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Immunoblot Analysis (immunoblot + analysis)
Kinds of Immunoblot Analysis Selected AbstractsImmunoblot Analysis as an Alternative Method to Diagnose Enterohepatic Helicobacter InfectionsHELICOBACTER, Issue 3 2009Torkel Wadström Abstract Introduction: Enterohepatic Helicobacter species have been associated with chronic infections of the hepatobiliary tract and lower bowel in naturally and experimentally infected mice, Helicobacter -infected animals should thus not be used in studies of diseases associated with chronic inflammation. Helicobacter species induce inflammation and modulate host immune responses, thus emphasizing the need to diagnose these infections in laboratory animals. Materials and Methods: An immunoblot assay was developed to analyze antibodies to enterohepatic Helicobacter species in naturally colonized laboratory mouse colonies. We evaluated the serum antibody responses to cell surface proteins of H. bilis, H. hepaticus, and H. ganmani in 188 mouse sera from four different university animal facilities. Lower bowel tissue specimens from 56 of these animals were available and analyzed by polymerase chain reaction-denaturing gradient gel electrophoresis (PCR-DGGE) and the results compared with matched immunoblot patterns. Results: Specific antibody reactivity to H. bilis was detected in 8 of 186 (4.3%) sera, to H. hepaticus in 45 of 184 (24%) sera, and to H. ganmani in 51 of 188 (27%) of tested sera. These results were compared with PCR-DGGE analyses of tissue samples of corresponding animals, and concordance between the two diagnostic tests was found in 96% for H. bilis, in 91% for H. hepaticus, and in 82% for H. ganmani. The PCR-DGGE also detected DNA of H. typhlonius, H. sp. flexispira, and H. rodentium. Conclusions: Infection with enterohepatic species was common in the laboratory mouse colonies tested, independent of strain and stock. Immunoblot analysis seems to be a promising diagnostic tool to monitor enterohepatic Helicobacter species infections of laboratory rodents. [source] Enhancement of poly-adenosine diphosphate-ribosylation in human hepatocellular carcinomaJOURNAL OF GASTROENTEROLOGY AND HEPATOLOGY, Issue 5 2000Fumio Nomura Abstract Background: Poly-adenosine diphosphate (ADP)-ribosylation, catalysed by poly(ADP-ribose) polymerase (PARP), is a post-translational modification of nuclear proteins and is involved in a wide range of biological processes including DNA repair, cell proliferation and malignant transformation. Alteration of this reaction in human hepatocellular carcinoma (HCC) is of interest, but has not yet been explored. The aim of this study was to evaluate poly-ADP-ribosylation and to compare the expression of PARP in HCC and adjacent non-tumour tissues. Methods: Tumorous and adjacent non-tumorous tissues were obtained from five consecutive patients with HCC during surgery for tumour resection. Tissue homogenates were subjected to ADP-ribosylation with [32P]-nicotinamide adenine dinucleotide. The ADP-ribosylated proteins were separated by sodium dodecylsulfate,polyacrylamide gel electrophoresis, followed by autoradiography. Expression of PARP was also evaluated by western blotting. Results: Several proteins were ADP-ribosylated in human HCC tissues. Notably, the radiolabelling of a 116-kDa protein was remarkably greater than that in adjacent non-tumorous tissues (86.5 ± 35.2 arbitrary units by densitometry vs 12.2 ± 9.9, mean± SD, n = 5, P < 0.02). The radiolabelling of the 116-kDa protein was decreased in the presence of PARP inhibitors in a concentration-dependent manner. Immunoblot analyses revealed that the radiolabelled protein was PARP and that its expression was significantly greater in HCC than in adjacent non-tumorous tissues (333 ± 204% of non-tumorous tissue, P < 0.05). Conclusions: We found that poly-ADP-ribosylation and PARP expression were significantly increased in human HCC compared with those in adjacent non-tumorous tissues in surgically obtained specimens. [source] Identification of a heat-shock protein Hsp40, DjB1, as an acrosome- and a tail-associated component in rodent spermatozoaMOLECULAR REPRODUCTION & DEVELOPMENT, Issue 2 2007Masamichi Doiguchi Abstract Iba1 is a 17-kDa EF-hand protein highly expressed in the cytoplasm of elongating spermatids in testis. Using Iba1 as a bait, we performed yeast Two-hybrid screening and isolated a heat-shock protein Hsp40, DjB1, from cDNA library of mouse testis. To characterize DjB1 that is encoded by Dnajb1 gene, we carried out immunoblot analyses, in situ hybridization, and immunohistochemistry. Immunoblot analyses showed that DjB1was constitutively expressed in mouse testis and that its expression level was not changed by heat shock. Dnajb1 mRNA was exclusively expressed in spermatocytes and round spermatids in mouse testis, and Dnajb1 protein DjB1 was predominantly expressed in the cytoplasm of spermatocytes, round spermatids, and elongating spermatids. In mature mouse spermatozoa, DjB1 was localized in the middle and the end pieces of flagella as well as in association with the head (acrosomal region). Association of DjB1 with the acrosomal region in sperm head was also observed in rat spermatozoa. These data suggested that DjB1, which was constitutively expressed in postmeiotic spermatogenic cells in testis, was integrated into spermatozoa as at least two components, that is, sperm head and tail of rodent spermatozoa. Mol. Reprod. Dev. © 2006 Wiley-Liss, Inc. [source] Nox-2 Is a Modulator of Fibrogenesis in Kidney AllograftsAMERICAN JOURNAL OF TRANSPLANTATION, Issue 1 2009A. Djamali We studied the role of classical phagocytic NADPH oxidase (Nox) in the pathogenesis of kidney allograft tubulointerstitial fibrosis. Immunofluorescence studies showed that Nox-2 and p22phox (electron transfer subunits of Nox) colocalized in the tubulointerstitium of human kidney allografts. Tubular Nox-2 also colocalized with ,-SMA in areas of injury, suggestive of epithelial-to-mesenchymal transition (EMT). Interstitial macrophages (CD68+) and myofibroblasts (,-SMA+) expressed Nox-2 while graft infiltrating T cells (CD3+) and mature fibroblasts (S100A4+) were Nox-2,. These results were confirmed in the Fisher-to-Lewis rat kidney transplant model. Areas of tubulitis were associated with Nox-2 and ,-SMA, suggestive of EMT. Immunoblot analyses showed that Nox-2 upregulation was associated with oxidative stress (nitrotyrosine) and fibrogenesis (,-SMA and phospho-Smad2) at 3 weeks and 6 months. Allografts treated with Nox inhibitors (DPI or apocynin) for 1 week showed reduced fibronectin and phospho-Smad2 and increased E-cadherin levels. Cyclosporine A, TGF-,1 and angiotensin II increased Nox-2 mRNA levels 2- to 7-fold in vitro (NRK52E cells). Treatment with