Cutting edge: serotonin is a chemotactic factor for eosinophils and functions additively with eotaxin

Elevated levels of serotonin (5-hydroxytryptamine, 5-HT) are observed in the serum of asthmatics. Herein, we demonstrate that 5-HT functions independently as an eosinophil chemoattractant that acts additively with eotaxin. 5-HT2A receptor antagonists (including MDL-100907 and cyproheptadine (CYP)) were found to inhibit 5-HT-induced, but not eotaxin-induced migration. Intravital microscopy studies revealed that eosinophils roll in response to 5-HT in venules under conditions of physiological shear stress, which could be blocked by pretreating eosinophils with CYP. OVA-induced pulmonary eosinophilia in wild-type mice was significantly inhibited using CYP alone and maximally in combination with a CCR3 receptor antagonist. Interestingly, OVA-induced pulmonary eosinophilia in eotaxin-knockout (Eot-/-) mice was inhibited by treatment with the 5-HT2A but not CCR3 receptor antagonist. These results suggest that 5-HT is a potent eosinophil-active chemoattractant that can function additively with eotaxin and a dual CCR3/5-HT2A receptor antagonist may be more effective in blocking allergen-induced eosinophil recruitment.     

Dimeric rous sarcoma virus capsid protein structure relevant to immature Gag assembly

The structure of the N-terminal domain (NTD) of Rous sarcoma virus (RSV) capsid protein (CA), with an upstream 25 amino acid residue extension corresponding to the C-terminal portion of the Gag p10 protein, has been determined by X-ray crystallography. Purified Gag proteins of retroviruses can assemble in vitro into virus-like particles closely resembling in vivo-assembled immature virus particles, but without a membrane. When the 25 amino acid residues upstream of CA are deleted, Gag assembles into tubular particles. The same phenotype is observed in vivo. Thus, these residues act as a “shape determinant” promoting spherical assembly, when they are present, or tubular assembly, when they are absent. We show that, unlike the NTD on its own, the extended NTD protein has no β-hairpin loop at the N terminus of CA and that the molecule forms a dimer in which the amino-terminal extension forms the interface between monomers. Since dimerization of Gag has been inferred to be a critical step in assembly of spherical, immature Gag particles, the dimer interface may represent a structural feature that is essential in retrovirus assembly.     

Gastrointestinal helminths of Cuvier's beaked whales, Ziphius cavirostris, from the western Mediterranean

We examined the gastrointestinal helminth fauna of 2 Cuvier's beaked whales, Ziphius cavirostris, stranded on the Spanish Mediterranean coast. Information regarding intestinal parasites of this species is provided for the first time. Six helminth taxa were identified. Thirty type II larvae of the nematode Anisakis sp. were found in the stomach and the intestine of both hosts; 2 type I larvae of Anisakis sp. were found in the intestine of 1 host. One juvenile of the acanthocephalan Bolbosoma vasculosum was found in the intestine; the metacestode Scolex pleuronectis was found mainly in the terminal colon and the anal crypts of both hosts; adult cestodes of Tetrabothrius sp., which may represent a new species, were collected from the duodenum of 1 host. Composition of the intestinal parasitic community is similar to that of other oceanic cetaceans, which mostly include species of Bolbosoma and tetrabothriids (Cestoda).     

Hemizygosity at the NCF1 gene in patients with Williams-Beuren syndrome decreases their risk of hypertension

Williams-Beuren syndrome (WBS), caused by a heterozygous deletion at 7q11.23, represents a model for studying hypertension, the leading risk factor for mortality worldwide, in a genetically determined disorder. Haploinsufficiency at the elastin gene is known to lead to the vascular stenoses in WBS and is also thought to predispose to hypertension, present in approximately 50% of patients. Detailed clinical and molecular characterization of 96 patients with WBS was performed to explore clinical-molecular correlations. Deletion breakpoints were precisely defined and were found to result in variability at two genes, NCF1 and GTF2IRD2. Hypertension was significantly less prevalent in patients with WBS who had the deletion that included NCF1 (P=.02), a gene coding for the p47(phox) subunit of the NADPH oxidase. Decreased p47(phox) protein levels, decreased superoxide anion production, and lower protein nitrotyrosination were all observed in cell lines from patients hemizygous at NCF1. Our results indicate that the loss of a functional copy of NCF1 protects a proportion of patients with WBS against hypertension, likely through a lifelong reduced angiotensin II-mediated oxidative stress. Therefore, antioxidant therapy that reduces NADPH oxidase activity might have a potential benefit in identifiable patients with WBS in whom serious complications related to hypertension have been reported, as well as in forms of essential hypertension mediated by a similar pathogenic mechanism.     

