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Western Blotting (western + blotting)
Kinds of Western Blotting Terms modified by Western Blotting Selected AbstractsChronic administration of valproic acid inhibits PC3 cell growth by suppressing tumor angiogenesis in vivoINTERNATIONAL JOURNAL OF UROLOGY, Issue 9 2007Dexuan Gao Aim: Chromatin remodeling agents such as histone deacetylase inhibitors have been shown to modulate gene expression in tumor cells and inhibit tumor growth and angiogenesis. We investigated the mechanisms of chronic valproic acid (VPA) inhibiting PC3 cell growth in the study. Methods: We established tumor xenografts of the PC3 cell line and investigated the effect of VPA chronic administration on tumor growth. Apoptosis in tumor tissue was measured using the TUNEL Detection Kit. We detected the effect of VPA chronic administration on histone acetylation; p21CIP1/WAF1 gene expression; vascular endothelial growth factor (VEGF) expression by reverse-transcription Polymerase Chain Reaction (PCR) analysis; immunohistochemistry; and Western Blotting. Result: In mouse models with established subcutaneous prostate (PC3), VPA treatment induced 70% inhibition of tumor growth without overt toxicity. Our result showed that chronic administration of VPA has an effect on tumor growth arrest and the effect was associated with increased histone acetylation, p21CIP1/WAF1 up-regulation, and VEGF down-regulation. Conclusion: We conclude that chronic VPA results in profound decreases in the proliferation of PC3 cells, not only by increasing histone H3 acetylation and up-regulating p21CIP1/WAF1 expression, but also by down-regulating VEGF. [source] The protective action of scutellarin against immunological liver injury induced by concanavalin A and its effect on pro-inflammatory cytokines in miceJOURNAL OF PHARMACY AND PHARMACOLOGY: AN INTERNATI ONAL JOURNAL OF PHARMACEUTICAL SCIENCE, Issue 1 2007Zheng Huai Tan Scutellarin is a natural compound from a Chinese herb. The purpose of this paper was to study the protective effect of scutellarin on concanavalin A (Con A)-induced immunological liver injury and its effect on liver nuclear factor ,B (NF-,B), tumor necrosis factor , (TNF-,), interferon , (IFN-,), and inducible nitric oxide synthase (iNOS) expression in mice. Mouse liver injury was produced by injection of Con A 25 mg kg,1 via the tail vein. Scutellarin 50 or 100 mg kg,1 was peritoneally administered to mice 9 or 1 h before injection of Con A. The levels of serum alanine aminotransferase (ALT) and asparatate aminotransferase (AST), NO2,/NO3, and TNF - , were determined with biochemical kits, and ELISA using Quantikine Mouse TNF-, kit according the manufacturer's instructions. Liver lesions were examined by light microscope. The expression of TNF-,, IFN-,, iNOS and Fas mRNA in the livers was detected by RT-PCR; and the expression of c-Fos, c-Jun, iNOS and I,B proteins was measured by Western Blotting. As a result, pretreatment with scutellarin 100 mg kg,1 significantly decreased the serum ALT, AST, NO2,/NO3,and TNF-, levels, and also reduced liver lesions induced by Con A. Scutellarin 100 mg kg,1 down-regulated expression of TNF-, and iNOS mRNA, and c-Fos, c-Jun and iNOS protein, while scutellarin enhanced the degradation of I,B, in the livers of mice injected with Con A. The results suggest that scutellarin has a protective action against Con A-induced liver injury in mice, and its active mechanism may be related to the inhibition of the NF-,B-TNF-,-iNOS transduction pathway. [source] OCTN2 is associated with carnitine transport capacity of rat skeletal musclesACTA PHYSIOLOGICA, Issue 1 2010Y. Furuichi Abstract Aim:, Carnitine plays an essential role in fat oxidation in skeletal muscles; therefore carnitine influx could be crucial for muscle metabolism. OCTN2, a sodium-dependent solute carrier, is assumed to transport carnitine into various organs. However, OCTN2 protein expression and the functional importance of carnitine transport for muscle metabolism have not been studied. We tested the hypothesis that OCTN2 is expressed at higher levels in oxidative muscles than in other muscles, and that the carnitine uptake capacity of skeletal muscles depends on the amount of OCTN2. Methods:, Rat hindlimb muscles (soleus, plantaris, and the surface and deep portions of gastrocnemius) were used for Western blotting to detect OCTN2. Tissue carnitine uptake was examined by an integration plot analysis using l -[3H]carnitine as a tracer. Tissue carnitine content was determined by enzymatic cycling methods. The percentage of type I fibres was determined by histochemical analysis. Results:, OCTN2 was detected in all skeletal muscles although the amount was lower than that in the kidney. OCTN2 expression was significantly higher in soleus than in the other skeletal muscles. The amount of OCTN2 was positively correlated with the percentage of type I fibres in hindlimb muscles. The integration plot analysis revealed a positive correlation between the uptake clearance of l -[3H]carnitine and the amount of OCTN2 in skeletal muscles. However, the carnitine content in soleus was lower than that in other skeletal muscles. Conclusion:, OCTN2 is functionally expressed in skeletal muscles and is involved in the import of carnitine for fatty acid oxidation, especially in highly oxidative muscles. [source] Akt2/PKB,-sensitive regulation of renal phosphate transportACTA PHYSIOLOGICA, Issue 1 2010D. S. Kempe Abstract Aim:, The protein kinase B (PKB)/Akt is known to stimulate the cellular uptake of glucose and amino acids. The kinase is expressed in proximal renal tubules. The present study explored the influence of Akt/PKB on renal tubular phosphate transport. Methods:, The renal phosphate transporter NaPi-IIa was expressed in Xenopus oocytes with or without PKB/Akt and Na+ phosphate cotransport determined using dual electrode voltage clamp. Renal phosphate excretion was determined in Akt2/PKB, knockout mice (akt2,/,) and corresponding wild-type mice (akt2+/+). Transporter protein abundance was determined using Western blotting and phosphate transport by 32P uptake into brush border membrane vesicles. Results:, The phosphate-induced current in NaPi-IIa-expressing Xenopus oocytes was significantly increased by the coexpression of Akt/PKB. Phosphate excretion [,mol per 24 h per g BW] was higher by 91% in akt2,/, than in akt2+/+ mice. The phosphaturia of akt2,/, mice occurred despite normal transport activity and expression of the renal phosphate transporters NaPi-IIa, NaPi-IIc and Pit2 in the brush border