specific Nox inhibitors (DPI or apocynin) prevented the downregulation of E-cadherin and upregulation of fibronectin transcripts. In aggregate, these studies suggest that Nox-2 is involved in the pathogenesis of allograft tubulointerstitial fibrosis via activation transcription factor Smad2, EMT and myofibroblasts. [source] Impaired myofibrillar function in the soleus muscle of mice with collagen-induced arthritisARTHRITIS & RHEUMATISM, Issue 11 2009Takashi Yamada Objective Progressive muscle weakness is a common feature in patients with rheumatoid arthritis (RA). However, little is known about whether the intrinsic contractile properties of muscle fibers are affected in RA. This study was undertaken to investigate muscle contractility and the myoplasmic free Ca2+ concentration ([Ca2+]i) in the soleus, a major postural muscle, in mice with collagen-induced arthritis (CIA). Methods Muscle contractility and [Ca2+]i were assessed in whole muscle and intact single-fiber preparations, respectively. The underlying mechanisms of contractile dysfunction were assessed by investigating redox modifications using Western blotting and antibodies against nitric oxide synthase (NOS), superoxide dismutase (SOD), 3-nitrotyrosine (3-NT), carbonyl, malondialdehyde (MDA), and S-nitrosocysteine (SNO-Cys). Results The tetanic force per cross-sectional area was markedly decreased in the soleus muscle of mice with CIA, and the change was not due to a decrease in the amplitude of [Ca2+]i transients. The reduction in force production was accompanied by slowing of the twitch contraction and relaxation and a decrease in the maximum shortening velocity. Immunoblot analyses showed a marked increase in neuronal NOS expression but not in inducible or endothelial NOS expression, which, together with the observed decrease in SOD2 expression, favors peroxynitrite formation. These changes were accompanied by increased 3-NT, carbonyl, and MDA adducts content in myofibrillar proteins from the muscles of mice with CIA. Moreover, there was a significant increase in SNO-Cys content in myosin heavy-chain and troponin I myofibrillar proteins from the soleus muscle of mice with CIA. Conclusion These findings show impaired contractile function in the soleus muscle of mice with CIA and suggest that this abnormality is due to peroxynitrite-induced modifications in myofibrillar proteins. [source] Human GCIP interacts with CT847, a novel Chlamydia trachomatis type III secretion substrate, and is degraded in a tissue-culture infection modelCELLULAR MICROBIOLOGY, Issue 10 2007Blandine Chellas-Géry Summary The obligate intracellular bacterium Chlamydia trachomatis occupies a parasitophorous vacuole and employs a type III secretion mechanism to translocate host-interactive proteins. These proteins most likely contribute to pathogenesis through modulation of host cell mechanisms crucial for the establishment and maintenance of a permissive intracellular environment. Using a surrogate Yersinia type III secretion system (T3SS), we have identified the conserved gene product CT847 as a chlamydial T3SS substrate. Yeast two-hybrid studies using CT847 as bait to screen a HeLa cell cDNA library identified an interaction with mammalian Grap2 cyclin D- interacting protein (GCIP). Immunoblot analyses of C. trachomatis -infected HeLa cells showed that GCIP levels begin to decrease (as compared with mock-infected HeLa cells) between 8 h and 12 h post infection. GCIP was virtually undetectable in 24 h time point material. This decrease was inhibited by proteasome inhibitors lactacystin and MG-132, and the T3SS inhibitor Compound 1. CT847 was detectible in purified reticulate body but not elementary body lysates, and reverse transcription polymerase chain reaction (RT-PCR) expression analyses indicate a mid-cycle expression pattern. Both of these findings are consistent with CT847 contributing to the observed effect on GCIP. Given the established roles of GCIP, we believe that we have discovered a novel C. trachomatis antihost protein whose activity is relevant to chlamydial pathogenesis. [source] Expression patterns of focal adhesion associated proteins in the developing retinaDEVELOPMENTAL DYNAMICS, Issue 4 2002Ming Li Abstract Adhesive interactions between integrin receptors and the extracellular matrix (ECM) are intimately involved in regulating development of a variety of tissues within the organism. In the present study, we have investigated the relationships between ,1 integrin receptors and focal adhesion associated proteins during eye development. We used specific antibodies to examine the distribution of ,1 integrin ECM receptors and the cytoplasmic focal adhesion associated proteins, talin, vinculin, and paxillin in the developing Xenopus retina. Immunoblot analysis confirmed antibody specificity and indicated that ,1 integrins, talin, vinculin, and paxillin were expressed in developing retina and in the retinal-derived Xenopus XR1 glial cell line. Triple-labeling immunocytochemistry revealed that talin, vinculin, paxillin, and phosphotyrosine proteins colocalized with ,1 integrins at focal adhesions located at the termini of F-actin filaments in XR1 cells. In the retina, these focal adhesion proteins exhibited developmentally regulated expression patterns during eye morphogenesis. In the embryonic retina, immunoreactivities for focal adhesion proteins were expressed in neuroepithelial cells, and immunoreactivity was especially strong at the interface between the optic vesicle and overlying ectoderm. At later stages, these proteins were expressed throughout all retinal layers with higher levels of expression observed in the plexiform layers, optic fiber layer, and in the region of the inner and outer limiting membrane. Strong immunoreactivities for ,1 integrin, paxillin, and phosphotyrosine were expressed in the radially oriented Müller glial cells at later stages of development. These results suggest that focal adhesion-associated proteins are involved in integrin-mediated adhesion and signaling and are likely to be essential in regulating retinal morphogenesis. © 2002 Wiley-Liss, Inc. [source] Expression of cathepsins B, D and L in mouse corneas infected with Pseudomonas aeruginosaFEBS JOURNAL, Issue 24 2001Zhong Dong C57BL/6J naïve and immunized mice were intracorneally infected with Pseudomonas aeruginosa. Semi-quantitative RT-PCR was performed to detect cathepsin gene expression and the results were further confirmed by immunoblot analysis. The enzymatic activities of cathepsins B, D and L were measured by peptidase assays. Immunohistochemical staining was carried out to localize the expression of the cathepsins. Cathepsins B, D and L were detected in the normal cornea by RT-PCR. A peptidase assay revealed activities of all three cathepsins