Objective To provide the acute toxicity data of hygromycin B phosphotransferase (HPT) using recombinant protein purified from E. coli. Methods Recombinant HPT protein was expressed and purified from E. coli. To exclud...Objective To provide the acute toxicity data of hygromycin B phosphotransferase (HPT) using recombinant protein purified from E. coli. Methods Recombinant HPT protein was expressed and purified from E. coli. To exclude the potential adverse effect of bacteria protein in recombinant HPT protein, bacterial control plasmid was constructed, and bacteria control protein was extracted and prepared as recombinant HPT protein. One hundred mice, randomly assigned to 5 groups, were administrated 10 g/kg, 5 g/kg, or 1 g/kg body weight of HPT or 5 g/kg body weight of bacterial control protein or phosphate-buffered saline (PBS) respectively by oral gavage. Results All animals survived with no significant change in body weight gain throughout the study. Macroscopic necropsy examination on day 15 revealed no gross pathological lesions in any of the animals. The maximum tolerated dose (MTD) of HPT was 10 g/kg body weight in mice and could be regarded as nontoxic. Conclusion HPT protein does not have any safety problems to human health.展开更多
Recognizing the composition and modulation of the microbiome, a viable therapeutic tool for multi-targeted therapy is a new strategy that has recently been explored. Glucosamine (GS) is being studied for its prebiotic...Recognizing the composition and modulation of the microbiome, a viable therapeutic tool for multi-targeted therapy is a new strategy that has recently been explored. Glucosamine (GS) is being studied for its prebiotic potential in addition to being the most abundant and naturally occurring amino monosaccharide. The current study focuses on glucosamine’s prebiotic potential by assessing the stability of various GS concentrations (1% - 5%) in the gastrointestinal tract (GIT) and its ability to be fermented by the gut microbiota. The results showed that GS stimulated the most growth in L. acidophilus even after a longer incubation time than B. bifidum and L. acidophilus growth was concentration-dependent, with maximum growth at 3% with a simultaneous decrease in pH (5.6 - 1.7). The decrease in GS concentration with time also represented the growth of bacterial species, demonstrating the species’ utilization of GS. Furthermore, at 3%, GS also represented the prebiotic index of 1.9. In addition, the concentration of GS in various simulated GIT fluids was estimated in both fast and fed conditions to examine GS stability at various levels in the gut. The results showed that GS remained unaffected and non-digestible in all of the simulated GIT fluids (salivary, gastric, intestinal, and colonic), but there was a slight decrease in GS concentration (2.8%) in the fasted state of gastric fluid due to low pH levels (1.6). As a result, the findings are conclusive and suggest that GS possesses prebiotic properties.展开更多
Inositol pyrophosphates(PP-InsPs)are important signaling molecules that regulate diverse cellular processes in eukaryotes,including energy homeostasis,phosphate(Pi)signaling,and phytohormone perception.Yet,in plants,t...Inositol pyrophosphates(PP-InsPs)are important signaling molecules that regulate diverse cellular processes in eukaryotes,including energy homeostasis,phosphate(Pi)signaling,and phytohormone perception.Yet,in plants,the enzymes responsible for their turnover remain largely unknown.Using a non-hydrolysable PP-InsP analog in a pull-down approach,we identified a family of Arabidopsis NUDIX-type hydrolases(NUDTs)that group into two closely related subclades.Through in vitro assays,heterologous expression systems,and higher order gene-edited mutants,we explored the substrate specificities and physiological roles of these hydrolases.Using a combination of strong anion exchange high-performance liquid chromatography(SAX-HPLC),polyacrylamide gel electrophoresis(PAGE),and capillary electrophoresis electrospray ionization mass spectrometry(CE-ESI-MS),we found that their PP-InsP pyrophosphatase activity is enantiomer selective and Mg^(2+)dependent.Specifically,SubcladeⅠNUDTs preferentially hydrolyze 4-InsP_(7),while SubcladeⅡNUDTs target 3-InsP_(7),with minor activity against other PP-InsPs,including5-InsP_(7).In higher order mutants of SubcladeⅡNUDTs,we observed defects in both Piand iron homeostasis,accompanied by increased levels of 1/3-InsP_(7)and 5-InsP_(7),with a markedly larger increase in 1/3-InsP_(7).Ectopic expression of NUDTs from both subclades induced local Pi starvation responses(PSRs),while