AIM: To study preliminarily the properties of myosin light chain kinase (MLCK) in rabbit liver. METHODS: The expression of MLCK was detected by reverse transcription-polymerase chain reaction(RT-PCR); the MLCK was obt...AIM: To study preliminarily the properties of myosin light chain kinase (MLCK) in rabbit liver. METHODS: The expression of MLCK was detected by reverse transcription-polymerase chain reaction(RT-PCR); the MLCK was obtained from rabbit liver, and its activity was analyzed by gamma-(32)P incorporation technique to detect the phosphorylation of myosin light chain. RESULTS: MLCK was expressed in rabbit liver, and the activity of the enzyme was similar to rabbit smooth muscle MLCK, and calmodulin-dependent. When the concentration was 0.65 mg x L(-1), the activity was at the highest level. CONCLUSION: MLCK expressed in rabbit liver may catalyze the phosphorylation of myosin light chain, which may play important roles in the regulation of hepatic cell functions.展开更多
The aim of this study is to investigate the functional relationship between filamin, a known actin binding protein, and myosin and the effects of filamin on the interaction between myosin and actin. Methods.Ultra cent...The aim of this study is to investigate the functional relationship between filamin, a known actin binding protein, and myosin and the effects of filamin on the interaction between myosin and actin. Methods.Ultra centrifugation method was used to investigate the binding of filamin to both phosphorylated and unphosphorylated myosins. Mg ATPase activities of both phosphorylated and unphosphorylated myosins in the presence and absence of actin were measured to observe the effects resulted from filamin actin and filamin myosin interactions. Results. It was found that filamin is also a myosin binding protein. Filamin inhibited the actin activated Mg ATPase activity of phosphorylated myosin and stimulated Mg ATPase of phosphorylated myosin in the absence of actin; in addition, filamin stimulated Mg ATPase activity of unphosphorylated myosin in both the presence or absence of actin. Conclusion. The results suggest that the effects of filamin on the myosin Mg ATPase activities are bi directional, i.e., stimulatory via binding to myosin and inhibitory via binding to actin.展开更多
Objective: To reveal the special feature of calponin (CaP) on myosins of different states. Methods: Myosin phosphorylation determination, myosin Mg^(2+)-ATPase measurement and protein binding assay were used in this s...Objective: To reveal the special feature of calponin (CaP) on myosins of different states. Methods: Myosin phosphorylation determination, myosin Mg^(2+)-ATPase measurement and protein binding assay were used in this study. The lowest CaP/myosin ratio used in the assay was 1/10000(mol/mol), which was 10 thousands-fold lower than the CaP/myosin ratio used in previous studies. Results: In the absence of actin, micro-amount of calponin (MAC) stimulated the Mg^(2+)-ATPase activities of myosin in different states slightly but significantly; and more importantly, MAC significantly increased the precipitations of unphosphorylated myosin, Ca^(2+)-dependently and independently phosphorylated myosins by MLCK but not the myosin phosphorylated by PKA. Conclusion: MAC has a high efficient and selective effect on myosin in the absence of actin.展开更多
The molecular structure of a higher plant myosin with two 174 kD heavy chains purified from the tendrils of Luffa cylindrica (L.) Roem. was viewed by electron microscopy. The myosin exhibited actin_activated MgATP...The molecular structure of a higher plant myosin with two 174 kD heavy chains purified from the tendrils of Luffa cylindrica (L.) Roem. was viewed by electron microscopy. The myosin exhibited actin_activated MgATPase activity and could be recognized immunologically by a monoclonal antibody against the skeletal muscle myosin. Electron micrographs of rotary shadowed images of this protein revealed that it had two heads with size and shape similar to those of the skeletal muscle myosin and a relatively short tail in comparison with the conventional myosin. Luffa tendril actin filaments were also visualized and occasionally other Luffa myosin_like proteins with globular structure at the tail ends were also observed. The structural similarity and immunological cross reactivity with antibodies against muscle myosin demonstrate that the 174 kD Luffa tendril myosin is a double_headed myosin. The possible involvement of myosin_actin interactions in Luffa tendril contact coiling will be the subject of further research.展开更多
