Home About us Contact
|
Home About us Contact | ||
|
|
||
Tissue Transglutaminase (tissue + transglutaminase)
Selected AbstractsCharacterization of Tissue Transglutaminase in Human Osteoblast-like CellsJOURNAL OF BONE AND MINERAL RESEARCH, Issue 8 2001Deborah J. Heath Abstract Tissue transglutaminase (tTG) is a calcium-dependent and guanosine 5,-triphosphate (GTP) binding enzyme, which catalyzes the post-translational modification of proteins by forming intermolecular ,(,-glutamyl)lysine cross-links. In this study, human osteoblasts (HOBs) isolated from femoral head trabecular bone and two osteosarcoma cell lines (HOS and MG-63) were studied for their expression and localization of tTG. Quantitative evaluation of transglutaminase (TG) activity determined using the [1,414C]-putrescine incorporation assay showed that the enzyme was active in all cell types. However, there was a significantly higher activity in the cell homogenates of MG-63 cells as compared with HOB and HOS cells (p < 0.001). There was no significant difference between the activity of the enzyme in HOB and HOS cells. All three cell types also have a small amount of active TG on their surface as determined by the incorporation of biotinylated cadaverine into fibronectin. Cell surface-related tTG was further shown by preincubation of cells with tTG antibody, which led to inhibition of cell attachment. Western blot analysis clearly indicated that the active TG was tTG and immunocytochemistry showed it be situated in the cytosol of the cells. In situ extracellular enzyme activity also was shown by the cell-mediated incorporation of fluorescein cadaverine into extracellular matrix (ECM) proteins. These results clearly showed that MG-63 cells have high extracellular activity, which colocalized with the ECM protein fibronectin and could be inhibited by the competitive primary amine substrate putrescine. The contribution of tTG to cell surface/matrix interactions and to the stabilization of the ECM of osteoblast cells therefore could by an important factor in the cascade of events leading to bone differentiation and mineralization. [source] Mucosal tissue transglutaminase expression in celiac diseaseJOURNAL OF CELLULAR AND MOLECULAR MEDICINE, Issue 2 2009Vincenzo Villanacci Abstract Tissue transglutaminase (tTG) plays an important role in celiac disease pathogenesis and antibodies to tTG are a diagnostic marker of gluten-sensitive enteropathy. The aim of this study was to investigate the localization of tTG in the duodenal mucosa in control tissues and in different histological stages of celiac disease by using a commercial and a novel set of anti-tTG monoclonal antibodies, to see whether this assessment can be useful for diagnostic purpose. The distribution of tTG was firstly evaluated in 18 untreated celiac patients by using a commercial monoclonal antibody (CUB7402) against tissue transglutaminase enzyme and directed against the loop-core region of the enzyme. Thereafter, in further 30 untreated celiac patients we employed three newly characterized anti-tTG monoclonal antibodies produced against recombinant human-tTG. The epitopes recognized are located in three distinct domains of the protein corresponding to the core, C1 and C2 protein structure. Eleven age- and sex-matched patients with chronic duodenitis acted as controls. All subjects underwent upper endoscopy to obtain biopsy samples from the duodenum. Overall, we found that (i) tTG is equally expressed in CD at different stages of disease; (ii) tTG is expressed, at similar level, in CD and controls with duodenitis. Assessment of tTG level in biopsy samples by immunohistochemical methods is not useful in the clinical diagnostic work-up of CD. [source] Does tissue transglutaminase play a role in Huntington's disease?JOURNAL OF NEUROCHEMISTRY, Issue 2002G. V. W. Johnson Tissue transglutaminase (tTG) catalyzes the incorporation of polyamines into substrates, or the formation of isopeptide bonds. tTG also binds and hydrolyzes GTP/ATP. Huntington's disease (HD) is caused by a pathological expansion of the polyglutamine domain in the protein huntingtin (htt). Because a polypeptide bound Gln residue is the primary determining factor for a tTG substrate, it has been hypothesized that due to the increase in Gln content, mutant htt may