under normal physiological conditions. In naïve mice, enzymatic activities of cathepsins B, D and L were all significantly enhanced when the corneas were infected with P. aeruginosa and the peak of the induction appeared around day 6 postinfection. Immunoblot analysis showed increased expression of cathepsins B, D and L. The infected corneal samples from immunized mice exhibited much lower induction of enzymatic activities compared to those from naïve mice. Immunohistochemistry showed that the expression of cathepsins in the normal cornea was restricted to the epithelial tissue while the induced expression of cathepsins was predominantly in the substantia propria. Our data revealed up-regulated enzymatic activities of cathepsins B, D and L in the naïve corneas infected with P. aeruginosa, which correlated well with the inflammatory response. Immunization of mice against P. aeruginosa attenuated the inducing effect on cathepsin expression caused by infection. The time sequence for induction of cathepsin proteins and enzymatic activities suggests a mechanism of host proteolytic degradation of the extracellular matrix resulting in corneal destruction after P. aeruginosa infection. [source] Adenylate cyclase influences filamentous haemagglutinin-mediated attachment of Bordetella pertussis to epithelial alveolar cellsFEMS IMMUNOLOGY & MEDICAL MICROBIOLOGY, Issue 1 2006Maria L.A. Perez Vidakovics Abstract Attachment to epithelial cells in the respiratory tract is a key event in Bordetella pertussis colonization. Filamentous haemagglutinin (FHA) is an important virulence factor mediating adhesion to host cells. In this study, the relevance of the interaction between FHA and adenylate cyclase toxin (ACT) during bacterial attachment was investigated. Mutants lacking either FHA or ACT showed significantly decreased adherence to epithelial respiratory cells. The use of several ACT-specific monoclonal antibodies and antiserum showed that the decrease in attachment of strains lacking ACT expression could not be explained by the adhesin-like activity of ACT, or a change of any of the biological activities of ACT. Immunoblot analysis showed that the lack of ACT expression did not interfere with FHA localization. An heparin-inhibitable carbohydrate-binding site is crucial in the process of FHA-mediated bacterial binding to epithelial cells. In the presence of heparin attachment of wild-type B. pertussis, but not of the isogenic ACT defective mutant, to epithelial cells was significantly decreased. These results suggest that ACT enhances the adhesive functions of FHA, and modifies the performance of the FHA heparin-inhibitable carbohydrate binding site. We propose that the presence of ACT in the outer membrane of B. pertussis to play a role in the functionality of FHA. [source] Accumulation of multiple forms of lamin A with down-regulation of FACE-1 suppresses growth in senescent human cellsGENES TO CELLS, Issue 3 2007Ryo Ukekawa 5-Bromodeoxyuridine (BrdU) clearly induces a senescence-like phenomenon in every cell type. Proteome analysis revealed that lamin A and C were most highly increased in the nuclei of HeLa cells upon addition of BrdU. Immunoblot analysis also revealed marked accumulation of nuclear prelamin A. Consistently, farnesylated-proteins converting enzyme 1 (FACE-1) was markedly down-regulated in the same cells. Similar phenomena were also observed in normal human fibroblasts undergoing replicative senescence. Immunochemical analysis confirmed the above results. Lamin A is a major component of lamina and responsible for several genetic diseases. Thus, we ectopically expressed a wild-type, a mature type and a premature type of lamin in HeLa cells. All of these forms similarly inhibited colony formation and delayed cell cycle progression mainly through G2 phase. These results suggest that a change in the amount of lamin A, rather than appearance of its truncated form, is responsible for growth retardation in affected cells. [source] Neutrophil elastase in pressure ulcer fluid degrades fibronectin in the exudatesGERIATRICS & GERONTOLOGY INTERNATIONAL, Issue 3 2004Shingo Ai Background: Pressure ulcers are classified as chronic wounds, which do not heal in a timely fashion. Fibronectin is condensed in granulation tissue, and essential glycoprotein of wound healing. It has been proposed that fibronectin degradation may be involved in delaying wound healing. We have investigated whether pressure ulcer fluid (PUF) contains degraded fibronectin. In addition, we tried to identify the proteinase which contributes to fibronectin degradation in PUF. Methods: Fibronectin degradation and the presence of neutrophil elastase (NE) in PUF were determined by immunoblot analysis. Fibronectin degradation activity in PUF was determined in the presence of various proteinase inhibitors. NE activity was assessed using NE specific substrate. Results: Immunoblot analysis revealed that degraded fibronectin was observed in PUF samples but not in acute wound fluid (AWF). The PUF contained a proteinase capable of degrading freshly added fibronectin and its activity in PUF was blocked by a broad-spectrum serine proteinase inhibitor or sivelestat, a specific neutrophil elastase inhibitor, but not by metalloproteinase and cysteine proteinase inhibitors. Immunoblot analysis of PUF using an antineutrophil elastase antibody revealed that neutrophil elastase was detected as three bands at molecular weights of ,30 kDa, ,38 kDa, and ,54 kDa, indicating that neutrophil elastase in the exudates existed not only as free monomers, but also in polymers or complexes with other molecules. Conclusion: These results suggest that PUF contains a high level of neutrophil elastase which may be involved in the delay of the healing of pressure ulcer through the fibronectin degradation. [source] Immunoblot Analysis as an Alternative Method to Diagnose Enterohepatic Helicobacter InfectionsHELICOBACTER, Issue 3 2009Torkel Wadström Abstract Introduction: Enterohepatic Helicobacter species have been associated with chronic infections of the hepatobiliary tract and lower bowel in naturally and experimentally infected mice, Helicobacter -infected animals should thus not be used in studies of diseases associated with chronic inflammation. Helicobacter species induce inflammation and modulate host immune responses, thus emphasizing the need to diagnose these infections in laboratory animals. Materials and Methods: An immunoblot assay was developed to analyze antibodies to enterohepatic Helicobacter species in naturally colonized laboratory mouse colonies. We evaluated the serum antibody responses to cell surface proteins of H. bilis, H. hepaticus, and H. ganmani in 188 mouse sera from four different university animal facilities. Lower bowel tissue specimens