Expression of Recombinant Potato leafroll virus Structural and Non‐structural Proteins for Antibody Production

Vector pMPM‐A4Ω and vectors pQE‐30 and pET‐45b(+) containing the 6x His‐tag sequence were used for expression of Potato leafroll virus (PLRV) structural and non‐structural proteins in Escherichia coli. Coat protein (CP) and RNA‐dependent RNA polymerase (RdRp)–fragments RdRp_43‐616 and RdRp_304‐537 were chosen for expression. A high level of CP and RdRp_304‐537 was obtained only in an expression system using pET‐45b(+) vector and E. coli Rosetta‐gami 2(DE3) cells. After purification, the His‐tagged PLRV proteins were used for immunization of rabbits.     

Different vanilloid agonists cause different patterns of calcium response in CHO cells heterologously expressing rat TRPV1

The vanilloid receptor subtype 1 (TRPV1 or VR1) is expressed in nociceptive primary afferents of the C-fiber 'pain' pathway and has attracted considerable attention as a therapeutic target. Here, using rat TRPV1 heterologously expressed in Chinese hamster ovary cells, we show that different agonists show different patterns of modulation of the intracellular Ca2+ concentration, monitored in individual cells by fura-2 Ca2+ imaging. We identified 5 parameters (potency, maximal response, latency of response, variability of latency of response among individual cells, and desensitization) which behaved differently for different compounds. The potencies of the compounds examined ranged from EC50 values of 80 pM to 9 microM. Peak levels of induced [Ca2+]i were observed either higher (RTX) or lower (anandamide) than for capsaicin. Significant latencies of response were observed for some (e.g. RTX) but not other derivatives, with great variation among individual cells in this latency. Marked desensitization after stimulation was detected in some cases (e.g. anandamide, capsaicin); for others, no desensitization was observed. We conclude that structurally diverse vanilloid agonists induce marked diversity in the patterns of Ca2+ response. This diversity of response may provide opportunities for pharmacological exploitation.     

Efficient bacterial expression of recombinant potato mop-top virus non-structural triple gene block protein 1 modified by progressive deletion of its N-terminus

To obtain strong bacterial expression of proteins that seem to be hard to express in bacteria or are highly toxic for bacteria, it is possible to create a palette of similar constructs, differing only by several nucleotides, gradually deleted from the full-length clone by exonuclease III. When a construct is equipped with the 6xHis tag, a simple colony-blot procedure can be performed and a colony giving strong and efficient expression can easily be selected for high range protein expression. We utilized this procedure to produce one of potato mop-top virus (PMTV) movement proteins, namely triple gene block protein 1 (TGBp1) which was very hard to express in bacteria in its original length. The TGBp1 gene was digested with exonuclease III and nuclease S1 from its 5' terminus, leaving 6xHis tag intact. The clone that showed the strongest signal with anti-His antibodies in colony-blot procedure was found to have 44 amino acids (of total 463) deleted. The SDS-PAGE and Western blot of high range bacterial culture lysate confirmed the efficient expression of this deleted 6xHis tagged TGBp1 fragment.     

SMG7 is a 14-3-3-like adaptor in the nonsense-mediated mRNA decay pathway

In metazoa, regulation of the phosphorylation state of UPF1 is crucial for nonsense-mediated mRNA decay (NMD), a process by which aberrant mRNAs containing nonsense mutations are degraded. UPF1 is targeted for dephosphorylation by three related proteins, SMG5, SMG6, and SMG7. We report here the crystal structure of the N-terminal domain of SMG7. The structure reveals that SMG7 contains a 14-3-3-like domain. Residues that bind phosphoserine-containing peptides in 14-3-3 are conserved at the equivalent positions in SMG7. Mutation of these residues impairs UPF1 binding to SMG7 in vitro and UPF1 recruitment to cytoplasmic mRNA decay foci in vivo, suggesting that SMG7 acts as an adaptor in targeting mRNAs associated with phosphorylated UPF1 for degradation. The 14-3-3 site of SMG7 is conserved in SMG5 and SMG6. These data also imply that the homologous human Est1 might have a 14-3-3 function at telomeres, and that phosphorylation events may be important for telomerase regulation.     

Defective CD3zeta chain expression in Herpesvirus saimiri (HVS)-derived T-cell lines in gastric adenocarcinoma

Low expression of the CD3zeta chain has been reported in patients with cancer and it has been suggested that tumor-derived factors are involved in its downregulation. The expression of CD3zeta chain was measured in T-cell lines from patients with gastric adenocarcinoma and healthy volunteers and grown in vitro for several months and, hence, in the absence of any tumor-derived factors. T-cell lines of mucosal origin were obtained by Herpesvirus saimiri transformation from gastric cancer patients. The expression of CD3zeta and CD3epsilon was measured by flow cytometry and Western-blot analysis. Calcium mobilization and apoptosis rate were also measured. The levels of CD3zeta, but not CD3epsilon, chain on the cell surface were significantly reduced in T-cell lines derived from patients with gastric cancer when cultured in the absence of IL-2. Western-blot analysis of total cell extracts or lipid raft fractions confirmed this finding. Calcium mobilization, a measure of signal transduction, was reduced in T cell lines from patients with gastric cancer. We conclude that T cells from patients with cancer express lower levels of CD3zeta. This downregulation is not caused by a direct effect of tumor-derived factors but, rather, it appears to be inherent to the patient cells. The low CD3zeta expression would render T lymphocytes unable to control the growth of tumor cells.     