modified by tTG and this event may contribute to the pathogenesis of HD, possibly by facilitating the formation of htt aggregates. tTG is increased in HD, suggesting involvement in the pathogenic process. However, tTG is not required for aggregate formation. Further, tTG is excluded from htt aggregates and increasing or decreasing tTG has no effect on the frequency or localization of the aggregates. Considering these and other data, tTG is unlikely to play a major role in the formation of htt inclusions in HD brain. tTG may play a role in modulating neuronal cell death in response to specific stressors. If a stress increases the transamidating activity of tTG (e.g. increases in Ca++ levels), then tTG facilitates the cell death process. In contrast, if a stress does not result in an increase in the transamidating activity of tTG, then tTG protects against cell death. The protective effects of tTG are independent of its transamidating and hence likely dependent on its GTP/ATP binding and hydrolytic activity. Therefore the increase in tTG levels in HD brain could either be helpful or harmful depending on the cellular mechanisms that contribute to neuronal death. Acknowledgements:, Supported by NIH grant AG12396. [source] Impaired Mitochondrial Function Results in Increased Tissue Transglutaminase Activity In SituJOURNAL OF NEUROCHEMISTRY, Issue 5 2000Mathieu Lesort Abstract: Tissue transglutaminase (tTG) is a transamidating enzyme that is elevated in Huntington's disease (HD) brain and may be involved in the etiology of the disease. Further, there is evidence of impaired mitochondrial function in HD. Therefore, in this study, we examined the effects of mitochondrial dysfunction on the transamidating activity of tTG. Neuroblastoma SH-SY5Y cells stably overexpressing human tTG or mutated inactive tTG were treated with 3-nitropropionic acid (3-NP), an irreversible inhibitor of succinate dehydrogenase. 3-NP treatment of tTG-expressing cells resulted in a significant increase of TG activity in situ. In vitro measurements demonstrated that 3-NP had no direct effect on tTG activity. However, 3-NP treatment resulted in a significant decrease of the levels of GTP and ATP, two potent inhibitors of the transamidating activity of tTG. No significant changes in the intracellular levels of calcium were observed in 3-NP-treated cells. Treatment with 3-NP in combination with antioxidants significantly reduced the 3-NP-induced increase in in situ TG activity, demonstrating that oxidative stress is a contributing factor to the increase of TG activity. This study demonstrates for the first time that impairment of mitochondrial function significantly increases TG activity in situ, a finding that may have important relevance to the etiology of HD. [source] Tissue transglutaminase modulates ,-synuclein oligomerizationPROTEIN SCIENCE, Issue 8 2008Ine M.J. Segers-Nolten Abstract We have studied the interaction of the enzyme tissue transglutaminase (tTG), catalyzing cross-link formation between protein-bound glutamine residues and primary amines, with Parkinson's disease-associated ,-synuclein protein variants at physiologically relevant concentrations. We have, for the first time, determined binding affinities of tTG for wild-type and mutant ,-synucleins using surface plasmon resonance approaches, revealing high-affinity nanomolar equilibrium dissociation constants. Nanomolar tTG concentrations were sufficient for complete inhibition of fibrillization by effective ,-synuclein cross-linking, resulting predominantly in intramolecularly cross-linked monomers accompanied by an oligomeric fraction. Since oligomeric species have a pathophysiological relevance we further investigated the properties of the tTG/,-synuclein oligomers. Atomic force microscopy revealed morphologically similar structures for oligomers from all ,-synuclein variants; the extent of oligomer formation was found to correlate with tTG concentration. Unlike normal ,-synuclein oligomers the resultant structures were extremely stable and resistant to GdnHCl and SDS. In contrast to normal ,-sheet-containing oligomers, the tTG/,-synuclein oligomers appear to be unstructured and are unable to disrupt phospholipid vesicles. These data suggest that tTG binds equally effective to wild-type and disease mutant ,-synuclein variants. We propose that tTG cross-linking imposes structural constraints on ,-synuclein, preventing the assembly of structured oligomers required for disruption of membranes and for progression into fibrils. In general, cross-linking of amyloid forming proteins by tTG may prevent the progression into pathogenic species. [source] Novel MRI and fluorescent probes responsive to the Factor XIII transglutaminase activityCONTRAST MEDIA & MOLECULAR IMAGING, Issue 4 2010Lorenzo Tei Abstract Transglutaminases, including factor XIII and tissue transglutaminase, participate in multiple extracellular processes associated with remodeling of the extracellular matrix during wound repair, blood clotting, tumor progression and fibrosis of ischemic injuries. The aim of this work was to evaluate a novel substrate analog for transglutaminase optimized by molecular modeling calculations (DCCP16), which can serve for molecular imaging of transglutaminase activity by magnetic resonance imaging and by near-infrared imaging. Experimental data showed covalent binding of Gd,DCCP16 and DCCP16-IRIS Blue to human clots, to basement membrane components and to casein in purified systems as well as in three-dimensional multicellular spheroids. In vivo, DCCP16 showed enhancement with a prolonged retention in clots and tumors, demonstrating the ability to detect both factor XIII and tissue transglutaminase mediated covalent binding of the contrast material. Copyright © 2010 John Wiley & Sons, Ltd. [source] Viability study of HL60 cells in contact with commonly used microchip materialsELECTROPHORESIS, Issue 24 2006Floor Wolbers Abstract This paper presents a study in which different commonly used microchip materials (silicon oxide, borosilicate glass, and PDMS) were analyzed for their effect on human promyelocytic leukemic (HL60) cells. Copper-coated silicon was analyzed for its toxicity and therefore served as a positive control. With quantitative PCR, the expression of the proliferation marker Cyclin D1 and the apoptosis marker tissue transglutaminase were measured. Flow cytometry was used to analyze the distribution through the different phases of the cell cycle (propidium iodide, PI) and the apoptotic cascade (Annexin V in combination with PI). All microchip materials, with the exception of Cu, appeared to be suitable for HL60 cells, showing a ratio apoptosis/proliferation (Rap) comparable to materials used in conventional cell culture (polystyrene). These results were confirmed with cell cycle analysis and apoptosis studies. Precoating the microchip material surfaces with serum favor the proliferation, as demonstrated by a lower Rap as compared to uncoated surfaces. The Cu-coated surface appeared to be toxic for HL60 cells, showing over 90% decreased viability within 24,h. From these results, it can be concluded that the chosen protocol is suitable for selection of the cell culture material, and that the most commonly used microchip materials are compatible with HL60 culturing. [source] The analysis of the fine specificity of celiac disease antibodies using tissue transglutaminase fragmentsFEBS JOURNAL, Issue 21 2002Daniele Sblattero Celiac disease is an intestinal malabsorption characterized by an intolerance to cereal proteins accompanied by immunological responses to dietary gliadins and an autoantigen located in the endomysium. The latter has been identified as the enzyme tissue transglutaminase which belongs to a family of enzymes that catalyze protein cross-linking reactions and is constitutively expressed in many tissues as well as being activated during apoptosis. In a recent paper, we described the selection and characterization of anti-transglutaminase Igs from phage antibody libraries created from intestinal lymphocytes from celiac disease patients. In this work, using transglutaminase gene fragments, we identify a region of tissue transglutaminase recognized by these antibodies as being conformational and located in the core domain of the enzyme. This is identical to the region recognized by anti-transglutaminase Igs found in the serum of celiac disease patients. [source] Non-axial bone fracture but not depression as a risk factor for coeliac diseaseINTERNAL MEDICINE JOURNAL, Issue 3 2010V. P. Tan Abstract Screening for coeliac disease is confined to