The effect of calcium on the conformation of cobalamin transporter BtuB

BtuB is a β‐barrel membrane protein that facilitates transport of cobalamin (vitamin B12) from the extracellular medium across the outer membrane of Escherichia coli. It is thought that binding of B12 to BtuB alters the conformation of its periplasm‐exposed N‐terminal residues (the TonB box), which enables subsequent binding of a TonB protein and leads to eventual uptake of B12 into the cytoplasm. Structural studies determined the location of the B12 binding site at the top of the BtuB's β‐barrel, surrounded by extracellular loops. However, the structure of the loops was found to depend on the method used to obtain the protein crystals, which—among other factors—differed in calcium concentration. Experimentally, calcium concentration was found to modulate the binding of the B12 substrate to BtuB. In this study, we investigate the effect of calcium ions on the conformation of the extracellular loops of BtuB and their possible role in B12 binding. Using all‐atom molecular dynamics, we simulate conformational fluctuations of several X‐ray structures of BtuB in the presence and absence of calcium ions. These simulations demonstrate that calcium ions can stabilize the conformation of loops 3–4, 5–6, and 15–16, and thereby prevent occlusion of the binding site. Furthermore, binding of calcium ions to extracellular loops of BtuB was found to enhance correlated motions in the BtuB structure, which is expected to promote signal transduction. Finally, we characterize conformation dynamics of the TonB box in different X‐ray structures and find an interesting correlation between the stability of the TonB box structure and calcium binding. Proteins 2010. © 2009 Wiley‐Liss, Inc.     

Value of human amniotic epithelial cells in tissue engineering for cornea

Human amniotic epithelial cells (hAECs) are potentially one of the key players in tissue engineering due to their easy availability. The aim of the present study was to develop an optimal isolation and transportation technique, as well as to determine the immunophenotype and epithelial gene expression of hAECs. Amnion was mechanically peeled off from the chorion and digested with trypsin-ethylenediaminetetraacetic acid. The isolated hAECs were cultured in medium containing 10 ng/mL epidermal growth factor until P4. The epithelial gene expression, cell surface antigen and protein expression of hAECs were analyzed by quantitative polymerase chain reaction, flow cytometry and immunocytochemistry. hAECs were also cultured in adipogenic, osteogenic and neurogenic induction media. The best cell yield of hAECs was seen in the digestion of 15 pieces of amnion (2 × 2 cm) and isolated 30 min after digestion with trypsin. F12:Dulbecco's modified eagle medium was the best medium for short term storage at 4 °C. hAECs expressed CD9, CD44, CD73 and CD90, and negligibly expressed CD31, CD34, CD45 and CD117. After serial passage, CK3, CK19 and involucrin gene expressions were upregulated, while p63, CK1 and CK14 gene expressions were downregulated. Sustained gene expressions of integrin β1 and CK18 were observed. At initial culture, these cells might have stem-like properties. However, they differentiated after serial passage. Nonetheless, hAECs have epithelial stem cell characteristics and have the potential to differentiate into corneal epithelial cells. Further investigations are still needed to elucidate the mechanism of differentiation involved and to optimize the culture condition for long term in vitro culture.     

Differentiation of primate ES cells into retinal cells induced by ES cell-derived pigmented cells

Purpose: Photoreceptors cannot regenerate and recover their functions once disordered. Transplantation of retinal pigment epithelium (RPE) has recently become a possible therapeutic approach for retinal degeneration. In the present study, we investigated the induction of photoreceptors by coculturing primate embryonic stem cells (ESCs) with ESC-derived RPE cells. Methods: RPE cells were derived by coculturing ESCs and Sertoli cells. Photoreceptors were then induced by using ESC-derived RPE cells and retinoic acid (RA) Results: RPE cell generation was confirmed by morphological analysis, which revealed highly pigmented polygonal cells with a compact cell-cell arrangement. After coculturing ESCs and RPE cells, some ESC derivatives became immunopositive for rhodopsin. RT-PCR analysis demonstrated the expression of retina-related gene markers such as Pax6, CRX, IRBP, rhodopsin, rhodopsin kinase, and Muschx10A. When RA was added, a distinct increase in the expression of photoreceptor-specific proteins and genes was found. In addition, the differentiation of bipolar horizontal cells was demonstrated by protein and gene expression. The ESCs that were cocultured with RPE cells and treated with RA were transplanted into the renal capsule or intra-vitreal space of nude mice. Grafted ESC derivatives demonstrated extensive rhodopsin expression, and they survived and organized into recipient tissues, although they formed teratomas. Conclusion: These results indicate that coculturing ESCs with ESC-derived RPE cells is a useful and efficient method for inducing photoreceptors and providing an insight into the use of ESCs for retina regeneration.     

