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Immunoblotting
Kinds of Immunoblotting Terms modified by Immunoblotting Selected AbstractsActin-dependent motility of melanosomes from fish retinal pigment epithelial (RPE) cells investigated using in vitro motility assaysCYTOSKELETON, Issue 2 2004E. L. McNeil Melanosomes (pigment granules) within retinal pigment epithelial (RPE) cells of fish and amphibians undergo massive migrations in response to light conditions to control light flux to the retina. Previous research has shown that melanosome motility within apical projections of dissociated fish RPE cells requires an intact actin cytoskeleton, but the mechanisms and motors involved in melanosome transport in RPE have not been identified. Two in vitro motility assays, the Nitella assay and the sliding filament assay, were used to characterize actin-dependent motor activity of RPE melanosomes. Melanosomes applied to dissected filets of the Characean alga, Nitella, moved along actin cables at a mean rate of 2 ,m/min, similar to the rate of melanosome motility in dissociated, cultured RPE cells. Path lengths of motile melanosomes ranged from 9 to 37 ,m. Melanosome motility in the sliding filament assay was much more variable, ranging from 0.4,33 ,m/min; 70% of velocities ranged from 1,15 ,m/min. Latex beads coated with skeletal muscle myosin II and added to Nitella filets moved in the same direction as RPE melanosomes, indicating that the motility is barbed-end directed. Immunoblotting using antibodies against myosin VIIa and rab27a revealed that both proteins are enriched on melanosome membranes, suggesting that they could play a role in melanosome transport within apical projections of fish RPE. Cell Motil. Cytoskeleton 58:71,82, 2004. © 2004 Wiley-Liss, Inc. [source] Effects of Antrodia camphorata on viability, apoptosis, [Ca2+]i, and MAPKs phosphorylation in MG63 human osteosarcoma cellsDRUG DEVELOPMENT RESEARCH, Issue 2 2007Yih-Chau Lu Abstract The present study explored the effect of Antrodia camphorata (AC) on viability, apoptosis, mitogen-activated protein kinases (MAPKs) phosphorylation, and Ca2+ regulation in MG63 human osteosarcoma cells. AC (25,50,µg/ml) did not affect cell viability, but at 100,200,µg/ml decreased viability and induced apoptosis in a concentration-dependent manner. AC at concentrations of 25,200,µg/ml did not alter basal [Ca2+]i, but at 25,µg/ml decreased [Ca2+]i increases induced by ATP, bradykinin, histamine, and thapsigargin. ATP, bradykinin, and histamine increased cell viability while thapsigargin decreased it. AC (25,µg/ml) pretreatment failed to alter bradykinin- and thapsigargin-induced effects on viability, but potentiated ATP- and histamine-induced increases in viability. Immunoblotting showed that MG63 cells did not have background phospho-JNK and phospho-p38 mitogen-activated protein kinases (MAPKs); and AC did not induce the phosphorylation of these two MAPKs. Conversely, the cells had significant background phospho-ERK MAPK that was inhibited by 200,µg/ml AC. The ERK-specific inhibitor PD98059 also induced cell death. Collectively, in MG63 cells, AC exerted multiple effects on viability and [Ca2+]i, caused apoptosis probably via inhibition of ERK MAPK phosphorylation. Drug Dev Res 68:71,78, 2007. © 2007 Wiley-Liss, Inc. [source] Retina expresses a novel variant of the ryanodine receptorEUROPEAN JOURNAL OF NEUROSCIENCE, Issue 11 2007Varda Shoshan-Barmatz Abstract Calcium released from intracellular stores via the ryanodine receptor (RyR) mediates a variety of signalling processes. We previously showed that retina expresses the three known types of RyR, but retinal membrane preparations exhibit unique characteristics such as Ca2+ -independent [3H]ryanodine-binding and inhibition by caffeine. We have heretofore suggested that the major retinal RyR isoform is novel. The present study aimed to identify this receptor isoform and to localize RyR in mammalian retina. Immunoblotting with specific and pan-antibodies showed that the major retinal RyR has a mobility similar to that of RyR2 or RyR3. Real-time PCR revealed that the major type is RyR2, and RT-PCR followed by sequencing showed a transcript that encodes a protein with ~ 99% identity to RyR2, yet lacking two regions of seven and 12 amino acids and including an additional insertion of eight amino acids. An antibody against RyR2 localized this type to somas and primary dendrites of most retinal neurons. An antibody against RyR1 localized RyR to most somas but also revealed staining in photoreceptor outer segments, concentrated on the disk membranes at their rim. The ryanodine-binding properties and the electrophoretic mobility of RyR from the outer segments were similar to those of the whole retinal preparation. The results thus identify a novel variant of RyR2 which can contribute to regulating photoreceptor Ca2+ concentrations. The restricted localization of the outer segment RyR to the disk rim suggests that its activation mechanism involves a coupling between retinal RyR and the cGMP-gated channel. [source] AMPA/kainate and NMDA-like glutamate receptors at the chromatophore neuromuscular junction of the squid: role in synaptic transmission and skin patterningEUROPEAN JOURNAL OF NEUROSCIENCE, Issue 3 2003Pedro A. Lima Abstract Glutamate receptor types were examined at the chromatophore synapses of the squids Alloteuthis subulata and Loligo vulgaris, where nerve-induced muscle contraction causes chromatophore expansion. Immunoblotting with antibody raised against a squid AMPA receptor (sGluR) demonstrated that AMPA/kainate receptors are present in squid skin. Application of l -glutamate evoked chromatophore muscle contractions in both ventral and dorsal skins, while NMDA was only active on a subpopulation of dorsal chromatophores. In dorsal skin, neurotransmission was partly blocked by either AMPA/kainate receptor antagonists (CNQX and DNQX) or NMDA receptor antagonists (AP-5 and MK-801) or completely blocked by simultaneous application of both classes of antagonists. In isolated muscle fibres, ionophoretic application of l -glutamate evoked fast inward CNQX- and DNQX-sensitive currents with reversal potentials around +14 mV and a high conductance to Na+. In fibres from dorsal skin only, a slower outward glutamate-sensitive current appeared at positive holding potentials. At negative potentials, currents were potentiated by glycine or by removing external Mg2+ and were blocked by AP-5 and MK-801. Glutamate caused a fast, followed by a slow, transient increase in cytoplasmic Ca2+. The