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ABSTRACT: Wheat coleoptile tips generate superoxide radical as a part of the phototropic response to blue light, but the source of this
free radical generation is not known. We evaluated the presence and involvement of homologs of neutrophil NADPH oxidase (NOX),
including gp91phox, p22phox, p67phox, p47phox, and p40phox, in wheat coleoptiles using Western blot analysis and immunofluorescence
microscopy. Blue light augmented the expression levels of all these subunits and targeted NOX subunits onto the plasma membrane
and to the nucleus. gp91phox, p22phox, p67phox, and p40phox showed entry into the nucleus and exhibited physical closeness
with DNA. CuZnSOD was also present in the coleoptile tip, which also showed a blue-light-dependent elevation in expression.
Superoxide production and phototropic response were both abrogated by DPIC and staurosporine, indicating their cause-and-effect
relationship. We conclude that blue light mediates a phototropic response in wheat coleoptiles through modulation of expression
of NOX and SOD as well as the translocation of NOX subunits onto the plasma membrane and nuclear membrane. Thus, this study
provides a mechanistic explanation for superoxide production during the photoresponse in wheat coleoptiles.
KeywordsColeoptile-Blue light-Superoxide-Signal transduction NOX
Journal of Plant Growth Regulation 04/2012; 29(2):232-241. · 2.86 Impact Factor
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ABSTRACT: The autoimmune regulator gene Aire shows predominant expression in thymus and other immunologically relevant tissues, and is assigned the major function of programming autoreactive T-cell deletion. However, the expression of this gene in tissues outside the immune system raises a question about its possible function beyond the T-cell deletion dogma. We detected Aire in mouse testis, and the expression of AIRE protein was remarkably high in postmeiotic germ cells. Sequencing results indicate that testis expressed Aire variant 1a. AIRE could be detected in spermatozoa, with heavy localization on the principal acrosomal domains. Mouse oocytes stained negatively for AIRE before fertilization, but stained positively for AIRE 30 min after fertilization. In the zygote, the levels of AIRE correlated negatively with cyclin B2 levels. Goat testicular lysates spiked with recombinant human AIRE exhibited augmented cyclin B2 degradation in the presence of protease inhibitors, which was inhibited by MG-132, indicating the operation of proteasomal pathways. Thus, this study identifies a correlation between the presence of AIRE and proteasomal breakdown of cyclin B2, which leads us to speculate that cyclin B2 could be a target of AIRE's E3-ubiquitin ligase activity.
Biochemistry and Cell Biology 08/2011; 89(4):411-22. · 2.67 Impact Factor
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ABSTRACT: Signaling via estrogen receptor (ER) occurs by interacting with many proteins. Nuclear interactome analysis of ERα in an embryo implantation model revealed the association of chicken tumor virus no. 10 regulator of kinase like (CrkL) with ERα, which was further validated by mammalian two-hybrid assay as well as coimmunoprecipitation and colocalization. Mutation in LPALL motif of CrkL disrupts the ERα-CrkL interaction and its transactivation potential, thereby suggesting that the interaction is mediated via its single ER binding motif, Leu-Pro-Ala-Leu-Leu (LXXLL) motif in the sarcoma homology (SH)2 domain. CrkL deletion constructs of SH2 domain target to the nucleus due to presence of nuclear localization signal. Interestingly, the SH2-SH3 (N terminal) construct shows an increased transactivation potential like CrkI. Weak interaction capability of mutated ERα-Y538F with CrkL validates that CrkL interacts with ERα via its YDLL motif at Tyr 541. In an attempt to understand the physiological relevance of this association, we investigated the impact on cell proliferation using a cancer model, because events associated in the process of pregnancy and cancer are analogous. Also, overexpression of CrkL is frequently associated with tumorigenesis. However, its significance in hormone-regulated cancers still remains obscure. Here, we demonstrate that association of ERα and CrkL directly enhances the tumorigenic potential of CrkL, thus pointing to its role in cell proliferation. In human endometrial cancers, we observed a strong association between ERα and CrkL levels. Thus, the molecular signaling set off by ERα and CrkL association may have a central role in pregnancy and cancer, two events which share parallels in growth, invasion, and immune tolerance.