membrane, a significantly decreased plasma PTH concentration (by 46%) and a significantly enhanced plasma 1,25-dihydroxyvitamin D3 concentration (by 46%). Moreover, fractional renal Ca2+ excretion was significantly enhanced (by 53%) and bone density significantly reduced (by 11%) in akt2,/, mice. Conclusions:, Akt2/PKB, plays a role in the acute regulation of renal phosphate transport and thus contributes to the maintenance of phosphate balance and adequate mineralization of bone. [source] TRAF6 knockdown promotes survival and inhibits inflammatory response to lipopolysaccharides in rat primary renal proximal tubule cellsACTA PHYSIOLOGICA, Issue 3 2010S. Liu Abstract Aim:, TRAF6 is a unique adaptor protein of the tumour necrosis factor receptor-associated factor family that mediates both tumour necrosis factor receptor (TNFR) and interleukin-1 receptor/Toll-like receptor (IL-1R/TLR) signalling. Activation of IL-1R/TLR and TNFR pathways in renal tubular cells contributes to renal injury. This study aimed to investigate if blockade of lipopolysaccharide (LPS)-triggered TLR4 signalling by small interfering RNA (siRNA) targeting TRAF6 protects survival and inhibits inflammatory response in isolated rat renal proximal tubular cells (PTCs). Methods:, PTCs isolated from F344 rat kidneys were transfected with chemically synthesized siRNA targeting TRAF6 mRNA. Real-time quantitative PCR was applied to measure mRNA level of TRAF6, TNF-,, IL-6 and monocyte chemoattractant protein-1 (MCP-1). Protein levels of extracellular signal-regulated kinase (ERK), c-jun N-terminal kinase (JNK), p38 mitogen-activated protein kinase, caspase 3 and cleaved caspase 3 were evaluated by Western blotting. Cell viability was analysed with XTT reagents. Results:, We found that the TRAF6 gene was effectively silenced in PTCs using siRNA. TRAF6 knockdown resulted in reduced TNF-, and IL-6 mRNA expression upon LPS challenge. LPS-induced phosphorylation of JNK and p38 was attenuated in TRAF6 siRNA-transfected cells while the change in the phosphorylation of ERK was not remarkable. TRAF6 knockdown was associated with increased cell viability and reduced protein level of cleaved caspase-3, both, in the absence and presence of LPS. Conclusion:, Our studies suggest that TRAF6 knockdown may inhibit inflammatory response and promote cell survival upon LPS challenge in primary rat proximal renal tubular cells. [source] Nucleotides and epidermal growth factor induce parallel cytoskeletal rearrangements and migration in cultured adult murine neural stem cellsACTA PHYSIOLOGICA, Issue 2 2010I. Grimm Abstract Aim:, The adult subventricular zone (SVZ) contains neural stem cells that generate neuroblasts migrating to the olfactory bulb (OB) and differentiating into interneurones. The molecular cues controlling essential functions within the neurogenesis pathway such as proliferation, short and long distance migration, functional integration and cell survival are poorly understood. We have previously shown that cultured adult neural stem cells express a considerable variety of nucleotide receptors and that nucleotides and epidermal growth factor (EGF) induce converging intracellular signalling pathways that carry potential for synergism in the control of neural stem cell proliferation and cell survival. Here we investigate the role of EGF and the nucleotides ATP, ADP,S and UTP in neural stem cell migration. Methods:, Neural stem cells were prepared from adult mice and subjected to adherent culture. Labelling of F-actin was performed with tetramethylrhodamine isothiocyanate-phalloidin. Images were processed for quantitative evaluation of fluorescence labelling. Agonist-induced phosphorylation of AKT and focal adhesion kinase was analysed by quantitative Western blotting. Agonist-dependent cell migration was assayed using 48-well microchemotaxis chambers. Results:, Nucleotides and EGF induce the formation of stress fibres, an increase in the cortical actin cytoskeleton and in cell spreading. This is associated with increased phosphorylation of AKT and focal adhesion kinase. Using microchemotaxis chambers we demonstrate a parallel increase in cell migration. Conclusion:, Our results suggest that nucleotides and EGF acting as paracrine or autocrine signalling substances can be of relevance for structuring and maintaining the cytoarchitecture of the SVZ and the stream of neuroblasts migrating to the OB. [source] Characterization of sleep,wake patterns in a novel transgenic mouse line overexpressing human prepro-orexin/hypocretinACTA PHYSIOLOGICA, Issue 3 2010K. A. Mäkelä Abstract Aim:, Orexin/hypocretin peptides are expressed in the lateral hypothalamus and involved in the regulation of autonomic functions, energy homeostasis and arousal states. The sleep disorder narcolepsy, which is characterized by excessive daytime sleepiness and occurrence of sudden rapid eye movement (REM) sleep, is associated with a loss of orexin neurones. Our study investigated the effects of orexins on sleep,wake patterns in a novel transgenic mouse line overexpressing the human prepro-orexin (hPPO) gene under the control of its endogenous promoter. Methods:, Orexin overexpression was investigated by PCR, Southern and Western blotting as well as immunohistochemistry. Polysomnographic recordings were performed for analyses of sleep,wake patterns and for electroencephalographic activity during 24 h baseline and during and after 6 h of sleep deprivation (SD). Results:, Transgenic hPPO mice had increased expression of human prepro-orexin (hPPO) and orexin-A in the hypothalamus. Transgene expression decreased endogenous orexin-2 receptors but not orexin-1 receptors in the hypothalamus without affecting orexin receptor levels in the basal forebrain, cortex or hippocampus. Transgenic mice compared with their wild type littermates showed small but significant differences in the amount of waking and slow wave sleep, particularly during the light,dark transition periods, in addition to a slight reduction in REM sleep during baseline and during recovery sleep after SD. Conclusion:, The hPPO-overexpressing mice show a small reduction in REM sleep, in addition to differences in vigilance state amounts in the light/dark transition periods, but overall the sleep,wake patterns of hPPO-overexpressing mice do not significantly differ from their wild type littermates. [source] Cardioprotection of bradykinin at reperfusion involves transactivation of the epidermal growth factor receptor via matrix metalloproteinase-8ACTA PHYSIOLOGICA, Issue 4 2009C. Methner Abstract Aim:, The endogenous