slow component was increased in amplitude and duration by glycine or by lowering external Mg2+ and decreased by AP-5 and MK-801. In cells from ventral skin, no ,NMDA-like responses' were detected. Thus, while AMPA/kainate receptors mediated fast excitatory synaptic transmission and rapid colour change over the whole skin, activation of both AMPA/kainate and NMDA-like receptors in a subpopulation of dorsal chromatophores prolonged the postsynaptically evoked Ca2+ elevation causing temporally extended colour displays with behavioural significance. [source] Fluoride down-regulates the expression of matrix metalloproteinase-20 in human fetal tooth ameloblast-lineage cells in vitroEUROPEAN JOURNAL OF ORAL SCIENCES, Issue 2006Yan Zhang Fluoride is associated with a decrease in the incidence of dental caries, but excessive fluoride intake during tooth enamel formation can result in enamel fluorosis. Fluorosed enamel has increased porosity, which has been related to a delay in the removal of amelogenin proteins as the enamel matures. This delay in protein removal suggests that fluoride may affect either the amount or the activity of enamel matrix proteinases. In this study, we investigated the role of fluoride in the synthesis and secretion of matrix metalloproteinase-20 (MMP-20), the proteinase primarily responsible for the initial hydrolysis of amelogenin during the secretory stage of enamel formation. Cultured human fetus tooth organ ameloblast-lineage cells were exposed to 10 µM fluoride and analyzed for synthesis of MMP-20. Immunoblotting showed that 10 µM NaF down-regulated the synthesis of MMP-20 by 21% compared with control cells, but did not alter the amount of amelogenin or kalikrein-4 (KLK-4) synthesized by the cells. Real-time polymerase chain reaction (PCR) showed that 10 µM NaF down-regulated MMP-20 mRNA expression to 28% of the levels found in the non-treated cells. These in vitro results suggest that fluoride can alter the expression of MMP-20 by ameloblasts, resulting in a disturbance of the balance between MMP-20 and its substrate that may contribute to the retention of amelogenins in the formation of fluorosed enamel. [source] Alcohol-induced alterations in hepatic microtubule dynamics can be explained by impaired histone deacetylase 6 function,HEPATOLOGY, Issue 5 2008Blythe D. Shepard We have been using polarized, hepatic WIF-B cells to examine ethanol-induced liver injury. These cells polarize in culture and maintain numerous liver-specific activities including the ability to metabolize alcohol. Previously, we found that microtubules were more highly acetylated and more stable in ethanol-treated WIF-B cells and that increased microtubule acetylation required ethanol metabolism and was likely mediated by acetaldehyde. This study was aimed at identifying the mechanism responsible for increased microtubule acetylation. We examined the expression of two known microtubule deacetylases, histone deacetylase 6 (HDAC6) and Sirtuin T2 (SirT2), in WIF-B cells. Immunoblotting, immunofluorescence microscopy, and assays using the SirT2 inhibitor nicotinamide revealed that WIF-B cells do not express SirT2. In contrast, HDAC6 was highly expressed in WIF-B cells. Addition of trichostatin A (TSA), an HDAC6 inhibitor, induced microtubule acetylation to the same extent as in ethanol-treated cells (approximately threefold). Although immunofluorescence labeling revealed that HDAC6 distribution did not change in ethanol-treated cells, immunoblotting showed HDAC6 protein levels slightly decreased. HDAC6 solubility was increased in nocodazole-treated cells, suggesting impaired microtubule binding. Direct microtubule binding assays confirmed this hypothesis. The decreased microtubule binding was partially prevented by 4-methyl pyrazole, indicating the effect was in part mediated by acetaldehyde. Interestingly, HDAC6 from ethanol-treated cells was able to bind and deacetylate exogenous tubulin to the same extent as control, suggesting that ethanol-induced tubulin modifications prevented HDAC6 binding to endogenous microtubules. Conclusion: We propose that lower HDAC6 levels combined with decreased microtubule binding lead to increased tubulin acetylation in ethanol-treated cells. (HEPATOLOGY 2008.) [source] Sustained calpain activation associated with lysosomal rupture executes necrosis of the postischemic CA1 neurons in primatesHIPPOCAMPUS, Issue 7 2003Tetsumori Yamashima Abstract Because of the paucity of primate experimental models, the precise molecular mechanism of ischemic neuronal death remains unknown in humans. This study focused on nonhuman primates to determine which cascade necrosis or apoptosis is predominantly involved in the development of delayed (day 5) neuronal death in the hippocampal CA1 sector undergoing 20 min ischemia. We investigated expression, activation, and/or translocation of ,-calpain, lysosome-associated membrane protein-1 (LAMP-1), caspase-3, and caspase-activated DNase (CAD), as well as morphology of the postischemic CA1 neurons and DNA electrophoresis pattern. Immunoblotting showed sustained (immediately after ischemia until day 5) and maximal (day 3) activation of ,-calpain. The immunoreactivity of activated ,-calpain became remarkable as coarse granules at lysosomes on day 2, while it translocated throughout the perikarya on day 3. The immunoreactivity of LAMP-1 also showed a dynamic and concomitant translocation that was maximal on days 2,3, indicating calpain-mediated disruption of the lysosomal membrane after ischemia. In contrast, immunoblotting demonstrated essentially no increase in the activated caspase-3 at any time points after ischemia, despite upregulation of pro-caspase-3. Although expression of CAD was slightly upregulated on day 1 or 2, or both, it was much less compared with lymph node or intestine tissues. Furthermore, light and electron microscopy showed eosinophilic coagulation necrosis and membrane disruption without apoptotic body formation, while DNA electrophoresis did not show a ladder pattern, but rather a smear pattern. Sustained calpain activation and the resultant lysosomal rupture, rather than CAD-mediated apoptosis, may cause ischemic neuronal necrosis in primates. © 2003 Wiley-Liss, Inc. [source] Signal transducers and activators of transcription 3 signaling pathway.INFLAMMATORY BOWEL DISEASES, Issue 2 2005An Essential Mediator of Inflammatory Bowel Disease, Other Forms of Intestinal Inflammation Abstract Crohn's disease (CD) and ulcerative colitis (UC), the two major forms of chronic inflammatory bowel disease (IBD), are