Phytases belong to the class of phosphohydrolases that begin the step-wise hydrolysis of phosphates from phytates. Phytates are a derivative of myo-inositol, which is the primary storage form of organic phosphorus in ...Phytases belong to the class of phosphohydrolases that begin the step-wise hydrolysis of phosphates from phytates. Phytates are a derivative of myo-inositol, which is the primary storage form of organic phosphorus in plant cells. Phytase has been used globally to diminish phosphorus pollution and to enhance nutrition in monogastrics. In this review, the classification, sources, and diversity of microbial phytases, and their practical applications, as well as supplementation of the soil with transgenic and wild types of microbial strains, which can release phytase to enhance phosphorus availability for plant uptake and reduce the need for fertilizers, are discussed. The overexpressed microbial phytases in transgenic plants enhance the growth capacity of co-cultivated plants and can therefore be employed in agricultural and biotechnological practices, such as intercropping. The introduction of phytases into the soil for improved plant growth and enhanced crop yield can be accomplished without extra cost. A diverse group of photoautotrophic microalgae can synthesize phytase and will likely be useful in many human food and animal industries.展开更多
本文报道了1例利用微滴式数字聚合酶链反应(droplet digital polymerase chain reaction,ddPCR)技术无创产前检测MFN2基因变异致胎儿腓骨肌萎缩症的病例。先证者为孕妇配偶,外院基因检测结果为MFN2基因(NM_014874)c.919A>G(p.K307E)...本文报道了1例利用微滴式数字聚合酶链反应(droplet digital polymerase chain reaction,ddPCR)技术无创产前检测MFN2基因变异致胎儿腓骨肌萎缩症的病例。先证者为孕妇配偶,外院基因检测结果为MFN2基因(NM_014874)c.919A>G(p.K307E)杂合变异,诊断为轴索型腓骨肌萎缩症2A2A型。孕妇孕8周采集外周血提取胎儿游离DNA,采用ddPCR技术进行无创产前检测,检出胎儿携带父源性致病基因变异位点。孕11周采集绒毛进行Sanger测序验证,证实胎儿携带与父亲一致的c.919A>G(p.K307E)杂合变异。由本例提示ddPCR技术也许可以用于无创产前检测孕妇外周血中胎儿游离DNA是否携带父源性致病基因变异。展开更多
Lipid biosynthesis is essential for eukaryotic cells, but the mechanisms of the process in microalgae remain poorly understood. Phosphatidic acid phosphohydrolase or 3-sn-phosphatidate phosphohydrolase(PAP) catalyzes ...Lipid biosynthesis is essential for eukaryotic cells, but the mechanisms of the process in microalgae remain poorly understood. Phosphatidic acid phosphohydrolase or 3-sn-phosphatidate phosphohydrolase(PAP) catalyzes the dephosphorylation of phosphatidic acid to form diacylglycerols and inorganic orthophosphates. This reaction is integral in the synthesis of triacylglycerols. In this study, the mRNA level of the PAP isoform CrPAP2 in a species of Chlamydomonas was found to increase in nitrogen-free conditions. Silencing of the CrPAP2 gene using RNA interference resulted in the decline of lipid content by 2.4%–17.4%. By contrast, over-expression of the CrPAP2 gene resulted in an increase in lipid content by 7.5%–21.8%. These observations indicate that regulation of the CrPAP2 gene can control the lipid content of the algal cells. In vitro CrPAP2 enzyme activity assay indicated that the cloned CrPAP2 gene exhibited biological activities.展开更多
OBJECTIVE:To study whether Shenshuai recipe(肾衰方,SSR)can play a protective role on chronic kidney disease myocardial injury model through phosphatase and tensin homolog-induced putative kinase 1(PINK1)/E3 ubiquitin ...OBJECTIVE:To study whether Shenshuai recipe(肾衰方,SSR)can play a protective role on chronic kidney disease myocardial injury model through phosphatase and tensin homolog-induced putative kinase 1(PINK1)/E3 ubiquitin ligase Parkin(Parkin)mitochondrial autophagy pathway.METHODS:Forty-eight nephrectomized rats were randomly divided into six groups:sham-operated group,model group,Benazepril group,low,medium and highdose groups of SSR.The rats were given the corresponding intervention for six weeks,then were sacrificed.Serum was examined by enzyme linked immunosorbent assay(ELISA).Cardiac ultrasound was used to detect cardiac function in 5/6 nephrectomized rats.Myocardial tissue was examined by light and electron microscopy;PINK1,Parkin,microtubule-associated protein 1 light chain 3 II(LC3B),sequestosome 1(P62),BECN1(Beclin-1)and dynamin-related protein 1(Drp-1)were measured by real time polymerase chain reaction(RT-PCR),Western blot(WB)and immunohistochemistry(IHC).RESULTS:The expression levels of blood urea nitrogen(BUN)and creatinine(SCr)in the model group were significantly higher than those in the sham-operated group,indicating that modeling was successful.SSR can protect myocardium by reducing the relative expression of creatine kinase myocardial isoenzyme and hypersensitivity cardiac troponin I(P<0.05).SSR can improve cardiac function in rats after ultrasound testing.SSR can improve the pathological manifestations of myocardial tissue after Masson staining.SSR can increase the number of autophagosomes and autophagiclysosomes in 5/6 nephrectomized rats(P<0.05).Determined by RT-PCR,WB and IHC,SSR can increase the relative expression of PINK1,Parkin,and LC3B(P<0.05),and decrease the relative expression of P62,Beclin-1 and Drp-1(P<0.05).CONCLUSIONS:The PINK1/Parkin mitochondrial autophagy pathway in myocardial tissues in 5/6 nephrectomy CKD myocardial injury rats was inhibited.SSR can activate PINK1/Parkin mitochondrial autophagy to enhance mitochondrial autophagy,and play a protective role in myocardial tissue展开更多
