DNA data and morphology reveal the existence of Kentrochrysalis streckeri Staudinger, 1880 (Lepidoptera: Sphingidae) instead of K. consimilis in South Korea

In the present study, we determined that specimens of Kentrochrysalis consimilis collected from South Korea were K. streckeri, rather than K. consimilis, based on morphology, DNA barcodes and nuclear elongation factor 1 alpha (EF‐1α) sequences. The major morphological differences between K. streckeri and K. consimilis include the shape of forewing and hind‐wing pattern elements and male and female genitalia. The DNA barcode analysis of the South Korean specimens and the Russia‐originated K. streckeri showed a maximum sequence divergence of only 0.659% (4 bp), whereas that of the South Korean specimens and Japan‐originated K. consimilis showed a minimum sequence divergence of 2.965% (18 bp), indicating that the Korean specimens are, in fact, K. streckeri and not K. consimilis. Phylogenetic analyses both by Bayesian inference and maximum likelihood methods strongly clustered the South Korean specimens and Russian K. streckeri into one group, excluding K. consimilis. The EF‐1α‐based sequence and phylogenetic analyses of the two species also supported data from the DNA barcode, indicating the distribution of K. streckeri in South Korea, instead of K. consimilis.     

The F-Box Domain-Dependent Activity of EMI1 Regulates PARPi Sensitivity in Triple-Negative Breast Cancers

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Synergistic action of the Saccharomyces cerevisiae homologous recombination factors Rad54 and Rad51 in chromatin remodeling

Rad54, a member of the Swi2/Snf2 protein family, works in concert with the RecA-like recombinase Rad51 during the early and late stages of homologous recombination. Rad51 markedly enhances the activities of Rad54, including the induction of topological changes in DNA and the remodeling of chromatin structure. Reciprocally, Rad54 promotes Rad51-mediated DNA strand invasion with either naked or chromatinized DNA. Here, using various Saccharomyces cerevisiae rad51 and rad54 mutant proteins, mechanistic aspects of Rad54/Rad51-mediated chromatin remodeling are defined. Disruption of the Rad51-Rad54 complex leads to a marked attenuation of chromatin remodeling activity. Moreover, we present evidence that assembly of the Rad51 presynaptic filament represents an obligatory step in the enhancement of the chromatin remodeling reaction. Interestingly, we find a specific interaction of the N-terminal tail of histone H3 with Rad54 and show that the H3 tail interaction domain resides within the amino terminus of Rad54. These results suggest that Rad54-mediated chromatin remodeling coincides with DNA homology search by the Rad51 presynaptic filament and that this process is facilitated by an interaction of Rad54 with histone H3.     

Role of Dnl4-Lif1 in nonhomologous end-joining repair complex assembly and suppression of homologous recombination

Nonhomologous end joining (NHEJ) eliminates DNA double-strand breaks (DSBs) in bacteria and eukaryotes. In Saccharomyces cerevisiae, there are pairwise physical interactions among the core complexes of the NHEJ pathway, namely Yku70-Yku80 (Ku), Dnl4-Lif1 and Mre11-Rad50-Xrs2 (MRX). However, MRX also has a key role in the repair of DSBs by homologous recombination (HR). Here we have examined the assembly of NHEJ complexes at DSBs biochemically and by chromatin immunoprecipitation. Ku first binds to the DNA end and then recruits Dnl4-Lif1. Notably, Dnl4-Lif1 stabilizes the binding of Ku to in vivo DSBs. Ku and Dnl4-Lif1 not only initiate formation of the nucleoprotein NHEJ complex but also attenuate HR by inhibiting DNA end resection. Therefore, Dnl4-Lif1 plays an important part in determining repair pathway choice by participating at an early stage of DSB engagement in addition to providing the DNA ligase activity that completes NHEJ.     

