Dermal application of nitric oxide releasing acidified nitrite-containing liniments significantly reduces blood pressure in humans

Vascular ischemic diseases, hypertension, and other systemic hemodynamic and vascular disorders may be the result of impaired bioavailability of nitric oxide (NO). NO but also its active derivates like nitrite or nitroso compounds are important effector and signal molecules with vasodilating properties. Our previous findings point to a therapeutical potential of cutaneous administration of NO in the treatment of systemic hemodynamic disorders. Unfortunately, no reliable data are available on the mechanisms, kinetics and biological responses of dermal application of nitric oxide in humans in vivo. The aim of the study was to close this gap and to explore the therapeutical potential of dermal nitric oxide application. We characterized with human skin in vitro and in vivo the capacity of NO, applied in a NO-releasing acidified form of nitrite-containing liniments, to penetrate the epidermis and to influence local as well as systemic hemodynamic parameters. We found that dermal application of NO led to a very rapid and significant transepidermal translocation of NO into the underlying tissue. Depending on the size of treated skin area, this translocation manifests itself through a significant systemic increase of the NO derivates nitrite and nitroso compounds, respectively. In parallel, this translocation was accompanied by an increased systemic vasodilatation and blood flow as well as reduced blood pressure. We here give evidence that in humans dermal application of NO has a therapeutic potential for systemic hemodynamic disorders that might arise from local or systemic insufficient availability of NO or its bio-active NO derivates, respectively.     

High yield expression of non-phosphorylated protein tyrosine kinases in insect cells

The key role of kinases in signal transduction and cell growth regulation has been a long standing interest among academics and the pharmaceutical industry. Recombinant enzymes have been used to understand the mechanism of action as well as to screen for chemical inhibitors. The baculo-insect system has been the primary method used to obtain soluble and active kinases, usually producing a mixture of the kinase in various phosphorylation states in different conformations. To obtain a homogenous preparation of non-phosphorylated kinases is critical for biochemical, biophysical and kinetic studies aimed at understanding the mechanism of kinase activation. Taking advantage of the eukaryotic expression property of insect cells, we were able to obtain high yield expression of non-phosphorylated protein tyrosine kinases BTK, JAK3 and Eph2A through coexpression with the tyrosine phosphatase YopH, which suggests that this method can be applied to protein tyrosine kinases in general. We have demonstrated that the fully non-phosphorylated BTK obtained with this method is suitable for various biochemical and kinetic studies.     

Hydrogen exchange of monomeric alpha-synuclein shows unfolded structure persists at physiological temperature and is independent of molecular crowding in Escherichia coli

Amide proton NMR signals from the N-terminal domain of monomeric α-synuclein (αS) are lost when the sample temperature is raised from 10°C to 35°C at pH 7.4. Although the temperature-induced effects have been attributed to conformational exchange caused by an increase in α-helix structure, we show that the loss of signals is due to fast amide proton exchange. At low ionic strength, hydrogen exchange rates are faster for the N-terminal segment of αS than for the acidic C-terminal domain. When the salt concentration is raised to 300 mM, exchange rates increase throughout the protein and become similar for the N- and C-terminal domains. This indicates that the enhanced protection of amide protons from the C-terminal domain at low salt is electrostatic in nature. Cα chemical shift data point to <10% residual α-helix structure at 10°C and 35°C. Conformational exchange contributions to R2 are negligible at both temperatures. In contrast to the situation in vitro, the majority of amide protons are observed at 37°C in ¹H-¹⁵N HSQC spectra of αS encapsulated within living Escherichia coli cells. Our finding that temperature effects on αS NMR spectra can be explained by hydrogen exchange obviates the need to invoke special cellular factors. The retention of signals is likely due to slowed hydrogen exchange caused by the lowered intracellular pH of high-density E. coli cultures. Taken together, our results emphasize that αS remains predominantly unfolded at physiological temperature and pH—an important conclusion for mechanistic models of the association of αS with membranes and fibrils.     

Microsatellite markers in the endangered Australian northern corroboree frog, Pseudophryne pengilleyi (Anura: Myobatrachidae) and amplification in other Pseudophryne species

Seven microsatellite primer pairs were isolated and characterized in the endangered Australian northern corroboree frog (Pseudophryne pengilleyi). All seven were polymorphic (2–14 alleles) and displayed high heterozygosity (0.036–0.964) in 28 sampled individuals. We also tested the microsatellites on two closely related species. Four were polymorphic in the southern corroboree frog (P. corroboree) and Bibron’s toadlet (P. bibronii). These primers will be useful in studies of conservation genetics and mating systems in Pseudophryne species.     