OBJECTIVE:To investigate the effect of acupotomy,on mitophagy and the Pink1-Parkin pathway in chondrocytes from rabbits with knee osteoarthritis(KOA).METHODS:A KOA model was established via the modified Videman method...OBJECTIVE:To investigate the effect of acupotomy,on mitophagy and the Pink1-Parkin pathway in chondrocytes from rabbits with knee osteoarthritis(KOA).METHODS:A KOA model was established via the modified Videman method.Rabbits were randomly divided into a control group(CON),KOA group and KOA+acupotomy group(Acu).Rabbits in the acupotomy group were subjected to acupotomy for 4 weeks after model establishment.The behavior of the rabbits before and after intervention was recorded.Cartilage degeneration was evaluated by optical microscopy and fluorescence microscopy.The level of mitophagy was evaluated by transmission electron microscopy,immunofluorescence and enzyme-linked immunosorbent assay(ELISA).The expression of phosphatase and tensin homolog(PTEN)-induced kinase 1(Pink1)-Parkin mitophagy pathway components was evaluated by immunofluorescence,Western blotting and real-time polymerase chain reaction.RESULTS:In rabbits with KOA,joint pain,mobility disorders and cartilage degeneration were observed,the Mankin score was increased,collagen typeⅡ(Col-Ⅱ)expression was significantly decreased,mitophagy was inhibited,mitochondrial function was impaired,and factors associated with the Pink1-Parkin pathway were inhibited.Acupotomy regulated the expression of Pink1-Parkin pathway-related proteins,the mitophagy-related protein microtubule-associated protein-1 light chain-3,the translocase of the outer membrane,and the inner mitochondrial membrane 23;increased the colocalization of mitochondria and autophagosomes;promoted the removal of damaged mitochondria;restored mitochondrial adenosine-triphosphate(ATP)production;and alleviated cartilage degeneration in rabbits with KOA.CONCLUSIONS:Acupotomy played a role in alleviating KOA in rabbits by activating mitophagy in chondrocytes via the regulation of proteins that are related to the Pink1-Parkin pathway.展开更多
Phosphatidic acid phosphohydrolase (PAP), EC 3.1.3.4, is the penultimate step in the Kennedy pathway of triacyl glycerol (TAG) synthesis leading to the formation of diacylglycerol (DAG), which is a key intermediate in...Phosphatidic acid phosphohydrolase (PAP), EC 3.1.3.4, is the penultimate step in the Kennedy pathway of triacyl glycerol (TAG) synthesis leading to the formation of diacylglycerol (DAG), which is a key intermediate in TAG synthesis. We partially purified a soluble PAP from mid maturing seeds of bottle gourd, Lagenaria siceraria. The steps include both anionic and cationic ion exchanger columns. Catalytic characterization of the partially purified PAP revealed that the optimum pH and temperature for activity were at 5.5?C and 45?C. Under optimum assay condition using dioleoyl phosphatidic acid (DPA) as the substrate, the Vmax and Km were 0.36 ηkat/mg of protein and 200 μM, respectively. For the synthetic substrate, ρ-nitrophenylphosphate, ρ-NPP, the Vmax and Km were 33.0 nkat/mg of protein and 140 μM, respectively. The activity was neither inhibited nor enhanced by the presence of Mg2+ at a concentration range of 0 to 10 mM. Two major protein bands at 42-kDa and 27-kDa were visible in SDS-PAGE after partial purification. Bioinformatics analysis of tryp-sinized protein fractions containing PAP activity showed peptide sequences with sequence homology to various phosphate metabolizing enzymes including cucumber and castor bean purple acid phosphatase, polyphosphate kinase, fructose biphosphate aldolase, and enolase from various dicotyledonous plants including rice, corn, grape, and Arabidopsis lyrata.展开更多
Phosphatidic acid phosphohydrolase (3-sn-phosphatidate phosphohydrolase, EC 3.1.3.4), also known as PAP, catalyzes the dephosphorylation of phosphatidic acid (PtdOH) to form diacylglycerol (DAG) and inorganic orthopho...Phosphatidic acid phosphohydrolase (3-sn-phosphatidate phosphohydrolase, EC 3.1.3.4), also known as PAP, catalyzes the dephosphorylation of phosphatidic acid (PtdOH) to form diacylglycerol (DAG) and inorganic orthophosphate. In eukaryotes, the PAP driven reaction is the committed step in the synthesis of triacylglycerol (TAG). Existing methods for measuring PAP activity rely on the use of radioactive PtdOH. These methods are costly and cumbersome. In this report, we describe a simple assay procedure to measure released inorganic orthophosphate, which is a coproduct of the PAP reaction. Each molecule of PtdOH would release one molecule of DAG and one molecule of inorganic orthophosphate (Pi) when subjected to enzymatic breakdown under optimal conditions. Given the published rates of in vitro PAP enzymatic activity from various sources, we proposed that colorimetric determination of released Pi is possible. With this view, we performed in vitro PAP activity assays using freshly isolated enzyme from bitter gourd, Momordica charantia, and measured the released Pi using two spectrophotometric methods. Both methods gave about 2.0 to 2.25 ηkat per mg of protein. Thus, it is now possible to perform PAP activity using a simple procedure that uses nonradioactive substrates, provided the sample is dialyzed extensively to lower the intrinsic concentration of free phosphate. The kinetics data presented in this study is comparable to that of other PAP enzymes reported elsewhere, which gives credence to the notion that non-radioactive methods can be used to perform PAP activity.展开更多