from 56 of these animals were available and analyzed by polymerase chain reaction-denaturing gradient gel electrophoresis (PCR-DGGE) and the results compared with matched immunoblot patterns. Results: Specific antibody reactivity to H. bilis was detected in 8 of 186 (4.3%) sera, to H. hepaticus in 45 of 184 (24%) sera, and to H. ganmani in 51 of 188 (27%) of tested sera. These results were compared with PCR-DGGE analyses of tissue samples of corresponding animals, and concordance between the two diagnostic tests was found in 96% for H. bilis, in 91% for H. hepaticus, and in 82% for H. ganmani. The PCR-DGGE also detected DNA of H. typhlonius, H. sp. flexispira, and H. rodentium. Conclusions: Infection with enterohepatic species was common in the laboratory mouse colonies tested, independent of strain and stock. Immunoblot analysis seems to be a promising diagnostic tool to monitor enterohepatic Helicobacter species infections of laboratory rodents. [source] Hepatic expression of gamma-glutamyltranspeptidase in the human liver of patients with alcoholic liver diseaseHEPATOLOGY RESEARCH, Issue 11 2007Makoto Irie Background:, Gamma-glutamyltranspeptidase (GGT) has been recognized as an enzyme that converts glutathione into cysteine, and it is localized predominantly within the liver. Serum GGT is clinically recognized as the most useful marker for diagnosis of alcoholic liver disease (ALD). Methods:, GGT localization within the liver was examined immunohistochemically using an anti-GGT antibody and was visualized by confocal laser scanning microscopy in ALD and normal livers. Double immunostaining for GGT and dipeptidylpeptidase-IV (DPP-IV) was carried out to evaluate GGT localization in greater detail. Results:, Expression of GGT protein and mRNA was studied with immunoblot analysis and in situ hybridization, respectively. Immunohistochemically, the expression of GGT in the normal liver was faintly demonstrated in the bile canaliculi of hepatocytes and in biliary epithelial cells. In ALD livers, GGT was clearly demonstrated at the same sites. Double immunostaining demonstrated that GGT and DPP-IV were colocalized in hepatocytes in the ALD liver. In situ hybridization clearly demonstrated GGT-mRNA within the cytoplasm of hepatocytes and biliary epithelial cells. Immunoblot analysis revealed that GGT protein expression was increased in the ALD livers compared with that seen in the normal livers. Conclusion:, These findings indicate that GGT in control and alcoholic livers is synthesized in hepatocytes and biliary epithelial cells, and is localized within the bile canalicular membrane and the luminal membrane in those cells, respectively. In conclusion, GGT synthesis and protein expression are increased in ALD livers, leading to the elevation of serum levels of GGT that are commonly noted in patients with the disease. [source] N-terminal CFTR missense variants severely affect the behavior of the CFTR chloride channel,HUMAN MUTATION, Issue 5 2008G.G. Gené Abstract Over 1,500 cystic fibrosis transmembrane conductance regulator (CFTR) gene sequence variations have been identified in patients with cystic fibrosis (CF) and related disorders involving an impaired function of the CFTR chloride channel. However, detailed structure,function analyses have only been established for a few of them. This study aimed evaluating the impact of eight N-terminus CFTR natural missense changes on channel behavior. By site-directed mutagenesis, we generated four CFTR variants in the N-terminal cytoplasmic tail (p.P5L, p.S50P, p.E60K, and p.R75Q) and four in the first transmembrane segment of membrane-spanning domain 1 (p.G85E/V, p.Y89C, and p.E92K). Immunoblot analysis revealed that p.S50P, p.E60K, p.G85E/V, and p.E92K produced only core-glycosylated proteins. Immunofluorescence and whole cell patch-clamp confirmed intracellular retention, thus reflecting a defect of CFTR folding and/or trafficking. In contrast, both p.R75Q and p.Y89C had a glycosylation pattern and a subcellular distribution comparable to the wild-type CFTR, while the percentage of mature p.P5L was considerably reduced, suggesting a major biogenesis flaw on this channel. Nevertheless, whole-cell chloride currents were recorded for all three variants. Single-channel patch-clamp analyses revealed that the channel activity of p.R75Q appeared similar to that of the wild-type CFTR, while both p.P5L and p.Y89C channels displayed abnormal gating. Overall, our results predict a major impact of the CFTR missense variants analyzed, except p.R75Q, on the CF phenotype and highlight the importance of the CFTR N-terminus on channel physiology. Hum Mutat 29(5), 738,749, 2008. © 2008 Wiley-Liss, Inc. [source] Molecular cloning, characterization, expression pattern and cellular distribution of an ovarian lipophorin receptor in the cockroach, Leucophaea maderaeINSECT MOLECULAR BIOLOGY, Issue 3 2009M. Tufail Abstract A cDNA that encodes a lipophorin receptor (LpR) with a predicted structure similar to that of the low density lipoprotein receptor (LDLR) gene superfamily was cloned from ovaries of the cockroach, Leucophaea maderae (Lem) and characterized. This is the first LpR sequenced from the order Dictyoptera. The cDNA has a length of 3362 bp coding for an 888-residue mature protein with a predicted molecular mass of ~99.14 kDa and a pI value of 4.68. The deduced amino acid sequence showed that the LemLpR harbours eight ligand-binding repeats (LBRs) at the N-terminus similar to the other insect LpRs, and thus resembles vertebrate VLDLRs. In addition to eight tandemly arranged LBRs, the five-domain receptor contains an O -linked sugar region and the classic LDLR internalization signal, FDNPVY. Northern blot analysis revealed the presence of ~4.0 kb ovarian mRNA that was transcribed throughout oogenesis with its peak especially during late previtellogenic and vitellogenic periods (from days 3 to 11). LpR transcript(s) or homologues of LDLRs were also detected in the head, midgut, Malpighian tubules, muscles and in the fat body. RNA in situ hybridization and immunocytochemistry localized the LpR mRNA and protein to germ line-derived cells, the oocytes, and revealed that LpR gene transcription and translation starts very early during oocyte differentiation in the germarium. LpR protein was evenly distributed throughout the cytoplasm during previtellogenic periods of oogenesis. However, during vitellogenic stages, the receptor was accumulated mainly in the cortex of the oocyte. Immunoblot analysis probed an ovarian LpR protein of ~115 and 97 kDa under reducing and nonreducing conditions, respectively. The protein signal appeared on day 2, increased every day and was high during vitellogenic periods from day 4 to day 7. Southern blot analysis suggested the presence of a single