Competition between bound and free peptides in an ELISA-based procedure that assays peptides derived from protein digests

Background  

We describe an ELISA-based method that can be used to identify and quantitate proteins in biological samples. In this method, peptides in solution, derived from proteolytic digests of the sample, compete with substrate-attached synthetic peptides for antibodies, also in solution, generated against the chosen peptides. The peptides used for the ELISA are chosen on the basis of their being (i) products of the proteolytic (e.g. tryptic) digestion of the protein to be identified and (ii) unique to the target protein, as far as one can know from the published sequences.     

Induction of osteoclastogenesis in an in vitro model of Gaucher disease is mediated by T cells via TNF-α

Gaucher disease is a lysosomal storage disorder caused by deficiency of glucocerebrosidase enzymatic activity leading to accumulation of its substrate glucocerebrosidase mainly in macrophages. Skeletal disorder of Gaucher disease is the major cause of morbidity and is highly refractory to enzyme replacement therapy. However, pathological mechanisms of bone alterations in Gaucher disease are still poorly understood. We hypothesized that cellular alteration in Gaucher disease produces a proinflammatory milieu leading to bone destruction through enhancement of monocyte differentiation to osteoclasts and osteoclasts resorption activity. Against this background we decided to investigate in an in vitro chemical model of Gaucher disease, the capacity of secreted soluble mediators to induce osteoclastogenesis, and the mechanism responsible for this phenomena. We demonstrated that soluble factors produced by CBE-treated PBMC induced differentiation of osteoclasts precursors into mature and active osteoclasts that express chitotriosidase and secrete proinflammatory cytokines. We also showed a role of TNF-α in promoting osteoclastogenesis in Gaucher disease chemical model. To analyze the biological relevance of T cells in osteoclastogenesis of Gaucher disease, we investigated this process in T cell-depleted PBMC cultures. The findings suggest that T cells play a role in osteoclast formation in Gaucher disease. In conclusion, our data suggests that in vitro GCASE deficiency, along with concomitant glucosylceramide accumulation, generates a state of osteoclastogenesis mediated in part by pro-resorptive cytokines, especially TNF-α. Moreover, T cells are involved in osteoclastogenesis in Gaucher disease chemical model.     

Application of expert networks for predicting proteins secondary structure

The present study utilizes expert neural networks for the prediction of proteins secondary structure. We use three independent networks, one for each structure (alpha, beta and coil) as the first-level processing unit; decision upon the chosen structure for each residue is carried out by a second-level, post-processing unit, which utilizes the Chou and Fasman frequency values Falpha and Fbeta in order to strengthen and/or deplete the probability of the specific structure under investigation. The highest prediction case was 76%. Our method requires primitive computational means and a relatively small training set, while still been comparable to previous work. It is not meant to be an alternative to the determination of secondary structure by means of free energy minimization, integration of dynamic equations of motion or crystallography, which are expensive, time-consuming and complicated, but to provide additional constrains, which might be considered and incorporated into larger computing setups in order to reduce the initial search space for the above methods.     

Maintenance of redox state and pancreatic beta-cell function: role of leptin and adiponectin

Whereas oxidative stress is linked to cellular damage, reactive oxygen species (ROS) are also believed to be involved in the propagation of signaling pathways. Studies on the role of ROS in pancreatic beta‐cell physiology, in contrast to pathophysiology, have not yet been reported. In this study we investigate the importance of maintaining cellular redox state on pancreatic beta‐cell function and viability, and the effects of leptin and adiponectin on this balance. Experiments were conducted on RINm and MIN6 pancreatic beta‐cells. Leptin (1–100 ng/ml) and adiponectin (1–100 nM) increased ROS accumulation, as was determined by DCFDA fluorescence. Using specific inhibitors, we found that the increase in ROS levels was mediated by NADPH oxidase (Nox), but not by AMP kinase (AMPK) or phosphatidyl inositol 3 kinase (PI3K). Leptin and adiponectin increased beta‐cell number as detected by the XTT method, but did not affect apoptosis, indicating that the increased cell number results from increased proliferation. The adipokines‐induced increase in viability is ROS dependent as this effect was abolished by N‐acetyl‐L‐cysteine (NAC) or PEG‐catalase. In addition, insulin secretion was found to be regulated by alterations in redox state, but not by adipokines. Finally, the effects of the various treatments on activity and mRNA expression of several antioxidant enzymes were determined. Both leptin and adiponectin reduced mRNA levels of superoxide dismutase (SOD)1. Adiponectin also decreased SOD activity and increased catalase and glutathione peroxidase (GPx) activities in the presence of H₂O₂. The results of this study show that leptin and adiponectin, by inducing a physiological increase in ROS levels, may be positive regulators of beta‐cell mass. J. Cell. Biochem. 113: 1966–1976, 2012. © 2012 Wiley Periodicals, Inc.     

