The neuromuscular junction becomes progressively less receptive to regenerating axons if nerve repair is delayed for a long period of time. It is difficult to ascertain the denervated muscle's residual receptivity by...The neuromuscular junction becomes progressively less receptive to regenerating axons if nerve repair is delayed for a long period of time. It is difficult to ascertain the denervated muscle's residual receptivity by time alone. Other sensitive markers that closely correlate with the extent of denervation should be found. After a denervated muscle develops a fibrillation potential, muscle fiber conduction velocity, muscle fiber diameter, muscle wet weight, and maximal isometric force all decrease; remodeling increases neuromuscular junction fragmentation and plantar area, and expression of myogenesis-related genes is initially up-regulated and then down-regulated. All these changes correlate with both the time course and degree of denervation. The nature and time course of these denervation changes in muscle are reviewed from the literature to explore their roles in assessing both the degree of detrimental changes and the potential success of a nerve repair. Fibrillation potential amplitude, muscle fiber conduction velocity, muscle fiber diameter, mRNA expression levels of myogenic regulatory factors and nicotinic acetylcholine receptor could all reflect the severity and length of denervation and the receptiveness of denervated muscle to regenerating axons, which could possibly offer an important clue for surgical choices and predict the outcomes of delayed nerve repair.展开更多
Triple immunofluorescence staining has recently been developed to simultaneously identify all muscle fibers on a single cryosection which is helpful for clinical and basic research, but it has disadvantages such as fa...Triple immunofluorescence staining has recently been developed to simultaneously identify all muscle fibers on a single cryosection which is helpful for clinical and basic research, but it has disadvantages such as fast photobleaching and unclear outlines of muscle fibers. Triple immunoenzyme staining(TIE) is likely to avoid these disadvantages. In this study, we aimed to establish a sensitive and specific TIE technique to identify fiber types in normal, denervated, and reinnervated rat muscles, and to develop a systematic sampling method for muscle fiber quantification. Tibialis anterior and soleus from normal, denervated, and reinnervated Lewis rat hind limbs were used. Five consecutive cryosections were cut from each muscle, including one for TIE and four for single immunoenzyme staining(SIE). The TIE was performed using the polymerized reporter enzyme staining system for the first two antigens(A4.74 for My HC-IIA, BA-F8 for My HC-I) and alkaline phosphatase staining system for the third antigen(BF-F3 for My HC-IIB), followed by corresponding detective systems and respective chromogens. The type of muscle fibers was quantified by systematic sampling at 12.5%, 25%, 33% and 50% of all muscle fibers, and was compared with that acquired from counting all the fibers(100%). All muscle fiber phenotypes, including pure and hybrid, could be simultaneously identified on a single TIE cryosection with clear outlines. The fiber types on TIE slides matched well with their respective counterpart on the consecutive SIE slides with a 95% match rate. Systematic sampling of 12.5% fibers could represent the true fiber type distribution of the entire muscle section. Our results suggest that novel TIE can effectively visualize fiber types in normal, denervated or reinnervated rat muscles.展开更多