characterized by mucosal immune cell activation that is driven by a cytokine imbalance. Several cytokines involved in IBD act through the activation of the signal transducers and activators of transcription (STAT) family. We investigated the activation of STAT3 in the mucosa of CD and UC patients, and evaluated whether this event is specific for IBD patients. Using immunofluorescence and immunoblotting, total and phosphorylated STAT3 levels were assessed in biopsy specimens, isolated lamina propria mononuclear cells, and peripheral blood mononuclear cells from patients with CD, UC, other forms of intestinal inflammation, and control subjects. Immunoblotting revealed phosphorylated STAT3 in mucosal biopsy specimens from patients with CD, UC, celiac disease, and acute self-limited colitis, but not in the normal mucosa of control subjects. In IBD patients, STAT3 activation was confined to actively inflamed areas. Accordingly, activated STAT3 was detected in isolated lamina propria mononuclear cells from inflamed IBD tissues, but not in peripheral blood mononuclear cells from control subjects or IBD patients. Immunofluorescence demonstrated that the sources of activated STAT3 were macrophages and T lymphocytes, but not neutrophils. STAT3 activation also was detected in T cells infiltrating the duodenal mucosa of celiac disease patients. We conclude that STAT3 signaling occurs in both CD and UC, where it is strictly confined to areas of active inflammation and is limited to infiltrating macrophages and T cells. The occurrence of STAT3 signaling in other acute and chronic intestinal inflammatory conditions suggests that, rather than a specific feature of IBD, it represents a fundamental signaling pathway that is shared by multiple forms of gut inflammation. [source] Degradation of transgenic Cry1Ab DNA and protein in Bt-176 maize during the ensiling processJOURNAL OF ANIMAL PHYSIOLOGY AND NUTRITION, Issue 3-4 2006B. Lutz Summary Maize silage is commonly used as feed for farm animals. The aim of this study was to monitor the time-dependent degradation of non-recombinant chloroplast DNA (exemplified by the rubisco gene) in comparison with the recombinant cry1Ab gene in the course of the ensiling process. In parallel, the Cry1Ab protein content and fragment sizes were determined. Fragments of the rubisco (173, 896, 1197, 1753 and 2521 bp) and of the cry1Ab gene (211, 420, 727 and 1423 bp) were selected to investigate the DNA degradation process. The detection of the Cry1Ab protein was performed using an enzyme-linked immunosorbent assay (ELISA) and immunoblotting. Rubisco gene fragments of 173 bp were still detectable after 61 days, while fragments of 1197 and 2521 bp were detectable up to 30 days and on the first day only respectively. Polymerase chain reaction (PCR) analyses revealed that fragments of the cry1Ab gene with sizes of 211 and 420 bp were detectable up to 61 days, fragments with sizes of 727 and 1423 bp, 30 and 6 days respectively. The ELISA showed a decrease of the Cry1Ab protein in maize silage during the ensiling process. No marked degradation was observed during the first 43 h. Thereafter, a sharp decrease was measured. After 61 days, 23.6 ± 0.9% of the initial Cry1Ab protein was still detectable. Immunoblotting confirmed the results of the ELISA showing a positive signal of approximately 60 kDa size for 8 days of ensiling; no further immunoactive fragments were detectable by immunoblotting. In conclusion, the ensiling process markedly decreases the presence of long functional cry1Ab gene fragments and full size Cry1Ab protein. [source] Modulation of expression of LDH isoenzymes in endothelial cells by laminin: Implications for angiogenesisJOURNAL OF CELLULAR BIOCHEMISTRY, Issue 6 2008V.B. Sameer Kumar Abstract Endothelial cell (EC) matrix interaction is critical in angiogenesis. Although matrix components can regulate the process of angiogenesis by acting as a reservoir of various cytokines, it is not clear if extracellular matrix (ECM) can modulate the production and activity of angiogenic cytokines. Investigations were therefore carried out to study the influence of the basement membrane (BM) protein, laminin (Ln) on the activity of vascular endothelial growth factor (VEGF), the major angiogenic cytokine, using isolated human umbilical vein ECs (HUVECs) in culture. Analysis of the biochemical markers of angiogenesis confirmed proangiogenic effect of Ln. The levels of VEGF protein and mRNA were not different in cells maintained on Ln, collagen I or polylysine substrata. Chorioallantoic membrane assay using VEGF isolated from cell extracts however revealed that Ln increased its angiogenic potency. Immunoblotting and HPLC analysis showed considerable reduction in poly adenosyl ribosylation of VEGF associated with a significant decrease in the levels of NAD+, in cells maintained on Ln substrata. Further, a shift in the isoenzymic pattern of LDH towards the B rich forms and an upregulation of LDH B gene were observed in cells maintained on Ln. Ln modulates expression of LDH gene through ,6,4 integrin mediated downstream signaling involving p38 mitogen activated protein kinases (MAPK) pathway. It thus appears that Ln can affect aerobic metabolism of ECs by modulating the expression of LDH isoenzymes resulting in a decrease in the level of NAD+ that can cause a reduction in the poly adenosyl ribosylation of VEGF altering its angiogenic potency. J. Cell. Biochem. 103: 1808,1825, 2008. © 2007 Wiley-Liss, Inc. [source] Functional analysis of polyomavirus BK non-coding control region quasispecies from kidney transplant recipientsJOURNAL OF MEDICAL VIROLOGY, Issue 11 2009Gunn-Hege Olsen Abstract Replication of the human polyomavirus BK (BKV) in renal tubular epithelial cells causes viruria and BKV-nephropathy in kidney transplant recipients. Following prolonged high-level BKV replication, rearrangement of the archetype non-coding control region (NCCR) leads to a mixture of BKV variants. The aim of this study was to compare potential functional differences of 12 rearranged (rr)-NCCR variants with the archetype (ww)-NCCR (WWT) found in allograft biopsies or urine from three kidney transplant recipients including two with BKV-nephropathy. Twelve different rr-NCCRs and one archetype ww-NCCR were inserted between the early and late protein coding region of BKV(Dunlop) to make recombinant BKV genomes for transfection into Vero cells. Immunoblotting, immunofluorescence staining, and quantitative PCR demonstrated that viral protein expression and extracellular BKV loads of 10 rr-NCCR variants were similar or higher than observed for the ww-NCCR BKV. Two rr-NCCR variants (RH-2 and RH-19) were non-functional. The functional rr-NCCRs produced infectious progeny successfully infecting primary renal proximal tubular epithelial cells. The number of infected cells and extracellular BKV loads corresponded to the activity seen in Vero cells. Three rr-NCCR variants (RH-1, RH-10, RH-13) only gave rise to a few infected cells similar to ww-NCCR, whereas seven variants had intermediate activity (RH-5, RH-6, RH-8, RH-9, RH-11) or high replication activity (RH-7 and RH-18) with several hundred infected cells per well. The results indicate that both functional and non-functional BKV rr-NCCR variants arise during BKV replication in kidney transplant recipients and that most functional rr-NCCR variants confer a higher replication capacity than archetype ww-NCCR. J. Med. Virol. 