低氧诱导因子和角质细胞生长因子双基因重组腺病毒的构建及其在肺泡上皮细胞中的表达

目的：构建同时携带低氧诱导因子-1α（HIF-1α）和角质细胞生长因子（KGF）N腺病毒载体（pAdxsi-GFP-HIF-KGF）,观察其在防治肺损伤潜在的应用前景。方法：低氧处理A549细胞后提取总RNA并逆转录为eDNA作为模板,依据GeneBank公布的HIF-1α cDNA设计引物,并分别引入KpnI和BamHI酶切位点,PCR扩增后将目的基因HIF-1α连接到载体pShuttle-CMV-EGFP上,构建重组质粒pShuttle-GFP—HIF。然后以质粒plRES2-EGFP-KGF为模板,用引入NheI和PmeI酶切位点的引物PCR扩增KGF基因并克隆到重组质粒pShuttle-GFP-HIF上,获得穿梭质粒重组质粒pShuttle—GFP-HIF—KGF。采用细菌内重组方法将目的序列重组到pAdxsi病毒骨架栽体上构建携带HIF．10t和KGF双基因的重组腺病毒载体pAdxsi-GFP-HIF-KGF。检测重组腺病毒滴度后,转染人肺泡上皮细胞A549,检测目的基因的转染表达。结果：通过对构建质粒克隆进行测序及酶切,证实携带HIF—lot和KGF双基因的重组腺病毒载体pAdxsi-GFP-HIF-KGF构建成功,且构建的重组腺病毒纯度好、滴度高。用pAdxsi-GFP-HIF-KGF以100MOI转染A549细胞后24h后在荧光显微镜下可观察到细胞有较强的绿色荧光表达,48h时荧光更强;转染48hELISA法检测培养上清中HIF-1蛋白表达水平为（56．36±4．53）ng／mL,KGF蛋白表达水平为（60．20±2．92）ng／mL。结论：成功构建了腺病毒栽体pAdxsi-GFP-HIF-KGF,其转染效率及目的基因的蛋白表达水平较高,具有潜在的进一步在肺损伤局部应用的前景,为后期制备可以同时发挥KGF、HIF-1作用的基因治疗药物打下基础,同时为高海拔地区应激性急性肺损伤的有效防治提供实验基础。     

基因枪在大鼠闭塞性血管病基因治疗中的应用

探讨利用基因枪技术转移肝细胞生长因子基因治疗大鼠肢体闭塞性血管病的可行性,构建了携带人肝细胞生长因子（HGF）基因的重组真核表达载体（pUDKH),在制备好大鼠下肢闭塞性血管病模型后,通过基因枪或肌肉直接注射法,向局部缺血部位肌肉中转移pUDKH,每只5ug(基因枪）和12ug(肌肉注射）,应用常规组织病理切片（H．E．染色）及免疫组织化学方法观察血管形成及基因表达,转移pUDKH后第10天,用基因枪和直接注射法转移的局部肌肉组织的HGF的表达明显高于转移空白质粒（pUDK)的对照组,pUDKH组可见明显的小血管新生,而pUKD组至20天时仍未观察到或仅见到极少量的新生血管,基因枪与肌肉注射两组相比血管密度无明显差异,采用基因枪直接转移pUDKH裸露质粒子肢体缺血局部的方法是可行的,转移的基因可在局部有效表达,达到促进血管形成的预期目的。     

大豆疫霉菌的EMS化学诱变

以甲基磺酸乙酯(ethylmethane sulfonate,EMS)为诱变剂,通过其对大豆疫霉菌Phytophthora sojae休止孢萌发的影响,确定化学诱变条件。通过收集单卵孢子,建立了包含640个单卵孢子系的突变体库,其中约有50%的诱变菌系在培养性状和菌落形态方面发生了明显变化,菌落形态多样,表现出较紧密或松散,近圆形或不规则;气生菌丝减少,生长速度较慢或快;在卵孢子产量方面,8.13%的菌系有增加,20.41%的菌系减少,27.82%的菌系极少或者没有卵孢子产生,43.64%的菌系卵孢子产量类似野生型。以质膜氢离子泵蛋白基因PsPMA1(plasma membrane H+-ATPase1)为对象,通过TILLING技术,从320个大豆疫霉菌突变体中获得9个突变体,进一步确认了EMS对大豆疫霉菌的诱变效果,并且估算EMS对大豆疫霉菌的诱变频率至多每115kb发生一个核苷酸变异。新构建的突变体库为开展大豆疫霉病菌的功能基因组研究奠定了遗传材料基础。     