subgroups at greater risk for the disease, including type 1 diabetes mellitus, autoimmune thyroid disease and family members of affected individuals. This study examined the hypothesis that patients taking antidepressants or presenting with fractures could represent new subgroups at higher risk for coeliac disease. A total of 105 and 199 consecutive patients presenting to hospital taking antidepressants and/or with a fracture was screened with IgA tissue transglutaminase and had their IgA serum levels quantified. Patients with positive serology were offered further diagnostic and management follow up. No patients taking antidepressants had positive serology. Seven with fractures had elevated titres of IgA tissue transglutaminase. All of these patients had presented with non-axial fractures, representing a prevalence of 5.2% (95% confidence interval: 1.4,8.9%). Uptake of further investigation and management was poor. Patients presenting with non-axial fractures may be a subgroup in whom coeliac screening may be indicated. There needs to be greater awareness of atypical presentations of coeliac disease. [source] Compact spheroid formation by ovarian cancer cells is associated with contractile behavior and an invasive phenotypeINTERNATIONAL JOURNAL OF CANCER, Issue 9 2009Katharine L. Sodek Abstract Ovarian cancer cells are present in malignant ascites both as individual cells and as multicellular spheroid aggregates. Although spheroid formation affords protection of cancer cells against some chemotherapeutic agents, it has not been established whether a relationship exists between invasive behavior and predisposition to spheroid formation. Aspects of spheroid formation, including cell-matrix adhesion, remodeling and contractility are characteristic myofibroblast-like behaviors associated with fibrosis that contribute to tumor growth and dissemination. We explored the possibility that cell behaviors that promote spheroid formation also facilitate invasion. Our analysis of 6 human ovarian cancer cell lines indicated that ovarian cancer cells possessing myofibroblast-like properties formed compact spheroids and invaded 3D matrices. These cells readily contracted collagen I gels, possessed a spindle-like morphology, and had elevated expression of genes associated with the TGF,-mediated fibrotic response and/or ,1 integrin function, including fibronectin (FN), connective tissue growth factor (CTGF/CCN2), lysyl oxidase (LOX1), tissue transglutaminase 2 (TGM2) and urinary plasminogen activator receptor (uPAR). Whereas cell aggregation was induced by TGF,, and by ,1-integrin overexpression and activation, these treatments did not stimulate the contractile activity required for spheroid compaction. The positive relationship found between compact spheroid formation and invasive behavior implies a preferential survival of an invasive subpopulation of ovarian cancer cells, as cells in spheroids are more resistant to several chemotherapeutics. Preventing the formation of ovarian cancer spheroids may represent a novel strategy to improve the efficacy of existing therapeutics. © 2008 Wiley-Liss, Inc. [source] Mechanically Strained Cells of the Osteoblast Lineage Organize Their Extracellular Matrix Through Unique Sites of ,V,3 -Integrin ExpressionJOURNAL OF BONE AND MINERAL RESEARCH, Issue 9 2000Magdalena Wozniak Abstract Bone cells transduce mechanical signals into anabolic biochemical responses. However, the mechanisms of mechanotransduction are unknown. To address this issue, we performed studies in primary cells of the human osteoblast lineage grown on collagen/vitronectin-coated supports. We discovered that mechanical strain stimulated a redistribution of the ,v,3 -integrin to irregular plaque-like areas at the cell-extracellular matrix surface. Proteins involved in integrin-matrix interactions in focal adhesions, vinculin and talin, did not localize to the plaque-like areas of ,v,3 -expression, but signaling molecules such as focal adhesion kinase (FAK) did. Mechanical strain increased the number and size of the plaques defined by surface expression of ,v,3 -integrin. Osteopontin was secreted as a cross-linked macromolecular complex, likely through the action of tissue transglutaminase that also was found in the plaques of ,v,3 -integrin cell-matrix