81:1959,1967, 2009. © 2009 Wiley-Liss, Inc. [source] Molecular epidemiology of molluscum contagiosum virus and analysis of the host-serum antibody response in Spanish HIV-negative patientsJOURNAL OF MEDICAL VIROLOGY, Issue 2 2002Monica Agromayor Abstract Molluscum contagiosum virus (MCV) lesions from Spanish human immunodeficiency virus (HIV)-negative patients were clinically examined and analyzed for virus detection and typing. In a study of 147 patients, 97 (66%) were children under 10 years, of whom 49% had atopic dermatitis. MCV lesions were morphologically indistinguishable among the different age groups, but atopic patients presented larger lesions compared with patients without the disorder. In adults, lesions were observed mainly on the genitals. MCVI was the predominant subtype. The deduced MCVI/MCVII ratio (146:1) was much higher than that found in other geographical areas. Protein preparations of the virus-induced lesions were immunoblotted with sera from 25 MCVI patients. The host-serum antibody response was weak and variable, although no significant differences were found between atopic and nonatopic patients. Three immunoreactive proteins of 74/80, 60, and 35 kDa were detected in almost all the analyzed sera. The 35 and 74/80-kDa proteins were virus specific, whereas the 60-kDa protein band was composed of a mix of human keratins. Immunoblotting of MCV lesions and vaccinia virus-infected cell extracts with either MCV patient serum or a rabbit antiserum against vaccinia virus showed no cross-reactivity of these two human poxviruses at the antigenic level. J. Med. Virol. 66:151,158, 2002. © 2002 Wiley-Liss, Inc. [source] GAT-1 regulates both tonic and phasic GABAA receptor-mediated inhibition in the cerebral cortexJOURNAL OF NEUROCHEMISTRY, Issue 5 2008Luca Bragina Abstract ,-Aminobutyric acid 1 (GAT-1) is the most copiously expressed GABA transporter; we studied its role in phasic and tonic inhibition in the neocortex using GAT-1 knockout (KO) mice. Immunoblotting and immunocytochemical studies showed that GAT-2 and GAT-3 levels in KOs were unchanged and that GAT-3 was not redistributed in KOs. Moreover, the expression of GAD65/67 was increased, whereas that of GABA or VGAT was unchanged. Microdialysis studies showed that in KOs spontaneous extracellular release of GABA and glutamate was comparable in WT and KO mice, whereas KCl-evoked output of GABA, but not of glutamate, was significantly increased in KOs. Recordings from layer II/III pyramids revealed a significant increase in GABAAR-mediated tonic conductance in KO mice. The frequency, amplitude and kinetics of spontaneous inhibitory post-synaptic currents (IPSCs) were unchanged, whereas the decay time of evoked IPSCs was significantly prolonged in KO mice. In KO mice, high frequency stimulation of GABAergic terminals induced large GABAAR-mediated inward currents associated with a reduction in amplitude and decay time of IPSCs evoked immediately after the train. The recovery process was slower in KO than in WT mice. These studies show that in the cerebral cortex of GAT-1 KO mice GAT-3 is not redistributed and GADs are adaptively changed and indicate that GAT-1 has a prominent role in both tonic and phasic GABAAR-mediated inhibition, in particular during sustained neuronal activity. [source] Up-regulation of cerebral carbonic anhydrase by anoxic stress in pigletsJOURNAL OF NEUROCHEMISTRY, Issue 4 2003Antal Nógrádi Abstract The resuscitation of asphyxiated babies is associated with changes in cerebral protein synthesis that can influence the neurological outcome. Insufficient gas exchange results in rapid shifts in extracellular and intracellular pH. Carbonic anhydrase (CA) plays an important role in buffering acute changes in pH in the brain. We investigated whether asphyxia/re-ventilation influences the expression of cerebral CA isoforms (CA-II, CA-III and CA-IV) in anaesthetized newborn pigs. The cerebral cortex, hippocampus, cerebellum and retina were sampled, and prepared for either CA immunohistochemistry or CA immunoblotting from piglets subjected to asphyxia (10 min) followed by 2,4 h of re-ventilation, and also from normoxic controls. The CA immunoreactivity (IR) of all the isoforms studied was weak in the controls, apart from staining of a few oligodendrocytes in the subcortical white matter, some astrocytes in the superficial layer of the cerebral cortex, the cerebellar Purkinje cells and the retinal Müller cells that possessed moderate CA-II IR. However, asphyxia induced a marked increase in the CA IR of all isoforms in all the cerebral regions investigated and the retina after 4 h of survival. The pyramidal cells of the frontal cortex and hippocampus displayed the most conspicuous increase in CA IR. Immunoblotting confirmed increased levels of all the CA isoenzymes. We conclude that raised CA levels after asphyxia may contribute to the compensatory mechanisms that protect against the pathological changes in the neonatal CNS. [source] Glycoprotein (GP) VI dimer as a major collagen-binding site of native platelets: direct evidence obtained with dimeric GPVI-specific FabsJOURNAL OF THROMBOSIS AND HAEMOSTASIS, Issue 8 2009S. M. JUNG Summary.,Background: The platelet collagen receptor glycoprotein (GP) VI is suggested to exist as a dimer on the platelet surface, but no direct proof of the functional importance of dimer formation has been provided. Objectives: To obtain direct evidence for GPVI dimers on the platelet membrane and their functional importance, Fab antibodies were developed that bind to GPVI dimer (GPVI-Fc2) but not to GPVI monomer (GPVIex) through a phage display method. Results: Ssix Fabs were found: B,F, only