Molecular definition of high-resolution multicolor banding probes: first within the human DNA sequence anchored FISH banding probe set.

Fluorescence in situ hybridization (FISH) banding approaches are standard for the exact characterization of simple, complex, and even cryptic chromosomal aberrations within the human genome. The most frequently applied FISH banding technique is the multicolor banding approach, also abbreviated as m-band, MCB, or in its whole genomic variant multitude MCB (mMCB). MCB allows the differentiation of chromosome region-specific areas at the GTG band and sub-band level and is based on region-specific microdissection libraries, producing changing fluorescence intensity ratios along the chromosomes. The latter are used to assign different pseudocolors to specific chromosomal regions. Here we present the first bacterial artificial chromosome (BAC) array comparative genomic hybridization (aCGH) mapped, comprehensive, genome-wide human MCB probe set. All 169 region-specific microdissection libraries were characterized in detail for their size and the regions of overlap. In summary, the unique possibilities of the MCB technique to characterize chromosomal breakpoints in one FISH experiment are now complemented by the feature of being anchored within the human DNA sequence at the BAC level.     

Mixed-valence porphyrin π-cation radical derivatives: Electrochemical investigations

The electrochemistry of [Cu(OEP)] and [Ni(OEP)] are compared with the mixed-valence π-cations and . These electrochemical studies, carried out with cyclic voltammetry and hydrodynamic voltammetry, show that the mixed valence π-cations have distinct electrochemical properties, although the differences between the [M(OEP)]^+/0 and processes are subtle.     

Intracellular sodium regulates proteolytic activation of the epithelial sodium channel

Na(+) transport across epithelia is mediated in part by the epithelial Na(+) channel ENaC. Previous work indicates that Na(+) is an important regulator of ENaC, providing a negative feedback mechanism to maintain Na(+) homeostasis. ENaC is synthesized as an inactive precursor, which is activated by proteolytic cleavage of the extracellular domains of the alpha and gamma subunits. Here we found that Na(+) regulates ENaC in part by altering proteolytic activation of the channel. When the Na(+) concentration was low, we found that the majority of ENaC at the cell surface was in the cleaved/active state. As Na(+) increased, there was a dose-dependent decrease in ENaC cleavage and, hence, ENaC activity. This Na(+) effect was dependent on Na(+) permeation; cleavage was increased by the ENaC blocker amiloride and by a mutation that decreases ENaC activity (alpha(H69A)) and was reduced by a mutation that activates ENaC (beta(S520K)). Moreover, the Na(+) ionophore monensin reversed the effect of the inactivating mutation (alpha(H69A)) on ENaC cleavage, suggesting that intracellular Na(+) regulates cleavage. Na(+) did not alter activity of Nedd4-2, an E3 ubiquitin ligase that modulates ENaC cleavage, but Na(+) reduced ENaC cleavage by exogenous trypsin. Our findings support a model in which intracellular Na(+) regulates cleavage by altering accessibility of ENaC cleavage sites to proteases and provide a molecular explanation for the earlier observation that intracellular Na(+) inhibits Na(+) transport via ENaC (Na(+) feedback inhibition).     

Intestinal trefoil factor/TFF3 promotes re-epithelialization of corneal wounds

Disorders of wound healing characterized by impaired or delayed re-epithelialization are a serious medical problem. These conditions affect many tissues, are painful, and are difficult to treat. In this study using cornea as a model, we demonstrate the importance of trefoil factor 3 (TFF3, also known as intestinal trefoil factor) in re-epithelialization of wounds. In two different models of corneal wound healing, alkali- and laser-induced corneal wounding, we analyzed the wound healing process in in vivo as well as in combined in vivo/in vitro model in wild type (Tff3(+)(/)(+)) and Tff3-deficient (Tff3(-)(/)(-)) mice. Furthermore, we topically applied different concentrations of recombinant human TFF3 (rTFF3) peptide on the wounded cornea to determine the efficacy of rTFF3 on corneal wound healing. We found that Tff3 peptide is not expressed in intact corneal epithelium, but its expression is extensively up-regulated after epithelial injury. Re-epithelialization of corneal wounds in Tff3(-/-) mice is significantly prolonged in comparison to Tff3(+/+) mice. In addition, exogenous application of rTFF3 to the alkali-induced corneal wounds accelerates significantly in in vivo and in combined in vivo/in vitro model wound healing in Tff3(+/+) and Tff3(-/-) mice. These findings reveal a pivotal role for Tff3 in corneal wound healing mechanism and have broad implications for developing novel therapeutic strategies for treating nonhealing wounds.     

3D reconstruction of the ribs from lateral and frontal X-rays in comparison to 3D CT-scan reconstruction

Subject-specific three-dimensional (3D) reconstructions of the ribs can be obtained from biplanar X-rays. The goal of this study was to evaluate the accuracy and the inter-observer reproducibility of this technique in comparison to CT-scan reconstructions. CT scans and biplanar X-rays were obtained from 50 ribs (from three cadaveric rib cages). Three experienced experimenters reconstructed each rib from biplanar X-rays. Morphometric parameters were then computed from the rib midlines. Differences were computed between parameters obtained from the 3D reconstructions based on biplanar X-rays and from CT scans. The accuracy was computed as the mean of this difference for the 50 ribs from all three experimenters. The inter-observer variability was assessed using the coefficient of variation (CV) between the three observers. The CT-scan reconstructions were considered to be the gold standard in spite of their limitations for rib reconstructions. According to the different linear parameters, the accuracy of the reconstructions was found to be between -6mm (-2%) and 3mm, (4%). The accuracy of the current method was close to that of CT-scan reconstructions. The inter-observer variability was between 3% and 6%. Frontal and lateral X-rays are commonly obtained clinically, so 3D reconstructions can be used without increased radiation exposure to the patient.     

