AIM： To investigate the prognostic role of thymidylate synthase （TS） and thymidine phosphorylase （TP） mRNA levels in T3 or T4 gastric cancer treated with 5-fluorouraci-based adjuvant chemotherapy. METHODS： Fifty...AIM： To investigate the prognostic role of thymidylate synthase （TS） and thymidine phosphorylase （TP） mRNA levels in T3 or T4 gastric cancer treated with 5-fluorouraci-based adjuvant chemotherapy. METHODS： Fifty-one patients with T3 or T4 gastric cancer received systemic 5-fluorouraci-based adjuvant chemotherapy, and intratumoral expression of TS and TP in 51 gastric cancer tissue samples was tested by realtime quantitative PCR.RESULTS： The median disease-free survival （DFS） time was 10.2 mo in the patients. There were no significant differences in DFS between the groups with high and low levels of TP. However, the group with low level of TS had a longer DFS （14.4 mo vs 8.3 mo, P = 0.017）. The median overall survival （OS） time was 18.5 mo, and there were significant differences in OS between the groups with high and low levels of TS or TP （for TS, 17.0 mo vs 21.3 mo, P = 0.010; for TP, 16.6 mo vs 22.5 too, P = 0.009）. Moreover, the coupled low expression of these two genes was strongly associated with a longer survival time of patients as compared with that of a single gene.CONCLUSION： Expression of TS and TP mRNA is a useful predictive parameter for the survival of postoperative gastric cancer patients after 5-fluorouracilbased adjuvant chemotherapy.展开更多
Nucleoside phosphorylases (NPases) were found to be induced in Enterobacter aerogenes DGO-04, and cytidine and cytidine 5′-monophosphate (CMP) were the best inducers. Five mmol/L to fifteen mmol/L cytidine or CMP cou...Nucleoside phosphorylases (NPases) were found to be induced in Enterobacter aerogenes DGO-04, and cytidine and cytidine 5′-monophosphate (CMP) were the best inducers. Five mmol/L to fifteen mmol/L cytidine or CMP could distinctly increase the activities of purine nucleoside phosphorylase (PNPase), uridine phosphorylase (UPase) and thymidine phosphorylase (TPase) when they were added into medium from 0 to 8 h. In the process of enzymatic synthesis of adenine arabinoside from adenine and uracil arabinoside with wet cells of Enterobacter aerogenes DGO-04 induced by cytidine or CMP, the reaction time could be shortened from 36 to 6 h. After enzymatic reaction the activity of NPase in the cells induced remained higher than that in the cells uninduced.展开更多
Thymidine phosphorylase (TP) is a key enzyme that contributes to the composition and decomposition of pyrimidine nucleotides. TP seems homologous to platelet-derived endothelial cell growth factor, and its effects on ...Thymidine phosphorylase (TP) is a key enzyme that contributes to the composition and decomposition of pyrimidine nucleotides. TP seems homologous to platelet-derived endothelial cell growth factor, and its effects on inducing vascularization and anti-apoptosis are closely related to growth and metastasis of colorectal carcinoma. In addition, TP is a key enzyme that catalyzes the transformation from 5-fluorouracil (FU) prodrugs of 5′-deoxy-5-fluorouridine (5′- DFUR) to 5-FU. The activity of TP is closely related to the sensitivity of colorectal carcinoma cells to fluorouracil drugs and targeted therapy. Given the important functions of TP in growth, metastasis, tumor treatment, and prognosis, determining its expression mechanism is significant. This article summarizes the research development of TP expression in colorectal carcinoma, tumor neovascularization, cytotoxicity activation of 5′-DFUR, and colorectal carcinoma therapy.展开更多
The mRNA and protein expression of thymidylate synthase (TS), thymidine phosphorylase (TP) and dihydropyrimidine dehydrogenase (DPD) and their relationship with prognosis were investigated. Real-time quantitative RT-P...The mRNA and protein expression of thymidylate synthase (TS), thymidine phosphorylase (TP) and dihydropyrimidine dehydrogenase (DPD) and their relationship with prognosis were investigated. Real-time quantitative RT-PCR (Taqman) was used to detect the mRNA expression of TS, TP and DPD in formalin-fixed and paraffin-embedded 106 samples of epithelial ovarian cancer and 29 normal ovaries. A TATA box-binding protein (TBP) was used as an endogenous reference gene. A relationship between TS, TP, DPD expression and clinicopathologic features was investigated. The protein location and expression of TS, TP and DPD was examined in the same patients by an avidin-biotin-peroxidase immunohistochemistry. TS and TP mRNA expression levels were significantly higher in tumor group than in normal controls, with