Comparative proteomic analysis of mesenchymal stem cells derived from human bone marrow,umbilical cord,and placenta: Implication in the migration

Umbilical cord (UC) and placenta (P) have been suggested as alternatives to bone marrow (BM) as sources of mesenchymal stem cells (MSC) for cell therapy, with both UC‐ and P‐MSC possess immunophenotypic and functional characteristics similar to BM‐MSC. However, their migration capacity, which is indispensable during tissue regeneration process, is unclear. Under defined conditions, the migration capacity of BM‐ and P‐MSC was found 5.9‐ and 3.2‐folds higher than that of UC‐MSC, respectively. By the use of 2‐DE and combined MS and MS/MS analysis, six differentially expressed proteins were identified among these MSC samples, with five of them known to be involved in cell migration as migration enhancing or inhibiting proteins. Consistent with their migration capacity, the levels of migration enhancing proteins including cathepsin B, cathepsin D and prohibitin,were significantly lower in UC‐MSC when compared with those in BM‐ and P‐MSC. For the migration inhibiting proteins such as plasminogen activator inhibitor‐1 (PAI‐1) and manganese superoxide dismutase, higher expression was found in the UC‐MSC. We also showed that the overexpression of the PAI‐1 impaired the migration capacity of BM‐ and P‐MSC while silencing of PAI‐1 enhanced the migration capacity of UC‐MSC. Our study indicates that PAI‐1 and other migration‐related proteins are pivotal in governing the migration capacity of MSC.     

蔗糖磷酸合成酶(SPS)与果实品质及成熟衰老的研究进展

从蔗糖磷酸合成酶（sucrose phosphate synthase,SPS）基本性质、SPS与果实糖的积累、相关酶类和外源刺激、乙烯和呼吸、果实软化的火系及SPS基因的克隆与表达调控等几方面综述了国内外近年来对SPS的研究进展。     

蝇蛆几丁低聚糖咀嚼片的调节血脂作用

研究和评价了蝇蛆几丁低聚糖咀嚼片的调节血脂作用及其安全性。将50只实验动物（大鼠）随机分为普通饲料对照组、高脂饲料对照组和3个实验组,实验组分别喂以不同剂量蝇蛆几丁低聚糖咀嚼片和高脂饲料。饲养28天后,分别测定大鼠血清总胆固醇（total cholesterol, TC）、血清总甘油三酯（triglyceride, TG）、血清高密度脂蛋白胆固醇（high density lipoprotein cholesterol, HDLC）的含量。用Horn's法对蝇蛆几丁低聚糖咀嚼片的急性毒性进行研究,连续观察7天,记录各组动物的中毒反应情况和死亡只数,计算咀嚼片对小鼠的半数致死剂量。同时对几丁低聚糖咀嚼片作了调节血脂的动物试验,研究结果表明,蝇蛆几丁低聚糖咀嚼片对大鼠血清总胆固醇、总甘油三酯有明显降低和对血清高密度脂蛋白胆固醇有明显稳定作用,具有辅助降血脂作用;对咀嚼片进行急性毒性实验表明,蝇蛆几丁低聚糖咀嚼片对小鼠的半数致死剂量大于10 g/kg·bw,提示其基本无毒。
     

双效表达载体的构建及其U6启动子的功能效率鉴定

利用pBudcE4.1双表达载体构建shRNA与蛋白共表达载体,为双效疫苗的研制提供新的研究思路.以含U6启动子的载体为模板,PCR扩增得到U6启动子,用其置换载体pBudcE4.1内的CMV启动子的核心部分构建shRNA与蛋白共表达载体.用干扰绿色荧光蛋白表达的方法鉴定重组载体中的U6启动子能否启动shRNA的表达.经PCR扩增、双酶切鉴定及DNA测序证明成功构建了载体pBudcE4.1-U6.用干扰载体pBudcE4.1-U6-eGFPshRNA与含eGFP的载体共转染293T细胞后,荧光显微镜观察显示eGFP的表达量下降;流式细胞仪检测细胞的转染效率降低.研究结果证明U6启动子正常发挥作用. 成功构建RNAi与蛋白共表达载体,为利用该载体研制动物双效疫苗奠定了基础.     

萘乙酸与多效唑对茉莉成花及新梢内源激素含量的影响

在茉莉开花前期分别使用不同浓度NAA与PP333均匀喷施于植株茎、叶片等生长部位,对其新梢与花蕾生长及其4种内源激素--生长素(IAA)、赤霉素(GA)、脱落酸(ABA)、玉米素核苷(ZR)的含量变化进行分析.结果表明:(1)NAA处理使茉莉新梢徒长,成花比对照推迟2～4 d;PP333处理的茉莉新梢矮化,而成花比对照提早2～4 d.(2)NAA处理后,茉莉新梢中IAA与ABA含量处理初期较高,后快速下降,后期稳定在较高水平;GA含量稍低于对照,ZR含量降低并稍低于对照.PP333处理后,茉莉新梢中IAA与ABA含量初期较高,而后缓慢下降;GA含量与对照一样快速上升;ZR含量在初期含量较高,后缓慢下降,但较对照稳定在较高水平.(3)PP333处理的茉莉植株新梢中ABA/IAA、GA/IAA、ZR/IAA比值在处理后迅速上升,特别是GA/IAA、ZR/IAA比值明显高于对照,而相应NAA处理的3个比值与对照无明显差异.可见,经NAA与PP333处理能明显调节茉莉新梢及花蕾生长进程,保持较高水平的内源激素GA、ZR、GA/IAA、ZR/IAA比值在茉莉新梢及花蕾生长过程中起关键作用.     