Genetic variation at hair length candidate genes in elephants and the extinct woolly mammoth

Background  

Like humans, the living elephants are unusual among mammals in being sparsely covered with hair. Relative to extant elephants, the extinct woolly mammoth, Mammuthus primigenius, had a dense hair cover and extremely long hair, which likely were adaptations to its subarctic habitat. The fibroblast growth factor 5 (FGF5) gene affects hair length in a diverse set of mammalian species. Mutations in FGF5 lead to recessive long hair phenotypes in mice, dogs, and cats; and the gene has been implicated in hair length variation in rabbits. Thus, FGF5 represents a leading candidate gene for the phenotypic differences in hair length notable between extant elephants and the woolly mammoth. We therefore sequenced the three exons (except for the 3' UTR) and a portion of the promoter of FGF5 from the living elephantid species (Asian, African savanna and African forest elephants) and, using protocols for ancient DNA, from a woolly mammoth.     

Antirheumatic drug response signatures in human chondrocytes: potential molecular targets to stimulate cartilage regeneration

Introduction  

Rheumatoid arthritis (RA) leads to progressive destruction of articular cartilage. This study aimed to disclose major mechanisms of antirheumatic drug action on human chondrocytes and to reveal marker and pharmacological target genes that are involved in cartilage dysfunction and regeneration.     

Geographic patterns of herbivory and resource allocation to defense, growth, and reproduction in an invasive biennial, Alliaria petiolata

We investigated geographic patterns of herbivory and resource allocation to defense, growth, and reproduction in an invasive biennial, Alliaria petiolata, to test the hypothesis that escape from herbivory in invasive species permits enhanced growth and lower production of defensive chemicals. We quantified herbivore damage, concentrations of sinigrin, and growth and reproduction inside and outside herbivore exclusion treatments, in field populations in the native and invasive ranges. As predicted, unmanipulated plants in the native range (Hungary, Europe) experienced greater herbivore damage than plants in the introduced range (Massachusetts and Connecticut, USA), providing evidence for enemy release, particularly in the first year of growth. Nevertheless, European populations had consistently larger individuals than US populations (rosettes were, for example, eightfold larger) and also had greater reproductive output, but US plants produced larger seeds at a given plant height. Moreover, flowering plants showed significant differences in concentrations of sinigrin in the invasive versus native range, although the direction of the difference was variable, suggesting the influence of environmental effects. Overall, we observed less herbivory, but not increased growth or decreased defense in the invasive range. Geographical differences in performance and leaf chemistry appear to be due to variation in the environment, which could have masked evolved differences in allocation.     

Murine model for dengue virus-induced lethal disease with increased vascular permeability

Lack of an appropriate animal model for dengue virus (DEN), which causes dengue fever and dengue hemorrhagic fever/dengue shock syndrome (DHF/DSS), has impeded characterization of the mechanisms underlying the disease pathogenesis. The cardinal feature of DHF/DSS, the severe form of DEN infection, is increased vascular permeability. To develop a murine model that is more relevant to DHF/DSS, a novel DEN strain, D2S10, was generated by alternately passaging a non-mouse-adapted DEN strain between mosquito cells and mice, thereby mimicking the natural transmission cycle of the virus between mosquitoes and humans. After infection with D2S10, mice lacking interferon receptors died early without manifesting signs of paralysis, carried infectious virus in both non-neuronal and neuronal tissues, and exhibited signs of increased vascular permeability. In contrast, mice infected with the parental DEN strain developed paralysis at late times after infection, contained detectable levels of virus only in the central nervous system, and displayed normal vascular permeability. In the mice infected with D2S10, but not the parental DEN strain, significant levels of serum tumor necrosis factor alpha (TNF-alpha) were produced, and the neutralization of TNF-alpha activity prevented early death of D2S10-infected mice. Sequence analysis comparing D2S10 to its parental strain implicated a conserved region of amino acid residues in the envelope protein as a possible source for the D2S10 phenotype. These results demonstrate that D2S10 causes a more relevant disease in mice and that TNF-alpha may be one of several key mediators of severe DEN-induced disease in mice. This report represents a significant advance in animal models for severe DEN disease, and it begins to provide mechanistic insights into DEN-induced disease in vivo.     

Cardiolipin metabolism and Barth Syndrome

Many advances have occurred in the field of Barth Syndrome biology in the 26 years since it was first described as an X-linked cardiomyopathy. Barth Syndrome is the first human disease recognized in which the primary causative factor is an alteration in cardiolipin remodeling. Cardiolipin is required for the optimal function of many proteins within the mitochondria, particularly in the respiratory chain and is involved in the mitochondrial-mediated apoptotic process. The appropriate content of cardiolipin appears to be critical for these functions. Cardiolipin is synthesized de novo in mitochondria and is rapidly remodeled to produce CL enriched in linoleic acid. The Barth Syndrome gene TAZ has been identified and expression of the gene yields proteins known as tafazzins. Mutations in TAZ result in a decrease in tetra-linoleoyl species of cardiolipin and an accumulation of monolysocardiolipin within cells from Barth Syndrome patients. Although the protein product of the TAZ gene shows sequence homology to the glycerolipid acyltransferase family of enzymes, its precise biochemical function remains to be elucidated. In this review we highlight some of the recent literature on cardiolipin metabolism and Barth Syndrome.