autacoid bradykinin (BK) reportedly reduces myocardial infarct size when given exogenously at reperfusion. Muscarinic and opioid G-protein-coupled receptors are equally protective and have been shown to couple through a matrix metalloproteinase (MMP)-dependent transactivation of the epidermal growth factor receptor (EGFR). Here we test whether BK protects the rat heart through the EGFR by an MMP-dependent pathway. Methods:, Infarct size was measured in isolated perfused rat hearts undergoing 30 min regional ischaemia followed by 120 min reperfusion. In additional studies HL-1 cardiomyocytes were loaded with tetramethylrhodamine ethyl to measure their mitochondrial membrane potential (,m). Adding the calcium ionophore calcimycin, causes ,m-collapse presumably due to calcium-induced mitochondrial permeability transition. Results:, As expected, BK (100 nmol L,1) started 5 min prior to reperfusion reduced infarct size from 38.9 ± 2.0% of the ischaemic zone in control hearts to 22.2 ± 3.3% (P < 0.001). Co-infusing the EGFR inhibitor AG1478, the broad-spectrum MMP-inhibitor GM6001, or a highly selective MMP-8 inhibitor abolished BK's protection, thus suggesting an MMP-8-dependent EGFR transactivation in the signalling. Eighty minutes of exposure to calcimycin reduced the mean cell fluorescence to 37.4 ± 1.8% of untreated cells while BK could partly preserve the fluorescence and, hence, protect the cells (50.5 ± 2.3%, P < 0.001). The BK-induced mitochondrial protection could again be blocked by AG1478, GM6001 and MMP-8 inhibitor. Finally, Western blotting revealed that BK's protection was correlated with increased phosphorylation of EGFR and its downstream target Akt. Conclusion:, These results indicate that BK at reperfusion triggers its protective signalling pathway through MMP-8-dependent transactivation of the EGFR. [source] Muscle type-specific response of PGC-1, and oxidative enzymes during voluntary wheel running in mouse skeletal muscleACTA PHYSIOLOGICA, Issue 3-4 2006S. Ikeda Abstract Aim:, It is generally accepted that endurance exercise increases the expression of peroxisome proliferator-activated receptor , coactivator-1, (PGC-1,), which governs the expression of oxidative metabolic enzymes. A previous report demonstrated that the regulation of mitochondrial protein expression in skeletal muscles in response to cold exposure depends on muscle fibre type. Cold exposure and endurance exercise are both metabolic challenges that require adjustments in mitochondrial energy metabolism, we hypothesized that the exercise-induced increase in oxidative enzymes and PGC-1, expression is higher in fast-type than in slow-type muscle. Methods:, Female ICR mice were individually housed in cages equipped with running wheel for 1, 2, 4, 6 or 8 weeks. The soleus, plantaris (PLA) and tibialis anterior (TA) muscles were then prepared from each mouse. The expression levels of PGC-1,, mitochondrial proteins and GLUT4 were evaluated by Western blotting. Results:, The expression level of PGC-1, was increased only in the PLA muscle. Furthermore, the expression levels of all mitochondrial proteins and GLUT4 in the PLA muscle were increased. In the TA muscle, although there was no increase in PGC-1, expression, the expression levels of mitochondrial proteins and GLUT4 were increased. Conclusions:, These results suggest that muscle type-specific responses occur during endurance exercise, and that the increase in PGC-1, expression is not the only factor that promotes oxidative capacity as a result of endurance exercise. [source] Expression of Na+/HCO3, co-transporter proteins (NBCs) in rat and human skeletal muscleACTA PHYSIOLOGICA, Issue 1 2004J. M. Kristensen Abstract Aim:, Sodium/bicarbonate co-transport (NBC) has been suggested to have a role in muscle pH regulation. We investigated the presence of NBC proteins in rat and human muscle samples and the fibre type distribution of the identified NBCs. Methods and results:, Western blotting of muscle homogenates and sarcolemmal membranes (sarcolemmal giant vesicles) were used to screen for the presence of NBCs. Immunohistochemistry was used for the subcellular localization. The functional test revealed that approximately half of the pH recovery in sarcolemmal vesicles produced from rat muscle is mediated by bicarbonate-dependent transport. This indicates that the NBCs are preserved in the vesicles. The western blotting experiments demonstrated the existence of at least two NBC proteins in skeletal muscle. One NBC protein (approximately 150 kDa) seems to be related to the kidney/pancreas/heart isoform NBC1, whereas the other protein (approximately 200 kDa) is related to the NBC4 isoform. The two NBC proteins represent the electrogenic isoforms named NBCe1 and NBCe2. Membrane fractionation and immunofluorescence techniques confirmed that the two NBCs are located in the sarcolemmal membrane as well as in some internal membranes, probably the T-tubules. The two NBCs localized in muscle have distinct fibre type distributions. Conclusions:, Skeletal muscle possesses two variants of the sodium/bicarbonate co-transporter (NBC) isoforms, which have been called NBCe1 and NBCe2. [source] MAP65 in tubulin/colchicine paracrystals of Vigna sinensis root cells: Possible role in the assembly and stabilization of atypical tubulin polymers,CYTOSKELETON, Issue 3 2010Emmanuel Panteris Abstract Members of the MAP65 family, colocalizing with microtubule arrays, have been identified in Vigna sinensis root cells by Western blotting and immunofluorescence. MAP65 proteins were also found in tubulin/colchicine paracrystals, which were formed during colchicine treatment by both immunofluorescence and immunogold microscopy. During recovery from colchicine, MAP65 signal was depleted from disintegrating paracrystals appearing in the reinstating microtubule arrays. MAP65-free perinuclear tubulin/colchicine aggregates were observed in plasmolyzed colchicine-treated cells. Deplasmolysis of the above cells resulted in the formation of MAP65-decorated paracrystals. As confirmed by appropriate biochemical assays with the Phos-tag reagent, MAP65 proteins underwent phosphorylation during plasmolysis, which was reversible by deplasmolysis. According to the effect of the mitogen-activated protein kinase (MAPK) inhibitor UO126, the phosphorylation status of MAP65, as well as its presence in tubulin/colchicine polymers is probably controlled by MAPK-mediated phosphorylation. According to the above, it seems likely that apart from binding to microtubules, MAP65 proteins may act as "tubulin associated proteins" in