Objective:To investigate the multienzyine complex formation of human malaria parasite Plasmodium falciparum[P.falciparum)orotate phosphoribosyltransferase(OPRT)and orotidine5'-monophosphate decarboxylase(OMPDC),th...Objective:To investigate the multienzyine complex formation of human malaria parasite Plasmodium falciparum[P.falciparum)orotate phosphoribosyltransferase(OPRT)and orotidine5'-monophosphate decarboxylase(OMPDC),the fifth and sixth enzyme of the de novo pyrimidine biosynthetic palhway.Previously,we have clearly established that the two enzymes in the malaria parasite exist physically as a heterotetrameric(OPRT)_2(OMPDG)_2 complex containing two subunits each of OPRT and OMPDC.and that the complex have catalytic kinetic advantages over the monofunetional enzyme.Methods:Both enzymes were cloned and expressed as recombinant proteins.The protein-protein interaction in the enzyme complex was identified using bifunctionul chemical cross-linker,liquid chromatography-mass spectrometric analysis and homology modeling,Results:The unique insertions of low complexity region at the a 2 and a 5 helices of the parasite OMPDC,characterized by single amino acid repeat sequence which was not found in homologous proteins from other organisms,was located on the OPRT-OMPDC interface.The structural models for the protein-prolein interaction of the helerotetrameric(OPRT)_2(OMPDC)_2multienzyme complex were proposed.Conclusions:Based on the proteomic data and structural modeling,it is surmised that the human malaria parasite low complexity region is responsible for the OPRT-OMPDC interaction.The structural complex of the parasite enzymes,thus,represents an efficient functional kinetic advantage,which in line with co-localization principles of evolutional origin,and allosteric control in protein-protein-interactions.展开更多
Hypoxanthine-guanine phosphoribosyltransferase （ HGPRT, EC 2.4.2.8） is a key enzyme of the purine salvage pathway, which allows recycling of purine bases into DNA and RNA. It is widely distributed in nature and has ...Hypoxanthine-guanine phosphoribosyltransferase （ HGPRT, EC 2.4.2.8） is a key enzyme of the purine salvage pathway, which allows recycling of purine bases into DNA and RNA. It is widely distributed in nature and has been studied both in prokaryotes and eukaryotes. In humans, a complete lack of HGPRT activity causes the Lesch-Nyhan syndrome, which is characterized by hyperuricaemia and neural disorders,展开更多
To elucidate the molecular mechanisms underlying cellular radioresistance, clinically relevant radioresistant cell lines were established via long-term exposure to X-rays with stepwise dose escalation. Established cel...To elucidate the molecular mechanisms underlying cellular radioresistance, clinically relevant radioresistant cell lines were established via long-term exposure to X-rays with stepwise dose escalation. Established cells continue to proliferate despite exposure to 2 Gy X-rays/day for more than 30 days, a standard protocol in cancer radiotherapy. DNA repair fidelity in radioresistant and the parental cells by evaluating the mutation frequency at the hypoxanthine phosphoribosyltransferase (HPRT) locus after exposure to X-rays was determined. Mutation spectrum at the HPRT locus was examined by multiplex polymerase chain reaction. Rejoining kinetics of X-ray-induced DNA double strand breaks (dsbs) was evaluated by the detection of phosphorylated histone H2AX (γH2AX) after X-irradiation. The fold increase in the HPRT mutation frequency due to acute radiation was similar between radioresistant and the parental cell lines. However, fractionated radiation (FR) consisting of 2 Gy X-rays/day increased the mutation frequency at the HPRT locus in parental but not in radioresistant cells. Analysis of the FR-induced mutations at the HPRT locus revealed a high frequency of deletion mutations (>70%) in parental but not in radioresistant cells. As assessed by γH2AX immunostaining, DNA dsbs induced by acute exposure to 10 Gy of X-rays were repaired to the control level within 7 days in radioresistant but not in the parental cells. Moreover, 2 Gy × 5 FR increased the number of γH2AX-positive cells in parental cultures but not in radioresistant cultures. DNA dsbs induced by 2 Gy/day FR are repaired with fidelity in radioresistant but not in parental cells.展开更多
Adenine phosphoribosyltransferase(APRT)deficiency is a rare autosomal recessive disease leading to generation of large amounts of 2,8-dihydroxyadenine(DHA).DHA is excreted in urine,where it precipitates into crystals ...Adenine phosphoribosyltransferase(APRT)deficiency is a rare autosomal recessive disease leading to generation of large amounts of 2,8-dihydroxyadenine(DHA).DHA is excreted in urine,where it precipitates into crystals due to its low solubility.DHA crystals can aggregate into stones or cause injury to the renal parenchyma(DHA nephropathy).Recurrent urolithiasis and DHA nephropathy are the two clinical manifestations of APRT deficiency.Diagnosis of APRT deficiency can be made during childhood as well as adulthood.Diagnosis mainly relies on the recognition of DHA in stones or urine crystals.Measurement of APRT activity and genetic testing are useful for confirmation of diagnosis,for family screening and should be considered in difficult cases of urolithiasis or crystalline nephropathy.Allopurinol therapy is the cornerstone of treatment and is highly effective in preventing recurrence of stones and kidney disease.High fluid intake and dietary modifications are also recommended.Early diagnosis and treatment are of paramount importance to prevent renal damage.Unfortunately,diagnosis of APRT deficiency is often overlooked and irreversible renal failure still occurs in a substantial proportion of patients.Clinicians must be alert to the possibility of APRT deficiency and consider the appropriate diagnostic tests in certain cases.This review discusses the genetic and biochemical mechanisms of APRT deficiency,and the issues of diagnosis and management.展开更多