Molecular Endocrinology 06/2011; 25(9):1499-512. · 4.54 Impact Factor
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ABSTRACT: Superoxide anion radical, produced in low quantities, plays a positive role in sperm function. Spermatozoa produce superoxide anion radical during posttesticular development, which shows an abrupt increase during capacitation. The NAD phosphate oxidase (NOX) family members NOX2 and NOX5 are the 2 enzymes implicated in superoxide production in spermatozoa. We examined the organization of NOX2 in goat spermatozoa during epididymal maturation, capacitation, and acrosome reaction. Spermatozoa from testis, caput epididymidis, corpus epididymidis, and cauda epididymidis possessed components of the phagocytic oxidase (PHOX; i.e., gp91phox, p22phox, p67phox, p47phox, p40phox), and ras-related C3 botulinum toxin substrate 1/2 (Rac1/2) on spermatozoa, and their concentrations did not show significant alterations during epididymal maturation. During capacitation in vitro, p22phox underwent Thr-phosphorylation, which resulted in a mobility shift of the corresponding band toward greater molecular mass. The Rac1/2 also showed a mobility shift from 32 to 23 kDa during capacitation. During progesterone-induced acrosome reaction, the spermatozoa experienced a total loss of p22phox and p47phox. The p47phox, but not p22phox, was detected in the exocytic vesicles of the acrosome. The Thr-phosphorylated form of p22phox was ubiquitinated and degraded through proteasome-mediated pathways in goat sperm cell lysates. Thus, Thr phosphorylation of p22phox acts as a regulatory switch in goat spermatozoa that transiently activates the NOX2 system during capacitation and subsequently directs it for degradation through the ubiiquitin-proteasomal pathway during progesterone-induced acrosome reaction.
Journal of Animal Science 05/2011; 89(10):2995-3007. · 2.10 Impact Factor
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Asian Journal of Andrology. 01/2010;
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ABSTRACT: Silkworms show high variability in silk quality and disease resistance. Attempts are on to combine the disease tolerance of multivoltine races and the silk quality of bivoltine races to generate new races with desirable phenotypic traits. We report the identification of a 26.5-kDa protein that is overexpressed in the gut juice of disease-resistant multivoltine races and that has anti-BmNPV activity. We have characterized this protein as a soluble NADH-oxidoreductase-like protein (BmNOX). Treatment of live BmNPV particles with BmNOX inhibited the capability of the viral particles to infect BmN cells in vitro.
Bioscience Biotechnology and Biochemistry 02/2007; 71(1):200-5. · 1.28 Impact Factor
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ABSTRACT: Successful implantation is dependent on precisely orchestrated and reciprocal signaling between the implanting blastocyst and the receptive uterus. A key signaling mechanism that is operative during implantation is the Wnt/ Beta -catenin signaling pathway. Secreted frizzled-related proteins (sFRPs) are reported to be antagonist to these pathways and are group of secreted glycoproteins, structurally similar to Wnt receptors [frizzled (FZD) proteins] but lack the transmembrane domains. SFRPs inhibit Wnt action through competitive binding to the ligand-binding domain of the frizzled receptor complex. In silico analysis using PSORTII has revealed mouse sFRP4 to be predominantly mitochondrial (43.5%) and nuclear (34.8%) and not extracellular like human sFRP4 (44.4 %). Our western blotting and immunohistochemical studies unraveled the sub-cellular localization of the sFRP4 molecule and its significant presence in the nucleus and the mitochondrial fraction during the peri-implantation stage. The nuclear presence of sFRP4 during pregnancy adds new dimension to its potential modes of action and biological function. Studies are underway to explore the structure and function of sFRP4 using molecular modeling.
J Endocrinol Reprod. 01/2007; 11(1):41-44.
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ABSTRACT: Establishment of early pregnancy is promoted by a complex network of signalling molecules that mediate cell-to-cell and cell-to-extracellular matrix communications between the receptive endometrium and the invasive trophectoderm. In this study, we have attempted to evaluate the expression profiles of cadherin and catenin during embryo implantation in the mouse. Western blotting studies along with immunocytochemical analysis revealed that E-cadherin is expressed rather ubiquitously in the uterine epithelial cells, distinct enrichment is observed on the apical membrane in the endometrium of peri-implantation uterus specifically at the implantation sites and not at the inter-implanation sites. beta-Catenin also is upregulated and is specifically restricted to apical membrane of epithelial cells of implantation sites. Progesterone induced expression of E-cadherin and 17beta-estradiol regulated the expression of catenin in implantation-delayed uteri. Interestingly, estradiol imparted negative modulation on cadherin expression when co-administered with progesterone. On the contrary, trophoblast exhibits a striking down regulation of cadherin, catenin and Ca(2+) at peri implanting stage. These observations suggest that the trophoblasts exhibited an invasive phenotype while the endometrial epithelium displayed an adhesive phenotype during the window of implantation. Thus, embryo implantation presents an instance where two interacting surfaces showed mutually complementing interaction phenotypes.