OBJECTIVE: To observe the inhibitory effect of the Chinese herbal compound Guizhu capsule(CGZC)on lung cancer and explore the possible mechanism underlying its actions by evaluating its effects on the expression of ph...OBJECTIVE: To observe the inhibitory effect of the Chinese herbal compound Guizhu capsule(CGZC)on lung cancer and explore the possible mechanism underlying its actions by evaluating its effects on the expression of phosphate and tension homology deleted on chromsome ten(PTEN) and murine double mimute 2(MDM2) in lung cancer model mice.METHODS: A mouse model of transplanted lung cancer was established by subcutaneous inoculation of cancer cells into the axillae of mice. Twenty-four hours later, the mice were weighed and randomly divided into the model control group, cisplatin group(DDP group), and high, moderate and low dosage CGZC groups, with ten mice in each group. The control mice received an equal volume of distilled water. DDP was intraperitoneally injected at 1 mg/kg in the DDP group, once a day for 3days. CGZC diluted with distilled water was admin-istered at 20 g/kg(10 times the clinical adult dosage) in the high-dose group, 10 g/kg(5 times the clinical adult dosage) in the moderate-dose group and 5 g/kg(2.5 times the clinical adult dosage) in the low-dose group once a day for 10 days. On the11 th day, the mice were weighed and killed. The tumor tissues were weighed and the tumor inhibition rate was calculated. PTEN and MDM2 protein expression were detected by immunohistochemical analysis of tumor tissues.RESULTS: The CGZC high-and moderate-dose groups showed a significant inhibitory effect on tumor growth(P < 0.05); there was no significant difference between the high dose group and the DDP group(P > 0.05). The CGZC high-dose group also showed enhanced expression of PTEN protein(P <0.01) and decreased expression of MDM2 protein(P < 0.01) in lung cancer cells of the mice. There was no significant difference between the high dose group and the DDP group.CONCLUSION: CGZC has a significant inhibitory effect on transplanted lung cancer in mice. The mechanism may involve reducing expression of PTEN and decreasing the expression of MDM2 in the lung cancer tissues of the mice.展开更多
OBJECTIVE:To investigate the protective effect of resveratrol on cardiomyocytes after hypoxia/reoxygenation intervention based on PTEN-induced putative kinase protein 1/Parkinson disease protein 2(PINK1/PARKIN)signali...OBJECTIVE:To investigate the protective effect of resveratrol on cardiomyocytes after hypoxia/reoxygenation intervention based on PTEN-induced putative kinase protein 1/Parkinson disease protein 2(PINK1/PARKIN)signaling pathway.METHODS:3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide was used to detect the effect of resveratrol on the viability of H9C2 cells;the hypoxia/reoxygenation(H/R)model was established in tri-gas incubator;2’,7’-Dichlorofluorescin diacetate staining was used to measure the content of reactive oxygen species(ROS);the changes of mitochondrial membrane potential was determined by 5,5’,6,6’-Tetrachloro-1,1’,3,3’-tetraethyl-imidacarbocyanine iodide staining;the changes of mitochondrial respiratory chain complex activity was evaluated by enzyme activity kits;flow cytometry was used to detect the ratio of apoptotic cells;transmission electron microscope was used to observe the ultrastructure of H9C2 cells;Western blot was used to detect the protein changes of mitochondrial 20 k Da outer membrane protein(TOM20),translocase of inner mitochondrial membrane 23(TIM23),presenilins associated rhomboid-like protein(PARL),PINK1,PARKIN and mitofusin 1(Mfn1),mitofusin 2(Mfn2),phosphotyrosine independent ligand for the Lck SH2 domain of 62 k Da(P62),microtubule-associated protein 1 light chain 3 beta(LC3B);the m RNA levels of PINK1 and PARKIN was detected by quantitative polymerase chain reaction;immunoprecipitation assay was used to detect the interaction between PARKIN and Ubiquitin.RESULTS:Resveratrol could inhibit the proliferation of H9C2 cells in a time-and concentration-dependent manner;however,pretreatment with low cytotoxic resveratrol could reduce the H/R-induced increase in cellular ROS levels,alleviate the loss of mitochondrial membrane potential induced by H/R,inhibit H/R-induced apoptosis of H9C2 cells,and protect the mitochondrial structure and respiratory chain of H9C2 cells from H/R damage.Resveratrol could further increase the levels of p62,PINK1,PARKIN protein,the e展开更多
