Understanding the role of Rho GTPase Rac1 in signaling pathways using antioxidants.

Antioxidants are intimately involved in the prevention of cellular damage, the common pathway for cancer, aging, and a variety of diseases. Antioxidants are molecules, which can safely interact with free radicals and terminate the chain reaction before vital molecules are damaged. Although there are several enzyme systems within the body that scavenge free radicals, the principle micronutrient antioxidants are vitamin E, beta-carotene, and vitamin C. Additionally, selenium, a trace metal that is required for proper function of one of the body's antioxidant enzyme systems, is sometimes included in this category. The body cannot manufacture these micronutrients so they must be supplied in the diet. Antioxidant is capable of slowing or preventing the oxidation of other molecules. Oxidation is a chemical reaction that transfers electrons from a substance to an oxidizing agent. Oxidation reactions can produce free radicals, which start chain reactions that damage cells. Antioxidants terminate these chain reactions by removing free radical intermediates, and inhibit other oxidation reactions by being oxidized themselves. As a result, antioxidants are often reducing agents such as thiols or polyphenols. Although oxidation reactions are crucial for life, they can also be damaging; hence, plants and animals maintain complex systems of multiple types of antioxidants, such as glutathione, Vitamin C, and vitamin E as well as enzymes such as catalase, super oxide dismutase and various peroxidases. Low levels of antioxidants, or inhibition of the antioxidant enzymes, causes oxidative stress and may damage or kill cells. As oxidative stress might be an important part of many human diseases, the use of antioxidants in pharmacology is intensively studied, particularly as treatments for stroke and neurodegenerative diseases. However, it is unknown whether oxidative stress is the cause or the consequence of disease.Digital copy of Thesis.University of Kashmir

Hydrogen peroxide regulation of endothelial exocytosis by inhibition of N-ethylmaleimide sensitive factor

Although an excess of reactive oxygen species (ROS) can damage the vasculature, low concentrations of ROS mediate intracellular signal transduction pathways. We hypothesized that hydrogen peroxide plays a beneficial role in the vasculature by inhibiting endothelial exocytosis that would otherwise induce vascular inflammation and thrombosis. We now show that endogenous H2O2 inhibits thrombin-induced exocytosis of granules from endothelial cells. H2O2 regulates exocytosis by inhibiting N-ethylmaleimide sensitive factor (NSF), a protein that regulates membrane fusion events necessary for exocytosis. H2O2 decreases the ability of NSF to hydrolyze adenosine triphosphate and to disassemble the soluble NSF attachment protein receptor complex. Mutation of NSF cysteine residue C264T eliminates the sensitivity of NSF to H2O2, suggesting that this cysteine residue is a redox sensor for NSF. Increasing endogenous H2O2 levels in mice decreases exocytosis and platelet rolling on venules in vivo. By inhibiting endothelial cell exocytosis, endogenous H2O2 may protect the vasculature from inflammation and thrombosis

P66shc and its downstream Eps8 and Rac1 proteins are upregulated in esophageal cancers

Members of Shc (src homology and collagen homology) family, p46shc, p52shc, p66shc have known to be related to cell proliferation and carcinogenesis. Whereas p46shc and p52shc drive the reaction forward, the role of p66shc in cancers remains to be understood clearly. Hence, their expression in cancers needs to be evaluated carefully so that Shc analysis may provide prognostic information in the development of carcinogenesis. In the present study, the expression of p66shc and its associate targets namely Eps8 (epidermal pathway substrate 8), Rac1 (ras-related C3 botulinum toxin substrate1) and Grb2 (growth factor receptor bound protein 2) were examined in fresh tissue specimens from patients with esophageal squamous cell carcinoma and esophageal adenocarcinoma using western blot analysis. A thorough analysis of both esophageal squamous cell carcinoma and adenocarcinoma showed p66shc expression to be significantly higher in both types of carcinomas as compared to the controls. The controls of adenocarcinoma show a higher basal expression level of p66shc as compared to the controls of squamous cell carcinoma. The expression level of downstream targets of p66shc i.e., eps8 and rac1 was also found to be consistently higher in human esophageal carcinomas, and hence correlated positively with p66shc expression. However the expression of grb2 was found to be equal in both esophageal squamous cell carcinoma and adenocarcinoma. The above results suggest that the pathway operated by p66shc in cancers does not involve the participation of Ras and Grb2 as downstream targets instead it operates the pathway involving Eps8 and Rac1 proteins. From the results it is also suggestive that p66shc may have a role in the regulation of esophageal carcinomas and represents a possible mechanism of signaling for the development of squamous cell carcinoma and adenocarcinoma of esophagus