reactive with GPVI-Fc2, and A, mainly reactive with GPVI-Fc2, with some reactivity towards GPVIex; each Fab (Fab-dHLX-MH) forms a bivalent dimer (b-Fab) by dimerizing the dHLX domains from two Fab molecules. Fab F was subcloned to a monovalent format by deleting its dHLX domain. All b-Fabs induced platelet aggregation, but the monomeric form of Fab F (m-Fab-F) specifically inhibited collagen-induced aggregation. All b-Fabs and m-Fab-F inhibited GPVI-Fc2 binding to fibrous collagen. Immunoblotting showed that b-Fab-F and m-Fab-F bound weakly to GPVI-Fc2. Adding the anti-GPVI monoclonal antibody 204-11 increased the Bmax of m-Fab-F binding to GPVI-Fc2, suggesting that 204-11 binds to GPVI-Fc2 molecules not already in the appropriate conformation to recognize the Fab, converting them to a conformation reactive to the Fab. Conclusions: GPVI forms a specific structure by dimerization that is necessary for the binding of this receptor to collagen fibrils. The binding of m-Fab-F to platelets directly demonstrates that GPVI is present as a functionally relevant dimer on the platelet surface. [source] Diversity of Glanzmann thrombasthenia in southern India: 10 novel mutations identified among 15 unrelated patientsJOURNAL OF THROMBOSIS AND HAEMOSTASIS, Issue 8 2006E. J. R. NELSON Summary.,Background: Glanzmann thrombasthenia (GT) is a congenital bleeding disorder caused by either a lack or dysfunction of the platelet integrin ,IIb,3. Objectives: To determine the molecular basis of GT in patients from southern India. Patients: Fifteen unrelated patients whose diagnosis was consistent with GT were evaluated. Results: Platelet surface expression of ,IIb,3 was < 10%, 10%,50%, and > 50% of controls in five, nine, and one patient(s), respectively. Immunoblotting of the platelet lysates showed no ,IIb in 14 patients, and no ,3 in 10 patients, although severely reduced in four patients. Platelet fibrinogen was undetectable in 13 patients, and severely reduced in one patient. One patient showed normal surface ,IIb,3 expression, and normal ,IIb, ,3 and fibrinogen levels in the lysate. Ten novel candidate disease-causing mutations were identified in 11 patients. The missense mutations included Gly128Ser, Ser287Leu, Gly357Ser, Arg520Trp, Leu799Arg in ,IIb, and Cys575Gly in ,3. We have already shown that Gly128Ser, Ser287Leu, and Gly357Ser mutations variably affect ,IIb,3 surface expression. The Cys575Gly mutation may disrupt the disulphide link with Cys586 to cause the GT phenotype. The molecular pathology of the other missense mutations is not clear. Two nonsense mutations, Trp-16Stop and Glu715Stop in ,IIb, and a 7-bp deletion (330-336TCCCCAG) in ,3 are predicted to result in truncated proteins. An IVS15(,1)G , A mutation in ,IIb induced a cryptic splice site as confirmed by reverse transcription-polymerase chain reaction (RT-PCR) analysis. Thirteen polymorphisms were also identified (five in ,IIb and eight in ,3), among which five were novel. Conclusions: While identifying a significant number of novel mutations causing GT, this study confirms the genetic heterogeneity of the disorder in southern India. [source] Acetaminophen UDP-glucuronosyltransferase in ferrets: species and gender differences, and sequence analysis of ferret UGT1A6JOURNAL OF VETERINARY PHARMACOLOGY & THERAPEUTICS, Issue 6 2001M. H. Court The principal objective of this study was to determine whether ferrets glucuronidate acetaminophen more slowly compared with other species, and if so investigate the molecular basis for the difference. Acetaminophen-UDP-glucuronosyltransferase (UGT) activities were measured using hepatic microsomes from eight ferrets, four humans, four cats, four dogs, rat, mouse, cow, horse, monkey, pig and rabbit. Gender differences between male and female ferret livers were explored using enzyme kinetic analysis. Immunoblotting of microsomal proteins was also performed using UGT-specific antibodies. Finally, the exon 1 region of UGT1A6, a major acetaminophen-UGT, was sequenced. Glucuronidation of acetaminophen was relatively slow in ferret livers compared with livers from all other species except cat. Gender differences were also apparent, with intrinsic clearance (Vmax/Km) values significantly higher in male compared with female ferret livers. Furthermore, Vmax values correlated with densitometric measurements of two protein bands identified with a UGT1A subfamily-specific antibody. No deleterious mutations were identified in the exon 1 or flanking regions of the ferret UGT1A6 gene. In conclusion, like cats, ferret livers glucuronidate acetaminophen relatively slowly. However, unlike cats, in which UGT1A6 is encoded by a pseudogene and dysfunctional, there are no defects in the ferret UGT1A6 gene which could account for the low activity. [source] Molecular and immunological characterization of the C-terminal region of a new Echinococcus granulosus Heat Shock Protein 70PARASITE IMMUNOLOGY, Issue 3 2003E. Ortona SUMMARY By screening an expression library of Echinococcus granulosus with IgE from sera of patients with cystic echinococcosis (CE) and allergic reactions, we isolated the C-terminal region of a new heat shock protein (HSP)70 of E. granulosus. The protein, named Eg2HSP70, has close homology with the C-terminal region of Dermatophagoides farinae and human HSP70. We investigated the humoral and cell-mediated immune responses to this antigen in patients with CE grouped according to the presence of allergic reactions. Immunoblotting detected total IgG, IgG4 and IgE specific to Eg2HSP70 (83% of sera contained IgG, 31% IgG4 and 57% IgE). No significant difference was found in immunoglobulin percentages according to the presence of allergic reactions. Immunoblotting inhibition showed that no IgG or IgE specific to Eg2HSP70 cross-reacted with D. farinae or human HSP70. Eg2HSP70-stimulated PBMC from 26 patients produced significantly greater amounts of TNF-,, IFN-,, and IL-10 than unstimulated cultures in all patients, irrespective of the presence of allergic reactions (P < 0·05). They also produced significantly greater amounts of IL-4 than unstimulated cultures exclusively in patients with allergic reactions (P < 0·05). These findings show that Eg2HSP70 is a new antigenic molecule inducing both B and T cell responses. [source] Expression of ADAMTS4 (aggrecanase-1) in human osteoarthritic cartilagePATHOLOGY INTERNATIONAL, Issue 11 2007Satoko Naito A disintegrin and metalloproteinase with thrombospondin motifs (ADAMTS)1, 4, 5, 8, 9 and 15, members of the ADAMTS gene family, have the ability to degrade a major