A fusion gene coding for two different δ-endotoxinsof Bacillus thuringiensis toxic to Plutella xylostella and usefulfor resistance management

Two truncated Bacillus thuringiensis -endotoxin genes, belonging to the classes cry1Ab and cry1B, and both coding for N-terminal toxic fragments of the corresponding crystal proteins, were translationally fused. Expression of the fusion gene driven by the cry1C promoter in Escherichia coli at a very high level resulted in a protein with enhanced toxicity to the diamondback moth (Plutella xylostella).     

An Efficiently Cleaved HIV-1 Clade C Env Selectively Binds to Neutralizing Antibodies

An ideal HIV-1 Env immunogen is expected to mimic the native trimeric conformation for inducing broadly neutralizing antibody responses. The native conformation is dependent on efficient cleavage of HIV-1 Env. The clade B isolate, JRFL Env is efficiently cleaved when expressed on the cell surface. Here, for the first time, we report the identification of a native clade C Env, 4-2.J41 that is naturally and efficiently cleaved on the cell surface as confirmed by its biochemical and antigenic characteristics. In addition to binding to several conformation-dependent neutralizing antibodies, 4-2.J41 Env binds efficiently to the cleavage-dependent antibody PGT151; thus validating its native cleaved conformation. In contrast, 4-2.J41 Env occludes non-neutralizing epitopes. The cytoplasmic-tail of 4-2.J41 Env plays an important role in maintaining its conformation. Furthermore, codon optimization of 4-2.J41 Env sequence significantly increases its expression while retaining its native conformation. Since clade C of HIV-1 is the prevalent subtype, identification and characterization of this efficiently cleaved Env would provide a platform for rational immunogen design.     

Reorganization of cytoskeletal proteins by Escherichia coli heat-stable enterotoxin (STa)-mediated signaling cascade

Background

IP₃-mediated calcium mobilization from intracellular stores activates and translocates PKC-α from cytosol to membrane fraction in response to STa in COLO-205 cell line. The present study was undertaken to determine the involvement of cytoskeleton proteins in translocation of PKC-α to membrane from cytosol in the Escherichiacoli STa-mediated signaling cascade in a human colonic carcinoma cell line COLO-205.

Methods

Western blots and consequent densitometric analysis were used to assess time-dependent redistribution of cytoskeletal proteins. This redistribution was further confirmed by using confocal microscopy. Pharmacological reagents were applied to colonic carcinoma cells to disrupt the microfilaments (cytochalasin D) and microtubules (nocodazole).

Results

STa treatment in COLO-205 cells showed dynamic redistribution and an increase in actin content in the Triton-insoluble fraction, which corresponds to an increase in polymerization within 1 min. Moreover, pharmacological disruption of actin-based cytoskeleton greatly disturbed PKC-α translocation to the membrane.

Conclusions

These results suggested that the organization of actin cytoskeleton is rapidly rearranged following E. coli STa treatment and the integrity of the actin cytoskeleton played a crucial role in PKC-α movement in colonic cells. Depolymerization of tubulin had no effect on the ability of the kinase to be translocated to the membrane.

General significance

In the present study, we have shown for the first time that in colonic carcinoma cells, STa-mediated rapid changes of actin cytoskeleton arrangement might be involved in the translocation of PKC-α to membrane.     

Differential expression and localization of 12/15 lipoxygenases in adipose tissue in human obese subjects

Adipose tissue inflammation in obesity is a major factor leading to cardiovascular disease and type 2 diabetes.12/15 lipoxygenases (ALOX) play an important role in the generation of inflammatory mediators, insulin resistance and downstream immune activation in animal models of obesity. However, the expression and roles of 12/15ALOX isoforms, and their cellular sources in human subcutaneous (sc) and omental (om) fat in obesity is unknown. The objective of this study was to examine the gene expression and localization of ALOX isoforms and relevant downstream cytokines in subcutaneous (sc) and omental (om) adipose tissue in obese humans. Paired biopsies of sc and om fat were obtained during bariatric surgeries from 24 morbidly obese patients. Gene and protein expression for ALOX15a, ALOX15b and ALOX 12 were measured by real-time PCR and western blotting in adipocytes and stromal vascular fractions (SVF) from om and sc adipose tissue along with the mRNA expression of the downstream cytokines IL-12a, IL-12b, IL-6, IFNγ and the chemokine CXCL10. In a paired analysis, all ALOX isoforms, IL-6, IL-12a and CXCL10 were significantly higher in om vs. sc fat. ALOX15a mRNA and protein expression was found exclusively in om fat. All of the ALOX isoforms were expressed solely in the SVF. Further fractionation of the SVF in CD34+ and CD34- cells indicated that ALOX15a is predominantly expressed in the CD34+ fraction including vascular and progenitor cells, while ALOX15B is mostly expressed in the CD34- cells containing various leucocytes and myeloid cells. This result was confirmed by immunohistochemistry showing exclusive localization of ALOX15a in the om fat and predominantly in the vasculature and non-adipocyte cells. Our finding is identifying selective expression of ALOX15a in human om but not sc fat. This is a study showing a major inflammatory gene exclusively expressed in visceral fat in humans.     