Ecotope mapping for landscape ecological assessment of habitat and ecosystem

An ecotope (spatial eco-space) map that considers topography and bio-organism-relevant variables emerges as an important basic framework when landscape-scale characteristics for ecosystem management and wildlife conservation are needed. A spatio-geoecological framework based on geographic information systems (GIS) and a vegetation survey were developed for wildlife habitat evaluation of national parks and applied to a representative rugged valley area of Mt. Sorak National Park in Korea. An ecotope map was classified into hundreds of types and dozens of groups by combining biological and geophysical variables. Variables included: forest vegetation type, topographic solar radiation, normalized difference vegetation index (NDVI), elevation, and anthropogenic factors, such as, streams and roads. Layers of GIS variables were produced by field surveys, modeling, satellite images, or digitalization. Vegetation surveys were carried out to identify finer-scale distribution of vegetation types in the rugged valley area. Digital forest vegetation maps from the Forestry Administrator were then modified using the field-surveyed vegetation maps. Topographic solar radiation was predicted with a daily topographic radiation model. The NDVI was calculated from the satellite imagery of a Landsat Thematic Mapper. A digital elevation model (DEM) was used and the other layers were digitized using topographical maps with a scale of 1:25000. The aim of this study is to determine the geoecological factors relating to the spatial pattern of plant community. It was cleared by the spatial pattern of environmental variables and vegetation characteristics by detrended correspondence analysis using plant species and the environmental variables of each plot. The ordination component value of the first axis shows significant regression to some environmental variables. A case study of habitat evaluation was carried out using the resultant ecotope map. The spatial distribution of potential goral habitat and vegetation characteristics were predicted and the impact of human trails on the neighboring vegetation was also examined for restoration planning. The GIS-based framework developed for wildlife habitat evaluation is useful for natural resource management and human activity control in national parks in Korea.     

Vascular endothelial growth factor regulates focal adhesion assembly in human brain microvascular endothelial cells through activation of the focal adhesion kinase and related adhesion focal tyrosine kinase

Vascular endothelial growth factor (VEGF) plays a significant role in blood-brain barrier breakdown and angiogenesis after brain injury. VEGF-induced endothelial cell migration is a key step in the angiogenic response and is mediated by an accelerated rate of focal adhesion complex assembly and disassembly. In this study, we identified the signaling mechanisms by which VEGF regulates human brain microvascular endothelial cell (HBMEC) integrity and assembly of focal adhesions, complexes comprised of scaffolding and signaling proteins organized by adhesion to the extracellular matrix. We found that VEGF treatment of HBMECs plated on laminin or fibronectin stimulated cytoskeletal organization and increased focal adhesion sites. Pretreating cells with VEGF antibodies or with the specific inhibitor SU-1498, which inhibits Flk-1/KDR receptor phosphorylation, blocked the ability of VEGF to stimulate focal adhesion assembly. VEGF induced the coupling of focal adhesion kinase (FAK) to integrin alphavbeta5 and tyrosine phosphorylation of the cytoskeletal components paxillin and p130cas. Additionally, FAK and related adhesion focal tyrosine kinase (RAFTK)/Pyk2 kinases were tyrosine-phosphorylated by VEGF and found to be important for focal adhesion sites. Overexpression of wild type RAFTK/Pyk2 increased cell spreading and the migration of HBMECs, whereas overexpression of catalytically inactive mutant RAFTK/Pyk2 markedly suppressed HBMEC spreading ( approximately 70%), adhesion ( approximately 82%), and migration ( approximately 65%). Furthermore, blocking of FAK by the dominant-interfering mutant FRNK (FAK-related non-kinase) significantly inhibited HBMEC spreading and migration and also disrupted focal adhesions. Thus, these studies define a mechanism for the regulatory role of VEGF in focal adhesion complex assembly in HBMECs via activation of FAK and RAFTK/Pyk2.     

Expression and purification of enzymatically active forms of the human lysyl oxidase-like protein 4

The lysyl oxidase-like protein 4 (LOXL4) is the latest member of the emerging family of lysyl oxidases, several of which were shown to function as copper-dependent amine oxidases catalyzing lysine-derived cross-links in extracellular matrix proteins. LOXL4 contains four scavenger receptor cysteine-rich domains in addition to the characteristic domains of the LOX family, including the copper-binding domain, the cytokine receptor-like domain, and the residues of the lysyl-tyrosyl quinone cofactor. In an effort to assess its amine oxidase activity, we expressed LOXL4 as recombinant forms attached with hexa-histidine residues at the carboxyl terminus by using an Escherichia coli expression system. The recombinant proteins were purified with nickel-chelating affinity chromatography and converted into enzymatically active forms by stepwise dialysis. The purified LOXL4 proteins showed beta-aminopropionitrile-inhibitable activity of 0.022-0.032 units/mg toward a nonpeptidyl substrate, benzylamine. These results indicate that LOXL4, with the four scavenger receptor cysteine rich domains, may also function as an active amine oxidase. Availability of the pure and active forms of LOXL4 will be significantly helpful in functional studies related to substrate specificity and crystal structure of this amine oxidase, which should provide significant insights into functional differences within the LOX family members.