Sos-mediated activation of rac1 by p66shc

The Son of Sevenless 1 protein (sos1) is a guanine nucleotide exchange factor (GEF) for either the ras or rac1 GTPase. We show that p66shc, an adaptor protein that promotes oxidative stress, increases the rac1-specific GEF activity of sos1, resulting in rac1 activation. P66shc decreases sos1 bound to the growth factor receptor bound protein (grb2) and increases the formation of the sos1–eps8–e3b1 tricomplex. The NH2-terminal proline-rich collagen homology 2 (CH2) domain of p66shc associates with full-length grb2 in vitro via the COOH-terminal src homology 3 (C-SH3) domain of grb2. A proline-rich motif (PPLP) in the CH2 domain mediates this association. The CH2 domain competes with the proline-rich COOH-terminal region of sos1 for the C-SH3 domain of grb2. P66shc-induced dissociation of sos1 from grb2, formation of the sos1–eps8–e3b1 complex, rac1-specific GEF activity of sos1, rac1 activation, and oxidative stress are also mediated by the PPLP motif in the CH2 domain. This relationship between p66shc, grb2, and sos1 provides a novel mechanism for the activation of rac1

Fibonacci Wavelet Method for the Numerical Solution of Nonlinear Reaction-Diffusion Equations of Fisher-Type

This article aims to propose an efficient Fibonacci wavelet-based collocation method for solving the nonlinear reaction-diffusion equation of Fisher-type. The underlying numerical scheme starts by formulating operational matrices of integration corresponding to the Fibonacci wavelets. Besides, we study the error analysis and convergence theorem of the proposed technique. Subsequently, a set of algebraic equations are formed corresponding to the given problem, which could be handled via any conventional method, for instance, the Newton iteration technique. To demonstrate the efficiency of the proposed wavelet-based numerical method, we compare the obtained absolute, L∞, L2, and root mean square (RMS) error norms with the existing Lie symmetry method and cubic trigonometric B-spline (CTB) differential quadrature method in tabular form. From the numerical outcomes, it is ascertained that the proposed numerical technique is computationally more effective and yields precise outcomes in comparison to the existing ones

E3B1/ABI-1 Isoforms Are Down-Regulated in Cancers of Human Gastrointestinal Tract

The expression of E3B1/ABI-1 protein and its role in cancer progression and prognosis are largely unknown in the majority of solid tumors. In this study, we examined the expression pattern of E3B1/ABI-1 protein in histologically confirmed cases of esophageal (squamous cell carcinoma and adenocarcinoma), gastro-esophageal junction, colorectal cancers and corresponding normal tissues freshly resected from a cohort of 135 patients, by Western Blotting and Immunofluorescence Staining. The protein is present in its phosphorylated form in cells and tissues. Depending on the extent of phosphorylation it is either present in hyper-phosphorylated (M. Wt. 72 kDa) form or in hypo-phosphorylated form (M. Wt. 68 kDa and 65 kDa). A thorough analysis revealed that expression of E3B1/ABI-1 protein is significantly decreased in esophageal, gastro-esophageal junction and colorectal carcinomas irrespective of age, gender, dietary and smoking habits of the patients. The decrease in expression of E3B1/ABI-1 was consistently observed for all the three isoforms. However, the decrease in the expression of isoforms varied with different forms of cancers. Down-regulation of E3B1/ABI-1 expression in human carcinomas may play a critical role in tumor progression and in determining disease prognosis

Rac1 Leads to Phosphorylation-dependent Increase in Stability of the p66shc Adaptor Protein: Role in Rac1-induced Oxidative Stress

The rac1 GTPase and the p66shc adaptor protein regulate intracellular levels of reactive oxygen species (ROS). We examined the relationship between rac1 and p66shc. Expression of constitutively active rac1 (rac1V12) increased phosphorylation, reduced ubiquitination, and increased stability of p66shc protein. Rac1V12-induced phosphorylation and up-regulation of p66shc was suppressed by inhibiting p38MAPK and was dependent on serine 54 and threonine 386 in p66shc. Phosphorylation of recombinant p66shc by p38MAPK in vitro was also partly dependent on serine 54 and threonine 386. Reconstitution of p66shc in p66shc-null fibroblasts increased intracellular ROS generated by rac1V12, which was significantly dependent on the integrity of residues 54 and 386. Overexpression of p66shc increased rac1V12-inducd apoptosis, an effect that was also partly dependent on serine 54 and threonine 386. Finally, RNA interference-mediated down-regulation of endogenous p66shc suppressed rac1V12-induced cell death. These findings identify p66shc as a mediator of rac1-induced oxidative stress. In addition, they suggest that serine 54 and threonine 386 are novel phosphorylatable residues in p66shc that govern rac1-induced increase in its expression, through a decrease in its ubiquitination and degradation, and thereby mediate rac1-stimulated cellular oxidative stress and death