Conformation dependence of the CαDα stretch mode in peptides: Side‐chain influence in dipeptide structures

We have shown by theoretical studies of alanine peptides that the C^αD^α stretch frequency could be particularly useful for determining peptide structure because of its sensitivity to the φ,ψ torsion angles at the C^α atom. To demonstrate that this is a robust methodology worthy of experimental exploration, we have also shown that this mode is even more determinative of conformation in aqueous solution, mainly as a result of the development of differential C^α? D^α···O(water) interactions. As further assurance, we now determine the influence of the side chain on this band, showing for aliphatic, a polar, and an aromatic side chains that the dependence is minor and explaining why this is also expected for other side chains. These results should stimulate new experimental methodologies in the field of peptide structure determination. © 2010 Wiley Periodicals, Inc. Biopolymers 93: 1065–1071, 2010. This article was originally published online as an accepted preprint. The “Published Online” date corresponds to the preprint version. You can request a copy of the preprint by emailing the Biopolymers editorial office at biopolymers@wiley.com     

Progesterone-induced blocking factor activates STAT6 via binding to a novel IL-4 receptor

Progesterone-induced blocking factor (PIBF) induces Th2-dominant cytokine production. Western blotting and EMSA revealed phosphorylation as well as nuclear translocation of STAT6 and inhibition of STAT4 phosphorylation in PIBF-treated cells. The silencing of STAT6 by small interfering RNA reduced the cytokine effects. Because the activation of the STAT6 pathway depends on the ligation of IL-4R, we tested the involvement of IL-4R in PIBF-induced STAT6 activation. Although PIBF does not bind to IL-4R, the blocking of the latter with an Ab abolished PIBF-induced STAT6 activation, whereas the blocking of the IL-13R had no effect. PIBF activated suppressor of cytokine signaling-3 and inhibited IL-12-induced suppressor of cytokine signaling-1 activation. The blocking of IL-4R counteracted all the described effects, suggesting that the PIBF receptor interacts with IL-4R alpha-chain, allowing PIBF to activate the STAT6 pathway. PIBF did not phosphorylate Jak3, suggesting that the gamma-chain is not needed for PIBF signaling. Confocal microscopic analysis revealed a colocalization and at 37 degrees C a cocapping of the FITC PIBF-activated PIBF receptor and PE anti-IL-4R-labeled IL-4R. After the digestion of the cells with phosphatidylinositol-specific phospholipase C, the STAT6-activating effect of PIBF was lost, whereas that of IL-4 remained unaltered. These data suggest the existence of a novel type of IL-4R composed of the IL-4R alpha-chain and the GPI-anchored PIBF receptor.     

Iron Reduction and Soil Phosphorus Solubilization in Humid Tropical Forests Soils: The Roles of Labile Carbon Pools and an Electron Shuttle Compound

The affinity of iron oxides and hydroxides for phosphorus is thought to contribute to phosphorus limitation to net primary productivity in humid tropical forests on acidic, highly weathered soils. Perennially warm, humid conditions and high biological activity in these soils can result in fluctuating redox potential that in turn leads to considerable iron reduction in the presence of labile carbon and humic substances. We investigated the effects of reducing conditions in combination with the addition of labile carbon substrates (glucose and acetate) and an electron shuttle compound on iron reduction and phosphorus release in a humid tropical forest soil. Glucose or acetate was added to soils as a single dose at the beginning of the experiment, and as pulsed inputs over time, which more closely mimics patterns in labile carbon availability. Iron reduction and phosphorus mobilization were weakly stimulated by a single low level addition of carbon, and the addition of the electron shuttle compound with or without added carbon. Pulsed labile carbon additions produced a significant increase in soil pH, soluble iron, and phosphorus concentrations. Pulsed labile carbon inputs also promoted the precipitation of ferrous hydroxide complexes which could increase the capacity for P sorption, although our results suggest that rates of P solubilization exceeded re-adsorption. Plant and microbial P demand are also likely to serve as an important sinks for released P, limiting the role of P re-adsorption. Our results suggest that reducing conditions coupled with periodic carbon inputs can stimulate iron reduction and a corresponding increase in soil phosphorus mobilization, which may provide a source of phosphorus to plants and microorganisms previously undocumented in these ecosystems.     

Trypanosoma cruzi disrupts myofibrillar organization and intracellular calcium levels in mouse neonatal cardiomyocytes

Immunofluorescence studies of normal and Trypanosoma cruzi-infected primary cultures of heart muscle cells were performed to gather information about the arrangement of myofibrillar components during the intracellular life cycle of this parasite. By using a panel of monoclonal antibodies against various myofibrillar proteins, a progressive disruption and loss of contractile proteins (such myosin and actin) of the host cell was detected during infection. The host cell formed a loose network of myofibrillar proteins around the parasites. Breakdown of the myofibrils occurred in regions where the parasites were present, and heavily infected cells showed myofibrillar proteins at their periphery. In parallel, we investigated the effect of T. cruzi infection on intracellular calcium levels by using a Ca²⁺ fluorescent indicator (confocal microscopy). Infected cardiomyocytes displayed a marked impairment in contractility, and calcium influxes became irregular and less intense when compared with those of non-infected cells. Our results demonstrate that T. cruzi infection dramatically affects calcium fluxes and causes myofibrillar breakdown disturbing cardiomyocyte contractility.Financial support through grants and scholarships from the Brazilian funding agencies FAPESP, CNPq, and CAPES is gratefully acknowledged.