Linkage Analysis Reveals the Independent Origin of Poeciliid Sex Chromosomes and a Case of Atypical Sex Inheritance in the Guppy (Poecilia reticulata)

Among different teleost fish species, diverse sex-determining mechanisms exist, including environmental and genetic sex determination, yet chromosomal sex determination with male heterogamety (XY) prevails. Different pairs of autosomes have evolved as sex chromosomes among species in the same genus without evidence for a master sex-determining locus being identical. Models for evolution of Y chromosomes predict that male-advantageous genes become linked to a sex-determining locus and suppressed recombination ensures their co-inheritance. In the guppy, Poecilia reticulata, a set of genes responsible for adult male ornaments are linked to the sex-determining locus on the incipient Y chromosome. We have identified >60 sex-linked molecular markers to generate a detailed map for the sex linkage group of the guppy and compared it with the syntenic autosome 12 of medaka. We mapped the sex-determining locus to the distal end of the sex chromosome. We report a sex-biased distribution of recombination events in female and male meiosis on sex chromosomes. In one mapping cross, we observed sex ratio and male phenotype deviations and propose an atypical mode of genetic sex inheritance as its basis.     

Dispersal failure contributes to plant losses in NW Europe

The ongoing decline of many plant species in Northwest Europe indicates that traditional conservation measures to improve the habitat quality, although useful, are not enough to halt diversity losses. Using recent databases, we show for the first time that differences between species in adaptations to various dispersal vectors, in combination with changes in the availability of these vectors, contribute significantly to explaining losses in plant diversity in Northwest Europe in the 20th century. Species with water- or fur-assisted dispersal are over-represented among declining species, while others (wind- or bird-assisted dispersal) are under-represented. Our analysis indicates that the 'colonization deficit' due to a degraded dispersal infrastructure is no less important in explaining plant diversity losses than the more commonly accepted effect of eutrophication and associated niche-based processes. Our findings call for measures that aim to restore the dispersal infrastructure across entire regions and that go beyond current conservation practices.     

Genome sequence of the probiotic strain Bifidobacterium animalis subsp. lactis CNCM I-2494

Bifidobacterium animalis subsp. lactis CNCM I-2494 is part of a commercialized fermented dairy product with documented health benefits revealed by multiple randomized placebo-controlled clinical trials. Here we report the complete genome sequence of this strain, which has a circular genome of 1,943,113 bp with 1,660 open reading frames and 4 ribosomal operons.     

Oligomerization of ICP4 and rearrangement of heat shock proteins may be important for herpes simplex virus type 1 prereplicative site formation

Herpes simplex virus type 1 (HSV-1) DNA replication occurs in replication compartments that form in the nucleus by an ordered process involving a series of protein scaffold intermediates. Following entry of viral genomes into the nucleus, nucleoprotein complexes containing ICP4 can be detected at a position adjacent to nuclear domain 10 (ND10)-like bodies. ND10s are then disrupted by the viral E3 ubiquitin ligase ICP0. We have previously reported that after the dissociation of ND10-like bodies, ICP8 could be observed in a diffuse staining pattern; however, using more sensitive staining methods, we now report that in addition to diffuse staining, ICP8 can be detected in tiny foci adjacent to ICP4 foci. ICP8 microfoci contain UL9 and components of the helicase-primase complex. HSV infection also results in the reorganization of the heat shock cognate protein 70 (Hsc70) and the 20S proteasome into virus-induced chaperone-enriched (VICE) domains. In this report we show that VICE domains are distinct but adjacent to the ICP4 nucleoprotein complexes and the ICP8 microfoci. In cells infected with an ICP4 mutant virus encoding a mutant protein that cannot oligomerize on DNA, ICP8 microfoci are not detected; however, VICE domains could still be formed. These results suggest that oligomerization of ICP4 on viral DNA may be essential for the formation of ICP8 microfoci but not for the reorganization of host cell chaperones into VICE domains.     

Ca2+/Calmodulin-dependent kinase II signaling causes skeletal overgrowth and premature chondrocyte maturation

The long bones of vertebrate limbs originate from cartilage templates and are formed by the process of endochondral ossification. This process requires that chondrocytes undergo a progressive maturation from proliferating to postmitotic prehypertrophic to mature, hypertrophic chondrocytes. Coordinated control of proliferation and maturation regulates growth of the skeletal elements. Various signals and pathways have been implicated in orchestrating these processes, but the underlying intracellular molecular mechanisms are often not entirely known. Here we demonstrated in the chick using replication-competent retroviruses that constitutive activation of Calcium/Calmodulin-dependent kinase II (CaMKII) in the developing wing resulted in elongation of skeletal elements associated with premature differentiation of chondrocytes. The premature maturation of chondrocytes was a cell-autonomous effect of constitutive CaMKII signaling associated with down-regulation of cell-cycle regulators and up-regulation of chondrocyte maturation markers. In contrast, the elongation of the skeletal elements resulted from a non-cell autonomous up-regulation of the Indian hedgehog responsive gene encoding Parathyroid-hormone-related peptide. Reduction of endogenous CaMKII activity by overexpressing an inhibitory peptide resulted in shortening of the skeletal elements associated with a delay in chondrocyte maturation. Thus, CaMKII is an essential component of intracellular signaling pathways regulating chondrocyte maturation.