Phylogeography of northern Dolly Varden Salvelinus malma (Salmoniformes: Salmonidae) from Asia and North America: An analysis based on the mitochondrial DNA genealogy

Spawning in habitats affected by Pleistocene glacial advances over most of its natural range, northern Dolly Varden Salvelinus malma malma typifies Arctic fauna distributed in northeastern Asia and northwestern North America. We reconstructed a genealogy of mtDNA haplotypes from 27 Alaskan and Asian populations to study the influence of historical events on the phylogeography and contemporary population genetic structure. Analysis of molecular variance partitioned most of the mtDNA variability to the intrapopulation component (72.5%) with much reduced differences between populations (21.1%) and regions (6.4%). Similar patterns of variation apparent from hierarchical diversity and nested clade phylogeographical analysis (NCPA) of mtDNA haplotypes identify weak spatial differentiation and low levels of divergence. These findings suggest (1) that demographic history has been influenced by historical range expansions and recent isolation by distance, (2) that present populations from Asia and North America were colonized from one main Beringian Refugium, and (3) that this taxon’s ancestral population probably experienced a bottleneck in the Beringian Refugium during the late Pleistocene (Wisconsin) glacial period. The genealogical and NCPA analyses, and mismatch distribution of S. m. malma mtDNA haplotypes do not confirm the assumptions about presence of the two refugia on the territories of the Beringian Land, in which allopatric S. m. malma ancestral populations evolved, and independent origin of the Sea of Okhotsk populations.     

Structural and gene expression analyses of uptake hydrogenases and other proteins involved in nitrogenase protection in Frankia

The actinorhizal bacterium Frankia expresses nitrogenase and can therefore convert molecular nitrogen into ammonia and the by-product hydrogen. However, nitrogenase is inhibited by oxygen. Consequently, Frankia and its actinorhizal hosts have developed various mechanisms for excluding oxygen from their nitrogen-containing compartments. These include the expression of oxygen-scavenging uptake hydrogenases, the formation of hopanoid-rich vesicles, enclosed by multi-layered hopanoid structures, the lignification of hyphal cell walls, and the production of haemoglobins in the symbiotic nodule. In this work, we analysed the expression and structure of the so-called uptake hydrogenase (Hup), which catalyses the in vivo dissociation of hydrogen to recycle the energy locked up in this ‘waste’ product. Two uptake hydrogenase syntons have been identified in Frankia: synton 1 is expressed under free-living conditions while synton 2 is expressed during symbiosis. We used qPCR to determine synton 1 hup gene expression in two Frankia strains under aerobic and anaerobic conditions. We also predicted the 3D structures of the Hup protein subunits based on multiple sequence alignments and remote homology modelling. Finally, we performed BLAST searches of genome and protein databases to identify genes that may contribute to the protection of nitrogenase against oxygen in the two Frankia strains. Our results show that in Frankia strain ACN14a, the expression patterns of the large (HupL1) and small (HupS1) uptake hydrogenase subunits depend on the abundance of oxygen in the external environment. Structural models of the membrane-bound hydrogenase subunits of ACN14a showed that both subunits resemble the structures of known [NiFe] hydrogenases (Volbeda et al. 1995), but contain fewer cysteine residues than the uptake hydrogenase of the Frankia DC12 and Eu1c strains. Moreover, we show that all of the investigated Frankia strains have two squalene hopane cyclase genes (shc1 and shc2). The only exceptions were CcI3 and the symbiont of Datisca glomerata, which possess shc1 but not shc2. Four truncated haemoglobin genes were identified in Frankia ACN14a and Eu1f, three in CcI3, two in EANpec1 and one in the Datisca glomerata symbiont (Dg).     

Binding and purification of plasmid DNA using multi-layered carbon nanotubes

We propose a new method for the separation of nucleic acids using multi-layered carbon nanotubes (CNTs) as an adsorbent. According to agarose gel electrophoresis, oxidized water-stable CNTs adsorb certain forms of nucleic acids, such as high molecular weight RNA, chromosomal DNA, linear and denatured forms of plasmid DNA. However, CNTs do not adsorb supercoiled form of plasmid DNA. Nucleic acids bound to CNTs can be readily removed by centrifugation whereas supercoiled plasmid DNA remains in solution. Upon the addition of divalent metal ions supercoiled plasmid DNA forms relatively stable complexes with CNTs due to chelation. Thus, new details about association of nucleic acids with CNTs were revealed and stoichiometry of the complexes was estimated. Our results can be used for fine purification of supercoiled plasmid DNA for gene therapy applications as well as manipulation of nucleic acids for biosensor design.     