Cotton (Gossypium hirsutum L.) provides a major source of oil for food and feed industries, but little was known about the enzymes in the oil biosynthesis pathway in cottonseed. We are interested in a better understan...Cotton (Gossypium hirsutum L.) provides a major source of oil for food and feed industries, but little was known about the enzymes in the oil biosynthesis pathway in cottonseed. We are interested in a better understanding of enzymatic components for oil accumulation in cottonseed. The objective of this study was to identify one key enzyme in oil biosynthesis pathway: phosphatidic acid phosphatase (PAP, 3-sn-phosphatidate phosphohydrolase, EC 3.1.3.4). PAP hydrolyzes the phosphomonoester bond in phosphatidate yielding diacylglycerol and Pi. PAPs are generally categorized into Mg<sup>2+</sup>-dependent soluble PAP and Mg<sup>2+</sup>-independent membrane-associated PAP. Cottonseed from 25 - 30 days post anthesis was used for the study. The results showed that an Mg<sup>2+</sup>-independent soluble PAP activity was identified from the cottonseed. While the microsomal fraction of the extract provided only 9% of the PAP activity, 69% of the PAP activity was associated with the cytosol. The PAP activity correlated well with enzyme concentration and incubation time. The pH and temperature optima of the enzyme were pH 5 and 55℃, respectively. Under optimized assay conditions, the V<sub>max</sub> and K<sub>m</sub> values of cottonseed PAP for dioleoyl phosphatidic acid as the substrate were 2.8 nkat/mg of protein and 539 μM, respectively. Inclusion of the detergent Triton X-100 (0% - 0.5%) or magnesium chloride (1 mM) in the reaction mix did not alter activity to a significant degree. This is the first report of a PAP activity in the seeds of Gossipium hirsutum. This study should provide a basis for purification and characterization of this important enzyme from cottonseed in the future.展开更多
OBJECTIVE:To elucidate the potential molecular mechanism by which Fuzheng Kang’ai decoction(扶正抗癌方,FZKA)inhibits proliferation,migration,and invasion of lung cancer cells.METHODS:Varying FZKA concentrations were ...OBJECTIVE:To elucidate the potential molecular mechanism by which Fuzheng Kang’ai decoction(扶正抗癌方,FZKA)inhibits proliferation,migration,and invasion of lung cancer cells.METHODS:Varying FZKA concentrations were used to manage lung cancer cells(A549 and PC9).We employed:cell counting kit-8(CCK-8)and plate clone formation assays to examine the cell viability;flow cytometry(FCM)to analyze the cycle arrest;transwell and woundhealing assays to assess the cell invasion and migration,respectively.Further,a quantitative real-time polymerase chain reaction(q RT-PCR)assay was adopted to evaluate the miR-21-5p expression.For protein expression analysis,we employed the Western blot technique.Recombinant miR-21-5p overexpression adenovirus vector harboring GFP was constructed and transfected into A549 and PC9,after which we explored the effect of FZKA on miR-21-5p overexpression.RESULTS:Notably,treatment with FZKA inhibited viability,clone-formation ability,invasion,and migration of lung cancer cells.Mechanistically,FZKA markedly suppressed miR-21-5p expression but elevated the human phosphatase and tensin homology deleted on chromosome ten(PTEN)protein level in both A549 and PC9 cells.Over-expression of miR-21-5p lowered PTEN protein expression.Besides,overexpressed miR-21-5p levels with adenovirus antagonized FZKA-upregulated PTEN protein expression.CONCLUSION:The present study demonstrates how FZKA modulates cell biological behaviors,for instance,it impedes the proliferation by upregulating PTEN expression with miR-21-5p as the target.These findings unveil the potential novel molecular mechanisms from the micro RNA aspect by which FZKA suppresses the growth of human lung cancer cells.展开更多
文摘Phytases belong to the class of phosphohydrolases that begin the step-wise hydrolysis of phosphates from phytates. Phytates are a derivative of myo-inositol, which is the primary storage form of organic phosphorus in plant cells. Phytase has been used globally to diminish phosphorus pollution and to enhance nutrition in monogastrics. In this review, the classification, sources, and diversity of microbial phytases, and their practical applications, as well as supplementation of the soil with transgenic and wild types of microbial strains, which can release phytase to enhance phosphorus availability for plant uptake and reduce the need for fertilizers, are discussed. The overexpressed microbial phytases in transgenic plants enhance the growth capacity of co-cultivated plants and can therefore be employed in agricultural and biotechnological practices, such as intercropping. The introduction of phytases into the soil for improved plant growth and enhanced crop yield can be accomplished without extra cost. A diverse group of photoautotrophic microalgae can synthesize phytase and will likely be useful in many human food and animal industries.
