Prokaryotic Expression,Purification,Antibody Production of a NH_2-Terminal Fragment of mCLCA3 Protein and Analysis of mClCA3 Protein Expression in Asthmatic Mouse Lung

mCLCA3 is a member of calcium activated chloride channel（CACC） family that may play an important role in mucin packaging and secretion in asthmatic and cystic fibrosis lung. To study the protein structure and expression of mCLCA3 in asthmatic mouse lung, an N-terminal 269 amino acid peptide of mCLCA3 was expressed in E. coli, purified to homogeneity and rabbit polyclonal antibodies against this peptide were generated. Immunohistochemistry of asthmatic mouse lung using the antibody indicated exclusive mCLCA3 expression in mucin granules of goblet cells in airway surface and lumen, Immunoblot analysis of lavage fluid from asthmatic mouse lung revealed a single 90 kDa protein form of mClCA3. The results demonstrate that the 90 kDa N-terminal peptide, neither the flail-length protein nor the reported N-terminal 35 kDa cleaved form of mClCA3 is the major functional form involved in the packaging and exocytosis of mucin granules in asthmatic goblet cells.

Aquaporin 1 Facilitated Hepatocellular Carcinoma SMMC7221 Cell Migration Associated with Water Permeability

The authors investigated the regulation of human aquaporin I（hAQP1） and the involvement of aquaporin 1 （AQP 1） in the migration of human hepatocellular carcinoma SMMC-7221 cells using RNA intereference technology Firstly, two short hairpin RNA（shRNA） constructs in PBSU6 vector were reconstructed and their knockdown effects were identified in SMMC-7221 cells. Next, the involvement of endogenous hAQP1 in regulating the migration of SMMC-7221 cells was investigated via siRNA technology. HAQPI-shRNA can specifically inhibit AQP1 dependent osmotic water permeability. Meanwhile the migration of SMMC-7221 cells was inhibited remarkably after silencing AQP1 by performing transwell cell migration assay and in vitro wound healing assay. Furthermore, in the presence of an inhibitor HgCl2, the water permeability of the cell membrane was remarkably decreased, the expression of AQP1 was upregulated after HgCla treatment and the cell movement was decreased at the moment. Increased AQP1 cannot attenuate cell migration ability when cell membrane loses its water permeability function. This demonstrates that the cell migration was remarkably related to the transporting water function of cell membrane.

Association of Matrix Metalloproteinase-9 and p53 Gene Polymorphisms with Genetic Susceptibility to No-small-cell Lung Cancer

Matrix metalloproteinase-9（MMP-9） and p53 genes play an essential role in the multi-step process of tumorigenesis in lung cancer. Single nucleotide polymorphisms（SNPs） of MMP-9 and p53 genes are associated with the risk and progression of many cancers. In this study, we evaluated the association of the R279Q polymorphism of MMP-9 or the A1/A2 polymorphism of p53 gcne with the risk of no-small-cell lung cancer（NSCLC） in Han population of Northeast China. We examined the frequency of SNPs in the two kinds of genes of 50 patients with NSCLC and 50 cancer-free controls frequency-matched by age and sex. Polymerase chain reaction-restriction fragment length polymorphism（PCR-RFLP） technique was used to determine the genotypes. The results indicate that the 279RR genotype in MMP-9 gene and the A1/A2 genotype in p53 gene show a significantly increased risk of NSCLC. Therefore, the MMP-9 279RR and p53 A1/A2 genotypes may be used as markers for susceptibility to NSCLC in Han population of Northeast China.

Remarkably Decreased CD11b Positive Splenocytes in Aquaporin 3-Null Mice

Aquaporins（AQPs） are molecular water channels that play important physiological roles in fluid trans-porting organs. The expression and function of AQPs in the immune system are largely unknown. CD 11 （a-d）/CD18 integrins are adhesion molecules expressed on leukocytes, which play a critical role in leukocyte adhesion, migration and host defense. In the present study, we discovered the expression of aquaporin-3（AQP3） on spleen CD1 lb positive cells, and the content of CDllb positive splenocytes in aquaporin 3-null mice is significantly decreased. Further analysis suggested remarkably decreased monocyte/macrophage subpopulation and significantly decreased granulocyte subpopulation. It is the first report suggesting an important role of AQP in the development and maturation of imrnunocytes.