A model for binding of an antifreeze polypeptide to ice.

A model is proposed, based on recent peptide analog and ice crystal etching studies, whereby an alanine-rich, alpha-helical antifreeze polypeptide (AFP) from the winter flounder inhibits the growth of ice crystals by hydrogen bonding of Thr, Asn, and Asp side chains in a specific pattern to the [2021] hexagonal bipyramidal planes of ice. It is further suggested that this mode of binding is unidirectional, maximizing opportunities for packing of AFPs on the ice surface, and that ice crystal growth inhibition occurs by a two-step mechanism involving hydrogen bonding and hydrophobic interpeptide interactions.     

R54C Mutation of NOTCH3 Gene in the First Rungus Family with CADASIL

Cerebral autosomal dominant arteriopathy with subcortical infarcts and leukoencephalopathy (CADASIL) is a rare hereditary stroke caused by mutations in NOTCH3 gene. We report the first case of CADASIL in an indigenous Rungus (Kadazan-Dusun) family in Kudat, Sabah, Malaysia confirmed by a R54C (c.160C>T, p.Arg54Cys) mutation in the NOTCH3. This mutation was previously reported in a Caucasian and two Korean cases of CADASIL. We recruited two generations of the affected Rungus family (n = 9) and found a missense mutation (c.160C>T) in exon 2 of NOTCH3 in three siblings. Two of the three siblings had severe white matter abnormalities in their brain MRI (Scheltens score 33 and 50 respectively), one of whom had a young stroke at the age of 38. The remaining sibling, however, did not show any clinical features of CADASIL and had only minimal changes in her brain MRI (Scheltens score 17). This further emphasized the phenotype variability among family members with the same mutation in CADASIL. This is the first reported family with CADASIL in Rungus subtribe of Kadazan-Dusun ethnicity with a known mutation at exon 2 of NOTCH3. The penetrance of this mutation was not complete during the course of this study.     

Sevoflurane Inhibits Nuclear Factor-κB Activation in Lipopolysaccharide-Induced Acute Inflammatory Lung Injury via Toll-Like Receptor 4 Signaling

Background

Infection is a common cause of acute lung injury (ALI). This study was aimed to explore whether Toll-like receptors 4 (TLR₄) of airway smooth muscle cells (ASMCs) play a role in lipopolysaccharide (LPS)-induced airway hyperresponsiveness and potential mechanisms.

Methods

In vivo: A sensitizing dose of LPS (50 µg) was administered i.p. to female mice before anesthesia with either 3% sevoflurane or phenobarbital i.p. After stabilization, the mice were challenged with 5 µg of intratracheal LPS to mimic inflammatory attack. The effects of sevoflurane were assessed by measurement of airway responsiveness to methacholine, histological examination, and IL-1, IL-6, TNF-α levels in bronchoalveolar lavage fluid (BALF). Protein and gene expression of TLR₄ and NF-κB were also assessed. In vitro: After pre-sensitization of ASMCs and ASM segments for 24h, levels of TLR₄ and NF-κB proteins in cultured ASMCs were measured after continuous LPS exposure for 1, 3, 5, 12 and 24h in presence or absence of sevoflurane. Constrictor and relaxant responsiveness of ASM was measured 24 h afterwards.

Results

The mRNA and protein levels of NF-κB and TLR₄ in ASM were increased and maintained at high level after LPS challenge throughout 24h observation period, both in vivo and in vitro. Sevoflurane reduced LPS-induced airway hyperresponsiveness, lung inflammatory cell infiltration and proinflammatory cytokines release in BALF as well as maximal isometric contractile force of ASM segments to acetylcholine, but it increased maximal relaxation response to isoproterenol. Treatment with specific NF-κB inhibitor produced similar protections as sevoflurane, including decreased expressions of TLR₄ and NF-κB in cultured ASMCs and improved pharmacodynamic responsiveness of ASM to ACh and isoproterenol.

Conclusions

This study demonstrates the crucial role of TLR₄ activation in ASMCs during ALI in response to LPS. Sevoflurane exerts direct relaxant and anti-inflammatory effects in vivo and in vitro via inhibition of TLR₄/NF-κB pathway.     

Inhibition of the Inositol Kinase Itpkb Augments Calcium Signaling in Lymphocytes and Reveals a Novel Strategy to Treat Autoimmune Disease

Emerging approaches to treat immune disorders target positive regulatory kinases downstream of antigen receptors with small molecule inhibitors. Here we provide evidence for an alternative approach in which inhibition of the negative regulatory inositol kinase Itpkb in mature T lymphocytes results in enhanced intracellular calcium levels following antigen receptor activation leading to T cell death. Using Itpkb conditional knockout mice and LMW Itpkb inhibitors these studies reveal that Itpkb through its product IP4 inhibits the Orai1/Stim1 calcium channel on lymphocytes. Pharmacological inhibition or genetic deletion of Itpkb results in elevated intracellular Ca²⁺ and induction of FasL and Bim resulting in T cell apoptosis. Deletion of Itpkb or treatment with Itpkb inhibitors blocks T-cell dependent antibody responses in vivo and prevents T cell driven arthritis in rats. These data identify Itpkb as an essential mediator of T cell activation and suggest Itpkb inhibition as a novel approach to treat autoimmune disease.