copy of the LpR gene in the genome of Le. maderae. [source] Epstein-Barr virus infection and risk of lymphoma: Immunoblot analysis of antibody responses against EBV-related proteins in a large series of lymphoma subjects and matched controlsINTERNATIONAL JOURNAL OF CANCER, Issue 8 2007Silvia de Sanjosé Abstract Epstein-Barr Virus (EBV) is consistently associated with distinct lymphoproliferative malignancies and aberrant EBV antibody patterns are found in most EBV cancer patients. We evaluate the detection of an abnormal reactive serological pattern to EBV (ab_EBV) infection and the risk of lymphoma in a multicentric case,control study. Serum samples were collected at study entry from 1,085 incident lymphoma cases from Spain, France, Germany, Czech Republic, Italy and 1,153 age, sex and country matched controls. EBV immunoglobulin G (IgG) serostatus was evaluated through a peptide-based ELISA combining immunodominant epitopes of EBNA1 (BKRF1) and VCA-p18 (BFRF3). Further, immunoblot analysis was performed to evaluate distinct antibody diversity patterns to EBV early antigens (EA), besides EBNA1, VCA-p18, VCA-p40 (BdRF1) and Zebra (BZLF1). Patients with chronic active EBV infection and aberrant EBV activity were characterized as having an abnormal reactive pattern (ab_EBV). Ab_EBV was observed in 20.9% of 2,238 included subjects with an increased proportion of cases presenting ab_EBV as compared to the control population (23.9% vs. 18.0% p = 0.001). Ab_EBV positivity was a risk factor for all lymphomas combined (odds ratio [OR] = 1.42, 95% confidence interval [CI]=1.15,1.74), and specifically for chronic lymphocytic leukaemia (OR = 2.96, 95%CI = 2.22,3.95). Lower levels of ab_EBV were observed for follicular lymphoma (OR = 0.38, 95%CI = 0.15,0.98). EBV may be involved in a larger subset of lymphomas among clinically immunocompetent subjects than previously thought, probably explained by an underlying loss of immune control of EBV latent infection. Ab_EBV is a useful tool to explore EBV imbalances preceeding or paralleling possible EBV associated oncogenic events. © 2007 Wiley-Liss, Inc. [source] Methoxychlor-induced alteration in the levels of HSP70 and clusterin is accompanied with oxidative stress in adult rat testisJOURNAL OF BIOCHEMICAL AND MOLECULAR TOXICOLOGY, Issue 1 2009S. Vaithinathan Abstract Methoxychlor, an organochlorine pesticide, has been reported to induce abnormalities in male reproductive tract. However, the insight into the mechanisms of gonadal toxicity induced by methoxychlor is not well known. We investigated whether treatment with methoxychlor would alter the levels of stress proteins, heat shock proteins (HSP), and clusterin (CLU), and oxidative stress-related parameters in the testis of adult male rats. Animals were exposed to a single dose of methoxychlor (50 mg/kg body weight) orally and were terminated at various time points (0, 3, 6, 12, 24, and 72 h) using anesthetic ether. The levels of HSP70, CLU, and the activities of superoxide dismutase (SOD), catalase, and lipid peroxidation levels were evaluated in a 10% testis homogenate. A sequential reduction in the activities of catalase and SOD with concomitant increase in the levels of thiobarbituric acid reactive substance (TBARS) was observed. These changes elicited by methoxychlor were very significant between 6,12 h of posttreatment. Immunoblot analysis of HSP revealed the expression of HSP72, an inducible form of HSP, at certain time points (3,24 h) following exposure to methoxychlor. Similarly, the levels of secretory CLU (sCLU) were also found to be elevated between 3,24 h of treatment. The present data demonstrate methoxychlor-elicited increase in the levels of inducible HSP72 and sCLU, which could be a part of protective mechanism mounted to reduce cellular oxidative damage. © 2009 Wiley Periodicals, Inc. J Biochem Mol Toxicol 23:29,35, 2009; Published online in Wiley InterScience (www.interscience.wiley.com). DOI 10.1002/jbt.20262 [source] Formation of lipid raft redox signalling platforms in glomerular endothelial cells: an early event of homocysteine-induced glomerular injuryJOURNAL OF CELLULAR AND MOLECULAR MEDICINE, Issue 9b 2009Fan Yi Abstract The present study tested the hypothesis that homocysteine (Hcys)-induced ceramide production stimulates lipid rafts (LRs) clustering on the membrane of glomerular endothelial cells (GECs) to form redox signalling platforms by aggregation and activation of NADPH oxidase subunits and thereby enhances superoxide (O2.,) production, leading to glomerular endothelial dysfunction and ultimate injury or sclerosis. Using confocal microscopy, we first demonstrated a co-localization of LR clusters with NADPH oxidase subunits, gp91phox and p47phox in the GECs membrane upon Hcys stimulation. Immunoblot analysis of floated detergent-resistant membrane fractions found that in LR fractions NADPH oxidase subunits gp91phox and p47phox are enriched and that the activity of this enzyme dramatically increased. We also examined the effect of elevated Hcys on the cell monolayer permeability in GECs. It was found that Hcys significantly increased GEC permeability, which was blocked by inhibition of LR redox signalling platform formation. Finally, we found that Hcys-induced enhancement of GEC permeability is associated with the regulation of microtubule stability through these LR-redox platforms. It is concluded that the early injurious effect of Hcys on the glomerular endothelium is associated with the formation of redox signalling platforms via LR clustering, which may lead to increases in glomerular permeability by disruption of microtubule network in GECs. [source] Characterization of tissue-specific LIM domain protein (FHL1C) which is an alternatively spliced isoform of a human LIM-only protein (FHL1)JOURNAL OF CELLULAR BIOCHEMISTRY, Issue 1 2001Enders Kai On Ng Abstract We have cloned and characterized another alternatively spliced isoform of the human four-and-a-half LIM domain protein 1 (FHL1), designated FHL1C. FHL1C contains a single zinc finger and two tandem repeats of LIM domains at the N-terminus followed by a putative RBP-J binding region at the C-terminus. FHL1C shares the same N-terminal two-and-a-half LIM domains with FHL1 but different C-terminal protein sequences. Due to the absence of the exon 4 in FHL1C, there is a frame-shift in the 3, coding region. Sequence analysis indicated that FHL1C is the human homolog of murine KyoT2. The Northern blot and RT-PCR results revealed that FHL1 is widely expressed in human tissues, including skeletal muscle and heart at a high level, albeit as a relatively low abundance transcript in brain, placenta, lung, liver, kidney, pancreas, and testis. In contrast, FHL1C is specifically expressed in testis, skeletal muscle, and heart at a relatively low level compared with FHL1. The expression of FHL1C transcripts was also seen in aorta, left atrium, left, and right ventricles of human heart at low level. Immunoblot analysis using affinity-purified anti-FHL1C antipeptide antibodies confirmed a 20 kDa protein of FHL1C in human skeletal muscle and heart. Unlike FHL1B, which is another FHL1 isoform recently reported by our group and localized predominantly in the nucleus [Lee et al., 1999], FHL1C is localized both in the nucleus and cytoplasm of mammalian cell. J. Cell. Biochem. 