Design and synthesis of novel hydrophilic spacers for the reduction of nonspecific binding proteins on affinity resins

Tubulin and actin often bind nonspecifically to affinity chromatography resins, complicating research toward identifying the cellular targets. Reduction of nonspecific binding proteins is important for success in finding such targets. We herein disclose the design, synthesis, and effectiveness in reduction of nonspecific binding proteins, of novel hydrophilic spacers (2-5), which were introduced between matrices and a ligand. Among them, tartaric acid derivative (5) exhibited the most effective reduction of nonspecific binding proteins, whilst maintaining binding of the target protein. Introduction of 5 on TOYOPEARL reduced tubulin and actin by almost 65% and 90% compared to that without the hydrophilic spacer, respectively, with effective binding to the target protein, FKBP12.     

Phylogeny and Biogeography of Hawkmoths (Lepidoptera: Sphingidae): Evidence from Five Nuclear Genes

Background

The 1400 species of hawkmoths (Lepidoptera: Sphingidae) comprise one of most conspicuous and well-studied groups of insects, and provide model systems for diverse biological disciplines. However, a robust phylogenetic framework for the family is currently lacking. Morphology is unable to confidently determine relationships among most groups. As a major step toward understanding relationships of this model group, we have undertaken the first large-scale molecular phylogenetic analysis of hawkmoths representing all subfamilies, tribes and subtribes.

Methodology/Principal Findings

The data set consisted of 131 sphingid species and 6793 bp of sequence from five protein-coding nuclear genes. Maximum likelihood and parsimony analyses provided strong support for more than two-thirds of all nodes, including strong signal for or against nearly all of the fifteen current subfamily, tribal and sub-tribal groupings. Monophyly was strongly supported for some of these, including Macroglossinae, Sphinginae, Acherontiini, Ambulycini, Philampelini, Choerocampina, and Hemarina. Other groupings proved para- or polyphyletic, and will need significant redefinition; these include Smerinthinae, Smerinthini, Sphingini, Sphingulini, Dilophonotini, Dilophonotina, Macroglossini, and Macroglossina. The basal divergence, strongly supported, is between Macroglossinae and Smerinthinae+Sphinginae. All genes contribute significantly to the signal from the combined data set, and there is little conflict between genes. Ancestral state reconstruction reveals multiple separate origins of New World and Old World radiations.

Conclusions/Significance

Our study provides the first comprehensive phylogeny of one of the most conspicuous and well-studied insects. The molecular phylogeny challenges current concepts of Sphingidae based on morphology, and provides a foundation for a new classification. While there are multiple independent origins of New World and Old World radiations, we conclude that broad-scale geographic distribution in hawkmoths is more phylogenetically conserved than previously postulated.     

Dock8 interacts with Nck1 in mediating Schwann cell precursor migration

During embryonic development of the peripheral nervous system (PNS), Schwann cell precursors migrate along neuronal axons to their final destinations, where they will myelinate the axons after birth. While the intercellular signals controlling Schwann cell precursor migration are well studied, the intracellular signals controlling Schwann cell precursor migration remain elusive. Here, using a rat primary cell culture system, we show that Dock8, an atypical Dock180-related guanine-nucleotide exchange factor (GEF) for small GTPases of the Rho family, specifically interacts with Nck1, an adaptor protein composed only of Src homology (SH) domains, to promote Schwann cell precursor migration induced by platelet-derived growth factor (PDGF). Knockdown of Dock8 or Nck1 with its respective siRNA markedly decreases PDGF-induced cell migration, as well as Rho GTPase activation, in precursors. Dock8, through its unique N-terminal proline-rich motif, interacts with the SH3 domain of Nck1, but not with other adaptor proteins composed only of SH domains, e.g. Grb2 and CrkII, and not with the adaptor protein Elmo1. Reintroduction of the proline-rich motif mutant of Dock8 in Dock8 siRNA-transfected Schwann cell precursors fails to restore their migratory abilities, whereas that of wild-type Dock8 does restore these abilities. These results suggest that Nck1 interaction with Dock8 mediates PDGF-induced Schwann cell precursor migration, demonstrating not only that Nck1 and Dock8 are previously unanticipated intracellular signaling molecules involved in the regulation of Schwann cell precursor migration but also that Dock8 is among the genetically-conservative common interaction subset of Dock family proteins consisting only of SH domain adaptor proteins.     