血清miRNA-210、miRNA-223预测新生儿缺血缺氧性脑病预后的临床价值

目的探讨血清miRNA-210、miRNA-223预测新生儿缺血缺氧性脑病预后的临床价值。方法选取新生儿缺血缺氧性脑病患儿124例作为观察组,根据新生儿磁共振成像(magnetic resonance imaging,MRI)病情严重程度将其分为轻度组51例、中度组40例、重度组33例,并选取同期60例健康新生儿作为对照组。然后根据患儿出生后28 d内预后情况将患儿分为生存组81例与死亡组43例。采用实时荧光定量聚合酶链式反应(polymerasechainreaction,PCR)检测血清微小RNA-210(mircoRNA-210,miRNA-210)、微小RNA-223(mircoRNA-223,miRNA-210)水平,分析其水平对预测新生儿缺血缺氧性脑病预后的临床价值。结果新生儿缺血缺氧性脑病患儿血清miRNA-210、miRNA-223表达量均高于健康对照组(P<0.05),重度组患儿血清miRNA-210、miRNA-223水平高于轻度组和中度组患儿,差异有统计学意义(P<0.05)。单因素分析结果显示,开始治疗日龄、Apgar评分、缺血缺氧性脑病病情严重程度、白细胞介素6、C反应蛋白、血清miRNA-210表达量、血清miRNA-223表达量与新生儿缺血缺氧性脑病患儿预后有关(P<0.05)。多因素分析结果显示,开始治疗日龄高于2.25 d(OR=3.554,95%CI:1.300~9.713)、病情严重程度未重度(OR=4.450,95%CI:2.189~9.048)、血清miRNA-210升高(OR=3.117,95%CI:1.598~6.082)、血清miRNA-223升高(OR=3.384,95%CI:1.790~6.398)是影响新生儿缺血缺氧性脑病患儿预后的独立危险因素(P<0.05)。ROC曲线分析结果显示,血清miRNA-210最佳分界值为1.71时,预测新生儿缺血缺氧性脑病患儿预后的曲线下面积为0.76,此时敏感度为78.62%,特异度为76.35%;miRNA-223最佳分界值为1.48时,预测新生儿缺血缺氧性脑病患儿预后的曲线下面积为0.79,此时敏感度为81.54%,特异度为76.18%;二者联合检测预测新生儿缺血缺氧性脑病患儿预后的曲线下面积为0.85,此时敏感度为86.73%,特异度为75.49%。结论新生儿缺血缺氧性脑病患儿miRNA-210、miRNA-223升高,其水平升高与患儿病情严重程度及预后密切相关,miRNA-210、miRNA-223升高对预测新生儿缺血缺氧性脑病患儿预后具有较好预测价值。

红毛藻抗氧化肽的菠萝蛋白酶酶解制备工艺优化及其活性评价

以红毛藻(Bangia fuscopurpurea (Dillw.) Lyngb.)蛋白为原料,经菠萝蛋白酶水解制备抗氧化肽,为红毛藻的深加工提供理论依据.以酶解液对DPPH·的?清除率为响应值,考察酶解温度、酶解时间、酶解pH、酶底比对酶解液抗氧化活性的影响.在单因素试验基础上,通过L_9(3~4)正交试验优化红毛藻抗氧化肽的酶解制备工艺,并以酶解产物对O_2~ˉ·的清除能力和还原力为指标评价酶解产物的抗氧化活性.结果表明,用菠萝蛋白酶酶解红毛藻蛋白制备抗氧化肽的最优工艺参数为:酶解温度45℃、酶解时间90 min、酶解pH7.5、酶底比1200U/g.在最优酶解工艺条件下,红毛藻蛋白的水解度达到(10.74±0.13)%,酶解液中多肽浓度为(6.98±0.07) mg/mL,酶解产物对DPPH·和O_2~ˉ·的半数清除浓度(IC_(50))值分别为(0.4403±0.0142) mg/mL和(4.2600±0.0180) mg/mL,且具备一定的还原力.

Molecular Cloning of a Glycyrrhiza uralensis F.Aquaporin GuPIP1 Up-regulated in Response to Drought,Salt and ABA Stress

A PCR-bosed homologous cloning strategy was used to identify aquaporin genes from the roots of Chinese licorice （ Glycyrrhiza uralertsis F. ）. A 1236 bp cDNA with 870 bp open reading frame encoding a 290 amino acids aquaporin ortholog, GuPIP1, was successfully cloned and characterized. The deduced GuPIP1 protein contains six putative transmembrane domains; two conserved NPA motifs as well as the MIP and PIP family signature sequences. A rabbit polyelonal antibody against N-terminal peptide of GuPIP1 corresponded to a 31 kDa GuPIP1 protein on Western blot of plasma membrane preparation of root tissue. RT-PCR and Western blot analysis indicated the expression of GuPIP1 in the root, leaf, and stem tissues. Thus far, GuPIP1 is the first Glycyrrhiza uralensis F. aquaporin that has been identified at a molecular level. Quantitative real-time PCR analysis showed that the expression of GuPIP1 was up-regulated in response to drought, ABA, and salt stress.

