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Objective To investigate the molecular mechanism of palmitoylation modification in regulating the activity of non-receptor tyrosine kinase Fyn. Methods The intracellular Fyn activity was detected by applying fluorescence resonance energy transfer (FRET) technology, and the mechanism was investigated by combining with Fyn palmitoylation deficiency and C-terminal Src kinase ( CSK ) plasmid co-expression. ResultsExperimental data showed that single loss of either of ( C3, C6) palmitoylation sites resulted in higher Fyn activity, and C6 seemed more significant. It is known that CSK membrane translocation occurred after activation. FRET assay confirmed that CSK could down-regulate the activity of Fyn in cells, but could not effectively regulate the activity of Fyn(GSS) with the loss of palmitoylation sites. Conclusions The results in this study support the hypothesis on Fyn regulation by spatial localization, namely, non-palmitoylated Fyn (GSS) is less effective in the inhibitory regulation by CSK on cell membrane, thus promoting constitutive high activity expression
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Objective To investigate the anti-injury and anti-inflammation protective effects of metformin in acute-liver-injury SD rat model induced by D-galactosamine and Pam3CSK4 .Methods Eighteen male Sprague-Dawley rats were treated with the mixture of D-galactosamine (350 mg/kg ) and Pam3CSK4 (50 μg/kg ) by intraperitoneal injection (i .p .) to construct acute liver injury model .The rats in intervention group were given PBS and metformin ,respectively .The liver and body weight were measured and the ratio of liver weight to body weight was calculated .HE staining was used to observe the pathological changes of the liver .Fasting serum was collected for detection of serological parameters .ELISA and RT-qPCR were used to determine the expression levels of IL-6 and TNF-α.Finally , activation of MAPK signal pathway in rat liver was detected by Western blot .Results Compared with those in control group , the ratio of body weight to liver weight , serum transaminase and proinflammatory cytokines IL-6 and TNF-a were all significantly increased in the two intervention groups .Meanwhile , hepatic degeneration and hepatic interstitial exudation indicated that D -galactosamine combined with Pam3CSK4 successfully constructed acute liver injury model in the SD rats.Compared with PBS group, the ratio of body weight to liver weight , hepatic damage , serum transaminase levels.and the expressions of proinflammatory cytokines IL-6 and TNF-a were significantly decreased in metformin-treated group.Meanwhile,the expressions of p-ERKl/2,p-SAPK/JNK and p-P38 MAPK decreased in liver tissues by metformin pretreatment,suggesting that metformin may play an anti-inflammatory effect by suppressing MAPK signaling pathway.Conclusion Metformin attenuated inflammatory reactions in SD rats with acute liver injury induced by D -galactosamine and Pam3CSK4.
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Objective:To observe whether pretreatment with Pam3CSK4,a TLR2 agonist,could decrease the inflammation response in kidney from mice with systemic MRSA infection,and to investigate the mechanism of the attenuation of inflammation with Pam3CSK4 pretreatment. Methods:BALB/c mice were pretreated with Pam3CSK4 (10 μg/100 μl/each mouse) or PBS via tail vein once daily for two consecutive days. All mice were infected with live MRSA (ATCC43300) at 2×107 CFU/each mouse (via tail vein) 24 h after the second treatment. The levels of cytokines in kidney were measured by ELISA and real-time PCR,respectively. The relative expression of TLR2,IRAKs etc. were detected by real-time PCR. Western blot was performed to detect the phosphorylation of NF-κB, the expression of IRAK-M and A20,respectively. Results:The level of TNF-α,IL-6,IL-1β,CCL3 and IFN-γ in renal tissue from mice pretreated with Pam3CSK4 was decreased significantly compared with that from PBS-treated mice,respectively. Pam3CSK4 pretreatment down-regulated the relative expression of TLR2, inhibited the expression of IRAK-1 and the phosphorylation of NF-κB post infection. The expression of IRAK-M,one of the negative regulators in TLRs signaling pathway was increased significantly in renal tissue from Pam3CSK4-treated mice post infection. Conclusion:Pam3CSK4 pretreatment attenuated the inflammation response in kidney from mice with systemic MRSA infection,and these attenuation is related with up-regulation of IRAK-M.
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Objective:To observe whether pretreatment with Pam3CSK4,a TLR2 agonist,could decrease the inflammation response in kidney from mice with systemic MRSA infection,and to investigate the mechanism of the attenuation of inflammation with Pam3CSK4 pretreatment. Methods:BALB/c mice were pretreated with Pam3CSK4 (10 μg/100 μl/each mouse) or PBS via tail vein once daily for two consecutive days. All mice were infected with live MRSA (ATCC43300) at 2×107 CFU/each mouse (via tail vein) 24 h after the second treatment. The levels of cytokines in kidney were measured by ELISA and real-time PCR,respectively. The relative expression of TLR2,IRAKs etc. were detected by real-time PCR. Western blot was performed to detect the phosphorylation of NF-κB, the expression of IRAK-M and A20,respectively. Results:The level of TNF-α,IL-6,IL-1β,CCL3 and IFN-γ in renal tissue from mice pretreated with Pam3CSK4 was decreased significantly compared with that from PBS-treated mice,respectively. Pam3CSK4 pretreatment down-regulated the relative expression of TLR2, inhibited the expression of IRAK-1 and the phosphorylation of NF-κB post infection. The expression of IRAK-M,one of the negative regulators in TLRs signaling pathway was increased significantly in renal tissue from Pam3CSK4-treated mice post infection. Conclusion:Pam3CSK4 pretreatment attenuated the inflammation response in kidney from mice with systemic MRSA infection,and these attenuation is related with up-regulation of IRAK-M.