Summary: To study whether there was an anti-cardiac myosin antibody (AMA) in serum of pa- tients with myocardial infarction (AMI), relationship between AMA and the prognosis in patients with AMI was investigated. In 6...Summary: To study whether there was an anti-cardiac myosin antibody (AMA) in serum of pa- tients with myocardial infarction (AMI), relationship between AMA and the prognosis in patients with AMI was investigated. In 67 patients with acute AMI, AMA was assayed by ELISA and left ventricular structure and cardiac function were examined by echocardiography at the end of the first week after infarction and during a 6-month follow-up. The patients with AMI were divided into AMA-positive group and AMA-negative group. The parameters of left ventricular end-dias- tolic function and prognosis were compared between the two groups. Results showed that the AMA was positive in 18 patients with AMI, with a positive rate of 26. 87 %, while it was negative in 20 health donors. The locations of myocardial infarction in the two groups were similar. There were significant differences in Killip class I (22. 22 % vs 55. 10 %, P<0. 05), decreasing of wall motion and ventricular aneurysm (92. 85 % vs 37. 5 %, P<0.01) between the positive group and the negative group. During a 6-month follow-up, the mortality was higher in AMA positive group than in AMA negative group (38. 89% vs 10. 20 %, P<0. 05). It is concluded that AMA can be detected in serum of patients with AMI and can serve as an important autoimmune marker. The autoimmune response might take place in AMI. AMA was associated with the left ventricular re- modeling and the prognosis of AMI.展开更多
The myosin heavy chain(MyHC)is one of the major structural and contracting proteins of muscle.We have isolated the cDNA clone encoding MyHC of the grass carp,Ctenopharyngodon idella. The sequence comprises 5 934 bp,in...The myosin heavy chain(MyHC)is one of the major structural and contracting proteins of muscle.We have isolated the cDNA clone encoding MyHC of the grass carp,Ctenopharyngodon idella. The sequence comprises 5 934 bp,including a 5 814 bp open reading frame encoding an amino acid sequence of 1 937 residues.The deduced amino acid sequence showed 69%homology to rabbit fast skeletal MyHC and 73%–76%homology to the MyHCs from the mandarin fish,walleye pollack,white croaker,chum salmon,and carp.The putative sequences of subfragment-1 and the light meromyosin region showed 61.4%–80%homology to the corresponding regions of other fish MyHCs.The tissue-specific and developmental stage-specific expressions of the MyHC gene were analyzed by quantitative real-time PCR.The MyHC gene showed the highest expression in the muscles compared with the kidney,spleen and intestine.Developmentally,there was a gradual increase in MyHC mRNA expression from the neural formation stage to the tail bud stage.The highest expression was detected in hatching larva.Our work on the MyHC gene from the grass carp has provided useful information for fish molecular biology and fish genomics.展开更多
Autoimmune is involved in the pathogenesis of ventricular remodeling in acute myocardial infarction (AMI).In the present study, we investigated the effect of anti-cardiac myosin heavy chain antibodies (AMHCA) from pat...Autoimmune is involved in the pathogenesis of ventricular remodeling in acute myocardial infarction (AMI).In the present study, we investigated the effect of anti-cardiac myosin heavy chain antibodies (AMHCA) from patients with AMI on rat cardiomyocyte apoptosis.Cardiomyocyte apoptosis was observed and measured by DNA end labeling and Annexin-Ⅴ/PI double-staining assay.The expression of apoptosis related p53 and Bcl-2 protein and the second messenger calcium were detected respectively by Western blotting, patch clamp and confocal calcium imaging.The results showed that AMHCA was able to induce cardiomyocyte apoptosis in a dose dependent manner.Apoptosis-accelerating nucleoprotein p53 was up-regulated, while apoptosis-inhibiting cytoplasmic protein Bcl-2 was down-regulated.In parallel, cytoplasmic calcium concentration was elevated.There was no effect on L-type calcium currents.It is concluded that AMHCA in patients with AMI as a novel triggering factor can induce cardiomyocyte apoptosis, which contributes to ventricular remodeling.展开更多