Objective: To examine the role of store-operated calcium entry(SOCE) and stromal interaction molecule 1(STIM1) in survival and migration of osteosarcoma cells and investigate what blockade of store-operated Ca^(2+)con...Objective: To examine the role of store-operated calcium entry(SOCE) and stromal interaction molecule 1(STIM1) in survival and migration of osteosarcoma cells and investigate what blockade of store-operated Ca^(2+)contributes to the regulation of osteosarcoma cells.Methods: First, we examined the expression levels of STIM1 in osteosarcoma cell lines by Western analysis and in tissue specimens by immunohistochemistry. Second, we investigated the effect of SOCE and STIM1 on osteosarcoma cell viability using MTS assays and on cell proliferation using colony formation. Third, we investigated the role of SOCE and STIM1 in cell migration using wound healing assays and Boyden chamber assays. Finally, we studied the effect of SOCE on the nuclear factor of activated T-cells cytoplasmic 1(NFATc1)activity by luciferase assays.Results: STIM1 was overexpressed in osteosarcoma cell lines and tissue specimens and was associated with poor survival of osteosarcoma patients. Also, inhibition of SOCE and STIM1 decreased the cell viability and migration of osteosarcoma cells. Furthermore, our results showed that blockade of store-operated Ca^(2+) channels involved down-regulation of NFATc1 in osteosarcoma cells.Conclusions: STIM1 is essential for osteosarcoma cell functions, and STIM1 and Ca^(2+) entry pathway could be further explored as molecular targets in the treatment of osteosarcoma.展开更多
Osteosarcoma is a primary bone tumor that affects children and young adults.The estrogen metabolite 2-methoxyestradiol(2-ME)induces cell death in osteosarcoma cells.To determine whether 2-ME actions involve the contro...Osteosarcoma is a primary bone tumor that affects children and young adults.The estrogen metabolite 2-methoxyestradiol(2-ME)induces cell death in osteosarcoma cells.To determine whether 2-ME actions involve the control of protein synthesis,we studied the effect of 2-ME on eukaryotic initiation factor 4E(eIF4E)and eIF4E-binding protein 1(4E-BP1)in MG63 osteosarcoma cells.Our results show that 2-ME treatment increases the association of eIF4E with 4E-BP1 in osteosarcoma cells.Also,2-ME decreases the binding of eIF4E protein to 7-methyl-guanosine cap structure,indicating that 2-ME treatment results in the inhibition of translational initiation.These findings are further supported by the inhibition of protein synthesis in 2-ME-treated osteosarcoma cells.Taken together,our studies show that 2-ME-mediated antitumor effects in osteosarcoma cells involve the regulation of protein synthesis,and translational machinery could serve as a target in the treatment of osteosarcoma.展开更多
基金sponsored by the Armed Forces Institute of Regenerative Medicine award number W81XWH-08-2-0034supported by the Sundt Fellowship fund,Department of Neurologic Surgery,Mayo Clinic,USA
文摘The neuromuscular junction becomes progressively less receptive to regenerating axons if nerve repair is delayed for a long period of time. It is difficult to ascertain the denervated muscle's residual receptivity by time alone. Other sensitive markers that closely correlate with the extent of denervation should be found. After a denervated muscle develops a fibrillation potential, muscle fiber conduction velocity, muscle fiber diameter, muscle wet weight, and maximal isometric force all decrease; remodeling increases neuromuscular junction fragmentation and plantar area, and expression of myogenesis-related genes is initially up-regulated and then down-regulated. All these changes correlate with both the time course and degree of denervation. The nature and time course of these denervation changes in muscle are reviewed from the literature to explore their roles in assessing both the degree of detrimental changes and the potential success of a nerve repair. Fibrillation potential amplitude, muscle fiber conduction velocity, muscle fiber diameter, mRNA expression levels of myogenic regulatory factors and nicotinic acetylcholine receptor could all reflect the severity and length of denervation and the receptiveness of denervated muscle to regenerating axons, which could possibly offer an important clue for surgical choices and predict the outcomes of delayed nerve repair.