文摘本文报道了1例利用微滴式数字聚合酶链反应(droplet digital polymerase chain reaction,ddPCR)技术无创产前检测MFN2基因变异致胎儿腓骨肌萎缩症的病例。先证者为孕妇配偶,外院基因检测结果为MFN2基因(NM_014874)c.919A>G(p.K307E)杂合变异,诊断为轴索型腓骨肌萎缩症2A2A型。孕妇孕8周采集外周血提取胎儿游离DNA,采用ddPCR技术进行无创产前检测,检出胎儿携带父源性致病基因变异位点。孕11周采集绒毛进行Sanger测序验证,证实胎儿携带与父亲一致的c.919A>G(p.K307E)杂合变异。由本例提示ddPCR技术也许可以用于无创产前检测孕妇外周血中胎儿游离DNA是否携带父源性致病基因变异。
基金supported by the National Natural Science Foundation of China(Nos.30960032 and 31000117)the Major Technology Project of Hainan(No.ZDZX2013023-1)+2 种基金the National Nonprofit Institute Research Grants(Nos.CATAS-ITBB 110507 and CATAS-ITBB130305)the Fundamental Scientific Research Funds for Chinese Academy of Tropical Agricultural Sciences(No.1630052013009)the Natural Science Foundation of Hainan Province(No.313077),China
文摘Lipid biosynthesis is essential for eukaryotic cells, but the mechanisms of the process in microalgae remain poorly understood. Phosphatidic acid phosphohydrolase or 3-sn-phosphatidate phosphohydrolase(PAP) catalyzes the dephosphorylation of phosphatidic acid to form diacylglycerols and inorganic orthophosphates. This reaction is integral in the synthesis of triacylglycerols. In this study, the mRNA level of the PAP isoform CrPAP2 in a species of Chlamydomonas was found to increase in nitrogen-free conditions. Silencing of the CrPAP2 gene using RNA interference resulted in the decline of lipid content by 2.4%–17.4%. By contrast, over-expression of the CrPAP2 gene resulted in an increase in lipid content by 7.5%–21.8%. These observations indicate that regulation of the CrPAP2 gene can control the lipid content of the algal cells. In vitro CrPAP2 enzyme activity assay indicated that the cloned CrPAP2 gene exhibited biological activities.
基金the National Natural Science Foundation of China:the Effect of Phosphatase and Tensin Homolog-induced Putative Kinase 1/Parkin Mediated Mitochondrial Autophagy on Myocardial Injury in Chronic Kidney Disease and the Intervention Mechanism of Shenshuai Recipe(No.81960843)Training Plan for Young and Middle-aged Key Talents Project of Traditional Chinese Medicine of Jiangxi Province(No.[2022]7)。
文摘OBJECTIVE:To study whether Shenshuai recipe(肾衰方,SSR)can play a protective role on chronic kidney disease myocardial injury model through phosphatase and tensin homolog-induced putative kinase 1(PINK1)/E3 ubiquitin ligase Parkin(Parkin)mitochondrial autophagy pathway.METHODS:Forty-eight nephrectomized rats were randomly divided into six groups:sham-operated group,model group,Benazepril group,low,medium and highdose groups of SSR.The rats were given the corresponding intervention for six weeks,then were sacrificed.Serum was examined by enzyme linked immunosorbent assay(ELISA).Cardiac ultrasound was used to detect cardiac function in 5/6 nephrectomized rats.Myocardial tissue was examined by light and electron microscopy;PINK1,Parkin,microtubule-associated protein 1 light chain 3 II(LC3B),sequestosome 1(P62),BECN1(Beclin-1)and dynamin-related protein 1(Drp-1)were measured by real time polymerase chain reaction(RT-PCR),Western blot(WB)and immunohistochemistry(IHC).RESULTS:The expression levels of blood urea nitrogen(BUN)and creatinine(SCr)in the model group were significantly higher than those in the sham-operated group,indicating that modeling was successful.SSR can protect myocardium by reducing the relative expression of creatine kinase myocardial isoenzyme and hypersensitivity cardiac troponin I(P<0.05).SSR can improve cardiac function in rats after ultrasound testing.SSR can improve the pathological manifestations of myocardial tissue after Masson staining.SSR can increase the number of autophagosomes and autophagiclysosomes in 5/6 nephrectomized rats(P<0.05).Determined by RT-PCR,WB and IHC,SSR can increase the relative expression of PINK1,Parkin,and LC3B(P<0.05),and decrease the relative expression of P62,Beclin-1 and Drp-1(P<0.05).CONCLUSIONS:The PINK1/Parkin mitochondrial autophagy pathway in myocardial tissues in 5/6 nephrectomy CKD myocardial injury rats was inhibited.SSR can activate PINK1/Parkin mitochondrial autophagy to enhance mitochondrial autophagy,and play a protective role in myocardial tissue