AIM: To identify and characterize the function of non-mu-scle myosin Ⅱ (NMM Ⅱ) isoforms in primary rat hepatic stellate cells (HSCs).METHODS: Primary HSCs were isolated from male Spra-gue-Dawley rats by pronase/coll...AIM: To identify and characterize the function of non-mu-scle myosin Ⅱ (NMM Ⅱ) isoforms in primary rat hepatic stellate cells (HSCs).METHODS: Primary HSCs were isolated from male Spra-gue-Dawley rats by pronase/collagenase digestion. Total RNA and protein were harvested from quiescent and culture-activated HSCs. NMM Ⅱ isoform (Ⅱ-A, Ⅱ-B and Ⅱ-C) gene and protein expression were measured by RealTime polymerase chain reaction and Western blot analyses respectively. NMM Ⅱ protein localization was visualized in vitro using immunocytochemical analysis. For in vivo assessment, liver tissue was harvested from bile duct-ligated (BDL) rats and NMM Ⅱisoform expression determined by immunohistochemistry. Using a selective myosin Ⅱ inhibitor and siRNA-mediated knockdown of each isoform, NMM Ⅱ functionality inprimary rat HSCs was determined by contraction and migration assays.RESULTS: NMM Ⅱ-A and Ⅱ-B mRNA expression was increased in culture-activated HSCs (Day 14) with sig-niflicant increases seen in all pairwise comparisons (Ⅱ-A: 12.67 ± 0.99 (quiescent) vs 17.36 ± 0.78 (Day 14), P < 0.05; Ⅱ-B: 4.94 ± 0.62 (quiescent) vs 13.90 ±0.85 (Day 14), P < 0.001). Protein expression exhibited similar expression patterns (Ⅱ-A: 1.87 ± 2.50 (quiescent) vs 58.64 ± 8.76 (Day 14), P < 0.05; Ⅱ-B: 1.17 ± 1.93 (quiescent) vs 103.71 ± 21.73 (Day 14), P < 0.05). No signif icant differences were observed in NMM Ⅱ-C mRNA and protein expression between quiescent and activated HSCs. In culture-activated HSCs, NMM Ⅱ-A and Ⅱ-B merged with F-actin at the cellular periphery and throughout cytoplasm respectively. In vitro stud-ies showed increased expression of NMM Ⅱ-B in HSCs activated by BDL compared to sham-operated animals. There were no apparent increases of NMM Ⅱ-A and Ⅱ-C protein expression in HSCs during hepatic BDL injury. To determine the contribution of NMM Ⅱ-A and Ⅱ-B to migration and contraction, NMM Ⅱ-A and Ⅱ-B expres-sion were downregulated with siRNA. NMM Ⅱ-A and/or Ⅱ-B siRNA inhibited HSC migration by approximately 25% compared to scramble siRNA-treated cells. Conversely, siRNA-mediated NMM Ⅱ-A and Ⅱ-B inhibition had no signif icant effect on HSC contraction; however, contraction was inhibited with the myosin Ⅱ inhibitor, blebbistatin (38.7% ± 1.9%).CONCLUSION: Increased expression of NMM Ⅱ-A and Ⅱ-B regulates HSC migration, while other myosin Ⅱclasses likely modulate contraction, contributing to development and severity of liver f ibrosis.展开更多
Actin and myosin were found to be associated with the cytoplasmic sleeve of plasmodesmata. As cytoskeletal proteins, actin and myosin are believed to regulate the conductivity of plasmodesmata （PDs） in higher plants...Actin and myosin were found to be associated with the cytoplasmic sleeve of plasmodesmata. As cytoskeletal proteins, actin and myosin are believed to regulate the conductivity of plasmodesmata （PDs） in higher plants. Using immunocytochemical methods, we found the two proteins to be co-localized - and closely linked to each other - in plasmodesmata and ectodesmata-like structure in ageing parenchymatous cells of Allium sativum L. We suggest that intercellular communication is affected by the interaction between actin and myosin.展开更多
基金Supported by the National Natural Science Foundation of China(39870324,YW)Key Innovation Project of Chinese Academy of Science,P.R.China(KSCX 2-2-01,XB Yao)Grant for Excellent Young Teachers of Ministry of Education of China(99044312,YW)
文摘AIM: To study preliminarily the properties of myosin light chain kinase (MLCK) in rabbit liver. METHODS: The expression of MLCK was detected by reverse transcription-polymerase chain reaction(RT-PCR); the MLCK was obtained from rabbit liver, and its activity was analyzed by gamma-(32)P incorporation technique to detect the phosphorylation of myosin light chain. RESULTS: MLCK was expressed in rabbit liver, and the activity of the enzyme was similar to rabbit smooth muscle MLCK, and calmodulin-dependent. When the concentration was 0.65 mg x L(-1), the activity was at the highest level. CONCLUSION: MLCK expressed in rabbit liver may catalyze the phosphorylation of myosin light chain, which may play important roles in the regulation of hepatic cell functions.
文摘The aim of this study is to investigate the functional relationship between filamin, a known actin binding protein, and myosin and the effects of filamin on the interaction between myosin and actin. Methods.Ultra centrifugation method was used to investigate the binding of filamin to both phosphorylated and unphosphorylated myosins. Mg ATPase activities of both phosphorylated and unphosphorylated myosins in the presence and absence of actin were measured to observe the effects resulted from filamin actin and filamin myosin interactions. Results. It was found that filamin is also a myosin binding protein. Filamin inhibited the actin activated Mg ATPase activity of phosphorylated myosin and stimulated Mg ATPase of phosphorylated myosin in the absence of actin; in addition, filamin stimulated Mg ATPase activity of unphosphorylated myosin in both the presence or absence of actin. Conclusion. The results suggest that the effects of filamin on the myosin Mg ATPase activities are bi directional, i.e., stimulatory via binding to myosin and inhibitory via binding to actin.