82: 1,10, 2001. © 2001 Wiley-Liss, Inc. [source] Quantitative Epstein-Barr virus (EBV) serology in lung transplant recipients with primary EBV infection and/or post-transplant lymphoproliferative diseaseJOURNAL OF MEDICAL VIROLOGY, Issue 2 2003Erik Verschuuren Abstract The Epstein-Barr virus (EBV)-specific antibody response was studied in lung transplant patients to assess their value in the diagnosis and prognosis of post-transplant lymphoproliferative disease. Recently developed synthetic peptides representing Epstein-Barr nuclear antigen-1 (EBNA-1), diffuse early antigen (EA(D)), and virus capsid antigen (VCA) were studied in a semiquantitative enzyme-linked immunosorbent assay (ELISA) to study antibody patterns in 12 seronegative lung transplant patients, of whom four developed a post-transplant lymphoproliferative disease, and seven seropositive lung transplant patients, all of whom developed a post-transplant lymphoproliferative disease. Immunoblot technique was used as a control. All 12 EBV-seronegative patients had a very limited antibody response that was restricted mainly to VCA antibodies. EA(D) antibodies became detectable in only two patients. Antibody response never preceded clinical diagnosis of post-transplant lymphoproliferative disease in the four EBV-seronegative patients who developed post-transplant lymphoproliferative disease. In the seven seropositive lung transplant patients with post-transplant lymphoproliferative disease, we found a rise in antibody titer in only two patients. Immunoblot analysis confirmed the serological results. In conclusion, EBV-specific antibody patterns after lung transplantation are highly restricted and variable and of limited value for the diagnosis or prognosis of post-transplant lymphoproliferative disease. J. Med. Virol. 69:258,266, 2003. © 2003 Wiley-Liss, Inc. [source] Identification of the isoforms of Ca2+/calmodulin-dependent protein kinase II and expression of brain-derived neurotrophic factor mRNAs in the substantia nigraJOURNAL OF NEUROCHEMISTRY, Issue 1 2006Akifumi Kamata Abstract Ca2+/calmodulin-dependent protein kinase (CaMK)II is highly expressed in the CNS and mediates activity-dependent neuronal plasticity. Four CaMKII isoforms, ,, ,, , and ,, have a large number of splicing variants. Here we identified isoforms of CaMKII in the rat substantia nigra (SN). Northern blot and RT,PCR analyses revealed that the , and , isoform mRNAs with several splicing variants were predominantly expressed in SN. Immunoblot analysis indicated that the major isoforms were ,A, ,C, ,1 and ,3. An immunohistochemical study also confirmed the preferential localization of , and , isoforms in SN dopaminergic neurons. In dopaminergic neurons, immunoreactivity against anti-CaMKII,1,4 antibody was detected in both nucleus and cytoplasm, in contrast to the predominant expression of , isoforms in the cytoplasm. Furthermore, we showed expression of brain-derived neurotrophic factor (BDNF) mRNAs with exons II and IV in SN. Taken together with our previous observations, the results suggest that the CaMKII,3 isoform is involved in the expression of BDNF in the SN. [source] Activation of MKK6, an upstream activator of p38, in Alzheimer's diseaseJOURNAL OF NEUROCHEMISTRY, Issue 2 2001Xiongwei Zhu Mitogen-activated protein kinase (MAPK) p38 has been implicated in the pathogenesis of Alzheimer's disease, but the upstream cascade leading to p38 activation has not been elucidated in the disease. In the present study, we focused on mitogen-activated protein kinase kinase 6 (MKK6), one of the upstream activators of p38 MAPK. We found that MKK6 was not only increased but also specifically associated with granular structures in the susceptible neurons in the hippocampus and cortex of Alzheimer's disease patients, but was only weakly diffuse in the cytoplasm in neurons in control cases. Immunoblot analysis demonstrated a significant increase of MKK6 level in Alzheimer's disease compared with age-matched controls. In this regard, in hippocampal and cortical regions of individuals with Alzheimer's disease, the activated phospho-MKK6 was localized exclusively in association with pathological alterations including neurofibrillary tangles, senile plaques, neuropil threads and granular structures, overlapping with activated p38 MAPK suggesting both a functional and mechanic link. By immunoblot analysis, phospho-MKK6 is also significantly increased in AD compared with control cases. Together, these findings lend further credence to the notion that the p38 MAPK pathway is dysregulated in Alzheimer's disease and also indicates an active role for this pathway in disease pathogenesis. [source] Bone morphogenetic proteins 4, 6, and 7 are up-regulated in mouse spinal cord during experimental autoimmune encephalomyelitisJOURNAL OF NEUROSCIENCE RESEARCH, Issue 1 2008Jahan Ara Abstract Although spontaneous remyelination occurs in multiple sclerosis (MS), the extent of myelin repair is often inadequate to restore normal function. Oligodendrocyte precursors remaining in nonremyelinating MS plaques may be restricted by an inhibitory signal. Bone morphogenetic proteins (BMPs) have been implicated as repressors of oligodendrocyte development and inducers of astrogliogenesis. We hypothesized that BMPs are up-regulated in MS lesions and play a role in demyelination and astrogliosis. We examined expression of BMPs in an animal model of MS, chronic experimental autoimmune encephalomyelitis (EAE) induced by the myelin oligodendrocyte glycoprotein (MOG) peptide in C57BL/6 mice. By 14 days postimmunization, compared to those of control mice, the lumbar spinal cords of MOG-peptide EAE mice demonstrated prominent astrogliosis, infiltration of inflammatory cells, and disrupted expression of myelin proteins. Quantitative RT-PCR showed that expression of BMP4, BMP6, and BMP7 mRNA increased 2- to 4-fold in the lumbar spinal cords of animals with symptomatic EAE versus in vehicle-treated and untreated controls on days 14, 21, and 42 postimmunization. BMP2 mRNA expression was not altered. BMP4 mRNA was much more abundant in the spinal cords of all animals than was mRNA encoding BMP2, BMP6, and BMP7. Immunoblot analysis confirmed the increased expression of BMP4 in the