interaction. Mechanical strain increased mineralization of the extracellular matrix that developed in these plaques in ,v,3 -integrin-dependent manner. Because the plaque-like areas of cell-matrix interaction exhibit macromolecular assembly and mineralization, we conclude that they may represent subcellular domains of bone formation and that ,v,3 -integrin activation represents one mechanism by which mechanical strain stimulates bone formation. [source] Prevalence of undiagnosed coeliac syndrome in osteoporotic womenJOURNAL OF INTERNAL MEDICINE, Issue 4 2001R. Nuti Abstract.,Nuti R, Martini G, Valenti R, Giovani S, Salvadori S, Avanzati A (Institute of Internal Medicine, Metabolic Disease Unit, University of Siena, Siena, Italy). Prevalence of undiagnosed coeliac syndrome in osteoporotic women. J Intern Med 2001; 250: 361,366. Objectives.,The aims of the study were to quantify the prevalence of asymptomatic coeliac disease (CD) in a cohort of osteoporotic females, and to investigate the features of bone loss. Design and subjects.,We studied 255 women (mean age 66.6 ± 8.5 SD) with primary osteoporosis (WHO diagnostic criteria). After the first CD screening with the measure of serum IgG antigliadin antibodies (IgG-AGA), 53 women showed a positive test: antibodies to tissue transglutaminase (TG-ab) were subsequently determined to confirm the diagnosis of CD. Bone metabolism was evaluated by: serum and urinary calcium, serum and urinary phosphate, serum alkaline phosphatase, urinary crosslaps, serum 25(OH)D and serum parathyroid hormone. Results.,High levels of IgG-AGA and TG-ab were observed in 24 patients with a prevalence of serological disease of 9.4%. These women were characterized, in comparison with the other patients, by a statistically significant reduction in serum 25(OH)D (17.8 ± 7.2 vs. 55.1 ± 20.3 nmol L,1, P < 0.01) together with a significant increase of iPTH (65.1 ± 29.7 vs. 35.1 ± 20.0 pg mL,1; P < 0.01). Patients with high TG-ab levels showed also slightly raised values of urinary crosslaps (288 ± 88 vs. 270 ± 90 ,m mol,1 Cr). In IgG-AG positive patients a statistically significant inverse correlation was found between 25(OH)D serum levels and log-transformed TG-ab values (r: ,0.95, P < 0.001). Intestinal biopsies were obtained in 10 TG-ab positive women and verified CD in six patients. Conclusions.,These data support the hypothesis that patients with undiagnosed celiac disease develop high remodelling processes related to calcium malabsorption, secondary hyperparathyroidism and unavailability of vitamin D with a consequent more marked bone loss. [source] Does tissue transglutaminase play a role in Huntington's disease?JOURNAL OF NEUROCHEMISTRY, Issue 2002G. V. W. Johnson Tissue transglutaminase (tTG) catalyzes the incorporation of polyamines into substrates, or the formation of isopeptide bonds. tTG also binds and hydrolyzes GTP/ATP. Huntington's disease (HD) is caused by a pathological expansion of the polyglutamine domain in the protein huntingtin (htt). Because a polypeptide bound Gln residue is the primary determining factor for a tTG substrate, it has been hypothesized that due to the increase in Gln content, mutant htt may modified by tTG and this event may contribute to the pathogenesis of HD, possibly by facilitating the formation of htt aggregates. tTG is increased in HD, suggesting involvement in the pathogenic process. However, tTG is not required for aggregate formation. Further, tTG is excluded from htt aggregates and increasing or decreasing tTG has no effect on the frequency or localization of the aggregates. Considering these and other data, tTG is unlikely to play a major role in the formation of htt inclusions in HD brain. tTG may play a role in modulating neuronal cell death in response to specific stressors. If a stress increases the transamidating activity of tTG (e.g. increases in Ca++ levels), then tTG facilitates the cell death process. In contrast, if a stress does not result in an increase in the transamidating activity of tTG, then tTG protects against cell death. The protective effects of tTG are independent of its transamidating and hence likely dependent on its GTP/ATP binding and hydrolytic activity. Therefore the increase in tTG levels in HD brain could either be helpful or harmful depending on the cellular