the average value of TS and TP mRNA being 6.14±0.62 and 0.59±0.06 in tumor tissue, and 0.71±0.14 and 0.16±0.04 in normal tissue, respectively. DPD mRNA expression levels were significantly lower in tumor group (0.11±0.02) than in normal controls (0.38±0.05). There was statistically significant difference in TS and TP mRNA expression levels among different pathological grades and clinical stages (P<0.05), but histological subtype was not significantly associated with TS and TP mRNA expression. DPD gene expression was not significantly associated with any clinicopathological parameters. Immunohistochemistry revealed that TP protein was mainly distributed in nucleus, and TS and DPD mainly in cytoplasm. The protein expression intensity of TS, TP and DPD was coincided with the mRNA expression levels. It was concluded that TS, TP mRNA and protein expression levels were significantly higher in epithelial ovarian cancer, and DPD mRNA and protein expression levels were significantly lower. The expression levels of TS and DPD were related to the patients’ prognosis and survival. Combined gene expression levels of TS, TP and DPD represent a new variable to predict the clinical outcome in ovarian cancer. The association of TS, TP and DPD expression levels with survival suggests an importance of these genes for tumor occurrence and progression.展开更多
AIM: To evaluate the role of thymidine phosphorylase (TP) in cholangiocarcinoma using small interfering RNA (siRNA). METHODS: A human cholangiocarcinoma-derived cell line KKU-M139, which has a naturally high level of ...AIM: To evaluate the role of thymidine phosphorylase (TP) in cholangiocarcinoma using small interfering RNA (siRNA). METHODS: A human cholangiocarcinoma-derived cell line KKU-M139, which has a naturally high level of endogenous TP, had TP expression transiently knocked down using siRNA. Cell growth, migration, in vitro angiogenesis, apoptosis, and cytotoxicity were assayed in TP knockdown and wild-type cell lines. RESULTS: TP mRNA and protein expression were decreased by 87.1% ± 0.49% and 72.5% ± 3.2%, respectively, compared with control cells. Inhibition of TP significantly decreased migration of KKU-M139, and suppressed migration and tube formation of human umbilical vein endothelial cells. siRNA also reduced the ability of TP to resist hypoxia-induced apoptosis, while suppression of TP reduced the sensitivity of KKU-M139 to 5-fluorouracil. CONCLUSION: Inhibition of TP may be beneficial in decreasing angiogenesis-dependent growth and migration of cholangiocarcinoma but may diminish the response to 5-fluorouracil chemotherapy.展开更多
Recombinant Escherichia coli pUDP,which overexpressed uridine phosphorylase（UPase）,was constructed.0.5 mmol·L 1lactose had a similar induction effect as the commonly used inducer IPTG during 2.5-5.5 h of cell g...Recombinant Escherichia coli pUDP,which overexpressed uridine phosphorylase（UPase）,was constructed.0.5 mmol·L 1lactose had a similar induction effect as the commonly used inducer IPTG during 2.5-5.5 h of cell growth.The lactose-induced UPase was stable at 50°C.Wet cells of pUDP was used as catalyst to biosynthesize 5-fluorouridine from 30 mmol·L 1uridine and 5-fluorouracil in phosphate buffer（pH 7.0）catalyzed at 50°C for 1.5 h and the yield of 5-fluorouridine was higher than 68%.Under the optimum reaction conditions for production of 5-fluorouridine,5-methyluridine and azauridine were synthesized from uridine by pUDP,the yield was 61.7%and 47.2%respectively.Deoxynucleosides were also synthesized by pUDP,but the yield was only about 20%.展开更多
Wistar rats were exposed to trinitrotoluene (TNT) for 6 weeks. After initiation of TNT exposure, serum phosphorylase A activities and calcium contents were assayed for every 2 weeks. Both of these 2 parameters increas...Wistar rats were exposed to trinitrotoluene (TNT) for 6 weeks. After initiation of TNT exposure, serum phosphorylase A activities and calcium contents were assayed for every 2 weeks. Both of these 2 parameters increased in rats treated with 50 and 100 mg TNT/kg b.w. at 3 intervals. Serum phosphorylase A activities and calcium contents of TNT exposure worker increased too.展开更多
