Role of myosin light chain kinase in regulation of basal blood pressure and maintenance of salt-induced hypertension

Vascular tone, an important determinant of systemic vascular resistance and thus blood pressure, is affected by vascular smooth muscle (VSM) contraction. Key signaling pathways for VSM contraction converge on phosphorylation of the regulatory light chain (RLC) of smooth muscle myosin. This phosphorylation is mediated by Ca(2+)/calmodulin-dependent myosin light chain kinase (MLCK) but Ca(2+)-independent kinases may also contribute, particularly in sustained contractions. Signaling through MLCK has been indirectly implicated in maintenance of basal blood pressure, whereas signaling through RhoA has been implicated in salt-induced hypertension. In this report, we analyzed mice with smooth muscle-specific knockout of MLCK. Mesenteric artery segments isolated from smooth muscle-specific MLCK knockout mice (MLCK(SMKO)) had a significantly reduced contractile response to KCl and vasoconstrictors. The kinase knockout also markedly reduced RLC phosphorylation and developed force. We suggest that MLCK and its phosphorylation of RLC are required for tonic VSM contraction. MLCK(SMKO) mice exhibit significantly lower basal blood pressure and weaker responses to vasopressors. The elevated blood pressure in salt-induced hypertension is reduced below normotensive levels after MLCK attenuation. These results suggest that MLCK is necessary for both physiological and pathological blood pressure. MLCK(SMKO) mice may be a useful model of vascular failure and hypotension.     

Novel Bacillus thuringiensis δ-endotoxin active against Locusta migratoria manilensis

A novel δ-endotoxin gene was cloned from a Bacillus thuringiensis strain with activity against Locusta migratoria manilensis by PCR-based genome walking. The sequence of the cry gene was 3,432 bp long, and it encoded a Cry protein of 1,144 amino acid residues with a molecular mass of 129,196.5 kDa, which exhibited 62% homology with Cry7Ba1 in the amino acid sequence. The δ-endotoxin with five conserved sequence blocks in the amino-terminal region was designated Cry7Ca1 (GenBank accession no. {"type":"entrez-nucleotide","attrs":{"text":"EF486523","term_id":"1121787749","term_text":"EF486523"}}EF486523). Protein structure analysis suggested that the activated toxin of Cry7Ca1 has three domains: 227 residues forming 7 α-helices (domain I); 213 residues forming three antiparallel β-sheets (domain II); and 134 residues forming a β-sandwich (domain III). The three domains, respectively, exhibited 47, 44, and 34% sequence identity with corresponding domains of known Cry toxins. SDS-PAGE and Western blot analysis showed that Cry7Ca1, encoded by the full-length open reading frame of the cry gene, the activated toxin 1, which included three domains but without the N-terminal 54 amino acid residues and the C terminus, and the activated toxin 2, which included three domains and N-terminal 54 amino acid residues but without the C terminus, could be expressed in Escherichia coli. Bioassay results indicated that the expressed proteins of Cry7Ca1 and the activated toxins (toxins 1 and 2) showed significant activity against 2nd instar locusts, and after 7 days of infection, the estimated 50% lethal concentrations (LC₅₀s) were 8.98 μg/ml for the expressed Cry7Ca1, 0.87 μg/ml for the activated toxin 1, and 4.43 μg/ml for the activated toxin 2. The δ-endotoxin also induced histopathological changes in midgut epithelial cells of adult L. migratoria manilensis.     

The affinity of human RANK binding to its ligand RANKL

Receptor activator of nuclear factor-kappa B (RANK) and its ligand, RANKL play critical roles in bone re-modeling, immune function, vascular disease and mammary gland development. To study the interaction of RANK and RANKL, we have expressed both extracellular domain of RANK and ectodomain of RANKL using Escherichia coli expression system. RANK was expressed as an inclusion body first which properly refolded later, while RANKL was initially produced as a GST fusion protein, after which the GST was removed by enzyme digestion. Soluble RANK existed as a monomer while RANKL was seen as a trimer in solution, demonstrated by gel filtration chromatography and cross-linking experiment. The recombinant RANK and RANKL could bind to each other and the binding affinity of RANKL for RANK was measured with surface plasmon resonance technology and K_D value is about 1.09 × 10⁻¹⁰ M.     