a broader manner, promoting the polymerization and/or stabilization of atypical polymers such as tubulin/colchicine paracrystals. © 2010 Wiley-Liss, Inc. [source] Maternal hypoxia increases the activity of MMPs and decreases the expression of TIMPs in the brain of neonatal ratsDEVELOPMENTAL NEUROBIOLOGY, Issue 3 2010Wenni Tong Abstract A recent study has shown that increased activity of matrix metalloproteinases-2 and metalloproteinases-9 (MMP-2 and MMP-9) has detrimental effect on the brain after neonatal hypoxia. The present study determined the effect of maternal hypoxia on neuronal survivability and the activity of MMP-2 and MMP-9, as well as the expression of tissue inhibitors of metalloproteinase 1 and 2 (TIMP-1 and TIMP-2) in the brain of neonatal rats. Pregnant rats were exposed to 10.5% oxygen for 6 days from the gestation day 15 to day 21. Pups were sacrificed at day 0, 4, 7, 14, and 21 after birth. Body weight and brain weight of the pups were measured at each time point. The activity of MMP-2 and MMP-9 and the protein abundance of TIMP-1 and TIMP-2 were determined by zymography and Western blotting, respectively. The tissue distribution of MMPs was examined by immunofluorescence staining. The neuronal death was detected by Nissl staining. Maternal hypoxia caused significant decreases in body and brain size, increased activity of MMP-2 at day 0, and increased MMP-9 at day 0 and 4. The increased activity of the MMPs was accompanied by an overall tendency towards a reduced expression of TIMPs at all ages with the significance observed for TIMPs at day 0, 4, and 7. Immunofluorescence analysis showed an increased expression of MMP-2, MMP-9 in the hippocampus at day 0 and 4. Nissl staining revealed significant cell death in the hippocampus at day 0, 4, and 7. Functional tests showed worse neurobehavioral outcomes in the hypoxic animals. © 2009 Wiley Periodicals, Inc. Develop Neurobiol 2010 [source] Splice-isoform specific immunolocalization of neuronal nitric oxide synthase in mouse and rat brain reveals that the PDZ-complex-building nNOS, ,-finger is largely exposed to antibodiesDEVELOPMENTAL NEUROBIOLOGY, Issue 4 2007Kristina Langnaese Abstract Knock out mice deficient for the splice-isoform ,, of neuronal nitric oxide synthase (nNOS,,) display residual nitric oxide synthase activity and immunosignal. To attribute this signal to the two minor neuronal nitric oxide synthase splice variants, ,, and ,,, we generated isoform-specific anti-peptide antibodies against the nNOS,, specific ,,-finger motif involved in PDZ domain scaffolding and the nNOS,, specific N-terminus. The nNOS,, ,,-finger-specific antibody clearly recognized the 160-kDa band of recombinant nNOS,, on Western blots. Using immunocytochemistry, this antibody displayed, in rats and wild-type mice, a labeling pattern similar to but not identical with that obtained using a commercial pan-nNOS antibody. This similarity indicates that the majority of immunocytochemically detectable nNOS is not likely to be complexed with PDZ-domain proteins via the ,,-finger motif. This conclusion was confirmed by the inhibition of PSD-95/nNOS interaction by the nNOS,, ,,-finger antibody in pull-down assays. By contrast, nNOS,, ,,-finger labeling was clearly reduced in hippocampal and cortical neuropil areas enriched in NMDA receptor complex containing spine synapses. In nNOS,, knock out mice, nNOS,, was not detectable, whereas the pan-nNOS antibody showed a distinct labeling of cell bodies throughout the brain, most likely reflecting ,,/,,-isoforms in these cells. The nNOS,, antibody clearly detected bacterial expressed nNOS,, fusion protein and nNOS,, in overexpressing HEK cells by Western blotting. Immunocytochemically, individual cell bodies in striatum, cerebral cortex, and in some brain stem nuclei were labeled in knock out but not in wild-type mice, indicating an upregulation of nNOS,, in nNOS,, deficient animals. © 2007 Wiley Periodicals, Inc. Develop Neurobiol, 2007 [source] In vivo analysis reveals different apoptotic pathways in pre- and postmigratory cerebellar granule cells of rabbitDEVELOPMENTAL NEUROBIOLOGY, Issue 4 2004Laura Lossi Abstract Naturally occurring neuronal death (NOND) has been described in the postnatal cerebellum of several species, mainly affecting the cerebellar granule cells (CGCs) by an apoptotic mechanism. However, little is known about the cellular pathway(s) of CGC apoptosis in vivo. By immunocytochemistry, in situ detection of fragmented DNA, electron microscopy, and Western blotting, we demonstrate here the existence of two different molecular mechanisms of apoptosis in the rabbit postnatal cerebellum. These two mechanisms affect CGCs at different stages of their maturation and migration. In the external granular layer, premigratory CGCs undergo apoptosis upon phosphorylation of checkpoint kinase 1 (Chk1), and hyperphosphorylation of retinoblastoma protein. In postmigratory CGCs within the internal granular layer, caspase 3 and to a lesser extent 7 and 9 are activated, eventually leading to poly-ADP-ribose polymerase-1 (PARP-1) cleavage and programmed cell death. We conclude that NOND of premigratory CGCs is linked to activation of DNA checkpoint and alteration of normal cell cycle, whereas in postmigratory CGCs apoptosis is, more classically, dependent upon caspase 3 activation. © 2004 Wiley Periodicals, Inc. J Neurobiol 60: 437,452, 2004 [source] Transforming growth factor-beta 1, 2, 3 and receptor type I and II in diabetic foot ulcersDIABETIC MEDICINE, Issue 6 2002E. B. Jude Abstract Aims To study the distribution of transforming growth factor-beta (TGF-,) 1, 2 and 3, and TGF-, receptor types I and II in diabetic foot ulcers, diabetic skin and normal skin by immunohistochemistry, immunofluorescence and Western blotting. We also compared the TGF-,s with those of chronic venous ulcers. Methods Skin biopsies were obtained from the leg or the foot of non-diabetic and diabetic subjects, and from the edge of diabetic foot ulcers and chronic venous ulcers. Distribution (by immunofluorescence and immunocytochemistry) of TGF-, 1, 2 and 3 and TGF-, receptors (RI and RII) was done by staining 8-µm skin sections using appropriate antibodies. Protein levels of TGF-, were measured by Western blot analysis. Results TGF-,3 expression was increased in the epithelium at the edge of diabetic foot ulcers, being more intense than diabetic and normal skin (P = 0.03, 0.02, respectively), as was its expression in venous ulcers compared with normal skin. However, TGF-,1 expression was not increased in diabetic foot ulcers