FEBS Letters 11/2006; 580(24):5653-60. · 3.54 Impact Factor
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ABSTRACT: A point mutation in the Stat5b DNA binding domain in the nonobese diabetic (NOD) mouse was shown to have weaker DNA binding compared with the B6 Stat5b. Here, we assessed the binding ability of the mutant Stat5b in the B6 genetic background (B6.NOD-c11) and the wild-type Stat5b in the NOD background (NOD.Lc11). To our surprise, the binding ability of Stat5b is inconsistent with the presence or absence of the Stat5b mutation in these congenic mice but is correlated with the expression levels of the Crkl protein, which was coprecipitated by an anti-Stat5b antibody. Both the expression of Crkl and the Stat5b binding ability are the highest in B6.NOD-c11 and the lowest in NOD while intermediate in B6 and NOD.Lc11 mice. We demonstrated that the adapter molecule Crkl can bind Stat5b and that the Crkl protein is a Stat5b binding cofactor. More importantly, profection of Crkl recombinant protein significantly increased Stat5b binding ability and rescued the binding defect of the NOD mutant Stat5b, suggesting that Crkl is a key regulatory molecule for Stat5b binding. Therefore, the defective Crkl expression may contribute to the development of diabetes in the NOD mice by exacerbating the defective Stat5b binding ability.
Diabetes 04/2006; 55(3):734-41. · 8.29 Impact Factor
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ABSTRACT: Though the spermatozoa are known to produce superoxide anion radicals, the enzyme system(s) that produce superoxide in these cells are not yet identified. Using Western blot assays and confocal laser scan microscopy, we detected gp91(phox) and p67(phox) associated with spermatozoa from testis and epididymis. We could not detect p22(phox) in any of the sperm samples analyzed. While the expression of gp91(phox) p67(phox) appeared to be constitutive, p47(phox) was detectable only in spermatozoa from testis and vas deferens. Importantly, p40(phox) could be seen in very high quantities in testicular spermatozoa, which also showed the highest levels of NADPH-oxidase activity. Spermatozoa from cauda epididymidis and vas deferens also showed the presence of p40(phox), though the amount was low when compared with that of testicular spermatozoa. The absence of p22(phox) and the striking correlation between the presence of p40(phox) and the NADPH-oxidase activity suggest that the NADPH oxidase associated with spermatozoa is p22(phox)-independent and that its activity is positively modulated by p40(phox). Further, since the confocal imaging detected that the subunits of the NADPH oxidase are located significantly on the head domains, the spermatozoa appear to present a case with dominant non-mitochondrial superoxide anion producing capabilities.
Biochemical and Biophysical Research Communications 07/2005; 331(2):476-83. · 2.48 Impact Factor
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ABSTRACT: The human autoimmune regulator (AIRE) gene encodes a putative DNA-binding protein, which is mutated in patients affected by the autoimmune polyglandular syndrome type 1 or autoimmune polyendocrinopathy-candidiasis-ectodermal dystrophy. We have recently reported that AIRE can bind to two different DNA sequence motifs, suggesting the existence of at least two DNA-binding domains in the AIRE protein. By expressing a series of recombinant AIRE protein fragments, we demonstrate here that the two well-known plant homeodomains (PHD) domains in AIRE can bind to the ATTGGTTA sequence motif. The first ATTGGTTA-binding domain is mapped to amino acids 299-355 and the second ATTGGTTA-binding domain to amino acids 434-475. Furthermore, the SAND domain of AIRE is shown to bind to TTATTA motif. Results presented herein show that the residues at position 189-196 of AIRE (QRAVAMSS) are required for its binding to the TTATTA motif. The required sequence for DNA binding in the SAND domain of AIRE is remarkably different from other SAND-containing proteins such as Sp-100b and NUDR. Data presented in this paper indicate that the two PHD domains contained in AIRE, in addition to the SAND domain, can bind to specific DNA sequence motifs.
Biochemical and Biophysical Research Communications 03/2005; 327(3):939-44. · 2.48 Impact Factor
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ABSTRACT: The survival of an embryo bearing the paternal antigens within the immunocompetent environment of the maternal uterus renders 'pregnancy' to be a state of immunological paradox. The ratio of Th1/Th2 responses is crucial for pregnancy maintenance. Monocyte Chemotactic Protein-3 (MCP3) is a pro-inflammatory, CC chemokine and a Th1 effector which is capable of eliciting significant anti-tumoral immune responses.