Aquaporin-3 Deletion Reduces Glycerol and ATP Content in Mouse Sciatic Nerve

We reported the expression and function of aquaglyceroporin AQP3 in mouse peripheral nervous system.AQP3 mRNA was identified in freshly isolated mouse sciatic nerve by RT-PCR.Immunofluorescence using AQP3 antibody localized the protein expression in the myelin sheathe of sciatic nerve fibers.Although primary morpholo-gical evaluation of sciatic nerves from AQP3-knockout and wildtype mice revealed no significant difference,biochemical analysis demonstrated remarkably decreased glycerol and ATP contents in freshly isolated AQP3-knockout sciatic nerve.The study indicates an important role of facilitated glycerol transport mechanism in peripheral nerve energy metabolism.

Reduced Bone Mineral Density and Bone Metabolism in Aquaporin-1 Knockout Mice

An overt phenotype of aquaporin-1 knockout（AQP1 ko） mice is growth retardation, suggesting possible defects in bone development and metabolism. In the present study, we analyzed the bone mineral density（ BMD）, bone calcium and phosphorus contents, and bone metabolism in an AQP1 ko mouse model. The BMD of femurs in AQP1 ko mice was significantly lower than that of litter-matched wildtype mice as measured by dual energy X-ray absorptiometry. Consistently, the contents of bone total calcium and phosphorus were also significantly lower in AQP1 ko mice. The reduced BMD caused by AQP1 deficiency mainly affect male mice. Bone metabolic activity, as indicated by 99m^Tc-MDP absorption measurements, was remarkably reduced in AQP1 ko mice. These results provide the first evidence that AQP1 play an important role in bone structure and metabolism.

GST Fusion Protein Based Specific Polyclonal Antibody Preparation of Mouse Aquaporin 1

Aquaporins（AQPs） are specific membrane channels for water and other small nonionic molecules.In order to overcome the difficulties to generate the effictive antibody of membrane protein,we selected the cytoplasmic C-terminus of Aquaporin 1（AQP1） as an unique antigen.The long C-terminus of mouse AQP1 was overexpressed in the Glutathione S-tansferase Gene Fusion System.On the basis of the resonable amounts of soluable membrane protein peptides,we prepared the specific antibody.To pursure this object,we constructed pGEX-4T-1/mAQP1（DNA sequence from 700 to 801 bp） recombinant plasmid and transformed it into Escherichia coli BL21 cells.The GST-AQP1 C-terminal hydrophilic peptide fusion protein was induced by IPTG and further purified by Glutathione Sepharose 4B to obtain the right size fusion protein.Then we immunized the New Zealand rabbits to prepare the antiserum.The purified AQP1 antibody showed high sensitivity by ELISA assay and high specificity by Western blot with AQP1 null mice served as negative control.Finally,we also checked the AQP1 localization in the mouse renal tissues in wild type of mice and AQP1 null mice served as negative control.We demonstrated that AQP1 was highly expressed at the descending limb of Henle tube using our purified AQP1 antibody,which was consistent with previous report.The successful design and preparation of AQP1 antibody through GST technique is an example as making antibodies against a specific membrane protein.

Natural Compound Curcumin—a Channel Potentiator Rather Than a Corrector of the Defective Intracellular Processing of △F508 Mutant Cystic Fibrosis Transmembrane Conductance Regulator

Cystic fibrosis（CF） is a severe genetic disease caused by the gene mutation of the cystic fibrosis transmembrane conductance regulator（CFTR） chloride channel. The most common point mutation AF508, which leads to impaired intracellular processing and channel gating of CFTR, appears in about 90% CF patients. The natural compound curcumin was reported to correct the processing defect of AF508-CFTR and proposed as a potential therapeutic drug to cure CF. In the present study, we analyzed the effect of curcumin on AF508-CFTR and demonstrated that curcumin can restore the impaired chloride conductance of AF508 mutant CFTR. The activity is rapid, reversible and cAMP-dependent. However, we couldn＇t reproduce the previously reported correction of the defective membrane trafficking of AF508-CFTR by curcumin. Therefore, curcumin may not be a superior lead compound for developing anti-CF drugs.