Nucleoside phosphorylase is an important enzyme involved in the biosynthesis of nucleosides. In this study, purine nucleoside phosphorylase and pyrimidine nucleoside phosphorylase were co-expressed in Escherichia coli...Nucleoside phosphorylase is an important enzyme involved in the biosynthesis of nucleosides. In this study, purine nucleoside phosphorylase and pyrimidine nucleoside phosphorylase were co-expressed in Escherichia coli and the intact cells were used as a catalyst for the biosynthesis of nucleosides. For protein induction, lactose was used in place of isopropyl β-D-l-thiogalactopyranoside （IPTG）. When the concentration of lactose was above 0.5 mmol/L, the ability to induce protein expression was similar to that of IPTG. We determined that the reaction conditions of four bacterial strains co-expressing these genes （TUD, TAD, DUD, and DAD） were similar for the biosyntheses of 2,6-diaminopurine nucleoside and 2,6-diaminopurine deoxynucleoside. When the substrate concentration was 30 mmol/L and 0.5% of the recombinant bacterial cell volume was used as the catalyst （pH 7.5）, a greater than 90% conversion yield was reached after a 2-h incubation at 50℃. In addition, several other nucleosides and nucleoside derivatives were efficiently synthesized using bacterial strains co-expressing these recombinant enzymes.展开更多
Purine nucleoside phosphorylase is a key enzyme in the purine-salvage pathway and an attractive target for drug design. The crystal structure of Streptococcus mutants purine nucleoside phosphorylase（Smu PNP） has bee...Purine nucleoside phosphorylase is a key enzyme in the purine-salvage pathway and an attractive target for drug design. The crystal structure of Streptococcus mutants purine nucleoside phosphorylase（Smu PNP） has been solved by molecular replacement at 1.80 resolution and refined to R factors of 19.9%/23.7%（Rcryst/Rfree） . Sequence alignment and structural comparison show that Smu PNP has more similarity with PNPs isolated from human and malarial sources than the bacterial PNPs. The structure complexed with hypoxanthine（HPA） and sulfate ion was solved at 2.24A resolution and refined to R factors of 21.6%/24.1%（Rcryst/Rfree） . It is interesting to note that the resulting electron density indicated the product,HPA,presents in the active site although inosine was included in the crystallization mixture with Smu PNP. Asn233 and Glu191 are the important residues for ligand binding and recognition. Comparison with PNPs from different species gives detailed information about binding of small molecules on the active site,which is important for the studies of enzymatic mechanism and rational design of specific inhibitors for PNPs.展开更多
Methylthioadenosine phosphorylase, (MTAP) is a key enzyme in the adenine and methionine salvage pathways. MTAP is encoded on human chromosome 9p21 in close proximity to the p16INK4a and p14ARF tumor suppressor genes a...Methylthioadenosine phosphorylase, (MTAP) is a key enzyme in the adenine and methionine salvage pathways. MTAP is encoded on human chromosome 9p21 in close proximity to the p16INK4a and p14ARF tumor suppressor genes and is frequently co-deleted with p16INK4a in many cancers. Deletion of MTAP has been reported to create a reliance of MTAP–/– tumors on de novo purine synthesis to maintain adequate pools of AMP, leading to increased sensitivity to purine synthesis inhibitors, such as L-alanosine. The ‘Achilles heel’ created by the loss of MTAP in cancer cells provides a unique therapeutic opportunity whereby MTAP–/– tumors could be selectively targeted with purine synthesis inhibitors and normal tissues could be preferentially rescued with MTAP substrates, such as MTA. We demonstrate that, in contrast to published literature, MTAP–/– cells are not more sensitive to inhibition of de novo purine synthesis than MTAP+/+ cells. Although MTA can preferentially rescue MTAP+/+ cells from purine-synthesis inhibitor toxicity in vitro, MTA protects cells of both genotypes from L-alanosine equivalently in vivo. Our data demonstrate that in vivo, adenine salvaged from plasma and adjacent tissues is sufficient to protect MTAP–/– tumors from the effects of purine synthesis inhibitors. These results suggest targeting MTAP–/– tumors with de novo purine synthesis inhibitors is unlikely to provide significant benefit over other therapeutic strategies and may explain, at least in part, the lack of efficacy of L-alanosine in clinical trials.展开更多
Purine Nucleoside Phosphorylase (PNP) deficiency is a rare autosomal recessive metabolic disorder. In PNP-deficiency disorder, the deficient enzyme leads to accumulation of toxic metabolites, especially in lymphocytes...Purine Nucleoside Phosphorylase (PNP) deficiency is a rare autosomal recessive metabolic disorder. In PNP-deficiency disorder, the deficient enzyme leads to accumulation of toxic metabolites, especially in lymphocytes and the metabolites exert toxic effect on T-cell generation. Purine nucleoside phosphorylase deficiency causes decreased numbers of T cells and lymphopenia. The patients suffering from PNP-deficiency may be admitted with recurrent infections, as well as neurological and autoimmune findings. We hereby presented a case admitted with the symptom of hematuria in which we established the diagnosis of PNP-deficiency early on the basis of detection of lymphopenia and low level of uric acid.展开更多