MCP3 expression was investigated in the murine uterine tissue at different days of initial pregnancy and the effect of RU 486 in immature and delayed implantation model studied using Western blotting and Immunocytochemical techniques.
Our results show very high uterine MCP3 expression during pre-implantation followed by a significant MCP3 down-regulation at peri-implantation and low levels of MCP3 during post-implantation period. At the peri-implantation stage, embryos exhibited lowered MCP3 expression when compared with the pre-implantation stage. Ru486, a progesterone antagonist when given in a competitive mode with progesterone resulted in a massive surge in MCP3 expression in both immature mice and delayed implantation models. We hypothesize that it is imperative for MCP3 expression to be down-regulated for the success of pregnancy. The cross-talk between Ru486 and amplified MCP3 expression may be one of the mechanisms by way of which RU486 performs its abortificient and anti tumor role.
American journal of reproductive immunology (New York, N.Y.: 1989) 08/2004; 52(1):8-18. · 3.05 Impact Factor
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ABSTRACT: Blue-light-induced photomorphogenesis is the sum total of a sequence of phenomena involving absorption of light by specific receptors, generation of a signal, processing transmembrane transport of signal, and the activation of a cascade of reactions in the cell interior. Though four blue-light receptors cryptochrome1, cryptochrome2, phototropin1, and phototropin2 have been identified, the signal transduction events associated with blue-light receptor activation are not understood. In this report, we demonstrate the generation and spatiotemporal distribution of H(2)O(2) in wheat coleoptile in response to blue light. Interception of the free-radical generation pathways dithiothreitol and propyl gallate rendered wheat coleoptile tips phototropically non-responsive. Unilateral application of H(2)O(2) onto the sub-apical region of a growing coleoptile brought about curvature in dark. Blue light also caused lipid peroxidation and augmented membrane rigidity of coleoptile cell membranes. We conclude that H(2)O(2) can act as a translocating second messenger that could bring about coleoptile curvature, and the signaling events may trigger Ca(2+) signaling cascades, changes in gene expression, and protein modifications.
Biochemical and Biophysical Research Communications 08/2004; 319(4):1190-6. · 2.48 Impact Factor
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ABSTRACT: Crk family adaptors are widely expressed and mediate the timely formation of signal transduction protein complexes upon a variety of extracellular stimuli, including various growth and differentiation factors. The window of implantation is the favorable time period when the uterus develops a receptive approach to the invading embryo. Various signaling cascades are likely to become active at the window of implantation both in the uterus and the embryo. This helps create maternal embryo dialogue leading to successful embryo implantation. In this study we report for the first time the presence and nuclear translocation of the adaptor molecule CrkL both in the uterine and embryonic partners at the window of implantation. We also report that estrogen, which initiates and guides crucial changes in the uterus and the embryo at the window of receptivity, causes a massive surge in the expression and subsequent nuclear translocation of CrkL. We have also identified the existence of one LXXLL motif in the CrkL amino acid sequence and a single LXD is sufficient for activation by the estrogen receptor. This is suggestive that CrkL can bind to estrogen receptors and act as a coactivator.
Biochemical and Biophysical Research Communications 06/2004; 318(1):103-12. · 2.48 Impact Factor
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ABSTRACT: Acrosomal assembly during spermatogenesis and acrosome reaction during sperm-oocyte interaction are unique events of vesicle synthesis, transport, and fusion leading to fertilization. SNARE complex formation is essential for membrane fusion, and vesicle-associated (v-) SNARE intertwines with target membrane (t-) SNARE to form a coiled coil that bridges two membranes and facilitates fusion. We detected messages of Vam6P and SNAP in mammalian testis and epididymis. Vam6P and SNAP were detected in a temporally organized fashion on the spermatozoa from testis and epididymis, which showed accumulation on the principal acrosomal domains during capacitation. Vam6P and SNAP were shed off from the principal acrosomal domain after acrosome reaction, but the equatorial and the post-acrosomal domains retained these proteins. Antibodies to VAMP and SNAP inhibited sperm-zona pellucida interaction, suggesting their possible involvement in sperm membrane vesiculation.