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Objective:To evaluate immune response of murine peritoneal macrophage challenging by methicillin-resistant S.aureus(MRSA)after pretreatment with Pam3Csk4(TLR2 agonist).Methods: Murine peritoneal macrophage was pretreated with Pam3Csk4(1 μg/ml).Following pretreatment 12 h later,heat-killed MRSA( HK-MRSA) was added and incubated for another 2 or 6 hours.The protein and mRNA level of TNF-α, IL-6 and IL-1 were determined by ELISA and Q-PCR, respectively.To estimate phagocytosis of macrophage,HK-MRSA/MSSA labeled with FITC( FITC-HK-MRSA/MSSA) were added to well and incubated for 30 min.After washing 5 times with PBS,intracellular FITC-HK-MRSA was detected by flow cytometry.To estimate antimicrobal activity of macrophage,live MRSA and MSSA were added to well and incubated at indication time,the CFU of s.aureus was estimated via a 10-fold serial dilution on agar media.cDNA was further quantitative assessed using primers for mouse FCR-Ⅰ,FCR-Ⅲ,CR-1,CR-3,iNOS and LL37 by Q-PCR .Results: Compared with saline-pretreated cell, the protein and mRNA level of TNF-α, IL-6 and IL-1 were markely reduced, respectively.However, both the phagocytosis and antimicrobal activity to S.aureus were significantly increased in macrophages pretreated with Pam3Csk4.Further study found that the macrophages had higher FCR-Ⅰ,FCR-Ⅲ,CR-1,CR-3,iNOS and LL37 expression at 6 h and 12 h post-stimulation Pam3Csk4.Conclusion: The results suggest that Pam3Csk4 could activate murine antimicrobal activity of peritoneal macrophage challenging by methicillin-resistant Saureus via increasing opsonophagocytosis in depended antibodies, complements manners.The results suggest Pam3Csk4 probably be a novel immunotherapy candidate against MRSA.
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C-terminal SRC kinase (CSK) is a ubiquitously expressed, cytosolic enzyme that phosphorylates and inactivates several SRC family protein tyrosine kinases. Recent genomewide association studies have implicated CSK in the regulation of blood pressure. The current study aim is to determine the blood pressure association of the genes regulated by CSK down-regulation. The CSK mRNA expression was downregulated in vascular smooth muscle cells using small interfering RNA (siRNA). CSK mRNA levels fell by 90% in cells that were treated with CSK siRNA; the RNA from these cells was examined by microarray using the Illumina HumanRef-8 v3 platform, which comprises 24,526 reference mRNA probes. On treatment with CSK siRNA, 19 genes were downregulated by more than 2-fold and 13 genes were upregulated by more than 2-fold. Three (CANX, SLC30A7, and HMOX1) of them revealed more than 3 fold differential expression. Interestingly, the HMOX1 SNPs were associated with diastolic blood pressure in the 7551 Koreans using Korea Association REsource data, and the result was supported by the other reports that HMOX1 linked to blood vessel maintenance. Among the remaining 29 differentially expressed genes, seven (SSBP1, CDH2, YWHAE, ME2, PFTK1, G3BP2, and TUFT1) revealed association with both systolic and diastolic blood pressures. The CDH2 gene was linked to blood pressures. Conclusively, we identified 32 differentially expressed genes which were regulated by CSK reduction, and two (HOMX1 and CDH2) of them might influence the blood pressure regulation through CSK pathway.
Subject(s)
Humans , Blood Pressure , Blood Vessels , Cytosol , Down-Regulation , Glycosaminoglycans , Korea , Muscle, Smooth, Vascular , Phosphotransferases , Polymorphism, Single Nucleotide , Protein-Tyrosine Kinases , RNA , RNA, Messenger , RNA, Small Interfering , src-Family KinasesABSTRACT
OBJECTIVE: The purpose of this study was to investigate the transcriptional expression and translational localization of c-Src and Csk, related to signal transduction of cell growth, differentiation, death, and DNA synthesis in the human endometrium during the normal menstrual cycle. METHODS: The specimens were obtained from the hysterectomized uterus and endometrial biopsies that showed no endometrial pathology. The mRNA level of c-Src and Csk were examined by RT-PCR and the expression of these proteins were confirmed by immunohistochemistry. RESULTS: The c-Src mRNA was strongly expressed at all phases, except in late secretory phase. The Csk mRNA was highly expressed at all phases. In stromal cells and glandular cells, immunohistochemical expression of c-Src protein was moderate in proliferative phase and was strong in the secretory phase. The expression of Csk protein was strong at all phases and more intensive in glandular cell than stromal cell. CONCLUSION: It is suggested that c-Src may play an important role in the signaling of the differentiation of the human endometrium and the Csk may modulate the c-Src kinase activity.