Background: Phosphorylase kinase (PhK) activity is induced by injurious stimuli, which is known to precipitate psoriasis. We had previously reported that elevated PhK activity in psoriatic epidermis correlated with in...Background: Phosphorylase kinase (PhK) activity is induced by injurious stimuli, which is known to precipitate psoriasis. We had previously reported that elevated PhK activity in psoriatic epidermis correlated with increased psoriatic activity, and that suppression of PhK activity by its inhibitor, curcumin gel, correlated with disease resolution. Objective: We evaluated the efficacy of a strategy of combining PhK inhibition by topical curcumin with elimination of PhK-generating precipitating factors from various injurious stimuli in producing improvement of psoriatic activity, aiming at complete resolution. Patients and Methods: We studied a cohort of 647 consecutive patients with mild to severe psoriasis in a single center. Our therapeutic regimen consisted of curcumin gel, topical steroids, strict avoidance of contact allergens, avoidance of dairy products in lactose-intolerant patients, and treatment of infections to eliminate bacterial superantigens. Results: PASI scores at 0 wk was 24.7 +/– 17.1 (SD), n = 647. PASI scores improved significantly at 4 weeks to 11.5 +/– 8.1 (n = 638;p < 0.0001), at 8 weeks to 4.5 +/– 4.2 (n = 636, p < 0.0001), and at 16 weeks to 0.9 +/– 2.5 (n = 641, p < 0.0001). At 16 weeks, 72.2% of patients were completely clear of psoriatic activity (PASI = 0). Conclusion: Our results indicate that a regimen of PhK inhibition by topical curcumin with elimination of PhK-generating factors is effective in producing significant reduction of psoriatic activity at 16 weeks, with complete clearance of psoriasis in 72.2% of patients.展开更多
Initially it was believed that phosphorylase was responsible for both glycogen breakdown and synthesis in the living cell.The discovery of glycogen synthase and McArdle's disease(lack of phosphorylase activity),to...Initially it was believed that phosphorylase was responsible for both glycogen breakdown and synthesis in the living cell.The discovery of glycogen synthase and McArdle's disease(lack of phosphorylase activity),together with the high Pi/glucose 1-P ratio in skeletal muscle,demonstrated that glycogen synthesis could not be attributed to reversal of the phosphorylase reaction.Rather,glycogen synthesis was attributable solely to the activity of glycogen synthase,subsequent to the transport of glucose into the cell.However,the well-established observation that phosphorylase was inactivated(i.e.,dephosphorylated)during the initial recovery period after prior exercise,when the rate of glycogen accumulation is highest and independent of insulin,suggested that phosphorylase could play an active role in glycogen accumulation.But the quantitative contribution of phosphorylase inactivation was not established until recently,when studying isolated murine muscle preparations during recovery from repeated contractions at temperatures ranging from 25 to 35C.Thus,in both slow-twitch,oxidative and fast-twitch,glycolytic muscles,inactivation of phosphorylase accounted for 45%–75%of glycogen accumulation during the initial hours of recovery following repeated contractions.Such data indicate that phosphorylase inactivation may be the most important mechanism for glycogen accumulation under defined conditions.These results support the initial belief that phosphorylase plays a quantitative role in glycogen formation in the living cell.However,the mechanism is not via activation of phosphorylase,but rather via inactivation of the enzyme.展开更多
Starch phosphorylase is an industrially important enzyme used in the production of glucose-l-phosphate. Here, we extracted the enzyme from the germinating wheat seeds and partially purified using ammonium sulfate frac...Starch phosphorylase is an industrially important enzyme used in the production of glucose-l-phosphate. Here, we extracted the enzyme from the germinating wheat seeds and partially purified using ammonium sulfate fractionation. The partially purified enzyme showed maximum enzyme activity at pH 6.2 and pH 7.2 in the polysaccharide and glucose-l-phosphate formation directions, respectively. The enzyme showed maximum enzyme activity at 37 ℃ temperature with 50% of the enzyme activity at 32 ℃ and 