文摘Objective: To reveal the special feature of calponin (CaP) on myosins of different states. Methods: Myosin phosphorylation determination, myosin Mg^(2+)-ATPase measurement and protein binding assay were used in this study. The lowest CaP/myosin ratio used in the assay was 1/10000(mol/mol), which was 10 thousands-fold lower than the CaP/myosin ratio used in previous studies. Results: In the absence of actin, micro-amount of calponin (MAC) stimulated the Mg^(2+)-ATPase activities of myosin in different states slightly but significantly; and more importantly, MAC significantly increased the precipitations of unphosphorylated myosin, Ca^(2+)-dependently and independently phosphorylated myosins by MLCK but not the myosin phosphorylated by PKA. Conclusion: MAC has a high efficient and selective effect on myosin in the absence of actin.
文摘The molecular structure of a higher plant myosin with two 174 kD heavy chains purified from the tendrils of Luffa cylindrica (L.) Roem. was viewed by electron microscopy. The myosin exhibited actin_activated MgATPase activity and could be recognized immunologically by a monoclonal antibody against the skeletal muscle myosin. Electron micrographs of rotary shadowed images of this protein revealed that it had two heads with size and shape similar to those of the skeletal muscle myosin and a relatively short tail in comparison with the conventional myosin. Luffa tendril actin filaments were also visualized and occasionally other Luffa myosin_like proteins with globular structure at the tail ends were also observed. The structural similarity and immunological cross reactivity with antibodies against muscle myosin demonstrate that the 174 kD Luffa tendril myosin is a double_headed myosin. The possible involvement of myosin_actin interactions in Luffa tendril contact coiling will be the subject of further research.
文摘Summary: To study whether there was an anti-cardiac myosin antibody (AMA) in serum of pa- tients with myocardial infarction (AMI), relationship between AMA and the prognosis in patients with AMI was investigated. In 67 patients with acute AMI, AMA was assayed by ELISA and left ventricular structure and cardiac function were examined by echocardiography at the end of the first week after infarction and during a 6-month follow-up. The patients with AMI were divided into AMA-positive group and AMA-negative group. The parameters of left ventricular end-dias- tolic function and prognosis were compared between the two groups. Results showed that the AMA was positive in 18 patients with AMI, with a positive rate of 26. 87 %, while it was negative in 20 health donors. The locations of myocardial infarction in the two groups were similar. There were significant differences in Killip class I (22. 22 % vs 55. 10 %, P<0. 05), decreasing of wall motion and ventricular aneurysm (92. 85 % vs 37. 5 %, P<0.01) between the positive group and the negative group. During a 6-month follow-up, the mortality was higher in AMA positive group than in AMA negative group (38. 89% vs 10. 20 %, P<0. 05). It is concluded that AMA can be detected in serum of patients with AMI and can serve as an important autoimmune marker. The autoimmune response might take place in AMI. AMA was associated with the left ventricular re- modeling and the prognosis of AMI.
基金Supported by the National Natural Science Foundation of China(Nos.30972263,30771644)the Natural Science Foundation of HunanProvince(No.09jj6037)
文摘The myosin heavy chain(MyHC)is one of the major structural and contracting proteins of muscle.We have isolated the cDNA clone encoding MyHC of the grass carp,Ctenopharyngodon idella. The sequence comprises 5 934 bp,including a 5 814 bp open reading frame encoding an amino acid sequence of 1 937 residues.The deduced amino acid sequence showed 69%homology to rabbit fast skeletal MyHC and 73%–76%homology to the MyHCs from the mandarin fish,walleye pollack,white croaker,chum salmon,and carp.The putative sequences of subfragment-1 and the light meromyosin region showed 61.4%–80%homology to the corresponding regions of other fish MyHCs.The tissue-specific and developmental stage-specific expressions of the MyHC gene were analyzed by quantitative real-time PCR.The MyHC gene showed the highest expression in the muscles compared with the kidney,spleen and intestine.Developmentally,there was a gradual increase in MyHC mRNA expression from the neural formation stage to the tail bud stage.The highest expression was detected in hatching larva.Our work on the MyHC gene from the grass carp has provided useful information for fish molecular biology and fish genomics.