Activation of CFTR-mediated Cl^- Transport by Capsaicinoids in Cell Culture Model

Previous studies reported that capsaicin potentiates AF508 mutant cystic fibrosis transmembrane conductance regulator（CFTR） channel gating defect by transfected cell-based assays. It has been postulated that orally ingested capsaicin may conceptually be used to develop a therapeutic strategy to treat gastrointestinal disorders in CF patients. We tried to reproduce and extend those pre-clinical data of previous studies. Cell-based fluorescence func- tional measurements in Fischer thyroid epithelial cells（FRT） expressing CFTR showed no effect of capsaicin on potentiating AF508-CFTR, while genistein showed a strongly positive activity. Studies show that capsaicin and dihy- drocapsaicin activated cAMP-prestimulated wild-type CFTR in a dose-dependent manner with a maximal response of 70% of that activated by genistein, thus gave an apparent EC50 of （40.4±6.8）μmol/L and （150.2±7.4） μtmol/L respectively. Preliminary study shows that the binding sites for capsaicin and dihydrocapsaicin may be probably partially overlapped with that for genistein because the maximal activation of wild-type CFTR with genistein is partially blocked by caosaicin and dihydrocapsaicin.

cDNA Cloning, Prokaryotic and Eukaryotic Expression and Characterization of Porcine Leukemia Inhibitory Factor

Molecular cloning of the porcine leukemia inhibitor factor（pLIF） has not been reported. A full-length eDNA encoding pLIF was cloned, expressed and characterized. The full-length porcine LIF cDNA encodes a 202 amino acid protein that has an 84% sequence identity to mouse LIF and 86% sequence identity to human LIF. The deduced amino acid sequence of a pLIF protein contains six conserved consensus N-linked glycosylation sites and six cysteine groups to form potential disulfide bonds. The pLIF was expressed in E coli, as a mature form, and in CHO cells as a secreted form. Both the forms of the recombinant pLIFs can maintain murine embryonic stem cells in an undifferentiated state in a culture. The recombinant pLiFs will be useful in establishing a long-term culture of stable pluripotent porcine embryonic stem cells for further manipulation.

Natural Compound Curcumin Corrects the Gating Defect of G551DMutant Cystic Fibrosis Transmembrane Conductance Regulator

In the present study, we identified the natural compound curcumin to be an effective G551D-CFTR activator by cell-based fluorescent assay and electrophysiological measurement. We demonstrated that curcumin can restore the impaired chloride conductance of G551D mutant CFTR. The activity is rapid, reversible, and cAMP-dependent. Our study identified a new natural lead compound for the pharmacological therapy of cystic fibrosis caused by G551D mutation of CFTR.

Identification of Herbal Compound Imperatorin with Adverse Effects on ANO1 and CFTR Chloride Channels

Calcium-activated chloride channels（CaCCs） are the crucial regulators of transepithelial fluid secretion, smooth muscle contraction and sensory transduction. Recently, compelling evidence has indicated that TMEM16A（ANO1 or anoctamin-1） is a bona fide calcium-acvtivated chloride channel. A few small molecule CaCCs regulators are available for functional and therapeutic studies. We screened 126 natural compounds from Chinese herbs. Screening was performed with an iodide influx assay in Fischer rat thyroid epithelial cells to coexpress ANO1 and an iodide-sensitive fluorescent indicator（EYFP-H148Q/I152L）. Imperatorin, a coumarin compound, was identified to inhibit ANO1-mediated chloride transport activated by multiple calcium-elevating agonists. The inhibitory effect is dose-dependent with IC50～14.63 μmol/L. Interestingly, imperatorin activated CFTR chloride channel with EC50～35.52 μmol/L. The adverse effects of imperatorin on CaCC and CFTR chloride channels will make it useful in pharmacological dissection of chloride transport in airway and intestinal epithelium. Further studies are required to evaluate the therapeutic effects of imperatorin on hypertension, asthma and certain tumors.