基金sponsored by the Armed Forces Institute of Regenerative Medicine award number W81XWH-08-2-0034The U.S.Army Medical Research Acquisition Activity,820 Chandler Street,Fort Detrick MD 21702-5014 is the awarding and administering acquisition office+4 种基金The content of the manuscript does not necessarily reflect the position or the policy of the Government,and no official endorsement should be inferredsupported(AJW)by CTSA Grants Number UL1 TR000135 and TL1 TR000137 from the National Center for Advancing Translational Science(NCATS)supported by the Sundt Fellowship fund,Department of Neurological Surgery,Mayo Clinicsupported by the Helene Houle Career Development Award in Neurologic Surgery ResearchFund for the Center for Regenerative Medicine Program Director,Neuroregenerative Medicine,Mayo Clinic
文摘Triple immunofluorescence staining has recently been developed to simultaneously identify all muscle fibers on a single cryosection which is helpful for clinical and basic research, but it has disadvantages such as fast photobleaching and unclear outlines of muscle fibers. Triple immunoenzyme staining(TIE) is likely to avoid these disadvantages. In this study, we aimed to establish a sensitive and specific TIE technique to identify fiber types in normal, denervated, and reinnervated rat muscles, and to develop a systematic sampling method for muscle fiber quantification. Tibialis anterior and soleus from normal, denervated, and reinnervated Lewis rat hind limbs were used. Five consecutive cryosections were cut from each muscle, including one for TIE and four for single immunoenzyme staining(SIE). The TIE was performed using the polymerized reporter enzyme staining system for the first two antigens(A4.74 for My HC-IIA, BA-F8 for My HC-I) and alkaline phosphatase staining system for the third antigen(BF-F3 for My HC-IIB), followed by corresponding detective systems and respective chromogens. The type of muscle fibers was quantified by systematic sampling at 12.5%, 25%, 33% and 50% of all muscle fibers, and was compared with that acquired from counting all the fibers(100%). All muscle fiber phenotypes, including pure and hybrid, could be simultaneously identified on a single TIE cryosection with clear outlines. The fiber types on TIE slides matched well with their respective counterpart on the consecutive SIE slides with a 95% match rate. Systematic sampling of 12.5% fibers could represent the true fiber type distribution of the entire muscle section. Our results suggest that novel TIE can effectively visualize fiber types in normal, denervated or reinnervated rat muscles.
基金supported by Mayo Clinic, USA and Peking University People’s Hospital, Beijing, China
文摘Objective: To examine the role of store-operated calcium entry(SOCE) and stromal interaction molecule 1(STIM1) in survival and migration of osteosarcoma cells and investigate what blockade of store-operated Ca^(2+)contributes to the regulation of osteosarcoma cells.Methods: First, we examined the expression levels of STIM1 in osteosarcoma cell lines by Western analysis and in tissue specimens by immunohistochemistry. Second, we investigated the effect of SOCE and STIM1 on osteosarcoma cell viability using MTS assays and on cell proliferation using colony formation. Third, we investigated the role of SOCE and STIM1 in cell migration using wound healing assays and Boyden chamber assays. Finally, we studied the effect of SOCE on the nuclear factor of activated T-cells cytoplasmic 1(NFATc1)activity by luciferase assays.Results: STIM1 was overexpressed in osteosarcoma cell lines and tissue specimens and was associated with poor survival of osteosarcoma patients. Also, inhibition of SOCE and STIM1 decreased the cell viability and migration of osteosarcoma cells. Furthermore, our results showed that blockade of store-operated Ca^(2+) channels involved down-regulation of NFATc1 in osteosarcoma cells.Conclusions: STIM1 is essential for osteosarcoma cell functions, and STIM1 and Ca^(2+) entry pathway could be further explored as molecular targets in the treatment of osteosarcoma.
文摘Osteosarcoma is a primary bone tumor that affects children and young adults.The estrogen metabolite 2-methoxyestradiol(2-ME)induces cell death in osteosarcoma cells.To determine whether 2-ME actions involve the control of protein synthesis,we studied the effect of 2-ME on eukaryotic initiation factor 4E(eIF4E)and eIF4E-binding protein 1(4E-BP1)in MG63 osteosarcoma cells.Our results show that 2-ME treatment increases the association of eIF4E with 4E-BP1 in osteosarcoma cells.Also,2-ME decreases the binding of eIF4E protein to 7-methyl-guanosine cap structure,indicating that 2-ME treatment results in the inhibition of translational initiation.These findings are further supported by the inhibition of protein synthesis in 2-ME-treated osteosarcoma cells.Taken together,our studies show that 2-ME-mediated antitumor effects in osteosarcoma cells involve the regulation of protein synthesis,and translational machinery could serve as a target in the treatment of osteosarcoma.