and chronic venous ulcers, and was comparable to diabetic and normal skin. There was also no increase for the receptors in diabetic foot ulcers. Conclusion The lack of TGF-,1 up-regulation in both diabetic foot ulcers and venous ulcers may explain the impaired healing in these chronic wounds, and could represent a general pattern for chronicity. [source] Protective effect of curcumin, a Curcuma longa constituent, in early colonic inflammation in rats,DRUG DEVELOPMENT RESEARCH, Issue 6 2009Juan Manuel Sįnchez-Calvo Abstract Curcumin, a polyphenol derived from the plant, Curcuma longa, has a variety of pharmacological effects, including chemotherapeutic, anti-inflammatory, antiangiogenic, and antioxidant activities. To gain a better understanding of the effects and mechanisms of action of curcumin on the acute injury caused by intra-colonic administration of acetic acid (AA) in rats, inflammation was assessed by histology and myeloperoxidase activity (MPO; an index of neutrophil infiltration in the mucosa); Th1 and Th2 cytokine production; histological and histochemical analysis of the lesions; nitrite production in colon mucosa; and the expression of iNOS, COX-1 and -2 using Western blotting and inmmunohistochemistry. We also studied the involvement of the p38 MAPK/JNK signalling pathway in the protective effect of curcumin in acute colonic inflammation. Curcumin (50,100,mg/kg/day) reduced the degree of colonic injury, the index of neutrophil infiltration and Th1 cytokine secretion, and increased IL-10 production, reduced colonic levels of nitrites, and reduced COX-2 and iNOS overexpression. A reduction in the activation of p38 and JNK MAPKs was also observed. Thus, we show that the widely used food additive, curcumin reduced the development of AA-induced colitis and alleviated the inflammatory response. Inhibition of MAPK signalling by curcumin could explain the changes on the cytokine Th1/Th2 profile, the reduction of COX-2 and iNOS signaling, as well as the decreased nitrite production in colonic mucosa, suggesting that curcumin may be useful in the treatment of ulcerative colitis. Drug Dev Res, 2009. © 2009 Wiley-Liss, Inc. [source] Focused proteomics: Monoclonal antibody-based isolation of the oxidative phosphorylation machinery and detection of phosphoproteins using a fluorescent phosphoprotein gel stainELECTROPHORESIS, Issue 15 2004James Murray Abstract We have raised monoclonal antibodies capable of immunocapturing all five complexes involved in oxidative phosphorylation for evaluating their post-translational modifications. Complex I (NADH dehydrogenase), complex II (succinate dehydrogenase), complex III (cytochrome c reductase), complex IV (cytochrome c oxidase), and complex V (F1F0 ATP synthase) from bovine heart mitochondria were obtained in good yield from small amounts of tissue in more than 90% purity in one step. The composition and purity of the complexes was evaluated by Western blotting using monoclonal antibodies against individual subunits of the five complexes. In this first study, the phosphorylation state of the proteins without inducing phosphorylation or dephosphorylation was identified by using the novel Pro-Q Diamond phosphoprotein gel stain. The major phosphorylated components were the same as described before in sucrose gradient enriched complexes. In addition a few additional potential phosphoproteins were observed. Since the described monoclonal antibodies show cross reactivity to human proteins, this procedure will be a fast and efficient way of studying post-translational modifications in control and patient samples using only small amounts of tissue. [source] Cross-reactivity of antibodies to actin- depolymerizing factor/cofilin family proteins and identification of the major epitope recognized by a mammalian actin-depolymerizing factor/cofilin antibodyELECTROPHORESIS, Issue 15 2004Alisa E. Shaw Abstract Members of the actin-depolymerizing factor (ADF)/cofilin family of proteins are expressed in all eukaryotic cells. In higher vertebrates, cells often express as many as three different ADF/cofilin genes and each of these proteins may be phosphorylated on serine 3, giving rise to up to six different species. Also, many avian, amphibian, and invertebrate systems have been useful in studying different aspects of ADF/cofilin function. Antibodies have been prepared against different members of the ADF/cofilin family, but no systematic examination of their cross-reactivity has been reported. Although ADF and cofilins within a single vertebrate species have about a 70% sequence homology, antibodies often differentiate between these proteins. Here, Western blotting was used with chemiluminescence substrates of different sensitivities to determine the relative immunoreactivities of different polyclonal rabbit antibodies and a mouse monoclonal antibody to purified ADF/cofilins from plants, protists, nematodes, insects, echinoderms, birds, and mammals. From immunocross-reactivities and sequence alignments, the principal epitope in mammalian ADF and cofilin-1 recognized by an antibody raised against avian ADF was identified. The specificity of an antibody to the phosphopeptide epitope of metazoan ADF/cofilins was confirmed by two-dimensional (2-D) immunoblot analysis. Futhermore, this bank of antibodies was used to identify by Western blotting a putative member of the ADF/cofilin family in the sea slug, Aplysia californica. [source] Postendocytic Provitellin Processing in the Growing Oocyte of the Short Horned Grasshopper, Oxyajaponicajaponica (Orthoptera: Acrididae)ENTOMOLOGICAL RESEARCH, Issue 1 2004Sae Youll CHO ABSTRACT Polyclonal antibodies made against 86 kDa (86 k), 80 kDa (80 k) and 54 kDa (54 k) vitellins of Oxya japonica japonica are used for Western blotting. Anti-80k vitellin antibody is cross-reacted with a 95 kDa (95 k) vitellin. While 95 k vitellin is present both in the female hemolymph and in the oocyte, 80 k vitellin is detected only in the oocyte and the laid egg. In the growing oocytes, as 95 k vitellin is faded out gradually, 80 k vitellin is accumulated increasingly, indicating postendocytic processing of 95 k vitellin brings 80 k vitellin. Further conforming the hypothesis, partial digestion of 95 k vitellin with pepsin and ,-chymotrypsin makes several protein bands of molecular weight around 80 kDa. Thus, the 95k vitellin may have a cleavage site (s) to produce 80 k vitellin which forms fairly stable tertiary structure. In the reduced condition (20 mM glutathion), both 95 k and 80 k vitellins were digested throughly by endogenous proteinase at