Biochemical and Biophysical Research Communications 06/2004; 318(1):148-55. · 2.48 Impact Factor
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ABSTRACT: Cadherins comprise a family of calcium-dependent glycoproteins that function in mediating cell-cell adhesion in virtually all solid tissues of multicellular organisms. We have examined the presence of a cadherin on spermatozoon and its possible involvement in sperm-oocyte interaction. Spermatozoa from fertile human subjects showed the presence of E-cadherin on its head domains, detectable only after permeabilization of the surface membranes. On the contrary, spermatozoa from oligozoospermic subjects did not possess E-cadherin on their principal acrosomal and equatorial domains. Immunoprecipitation and Western blot studies also showed the presence of E-cadherin in spermatozoa from fertile males and its absence in oligozoospermic males. Using RT-PCR, we detected E-cadherin message in the round cells of fertile males, which was absent in the cells from oligozoospermic males. The presence of anti-E-cadherin antibody brought about quantitative reduction in the sperm-oocyte binding in vitro. These observations indicate the possibility of the interplay of a cadherin-dependent homophilic and/or heterophilic adhesion interaction between spermatozoa and oocyte during fertilization. The absence of a key adhesion molecule in a human male infertility disorder points towards genetic defects causing failure in gamete interactions.
Biochemical and Biophysical Research Communications 05/2004; 316(3):903-9. · 2.48 Impact Factor
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ABSTRACT: Capacitated mammalian spermatozoa undergo a fusion response of their head plasma membrane and the outer acrosomal membrane leading to vesiculation classically known as acrosome reaction. Acrosome reaction occurs in response to various acrosome reaction inducers including zona pellucida proteins, calcium ionophore, dibutyryl cAMP, progesterone, etc. All the acrosome reaction inducers cause a transient of calcium influx into the sperm through voltage-dependent cation channels. Efflux of chloride, stimulation of activity of phospholipases, and phosphorylation of proteins are other known changes introduced by acrosome reaction inducers. Macromolecular organization and dynamics of sperm membranes during the progression of this vesiculation are largely unexplored. In this study, we report that progesterone induced the formation of horizontal microdomains within the exofacial surfaces of sperm membranes, which showed progressive and independent alterations in molecular dynamics. In the light of this observation, we propose that sperm membrane rafts may contain both horizontal and vertical microdomains.
Biochemical and Biophysical Research Communications 04/2004; 315(3):763-70. · 2.48 Impact Factor
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ABSTRACT: A number of cytokines that finely regulate immune response have been implicated in the pathogenesis or protection of type 1 diabetes and other autoimmune diseases. It is, therefore, of pivotal importance to examine a family of proteins that serve as signal transducers and activators of transcription (STATs), which regulate the transcription of a variety of cytokines. We report here a defective gene (Stat5b) located on chromosome 11 within a previously mapped T1D susceptibility interval (Idd4) in the nonobese diabetic (NOD) mice. Our sequencing analysis revealed a unique mutation C1462A that results in a leucine to methionine (L327M) in Stat5b of NOD mice. Leu(327), the first residue in the DNA binding domain of STAT proteins, is conserved in all identified mammalian STAT proteins. Homology modeling predicted that the mutant Stat5b has a weaker DNA binding, which was confirmed by DNA-protein binding assays. The inapt transcriptional regulation ability of the mutated Stat5b is proved by decreased levels of RNA of Stat5b-regulated genes (IL-2Rbeta and Pim1). Consequently, IL-2Rbeta and Pim1 proteins were shown by Western blotting to have lower levels in NOD compared with normal B6 mice. These proteins have been implicated in immune regulation, apoptosis, activation-induced cell death, and control of autoimmunity. Therefore, the Stat5b pathway is a key molecular defect in NOD mice.
Journal of Biological Chemistry 04/2004; 279(12):11553-61. · 4.77 Impact Factor
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ABSTRACT: The autoimmune polyglandular syndrome type 1 (APS1), also known as autoimmune polyendocrinopathy-candidiasis-ectodermal dystrophy (APS1), is a monogenic autosomal disease with recessive inheritance. It is characterized by chronic mucocutaneous candidiasis, multiple autoimmune endocrinopathies, and ectodermal dystrophies. The defective gene responsible for this disease has been identified and named "autoimmune regulator" (AIRE). The AIRE gene is located on chromosome 21q22.3. At least 45 different disease-causing mutations in AIRE have been discovered. This review summarizes the global distribution of AIRE mutations and the relevance of major mutations to the clinical disorders associated with APS1. We also will review studies on the structure and DNA-binding ability of the AIRE protein and the possible malfunctions of the AIRE protein as a result of major disease-causing mutations.
Endocrinology & Metabolism Clinics of North America 07/2002; 31(2):321-38, vi. · 3.41 Impact Factor
