Detailed mechanisms behind regeneration after nerve injury, in particular signal transduction and the fate of Schwann cells （SCs）, are poorly understood. Here, we investigated axotomy-induced activation of extracell...Detailed mechanisms behind regeneration after nerve injury, in particular signal transduction and the fate of Schwann cells （SCs）, are poorly understood. Here, we investigated axotomy-induced activation of extracellular- signal-regulated kinase-1/2 （ERK1/2; important for proliferation） and m-calpain in vitro, and the relation to Ca2＋ deletion and Schwann cell proliferation and death after rat sciatic nerve axotomy. Nerve segments were cultured for up to 72 hours with and without ethylene glycol-bis（β-aminoethyl ether）- N,N,N＇,N＇-tetraacetic acid （EGTA）. In some experiments, 5-bromo-2′-deoxyuridine （BrdU） was added during the last 24 hours to detect proliferating cells and propidium iodide （PI） was added at the last hour to detect dead and/or dying cells. Immunohistochemistry of sections of the cultured nerve segments was performed to label m-calpain and the phosphorylated and activated form of ERK1/2. The experiments revealed that immunoreactivity for p-ERK1/2 increased with time in organotypically cultured SCs. p-ERK1/2 and m-calpain were also observed in axons. A significant increase in the number of dead or dying SCs was observed in nerve segments cultured for 24 hours. When deprived of Ca2＋, activation of axonal m-calpain was reduced, whereas p-ERK1/2 was increased in SCs. Ca2＋ deprivation also significantly reduced the number of proliferating SCs, and instead increased the number of dead or dying SCs. Ca2＋ seems to play an important role in activation of ERK1/2 in SCs and in SC survival and proliferation. In addition, extracellular Ca2＋ levels are also required for m-calpain activation and up-regulation in axons. Thus, regulation of Ca2＋ levels is likely to be a useful method to promote SC proliferation.展开更多
The economic value of a black walnut (Juglans nigra L.) tree is strongly determined by the quality and quantity of darkly colored heartwood in its stem. To understand the regulation of heartwood formation, we analyzed...The economic value of a black walnut (Juglans nigra L.) tree is strongly determined by the quality and quantity of darkly colored heartwood in its stem. To understand the regulation of heartwood formation, we analyzed the region of heartwood formation in walnut stems (i.e., the transition zone, TZ) for the expression of 80 ESTs. Semi-quantitative RT-PCR and real-time PCR was performed to detect expression changes of candidate genes in the TZ and sapwood of trees harvested in summer and fall. Results revealed that the transcript of a clone containing two presumed EF-hand motifs was expressed at higher levels in the TZ than in other xylem tissues. Analysis of the full-length coding sequence revealed that the black walnut gene JnCML-like is similar to grancalcin-like calcium-binding EF hand proteins in Arabidopsis thaliana (At3g10300) and Zea mays (NM 001153810). A model of the predicted structure of JnCML-like showed it is similar to grancalcin and m-calpain, penta-EF-hand family proteins associated with cell proliferation, differentiation and programmed cell death. JnCML-like transcript was detected in tissue from the region of the pith meristem, and in roots, embryogenic callus, vascular cambium, female flowers, male flowers, green leaves, and partially and fully senescent leaves of black walnut, although transcript abundance varied considerably among these tissues.展开更多
Purpose: The aim of the study is to reveal associations between NF-κB, HIF-1alpha, VEGF expres-sions, proteasome and calpain activities with tumor progression in patients with kidney cancers and to find molecular par...Purpose: The aim of the study is to reveal associations between NF-κB, HIF-1alpha, VEGF expres-sions, proteasome and calpain activities with tumor progression in patients with kidney cancers and to find molecular parameters, associated with the effective pazopanib therapy. 93 patients with clear cell kidney cancers are included in investigation. 