微生物群落结构探针杂交评价不同培养基从活性污泥分离优势菌群的能力

用ERICPCR （Enterobacterial Repetitive Intergenic ConsensusPCR）、苯酚羟化酶大亚基基因(LmPHs)扩增和群落结构探针分子杂交检测技术对LB、dCGY、MP和FWM 4种培养基从焦化废水处理厂2个曝气池活性污泥中分离优势功能菌群的能力进行了比较研究。LmPHs扩增显示7种回收菌群中均有以多亚基苯酚羟化酶为代谢途径的苯酚降解菌存在。用代表苯酚降解高峰期活性污泥优势菌组成的总DNA的ERICPCR产物经地高辛标记作为群落结构的混合探针M1和M8,对8种回收菌群的ERICPCR指纹图谱进行杂交检测,不同培养基回收优势菌的能力不同,以废水为基础的FWM培养基从活性污泥中回收到的优势菌种群最多(30.8%～42.9%)。本文建立了用微生物群落结构探针杂交技术对不同培养基回收分离优势菌能力进行评价的方法。     

Phylogenetic analysis of Nosema antheraeae (Microsporidia) isolated from Chinese oak silkworm, Antheraea pernyi

The microsporidian Nosema antheraeae is a pathogen that infects the Chinese oak silkworm, Antheraea pernyi. We sequenced the complete small subunit (SSU) rRNA gene and the internal transcribed spacer (ITS) of N. antheraeae, and compared the SSU rRNA sequences in other microsporidia. The results indicated that Nosema species, including N. antheraeae, formed two distinct clades, consistent with previous observations. Furthermore, N. antheraeae is clustered with N. bombycis with high bootstrap support. The organization of the rRNA gene of N. antheraeae is LSU-ITS1-SSU-ITS2-5S, also following a pattern similar to the Nosema type species, N. bombycis. Thus, N. antheraeae is a Nosema species and has a close relationship to N. bombycis.     

Combinatorial signals of activin/nodal and bone morphogenic protein regulate the early lineage segregation of human embryonic stem cells

Cell fate commitment of pre-implantation blastocysts, to either the inner cell mass or trophoblast, is the first step in cell lineage segregation of the developing human embryo. However, the intercellular signals that control fate determination of these cells remain obscure. Human embryonic stem cells (hESCs) provide a unique model for studying human early embryonic development. We have previously shown that Activin/Nodal signaling contributes to maintaining pluripotency of hESCs, which are derivatives of the inner cell mass. Here we further demonstrate that the inhibition of Activin/Nodal signaling results in the loss of hESC pluripotency and trophoblast differentiation, similar to BMP4-induced trophoblast differentiation from hESCs. We also show that the trophoblast induction effect of BMP4 correlates with and depends on the inhibition of Activin/Nodal signaling. However, the activation of BMP signaling is still required for trophoblast differentiation when Activin/Nodal signaling is inhibited. These data reveal that the early lineage segregation of hESCs is determined by the combinatorial signals of Activin/Nodal and BMP.     

买麻藤根的异常次生生长

买麻藤(Cnetum montanum)根的异常次生生长与茎的异常次生生长相似,位于维管束外围的薄壁组织细胞可以形成维管束,以这种方式使根加粗。并且在生长过程中以同样的方式,在维管束的外围不断形成新的维管束。这些新的维管束成环状排列,因此,在老根中呈多圈的维管束。与茎唯一不同的是根的异常次生生长为不均等的,在两个宽大的射线区外侧,没有异常的维管束形成,因此,根主要向着与两条宽大射线相垂直的方向扩展,故外形呈扁圆形。