pH 4. Both 86 k and 54 k vitellins, respectively, show no apparent molecular weight changes in the growing oocyte and in the hemolymph. [source] Interaction between halogenated aromatic compounds in the Ah receptor signal transduction pathwayENVIRONMENTAL TOXICOLOGY, Issue 5 2004Guosheng Chen Abstract Many toxic and biochemical responses to halogenated aromatic compounds (HACs) such as polychlorinated biphenyls (PCBs) and polychlorinated dibenzo- p -dioxins (PCDDs) are mediated through the aryl hydrocarbon receptor (AhR), which is an intracellular cytosolic target for HACs. Environmental exposure to HACs almost always involves complex mixtures of congeners, some of which can antagonize the action of potent HACs such as 2,3,7,8-tetrachlorodibenzo- p -dioxin (TCDD). In this work we studied TCDD and representative PCB congeners, alone and in mixture, for their effect on CYP1A gene transcription and protein levels in primary rat hepatocytes. Together with our previous work, our results suggest that formation of the Ah receptor-ligand-DRE (dioxin response element) complex is the principal point of divergence in the mechanism between an AhR agonist and an AhR antagonist. The coplanar PCBs 77 and 126 and the mono- ortho PCB 156 were full agonists toward CYP1A1 gene transcription and CYP1A protein levels, showing typical additive behavior with TCDD to the target molecule AhR. In contrast, the nonplanar PCB 153 antagonized the action of TCDD, even at concentrations that occupied a significant fraction of AhR molecules. Competitive inhibition explains the commonly reported decrease of ethoxyresorufin- O -deethylase (EROD) activity when PCBs are present in high concentrations and the antagonism of PCBs to the EROD activity of TCDD. The result is that Western blotting offers a much more reliable measure of CYP1A protein concentration than does the EROD assay, despite the greater convenience of the latter. © 2004 Wiley Periodicals, Inc. Environ Toxicol 19: 480,489, 2004. [source] NF,B, cytokines, TLR 3 and 7 expression in human end-stage HCV and alcoholic liver diseaseEUROPEAN JOURNAL OF CLINICAL INVESTIGATION, Issue 7 2010Peter Stärkel Eur J Clin Invest 2010; 40 (7): 575,584 Abstract Background/aims, Conflicting observations exist concerning the role of nuclear factor kappa B (NF,B) in alcoholic liver disease (ALD) in animal models. To date no studies have examined this aspect in human liver tissue. We here assessed cytokines and toll-like receptors (TLRs) expressions in conjunction with NF,B activation in non-active end-stage human ALD compared with normal livers and hepatitis C virus (HCV) related end-stage disease. Methods, mRNA and protein expression were examined by quantitative PCR and Western blotting, DNA-binding by electrophoretic mobility shift assays and NF,B sub-cellular localization by immunofluorescent staining of livers. Results, NF,B mRNA and protein expression as well as strong DNA-binding were preserved in ALD but significantly down-regulated in HCV compared with normal livers. P50 immunofluorescence was found in hepatocytes and bile ducts in ALD and normal livers, whereas a shift was observed in p65 staining from non-parenchymal cells in normal livers to hepatocytes in ALD. NF,B responsive genes mRNA levels IkB, and interleukin 6 were significantly higher in ALD compared with HCV. Tumour necrosis factor alpha (TNF,), TLRs 3 and 7 mRNA were up-regulated in ALD and HCV compared with normal liver with TNF, and TLR7 being the highest in HCV. Strong induction of interferon beta was found in HCV but not in ALD or normal liver tissue. Conclusions, Persistent NF,B activation together with high pro-inflammatory cytokine expression and upregulation of TLR3 and TLR7 is associated with end-stage ALD in humans and could contribute to disease progression even in absence of alcohol intake. [source] 8-isoprostane increases scavenger receptor A and matrix metalloproteinase activity in THP-1 macrophages, resulting in long-lived foam cellsEUROPEAN JOURNAL OF CLINICAL INVESTIGATION, Issue 7 2004H. Scholz Abstract Background, Oxidative stress is a key factor in atherogenesis, in which it is closely associated with the inflammation and formation of bioactive lipids. Although 8-isoprostane is regarded as a reliable marker of oxidative stress in vivo, the pathogenic role of this F2 -isoprostane in atherogenesis is far from clear. Based on the important role of foam cells in the initiation and progression of atherosclerosis we hereby examined the ability of 8-isoprostane to modulate oxidized (ox)LDL-induced foam cell formation and the function of these cells, particularly focusing on the effect on matrix degradation. Methods and results, 8-isoprostane (10 µM) augmented the oxLDL-induced (20 µg mL,1) lipid accumulation of THP-1 macrophages evaluated by Oil-Red-O staining and lipid mass quantification (colourimetric assay). Additionally, 8-isoprostane induced the expression of the scavenger receptor A type 1 (MSR-1) [mRNA and protein level], assessed by RT-PCR and Western blotting, respectively. Moreover, 8-isoprostane counteracted the oxLDL-induced apoptosis of these cells, involving both mitochondrial-protective and caspase-suppressive mechanisms. Along with these changes, 8-isoprostane increased the oxLDL-induced gene expression of matrix metalloproteinase (MMP)-9 and its endogenous inhibitor [i.e. tissue inhibitor of MMP (TIMP)-1] accompanied by enhanced total MMP activity. Conclusions, We show that 8-isoprostane increases foam cell formation at least partly by enhancing MSR-1 expression and by inhibiting apoptosis of these cells, inducing long-lived foam cells with enhanced matrix degrading capacity. Our findings further support a role for 8-isoprostane not only as a marker of oxidative stress in patients with atherosclerotic disorders, but also as a mediator in atherogenesis and plaque destabilization. [source] Fatty acid metabolism assessed by 125I-iodophenyl 9-methylpentadecanoic acid (9MPA) and expression of fatty acid utilization enzymes in volume-overloaded heartsEUROPEAN JOURNAL OF CLINICAL INVESTIGATION, Issue 3 2004T. Miyamoto Abstract Background, The peroxisome proliferator-activated receptor (PPAR) , is a member of the nuclear receptor superfamily and regulates gene expression of fatty acid utilization enzymes. In cardiac hypertrophy and heart failure by pressure-overload, myocardial energy utilization reverts to the fetal pattern, and metabolic substrate switches from fatty acid to glucose. However, myocardial metabolism in volume-overloaded hearts has not been rigorously studied. The aim of the present study was to examine fatty acid metabolism and protein expressions of PPAR, and fatty acid oxidation enzymes in volume-overloaded rabbit hearts. Methods, Volume-overload was induced by carotid-jugular shunt formation. Sham-operated rabbits were used as control. Chronic volume-overload increased left ventricular weight and ventricular cavity size, and relative wall thickness was decreased, indicating eccentric cardiac hypertrophy. 