An imaging system for standardized quantitative analysis of <Emphasis Type="Italic">C. elegans</Emphasis> behavior

Background  

The nematode Caenorhabditis elegans is widely used for the genetic analysis of neuronal cell biology, development, and behavior. Because traditional methods for evaluating behavioral phenotypes are qualitative and imprecise, there is a need for tools that allow quantitation and standardization of C. elegans behavioral assays.     

Nest architecture and genetic differentiation in a species complex of Australian stingless bees

We investigated the taxonomic significance of nest shape and its putative role in speciation in Trigona (Heterotrigona) carbonaria and T. (H.) hockingsi, two sibling species of stingless bee species from eastern Australia. These species are primarily distinguished by their nest architecture, as in all other respects they are nearly identical. We genotyped 130 colonies from six locations in Queensland at 13 microsatellite loci together with 106 additional colonies from six other Indo-Pacific Trigona species. Whether they were present in allopatry or in sympatry, colonies that displayed the T. carbonaria or the T. hockingsi nest architecture could be unambiguously differentiated at the genetic level. However, T. hockingsi colonies were classifiable into two highly differentiated paraphyletic and geographically separate populations, one in northern and one in southern Queensland. These two populations probably belong to two distinct species, T. hockingsi and T. davenporti nov. sp. Our results suggest that nest architecture characters are relevant but not sufficient criteria to identify species in this group. Consequently, modifications of nest architecture are probably not of prime importance in the speciation process of Australian stingless bees, although nest architecture differences probably result from relatively simple mechanisms. The rare interspecific hybrid colonies detected did not display a nest with an intermediate form between T. hockingsi and T. carbonaria.     

Isolation by distance and sharp discontinuities in gene frequencies: implications for the phylogeography of an alpine insect species, Carabus solieri

Analysis of genetic isolation by distance (IBD) is of prime importance for the study of processes responsible for spatial population genetic structure and is thus frequently used in case studies. However, the identification of a significant IBD pattern does not necessarily imply the absence of sharp discontinuities in gene frequencies. Therefore, identifying barriers to gene flow and/or secondary contact between differentiated entities remains a major challenge in population biology. Geographical genetic structure of 41 populations (1080 individuals) of an alpine insect species, Carabus solieri, was studied using 10 microsatellite loci. All populations were significantly differentiated and spatially structured according to IBD over the entire range. However, clustering analyses clearly identified three main clusters of populations, which correspond to geographical entities. Whereas IBD also occurs within each cluster, population structure was different according to which group of populations was considered. The southernmost cluster corresponds to the most fragmented part of the range. Consistently, it was characterized by relatively high levels of differentiation associated with low genetic diversity, and the slope of the regression of genetic differentiation against geographical distances was threefold those of the two other clusters. Comparisons of within-cluster and between-cluster IBD patterns revealed barriers to gene flow. A comparison of the two approaches, IBD and clustering analyses, provided us with valuable information with which to infer the phylogeography of the species, and in particular to propose postglacial colonization routes from two potential refugia located in Italy and in southeastern France. Our study highlights strongly the possible confounding contribution of barriers to gene flow to IBD pattern and emphasizes the utility of the model-based clustering analysis to identify such barriers.     

Design of bivalent ligands using hydrogen bond linkers: synthesis and evaluation of inhibitors for human beta-tryptase

We exploit the concept of using hydrogen bonds to link multiple ligands for maintaining simultaneous interactions with polyvalent binding sites. This approach is demonstrated by the syntheses and evaluation of pseudo-bivalent ligands as potent inhibitors of human β-tryptase.     

Cloning and expression of a putative transferrin cDNA of the spruce budworm, Choristoneura fumiferana

A spruce budworm (Choristoneura fumiferana) transferrin cDNA (CfTf) was isolated and cloned from a cDNA library that was constructed using mRNA from fifth to sixth instar larvae. CfTf cDNA encoded a predicted protein of 681 amino acids with a molecular mass of approximately 76 kDa. CfTf shared 72% and 74% identities at the amino acid level with transferrins of Manduca sexta and Bombyx mori, respectively. Like other transferrins, CfTf retains most of the N-terminal, iron-binding amino acid residues. Northern blot analyses indicated that CfTf mRNA was present at high levels after ecdysis, but that the expression level was low prior to ecdysis at the fourth-sixth instar stages. The highest level of CfTf expression was detected in the fat body. Relatively low levels of expression were detected in the epidermis and no expression was found in the midgut. Expression of CfTf mRNA could be induced by bacteria but not fungi. Expression of CfTf mRNA was suppressed by iron load.     

可视化动缘探测系统检测心肌细胞舒缩功能

目的:分离成年大鼠心室肌细胞,利用可视化动缘探测系统检测其收缩/舒张功能.方法:常规酶解法分离制备钙耐受心肌细胞;用Ca2 荧光探剂fura-2/AM(0.5μmol/L)负载心肌细胞30 min(25℃)后,以含氧台氏液灌流并予电场刺激(0.5 Hz,5 ms),采用动缘探测系统检测心肌细胞收缩/舒张功能及细胞内荧光信号变化.结果:可视化动缘探测系统可实时同步观察和记录心肌细胞机械功能和细胞内钙瞬变变化,并可计算得到细胞收缩/舒张和钙瞬变的变化幅度、时程及速率等一系列指标,从而直接反映细胞水平的心肌功能改变.结论:可视化动缘探测系统为人们提供了一个在单心肌细胞水平探讨心肌肌源性功能及其改变的新方法,是当今心血管研究的一项重要技术.     