26 patients with disseminated kidney cancer have the pazopanib therapy. Methods: Transcription factors, VEGF, VEGFR2 and p-m-TOR expression are measured by ELISA kits. Proteasome and calpain activity are determined using specific fluorogenic substrate. Results: It is found the increase of NF-κB, HIF-1 expression in cancer tissues followed the hematogenic metastasis development. Coefficient NF-κB р65/р50 and VEGF expression are increased in cancer tissues with single metastasis and are decreased in cancer tissues with multiple ones. It is observed in the low proteasome activity in metastatic cancer tissues. The partial cancer regression is revealed in 29.6% of patients treated with pazopanib, cancer stabilization—in 61.5% of patients and cancer progression—in 11.5% of patients. The increased level of transcription factors NF-κB, HIF-1, growth factor VEGF and high proteasome activity in cancer tissues before targeted therapy are associated with the effective treatment. It is obtained the significant decrease of investigated markers after pazopanib application in metastatic kidney cancer patients. Conclusion: Coefficient NF-κB р65/р50, VEGF expression and proteases activities are the potential prognostic molecular markers of hematogenic metastasis development in kidney cancers. NF-κB, HIF-1 and VEGF levels can be considered as additional molecular markers predicting the effective pazopanib therapy.展开更多
Virtual screening can be a helpful approach to propose treatments for COVID-19 by developing inhibitors for blocking the attachment of the virus to human cells. This study uses molecular docking, recovery time and dyn...Virtual screening can be a helpful approach to propose treatments for COVID-19 by developing inhibitors for blocking the attachment of the virus to human cells. This study uses molecular docking, recovery time and dynamics to analyze if potential inhibitors of main protease (M<sup>pro</sup>) of SARS-CoV-2 can interfere in the attachment of nanobodies, specifically Nb20, in the receptor binding domain (RBD) of SARS-CoV-2. The potential inhibitors are four compounds previously identified in a fluorescence resonance energy transfer (FRET)-based enzymatic assay for the SARS-CoV-2 M<sup>pro</sup>: Boceprevir, Calpain Inhibitor II, Calpain Inhibitor XII, and GC376. The findings reveal that Boceprevir has the higher affinity with the RBD/Nb20 complex, followed by Calpain Inhibitor XII, GC376 and Calpain Inhibitor II. The recovery time indicates that the RBD/Nb20 complex needs a relatively short time to return to what it was before the presence of the ligands. For the RMSD the Boceprevir and Calpain Inhibitor II have the shortest interaction times, while Calpain Inhibitor XII shows slightly more interaction, but with significant pose fluctuations. On the other hand, GC376 remains stably bound for a longer duration compared to the other compounds, suggesting that they can potentially interfere with the neutralization process of Nb20.展开更多
基金supported by the Research School in Pharmaceutical Science in Lund,The Royal Physiographic Society in LundThe Swedish Research Council(Medicine)+1 种基金the Craaford’s and Thure Nilsson’s Funds for Medical ResearchFunds for diabetic research,Lund University and Region Skane
文摘Detailed mechanisms behind regeneration after nerve injury, in particular signal transduction and the fate of Schwann cells （SCs）, are poorly understood. Here, we investigated axotomy-induced activation of extracellular- signal-regulated kinase-1/2 （ERK1/2; important for proliferation） and m-calpain in vitro, and the relation to Ca2＋ deletion and Schwann cell proliferation and death after rat sciatic nerve axotomy. Nerve segments were cultured for up to 72 hours with and without ethylene glycol-bis（β-aminoethyl ether）- N,N,N＇,N＇-tetraacetic acid （EGTA）. In some experiments, 5-bromo-2′-deoxyuridine （BrdU） was added during the last 24 hours to detect proliferating cells and propidium iodide （PI） was added at the last hour to detect dead and/or dying cells. Immunohistochemistry of sections of the cultured nerve segments was performed to label m-calpain and the phosphorylated and activated form of ERK1/2. The experiments revealed that immunoreactivity for p-ERK1/2 increased with time in organotypically cultured SCs. p-ERK1/2 and m-calpain were also observed in axons. A significant increase in the number of dead or dying SCs was observed in nerve segments cultured for 24 hours. When deprived of Ca2＋, activation of axonal m-calpain was reduced, whereas p-ERK1/2 was increased in SCs. Ca2＋ deprivation also significantly reduced the number of proliferating SCs, and instead increased the number of dead or dying SCs. Ca2＋ seems to play an important role in activation of ERK1/2 in SCs and in SC survival and proliferation. In addition, extracellular Ca2＋ levels are also required for m-calpain activation and up-regulation in axons. Thus, regulation of Ca2＋ levels is likely to be a useful method to promote SC proliferation.