125I-iodophenyl 9-methylpentadecanoic acid (9MPA) was intravenously administered, and animals were sacrificed at 5 min after injection. The 9MPA was rapidly metabolized to iodophenyl-3-methylnonanoic acid (3MNA) by ,-oxidation. Lipid extraction from the myocardium was performed by the Folch method, and radioactivity distribution of metabolites was assayed by thin-layer chromatography. The protein was extracted from the left ventricular myocardium, and levels of PPAR, and fatty acid oxidation enzymes were examined by Western blotting. Results, Myocardial distribution of 9MPA tended to be more heterogeneous in shunt than in sham rabbits (P = 0·06). In volume-overloaded hearts by shunt, the conversion from 9MPA to 3MNA by ,-oxidation was faster than the sham-control hearts (P < 0·05). However, protein levels of PPAR, and fatty acid utilization enzymes were unchanged in shunt rabbits compared with sham rabbits. Conclusions, These data suggest that myocardial fatty acid metabolism is enhanced in eccentric cardiac hypertrophy by volume-overload without changes in protein expressions of PPAR, and fatty acid utilization enzymes. Our data may provide a novel insight into the subcellular mechanisms for the pathological process of cardiac remodelling in response to mechanical stimuli. [source] Basic fibrobrast growth factor induces the secretion of vascular endothelial growth factor by human aortic smooth muscle cells but not by endothelial cellsEUROPEAN JOURNAL OF CLINICAL INVESTIGATION, Issue 10 2003F. Belgore Abstract Background, Endothelial cell dysfunction and smooth muscle cell (SMC) proliferation are major events in atherogenesis. Both cells are a source of growth factors that mediate cellular proliferation and chemotaxis. Inappropriate production of, and/or response to, these growth factors (such as vascular endothelial growth factor, VEGF, and basic fibroblast growth factor (bFGF)) may contribute to atherogenesis and therefore to disease progression. Methods, Production of VEGF and its soluble receptor (sFlt-1) by human SMCs and human umbilical endothelial cells (HUVECs) after stimulation with bFGF were examined by ELISA of cell culture media and by Western blotting. Results, Smooth muscle cells produced significantly more VEGF than HUVECs (P < 0·05) after 24 h of culture with bFGF levels , 0·001 µg mL,1. bFGF induced dose-dependent production of VEGF by SMCs, where maximum production was present in 1 µg mL,1 of bFGF. Conversely, the SMCs produced less sFlt-1 than HUVECs (P < 0·05). However, bFGF induced dose-dependent phosphorylation of Flt1 and another VEGF receptor, KDR, in HUVECs but not SMCs. There was no VEGF or sFLT-1 after 6 h of culture in any dose of bFGF in either type of cell. Conclusions, Differences in the production of VEGF and sFlt-1 by SMCs and HUVECs are consistent with the role of these cells in angiogenesis. Induction of VEGF production and expression by bFGF in these cells indicates that this growth factor may participate in angiogenesis indirectly by the induction of VEGF. The production of sFlt-1 by both cell types is in agreement with the notion that sFlt-1 may be involved in the regulation of VEGF activity. Additionally, the ability of bFGF to induce dose-dependent phosphorylation of KDR in HUVECs highlights the important role of bFGF in VEGF-mediated angiogenic processes. [source] Human platelets express hemochromatosis protein (HFE) and transferrin receptor 2EUROPEAN JOURNAL OF HAEMATOLOGY, Issue 4 2003Jokke Hannuksela Abstract: Objectives: While body iron status may influence platelets, little information is available about platelet expression of proteins regulating iron homeostasis. HFE, the protein defective in hereditary hemochromatosis, and transferrin receptor 2 (TfR2) are two novel protein candidates that could be involved in mechanisms of iron transport across the platelet plasma membrane. Methods: The expression and localization of HFE, TfR1 and TfR2 proteins in human platelets were examined using Western blotting and immunocytochemistry. Results: Human platelets expressed HFE and TfR2, whereas no signal for TfR1 was found. The positive reactions for HFE and TfR2 were mainly confined to the platelet plasma membrane. Conclusions: Expression of HFE and TfR2 proteins in human platelets may indicate that the mutations in the corresponding genes could influence platelet count, size and/or activation. The presence of TfR2 and absence of TfR1 suggests that HFE may serve a different function in platelets compared with the other HFE-positive cell types, e.g. enterocytes, macrophages and syncytiotrophoblasts. [source] Hypothermia treatment potentiates ERK1/2 activation after traumatic brain injuryEUROPEAN JOURNAL OF NEUROSCIENCE, Issue 4 2007Coleen M. Atkins Abstract Traumatic brain injury (TBI) results in significant hippocampal pathology and hippocampal-dependent memory loss, both of which are alleviated by hypothermia treatment. To elucidate the molecular mechanisms regulated by hypothermia after TBI, rats underwent moderate parasagittal fluid-percussion brain injury. Brain temperature was maintained at normothermic or hypothermic temperatures for 30 min prior and up to 4 h after TBI. The ipsilateral hippocampus was assayed with Western blotting. We found that hypothermia potentiated extracellular signal-regulated kinase 1/2 (ERK1/2) activation and its downstream effectors, p90 ribosomal S6 kinase (p90RSK) and the transcription factor cAMP response element-binding protein. Phosphorylation of another p90RSK substrate, Bad, also increased with hypothermia after TBI. ERK1/2 regulates mRNA translation through phosphorylation of mitogen-activated protein kinase-interacting kinase 1 (Mnk1) and the translation factor eukaryotic initiation factor 4E (eIF4E). Hypothermia also potentiated the phosphorylation of both Mnk1 and eIF4E. Augmentation of ERK1/2 activation and its downstream signalling components may be one molecular mechanism that hypothermia treatment elicits to improve functional outcome after TBI. [source] Aquaporin 4 changes in rat brain with severe hydrocephalusEUROPEAN JOURNAL OF NEUROSCIENCE, Issue 11 2006Xiaoyan Mao