Distinction of strains of bean common mosaic virus and blackeye cowpea mosaic virus using antibodies to N- and C- or N-terminal peptide domains of coat proteins

Earlier attempts to discriminate serologically strains NL1, NL3 and NY15 of bean common mosaic virus (BCMV) and strain W of blackeye cowpea mosaic virus (B1CMV) had been unsuccessful. Antibodies directed towards N- and C-, or N-terminal peptide regions of the coat proteins of the above strains enabled the distinction between B1CMV-W, BCMV-NY15 and BCMV-NL3 in electroblot immunoassay and in ELISA. The distinction was better with antibodies directed towards N-termini than with those to N- and C-termini. Strain NL1 of BCMV cross-reacted with both B1CMV-W and BCMV-NY15, but not with BCMV-NL3. Taxonomic implications of these findings are discussed.     

Origins of immunoglobulin heavy chain domains

Summary Using computer programs that analyze the evolutionary history and probability of relationship of protein sequences, we have investigated the gene duplication events that led to the present configuration of immunoglobulin C regions, with particular attention to the origins of the homology regions (domains) of the heavy chains. We conclude that all of the sequenced heavy chains share a common ancestor consisting of four domains and that the two shorter heavy chains, alpha and gamma, have independently lost most of the second domain. These conclusions allow us to align corresponding regions of these sequences for the purpose of deriving evolutionary trees. Three independent internal gene duplications are postulated to explain the observed pattern of relationships among the four domains: first a duplication of the ancestral single domain C region, followed by independent duplications of the resulting first and last domains. In these studies there was no evidence of crossing-over and recombination between ancestral chains of different classes; however, certain types of recombinations would not be detectable from the available sequence data.     

Energetic determinants of animal cell polarity regulator Par-3 interaction with the Par complex

The animal cell polarity regulator Par-3 recruits the Par complex (consisting of Par-6 and atypical PKC, aPKC) to specific sites on the cell membrane. Although numerous physical interactions have been reported between Par-3 and the Par complex, it is unclear how each of these interactions contributes to the overall binding. Using a purified, intact Par complex and a quantitative binding assay, here, we found that the energy required for this interaction is provided by the second and third PDZ protein interaction domains of Par-3. We show that both Par-3 PDZ domains bind to the PDZ-binding motif of aPKC in the Par complex, with additional binding energy contributed from the adjacent catalytic domain of aPKC. In addition to highlighting the role of Par-3 PDZ domain interactions with the aPKC kinase domain and PDZ-binding motif in stabilizing Par-3–Par complex assembly, our results indicate that each Par-3 molecule can potentially recruit two Par complexes to the membrane during cell polarization. These results provide new insights into the energetic determinants and structural stoichiometry of the Par-3–Par complex assembly.     

Structural insights into the role of the WW2 domain on tandem WW–PPxY motif interactions of oxidoreductase WWOX

Class I WW domains are present in many proteins of various functions and mediate protein interactions by binding to short linear PPxY motifs. Tandem WW domains often bind peptides with multiple PPxY motifs, but the interplay of WW–peptide interactions is not always intuitive. The WW domain–containing oxidoreductase (WWOX) harbors two WW domains: an unstable WW1 capable of PPxY binding and stable WW2 that cannot bind PPxY. The WW2 domain has been suggested to act as a WW1 domain chaperone, but the underlying mechanism of its chaperone activity remains to be revealed. Here, we combined NMR, isothermal calorimetry, and structural modeling to elucidate the roles of both WW domains in WWOX binding to its PPxY-containing substrate ErbB4. Using NMR, we identified an interaction surface between these two domains that supports a WWOX conformation compatible with peptide substrate binding. Isothermal calorimetry and NMR measurements also indicated that while binding affinity to a single PPxY motif is marginally increased in the presence of WW2, affinity to a dual-motif peptide increases 10-fold. Furthermore, we found WW2 can directly bind double-motif peptides using its canonical binding site. Finally, differential binding of peptides in mutagenesis experiments was consistent with a parallel N- to C-terminal PPxY tandem motif orientation in binding to the WW1–WW2 tandem domain, validating structural models of the interaction. Taken together, our results reveal the complex nature of tandem WW-domain organization and substrate binding, highlighting the contribution of WWOX WW2 to both protein stability and target binding.     