Lysozyme-modified probiotic components protect rats against polymicrobial sepsis: role of macrophages and cathelicidin-related innate immunity

Severe sepsis is associated with dysfunction of the macrophage/monocyte, an important cellular effector of the innate immune system. Previous investigations suggested that probiotic components effectively enhance effector cell functions of the immune system in vivo. In this study, we produced bacteria-free, lysozyme-modified probiotic components (LzMPC) by treating the probiotic bacteria, Lactobacillus sp., with lysozyme. We showed that oral delivery of LzMPC effectively protected rats against lethality from polymicrobial sepsis induced by cecal ligation and puncture. We found that orally administrated LzMPC was engulfed by cells such as macrophages in the liver after crossing the intestinal barrier. Moreover, LzMPC-induced protection was associated with an increase in bacterial clearance in the liver. In vitro, LzMPC up-regulated the expression of cathelicidin-related antimicrobial peptide (CRAMP) in macrophages and enhanced bactericidal activity of these cells. Furthermore, we demonstrated that surgical stress or cecal ligation and puncture caused a decrease in CRAMP expression in the liver, whereas enteral administration of LzMPC restored CRAMP gene expression in these animals. Using a neutralizing Ab, we showed that protection against sepsis by LzMPC treatment required endogenous CRAMP. In addition, macrophages from LzMPC-treated rats had an enhanced capacity of cytokine production in response to LPS or LzMPC stimulation. Together, our data suggest that the protective effect of LzMPC in sepsis is related to an enhanced cathelicidin-related innate immunity in macrophages. Therefore, LzMPC, a novel probiotic product, is a potent immunomodulator for macrophages and may be beneficial for the treatment of sepsis.     

Interference of Mycobacterium tuberculosis cell division by Rv2719c, a cell wall hydrolase

The genetic factors responsible for the regulation of cell division in Mycobacterium tuberculosis are largely unknown. We showed that exposure of M. tuberculosis to DNA damaging agents, or to cephalexin, or growth of M. tuberculosis in macrophages increased cell length and sharply elevated the expression of Rv2719c, a LexA-controlled gene. Overexpression of Rv2719c in the absence of DNA damage or of antibiotic treatment also led to filamentation and reduction in viability both in broth and in macrophages indicating a correlation between Rv2719c levels and cell division. Overproduction of Rv2719c compromised midcell localization of FtsZ rings, but had no effect on the intracellular levels of FtsZ. In vitro, the Rv2719c protein did not interfere with the GTP-dependent polymerization activity of FtsZ indicating that the effects of Rv2719c on Z-ring assembly are indirect. Rv2719c protein exhibited mycobacterial murein hydrolase activity that was localized to the N-terminal 110 amino acids. Visualization of nascent peptidoglycan (PG) synthesis zones by probing with fluoresceinated vancomycin (Van-FL) and localization of green fluorescent protein-Rv2719c fusion suggested that the Rv2719c activity is targeted to potential PG synthesis zones. We propose that Rv2719c is a potential regulator of M. tuberculosis cell division and that its levels, and possibly activities, are modulated under a variety of growth conditions including growth in vivo and during DNA damage, so that the assembly of FtsZ-rings, and therefore the cell division, can proceed in a regulated manner.     