APRT) deficiency is an uncommon genetic cause ofchronic kidney disease due to crystalline nephropathy.Methods: A case of a Chinese boy with APRT defi ciencypresenting with severe acute kidney injury secondaryto obstru...APRT) deficiency is an uncommon genetic cause ofchronic kidney disease due to crystalline nephropathy.Methods: A case of a Chinese boy with APRT defi ciencypresenting with severe acute kidney injury secondaryto obstructive uropathy from multiple renal calculi wasreviewed.Results: The patient underwent staged removal of thecalculi. Infrared spectrometry of the renal calculi showed2,8-dihydroxyadenine. APRT deficiency was confirmedwith abolished APRT enzyme activity in red blood cells.He was started on allopurinol and low purine diet withcomplete resolution of the residual calculi.Conclusion: APRT defi ciency should be considered inpatients with multiple radiolucent renal calculi.展开更多
OBJECTIVE:To further elucidate the mechanism underlying the anti-atherosclerotic effect of Dingxin recipe(DXR).METHODS:Fifty 6-week-old male Apo E^-/-mice were randomly divided into the following groups:model,simvasta...OBJECTIVE:To further elucidate the mechanism underlying the anti-atherosclerotic effect of Dingxin recipe(DXR).METHODS:Fifty 6-week-old male Apo E^-/-mice were randomly divided into the following groups:model,simvastatin(5 mg·kg^-1·d^-1),DXR low-dose(9.30 g·kg^-1·d^-1),DXR middle-dose(18.59 g·kg^-1·d^-1)and DXR high-dose(37.18 g·kg^-1·d^-1)(n=10).Ten male C57BL/6J mice were used as the control group.All Apo E^-/-mice were fed a high-fat diet(HFD)and the control mice received a common diet.After HFD for 12 weeks,the mice were treated with DXR or simvastatin for another 12 weeks.The expression of inflammatory cytokines and visfatin was determined in serum and atherosclerotic lesions by enzyme-linked immunosorbent assay.Visfatin expression was also assessed in aortic atherosclerotic plaques.Cultured vessel endothelial cells(VECs)were pretreated with DXR sera prior to visfatin.The effects of DXR were analyzed to elucidate its protective mechanism against visfatin-induced inflammation in VECs.RESULTS:DXR regulated blood lipids and reduced tumor necrosis factor-α(TNF-α),interleukin-6(IL-6),intercellular adhesion molecules-1(ICAM-1),vascular cell adhesion molecule-1(VCAM-1)and visfatin expression in Apo E^-/-mice,particularly at the higher doses.The areas of atherosclerotic lesions in the DXR groups were significantly smaller than those in the model group.DXR alleviated visfatin-induced VEC injury via downregulation of TNF-α,IL-6,ICAM-1 and VCAM-1 through mitogen-activated protein kinase pathways.CONCLUSION:DXR alleviated atherosclerosis injury via downregulation of visfatin expression and inhibition of the visfatin-induced inflammatory response in VECs.展开更多
Seed number per silique(SNPS)is one of seed yield components in rapeseed,but its genetic mechanism remains elusive.Here a double haploid(DH)population derived from a hybrid between female 6Q006with 35–40 SNPS and mal...Seed number per silique(SNPS)is one of seed yield components in rapeseed,but its genetic mechanism remains elusive.Here a double haploid(DH)population derived from a hybrid between female 6Q006with 35–40 SNPS and male 6W26 with 10–15 SNPS was investigated for SNPS in the year 2017,2018,2019 and 2021,and genotyped with Brassica 60K Illumina Infinium SNP array.An overlapping major QTL(qSNPS.C09)explaining 51.50%of phenotypic variance on average was narrowed to a 0.90 Mb region from 44.87 Mb to 45.77 Mb on chromosome C09 by BSA-seq.Subsequently,two DEGs in this interval were detected between extreme individuals in DH and F_2populations by transcriptome sequencing at7 and 14 days after pollination siliques.Of which,BnaC09g45400D encoded an adenine phosphoribosyltransferase 5(APT5)has a 48-bp InDel variation in the promoter of two parents.Candidate gene association analysis showed that this InDel variation was associated with SNPS in a nature population of rapeseed,where 54 accessions carrying the same haplotype as parent 6Q006 had higher SNPS than103 accessions carrying the same haplotype as parent 6W26.Collectively,the findings are helpful for rapeseed molecular breeding of SNPS,and provide new insight into the genetic and molecular mechanism of SNPS in rapeseed.展开更多
OBJECTIVE:To investigate the influence and possible targets of Dangua Fang(丹瓜方)on tricarboxylic acid(TCA)cycle and respiratory chain to enrich the prescription’s mechanism of effective intervention on glycolipid m...OBJECTIVE:To investigate the influence and possible targets of Dangua Fang(丹瓜方)on tricarboxylic acid(TCA)cycle and respiratory chain to enrich the prescription’s mechanism of effective intervention on glycolipid metabolic diseases such as type 2 diabetes.METHODS:After interventional rats were fed with high glucose and high fat diet ad libitum for 4 weeks,intraperitoneally injected streptozotocin to induce diabetic model.According to blood glucose level,28 diabetic rats were selected and continued to be fed with high glucose and high fat diet,were stratified by body weight,and divided randomly by blood glucose into Model group(was given sterile water by gastric perfusion and injected aquae pro injection intraperitoneally),Dangua group[Dangua liquor(丹瓜方液)20.5 g·kg^(-1)·d^(-1) by perfusion and aquae pro injection intraperitoneally],Inhibitor group[sterile water by perfusion and nicotinamide phosphoribosyl transferase(Nampt)specific blocker GEN-6171.25 mg/kg intraperitoneally],DanInhit group(Dangua liquor and