cartilage proteoglycan, aggrecan, at the specific sites, and thus are called ,aggrecanases'. The expression of these ADAMTS species was examined in human osteoarthritic articular cartilage on reverse transcription,polymerase chain reaction. The results demonstrated the predominant expression of ADAMTS4 in osteoarthritic cartilage, while ADAMTS5 was constitutively expressed in osteoarthritic and normal cartilage. ADAMTS9 was expressed mainly in normal cartilage, whereas no or negligible expression of ADAMTS1, 8 and 15 was observed in either osteoarthritic or normal cartilage. In situ hybridization for ADAMTS4 indicated that chondrocytes in osteoarthritic cartilage expressed the mRNA. Two monoclonal antibodies to ADAMTS4 were developed, and immunolocalized ADAMTS4 to chondrocytes in the proteoglycan-depleted zones of osteoarthritic cartilage, showing a direct correlation with the Mankin scores. Immunoblotting indicated a major protein band of 58 kDa in the chondrocyte culture media and osteoarthritic cartilage tissue homogenates. These data demonstrate that among the six ADAMTS species, ADAMTS4 is mainly expressed in an active form in osteoarthritic cartilage, and suggest that ADAMTS4 may play an important role in the degradation of aggrecan in human osteoarthritic cartilage. [source] Activity and concentration of non-proteolyzed phosphoenolpyruvate carboxykinase in the endosperm of germinating castor oil seeds: effects of anoxia on its activityPHYSIOLOGIA PLANTARUM, Issue 4 2007Mariana Martín Phosphoenolpyruvate carboxykinase (PEPCK; EC 4.1.1.49) catalyses the reversible decarboxylation of oxaloacetate to phosphoenolpyruvate in the gluconeogenic production of sugars from storage lipids in germinating oil seeds. The enzyme is quite susceptible to limited proteolysis during extraction. Immunoblotting was used to diagnose unwanted in vitro proteolytic activity against PEPCK from germinating castor oil seeds (COS) by following the disappearance of its native 74-kDa subunit and concomitant appearance of a truncated 64-kDa polypeptide. Alkaline pH and the inclusion of thiol protease inhibitors effectively prevented COS PEPCK proteolysis during incubation of clarified COS extracts at 4°C. The carboxylating and decarboxylating activities and concentration of non-proteolyzed COS PEPCK were investigated during germination. This is the first report in which both activities catalyzed by PEPCK were measured in vitro during a whole developmental process. Carboxylating activity and the level of immunoreactive 74-kDa PEPCK polypeptides rapidly increased in parallel to maximal values by day 5 and then significantly declined over the subsequent 2 days. In contrast, decarboxylating PEPCK activity was much higher over the 7 days of growth examined. In addition, the effect on PEPCK activity while changing the endosperm from aerobic (when gluconeogenesis predominates in the tissue) to anaerobic conditions (where the tissue becomes glycolytic) was studied. While PEPCK decarboxylating activity remained almost constant, carboxylating activity declined to undetectable levels in response to anaerobiosis. These and the developmental profile results suggest that COS PEPCK may be subject to a mechanism of post-translation control that selectively inhibits the carboxylating, but not the decarboxylating activity. [source] Proteomic analysis identifies in vivo candidate matrix metalloproteinase-9 substrates in the left ventricle post-myocardial infarctionPROTEINS: STRUCTURE, FUNCTION AND BIOINFORMATICS, Issue 11 2010Rogelio Zamilpa Abstract Matrix metalloproteinase-9 (MMP-9) deletion has been shown to improve remodeling of the left ventricle post-myocardial infarction (MI), but the mechanisms to explain this improvement have not been fully elucidated. MMP-9 has a broad range of in vitro substrates, but relevant in vivo substrates are incompletely defined. Accordingly, we evaluated the infarct regions of wild-type (wt) and MMP-9 null (null) mice using a proteomic strategy. Wt and null groups showed similar infarct sizes (48±3 in wt and 45±3% in null), indicating that both groups received an equal injury stimulus. Left ventricle infarct tissue was homogenized and analyzed by 2-DE and MS. Of 31 spot intensity differences, the intensities of 9 spots were higher and 22 spots were lower in null mice compared to wt (all p<0.05). Several extracellular matrix proteins were identified in these spots by MS, including fibronectin, tenascin-C, thrombospondin-1, and laminin. Fibronectin was observed on the gels at a lower than expected molecular weight in the wt group, which suggested substrate cleavage, and the lower molecular weight spot was observed at lower intensity in the MMP-9 null group, which suggested cleavage by MMP-9. Immunoblotting confirmed the presence of fibronectin cleavage products in the wt samples and lower levels in the absence of MMP-9. In conclusion, examining infarct tissue from wt and MMP-9 null mice by proteomic analysis provides a powerful and unique method to identify in vivo candidate MMP substrates. [source] Proteomic DIGE analysis of the mitochondria-enriched fraction from aged rat skeletal musclePROTEINS: STRUCTURE, FUNCTION AND BIOINFORMATICS, Issue 24 2009Kathleen O'Connell Abstract Skeletal muscle aging is associated with a loss in tissue mass and contractile strength, as well as fiber type shifting and bioenergetic adaptation processes. Since mitochondria represent the primary site for energy generation via oxidative phosphorylation, we investigated potential changes in the expression pattern of the mitochondrial proteome using the highly sensitive DIGE approach. The comparative analysis of the mitochondria-enriched fraction from young adult versus aged muscle revealed an age-related change in abundance for 39 protein species. MS technology identified the majority of altered proteins as constituents of muscle mitochondria. An age-dependent increase was observed for NADH dehydrogenase, the mitochondrial inner membrane protein mitofilin, peroxiredoxin isoform PRX-III, ATPase synthase, succinate dehydrogenase, mitochondrial fission protein Fis1, succinate-coenzyme A ligase, acyl-coenzyme A dehydrogenase, porin isoform VDAC2, ubiquinol-cytochrome c reductase core I protein and prohibitin. Immunoblotting, enzyme testing and confocal microscopy were used to validate proteomic findings. The DIGE-identified increase in key mitochondrial elements during aging agrees with the concept that sarcopenia is associated with a shift to a slower contractile phenotype and more pronounced aerobic-oxidative