EAE animals. Immunohistochemistry revealed increased BMP4 immunoreactivity in areas of inflammation in MOG-peptide EAE animals. BMP4 labeling was mostly limited to macrophages but was sometimes associated with astrocytes and oligodendrocytes. These results indicate that members of the BMP family are differentially expressed in adult spinal cord and are up-regulated during EAE. © 2007 Wiley-Liss, Inc. [source] Effect of interferons on P-glycoprotein-mediated rhodamine-123 efflux in cultured rat hepatocytesJOURNAL OF PHARMACEUTICAL SCIENCES, Issue 10 2002Yukiko Akazawa Abstract The effect of interferon (IFN)-, and IFN-, on P-glycoprotein (P-gp)-mediated efflux of rhodamin-123(Rho-123), a typical substrate of P-gp, was studied in rat hepatocytes in primary culture. After treatment with IFN-,, IFN-,, or both for 3 days, steady-state levels of Rho-123, incorporated into the hepatocytes, were measured to evaluate the P-gp activity. Whereas IFN-, did not affect the intracellular level of Rho-123, IFN-, treatment caused a significant increase of the level, suggesting that IFN-, treatment suppresses the expression of P-gp or its activity. A combination of the two types of IFN exhibited a similar effect to that of IFN-, alone. The effect of IFN-, was still observed in the presence of H2O2, which enhances the expression and activity of P-gp. Immunoblot analysis using a monoclonal antibody C219 revealed, however, that P-gp expression was increased after treatment with IFN-,, but only slightly by IFN-, treatment. These results suggest that the enhanced Rho-123 uptake of rat primary hepatocytes induced by IFN-, does not result from reduced expression of P-gp but, rather, from impaired maturation or dysfunction of the efflux transporter. © 2002 Wiley-Liss Inc. and the American Pharmaceutical Association J Pharm Sci 91:2110,2115, 2002 [source] Bioavailability and efficacy of antisense morpholino oligomers targeted to c- myc and cytochrome P-450 3A2 following oral administration in ratsJOURNAL OF PHARMACEUTICAL SCIENCES, Issue 4 2002Vikram Arora Abstract Antisense phosphorodiamidate Morpholino oligomers (PMO) are resistant to degradation by cellular hydrolases, DNases, RNases, and phosphodiesterases, but remain sensitive to prolonged exposure to low pH. The present studies evaluate the oral fractional bioavailability, stability, and efficacy of two distinct PMO sequences targeted to c- myc and cytochrome P-450 (CYP) 3A2. The c- myc antisense 20-mer, AVI-4126 (5,-ACGTTGAGGGGCATCGTCGC-3,), slowed the regenerative process in the rat liver after a 70% partial hepatectomy (PH). Rats were administered 3.0 mg/kg AVI-4126 in 0.1 mL saline via a bolus intravenous injection or in 0.5 mL sterile phosphate-buffered saline via gavage immediately following PH. The areas under the plasma concentration versus time curves revealed a fractional oral availability of 78.8% over a period of 10 min through 24 h. Immunoblot analysis of liver tissue from rats treated orally with AVI-4126 demonstrated a sequence-specific reduction in the target protein c-Myc, as well as secondary proliferation markers: proliferating cell nuclear antigen (PCNA), cyclin D1, and p53. The CYP3A2 antisense 22-mer AVI-4472 (5,-GAGCTGAAAGCAGGTCCATCCC-3,) caused a sequence-dependent reduction of approximately five-fold in the rat liver CYP3A2 protein levels and erythromycin demethylation activity in 24 h following oral administration at a dose of 2 mg/kg. It is concluded that oral administration of PMOs can inhibit c- myc and CYP3A2 gene expression in rat liver by an antisense-based mechanism of action. These studies highlight the potential for development of PMOs as orally administered therapeutic agents. © 2002 Wiley-Liss, Inc. and the American Pharmaceutical Association J Pharm Sci 91:1009,1018, 2002 [source] Acute Ethanol Inhibits Extracellular Signal,Regulated Kinase, Protein Kinase B, and Adenosine 3,:5,-Cyclic Monophosphate Response Element Binding Protein Activity in an Age- and Brain Region,Specific MannerALCOHOLISM, Issue 4 2005L Judson Chandler Background: As little as a single episode of exposure of the developing brain to ethanol can result in developmental neuropathology and mental retardation. Extracellular signal,regulated kinases (ERKs), protein kinase B (PKB), and adenosine 3,:5,-cyclic monophosphate response element binding protein (CREB) are messenger molecules that play important roles in neuronal plasticity and survival. This study was undertaken to examine the effects of acute ethanol on ERK, PKB, and CREB activation in the brain. Methods: Immunoblot analysis was used to determine the effects of a 1-hr exposure of ethanol on levels of phospho-ERC in primary cortical cultures and in the cerebral cortex, hippocampus, and cerebellum of postnatal day 5 (PN5), postnatal day 21 (PN21), and adult rats. Results: In cortical cultures, ethanol (100 mM) significantly reduced activity-dependent activation of phospho-ERK, phospho-PKB, and phospho-CREB by approximately 50%. In PN5 rats, ethanol (3.5 g/kg) inhibited both phospho-ERK and phospho-PKB in the cerebral cortex and hippocampus but was without effect in the cerebellum. A similar brain region,specific inhibition of phospho-ERK was observed in PN21 rats, whereas in adult rats, ethanol inhibited phospho-ERK in all three brain regions. In contrast, ethanol had no effect on phospho-PKB in either PN21 or adult rats. Without exception, ethanol inhibited phospho-CREB in an identical brain region, and age-dependent manner as was observed for phospho-ERK. Finally, administration of the NMDA antagonist MK-801 (0.5 mg/kg) to PN5 rats had no effect on phospho-ERK or phospho-PKB levels in any brain region. Conclusion: The results demonstrate that acute ethanol inhibits ERK/PKB/CREB signaling in brain. This inhibition occurs in an age- and brain region,specific manner, with inhibition of PKB restricted to a time during the brain growth-spurt period. Furthermore, the lack of effect of MK-801 suggests that inhibition of NMDA receptors is unlikely to play a major role in binge ethanol inhibition of ERK/PKB/CREB signaling in vivo. [source] Biochemical, immunological and clinical characterization of a cross-reactive nonspecific lipid transfer protein 1 from mulberryALLERGY, Issue 5 2010M. A. Ciardiello To cite this article: Ciardiello MA, Palazzo P, Bernardi ML, Carratore V, Giangrieco I, Longo V, Melis M, Tamburrini M, Zennaro D, Mari A, Colombo P. Biochemical, immunological and clinical characterization of a cross-reactive nonspecific lipid transfer protein 1 from mulberry. Allergy 2010; 65: 597,605. Abstract Background:, Mulberry (Morus spp.) is a genus comprising several species of deciduous trees whose fruits are commonly eaten in southern Europe. Subjects with severe systemic reaction have been described. The