GEN-617 synchronously).Control group were continuously fed with ordinary diet.The intervention was last for 10 weeks.Body weight(BW),liver index(LI),glycosylated hemoglobin(HbA1c),TC,TG,free fatty acids(FFA),creatinine(Cr),and A-ketoglutarate(α-KG),Iso-citric acid(ICA),oxaloacetic acid(OAA)were tested.The cytochrome C oxidase(COX)and Succinate dehydrogenase(SDH)were evaluated by Colorimetry;Nampt protein,Adenosine triphosphate(ATP)synthase(ATPs),Nicotinamide adenine dinucleotide(NAD^(+))and its reduced(NADH)in liver were measured by enzyme linked immunosorbent assay.The expressions of Nampt and mitochondrialnadhdehydrogenase-1(mt-ND1)gene in liver was assessed by real-time polymerase chain reaction.Hepatic tissue staining was also completed.RESULTS:The levels of BW,ICA,α-KG and Nampt-mRNA in the Model group are lower than that in the Normal group(P<0.05),conversely,liver weight,LI,TC,HbA1c,SDH and ATPs,mt-ND1-mRNA,and Nampt protein in the Model group are higher(P<0.01,P<0.05).Compared with Model group,the levels of ICA,Nampt-mRNA and Nampt in Dangua group are significantly increased,and FFA obviously raised(P<0.01 and P<0.05);liver weight,BW,SDH are obviously lower,and HbA1c decreased significantly(P<0.01,P<0.05).TG,FFA and Nampt protein increased in the DanInhit group,TC,TG,BW obviously increased in the Inhibitor group,but SDH is decreased in both the two groups(P<0.05,P<0.01).Compared with Dangua group,DanInhib group has the lower levels of ICA,mt-ND1-mRNA,Nampt-mRNA,and the higher level of BW,LI and HbA1c.In the Inhibitor group,ICA and Nampt protein decreased,BW and LI,HbA1c and TG increased(P<0.01 or P<0.05).Tissue staining display that,in the model group there is obvious pathologic changes ie:fibrosis,steatosis and inflammatory cell infiltration.Lesions in the Dangua group are mild,and those of Inhibitor group are more obvious than the Model group,and DanInhit group is intermediately affected compared to Dangua group and Inhibitor group.CONCLUSION:Dangua Fang increases the metabolic flux of TCA cycle and optimizes respiratory chain function by up-regulating Nampt expression.展开更多
Objective: To analyze the impact of mRNA expression of oral fluoropyrimidine (S-1) metabolism (thymidylate synthase, dihydropyrimidine dehydrogenase, thymidine phosphorylase, and orotate phosphoribosyltransferase [OPR...Objective: To analyze the impact of mRNA expression of oral fluoropyrimidine (S-1) metabolism (thymidylate synthase, dihydropyrimidine dehydrogenase, thymidine phosphorylase, and orotate phosphoribosyltransferase [OPRT]), on treatment outcomes in locally advanced gastric cancer patients receiving preoperative S-1 combined with oxaliplatin chemotherapy. Methods: Preoperative stage III gastric cancer patients received S-1 (80 mg/m2/day;days 1-14) and oxaliplatin (130 mg/m2;day 1) every 3 weeks and subsequently received gastrectomy with D1/D2 lymphadenectomy. Paired tumor and normal fresh frozen tissues were collected to evaluate mRNA levels of thymidylate synthase, thymidine phosphorylase, dihydropyrimidine dehydrogenase, and orotate phosphoribosyltransferase using quantitative reverse-transcriptase polymerase chain reaction. Results: Between December 2009 and October 2010, thirty-five patients were enrolled in this study. 24 (68.5%) patients had clinical tumor response and 10 (28.6%) patients achieved histological response. Quantitative reverse-transcriptase polymerase chain reaction results showed that orotate phosphoribo-syltransferase (OPRT) mRNA expression was significantly higher in histological responders than non-responders (3.75 vs. 1.81, P = 0.005). Diffuse-type gastric cancer patients demonstrated higher orotate phosphoribosyltransferase (OPRT) expression levels than intestinal-type ones (2.79 vs. 1.60, P = 0.014). Similar results were not found when comparing thymidylate synthase, thymidine phosphorylase and dihydropyrimidine dehydrogenase expression levels. Conclusion: Orotate phosphoribosyltransferase (OPRT) expression level may be a potential predictive biomarker in advanced gastric cancer patients treated with oral fluoropyrimidine (S-1) based chemotherapy. Mini Abstract: Orotate phosphoribosyltransferase (OPRT) expression level may be a potential predictive biomarker in advanced gastric cancer patients treated with oral fluoropyrimidine (S-1) based chemotherapy.展开更多
Background Cytosine deaminase （CD） converts 5-fluorocytosine （5-FC） to 5-fluorouracil （5-FU） in CD/5-FC gene therapy, 5-FU will be mostly converted into nontoxic 13-alanine without uracil phosphoribosyltransfera...Background Cytosine deaminase （CD） converts 5-fluorocytosine （5-FC） to 5-fluorouracil （5-FU） in CD/5-FC gene therapy, 5-FU will be mostly converted into nontoxic 13-alanine without uracil phosphoribosyltransferase （UPRT）. UPRT catalyzes the conversion of 5-FU to 5-fluorouridine monophosphate, which directly kills CD：：UPRT-expressing cells and surrounding cells via the bystander effect. But the pharmacokinetics and the bystander effect of CD：：UPRT/5-FC has not been verified in vivo and in vitro. Before the CD：：UPRT/5-FC bi-gene therapy system is used in clinical trial, it is essential to monitor the transgene expression and function in vivo. Thus, we developed a preclinical tumor model to investigate the feasibility of using ^19F-magnetic resonance spectroscopy （^19F-MRS） and optical imaging to measure non-invasive CD and UPRT expression and its bystander effect. Methods C6 and C6-CD：：UPRT cells were cultured with 5-FC. The medium, cells and their mixture were analyzed by ^19F-MRS. Rats with intracranial xenografted encephalic C6-CD：：UPRT glioma were injected intraperitoneally with 5-FC and their ^19F-MRS spectra recorded. Then the pharmacokinetics of 5-FC was proved. Mixtures of C6 and C6-CD：：UPRT cells at