mechanisms that contribute to neuronal death. Acknowledgements:, Supported by NIH grant AG12396. [source] Antibodies against deamidated gliadin peptides identify adult coeliac disease patients negative for antibodies against endomysium and tissue transglutaminaseALIMENTARY PHARMACOLOGY & THERAPEUTICS, Issue 2 2010C. Dahle Aliment Pharmacol Ther 2010; 32: 254,260 Summary Background, This study was done to evaluate the diagnostic utility of antibodies against deamidated gliadin peptides compared to traditional markers for coeliac disease. Aim, To evaluate diagnostic utility of antibodies against deamidated gliadin peptide (DGP). Methods, Sera from 176 adults, referred for endoscopy without previous analysis of antibodies against tissue transglutaminase (tTG) or endomysium (EmA), were retrospectively analysed by ELISAs detecting IgA/IgG antibodies against DGP or a mixture of DGP and tTG, and compared with IgA-tTG and EmA. Seventy-nine individuals were diagnosed with coeliac disease. Results, Receiver operating characteristic analyses verified the manufacturers' cut-off limits except for IgA/IgG-DGP/tTG. In sera without IgA deficiency, the sensitivity was higher for IgA/IgG-DGP (0.85,0.87) compared with IgA-tTg (0.76) and EmA (0.61). All tests showed high specificity (0.95,1.00). Eighteen coeliac disease-sera were negative regarding IgA-tTG, nine of which were positive for IgA/IgG-DGP. Sera from coeliac disease-patients >70 years were more often negative for IgA-tTG (50%) and IgA/IgG-DGP (36%) than younger patients (15% and 8% respectively) (P < 0.01). Three of the four IgA-deficient patients were positive in the IgA/IgG-DGP assay. Conclusions, In this study of patients unselected regarding IgA-tTg/EmA, thus unbiased in this respect, IgA/IgG-DGP identified adult coeliac disease patients negative for antibodies against endomysium and tissue transglutaminase. Serology is often negative in elderly patients with coeliac disease; a small bowel biopsy should therefore be performed generously before coeliac disease is excluded. [source] Anti-tissue transglutaminase antibodies in the follow-up of adult coeliac diseaseALIMENTARY PHARMACOLOGY & THERAPEUTICS, Issue 3 2009C. R. DIPPER Summary Background, The detection of auto antibodies directed against tissue transglutaminase (anti-tTG antibodies) has a well-established role in the diagnosis of coeliac disease, but the value of these antibodies in long-term follow-up is controversial. Aims, To determine if serial anti-tTG antibody measurements could confirm adherence to a gluten-free diet (GFD) and identify patients at risk of disease complications. Methods, In a 54-month cohort follow-up study, 182 adult patients were assessed. Data recorded included self-assessment of GFD adherence; anti-tTG antibody concentration and serum ferritin, vitamin B12 and folate. Where available, bone mineral density (BMD) and duodenal histology data were retrieved. Results, Persistently elevated anti-tTG antibody levels were significantly associated with abnormal duodenal histology (P < 0.001), low ferritin (P < 0.01) and poor adherence to the GFD (P < 0.001). The specificity was >85% while the sensitivity was 39,60%. Anti-tTG antibody concentrations fell rapidly following successful initiation of a GFD, and maintenance of normalization identified those who continued to be adherent to the diet. Conclusions, This study supports a strategy of using anti-tTG antibody concentrations to monitor newly diagnosed and established patients with coeliac disease, and to target dietetic intervention to reduce the risk of complication. [source] Meta-analysis: yield of diagnostic tests for coeliac disease in dyspepsiaALIMENTARY PHARMACOLOGY & THERAPEUTICS, Issue 1 2009A. C. FORD Summary Background, The prevalence of coeliac disease (CD) may be increased in individuals with dyspepsia, but evidence is conflicting. Aims, To conduct a systematic review and meta-analysis of studies reporting prevalence of CD in dyspepsia. Methods, MEDLINE, EMBASE, and CINAHL were searched up to February 2009. Case series and case-control studies applying serological tests and/or distal duodenal biopsy for CD to unselected adults with dyspepsia were eligible. Prevalence of positive coeliac serology and biopsy-proven CD were pooled for all studies and compared