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.     

Another small supernumerary marker chromosome (sSMC) derived from chromosome 2: towards a genotype/phenotype correlation.

Here we report a prenatally detected small supernumerary marker chromosome (sSMC) derived from chromosome 2 as demonstrated by cenM-FISH (centromere-specific multicolor fluorescence in situ hybridization). By application of a recently described subcentromere-specific probe set (subcenM-FISH) for chromosome 2, the presence of a small partial trisomy due to a karyotype 47,XX,+r(2)(::p11.1->q11.2::) was demonstrated. Including this case, a total of 11 patients with sSMC(2) are described throughout the literature. Based on that data, a first genotype/phenotype correlation according to the size and structure of the marker is suggested.     

Discovery of a potent and selective c-Kit inhibitor for the treatment of inflammatory diseases

A potent and selective c-Kit inhibitor 20 was identified through a structure–activity relationship study. In an in vivo mouse model of mast cell activation, 20 blocked the SCF-induced histamine release with an EC₅₀ of 26 nM.     

Latitudinal gradients in sea ice and primary production determine Arctic seabird colony size in Greenland

Sea ice loss will indirectly alter energy transfer through the pelagic food web and ultimately impact apex predators. We quantified spring-time trends in sea ice recession around each of 46 thick-billed murre (Uria lomvia) colonies in west Greenland across 20 degrees of latitude and investigated the magnitude and timing of the associated spring-time primary production. A geographical information system was used to extract satellite-based observations of sea ice concentration from the Nimbus-7 scanning multichannel microwave radiometer (SMMR, 1979-1987) and the Defence Meteorological Satellite Programs Special Sensor Microwave/Imager (SSMI, 1987-2004), and satellite-based observations of chlorophyll a from the moderate resolution imaging spectroradiometer (MODIS: EOS-Terra satellite) in weekly intervals in circular buffers around each colony site (150 km in radius). Rapid recession of high Arctic seasonal ice cover created a temporally predictable primary production bloom and associated trophic cascade in water gradually exposed to solar radiation. This pattern was largely absent from lower latitudes where little to no sea ice resulted in a temporally variable primary production bloom driven by nutrient cycling and upwelling uncoupled to ice. The relationship between the rate and variability of sea ice recession and colony size of thick-billed murres shows that periodical confinement of the trophic cascade at high latitudes determines the carrying capacity for Arctic seabirds during the breeding period.     

Studies toward the discovery of the next generation of antidepressants. Part 6: Dual 5-HT1A receptor and serotonin transporter affinity within a class of arylpiperazinyl-cyclohexyl indole derivatives

Based on the previously reported discovery lead, 3-(cis-4-(4-(1H-indol-4-yl)piperazin-1-yl)cyclohexyl)-5-fluoro-1H-indole (2), a series of related arylpiperazin-4-yl-cyclohexyl indole analogs were synthesized then evaluated as 5-HT transporter inhibitors and 5-HT(1A) receptor antagonists. The investigation of the structure-activity relationships revealed the optimal pharmacophoric elements required for activities in this series. The best example from this study, 5-(piperazin-1-yl)quinoline analog (trans-20), exhibited equal binding affinities at 5-HT transporter (K(i)=4.9nM), 5-HT(1A) receptor (K(i)=6.2nM) and functioned as a 5-HT(1A) receptor antagonist.     

Cloning, expression, purification, and characterization of a designer protein with repetitive sequences

An artificial protein containing alternating hydrophilic–hydrophobic blocks of amino acids was designed in order to mimic the structure of synthetic multiblock copolymers. The hydrophobic block consisted of the six amino acids Ala Ile Leu Leu Ile Ile (AILLII) and the hydrophilic block of the eight amino acids Thr Ser Glu Asp Asp Asn Asn Gln (TSEDDNNQ). The coding DNA sequence of the cluster was inserted into an commercial pET 30a(+) vector using a two step strategy. The expression of the artificial protein in Escherichia coli was optimized using a temperature shift strategy. Only at cultivation temperature of 24 °C after induction expression was observed, whereas at 30 and 37 °C no target protein could be detected. Cells obtained from a 15 L bioreactor cultivation of E. coli were disintegrated by mechanical methods. Interestingly, glass bead milling and high pressure homogenization resulted in a different solubility of the target protein. The further purification was carried out by affinity chromatography using the soluble homogenized protein. Extreme conditions (6 M urea, 0.5 M NaCl) were applied in order to prevent aggregation to insoluble particles. The designer protein showed an extremely high tendency to form dimers or trimers caused by intermolecular interactions which were even not broken under the conditions of SDS–polyacrylamide gel electrophoresis, rendering the behavior during purification different from proteins usually found in nature. The protein preparation was not completely pure according to SDS–PAGE stained by Coomassie blue or silver. In MALDI-TOF-MS, nano-ESI qTOF-MS of the entire protein preparation and nano-ESI-MS after digestion by trypsin and chymotrypsin impurities were not detectable.     

The effect of rottlerin in calcium regulation in HMC-1(560) cells is mediated by a PKC-delta independent effect

The human mast cell line (HMC-1(560)) is a good model for Ca(2+) signaling studies, because intracellular alkalinization is the mainly histamine release stimulus without changes in the intracellular Ca(2+) levels. This fact allows us to study Ca(2+) changes without degranulation, since this process can affected cellular viability. Ionomycin and thapsigargin have been fully used for induced Ca(2+) influx across SOC channels. When HMC-1(560) cells are incubated with rottlerin, 5 microM, for 5 min a strong inhibition of ionomycin-induced Ca(2+) influx is observed. However, when thapsigargin stimulates Ca(2+) influx, rottlerin did not show any effect on Ca(2+) levels. This fact point two possibilities, ionomycin and thapsigargin might activate different SOC channels or that these drugs might activate the same channel but in a different way in HMC-1(560) cells. The rottlerin inhibition of ionomycin-induced Ca(2+) influx is PKC-delta independent and this effect is not related with the store depletion, since rottlerin has the same effect when it is added before or after the stores are empty. FCCP, a know uncoupler of oxidative phosphorylation in mitochondria, induces the same inhibition in ionomycin Ca(2+) influx than rottlerin which point to the mitochondria as a cellular target to rottlerin.