different ratios were cultured with 5-FC and the cytotoxic efficacy and survival rate of cells recorded. To determine the mechanism of the bystander effect, the culture media from cell comprising 25% and 75% C6-CD：：UPRT cells were examined by ^19F-MRS. A comparative study of mean was performed using analysis of variance （ANOVA）. Results ^19F-MRS on samples from C6-CD：：UPRT cells cultured with 5-FC showed three broad resonance signals corresponding to 5-FC, 5-FU and fluorinated nucleotides （F-Nuctd）. For the C6 mixture, only the 5-FC peak was detected. In vivo serial ^19F-MRS spectra showed a strong 5-FC peak and a weak 5-FU peak at 20 minutes after 5-FC injection. The 5-FU concentration reached a maximum at about 50 minutes. The F-Nuctd signal appeared after about 1 hour, reached a maximum at around 160 minutes, and was detectable for several hours. At a 10% ratio of C6-CD：：UPRT cells, the survival rate was （79.55±0.88）% （P 〈0.01）. As the C6-CD：：UPRT ratio increased, the survival rate of the cells decreased. ^19F-MRS showed that the signals for 5-FU and F-Nuctd in the culture medium increased as the ratio of C6-CD：：UPRT in the mixture increased. Conclusions ^19F-MRS studies indicated that C6-CD：：UPRT cells could effectively express CD and UPRT enzymes. The CD：：UPRT/5-FC system showed an obvious bystander effect. This study demonstrated that CD：：UPRT/5-FC gene therapy is suitable for 5-FC to F-Nuctd metabolism; and ^19F-MRS can monitor transferred CD：：UPRT gene expression and catalysis of substrates noninvasively, dynamically and quantitatively.展开更多
Background Toxoplasma gondii is an obligate intracellular apicomplexan parasite and is responsible for zoonotic toxoplasmosis.It is essential to develop an effective anti-T.gondii vaccine for the control of toxoplasmo...Background Toxoplasma gondii is an obligate intracellular apicomplexan parasite and is responsible for zoonotic toxoplasmosis.It is essential to develop an effective anti-T.gondii vaccine for the control of toxoplasmosis,and this study is to explore the immunoprotective effects of a live attenuated vaccine in mice and cats.Methods First,theompdc anduprt genes of T.gondii were deleted through the CRISPR-Cas9 system.Then,the intracellular proliferation and virulence of this mutant strain were evaluated.Subsequently,the immune responses induced by this mutant in mice and cats were detected,including antibody titers,cytokine levels,and subsets of T lymphocytes.Finally,the immunoprotective effects were evaluated by challenge with tachyzoites of different strains in mice or cysts of the ME49 strain in cats.Furthermore,to discover the effective immune element against toxoplasmosis,passive immunizations were carried out.GraphPad Prism software was used to conduct the log-rank(Mantel–Cox)test,Student’st test and one-way ANOVA.Results The RHΔompdcΔuprt were constructed by the CRISPR-Cas9 system.Compared with the wild-type strain,the mutant notably reduced proliferation(P<0.05).In addition,the mutant exhibited virulence attenuation in both murine(BALB/c and BALB/c-nu)and cat models.Notably,limited pathological changes were found in tissues from RHΔompdcΔuprt-injected mice.Furthermore,compared with nonimmunized group,high levels of IgG(IgG1 and IgG2a)antibodies and cytokines(IFN-γ,IL-4,IL-10,IL-2 and IL-12)in mice were detected by the mutant(P<0.05).Remarkably,all RHΔompdcΔuprt-vaccinated mice survived a lethal challenge with RHΔku80 and ME49 and WH6 strains.The immunized sera and splenocytes,especially CD8^(+)T cells,could significantly extend(P<0.05)the survival time of mice challenged with the RHΔku80 strain compared with naïve mice.In addition,compared with nonimmunized cats,cats immunized with the mutant produced high levels of antibodies and cytokines(P<0.05),and notably decreased the shedding numbers of oocysts in feces(95.3%).Conclusions The avirulent RHΔompdcΔuprt strain can provide strong anti-T.gondii immune responses,and is a promising candidate for developing a safe and effective live attenuated vaccine.展开更多
基金supported in part by Faculty of Graduate School(to W.L)Faculty of Medicine(contract no. RAH/54(1) to J.K.),Chulalongkorn University
文摘Objective:To investigate the multienzyine complex formation of human malaria parasite Plasmodium falciparum[P.falciparum)orotate phosphoribosyltransferase(OPRT)and orotidine5'-monophosphate decarboxylase(OMPDC),the fifth and sixth enzyme of the de novo pyrimidine biosynthetic palhway.Previously,we have clearly established that the two enzymes in the malaria parasite exist physically as a heterotetrameric(OPRT)_2(OMPDG)_2 complex containing two subunits each of OPRT and OMPDC.and that the complex have catalytic kinetic advantages over the monofunetional enzyme.Methods:Both enzymes were cloned and expressed as recombinant proteins.The protein-protein interaction in the enzyme complex was identified using bifunctionul chemical cross-linker,liquid chromatography-mass spectrometric analysis and homology modeling,Results:The unique insertions of low complexity region at the a 2 and a 5 helices of the parasite OMPDC,characterized by single amino acid repeat sequence which was not found in homologous proteins from other organisms,was located on the OPRT-OMPDC interface.The structural models for the protein-prolein interaction of the helerotetrameric(OPRT)_2(OMPDC)_2multienzyme complex were proposed.Conclusions:Based on the proteomic data and structural modeling,it is surmised that the human malaria parasite low complexity region is responsible for the OPRT-OMPDC interaction.The structural complex of the parasite enzymes,thus,represents an efficient functional kinetic advantage,which in line with co-localization principles of evolutional origin,and allosteric control in protein-protein-interactions.