Mapping functional connectivity

An objective and reliable assessment of wildlife movement is important in theoretical and applied ecology. The identification and mapping of landscape elements that may enhance functional connectivity is usually a subjective process based on visual interpretations of species movement patterns. New methods based on mathematical morphology provide a generic, flexible, and automated approach for the definition of indicators based on the classification and mapping of spatial patterns of connectivity from observed or simulated movement and dispersal events. The approach is illustrated with data derived from simulated movement on a map produced from satellite imagery of a structurally complex, multi-habitat landscape. The analysis reveals critical areas that facilitate the movement of dispersers among habitat patches. Mathematical morphology can be applied to any movement map providing new insights into pattern-process linkages in multi-habitat landscapes.     

Inflammatory mediators and cartilage biomarkers in synovial fluid after a single inflammatory insult: a longitudinal experimental study

Introduction  

Inflammation is an important feature of many joint diseases, and levels of cartilage biomarkers measured in synovial fluid may be influenced by local inflammatory status. Little is known about the magnitude and time course of inflammation-induced changes in cartilage tissue turnover as measured in vivo by synovial fluid markers. We aimed to study temporal changes in concentrations of inflammatory mediators, matrix metalloproteinase activity and cartilage biomarkers over 1 week in joints with experimentally induced inflammation.     

Genetic variation in TIMP1 but not MMPs predict excess FEV1 decline in two general population-based cohorts

Background

An imbalance in Matrix MetalloProteases (MMPs) and Tissue Inhibitors of MMPs (TIMPs) contributes to Chronic Obstructive Pulmonary Disease (COPD) development. Longitudinal studies investigating Single Nucleotide Polymorphisms (SNPs) in MMPs and TIMPs with respect to COPD development and lung function decline in the general population are lacking.

Methods

We genotyped SNPs in MMP1 (G-1607GG), MMP2 (-1306 C/T), MMP9 (3 tagging SNPs), MMP12 (A-82G and Asn357Ser) and TIMP1 (Phe124Phe and Ile158Ile) in 1390 Caucasians with multiple FEV₁ measurements from a prospective cohort study in the general population. FEV₁ decline was analyzed using linear mixed effect models adjusted for confounders. Analyses of the X-chromosomal TIMP1 gene were stratified according to sex. All significant associations were repeated in an independent general population cohort (n = 1152).

Results

MMP2 -1306 TT genotype carriers had excess FEV₁ decline (-4.0 ml/yr, p = 0.03) compared to wild type carriers. TIMP1 Ile158Ile predicted significant excess FEV₁ decline in both males and females. TIMP1 Phe124Phe predicted significant excess FEV₁ decline in males only, which was replicated (p = 0.10) in the second cohort. The MMP2 and TIMP1 Ile158Ile associations were not replicated. Although power was limited, we did not find associations with COPD development.

Conclusions

We for the first time show that TIMP1 Phe124Phe contributes to excess FEV₁ decline in two independent prospective cohorts, albeit not quite reaching conventional statistical significance in the replication cohort. SNPs in MMPs evidently do not contribute to FEV₁ decline in the general population.     