between cases and controls using an odds ratio (OR) and 95% confidence interval (CI). Results, Fifteen studies were identified. Prevalence of positive coeliac serology was higher in cases with dyspepsia (7.9%) compared with controls (3.9%), but not significantly so (OR for positive endomysial antibodies or tissue transglutaminase 1.89; 95% CI 0.90,3.99). Prevalence of biopsy-proven CD following positive serology was also higher (3.2% in cases vs. 1.3% in controls), but again this was not statistically significant (OR 2.85; 95% CI 0.60,13.38). Prevalence of biopsy-proven CD was 1% in ten studies performing duodenal biopsy first-line. Conclusion, Prevalence of biopsy-proven CD in subjects with dyspepsia was 1% and was higher than in controls, although this difference was not statistically significant. [source] Protease inhibitors derived from elafin and SLPI and engineered to have enhanced specificity towards neutrophil serine proteasesPROTEIN SCIENCE, Issue 3 2009Marie-Louise Zani Abstract The secretory leukocyte protease inhibitor (SLPI), elafin, and its biologically active precursor trappin-2 are endogeneous low-molecular weight inhibitors of the chelonianin family that control the enzymatic activity of neutrophil serine proteases (NSPs) like elastase, proteinase 3, and cathepsin G. These inhibitors may be of therapeutic value, since unregulated NSP activities are linked to inflammatory lung diseases. However SLPI inhibits elastase and cathepsin G but not proteinase 3, while elafin targets elastase and proteinase 3 but not cathepsin G. We have used two strategies to design polyvalent inhibitors of NSPs that target all three NSPs and may be used in the aerosol-based treatment of inflammatory lung diseases. First, we fused the elafin domain with the second inhibitory domain of SLPI to produce recombinant chimeras that had the inhibitory properties of both parent molecules. Second, we generated the trappin-2 variant, trappin-2 A62L, in which the P1 residue Ala is replaced by Leu, as in the corresponding position in SLPI domain 2. The chimera inhibitors and trappin-2 A62L are tight-binding inhibitors of all three NSPs with subnanomolar Kis, similar to those of the parent molecules for their respective target proteases. We have also shown that these molecules inhibit the neutrophil membrane-bound forms of all three NSPs. The trappin-2 A62L and elafin-SLPI chimeras, like wild-type elafin and trappin-2, can be covalently cross-linked to fibronectin or elastin by a tissue transglutaminase, while retaining their polypotent inhibition of NSPs. Therefore, the inhibitors described herein have the appropriate properties to be further evaluated as therapeutic anti-inflammatory agents. [source] Tissue transglutaminase modulates ,-synuclein oligomerizationPROTEIN SCIENCE, Issue 8 2008Ine M.J. Segers-Nolten Abstract We have studied the interaction of the enzyme tissue transglutaminase (tTG), catalyzing cross-link formation between protein-bound glutamine residues and primary amines, with Parkinson's disease-associated ,-synuclein protein variants at physiologically relevant concentrations. We have, for the first time, determined binding affinities of tTG for wild-type and mutant ,-synucleins using surface plasmon resonance approaches, revealing high-affinity nanomolar equilibrium dissociation constants. Nanomolar tTG concentrations were sufficient for complete inhibition of fibrillization by effective ,-synuclein cross-linking, resulting predominantly in intramolecularly cross-linked monomers accompanied by an oligomeric fraction. Since oligomeric species have a pathophysiological relevance we further investigated the properties of the tTG/,-synuclein oligomers. Atomic force microscopy revealed morphologically similar structures for oligomers from all ,-synuclein variants; the extent of oligomer formation was found to correlate with tTG concentration. Unlike normal ,-synuclein oligomers the resultant structures were extremely stable and resistant to GdnHCl and SDS. In contrast to normal ,-sheet-containing oligomers, the tTG/,-synuclein oligomers appear to be unstructured and are unable to disrupt phospholipid vesicles. These data suggest that tTG binds equally effective to wild-type and disease mutant ,-synuclein variants. We propose that tTG cross-linking imposes