Conformational spaces of the gastrointestinal antisecretory chiral drug omeprazole: stereochemistry and tautomerism

A study of the conformational spaces of the chiral proton pump inhibitor (PPI) drug omeprazole by semiempirical, ab-initio, and DFT methods is described. In addition to the chiral center at the sulfinyl sulfur atom, the chiral axis at the pyridine ring (due to the hindered rotation of the 4-methoxy substituents) was considered. The results were analyzed in terms of the 5-methoxy and 6-methoxy tautomers and the two pairs of enantiomers (R,P)/(S,M) and (R,M)/(S,P). Five torsion angles were systematically explored: the backbone rotations defined by D1 (N3-C2-S10-O11), D2 (C2-S10-C12-C13), and D3 (S10-C12-C13-N14) and two methoxy rotations defined by D4 (C6-C5-O8-C9) and D5 (C16-C17-O19-C20). Significant energy differences were revealed between the 5- and 6-methoxy tautomers, the extended and folded conformations, and the (S,M) and (S,P) diastereomers. The "extended M" conformation of the 6-methoxy tautomer of (S)-omeprazole was found to be the most stable conformer.     

Regulation and temporal expression patterns of Vibrio cholerae virulence genes during infection

The temporal expression patterns of the critical Vibrio cholerae virulence genes, tcpA and ctxA, were determined during infection using a recombinase reporter. TcpA was induced biphasically in two temporally and spatially separable events in the small intestine, whereas ctxA was induced monophasically only after, and remarkably, dependent upon, tcpA expression; however, this dependence was not observed during in vitro growth. The requirements of the virulence regulators, ToxR, TcpP, and ToxT, for expression of tcpA and ctxA were determined and were found to differ significantly during infection versus during growth in vitro. These results illustrate the importance of examining virulence gene expression in the context of bona fide host-pathogen interactions.     

Integrin regulation by vascular endothelial growth factor in human brain microvascular endothelial cells: role of alpha6beta1 integrin in angiogenesis

The precise role of vascular endothelial growth factor (VEGF) in regulating integrins in brain microvascular endothelial cells is unknown. Here, we analyzed VEGF effects on integrin expression and activation in human brain microvascular endothelial cells (HBMECs). Using human cDNA arrays and ribonuclease (RNase) protection assays, we observed that VEGF up-regulated the mRNA expression of alpha(6) integrin in HBMECs. VEGF significantly increased alpha(6)beta(1) integrin expression, but not alpha(6)beta(4) integrin expression in these cells. Specific down-regulation of alpha(6) integrin expression by small interfering RNA (siRNA) oligonucleotides inhibited both the capillary morphogenesis of HBMECs and their adhesion and migration. Additionally, VEGF treatment resulted in activation of alpha(6)beta(1) integrins in HBMECs. Functional blocking of alpha(6) integrin with its specific antibody inhibited the VEGF-induced adhesion and migration as well as in vivo angiogenesis, and markedly suppressed tumor angiogenesis and breast carcinoma growth in vivo. Thus, VEGF can modulate angiogenesis via increased expression and activation of alpha(6)beta(1) integrins, which may promote VEGF-driven tumor angiogenesis in vivo.     

Cdc42 and RhoB activation are required for mannose receptor-mediated phagocytosis by human alveolar macrophages

Human alveolar macrophages (AMs) phagocytose Pneumocystis (Pc) organisms predominantly through mannose receptors, although the molecular mechanism mediating this opsonin-independent process is not known. In this study, using AMs from healthy individuals, Pc phagocytosis was associated with focal F-actin polymerization and Cdc42, Rac1, and Rho activation in a time-dependent manner. Phagocytosis was primarily dependent on Cdc42 and RhoB activation (as determined by AM transfection with Cdc42 and RhoB dominant-negative alleles) and mediated predominantly through mannose receptors (as determined by siRNA gene silencing of AM mannose receptors). Pc also promoted PAK-1 phosphorylation, which was also dependent on RhoGTPase activation. HIV infection of AMs (as a model for reduced mannose receptor expression and function) was associated with impaired F-actin polymerization, reduced Cdc42 and Rho activation, and markedly reduced PAK-1 phosphorylation in response to Pc organisms. In healthy AMs, Pc phagocytosis was partially dependent on PAK activation, but dependent on the Rho effector molecule ROCK. These data provide a molecular mechanism for AM mannose receptor-mediated phagocytosis of unopsonized Pc organisms that appears distinct from opsonin-dependent phagocytic receptors. Reduced AM mannose receptor-mediated Cdc42 and Rho activation in the context of HIV infection may represent a mechanism that contributes to the pathogenesis of opportunistic pneumonia.