基金Key Project of Beijing University of Chinese Medicine:to Investigate the Effect of Acupotomy Mechanics on Oxidative Injury of Rabbit Chondrocytes with Knee Osteoarthritis Based on Mitophagy Mediated by Pink1/Parkin Signaling Pathway(2020-JYB-ZDGG-059)National Natural Science Foundation of China:to Investigate the Mechanism of Acupotomy"Regulating Tendon and Treating Bone"in the Treatment of Knee Osteoarthritis based on the Vascularization of Hypoxic Cartilage Mediated by Hypoxia-Inducible Factor 1alpha/Vascular Endothelial Growth Factor Pathway(82074523)。
文摘OBJECTIVE:To investigate the effect of acupotomy,on mitophagy and the Pink1-Parkin pathway in chondrocytes from rabbits with knee osteoarthritis(KOA).METHODS:A KOA model was established via the modified Videman method.Rabbits were randomly divided into a control group(CON),KOA group and KOA+acupotomy group(Acu).Rabbits in the acupotomy group were subjected to acupotomy for 4 weeks after model establishment.The behavior of the rabbits before and after intervention was recorded.Cartilage degeneration was evaluated by optical microscopy and fluorescence microscopy.The level of mitophagy was evaluated by transmission electron microscopy,immunofluorescence and enzyme-linked immunosorbent assay(ELISA).The expression of phosphatase and tensin homolog(PTEN)-induced kinase 1(Pink1)-Parkin mitophagy pathway components was evaluated by immunofluorescence,Western blotting and real-time polymerase chain reaction.RESULTS:In rabbits with KOA,joint pain,mobility disorders and cartilage degeneration were observed,the Mankin score was increased,collagen typeⅡ(Col-Ⅱ)expression was significantly decreased,mitophagy was inhibited,mitochondrial function was impaired,and factors associated with the Pink1-Parkin pathway were inhibited.Acupotomy regulated the expression of Pink1-Parkin pathway-related proteins,the mitophagy-related protein microtubule-associated protein-1 light chain-3,the translocase of the outer membrane,and the inner mitochondrial membrane 23;increased the colocalization of mitochondria and autophagosomes;promoted the removal of damaged mitochondria;restored mitochondrial adenosine-triphosphate(ATP)production;and alleviated cartilage degeneration in rabbits with KOA.CONCLUSIONS:Acupotomy played a role in alleviating KOA in rabbits by activating mitophagy in chondrocytes via the regulation of proteins that are related to the Pink1-Parkin pathway.
文摘Phosphatidic acid phosphohydrolase (PAP), EC 3.1.3.4, is the penultimate step in the Kennedy pathway of triacyl glycerol (TAG) synthesis leading to the formation of diacylglycerol (DAG), which is a key intermediate in TAG synthesis. We partially purified a soluble PAP from mid maturing seeds of bottle gourd, Lagenaria siceraria. The steps include both anionic and cationic ion exchanger columns. Catalytic characterization of the partially purified PAP revealed that the optimum pH and temperature for activity were at 5.5?C and 45?C. Under optimum assay condition using dioleoyl phosphatidic acid (DPA) as the substrate, the Vmax and Km were 0.36 ηkat/mg of protein and 200 μM, respectively. For the synthetic substrate, ρ-nitrophenylphosphate, ρ-NPP, the Vmax and Km were 33.0 nkat/mg of protein and 140 μM, respectively. The activity was neither inhibited nor enhanced by the presence of Mg2+ at a concentration range of 0 to 10 mM. Two major protein bands at 42-kDa and 27-kDa were visible in SDS-PAGE after partial purification. Bioinformatics analysis of tryp-sinized protein fractions containing PAP activity showed peptide sequences with sequence homology to various phosphate metabolizing enzymes including cucumber and castor bean purple acid phosphatase, polyphosphate kinase, fructose biphosphate aldolase, and enolase from various dicotyledonous plants including rice, corn, grape, and Arabidopsis lyrata.