基金supported by a grant from National Key Basic Research Program of China (No.2007CB512000,Sub-Project No.2007CB512005)
文摘Autoimmune is involved in the pathogenesis of ventricular remodeling in acute myocardial infarction (AMI).In the present study, we investigated the effect of anti-cardiac myosin heavy chain antibodies (AMHCA) from patients with AMI on rat cardiomyocyte apoptosis.Cardiomyocyte apoptosis was observed and measured by DNA end labeling and Annexin-Ⅴ/PI double-staining assay.The expression of apoptosis related p53 and Bcl-2 protein and the second messenger calcium were detected respectively by Western blotting, patch clamp and confocal calcium imaging.The results showed that AMHCA was able to induce cardiomyocyte apoptosis in a dose dependent manner.Apoptosis-accelerating nucleoprotein p53 was up-regulated, while apoptosis-inhibiting cytoplasmic protein Bcl-2 was down-regulated.In parallel, cytoplasmic calcium concentration was elevated.There was no effect on L-type calcium currents.It is concluded that AMHCA in patients with AMI as a novel triggering factor can induce cardiomyocyte apoptosis, which contributes to ventricular remodeling.
文摘AIM: To identify and characterize the function of non-mu-scle myosin Ⅱ (NMM Ⅱ) isoforms in primary rat hepatic stellate cells (HSCs).METHODS: Primary HSCs were isolated from male Spra-gue-Dawley rats by pronase/collagenase digestion. Total RNA and protein were harvested from quiescent and culture-activated HSCs. NMM Ⅱ isoform (Ⅱ-A, Ⅱ-B and Ⅱ-C) gene and protein expression were measured by RealTime polymerase chain reaction and Western blot analyses respectively. NMM Ⅱ protein localization was visualized in vitro using immunocytochemical analysis. For in vivo assessment, liver tissue was harvested from bile duct-ligated (BDL) rats and NMM Ⅱisoform expression determined by immunohistochemistry. Using a selective myosin Ⅱ inhibitor and siRNA-mediated knockdown of each isoform, NMM Ⅱ functionality inprimary rat HSCs was determined by contraction and migration assays.RESULTS: NMM Ⅱ-A and Ⅱ-B mRNA expression was increased in culture-activated HSCs (Day 14) with sig-niflicant increases seen in all pairwise comparisons (Ⅱ-A: 12.67 ± 0.99 (quiescent) vs 17.36 ± 0.78 (Day 14), P < 0.05; Ⅱ-B: 4.94 ± 0.62 (quiescent) vs 13.90 ±0.85 (Day 14), P < 0.001). Protein expression exhibited similar expression patterns (Ⅱ-A: 1.87 ± 2.50 (quiescent) vs 58.64 ± 8.76 (Day 14), P < 0.05; Ⅱ-B: 1.17 ± 1.93 (quiescent) vs 103.71 ± 21.73 (Day 14), P < 0.05). No signif icant differences were observed in NMM Ⅱ-C mRNA and protein expression between quiescent and activated HSCs. In culture-activated HSCs, NMM Ⅱ-A and Ⅱ-B merged with F-actin at the cellular periphery and throughout cytoplasm respectively. In vitro stud-ies showed increased expression of NMM Ⅱ-B in HSCs activated by BDL compared to sham-operated animals. There were no apparent increases of NMM Ⅱ-A and Ⅱ-C protein expression in HSCs during hepatic BDL injury. To determine the contribution of NMM Ⅱ-A and Ⅱ-B to migration and contraction, NMM Ⅱ-A and Ⅱ-B expres-sion were downregulated with siRNA. NMM Ⅱ-A and/or Ⅱ-B siRNA inhibited HSC migration by approximately 25% compared to scramble siRNA-treated cells. Conversely, siRNA-mediated NMM Ⅱ-A and Ⅱ-B inhibition had no signif icant effect on HSC contraction; however, contraction was inhibited with the myosin Ⅱ inhibitor, blebbistatin (38.7% ± 1.9%).CONCLUSION: Increased expression of NMM Ⅱ-A and Ⅱ-B regulates HSC migration, while other myosin Ⅱclasses likely modulate contraction, contributing to development and severity of liver f ibrosis.
基金supported by the National Natural Science Foundation of China (30070365, 30470861, 30971706)the Natural Science Foundation of Hebei Province, China (C2008000321)the Specialized Research Fund for the Doctoral Program of Higher Education, China (20060086003)
文摘Actin and myosin were found to be associated with the cytoplasmic sleeve of plasmodesmata. As cytoskeletal proteins, actin and myosin are believed to regulate the conductivity of plasmodesmata （PDs） in higher plants. Using immunocytochemical methods, we found the two proteins to be co-localized - and closely linked to each other - in plasmodesmata and ectodesmata-like structure in ageing parenchymatous cells of Allium sativum L. We suggest that intercellular communication is affected by the interaction between actin and myosin.