structural constraints on ,-synuclein, preventing the assembly of structured oligomers required for disruption of membranes and for progression into fibrils. In general, cross-linking of amyloid forming proteins by tTG may prevent the progression into pathogenic species. [source] Using radioligand-binding assays to measure tissue transglutaminase autoantibodies in young childrenACTA PAEDIATRICA, Issue 8 2004D Agardh Aim: To measure autoantibodies against tissue transglutaminase (tTG) in young children prospectively screened for coeliac disease (CD). Methods: In total, 652 children aged 2.9 (2.5,4.2) y were analysed for IgA-tTG and IgG-tTG with radioligand-binding assays and IgA endomysial antibodies (EMA) by indirect immunofluorescence. Antibody-positive children were retested after 1.2 (range 0.2,1.9) y. Intestinal biopsy was performed on children with persistently high antibody levels. Results: In total, 3.2% (95% CI: 1.9,4.6%) of the 652 children were positive for at least one antibody at baseline: 2.5% (95% CI: 1.3,3.7%) for IgA-tTG, 1.7% (95% CI: 0.7,2.7%) for IgG-tTG and 2.9% (95% CI: 1.6,4.2%) for IgA-EMA, respectively. Ten children were positive for all three antibodies, five for both IgA-tTG and EMA, four for EMA only, one for IgA-tTG and another for IgG-tTG. IgA-EMA titres correlated with IgA-tTG levels (r= 0.73, p= 0.0003). At follow-up, seven of 20 children remained positive for all three antibodies, three for IgA-tTG only, one for both IgA-tTG and EMA, one for IgA-tTG and IgG-tTG, and the remaining child refused further participation. Three biopsies showed villous atrophy, two increased intraepithelial lymphocytes and two normal findings. Biopsy was not performed in four children with low or declining tTG antibody levels at follow-up and in one child who declined. CD was evident in 0.5% (95% CI: 0.0,1.0%) (3/652). Conclusion: This study revealed a high number of young children positive for tTG antibodies as well as EMA, but the majority showed declining levels in both antibodies over time. We suggest using radioligand-binding assays for quantitative measurement of tTG antibodies when change in antibody levels is studied in young children. [source] Bcl-2 overexpression in hepatic stellate cell line CFSC-2G, induces a pro-fibrotic stateJOURNAL OF GASTROENTEROLOGY AND HEPATOLOGY, Issue 7 2010Viridiana Y González-Puertos Abstract Background and Aim:, Development of hepatic fibrosis is a complex process that involves oxidative stress (OS) and an altered balance between pro- and anti-apoptotic molecules. Since Bcl-2 overexpression preserves viability against OS, our objective was to address the effect of Bcl-2 overexpression in the hepatic stellate cells (HSC) cell-line CFSC-2G under acetaldehyde and H2O2 challenge, and explore if it protects these cells against OS, induces replicative senescence and/or modify extracellular matrix (ECM) remodeling potential. Methods:, To induce Bcl-2 overexpression, HSC cell line CFSC-2G was transfected by lipofection technique. Green fluorescent protein-only CFSC-2G cells were used as a control. Cell survival after H2O2 treatment and total protein oxidation were assessed. To determine cell cycle arrest, proliferation-rate, DNA synthesis and senescence were assessed. Matrix metalloproteinases (MMP), tissue-inhibitor of MMP (TIMP), transglutaminases (TG) and smooth muscle a-actin (,-SMA) were evaluated by western blot in response to acetaldehyde treatment as markers of ECM remodeling capacity in addition to transforming growth factor-, (TGF-,) mRNA. Results:, Cells overexpressing Bcl-2 survived , 20% more than control cells when exposed to H2O2 and , 35% proteins were protected from oxidation, but Bcl-2 did not slow proliferation or induced senescence. Bcl-2 overexpression did not change ,-SMA levels, but it increased TIMP-1 (55%), tissue transglutaminases (tTG) (25%) and TGF-, mRNA (49%), when exposed to acetaldehyde, while MMP-13 content decreased (47%). Conclusions:, Bcl-2 overexpression protected HSC against oxidative stress but it did not induce replicative senescence. It increased TIMP-1, tTG and TGF-, mRNA levels and decreased MMP-13 content, suggesting that Bcl-2 overexpression may play a key role in the progression of fibrosis in chronic liver diseases. [source] | ||||||||||||||||||||||||||||