Expression and Emmprin Regulated Function of Aquaporinl in SMMC7221 Cells

Herein lie the crosstalk and regulation between AQP1 and Emmprin in SMMC7221 cells by means of siRNA technology and deglycosylation method. Firstly, HAQP1, rather than hAQP3, was selectively upregulated in SMMC7221 cells by FBS, flollowed by the upregulated expression of Emmprin. Emmprin gene silencing caused a remarkable change in the expression ofAQP1 gene, just like its downstream gene, MMP9, meanwhile the water permeability and cell migration were also descended prominently. Furthermore, when treated with tunicamycin, Emmprin was deglycosylated, which made the expression of AQP 1 significantly declined, followed by remarkably decreased cell membrane water permeability and cell migration. Taken together, all the data indicates the expression level and the modification of Emmprin by glycosylation are the key factors in regulating the expression of AQP1.

Activation Effect of Cathartic Natural Compound Rhein to CFTR Chloride Channel

The cystic fibrosis transmembrane conductance regulator （CFFR） is a cAMP-activated chloride channel expressed in intestinal exoerine glands, which plays a key role in intestinal fluid secretion. A natural anthraquinone ac tivator of CFTR Cl^- channel, rhein, was identified by screening 217 single compounds from Chinese herbs via a cellbased halide-sensitive fluorescent assay. Rhein activates CFTR Cl^- transportation in a dose-dependent manner in the presence of cAMP with a physiological concentration. This study provides a novel molecular pharmacological mechanism for the laxative drugs in Treditional Chinese Medicine such as aloe, cascara and senna.

某市高中新生中结核分枝杆菌潜伏感染筛查情况及防治对策分析

目的:分析佳木斯市区高中新生中结核分枝杆菌潜伏感染筛查结果及其防治对策,为学校制定新生结核病管理工作提供参考。方法:收集市区内8所高中新生的结核菌素纯蛋白衍生物(tuberculin-purified protein derivative,TB-PPD)检测结果,分析和比较普通高中与职业高中新生强阳性、中度阳性和一般阳性发生率的差异。结果:8所高中新生中,3 563人学生经TB-PPD筛查阴性率仅为46.65%、一般阳性率为29.41%、中度阳性率为18.72%,强阳性率为5.22%;女生中强阳性率高于男生(χ^(2)=4.658,P<0.05),<15岁年龄组学生的强阳性率高于≥15岁年龄组,(χ^(2)=4.156,P<0.05)。结论:新生入学结核病筛查是控制学校结核病疫情的重要手段,学校应建立新生入学结核病筛查的长效机制;对TB-PPD筛查的强阳性者应进行健康宣教、预防性服药、后续的监测管理工作,从而降低学生结核病的患病率。

菠萝液泡膜水通道蛋白基因AcTIP1-1的克隆、重组载体构建

以菠萝(Ananas comosus(Linn.)Merr.)果实为材料,通过RT-PCR分别扩增液泡膜水通道蛋白基因(AcTIP1-1)(GenBank登录号:XM_020238793.1)的开放阅读框(ORF)和编码区(CDS)序列,序列长度分别为753 bp和750 bp.将ORF序列亚克隆到载体pET32a(+)上,CDS序列被亚克隆到载体pSP64 Poly(A)上,构建成重组载体pET32a(+)-AcTIP1-1和pSP64 Poly(A)-AcTIP1-1.pET32a(+)-AcTIP1-1可为诱导表达并纯化融合蛋白制备抗体,pSP64 Poly(A)-AcTIP1-1可为制备含有Poly(A)+的AcTIP1-1转录子奠定基础.

菠萝质膜水通道蛋白基因AcPIP2-4的克隆、重组载体构建

本研究以菠萝(Ananas comosus (L.) Merrill)果实为材料提取总RNA并合成cDNA.根据菠萝质膜水通道蛋白基因(AcPIP2-4)序列(GenBank登录号:XM020240565.1)设计1对特异性引物,通过RT-PCR从果实cDNA中扩增AcPIP2-4的ORF序列,并将该序列亚克隆至原核表达载体pET32a(+).经酶切鉴定和序列分析,结果表明,原核表达重组载体pET32a(+)-AcPIP2-4构建成功.以重组载体pET32a(+)-AcPIP2-4质粒为模板,通过RT-PCR扩增AcPIP2-4的CDS序列,再将其亚克隆至载体pSP64 Poly(A)上.经酶切鉴定及序列分析,结果表明,重组载体pSP64 Poly(A)-AcPIP2-4构建成功.重组载体pET32a(+)-AcPIP2-4和pSP64 Poly(A)-AcPIP2-4的成功构建,为后续制备抗AcPIP2-4的抗体和AcPIP2-4 c的RNA,进而进行AcPIP2-4的组织表达和功能研究奠定基础.