Abstract Hydrocephalus is characterized by impaired cerebrospinal fluid (CSF) flow with enlargement of the ventricular cavities of the brain and progressive damage to surrounding tissue. Bulk water movement is altered in these brains. We hypothesized that increased expression of aquaporins, which are water-permeable channel proteins, would occur in these brains to facilitate water shifts. We used quantitative (real-time) RT-PCR, Western blotting and immunohistochemistry to evaluate the brain expression of aquaporins (AQP) 1, 4, and 9 mRNA and protein in Sprague,Dawley rats rendered hydrocephalic by injection of kaolin into cistern magna. AQP4 mRNA was significantly up-regulated in parietal cerebrum and hippocampus 4 weeks and 9 months after induction of hydrocephalus (P < 0.05). Although Western blot analysis showed no significant change, there was more intense perivascular AQP4 immunoreactivity in cerebrum of hydrocephalic brains at 3,4 weeks after induction. We did not detect mRNA or protein changes in AQP1 (located in choroid plexus) or AQP9 (located in select neuron populations). Kir4.1, a potassium channel protein linked to water flux, exhibited enhanced immunoreactivity in the cerebral cortex of hydrocephalic rats; the perineuronal distribution was entirely different from that of AQP4. These results suggest that brain AQP4 up-regulation might be a compensatory response to maintain water homeostasis in hydrocephalus. [source] Decrease of Hsp25 protein expression precedes degeneration of motoneurons in ALS-SOD1 miceEUROPEAN JOURNAL OF NEUROSCIENCE, Issue 1 2004Arjen Maatkamp Abstract We have investigated the expression of Hsp25, a heat shock protein constitutively expressed in motoneurons, in amyotrophic lateral sclerosis (ALS) mice that express G93A mutant SOD1 (G93A mice). Immunocytochemistry and Western blotting showed that a decrease of Hsp25 protein expression occurred in motoneurons of G93A mice prior to the onset of motoneuron death and muscle weakness. This decrease in Hsp25 expression also preceded the appearance of SOD1 aggregates as identified by cellulose acetate filtration and Western blot analysis. In contrast to Hsp25 protein levels, Hsp25 mRNA as determined by in situ hybridization and RT-PCR, remained unchanged. This suggests that the decrease in Hsp25 protein levels occurs post-transcriptionally. In view of the cytoprotective properties of Hsp25 and the temporal relationship between decreased Hsp25 expression and the onset of motoneuron death, it is feasible that reduced Hsp25 concentration contributes to the degeneration of motoneurons in G93A mice. These data are consistent with the idea that mutant SOD1 may reduce the availability of the protein quality control machinery in motoneurons. [source] BDNF, NT-3 and NGF induce distinct new Ca2+ channel synthesis in developing hippocampal neuronsEUROPEAN JOURNAL OF NEUROSCIENCE, Issue 11 2000Pietro Baldelli Abstract Neurotrophins exert short- and long-term effects on synaptic transmission. The mechanism underlying these forms of synaptic plasticity is unknown although it is likely that intracellular Ca2+ and presynaptic Ca2+ channels play a critical role. Here we show that BDNF, NGF and NT-3 (10,100 ng/mL) exhibit a selective long-term up-regulation of voltage-gated Ca2+ current densities in developing hippocampal neurons of 6,20 days in culture. NGF and NT-3 appear more effective in up-regulating L-currents, while BDNF predominantly acts on non-L-currents (N, P/Q and R). The effects of the three neurotrophins were time- and dose-dependent. The EC50 was comparable for BDNF, NGF and NT-3 (10,16 ng/mL) while the time of half-maximal activation was significantly longer for NGF compared to BDNF (58 vs. 25 h). Despite the increased Ca2+ current density, the neurotrophins did not alter the voltage-dependence of channel activation, the kinetics parameters or the elementary properties of Ca2+ channels (single-channel conductance, probability of opening and mean open time). Neurotrophin effects were completely abolished by coincubation with the nonspecific Trk-receptor inhibitor K252a, the protein synthesis blocker anisomycin and the MAP-kinase inhibitor PD98059, while cotreatment with the PLC-, blocker, U73122, was without effect. Immunocytochemistry and Western blotting revealed that neurotrophins induced an increased MAP-kinase phosphorylation and its translocation to the nucleus. The present findings suggest that on a long time scale different neurotrophins can selectively up-regulate different Ca2+ channels. The action is mediated by Trk-receptors/MAP-kinase pathways and induces an increased density of newly available Ca2+ channels with unaltered gating activity. [source] Regulation of GluR2 promoter activity by neurotrophic factors via a neuron-restrictive silencer elementEUROPEAN JOURNAL OF NEUROSCIENCE, Issue 5 2000Stefan Brené Abstract The AMPA glutamate receptor subunit GluR2, which plays a critical role in regulation of AMPA channel function, shows altered levels of expression in vivo after several chronic perturbations. To evaluate the possibility that transcriptional mechanisms are involved, we studied a 1254-nucleotide fragment of the 5,-promoter region of the mouse GluR2 gene in neural-derived cell lines. We focused on regulation of GluR2 promoter activity by two neurotrophic factors, which are known to be altered in vivo in some of the same systems that show GluR2 regulation. Glial-cell line derived neurotrophic factor (GDNF) and brain-derived neurotrophic factor (BDNF) both induced GluR2 promoter activity. This was associated with increased expression of endogenous GluR2 immunoreactivity in the cells as measured by Western blotting. The effect of GDNF and BDNF appeared to be mediated via a NRSE (neuron-restrictive silencer element) present within the GluR2 promoter. The response to these neurotrophic factors was lost upon mutating or deleting this site, but not several other putative response elements present within the promoter. Moreover, overexpression of REST (restrictive element silencer transcription factor; also referred to as NRSF or neuron restrictive silencer factor), which is known to act on NRSEs in other genes to repress gene expression, blocked the ability of GDNF to induce GluR2 promoter activity. However, GDNF did not alter endogenous levels of REST in the cells. Together, these findings suggest that GluR2 expression can be regulated by neurotrophic factors via an apparently novel mechanism involving the NRSE present within the GluR2 gene promoter. 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