文摘Hypoxanthine-guanine phosphoribosyltransferase （ HGPRT, EC 2.4.2.8） is a key enzyme of the purine salvage pathway, which allows recycling of purine bases into DNA and RNA. It is widely distributed in nature and has been studied both in prokaryotes and eukaryotes. In humans, a complete lack of HGPRT activity causes the Lesch-Nyhan syndrome, which is characterized by hyperuricaemia and neural disorders,
文摘To elucidate the molecular mechanisms underlying cellular radioresistance, clinically relevant radioresistant cell lines were established via long-term exposure to X-rays with stepwise dose escalation. Established cells continue to proliferate despite exposure to 2 Gy X-rays/day for more than 30 days, a standard protocol in cancer radiotherapy. DNA repair fidelity in radioresistant and the parental cells by evaluating the mutation frequency at the hypoxanthine phosphoribosyltransferase (HPRT) locus after exposure to X-rays was determined. Mutation spectrum at the HPRT locus was examined by multiplex polymerase chain reaction. Rejoining kinetics of X-ray-induced DNA double strand breaks (dsbs) was evaluated by the detection of phosphorylated histone H2AX (γH2AX) after X-irradiation. The fold increase in the HPRT mutation frequency due to acute radiation was similar between radioresistant and the parental cell lines. However, fractionated radiation (FR) consisting of 2 Gy X-rays/day increased the mutation frequency at the HPRT locus in parental but not in radioresistant cells. Analysis of the FR-induced mutations at the HPRT locus revealed a high frequency of deletion mutations (>70%) in parental but not in radioresistant cells. As assessed by γH2AX immunostaining, DNA dsbs induced by acute exposure to 10 Gy of X-rays were repaired to the control level within 7 days in radioresistant but not in the parental cells. Moreover, 2 Gy × 5 FR increased the number of γH2AX-positive cells in parental cultures but not in radioresistant cultures. DNA dsbs induced by 2 Gy/day FR are repaired with fidelity in radioresistant but not in parental cells.
文摘Adenine phosphoribosyltransferase(APRT)deficiency is a rare autosomal recessive disease leading to generation of large amounts of 2,8-dihydroxyadenine(DHA).DHA is excreted in urine,where it precipitates into crystals due to its low solubility.DHA crystals can aggregate into stones or cause injury to the renal parenchyma(DHA nephropathy).Recurrent urolithiasis and DHA nephropathy are the two clinical manifestations of APRT deficiency.Diagnosis of APRT deficiency can be made during childhood as well as adulthood.Diagnosis mainly relies on the recognition of DHA in stones or urine crystals.Measurement of APRT activity and genetic testing are useful for confirmation of diagnosis,for family screening and should be considered in difficult cases of urolithiasis or crystalline nephropathy.Allopurinol therapy is the cornerstone of treatment and is highly effective in preventing recurrence of stones and kidney disease.High fluid intake and dietary modifications are also recommended.Early diagnosis and treatment are of paramount importance to prevent renal damage.Unfortunately,diagnosis of APRT deficiency is often overlooked and irreversible renal failure still occurs in a substantial proportion of patients.Clinicians must be alert to the possibility of APRT deficiency and consider the appropriate diagnostic tests in certain cases.This review discusses the genetic and biochemical mechanisms of APRT deficiency,and the issues of diagnosis and management.
文摘APRT) deficiency is an uncommon genetic cause ofchronic kidney disease due to crystalline nephropathy.Methods: A case of a Chinese boy with APRT defi ciencypresenting with severe acute kidney injury secondaryto obstructive uropathy from multiple renal calculi wasreviewed.Results: The patient underwent staged removal of thecalculi. Infrared spectrometry of the renal calculi showed2,8-dihydroxyadenine. APRT deficiency was confirmedwith abolished APRT enzyme activity in red blood cells.He was started on allopurinol and low purine diet withcomplete resolution of the residual calculi.Conclusion: APRT defi ciency should be considered inpatients with multiple radiolucent renal calculi.