An Iris-Like Mechanism of Pore Dilation in the CorA Magnesium Transport System

Magnesium translocation across cell membranes is essential for numerous physiological processes. Three recently reported crystal structures of the CorA magnesium transport system revealed a surprising architecture, with a bundle of giant α-helices forming a 60-Å-long pore that extends beyond the membrane before widening into a funnel-shaped cytosolic domain. The presence of divalent cations in putative intracellular regulation sites suggests that these structures correspond to the closed conformation of CorA. To examine the nature of the conduction pathway, we performed 110-ns molecular-dynamics simulations of two of these structures in a lipid bilayer with and without regulatory ions. The results show that a 15-Å-long hydrophobic constriction straddling the membrane-cytosol interface constitutes a steric bottleneck whose location coincides with an electrostatic barrier opposing cation translocation. In one of the simulations, structural relaxation after the removal of regulatory ions led to concerted changes in the tilt of the pore helices, resulting in iris-like dilation and spontaneous hydration of the hydrophobic neck. This simple and robust mechanism is consistent with the regulation of pore opening by intracellular magnesium concentration, and explains the unusual architecture of CorA.     

Akt activation and augmented fibronectin production in hyperhexosemia

Dysmetabolic state in diabetes may lead to augmented synthesis of extracellular matrix (ECM) proteins. In the endothelial cells, we have previously demonstrated that glucose-induced fibronectin (FN) production and that of its splice variant, EDB(+)FN, is regulated by protein kinase B (PKB, also known as Akt). In this study, we investigated the role of Akt1 in ECM protein production in the organs affected by chronic diabetic complications. We studied Akt1/PKBalpha knockout mice and wild-type control littermates. To avoid confounding effects of systemic insulin, we used 30% galactose feeding to induce hyperhexosemia for 8 wk starting at 6 wk of age. We investigated FN mRNA, EDB(+)FN mRNA, and transforming growth factor (TGF)-beta mRNA expression, Akt phosphorylation, Akt kinase activity, and NF-kappaB and AP-1 activation in the retina, heart, and kidney. Renal and cardiac tissues were histologically examined. Galactose feeding caused significant upregulation of FN, EDB(+)FN, and TGF-beta in all tissues. FN protein levels paralleled mRNA. Such upregulation were prevented in Akt1-deficient galactose-fed mice. Galactose feeding caused ECM protein deposition in the glomeruli and in the myocardium, which was prevented in the Akt knockout mice. NF-kappaB and AP-1 activation was pronounced in galactose-fed wild-type mice and prevented in the galactose-fed Akt1/PKBalpha-deficient group. In the retina and kidney, Ser473 was the predominant site for Akt phosphorylation, whereas in the heart it was Thr308. Parallel experiment in streptozotocin-induced diabetic animals showed similar results. The data from this study indicate that hyperhexosemia-induced Akt/PKB activation may be an important mechanism leading to NF-kappaB and AP-1 activation and increased ECM protein synthesis in the organs affected by chronic diabetic complications.     

Lysis of human immunodeficiency virus type 1 by a specific secreted human phospholipase A2

Phospholipase A2 (PLA2) proteins affect cellular activation, signal transduction, and possibly innate immunity. A specific secretory PLA2, sPLA2-X, is shown here to neutralize human immunodeficiency virus type 1 (HIV-1) through degradation of the viral membrane. Catalytic function was required for antiviral activity, and the target cells of infection were unaffected. sPLA2-X potently reduced gene transfer of HIV-1 Env-pseudotyped lentivirus vectors and inhibited the replication of both CCR5- and CXCR4-tropic HIV-1 in human CD4+ T cells. Virions resistant to damage by antibody and complement were sensitive to lysis by sPLA2-X, suggesting a novel mechanism of antiviral surveillance independent of the acquired immune system.     

Peptide segments in protein-protein interfaces

An important component of functional genomics involves the understanding of protein association. The interfaces resulting from protein-protein interactions - (i) specific, as represented by the homodimeric quaternary structures and the complexes formed by two independently occurring protein components, and (ii) non-specific, as observed in the crystal lattice of monomeric proteins - have been analysed on the basis of the length and the number of peptide segments. In 1000 A2 of the interface area, contributed by a polypeptide chain, there would be 3.4 segments in homodimers, 5.6 in complexes and 6.3 in crystal contacts. Concomitantly, the segments are the longest (with 8.7 interface residues) in homodimers. Core segments (likely to contribute more towards binding) are more in number in homodimers (1.7) than in crystal contacts (0.5), and this number can be used as one of the parameters to distinguish between the two types of interfaces. Dominant segments involved in specific interactions, along with their secondary structural features, are enumerated.     

Analysis and prediction of functionally important sites in proteins

The rapidly increasing volume of sequence and structure information available for proteins poses the daunting task of determining their functional importance. Computational methods can prove to be very useful in understanding and characterizing the biochemical and evolutionary information contained in this wealth of data, particularly at functionally important sites. Therefore, we perform a detailed survey of compositional and evolutionary constraints at the molecular and biological function level for a large set of known functionally important sites extracted from a wide range of protein families. We compare the degree of conservation across different functional categories and provide detailed statistical insight to decipher the varying evolutionary constraints at functionally important sites. The compositional and evolutionary information at functionally important sites has been compiled into a library of functional templates. We developed a module that predicts functionally important columns (FIC) of an alignment based on the detection of a significant "template match score" to a library template. Our template match score measures an alignment column's similarity to a library template and combines a term explicitly representing a column's residue composition with various evolutionary conservation scores (information content and position-specific scoring matrix-derived statistics). Our benchmarking studies show good sensitivity/specificity for the prediction of functional sites and high accuracy in attributing correct molecular function type to the predicted sites. This prediction method is based on information derived from homologous sequences and no structural information is required. Therefore, this method could be extremely useful for large-scale functional annotation.