Pleuston communities are buffered from regional flood pulses: the example of ostracods in the Paraná River floodplain, Brazil

1. It is widely acknowledged that sudden, large‐scale flood pulses are drivers of benthic and planktonic biodiversity change in floodplains. The impact of such pulses on pleuston (biotic communities associated with root systems of floating plants) remains to be demonstrated. Here, we investigate the effects of local and regional drivers on seasonal changes in abundance and diversity of ostracod communities in pleuston. 2. Temporal and spatial distribution patterns of species richness, abundance, diversity and evenness of ostracods associated with the floating water hyacinth, Eichhornia crassipes, in a lentic environment from the upper Paraná River floodplain, were investigated in relation to local, as well as regional, environmental factors. Ostracods were sampled monthly over an annual cycle (March 2004–February 2005). Twenty‐seven species were found, representing the families Cyprididae, Candonidae, Limnocytheridae and Darwinulidae. Both diversity and abundance of ostracod communities showed seasonal changes, although species turn‐over during the year was limited. 3. We tested two hypotheses concerning the causality of these fluctuations: seasonal recruitment and influx of allochthonous ostracods during the flood pulse. Our results indicate that seasonal recruitment is more likely to be the driver of fluctuations in relation to the flood pulse. We postulate that pleuston communities are buffered against possible detrimental effects of flood pulses.     

Reproductive patterns of selected understorey trees in the Malaysian rain forest: the apomictic species

HA, C. O., SANDS, V. E., SOEPADMO, E. & JONG, K., 1988. Reproductive patterns of selected understorey trees in the Malaysian raia forest: the apomictic species. Garcinia is the predominant representative of the Clusiaceae in the understorey of the lowland rain forest of Peninsular Malaysia. In Pasoh Forest Reserve the dioecious species G. paruifolia was investigated since no male trees, but only trees with structurally hermaphodite flowers which later set fruit, were found there. Pollination and embryological studies indicated a condition of non-pseudogamous agamospermy, with no viable pollen grains being formed in the staminodes, and the unfertilized egg cell giving rise to the embryo. Supportive studies were made of microspore development in G. forbesi , and of embryogenesis and seed development in G. malaccensis and G. scortechinii which also appear to be agamospermous. The role of either facultative or obligate apomixis in gene fixation in these dioecious species is discussed, and the possible adaptive significance of reproductive versatility among certain rain forest trees is examined.     

Regeneration of Asparagus officinalis L. through Callus Cultures Derived from Protoplasts

Undifferentiated callus derived from asparagus protoplast cultureshas been used for studies on organogenesis. Root and shoot formationhas been obtained with different hormonal balances. Adeninehas been found to be effective, together with a cytokinin, inpromoting the formation of somatic embryoids in this tissue.     

The oligomerization of a family of four genetically clustered human gastrointestinal mucins

Mucins are synthesized and secreted by many epithelia. They are complex glycoproteins that offer cytoprotection. In their functional configuration, mucins form oligomers by a biosynthetic process that is poorly understood. A family of four human gastrointestinal mucin genes (MUC2, MUC5AC, MUC5B, and MUC6) is clustered to chromosome 11p15.5. To study oligomerization of these related mucins, we performed metabolic labeling experiments with [35S]amino acids in LS174T cells, and isolated mucin precursors by specific immunoprecipitations that were analyzed on SDS-PAGE. Each of the precursors of MUC2, MUC5AC, MUC5B, and MUC6 formed a single species of disulfide-linked homo-oligomer within 1 h after pulse labeling. Based on apparent molecular masses, these oligomeric precursors were most likely dimers. Inhibition of vesicular RER-to-Golgi transport, with brefeldin A and CCCP, did not affect the dimerization of MUC2 precursors, localizing dimerization to the RER. O-Glycosylation of MUC2 followed dimerization. Inhibition of N- glycosylation by tunicamycin retarded, but did not inhibit, dimerization, indicating that N-glycans play a role in efficient dimerization of MUC2 precursors. Based on sequence homology, the ability of MUC2, MUC5AC, MUC5B and MUC6 to dimerize most likely resides in their C-terminal domains. Thus, the RER-localized dimerization of secretory mucins likely proceeds by similar mechanisms, which is an essential step in the formation of the human gastrointestinal mucus- gels.