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The nigrostriatal dopaminergic (DA) system, which includes DA neurons in the ventral and dorsal tiers of the substantia nigra pars compacta (vSNc, dSNc) and DA terminals in the dorsal striatum, is critically implicated in motor control. Accumulating studies demonstrate that both the nigrostriatal DA system and motor function are impaired in aged subjects. However, it is unknown whether dSNc and vSNc DA neurons and striatal DA terminals age in similar patterns, and whether these changes parallel motor deficits. To address this, we performed ex vivo patch-clamp recordings in dSNc and vSNc DA neurons, measured striatal dopamine release, and analyzed motor behaviors in rodents. Spontaneous firing in dSNc and vSNc DA neurons and depolarization-evoked firing in dSNc DA neurons showed inverse V-shaped changes with age. But depolarization-evoked firing in vSNc DA neurons increased with age. In the dorsal striatum, dopamine release declined with age. In locomotor tests, 12-month-old rodents showed hyperactive exploration, relative to 6- and 24-month-old rodents. Additionally, aged rodents showed significant deficits in coordination. Elevating dopamine levels with a dopamine transporter inhibitor improved both locomotion and coordination. Therefore, key components in the nigrostriatal DA system exhibit distinct aging patterns and may contribute to age-related alterations in locomotion and coordination.

Aim: To investigate the effects of Sonic hedgehog (shh) protein on bone marrow-derived endothelial progenitor cells (BM-EPC) proliferation, migration and vascular endothelial growth factor (VEGF) production, and the potential signaling pathways involved in these effects. Methods: Bone marrow-derived Flk-1 + cells were enriched using the MACS system from adult Kunming mice and then BM-EPC was cultured in gelatin-coated culture dishes. The effects of shh N-terminal peptide on BM-EPC proliferation were evaluated using the MTT colorimetric assay. Cell migration was assayed using a modified Boy den chamber technique. The production of VEGF was determined by ELISA and immunofluorescence analysis. The potential involvement of PKC and PI3K signaling pathways was explored using selective inhibitor or Western blot. Results: The proliferation, migration and VEGF production in BM-EPC could be promoted by endogenous shh N-terminal peptide at concentrations of 0.1 μg/mL to 10 μg/mL, and could be inhibited by anti-shh antibodies. Shh-mediated proliferation and migration in BM-EPC could be partly attenuated by anti-VEGF. Phospho-PI3-kinase expression in newly separated BM-EPC was low, and it increased significantly when exogenous shh N-terminal peptide was added, but could be attenuated by anti-human/mouse shh N-terminal peptide antibody. Moreover, the inhibitor of the PI3-kinase, but not the inhibitor of the PKC, significantly inhibited the shh-mediated proliferation, migration and VEGF production. Conclusion: Shh protein can stimulate bone marrow-derived BM-EPC proliferation, migration and VEGF production, which may promote neovascularization to ischemic tissues. This results also suggests that the PI3-kinase/Akt signaling pathways are involved in the angiogenic effects of shh.

Aim: To study the enzymological characterization of a fibrinolytic enzyme (FII a ) from Agkistrodon acutus venom. Methods: The fibrinogenolytic effect and the influences of several protease inhibitors, chelating agents, and metal ions on fibrinogenolytic activity were visualized by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The metal content of FII a was determined by atomic absorption spectroscopy. Results: After incubation with FII a (0.25 g/L), Aalpha-, Bbeta- and gamma-chains of fibrinogen disappeared within 5 min, 30 min, and 8 h, respectively. The molecular weights of major degradation products were 45 000 and 41 000, which were different from those bands produced by plasmin. The fibrinogenolytic activity of FIIa was strongly inhibited by ethylenediamine tetraacetic acid (EDTA), ethyleneglycol tetraacetic acid (EGTA), dithiothreitol and cysteine, but not by phenylmethyl-sulfonyl fluoride and soybean trypsin inhibitor. Zinc (3171plusminus25 mg/kg), potassium (489plusminus17 mg/kg) and calcium (319plusminus13 mg/kg) were found in FII a . Zn 2+ , Ca 2+ and Mg 2+ could recover the fibrinogenolytic activity of FII a , which was inhibited by EDTA. Only Ca 2+ could recover the fibrinogenolytic activity inhibited by EGTA. Conclusion: FII a can degrade the Aalpha-, Bbeta- and gamma-chains of fibrinogen. FIIa is a metalloproteinase, and Zn 2+ , Ca 2+ , and disulfide bonds are necessary for its fibrinogenolytic activity.