Effect of Enhanced UV-B Radiation on Polyamine Content and Membrane Permeability in Cucumber Leaves

Cucumber plants (Cucumis sativus L., cv. Jingchun 3) were grown in a greenhouse under PAR illumination of 400–600 mol/(m² s) at 30/15°C (day/night) temperature. Two enhanced biologically effective UV-B radiation levels per day were applied: 8.82 kJ/m² (T₁) and 12.6 kJ/m² (T₂). Cucumber seedlings were irradiated 7 h per day for 25 days under T₁ and T₂. A comparative study of growth, membrane permeability, and polyamine content in cucumber leaves under T₁ and T₂ treatments was conducted. UV-B radiation resulted in the dose-dependent decrease in leaf area, dry weight of foliage, and plant height. The T₁ and T₂ treatments caused an increase in the contents of putrescine, spermine, and spermidine. However, the total polyamine content declined slightly when electrolyte leakage increased dramatically on the 18th day of treatment, especially after T₂ treatment. It can be concluded that polyamine accumulation in the cucumber leaves is an adaptive mechanism to the stress caused by UV-B radiation.     

p38 MAP kinase-dependent regulation of endothelial cell permeability

We have previously shown that thrombin induces endothelial cell barrier dysfunction via cytoskeleton activation and contraction and have determined the important role of endothelial cell myosin light chain kinase (MLCK) in this process. In the present study we explored p38 MAP kinase as a potentially important enzyme in thrombin-mediated endothelial cell contractile response and permeability. Thrombin induces significant p38 MAP kinase activation in a time-dependent manner with maximal effect at 30 min, which correlates with increased phosphorylation of actin- and myosin-binding protein, caldesmon. Both SB-203580 and dominant negative p38 adenoviral vector significantly attenuated thrombin-induced declines in transendothelial electrical resistance. Consistent with these data SB-203580 decreased actin stress fiber formation produced by thrombin in endothelium. In addition, dominant negative p38 had no effect on thrombin-induced myosin light chain diphosphorylation. Thrombin-induced total and site-specific caldesmon phosphorylation (Ser789) as well as dissociation of caldesmon-myosin complex were attenuated by SB-203580 pretreatment. These results suggest the involvement of p38 MAP kinase activities and caldesmon phosphorylation in the MLCK-independent regulation of thrombin-induced endothelial cell permeability.     

Expression, purification, and antifreeze activity of carrot antifreeze protein and its mutants

Antifreeze proteins (AFPs) enable organisms to survive under freezing or sub-freezing conditions. AFPs have a great potential in the low temperature storage of cells, tissues, organs, and foods. This process will require a large number of recombinant AFPs. In the present study, the recombinant carrot AFP was highly expressed in Escherichia coli strain BL21 (DE3). The activity of the purified and refolded recombinant proteins was analyzed by measurement of thermal hysteresis (TH) activity and detection of in vitro antifreeze activity by measuring enhanced cold resistance of bacteria. Two carrot AFP mutants generated by site-directed mutagenesis were also expressed and purified under these conditions for use in parallel experiments. Recombinant DcAFP displayed a TH activity equivalent to that of native DcAFP, while mutants DcAFP-N130Q and rDcAFP-N130V showed 32 and 43% decreases in TH activity, respectively. Both the recombinant DcAFP and its mutants were able to enhance the cold resistance of bacteria, to degrees consistent with their respective TH activities.     

A single amino acid change (Asp 53 --> Ala53) converts Survivin from anti-apoptotic to pro-apoptotic

Survivin is a member of the inhibitor of apoptosis protein (IAP) family that has been implicated in both apoptosis inhibition and cell cycle control. Recently, Survivin has attracted growing attention because of its tumor-specific expression and potential applications in tumor therapy. However, its inhibitory mechanism and subcellular localization remain controversial. Here, we report a novel Survivin mutant Surv-D53A, which displays a function opposite to Survivin and a distinctive subcellular distribution compared with its wild-type counterpart. Surv-D53A was shown to induce apoptosis in a p53-independent manner, indicating that tumor suppressor p53 is not involved in its apoptosis pathway. Surv-D53A was shown to markedly sensitize apoptosis induced by TRAIL, doxorubicin, and RIP3. We also demonstrated that similar to wild-type Survivin, Surv-D53A was localized in cytoplasm in interphase and to midbody at telophase. However, it fails to colocalize in chromosomes with Aurora-B in metaphase as wt-Survivin. Surv-D53A mutant is less stable than wt-Survivin and is degraded more rapidly by ubiquitin-proteasome pathway. Additionally, we found that Surv-D53A interacts with wt-Survivin to form heterodimer or with itself to form mutant homodimer, which may account for the loss of its antiapoptotic function. Finally, unlike Survivin*Survivin, neither Surv-D53A*Survivin nor Surv-D53A*Surv-D53A is able to bind to Smac/DIABLO, which may explain the underlying mechanism for its abolishment of antiapoptotic activity of Survivin.