metabolism. This suggests that mitochondrial markers are reliable candidates that should be included in the future establishment of a biomarker signature of skeletal muscle aging. [source] Proteomic analysis identified N-cadherin, clusterin, and HSP27 as mediators of SPARC (secreted protein, acidic and rich in cysteines) activity in melanoma cellsPROTEINS: STRUCTURE, FUNCTION AND BIOINFORMATICS, Issue 22 2007María Soledad Sosa Abstract Secreted protein, acidic and rich in cysteines (SPARC) is a secreted protein associated with increased aggressiveness of different human cancer types. In order to identify downstream mediators of SPARC activity, we performed a 2-DE proteomic analysis of human melanoma cells following antisense-mediated downregulation of SPARC expression. We found 23/504 differential spots, 15 of which were identified by peptide fingerprinting analysis. Three of the differential proteins (N-cadherin (N-CAD), clusterin (CLU), and HSP27) were validated by immunoblotting, confirming decreased levels of N-CAD and CLU and increased amounts of HSP27 in conditioned media of cells with diminished SPARC expression. Furthermore, transient knock down of SPARC expression in melanoma cells following adenoviral-mediated transfer of antisense RNA confirmed these changes. We next developed two different RNAs against SPARC that were able to inhibit in vivo melanoma cell growth. Immunoblotting of the secreted fraction of RNAi-transfected melanoma cells confirmed that downregulation of SPARC expression promoted decreased levels of N-CAD and CLU and increased secretion of HSP27. Transient re-expression of SPARC in SPARC-downregulated cells reverted extracellular N-CAD, CLU, and HSP27 to levels similar to those in the control. These results constitute the first evidence that SPARC, N-CAD, CLU, and HSP27 converge in a unique molecular network in melanoma cells. [source] Changes in the rat heart proteome induced by exercise training: Increased abundance of heat shock protein hsp20PROTEINS: STRUCTURE, FUNCTION AND BIOINFORMATICS, Issue 10 2006Marvin O. Boluyt Dr. Abstract Chronic exercise training elicits adaptations in the heart that improve pump function and confer cardioprotection. To identify molecular mechanisms by which exercise training stimulates this favorable phenotype, a proteomic approach was employed to detect rat cardiac proteins that were differentially expressed or modified after exercise training. Exercise-trained rats underwent six weeks of progressive treadmill training five days/week, 0%,grade, using an interval training protocol. Sedentary control rats were age- and weight-matched to the exercise-trained rats. Hearts were harvested at various times (0,72,h) after the last bout of exercise and were used to generate 2-D electrophoretic proteome maps and immunoblots. Compared with hearts of sedentary rats, 26,protein spot intensities were significantly altered in hypertrophied hearts of exercise-trained rats (p,<0.05), and 12,spots appeared exclusively on gels from hearts of exercise-trained rats. Immunoblotting confirmed that chronic exercise training, but not a single bout of exercise, elicited a ,2.5-fold increase in the abundance of one of the candidate proteins in the heart, a ,20,kDa heat shock protein (hsp20) that persisted for at least 72,h of detraining. Thus, exercise training alters the cardiac proteome of the rat heart; the changes include a marked increase in the expression of hsp20. [source] CO2 limitation induces specific redox-dependent protein phosphorylation in Chlamydomonas reinhardtiiPROTEINS: STRUCTURE, FUNCTION AND BIOINFORMATICS, Issue 9 2006Maria V. Turkina Abstract Acclimation of the green alga Chlamydomonas reinhardtii to limiting environmental CO2 induced specific protein phosphorylation at the surface of photosynthetic thylakoid membranes. Four phosphopeptides were identified and sequenced by nanospray quadrupole TOF MS from the cells acclimating to limiting CO2. One phosphopeptide originated from a protein that has not been annotated. We found that this unknown expressed protein (UEP) was encoded in the genome of C.,reinhardtii. Three other phosphorylated peptides belonged to Lci5 protein encoded by the low-CO2 -inducible gene,5 (lci5). The phosphorylation sites were mapped in the tandem repeats of Lci5 ensuring phosphorylation of four serine and three threonine residues in the protein. Immunoblotting with Lci5-specific antibodies revealed that Lci5 was localized in chloroplast and confined to the stromal side of the thylakoid membranes. Phosphorylation of Lci5 and UEP occurred strictly at limiting CO2; it required reduction of electron carriers in the thylakoid membrane, but was not induced by light. Both proteins were phosphorylated in the low-CO2 -exposed algal mutant deficient in the light-activated protein kinase,Stt7. Phosphorylation of previously unknown basic proteins UEP and Lci5 by specific redox-dependent protein kinase(s) in the photosynthetic membranes reveals the early response of green algae to limitation in the environmental inorganic carbon. [source] Isolation, Partial Purification, and Immunogenicity of Flagella from Tritrichomonas foetusTHE JOURNAL OF EUKARYOTIC MICROBIOLOGY, Issue 3 2005PHILIP F. JEMILOHUN Abstract. Tritrichomonas foetus, the agent of bovine trichomoniasis, is a flagellate protozoan responsible for substantial economic losses to the dairy and calf industries worldwide. As yet, there is no approved treatment nor is there a sensitive diagnostic method. All these problems suggest that immunization is the best control strategy. In view of this, we isolated and partially purified flagella of the parasite by vortex homogenization followed by low-speed differential centrifugation. The resulting enriched flagellar preparation termed "crude flagellar prep" was purified further by sucrose and percoll gradients. Microscopic analysis showed that the flagellar membrane was intact. Analysis by sodium dodecyl-sulfate polyacrylamide gel electrophoresis revealed three prominent protein bands of 42, 49, and >250 kDa, and several minor bands. Immunoblotting of flagellar and whole-cell extracts revealed many flagellar antigens. [source] Prostatic stromal cells derived from benign prostatic hyperplasia specimens possess stem cell like propertyTHE PROSTATE, Issue 12 2007Victor K. Lin Abstract INTRODUCTION The hyper-proliferative activity of stromal smooth muscle (SM) cells is believed to be responsible for the pathogenesis of benign prostatic hyperplasia (BPH). We have observed that those stromal cells can differentiate into