Rice NTRC is a high-efficiency redox system for chloroplast protection against oxidative damage

One of the mechanisms plants have developed for chloroplast protection against oxidative damage involves a 2-Cys peroxiredoxin, which has been proposed to be reduced by ferredoxin and plastid thioredoxins, Trx x and CDSP32, the FTR/Trx pathway. We show that rice (Oryza sativa) chloroplast NADPH THIOREDOXIN REDUCTASE (NTRC), with a thioredoxin domain, uses NADPH to reduce the chloroplast 2-Cys peroxiredoxin BAS1, which then reduces hydrogen peroxide. The presence of both NTR and Trx-like domains in a single polypeptide is absolutely required for the high catalytic efficiency of NTRC. An Arabidopsis thaliana knockout mutant for NTRC shows irregular mesophyll cell shape, abnormal chloroplast structure, and unbalanced BAS1 redox state, resulting in impaired photosynthesis rate under low light. Constitutive expression of wild-type NTRC in mutant transgenic lines rescued this phenotype. Moreover, prolonged darkness followed by light/dark incubation produced an increase in hydrogen peroxide and lipid peroxidation in leaves and accelerated senescence of NTRC-deficient plants. We propose that NTRC constitutes an alternative system for chloroplast protection against oxidative damage, using NADPH as the source of reducing power. Since no light-driven reduced ferredoxin is produced at night, the NTRC-BAS1 pathway may be a key detoxification system during darkness, with NADPH produced by the oxidative pentose phosphate pathway as the source of reducing power.     

The molybdate-binding protein (ModA) of the plant pathogen Xanthomonas axonopodis pv. citri

The modABC operon of phytopathogen Xanthomonas axonopodis pv. citri (X. citri) encodes a putative ABC transporter involved in the uptake of the molybdate and tungstate anions. Sequence analyses showed high similarity values of ModA orthologs found in X. campestris pv. campestris (X. campestris) and Escherichia coli. The X. citri modA gene was cloned in pET28a and the recombinant protein, expressed in the E. coli BL21 (DE3) strain, purified by immobilized metal affinity chromatography. The purified protein remained soluble and specifically bound molybdate and tungstate with K(d) 0.29+/-0.12 microM and 0.58+/-0.14 microM, respectively. Additionally binding of molybdate drastically enhanced the thermal stability of the recombinant ModA as compared to the apoprotein. This is the first characterization of a ModA ortholog expressed by a phytopathogen and represents an important tool for functional, biochemical and structural analyses of molybdate transport in Xanthomonas species.     

Mitochondrial free cholesterol loading sensitizes to TNF- and Fas-mediated steatohepatitis

The etiology of progression from steatosis to steatohepatitis (SH) remains unknown. Using nutritional and genetic models of hepatic steatosis, we show that free cholesterol (FC) loading, but not free fatty acids or triglycerides, sensitizes to TNF- and Fas-induced SH. FC distribution in endoplasmic reticulum (ER) and plasma membrane did not cause ER stress or alter TNF signaling. Rather, mitochondrial FC loading accounted for the hepatocellular sensitivity to TNF due to mitochondrial glutathione (mGSH) depletion. Selective mGSH depletion in primary hepatocytes recapitulated the susceptibility to TNF and Fas seen in FC-loaded hepatocytes; its repletion rescued FC-loaded livers from TNF-mediated SH. Moreover, hepatocytes from mice lacking NPC1, a late endosomal cholesterol trafficking protein, or from obese ob/ob mice, exhibited mitochondrial FC accumulation, mGSH depletion, and susceptibility to TNF. Thus, we propose a critical role for mitochondrial FC loading in precipitating SH, by sensitizing hepatocytes to TNF and Fas through mGSH depletion.     

Allosteric inhibitors of Bcr-abl-dependent cell proliferation

Chronic myelogenous leukemia (CML) is a myeloproliferative disorder characterized at the molecular level by the expression of Bcr-abl, a 210-kDa fusion protein with deregulated tyrosine kinase activity. Encouraged by the clinical validation of Bcr-abl as the target for the treatment of CML by imatinib, we sought to identify pharmacological agents that could target this kinase by a distinct mechanism. We report the discovery of a new class of Bcr-abl inhibitors using an unbiased differential cytotoxicity screen of a combinatorial kinase-directed heterocycle library. Compounds in this class (exemplified by GNF-2) show exclusive antiproliferative activity toward Bcr-abl-transformed cells, with potencies similar to imatinib, while showing no inhibition of the kinase activity of full-length or catalytic domain of c-abl. We propose that this new class of compounds inhibits Bcr-abl kinase activity through an allosteric non-ATP competitive mechanism.