文摘The economic value of a black walnut (Juglans nigra L.) tree is strongly determined by the quality and quantity of darkly colored heartwood in its stem. To understand the regulation of heartwood formation, we analyzed the region of heartwood formation in walnut stems (i.e., the transition zone, TZ) for the expression of 80 ESTs. Semi-quantitative RT-PCR and real-time PCR was performed to detect expression changes of candidate genes in the TZ and sapwood of trees harvested in summer and fall. Results revealed that the transcript of a clone containing two presumed EF-hand motifs was expressed at higher levels in the TZ than in other xylem tissues. Analysis of the full-length coding sequence revealed that the black walnut gene JnCML-like is similar to grancalcin-like calcium-binding EF hand proteins in Arabidopsis thaliana (At3g10300) and Zea mays (NM 001153810). A model of the predicted structure of JnCML-like showed it is similar to grancalcin and m-calpain, penta-EF-hand family proteins associated with cell proliferation, differentiation and programmed cell death. JnCML-like transcript was detected in tissue from the region of the pith meristem, and in roots, embryogenic callus, vascular cambium, female flowers, male flowers, green leaves, and partially and fully senescent leaves of black walnut, although transcript abundance varied considerably among these tissues.
文摘Purpose: The aim of the study is to reveal associations between NF-κB, HIF-1alpha, VEGF expres-sions, proteasome and calpain activities with tumor progression in patients with kidney cancers and to find molecular parameters, associated with the effective pazopanib therapy. 93 patients with clear cell kidney cancers are included in investigation. 26 patients with disseminated kidney cancer have the pazopanib therapy. Methods: Transcription factors, VEGF, VEGFR2 and p-m-TOR expression are measured by ELISA kits. Proteasome and calpain activity are determined using specific fluorogenic substrate. Results: It is found the increase of NF-κB, HIF-1 expression in cancer tissues followed the hematogenic metastasis development. Coefficient NF-κB р65/р50 and VEGF expression are increased in cancer tissues with single metastasis and are decreased in cancer tissues with multiple ones. It is observed in the low proteasome activity in metastatic cancer tissues. The partial cancer regression is revealed in 29.6% of patients treated with pazopanib, cancer stabilization—in 61.5% of patients and cancer progression—in 11.5% of patients. The increased level of transcription factors NF-κB, HIF-1, growth factor VEGF and high proteasome activity in cancer tissues before targeted therapy are associated with the effective treatment. It is obtained the significant decrease of investigated markers after pazopanib application in metastatic kidney cancer patients. Conclusion: Coefficient NF-κB р65/р50, VEGF expression and proteases activities are the potential prognostic molecular markers of hematogenic metastasis development in kidney cancers. NF-κB, HIF-1 and VEGF levels can be considered as additional molecular markers predicting the effective pazopanib therapy.
文摘Virtual screening can be a helpful approach to propose treatments for COVID-19 by developing inhibitors for blocking the attachment of the virus to human cells. This study uses molecular docking, recovery time and dynamics to analyze if potential inhibitors of main protease (M<sup>pro</sup>) of SARS-CoV-2 can interfere in the attachment of nanobodies, specifically Nb20, in the receptor binding domain (RBD) of SARS-CoV-2. The potential inhibitors are four compounds previously identified in a fluorescence resonance energy transfer (FRET)-based enzymatic assay for the SARS-CoV-2 M<sup>pro</sup>: Boceprevir, Calpain Inhibitor II, Calpain Inhibitor XII, and GC376. The findings reveal that Boceprevir has the higher affinity with the RBD/Nb20 complex, followed by Calpain Inhibitor XII, GC376 and Calpain Inhibitor II. The recovery time indicates that the RBD/Nb20 complex needs a relatively short time to return to what it was before the presence of the ligands. For the RMSD the Boceprevir and Calpain Inhibitor II have the shortest interaction times, while Calpain Inhibitor XII shows slightly more interaction, but with significant pose fluctuations. On the other hand, GC376 remains stably bound for a longer duration compared to the other compounds, suggesting that they can potentially interfere with the neutralization process of Nb20.