RNA-seq analysis comparing wild-type(WT)and SubcladeⅡnudt12/13/16 loss-of-function plants indicates additional PSR-independent roles,potentially involving 1/3-InsP_(7) in the regulation of plant defense.Consistently,nudt12/13/16 mutants displayed enhanced resistance to Pseudomonas syringae infection,indicating a role in bacterial pathogen susceptibility.Expanding beyond SubcladeⅡNUDTs,we demonstrated susceptibility of the 3PP-position of PP-InsPs to enzymatic activities unrelated to NUDTs,and found that such activities are conserved across plants and humans.Additionally,we observed that NUDT effectors from pathogenic ascomycete fungi exhibit a substrate specificity similar to SubcladeⅠNUDTs.Collectively,our findings reveal new roles for NUDTs in PP-InsP signaling,plant nutrient and immune responses,and highlight a cross-kingdom conservation of PP-InsP-metabolizing enzymes.展开更多
OBJECTIVE: To study the changes in activity of phosphatidylinositol 4 kinase (PI 4 kinase), phosphatidylinositol 4 phosphate 5 kinase (PIP 5 kinase) and protein kinase C (PKC) during myocardial ischemia and elucidate ...OBJECTIVE: To study the changes in activity of phosphatidylinositol 4 kinase (PI 4 kinase), phosphatidylinositol 4 phosphate 5 kinase (PIP 5 kinase) and protein kinase C (PKC) during myocardial ischemia and elucidate the relationship between phosphatidylinositol signal pathways and prolonged myocardial ischemia. METHODS: In vivo an ischemic rat model was used. Activity of PI 4 kinase, PIP 5 kinase and PKC were measured at different times in postischemic heart cells using isotope analysis. RESULTS: The activity of PI kinase, PIP kinase and PKC in the myocardium increased to peak at 1 hour postischemia, with activities 6.1, 3.0 and 4.0 fold over control levels, respectively. Their activities declined to normal levels with time. CONCLUSION: The phosphatidylinositol signal pathway is involved in prolonged myocardial ischemia, but its mechanism needs further study.展开更多
基金supported by 973 Project(No.2007CB 109207)of Ministry of Science and Technology of China.
文摘Objective To provide the acute toxicity data of hygromycin B phosphotransferase (HPT) using recombinant protein purified from E. coli. Methods Recombinant HPT protein was expressed and purified from E. coli. To exclude the potential adverse effect of bacteria protein in recombinant HPT protein, bacterial control plasmid was constructed, and bacteria control protein was extracted and prepared as recombinant HPT protein. One hundred mice, randomly assigned to 5 groups, were administrated 10 g/kg, 5 g/kg, or 1 g/kg body weight of HPT or 5 g/kg body weight of bacterial control protein or phosphate-buffered saline (PBS) respectively by oral gavage. Results All animals survived with no significant change in body weight gain throughout the study. Macroscopic necropsy examination on day 15 revealed no gross pathological lesions in any of the animals. The maximum tolerated dose (MTD) of HPT was 10 g/kg body weight in mice and could be regarded as nontoxic. Conclusion HPT protein does not have any safety problems to human health.
文摘Recognizing the composition and modulation of the microbiome, a viable therapeutic tool for multi-targeted therapy is a new strategy that has recently been explored. Glucosamine (GS) is being studied for its prebiotic potential in addition to being the most abundant and naturally occurring amino monosaccharide. The current study focuses on glucosamine’s prebiotic potential by assessing the stability of various GS concentrations (1% - 5%) in the gastrointestinal tract (GIT) and its ability to be fermented by the gut microbiota. The results showed that GS stimulated the most growth in L. acidophilus even after a longer incubation time than B. bifidum and L. acidophilus growth was concentration-dependent, with maximum growth at 3% with a simultaneous decrease in pH (5.6 - 1.7). The decrease in GS concentration with time also represented the growth of bacterial species, demonstrating the species’ utilization of GS. Furthermore, at 3%, GS also represented the prebiotic index of 1.9. In addition, the concentration of GS in various simulated GIT fluids was estimated in both fast and fed conditions to examine GS stability at various levels in the gut. The results showed that GS remained unaffected and non-digestible in all of the simulated GIT fluids (salivary, gastric, intestinal, and colonic), but there was a slight decrease in GS concentration (2.8%) in the fasted state of gastric fluid due to low pH levels (1.6). As a result, the findings are conclusive and suggest that GS possesses prebiotic properties.