基金Supported by National Natural Science Foundation of China To Study the Mechanism of CXLZF on Atherosclerotic Plaque through the mi R-421/ACE2/Ang(1-7)Pathway(No.81774213)To Study the Mechanism of DXR on Atherosclerotic Vulnerable Plaque Based on the ACE2-Ang(1-7)-Mas Axis(No.81373574)To Study the Mechanism of Treatment from Heart for Atherosclerotic Vulnerable Plaque Based on the Web Regulated by CD4+CD25+Foxp3+Tregs(No.81403339)。
文摘OBJECTIVE:To further elucidate the mechanism underlying the anti-atherosclerotic effect of Dingxin recipe(DXR).METHODS:Fifty 6-week-old male Apo E^-/-mice were randomly divided into the following groups:model,simvastatin(5 mg·kg^-1·d^-1),DXR low-dose(9.30 g·kg^-1·d^-1),DXR middle-dose(18.59 g·kg^-1·d^-1)and DXR high-dose(37.18 g·kg^-1·d^-1)(n=10).Ten male C57BL/6J mice were used as the control group.All Apo E^-/-mice were fed a high-fat diet(HFD)and the control mice received a common diet.After HFD for 12 weeks,the mice were treated with DXR or simvastatin for another 12 weeks.The expression of inflammatory cytokines and visfatin was determined in serum and atherosclerotic lesions by enzyme-linked immunosorbent assay.Visfatin expression was also assessed in aortic atherosclerotic plaques.Cultured vessel endothelial cells(VECs)were pretreated with DXR sera prior to visfatin.The effects of DXR were analyzed to elucidate its protective mechanism against visfatin-induced inflammation in VECs.RESULTS:DXR regulated blood lipids and reduced tumor necrosis factor-α(TNF-α),interleukin-6(IL-6),intercellular adhesion molecules-1(ICAM-1),vascular cell adhesion molecule-1(VCAM-1)and visfatin expression in Apo E^-/-mice,particularly at the higher doses.The areas of atherosclerotic lesions in the DXR groups were significantly smaller than those in the model group.DXR alleviated visfatin-induced VEC injury via downregulation of TNF-α,IL-6,ICAM-1 and VCAM-1 through mitogen-activated protein kinase pathways.CONCLUSION:DXR alleviated atherosclerosis injury via downregulation of visfatin expression and inhibition of the visfatin-induced inflammatory response in VECs.
基金supported by the National Basic Research Program of China(2015CB150201)the Natural Science Foundation of Chongqing(cstc2019jcyj-bshX0055,cstc2019jcyj-zdxmX0012cstc2020jcyj-msxmX0461)。
文摘Seed number per silique(SNPS)is one of seed yield components in rapeseed,but its genetic mechanism remains elusive.Here a double haploid(DH)population derived from a hybrid between female 6Q006with 35–40 SNPS and male 6W26 with 10–15 SNPS was investigated for SNPS in the year 2017,2018,2019 and 2021,and genotyped with Brassica 60K Illumina Infinium SNP array.An overlapping major QTL(qSNPS.C09)explaining 51.50%of phenotypic variance on average was narrowed to a 0.90 Mb region from 44.87 Mb to 45.77 Mb on chromosome C09 by BSA-seq.Subsequently,two DEGs in this interval were detected between extreme individuals in DH and F_2populations by transcriptome sequencing at7 and 14 days after pollination siliques.Of which,BnaC09g45400D encoded an adenine phosphoribosyltransferase 5(APT5)has a 48-bp InDel variation in the promoter of two parents.Candidate gene association analysis showed that this InDel variation was associated with SNPS in a nature population of rapeseed,where 54 accessions carrying the same haplotype as parent 6Q006 had higher SNPS than103 accessions carrying the same haplotype as parent 6W26.Collectively,the findings are helpful for rapeseed molecular breeding of SNPS,and provide new insight into the genetic and molecular mechanism of SNPS in rapeseed.