Two-photon fluorescence vascular bioimaging with new bioconjugate probes selective toward the vascular endothelial growth factor receptor 2

We report the synthesis and characterization of two amine reactive fluorescent dyes with efficient two-photon absorption (2PA) properties and high fluorescence quantum yields. Bioconjugation of these dyes with the DC-101 antibody proved to be useful for selectively imaging the vascular endothelial growth factor receptor 2 (VEGFR-2) in cells expressing this receptor in vitro and in "whole" mounted excised tumors (ex vivo) by two-photon fluorescence microscopy (2PFM). The penetration depths reached within the tumors by 2PFM was over 800 μm. In addition, the concentration of dye required for incubation of these bioconjugates was in the picomolar domain, the probes possessed very good photostability, and the 2PFM setup did not require any additional means of increasing the collection efficiencies of fluorescent photons to achieve the relatively deep tissue imaging that was realized, due, in large part, to the favorable photophysical properties of the new probes.     

Proton-Coupled Dynamics in Lactose Permease

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Highlights? Deprotonation of Glu325 brings H⁺ translocation and sugar-binding sites together ? Deprotonation of Glu325 closes the internal water-filled cytoplasmic cavity ? Deprotonated LacY inserted into DMPC lipids shows no structural rearrangements ? The structural changes show remarkable similarities to experimental observations     

Copper-induced liver fibrosis affects the behavior of bone marrow cells in primary culture

Background

The present study investigated the effects of low-molecular-weight components of bovine colostrum (LMCC), which were administered per os, on the differentiation, growth, and survival of cells obtained from the bone marrow of rats in primary culture.

Methods

Bone marrow cells (BMCs) were obtained from both the rat femurs and were cultured in medium 199 supplemented with antibiotics (8% streptomycin and 8% gentamycin) and 20% inactivated fetal calf serum. In addition, the number of BMCs was counted and their morphotypes were determined.

Results

Animals were treated with copper (Cu) sulfate to induce liver fibrosis. Subsequent treatment with LMCC eliminated growth inhibition and normalized the bodyweight and temperature of animals with Cu-induced liver fibrosis. The number of lymphocytes in the bone marrow of animals with Cu-induced liver fibrosis was significantly higher than that in the bone marrow of control animals. The number of neutrophils in untreated animals with liver fibrosis and LMCC-treated animals with liver fibrosis was lower than that in control animals. Neutrophils obtained from untreated animals with liver fibrosis and LMCC-treated animals with liver fibrosis underwent two-times faster degradation in vitro than neutrophils obtained from control animals.

Conclusions

Our results indicate that LMCC affects the distribution of different morphological types of BMCs but does not prevent their degradation in vitro, which was two-times faster than that of BMCs obtained from control animals.

     

Analysis of <Emphasis Type="Italic">GJB6</Emphasis> (Сx30) and <Emphasis Type="Italic">GJB3</Emphasis> (Сx31) genes in deaf patients with monoallelic mutations in <Emphasis Type="Italic">GJB2</Emphasis> (Сx26) gene in the Sakha Republic (Yakutia)