Classification of rice (<Emphasis Type="Italic">Oryza sativa</Emphasis>l. japonica nipponbare) immunophilins (FKBPs,CYPs) and expression patterns under water stress

Background  

FK506 binding proteins (FKBPs) and cyclophilins (CYPs) are abundant and ubiquitous proteins belonging to the peptidyl-prolyl cis/trans isomerase (PPIase) superfamily, which regulate much of metabolism through a chaperone or an isomerization of proline residues during protein folding. They are collectively referred to as immunophilin (IMM), being present in almost all cellular organs. In particular, a number of IMMs relate to environmental stresses.     

Use of transposon promoter-probe vectors in the metabolic engineering of the obligate methanotroph Methylomonas sp. strain 16a for enhanced C40 carotenoid synthesis

The recent expansion of genetic and genomic tools for metabolic engineering has accelerated the development of microorganisms for the industrial production of desired compounds. We have used transposable elements to identify chromosomal locations in the obligate methanotroph Methylomonas sp. strain 16a that support high-level expression of genes involved in the synthesis of the C(40) carotenoids canthaxanthin and astaxanthin. with three promoterless carotenoid transposons, five chromosomal locations-the fliCS, hsdM, ccp-3, cysH, and nirS regions-were identified. Total carotenoid synthesis increased 10- to 20-fold when the carotenoid gene clusters were inserted at these chromosomal locations compared to when the same carotenoid gene clusters were integrated at neutral locations under the control of the promoter for the gene conferring resistance to chloramphenicol. A chromosomal integration system based on sucrose lethality was used to make targeted gene deletions or site-specific integration of the carotenoid gene cluster into the Methylomonas genome without leaving genetic scars in the chromosome from the antibiotic resistance genes that are present on the integration vector. The genetic approaches described in this work demonstrate how metabolic engineering of microorganisms, including the less-studied environmental isolates, can be greatly enhanced by identifying integration sites within the chromosome of the host that permit optimal expression of the target genes.     

Acute hyperglycemia exacerbates myocardial ischemia/reperfusion injury and blunts cardioprotective effect of GIK

There is a close association between hyperglycemia and increased risk of mortality after acute myocardial infarction (AMI). However, whether acute hyperglycemia exacerbates myocardial ischemia/reperfusion (MI/R) injury remains unclear. We observed the effects of acute hyperglycemia on MI/R injury and on the cardioprotective effect of glucose-insulin-potassium (GIK). Male rats were subjected to 30 min of myocardial ischemia and 6 h of reperfusion. Rats were randomly received one of the following treatments (at 4 ml.kg(-1).h(-1) iv): Vehicle, GIK (GIK during reperfusion; glucose: 200g/l, insulin: 60 U/l, KCL: 60 mmol/l), HG (high glucose during ischemia; glucose:500 g/l), GIK + HG (HG during I and GIK during R) or GIK + wortmannin (GIK during R and wortmannin 15 min before R). Blood glucose, plasma insulin concentration and left ventricular pressure (LVP) were monitored throughout the experiments. Hyperglycemia during ischemia not only significantly increased myocardial apoptosis (23.6 +/- 1.7% vs. 18.8 +/- 1.4%, P < 0.05 vs. vehicle), increased infarct size (IS) (45.6 +/- 3.0% vs. 37.6 +/- 2.0%, P < 0.05 vs. vehicle), decreased Akt and GSK-3beta phosphorylations (0.5 +/- 0.2 and 0.6 +/- 0.1% fold of vehicle, respectively, P < 0.05 vs. vehicle) following MI/R, but almost completely blocked the cardioprotective effect afforded by GIK, as evidenced by significantly increased apoptotic index (19.1 +/- 2.0 vs. 10.3 +/- 1.2%, P < 0.01 vs. GIK), increased myocardial IS (39.2 +/- 2.8 vs. 27.2 +/- 2.1%, P < 0.01 vs. GIK), decreased Akt phosphorylation (1.1 +/- 0.1 vs. 1.7 +/- 0.2%, P < 0.01 vs. GIK) and GSK-3beta phosphorylation (1.4 +/- 0.2 vs. 2.3 +/- 0.2%, P < 0.05 vs. GIK). Hyperglycemia significantly exacerbates MI/R injury and blocks the cardioprotective effect afforded by GIK, which is, at least in part, due to hyperglycemia-induced decrease of myocardial Akt activation.     