基金the National Natural Science Foundation of China:Based on the "miR34a/Nampt-NAD+-TAC" Pathway to Study the Mechanism of Simultaneously Treating the Phlegm and Blood Stasis in the Regulation of Glycolipid (No.81873213)Study on the Mechanism of Simultaneously Treating the Phlegm and Blood Stasis on Glycolipid Metabolism Based on Intestinal Fat Absorption Regulated by miR-34a/Stat3-Nfil3 Pathway (82074308)+1 种基金a New Mechanism of Regulating the Amino Acid Metabolism of Type 2 Diabetes Mellitus with Dissipating Phlegm-Stasis:Based on the tricarboxylic acid Cycle-Mediated Transformation of "α-KG→Glutamate"(8227150196)by Industry-University Cooperation Project for University in Fujian Province:Preparation of Monomeric Traditional Chinese Medicine Complexes Based on Nampt’s Activation of Tricarboxylic Acid Cycle and Respiratory Chain to Interfere with Glycolipid Metabolism (2022Y41010015)
文摘OBJECTIVE:To investigate the influence and possible targets of Dangua Fang(丹瓜方)on tricarboxylic acid(TCA)cycle and respiratory chain to enrich the prescription’s mechanism of effective intervention on glycolipid metabolic diseases such as type 2 diabetes.METHODS:After interventional rats were fed with high glucose and high fat diet ad libitum for 4 weeks,intraperitoneally injected streptozotocin to induce diabetic model.According to blood glucose level,28 diabetic rats were selected and continued to be fed with high glucose and high fat diet,were stratified by body weight,and divided randomly by blood glucose into Model group(was given sterile water by gastric perfusion and injected aquae pro injection intraperitoneally),Dangua group[Dangua liquor(丹瓜方液)20.5 g·kg^(-1)·d^(-1) by perfusion and aquae pro injection intraperitoneally],Inhibitor group[sterile water by perfusion and nicotinamide phosphoribosyl transferase(Nampt)specific blocker GEN-6171.25 mg/kg intraperitoneally],DanInhit group(Dangua liquor and GEN-617 synchronously).Control group were continuously fed with ordinary diet.The intervention was last for 10 weeks.Body weight(BW),liver index(LI),glycosylated hemoglobin(HbA1c),TC,TG,free fatty acids(FFA),creatinine(Cr),and A-ketoglutarate(α-KG),Iso-citric acid(ICA),oxaloacetic acid(OAA)were tested.The cytochrome C oxidase(COX)and Succinate dehydrogenase(SDH)were evaluated by Colorimetry;Nampt protein,Adenosine triphosphate(ATP)synthase(ATPs),Nicotinamide adenine dinucleotide(NAD^(+))and its reduced(NADH)in liver were measured by enzyme linked immunosorbent assay.The expressions of Nampt and mitochondrialnadhdehydrogenase-1(mt-ND1)gene in liver was assessed by real-time polymerase chain reaction.Hepatic tissue staining was also completed.RESULTS:The levels of BW,ICA,α-KG and Nampt-mRNA in the Model group are lower than that in the Normal group(P<0.05),conversely,liver weight,LI,TC,HbA1c,SDH and ATPs,mt-ND1-mRNA,and Nampt protein in the Model group are higher(P<0.01,P<0.05).Compared with Model group,the levels of ICA,Nampt-mRNA and Nampt in Dangua group are significantly increased,and FFA obviously raised(P<0.01 and P<0.05);liver weight,BW,SDH are obviously lower,and HbA1c decreased significantly(P<0.01,P<0.05).TG,FFA and Nampt protein increased in the DanInhit group,TC,TG,BW obviously increased in the Inhibitor group,but SDH is decreased in both the two groups(P<0.05,P<0.01).Compared with Dangua group,DanInhib group has the lower levels of ICA,mt-ND1-mRNA,Nampt-mRNA,and the higher level of BW,LI and HbA1c.In the Inhibitor group,ICA and Nampt protein decreased,BW and LI,HbA1c and TG increased(P<0.01 or P<0.05).Tissue staining display that,in the model group there is obvious pathologic changes ie:fibrosis,steatosis and inflammatory cell infiltration.Lesions in the Dangua group are mild,and those of Inhibitor group are more obvious than the Model group,and DanInhit group is intermediately affected compared to Dangua group and Inhibitor group.CONCLUSION:Dangua Fang increases the metabolic flux of TCA cycle and optimizes respiratory chain function by up-regulating Nampt expression.
文摘Objective: To analyze the impact of mRNA expression of oral fluoropyrimidine (S-1) metabolism (thymidylate synthase, dihydropyrimidine dehydrogenase, thymidine phosphorylase, and orotate phosphoribosyltransferase [OPRT]), on treatment outcomes in locally advanced gastric cancer patients receiving preoperative S-1 combined with oxaliplatin chemotherapy. Methods: Preoperative stage III gastric cancer patients received S-1 (80 mg/m2/day;days 1-14) and oxaliplatin (130 mg/m2;day 1) every 3 weeks and subsequently received gastrectomy with D1/D2 lymphadenectomy. Paired tumor and normal fresh frozen tissues were collected to evaluate mRNA levels of thymidylate synthase, thymidine phosphorylase, dihydropyrimidine dehydrogenase, and orotate phosphoribosyltransferase using quantitative reverse-transcriptase polymerase chain reaction. Results: Between December 2009 and October 2010, thirty-five patients were enrolled in this study. 24 (68.5%) patients had clinical tumor response and 10 (28.6%) patients achieved histological response. Quantitative reverse-transcriptase polymerase chain reaction results showed that orotate phosphoribo-syltransferase (OPRT) mRNA expression was significantly higher in histological responders than non-responders (3.75 vs. 1.81, P = 0.005). Diffuse-type gastric cancer patients demonstrated higher orotate phosphoribosyltransferase (OPRT) expression levels than intestinal-type ones (2.79 vs. 1.60, P = 0.014). Similar results were not found when comparing thymidylate synthase, thymidine phosphorylase and dihydropyrimidine dehydrogenase expression levels. Conclusion: Orotate phosphoribosyltransferase (OPRT) expression level may be a potential predictive biomarker in advanced gastric cancer patients treated with oral fluoropyrimidine (S-1) based chemotherapy. Mini Abstract: Orotate phosphoribosyltransferase (OPRT) expression level may be a potential predictive biomarker in advanced gastric cancer patients treated with oral fluoropyrimidine (S-1) based chemotherapy.
基金This study was supported by grants from Jiangsu Provincial Health Department （No. RC2002075） and Natural Science Foundation of Jiangsu Province （No. BK2007257）.