文摘Phosphatidic acid phosphohydrolase (3-sn-phosphatidate phosphohydrolase, EC 3.1.3.4), also known as PAP, catalyzes the dephosphorylation of phosphatidic acid (PtdOH) to form diacylglycerol (DAG) and inorganic orthophosphate. In eukaryotes, the PAP driven reaction is the committed step in the synthesis of triacylglycerol (TAG). Existing methods for measuring PAP activity rely on the use of radioactive PtdOH. These methods are costly and cumbersome. In this report, we describe a simple assay procedure to measure released inorganic orthophosphate, which is a coproduct of the PAP reaction. Each molecule of PtdOH would release one molecule of DAG and one molecule of inorganic orthophosphate (Pi) when subjected to enzymatic breakdown under optimal conditions. Given the published rates of in vitro PAP enzymatic activity from various sources, we proposed that colorimetric determination of released Pi is possible. With this view, we performed in vitro PAP activity assays using freshly isolated enzyme from bitter gourd, Momordica charantia, and measured the released Pi using two spectrophotometric methods. Both methods gave about 2.0 to 2.25 ηkat per mg of protein. Thus, it is now possible to perform PAP activity using a simple procedure that uses nonradioactive substrates, provided the sample is dialyzed extensively to lower the intrinsic concentration of free phosphate. The kinetics data presented in this study is comparable to that of other PAP enzymes reported elsewhere, which gives credence to the notion that non-radioactive methods can be used to perform PAP activity.
基金Supported by Science and Technology Project of Guangdong Province(No.73062)the Guangzhou Municipal Science and Technology Project(No.2007Z3-E5091)
文摘OBJECTIVE: To observe the inhibitory effect of the Chinese herbal compound Guizhu capsule(CGZC)on lung cancer and explore the possible mechanism underlying its actions by evaluating its effects on the expression of phosphate and tension homology deleted on chromsome ten(PTEN) and murine double mimute 2(MDM2) in lung cancer model mice.METHODS: A mouse model of transplanted lung cancer was established by subcutaneous inoculation of cancer cells into the axillae of mice. Twenty-four hours later, the mice were weighed and randomly divided into the model control group, cisplatin group(DDP group), and high, moderate and low dosage CGZC groups, with ten mice in each group. The control mice received an equal volume of distilled water. DDP was intraperitoneally injected at 1 mg/kg in the DDP group, once a day for 3days. CGZC diluted with distilled water was admin-istered at 20 g/kg(10 times the clinical adult dosage) in the high-dose group, 10 g/kg(5 times the clinical adult dosage) in the moderate-dose group and 5 g/kg(2.5 times the clinical adult dosage) in the low-dose group once a day for 10 days. On the11 th day, the mice were weighed and killed. The tumor tissues were weighed and the tumor inhibition rate was calculated. PTEN and MDM2 protein expression were detected by immunohistochemical analysis of tumor tissues.RESULTS: The CGZC high-and moderate-dose groups showed a significant inhibitory effect on tumor growth(P < 0.05); there was no significant difference between the high dose group and the DDP group(P > 0.05). The CGZC high-dose group also showed enhanced expression of PTEN protein(P <0.01) and decreased expression of MDM2 protein(P < 0.01) in lung cancer cells of the mice. There was no significant difference between the high dose group and the DDP group.CONCLUSION: CGZC has a significant inhibitory effect on transplanted lung cancer in mice. The mechanism may involve reducing expression of PTEN and decreasing the expression of MDM2 in the lung cancer tissues of the mice.
基金Supported by the Open Fund of Key Laboratory of Dunhuang Medicine,Ministry of Education(DHYX20-09)the Youth Research Foundation of the Gansu University of Chinese Medicine(No.ZQ2017-14)。
文摘OBJECTIVE:To investigate the protective effect of resveratrol on cardiomyocytes after hypoxia/reoxygenation intervention based on PTEN-induced putative kinase protein 1/Parkinson disease protein 2(PINK1/PARKIN)signaling pathway.METHODS:3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide was used to detect the effect of resveratrol on the viability of H9C2 cells;the hypoxia/reoxygenation(H/R)model was established in tri-gas incubator;2’,7’-Dichlorofluorescin diacetate staining was used to measure the content of reactive oxygen species(ROS);the changes of mitochondrial membrane potential was determined by 5,5’,6,6’-Tetrachloro-1,1’,3,3’-tetraethyl-imidacarbocyanine iodide staining;the changes of mitochondrial respiratory chain complex activity was evaluated by enzyme activity kits;flow cytometry was used to detect the ratio of apoptotic cells;transmission electron microscope was used to observe the ultrastructure of H9C2 cells;Western blot was used to detect the protein changes of mitochondrial 20 k Da outer membrane protein(TOM20),translocase of inner mitochondrial membrane 23(TIM23),presenilins associated rhomboid-like protein(PARL),PINK1,PARKIN