Identification and characterization of FHL3 as a novel angiogenin-binding partner

Angiogenin (Ang) is known to induce cell proliferation and inhibit apoptosis by cellular signaling pathways and by direct nuclear functions of Ang, but the mechanism of action for Ang is not yet clear. The aim of present study was to identify novel binding partner of Ang and to explore the underlying mechanism. With the use of yeast two-hybrid screening system, Ang was used as the bait to screen human fetal hepatic cDNA library for interacting proteins. Four and a half LIM domains 3 (FHL3) was identified as a novel Ang binding partner. The interaction between Ang and the full length FHL3 was further confirmed by yeast two-hybrid assay, co-immunoprecipitation and GST pull-down assays. Furthermore, FHL3 was required for Ang-mediated HeLa cell proliferation and nuclear translocation of Ang. These findings suggest that the interaction between Ang and FHL3 may provide some clues to the mechanisms of Ang-regulated cell growth and apoptosis.     

Structure and physiological importance of angiotensin converting enzyme domains

Somatic angiotensin converting enzyme (ACE) consists of two homologous catalytic domains (N- and C-domain), exhibiting different biochemical properties. The catalytically active ACE isoforms consisted of just one domain have been also detected in mammals. Substantial progress in ACE domain research was achieved during the last years, when their crystal structures were determined. The crystal structures of domains in complex with diverse potent ACE inhibitors provided new insights into structure-based differences of the domain active sites. Physiological functions of ACE are not limited by regulation of the cardiovascular system. Recent evidence suggests that the ACE domains may be also involved into control of different physiological functions. The C-terminal catalytic domain plays an important role in the regulation of blood pressure: it catalyzes angiotensin I cleavage in vivo. The N-domain contributes to the processing of other bioactive peptides for which it exhibits high affinity. The role of the N-domain is not ultimately associated with functioning of the rennin-angiotensin system and it contributes processing of other bioactive peptides for which it exhibits high affinity (goralatide, luliberin, enkephalin heptapeptide, beta-amyloid peptide). Domain-selective inhibitors selectively blocking either the N- or C-domain of ACE have been developed.     

P—选择素凝集素—EGF功能域的克隆、表达及其单克隆抗体制备

制备特异性抗人P 选择素的凝集素 表皮生长因子 (L EGF)功能域的单克隆抗体。利用特异引物 ,通过RT PCR从外周血血小板中扩增出人P 选择素的L EGF功能域基因 ,将其克隆至pET42b( )载体中 ,测序验证后转染大肠杆菌BL2 1,经诱导表达了C端融合 6×His的蛋白质。融合蛋白质经分离纯化后 ,免疫Balb/c小鼠 ,应用杂交瘤技术 ,通过间接ELISA筛选阳性克隆。获得 3株可稳定分泌抗L EGF功能域单抗的杂交瘤细胞株 (B10、F3和H5 )。其亚型分别为IgG2 、IgG1和IgG3;轻链均为κ型。所获的单抗对LPS刺激活化的人脐静脉内皮细胞均有特异性结合反应 ,并可在体外阻断经凝血酶激活的血小板与中性粒细胞间的粘附。表明所获的单抗可特异性识别结合天然P 选择素 ,具有体外抗活化血小板与中性粒细胞粘附的功能 ,为进一步应用此单抗进行P 选择素结构和功能及抗粘附治疗研究提供了实验基础。     

Phylogenetic scale in ecology and evolution

Aim

Many important patterns and processes vary across the phylogeny and depend on phylogenetic scale. Nonetheless, phylogenetic scale has never been formally conceptualized, and its potential remains largely unexplored. Here, we formalize the concept of phylogenetic scale, review how phylogenetic scale has been considered across multiple fields and provide practical guidelines for the use of phylogenetic scale to address a range of biological questions.

Innovation

We summarize how phylogenetic scale has been treated in macroevolution, community ecology, biogeography and macroecology, illustrating how it can inform, and possibly resolve, some of the longstanding controversies in these fields. To promote the concept empirically, we define phylogenetic grain and extent, scale dependence, scaling and the domains of phylogenetic scale. We illustrate how existing phylogenetic data and statistical tools can be used to investigate the effects of scale on a variety of well‐known patterns and processes, including diversification rates, community structure, niche conservatism or species‐abundance distributions.

Main conclusions

Explicit consideration of phylogenetic scale can provide new and more complete insight into many longstanding questions across multiple fields (macroevolution, community ecology, biogeography and macroecology). Building on the existing resources and isolated efforts across fields, future research centred on phylogenetic scale might enrich our understanding of the processes that together, but over different scales, shape the diversity of life.     

Condensin-Dependent Chromatin Compaction Represses Transcription Globally during Quiescence

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