GPAT2, a mitochondrial outer membrane protein,in piRNA biogenesis in germline stem cells

piRNA (PIWI-interacting RNA) is a germ cell–specific small RNA in which biogenesis PIWI (P-element wimpy testis) family proteins play crucial roles. MILI (mouse Piwi-like), one of the three mouse PIWI family members, is indispensable for piRNA production, DNA methylation of retrotransposons presumably through the piRNA, and spermatogenesis. The biogenesis of piRNA has been divided into primary and secondary processing pathways; in both of these MILI is involved in mice. To analyze the molecular function of MILI in piRNA biogenesis, we utilized germline stem (GS) cells, which are derived from testicular stem cells and possess a spermatogonial phenotype. We established MILI-null GS cell lines and their revertant, MILI-rescued GS cells, by introducing the Mili gene with Sendai virus vector. Comparison of wild-type, MILI-null, and MILI-rescued GS cells revealed that GS cells were quite useful for analyzing the molecular mechanisms of piRNA production, especially the primary processing pathway. We found that glycerol-3-phosphate acyltransferase 2 (GPAT2), a mitochondrial outer membrane protein for lysophosphatidic acid, bound to MILI using the cells and that gene knockdown of GPAT2 brought about impaired piRNA production in GS cells. GPAT2 is not only one of the MILI bound proteins but also a protein essential for primary piRNA biogenesis.     

Role of Caribbean Islands in the diversification and biogeography of Neotropical Heraclides swallowtails

Numerous hypotheses on the evolution of Neotropical biodiversity have stimulated research to provide a better understanding of diversity dynamics and distribution patterns of the region. However, few studies integrate molecular and morphological data with complete sampling of a Neotropical group, and so there has been little synthesis of the multiple processes governing biodiversity through space and time. Here, a total‐evidence phylogenetic approach is used to reconstruct the evolutionary history of the butterfly subgenus Heraclides. We used DNA sequences for two mitochondrial genes and one nuclear gene and coded 133 morphological characters of larvae and adults. A robust and well‐resolved phylogeny was obtained using several analytical approaches, while molecular dating and biogeographical analyses indicated an early Miocene origin (22 Mya) in the Caribbean Islands. We inferred six independent dispersal events from the Caribbean to the mainland, and three from the mainland to the Caribbean, and we suggest that cooling climates with decreasing sea levels may have contributed to these events. The time‐calibrated tree is best explained by a museum model of diversity in which both speciation and extinction rates remained constant through time. By assessing both continental and fine‐scale biodiversity patterns, this study provides new findings, for instance that islands may act as source of diversity rather than as a sink, to explain spatio‐temporal macroevolutionary processes within the Neotropical region.     

Leaf structural and hydraulic adjustment with respect to air humidity and canopy position in silver birch (Betula pendula)

Journal of Plant Research - Climate change scenarios predict an increase in air temperature and precipitation in northern temperate regions of Europe by the end of the century. Increasing...     

Attenuated stress-induced catecholamine release in mice lacking the vasopressin V1b receptor

Vasopressin V(1b) receptor is specifically expressed in the pituitary and mediates adrenocorticotropin release, thereby regulating stress responses via its corticotropin releasing factor-like action. In the present study we examined catecholamine release in response to two types of stress in mice lacking the V(1b) receptor gene (V(1b)R(-/-) mice) vs. wild-type mice. There were no significant differences in the basal plasma levels of catecholamines between the two genotypes. In response to stress induced by forced swimming, norepinephrine (NE), but not epinephrine (E) or dopamine (DA), was increased in wild-type mice, whereas the increases in NE and DA were not observed in V(1b)R(-/-) mice. In wild-type mice, E, but not NE or DA, was increased in response to social isolation stress, whereas the increase in E was not observed in V(1b)R(-/-) mice. These results suggest that the V(1b) receptor regulates stress-induced catecholamine release. Because it has been suggested that arginine-vasopressin (AVP) is related to the development of depression, we also evaluated immobility time in the forced swimming test, and we found no significant change in V(1b)R(-/-) mice. Taken together, these findings suggest that, in addition to the previously elucidated effect on the hypothalamic-pituitary-adrenal axis, vasopressin activity via V(1b) receptors regulates stress-induced catecholamine release.