and mitofusin 1(Mfn1),mitofusin 2(Mfn2),phosphotyrosine independent ligand for the Lck SH2 domain of 62 k Da(P62),microtubule-associated protein 1 light chain 3 beta(LC3B);the m RNA levels of PINK1 and PARKIN was detected by quantitative polymerase chain reaction;immunoprecipitation assay was used to detect the interaction between PARKIN and Ubiquitin.RESULTS:Resveratrol could inhibit the proliferation of H9C2 cells in a time-and concentration-dependent manner;however,pretreatment with low cytotoxic resveratrol could reduce the H/R-induced increase in cellular ROS levels,alleviate the loss of mitochondrial membrane potential induced by H/R,inhibit H/R-induced apoptosis of H9C2 cells,and protect the mitochondrial structure and respiratory chain of H9C2 cells from H/R damage.Resveratrol could further increase the levels of p62,PINK1,PARKIN protein,the e
文摘Cotton (Gossypium hirsutum L.) provides a major source of oil for food and feed industries, but little was known about the enzymes in the oil biosynthesis pathway in cottonseed. We are interested in a better understanding of enzymatic components for oil accumulation in cottonseed. The objective of this study was to identify one key enzyme in oil biosynthesis pathway: phosphatidic acid phosphatase (PAP, 3-sn-phosphatidate phosphohydrolase, EC 3.1.3.4). PAP hydrolyzes the phosphomonoester bond in phosphatidate yielding diacylglycerol and Pi. PAPs are generally categorized into Mg<sup>2+</sup>-dependent soluble PAP and Mg<sup>2+</sup>-independent membrane-associated PAP. Cottonseed from 25 - 30 days post anthesis was used for the study. The results showed that an Mg<sup>2+</sup>-independent soluble PAP activity was identified from the cottonseed. While the microsomal fraction of the extract provided only 9% of the PAP activity, 69% of the PAP activity was associated with the cytosol. The PAP activity correlated well with enzyme concentration and incubation time. The pH and temperature optima of the enzyme were pH 5 and 55℃, respectively. Under optimized assay conditions, the V<sub>max</sub> and K<sub>m</sub> values of cottonseed PAP for dioleoyl phosphatidic acid as the substrate were 2.8 nkat/mg of protein and 539 μM, respectively. Inclusion of the detergent Triton X-100 (0% - 0.5%) or magnesium chloride (1 mM) in the reaction mix did not alter activity to a significant degree. This is the first report of a PAP activity in the seeds of Gossipium hirsutum. This study should provide a basis for purification and characterization of this important enzyme from cottonseed in the future.
基金Supported by China Postdoctoral Science Foundation(No.2020M672745)the Research Project of Traditional Chinese Medicine Bureau of Guangdong Province(No.20201149,Ultrasound-delivery System to Deliver Solasonine to Regulate miR-141-3p and Enhance the Inhibition of Lung Cancer by Gefitinib)the National Natural Science Foundation of China of China(No.81503507:Exploring the Mechanism on Reversal of Gefitinib Resistance in Treating Non-Small Cell Lung Cancer by Fuzhengkangai Decoction base on miRNA-206/449a,81974543:Research on Fuzheng kang’ai Formula Reversing the Drug Resistance to Gefitinib in Non-Small Cell Lung Cancer(NSCLC)via inducing Ferroptosis by Regulating Ferritin and 81803919:the Molecular Mechanism of Fuzheng Kang-ai Decoction Reversing EMT based on Crosstalk between Tumor and Its Microenvironment)
文摘OBJECTIVE:To elucidate the potential molecular mechanism by which Fuzheng Kang’ai decoction(扶正抗癌方,FZKA)inhibits proliferation,migration,and invasion of lung cancer cells.METHODS:Varying FZKA concentrations were used to manage lung cancer cells(A549 and PC9).We employed:cell counting kit-8(CCK-8)and plate clone formation assays to examine the cell viability;flow cytometry(FCM)to analyze the cycle arrest;transwell and woundhealing assays to assess the cell invasion and migration,respectively.Further,a quantitative real-time polymerase chain reaction(q RT-PCR)assay was adopted to evaluate the miR-21-5p expression.For protein expression analysis,we employed the Western blot technique.Recombinant miR-21-5p overexpression adenovirus vector harboring GFP was constructed and transfected into A549 and PC9,after which we explored the effect of FZKA on miR-21-5p overexpression.RESULTS:Notably,treatment with FZKA inhibited viability,clone-formation ability,invasion,and migration of lung cancer cells.Mechanistically,FZKA markedly suppressed miR-21-5p expression but elevated the human phosphatase and tensin homology deleted on chromosome ten(PTEN)protein level in both A549 and PC9 cells.Over-expression of miR-21-5p lowered PTEN protein expression.Besides,overexpressed miR-21-5p levels with adenovirus antagonized FZKA-upregulated PTEN protein expression.CONCLUSION:The present study demonstrates how FZKA modulates cell biological behaviors,for instance,it impedes the proliferation by upregulating PTEN expression with miR-21-5p as the target.These findings unveil the potential novel molecular mechanisms from the micro RNA aspect by which FZKA suppresses the growth of human lung cancer cells.