基金funded by grants from the Deutsche Forschungsgemeinschaft(SCHA 1274/4-1,SCHA 1274/5-1,and under Germany's Excellence Strategy,EXC2070–390732324,Pheno Rob to G.S.,JE 572/4-1 and under Germany's Excellence Strategy,CIBSS–EXC-2189–Project ID 390939984 to H.J.J.,LA 1338/18-1 to T.L.,and TRR356/I(491090170),TP-B08 and Project No.451218338 to M.K.R.-L.)by the Marie Sklodowska-Curie Action(Grant Agreement ID 101108767)to S.W.by the Department of Biotechnology,Government of India(Grant No.BT/PR45561/AGIII/103/1386/2023)to S.B.Open Access funding enabled and organized by Projekt DEAL
文摘Inositol pyrophosphates(PP-InsPs)are important signaling molecules that regulate diverse cellular processes in eukaryotes,including energy homeostasis,phosphate(Pi)signaling,and phytohormone perception.Yet,in plants,the enzymes responsible for their turnover remain largely unknown.Using a non-hydrolysable PP-InsP analog in a pull-down approach,we identified a family of Arabidopsis NUDIX-type hydrolases(NUDTs)that group into two closely related subclades.Through in vitro assays,heterologous expression systems,and higher order gene-edited mutants,we explored the substrate specificities and physiological roles of these hydrolases.Using a combination of strong anion exchange high-performance liquid chromatography(SAX-HPLC),polyacrylamide gel electrophoresis(PAGE),and capillary electrophoresis electrospray ionization mass spectrometry(CE-ESI-MS),we found that their PP-InsP pyrophosphatase activity is enantiomer selective and Mg^(2+)dependent.Specifically,SubcladeⅠNUDTs preferentially hydrolyze 4-InsP_(7),while SubcladeⅡNUDTs target 3-InsP_(7),with minor activity against other PP-InsPs,including5-InsP_(7).In higher order mutants of SubcladeⅡNUDTs,we observed defects in both Piand iron homeostasis,accompanied by increased levels of 1/3-InsP_(7)and 5-InsP_(7),with a markedly larger increase in 1/3-InsP_(7).Ectopic expression of NUDTs from both subclades induced local Pi starvation responses(PSRs),while RNA-seq analysis comparing wild-type(WT)and SubcladeⅡnudt12/13/16 loss-of-function plants indicates additional PSR-independent roles,potentially involving 1/3-InsP_(7) in the regulation of plant defense.Consistently,nudt12/13/16 mutants displayed enhanced resistance to Pseudomonas syringae infection,indicating a role in bacterial pathogen susceptibility.Expanding beyond SubcladeⅡNUDTs,we demonstrated susceptibility of the 3PP-position of PP-InsPs to enzymatic activities unrelated to NUDTs,and found that such activities are conserved across plants and humans.Additionally,we observed that NUDT effectors from pathogenic ascomycete fungi exhibit a substrate specificity similar to SubcladeⅠNUDTs.Collectively,our findings reveal new roles for NUDTs in PP-InsP signaling,plant nutrient and immune responses,and highlight a cross-kingdom conservation of PP-InsP-metabolizing enzymes.
基金the National Great Foundamental Research Proiect (No．G2000057004) the National Nature Science Foundation of China(No．19890308)．
文摘OBJECTIVE: To study the changes in activity of phosphatidylinositol 4 kinase (PI 4 kinase), phosphatidylinositol 4 phosphate 5 kinase (PIP 5 kinase) and protein kinase C (PKC) during myocardial ischemia and elucidate the relationship between phosphatidylinositol signal pathways and prolonged myocardial ischemia. METHODS: In vivo an ischemic rat model was used. Activity of PI 4 kinase, PIP 5 kinase and PKC were measured at different times in postischemic heart cells using isotope analysis. RESULTS: The activity of PI kinase, PIP kinase and PKC in the myocardium increased to peak at 1 hour postischemia, with activities 6.1, 3.0 and 4.0 fold over control levels, respectively. Their activities declined to normal levels with time. CONCLUSION: The phosphatidylinositol signal pathway is involved in prolonged myocardial ischemia, but its mechanism needs further study.