Mechanical stretch inhibits oxidized low density lipoprotein-induced apoptosis in vascular smooth muscle cells by up-regulating integrin alphavbeta3 and stablization of PINCH-1

To determine the mechanisms involved in regulating the balance between apoptosis and survival in vascular smooth muscle cells (VSMC), we studied anti-apoptotic stimuli that can counteract pro-apoptotic events in the process of early atherosclerotic lesions formation. Such a process involves VSMC accumulation even in the presence of oxidized low density lipoprotein (Ox-LDL). In the arch of the aorta, we find that integrin beta3 is higher than in descending arteries. In the advanced atherosclerosis lesion, we found an inverse correlation between the level of integrin beta3 and apoptosis (deoxynucleotidyltransferase-mediated dUTP nick end labeling-positive). We also found an increase in integrin alphaVbeta3 (but not integrin beta1) expression in VSMC that are subjected to cyclic stretch. VSMC subjected to stretch as well as VSMC with forced expression of alphaVbeta3 were demonstrated to be resistant to Ox-LDL-induced cytoskeleton disruption and apoptosis. The anti-apoptotic effect of stretch was abolished by treatment of VSMC with small interfering RNA against integrin beta3 as well as VSMC isolated from integrin beta3 knock-out mice. Disruption of the cytoskeleton abolished the protective effect of stretch or alphaVbeta3 overexpression on Ox-LDL-induced activation of Bax and apoptosis. We also demonstrated that stretch-mediated protection of Ox-LDL-induced apoptosis involved stabilization of PINCH-1; Ox-LDL decreased the level of PINCH-1, but the application of mechanical stretch or overexpression of either integrin beta1 or integrin beta3 prevented its down-regulation. In the arteries of integrin beta3 null mice, there were lower levels of PINCH-1 and ILK-1. Moreover, deletion of integrin beta3 in VSMC abolished the stretch protective effect on PINCH-1. Small interfering RNA-mediated knockdown of PINCH-1 disrupted the cytoskeleton and caused apoptosis of VSMC. These findings provided experimental evidence that mechanical stretch acted as a survival factor in the arches of aortas. Furthermore, mechanical stretch prevented VSMC from apoptosis via a mechanism that involves alphaVbeta3 integrin expression, stabilization of PINCH-1, and remodeling of the cytoskeleton.     

出、入肺血中ET-1、VEGF含量与慢性低氧性肺动脉高压的相关性研究

目的：观察间断性减压低氧大鼠出、入肺血血浆、肺组织匀浆和体循环动脉匀浆中内皮素-1（ET-1）的含量变化,以及出、入肺血血清中血管内皮生长因子（VEGF）的含量变化及其与低氧性肺动脉高压的关系。方法：建立间断性减压低氧模型,采用插管法分别测定左侧颈总动脉压力（CAP）、右心室平均压分别代表体循环压力和平均肺动脉压力（mPAP）,放射免疫测定法测定ET-1含量,双抗体夹心ABC—ELISA法测定VEGF含量。结果：①低氧2周组（H2）、低氧3周组（H3）大鼠平均肺动脉压力、右心肥厚指数（RV／LV＋S）均分别显著高于对照组,而各组平均颈动脉压力之间无明显变化（P〉0．05）。②对照组大鼠出肺血中ET含量比入肺血中低（P〈0．05）。H2、H3组大鼠出、入肺血中ET-1含量较对照组均明显增高（P〈0．01）,且H2、H3组大鼠出肺血中ET-1含量均高于入肺血中ET-1含量（P〈0．05）,刚好与对照组相反。H2、H3组大鼠肺组织匀浆中ET—1含量与对照组肺组织和低氧组体循环动脉组织匀浆比较,均明显增高（P〈0.01）,而各组体循环动脉组织匀浆中ET-1含量间无明显差异（P〉0．05）：③对照组大鼠出、入肺血中VEGF含量无明显差异（P〉0．05）,H2、H3组大鼠出肺血血清中VEGF含量较对照组明显增高（P〈0.01）。结论：在慢性低氧可使肺组织产生ET-1和VEGF增多,这可能是慢性肺动脉高压发生、发展的重要机制之一。肺动脉局部缩血管物质ET-1增高,而体循环动脉局部ET-1不增高。可能是长期慢性低氧可导致肺动脉高压的形成,却不发生高血压的机制之一。