To investigate the effects of Sonic hedgehog (shh) protein on bone marrow- derived endothelial progenitor cells (BM-EPC) proliferation, migration and vascular endothelial growth factor (VEGF) production, and the potential signaling pathways involved in these effects.Bone marrow-derived Flk-1(+) cells were enriched using the MACS system from adult Kunming mice and then BM-EPC was cultured in gelatin-coated culture dishes. The effects of shh N-terminal peptide on BM-EPC proliferation were evaluated using the MTT colorimetric assay. Cell migration was assayed using a modified Boyden chamber technique. The production of VEGF was determined by ELISA and immunofluorescence analysis. The potential involvement of PKC and PI3K signaling pathways was explored using selective inhibitor or Western blot.The proliferation, migration and VEGF production in BM-EPC could be promoted by endogenous shh N-terminal peptide at concentrations of 0.1 microg/mL to 10 microg/mL, and could be inhibited by anti-shh antibodies. Shh-mediated proliferation and migration in BM-EPC could be partly attenuated by anti-VEGF. Phospho-PI3-kinase expression in newly separated BM-EPC was low, and it increased significantly when exogenous shh N-terminal peptide was added, but could be attenuated by anti-human/mouse shh N-terminal peptide antibody. Moreover, the inhibitor of the PI3-kinase, but not the inhibitor of the PKC, significantly inhibited the shh-mediated proliferation, migration and VEGF production.Shh protein can stimulate bone marrow-derived BM-EPC proliferation, migration and VEGF production, which may promote neovascularization to ischemic tissues. This results also suggests that the PI3-kinase/Akt signaling pathways are involved in the angiogenic effects of shh.

To study the enzymological characterization of a fibrinolytic enzyme (FII(a)) from Agkistrodon acutus venom.The fibrinogenolytic effect and the influences of several protease inhibitors, chelating agents, and metal ions on fibrinogenolytic activity were visualized by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The metal content of FII(a) was determined by atomic absorption spectroscopy.After incubation with FII(a) (0.25 g/L), Aalpha-, Bbeta- and gamma-chains of fibrinogen disappeared within 5 min, 30 min, and 8 h , respectively. The molecular weights of major degradation products were 45,000 and 41,000, which were different from those bands produced by plasmin. The fibrinogenolytic activity of FIIa was strongly inhibited by ethylenediamine tetraacetic acid (EDTA), ethyleneglycol tetraacetic acid (EGTA), dithiothreitol and cysteine, but not by phenylmethyl-sulfonyl fluoride and soybean trypsin inhibitor. Zinc (3171+/-25 mg/kg), potassium (489+/-17 mg/kg) and calcium (319+/-13 mg/kg) were found in FIIa. Zn2+, Ca2+ and Mg2+ could recover the fibrinogenolytic activity of FIIa, which was inhibited by EDTA. Only Ca2+ could recover the fibrinogenolytic activity inhibited by EGTA.FIIa can degrade the Aalpha-, Bbeta- and gamma-chains of fibrinogen. FII(a) is a metalloproteinase, and Zn2+, Ca2+, and disulfide bonds are necessary for its fibrinogenolytic activity.

To determine the mechanisms of interactions between different drugs and HERG channels.Various antiarrhythmic (dofetilide, quinidine, azimilide, RP58866) and non-antiarrhythmic (terfenadine, nicotine) agents were used on HERG channels expressed in Xenopus oocyte. Whole-cell voltage-clamp techniques were used.All drugs produced concentration-dependent block of HERG current. The inhibition was markedly facilitated with voltage protocols favoring channel inactivation (eg, less negative holding potentials). Maneuvers that weakened channel inactivation (eg, elevation of external K+), relieved HERG blockade by all drugs. Moreover, the inhibitory potency was reduced by at least 20-300 fold with varying compounds when rapid C-type inactivation was removed by a mutation located between the transmembrane domains 5 and 6 (S631A).The inactivation gating of HERG channels determines the blocking potency of drugs. This mechanism might be common to drugs of various classes.