Тhe DNA testing of autosomal recessive deafness type 1A (DFNB1A, MIM 220290) is complicated when deaf patients have only monoallelic (heterozygous) recessive mutations in the GJB2 (Сх26) gene that is uninformative for establishment of diagnosis. Such patients may be “random” heterozygous carriers of GJB2 mutations as well as have the mutant allele in a cis-regulatory region of GJB2 gene, in element genes encoding other connexins: GJB6 (Сх30) or GJB3 (Сх31). Previous studies of genetic causes of hearing loss in patients from Yakutia were directed to search for only mutations in the GJB2 gene, and the DNA diagnostics was uninformative for 9.7% (38/393) of the patients with monoallelic GJB2 mutations. In this work the search for mutations in genes GJB3 and GJB6 and two deletions с.del(GJB6-D13S1830) and с.del(GJB6-D13S1854) to the cis-regulatory region of GJB2 gene was conducted in 35 patients with GJB2 monoallelic mutations and in 104 normal hearing individuals. The genes studied are two synonymous substitution c.489G>A (р.Leu163Leu) (GJB6) and c.357C>T (р.Asn119Asn) (GJB3) have been found, probably do not have clinical significance, and two nonsynonymous substitution c.301G>A (p.Glu101Lys) (GJB6) and с.580G>A (p.Ala194Thr) (GJB3). Additional experimental evidences are needed for confirmation of pathogenic significance of detected nonsynonymous substitutions in development of hearing loss in studied patients. Diagnosis of the DFNB1A was confirmed in only one patient, who was discovered by the deletion с.del(GJB6-D13S1830) (GJB2) in combination with a recessive mutation с.35delG (GJB2). In general, our results indicate low contribution of mutations in genes GJB6 and GJB3 in hearing loss etiology in Yakutia.     

Effects of Light Spectral Quality on Morphogenesis and Source–Sink Relations in Radish Plants

The accumulation of dry matter and the content of major phytohormones in the aboveground and underground plant parts, as well as light curves and the diurnal course of photosynthesis in the leaves were studied in radish (Raphanus sativusL.) plants of different ages that were grown under red (RL) or blue (BL) light. As seen from the rapid increase in plant biomass, the development of storage organs (hypocotyl or tap root) started on the 14th day after the emergence of seedling of the BL plants and on the 21st day for the RL plants. Conversely, RL stimulated biomass accumulation in the aboveground parts (petioles and stems) already in the early stages of plant development. Light spectral quality only slightly affected the activity and the diurnal course of photosynthesis. The GA content was ten times higher in the aboveground parts of the RL plants than those of the BL plants. The hypocotyl of the BL plants contained much higher amounts of cytokinins and IAA than that of the RL plants. The specific responses of the source–sink relations to the light quality were related to the distribution of various phytohormones between the aboveground and underground parts of the plants: RL increased the content of gibberellins (GA) in the aboveground parts of plants, thus increasing their sink activity, whereas BL stimulated the synthesis of cytokinins and IAA in the hypocotyl and enhanced its development. Light quality-specific morphogenetic responses were reversed when plants were treated with exogenous GA or paclobutrazol, an inhibitor of GA synthesis. The treatment of the BL plants with exogenous GA stimulated petiole and hypocotyl elongation and induced stem formation. The treatment of the BL plants with paclobutrazol led to shortened petioles, the flattening of the storage organ, and the disappearance of the stem.     

The NC1 domain of collagen IV encodes a novel network composed of the alpha 1, alpha 2, alpha 5, and alpha 6 chains in smooth muscle basement membranes

Type IV collagen, the major component of basement membranes (BMs), is a family of six homologous chains (alpha1-alpha6) that have a tissue-specific distribution. The chains assemble into supramolecular networks that differ in the chain composition. In this study, a novel network was identified and characterized in the smooth muscle BMs of aorta and bladder. The noncollagenous (NC1) hexamers solubilized by collagenase digestion were fractionated by affinity chromatography using monoclonal antibodies against the alpha5 and alpha6 NC1 domains and then characterized by two-dimensional gel electrophoresis and Western blotting. Both BMs were found to contain a novel alpha1.alpha2.alpha5.alpha6 network besides the classical alpha1.alpha2 network. The alpha1.alpha2.alpha5.alpha6 network represents a new arrangement in which a protomer (triple-helical isoform) containing the alpha5 and alpha6 chains is linked through NC1-NC1 interactions to an adjoining protomer composed of the alpha1 and alpha2 chains. Re-association studies revealed that the NC1 domains contain recognition sequences sufficient to encode the assembly of both networks. These findings, together with previous ones, indicate that the six chains of type IV collagen are distributed in three major networks (alpha1.alpha2, alpha3.alpha4.alpha5, and alpha1.alpha2.alpha5.alpha6) whose chain composition is encoded by the NC1 domains. The existence of the alpha1.alpha2.alpha5.alpha6 network provides a molecular explanation for the concomitant loss of alpha5 and alpha6 chains from the BMs of patients with X-linked Alport's syndrome.