unrelated specialized cells. We thus hypothesize that stromal cells derived from adults prostate specimens may contain adult stem cells. To test this hypothesis, human prostate stromal primary cultures were established and used for characterization of their stem cell properties. METHODS Immunoblotting, immunohistochemistry, RT-PCR, and tissue culture techniques were used to characterize the primary cultured human prostate-derived stromal cells for their stem cell and differentiation properties. The plasticity of these stromal cells was analyzed using cell culture and histology techniques. RESULTS Primary cultured prostate stromal cells from BPH patient possess polygonal and elongated fibroblast/myofibroblast cellular morphology. They are positive in CD30, CD34, CD44, NSE, CD133, Flt-1, stem cell factor (SCF), and neuron-specific enolase (NSE), but negative in C-Kit, stem cell antigen (SCA), SH2, CD11b. Expression of SM myogenic markers in these cells may be induced by sodium butyrate (NaBu) treatment. Induction to osteogenic and adipogenic differentiation in these cells is also evident. CONCLUSIONS Our study on primary stromal cells from BPH patients have yielded many interesting findings that these prostate stroma cells possess: (1) mesenchymal stem cell (MSC) markers; (2) strong proliferative potential; and (3) ability to differentiate or transdifferentiate to myogenic, adipogenic, and osteogenic lineages. These cell preparations may serve as a potential tool for studies in prostate adult stem cell research and the regulation of benign prostatic hyperplasia. Prostate 67: 1265,1276, 2007. © 2007 Wiley-Liss, Inc. [source] Basement membrane laminin-5 is deposited in colorectal adenomas and carcinomas and serves as a ligand for ,3,1 integrinAPMIS, Issue 3 2000Jouni Lohi Interplay between laminin-5 (Ln-5) and its integrin (Int) receptors ,2,1, ,3,1 and ,6,4 has been implicated in the progression and invasion of carcinomas. In this study we found abundant immuno-reactivity for chains of Ln-5 (,3-,3-,2) and Ln-10 (,5-,1), as well as for type VII collagen, in basement membranes (BM) of colorectal adenomas. In carcinomas of all differentiation grades, Lns were seen in tumor BMs, whereas type VII collagen was almost absent. Ln-5 appeared to accumulate along the invading edges of carcinomas, while Ln-10 was mostly absent. Immunoreactivity for Ln ,1 chain, a component of Lns-1 and -3, was not seen in adenomas or carcinomas. Immunoreactivity for ,2, ,6, ,1 and ,4 Ints was found in all tumors and that for ,3 Int in all adenomas and most of the carcinomas, often in colocalization with Ln-5. Immunoblotting of carcinoma tissues showed that the ,2 chain of Ln-5 was present as typical Mr 105000 and 155000 isoforms. Immunoprecipitation experiments showed production of Ln-5 by cultured colon carcinoma cells. In quantitative cell adhesion experiments, function-blocking MAbs to ,3 and ,1 Int subunits, but not those to Int ,2 or ,6 subunits, significantly inhibited the adhesion of cells to Ln-5. Our results suggest that BM composition in colorectal adenomas reflects the properties of surface epithelial BM of colorectal mucosa. In invading carcinomas, trimeric Ln-5, produced by carcinoma cells, is a major BM component and the cells use the ,3,1 Int complex for adhesion to Ln-5. [source] Characterization of the trypsin-like protease (Ha-TLP2) constitutively expressed in the integument of the cotton bollworm, Helicoverpa armigera,ARCHIVES OF INSECT BIOCHEMISTRY AND PHYSIOLOGY (ELECTRONIC), Issue 2 2009Yang Liu Abstract Trypsins belong to the serine endoproteases. They are the most important proteases in insects because of their key roles in food digestion and zymogens activation. But there has been little study of the trypsins in the integuments of insects. In this work, we cloned a trypsin-like protease gene from Helicoverpa armigera and named it trypsin-like protease 2 (Ha-TLP2). Semi-quantitative reverse transcription PCR analysis showed that Ha-TLP2 is constitutively expressed in the integument and can be down-regulated by 20-hydroxyecdysone (20E) and up-regulated by the juvenile hormone (JH) analog methoprene. Immunohistochemistry showed that Ha-TLP2 is located not only in the epidermis, but also in new and old cuticles. Immunoblotting and gelatin-SDS-PAGE revealed that Ha-TLP2 is constitutively expressed with activity in the integument during larval feeding, molting, and metamorphosis. This evidence suggests that Ha-TLP2 is involved in the remodeling of the integument. © 2009 Wiley Periodicals, Inc. [source] Interleukin-7 stimulates secretion of S100A4 by activating the JAK/STAT signaling pathway in human articular chondrocytesARTHRITIS & RHEUMATISM, Issue 3 2009Raghunatha R. Yammani Objective S100A4 has been shown to be increased in osteoarthritic (OA) cartilage and to stimulate chondrocytes to produce matrix metalloproteinase 13 (MMP-13) through activation of the receptor for advanced glycation end products (RAGE). The aim of this study was to examine the mechanism of S100A4 secretion by chondrocytes. Methods Human articular chondrocytes isolated from ankle cartilage were stimulated with 10 ng/ml of interleukin-1, (IL-1,), IL-6, IL-7, or IL-8. Cells were pretreated with either a JAK-3 inhibitor, brefeldin A, or cycloheximide. Immunoblotting with phospho-specific antibodies was used to determine the activation of signaling proteins. Secretion of S100A4 was measured in conditioned media by immunoblotting, and MMP-13 was measured by enzyme-linked immunosorbent assay. Results Chondrocyte secretion of S100A4 was observed after treatment with IL-6 or IL-8 but was much greater in cultures treated with equal amounts of IL-7 and was not observed after treatment with IL-1,. IL-7 activated the JAK/STAT pathway, with increased phosphorylation of JAK-3 and STAT-3, leading to increased production of S100A4 and MMP-13. Overexpression of a dominant-negative RAGE construct inhibited the IL-7,mediated production of MMP-13. Pretreatment of chondrocytes with a JAK-3 inhibitor or with cycloheximide blocked the IL-7,mediated secretion of S100A4, but pretreatment with brefeldin A did not. Conclusion IL-7 stimulates chondrocyte secretion of S100A4 via activation of JAK/STAT signaling, and then S100A4 acts in an autocrine manner to stimulate MMP-13 production via RAGE. Since both IL-7 and S100A4 are up-regulated in OA cartilage and can stimulate MMP-13 production by chondrocytes, this signaling pathway could contribute to cartilage destruction during the development of OA. [source] | |||||||||||||||||||||||||||||||||||||