxon-pairs and allows variation across taxa in demographic parameters as well as heterogeneity in DNA mutation rates across loci. The method also allows a flexible sampling scheme: different numbers of loci of varying length can be sampled from different taxon-pairs.     

Predicting disease-associated substitution of a single amino acid by analyzing residue interactions

Background  

The rapid accumulation of data on non-synonymous single nucleotide polymorphisms (nsSNPs, also called SAPs) should allow us to further our understanding of the underlying disease-associated mechanisms. Here, we use complex networks to study the role of an amino acid in both local and global structures and determine the extent to which disease-associated and polymorphic SAPs differ in terms of their interactions to other residues.