文摘Background Cytosine deaminase （CD） converts 5-fluorocytosine （5-FC） to 5-fluorouracil （5-FU） in CD/5-FC gene therapy, 5-FU will be mostly converted into nontoxic 13-alanine without uracil phosphoribosyltransferase （UPRT）. UPRT catalyzes the conversion of 5-FU to 5-fluorouridine monophosphate, which directly kills CD：：UPRT-expressing cells and surrounding cells via the bystander effect. But the pharmacokinetics and the bystander effect of CD：：UPRT/5-FC has not been verified in vivo and in vitro. Before the CD：：UPRT/5-FC bi-gene therapy system is used in clinical trial, it is essential to monitor the transgene expression and function in vivo. Thus, we developed a preclinical tumor model to investigate the feasibility of using ^19F-magnetic resonance spectroscopy （^19F-MRS） and optical imaging to measure non-invasive CD and UPRT expression and its bystander effect. Methods C6 and C6-CD：：UPRT cells were cultured with 5-FC. The medium, cells and their mixture were analyzed by ^19F-MRS. Rats with intracranial xenografted encephalic C6-CD：：UPRT glioma were injected intraperitoneally with 5-FC and their ^19F-MRS spectra recorded. Then the pharmacokinetics of 5-FC was proved. Mixtures of C6 and C6-CD：：UPRT cells at different ratios were cultured with 5-FC and the cytotoxic efficacy and survival rate of cells recorded. To determine the mechanism of the bystander effect, the culture media from cell comprising 25% and 75% C6-CD：：UPRT cells were examined by ^19F-MRS. A comparative study of mean was performed using analysis of variance （ANOVA）. Results ^19F-MRS on samples from C6-CD：：UPRT cells cultured with 5-FC showed three broad resonance signals corresponding to 5-FC, 5-FU and fluorinated nucleotides （F-Nuctd）. For the C6 mixture, only the 5-FC peak was detected. In vivo serial ^19F-MRS spectra showed a strong 5-FC peak and a weak 5-FU peak at 20 minutes after 5-FC injection. The 5-FU concentration reached a maximum at about 50 minutes. The F-Nuctd signal appeared after about 1 hour, reached a maximum at around 160 minutes, and was detectable for several hours. At a 10% ratio of C6-CD：：UPRT cells, the survival rate was （79.55±0.88）% （P 〈0.01）. As the C6-CD：：UPRT ratio increased, the survival rate of the cells decreased. ^19F-MRS showed that the signals for 5-FU and F-Nuctd in the culture medium increased as the ratio of C6-CD：：UPRT in the mixture increased. Conclusions ^19F-MRS studies indicated that C6-CD：：UPRT cells could effectively express CD and UPRT enzymes. The CD：：UPRT/5-FC system showed an obvious bystander effect. This study demonstrated that CD：：UPRT/5-FC gene therapy is suitable for 5-FC to F-Nuctd metabolism; and ^19F-MRS can monitor transferred CD：：UPRT gene expression and catalysis of substrates noninvasively, dynamically and quantitatively.
基金supported by the National Natural Science Foundation of China(No.81871684,No.81802037)the Provincial Key R&D program of Zhejiang Department of Science and Technology(No.2019C03057)+3 种基金the Zhejiang Provincial Natural Science Foundation of China(No.LY22H190003)the Zhejiang Medical and Health Science and Technology Plan(WKJ-ZJ-2203)the Central Leading Local Science and Technology Development Fund Project(2023ZY1019)the Key Discipline of Zhejiang Province in Public Health and Preventive Medicine(First Class,Category A),Hangzhou Medical College.
文摘Background Toxoplasma gondii is an obligate intracellular apicomplexan parasite and is responsible for zoonotic toxoplasmosis.It is essential to develop an effective anti-T.gondii vaccine for the control of toxoplasmosis,and this study is to explore the immunoprotective effects of a live attenuated vaccine in mice and cats.Methods First,theompdc anduprt genes of T.gondii were deleted through the CRISPR-Cas9 system.Then,the intracellular proliferation and virulence of this mutant strain were evaluated.Subsequently,the immune responses induced by this mutant in mice and cats were detected,including antibody titers,cytokine levels,and subsets of T lymphocytes.Finally,the immunoprotective effects were evaluated by challenge with tachyzoites of different strains in mice or cysts of the ME49 strain in cats.Furthermore,to discover the effective immune element against toxoplasmosis,passive immunizations were carried out.GraphPad Prism software was used to conduct the log-rank(Mantel–Cox)test,Student’st test and one-way ANOVA.Results The RHΔompdcΔuprt were constructed by the CRISPR-Cas9 system.Compared with the wild-type strain,the mutant notably reduced proliferation(P<0.05).In addition,the mutant exhibited virulence attenuation in both murine(BALB/c and BALB/c-nu)and cat models.Notably,limited pathological changes were found in tissues from RHΔompdcΔuprt-injected mice.Furthermore,compared with nonimmunized group,high levels of IgG(IgG1 and IgG2a)antibodies and cytokines(IFN-γ,IL-4,IL-10,IL-2 and IL-12)in mice were detected by the mutant(P<0.05).Remarkably,all RHΔompdcΔuprt-vaccinated mice survived a lethal challenge with RHΔku80 and ME49 and WH6 strains.The immunized sera and splenocytes,especially CD8^(+)T cells,could significantly extend(P<0.05)the survival time of mice challenged with the RHΔku80 strain compared with naïve mice.In addition,compared with nonimmunized cats,cats immunized with the mutant produced high levels of antibodies and cytokines(P<0.05),and notably decreased the shedding numbers of oocysts in feces(95.3%).Conclusions The avirulent RHΔompdcΔuprt strain can provide strong anti-T.gondii immune responses,and is a promising candidate for developing a safe and effective live attenuated vaccine.