aim of this study was to isolate the allergens of this species. Methods:, A nonspecific lipid transfer protein 1 (ns-LTP1) was purified from black mulberry by ion exchange and reverse phase high-performance liquid chromatography, and the primary structure was elucidated by direct protein sequencing. Its allergenic activity was evaluated in vivo by skin prick test and in vitro by Western Blot, CD203c basophil activation assay and high throughput multiplex inhibition method on immunosolid-phase allergen chip (ISAC). Results:, Mulberry ns-LTP (Mor n 3) comprises 91 amino acids producing a molecular mass of 9246 Da. This protein shows high sequence identity with several allergenic ns-LTP1. Immunoblot analysis and CD203c activation assay demonstrated its allergenic activity in symptomatic subjects and in ns-LTP allergic patients who are not mulberry consumers. Immunological co-recognition was studied in vivo on a selected group of well-characterized ns-LTP allergic patients showing a high percentage of nMor n 3+ subjects (88.46%) even in patients who have never eaten mulberry before. IgE inhibition on ISAC micro-array demonstrated an almost complete cross-reactivity to nArt v 3, rCor a 8 and a very high percentage of inhibition to nPru p 3. Conclusions:, Mor n 3 is the first allergen isolated in black mulberry and immunologically characterized. It displayed allergenic activity among symptomatic and nonconsumer patients and a pattern of cross-reactivity to other plant-derived LTPs. [source] Long-term ethanol exposure causes human liver cancer cells to become resistant to mitomycin C treatment through the inactivation of bad-mediated apoptosis,MOLECULAR CARCINOGENESIS, Issue 8 2010Ching-Shui Huang Abstract The aim of this study was to test whether long-term ethanol consumption confers therapeutic resistance to human liver cancer patients infected with hepatitis B virus (HBV). Chronic ethanol-treated cells were established by consecutively culturing a human hepatocellular carcinoma cell line, Hep 3B, which contains integrated HBV sequences, for 20,40 passages with or without 10,mM ethanol (designated as E20,E40 and C20,C40, respectively). Flow cytometry analysis demonstrated that a growth promoting effect of long-term ethanol treatment was induced in the E40 cells through preferential acceleration of S-phase in these cells. Lower protein expression levels of p16, p21/Cip1, and p27/Kip1 were detected in the ethanol-treated E40 cells. We further demonstrated that long-term ethanol-treated E40 cells develop drug resistance in response to mitomycin C (MMC) treatment (>8,µM). Immunoblot analysis revealed that caspase-8-mediated mitochondrial apoptotic signals (such as Bad) were inactivated in the MMC-resistant E40 cells. Immunoprecipitation experiments demonstrated that the sequestration of phosphorylated Bad (Ser-112) through its binding with 14-3-3 was detected more profoundly in the MMC-resistant E40 cells. Next, we examined the therapeutic efficacy of MMC (10,mg MMC/kg body weight, three times per week) in severe combined immunodeficient (SCID) mice bearing E40- and C40-xenografted tumors. Significant reductions (>3-fold) in tumor growth were detected in MMC-treated C40-xenografted mice. In vivo and in vitro studies demonstrated that AKT- and extracellular signal-regulated kinase (ERK)-mediated survival factors inhibited the Bad-induced mitochondrial apoptotic signals that were involved in E40 tumor cells and that conferred resistance to MMC. © 2010 Wiley-Liss, Inc. [source] GPI-anchored aminopeptidase is involved in the acrosome reaction in sperm of the mussel mytilusedulisMOLECULAR REPRODUCTION & DEVELOPMENT, Issue 4 2004Tatsuru Togo Abstract The sperm of the mussel Mytilus had hydrolytic activities against substrates for aminopeptidase. Acrosome reaction (AR) was suppressed in the presence of aminopeptidase substrate, Phe-4-methylcoumaryl-7-amide (MCA), and an aminopeptidase inhibitor, bestatin. Treatment of sperm with phosphatidylinositol-specific phospholipase C (PI-PLC) released aminopeptidase activity from sperm and suppressed AR. These results suggest that the enzyme is located on the sperm surface via glycosylphosphatidylinositol (GPI)-anchor and is involved in the AR. Immunoblot analysis showed that tyrosine residues of 40, 59, 68, and 72 kDa proteins were phosphorylated during induction of the AR. The 40 kDa protein was also recognized by anti-c-Src antibody by immunoblotting. The tyrosine phosphorylation of these proteins was inhibited when sperm were inseminated in the presence of Phe-MCA, and by PI-PLC treatment. Treatment of sperm with tyrosine kinase activator, 9,10-dimethyl-1,2-benzanthracene, induced AR, and its inhibitor, genistein, suppressed AR. These results suggest that tyrosine phosphorylation of 40, 59, 68, and 72 kDa proteins, induced by the interaction of GPI-anchored aminopeptidase with oocyte surface, triggers AR in Mytilus sperm. Mol. Reprod. Dev. 67: 465,471, 2004. © 2004 Wiley-Liss, Inc. [source] Activation of STAT3 and inhibitory effects of pioglitazone on STAT3 activity in a mouse model of SOD1-mutated amyotrophic lateral sclerosisNEUROPATHOLOGY, Issue 4 2010Noriyuki Shibata Signal transducer and activator of transcription-3 (STAT3) is a member of the proinflammatory transcription factor STAT family. Several studies have documented implications for neuroinflammation in amyotrophic lateral sclerosis (ALS). We recently demonstrated activation of STAT3 in spinal cords obtained at autopsy from sporadic ALS patients. To determine the involvement of STAT3 and effects of pioglitazone on STAT3 activity in familial ALS with superoxide dismutase-1 (SOD1) mutation, we performed immunoblot and immunohistochemical analyses of the active form of STAT3 (p-STAT3) in spinal cords from mice overexpressing mutant SOD1 (ALS mice) and nontransgenic littermates (control mice). Immunoblot analysis delineated significant increases in nuclear p-STAT3 levels in non-treated ALS mice as compared with pioglitazone-treated ALS mice and non-treated and pioglitazone-treated control mice. Immunohistochemical analysis revealed prominent p-STAT3 accumulations in the nucleus of motor neurons, reactive astrocytes and activated microglia in non-treated ALS mice but not pioglitazone-treated ALS mice and non-treated and pioglitazone-treated control mice. The present results provide in vivo evidence for increased phosphorylative activation and nuclear translocation of STAT3 in motor neurons and glia in mouse motor neuron disease, suggesting a common pathological process between sporadic and SOD1-mutated familial forms of ALS. Moreover, it is likely that pioglitazone may exert inhibitory effects on STAT3-mediated proinflammtory mechanisms in this disease. [source] | |||||||||||||||||||||||||||||||||||||