42 ℃. The desalted ammonium sulfate fractionated enzyme has been immobilized on brick dust with nearly 60% enzyme activity retention. The specific activity of the immobilized starch phosphorylase increased from 0.410 to 0.925. There was a slight alkaline shift in the optimum pH when assayed in both the directions. The immobilized starch phosphorylase also displayed increased optimum temperature and thermo-stability and could be reused many times. The desalted ammonium sulfate fractionated enzyme exhibited a half life of 4 h at 30 ℃ and 30 rain at 50 ℃ whereas brick dust immobilized enzyme exhibited a half life of 7 h at 30 ℃ and 45 min at 50 ℃. The immobilized enzyme may be exploited for glucose-l-phosphate production.展开更多
A gene encoding the trehalose phosphorylase(TreP),which reversibly catalyzes trehalose degradation and synthesis fromα-glucose-1-phosphate(α-Glc-1-P)and glucose,was cloned from Thermoanaerobacter tengcongensis and s...A gene encoding the trehalose phosphorylase(TreP),which reversibly catalyzes trehalose degradation and synthesis fromα-glucose-1-phosphate(α-Glc-1-P)and glucose,was cloned from Thermoanaerobacter tengcongensis and successfully expressed in Escherichia coli.The overexpressed TreP,with a molecular mass of approximately 90 kDa,was determined by SDS-PAGE.It catalyzes trehalose synthesis and degradation optimally at 70℃(for 30 min),with the optimum pHs at 6.0 and 7.0,respectively.It is highly thermostable,with a 77%residual ac-tivity after incubation at 50℃for 7 h.Under the optimum reaction conditions,50μg crude en-zyme of the TreP is able to catalyze the synthesis of trehalose up to 11.6 mmol/L from 25 mmol/Lα-Glc-1-P and 125 mmol/L glucose within 30 min,while only 1.5 mmol/L out of 250 mmol/L tre-halose is degraded within the same time period.Dot blotting revealed that the treP gene in T.tengcongensis was upregulated in response to salt stress but downregulated when trehalose was supplied.Both results indicate that the dominant function of the T.tengcongensis TreP is catalyzing trehalose synthesis but not degradation.Thus it might provide a novel route for indus-trial production of trehalose.展开更多
Canceling grids accommodating probes in comparative molecular field analysis(CoMFA),the idea of flexibleness is introduced into the CoMFA,and in combination with swarm intelligent algorithm which attempts to optimize ...Canceling grids accommodating probes in comparative molecular field analysis(CoMFA),the idea of flexibleness is introduced into the CoMFA,and in combination with swarm intelligent algorithm which attempts to optimize distributions of diverse probes around drug molecules,a new 3D-QSAR method is proposed in this context as flexible comparative molecular field analysis(FCoMFA).In preliminary at-tempts to performing QSAR studies on 47 glycogen phosphorylase inhibitors,FCoMFA is employed and confirmed to be potent to exploring ligand-receptor interaction manners at active positions and thus to generating stable and predictable models.Simultaneously by an intuitive graphics regarding probe distribution patterns,impacts of different substituted groups on activities is also given an insight into.展开更多
Influenza A virus(IAV)poses a significant threat to human health.The outcome of IAV results from the viral-host interaction,with the underlying molecular mechanisms largely unknown.By integrating the plasma proteomics...Influenza A virus(IAV)poses a significant threat to human health.The outcome of IAV results from the viral-host interaction,with the underlying molecular mechanisms largely unknown.By integrating the plasma proteomics data of the IAV-infected patients into the viral-inflammation protein-protein interaction(VI-PPI)network created in this study,purine nucleoside phosphorylase(PNP),the critical enzyme in purine salvage,was identified as a potential hub gene that connected the different stages of IAV infection.Extended survival rates and reduced pulmonary inflammatory lesions were observed in alveolar epithelial cell(AEC)-specific PNP conditional knockout mice upon H1N1 infection.Mechanistically,PB1-F2 of IAV was revealed as a novel viral transcriptional factor to bind to the TATA box of PNP promoter,leading to enhanced purine salvage in H1N1-challenged AECs.The activation of PNP-mediated purine salvage was verified in IAV-infected patients and A549 cells.PNP knockdown elicited a purine metabolic shift from augmented salvage pathway to de novo synthesis,constraining both viral infection and pro-inflammatory signaling through APRT-AICAR-AMPK activation.Moreover,durdihydroartemisinin(DHA),predicted by VI-PPI as a novel PNP inhibitor,exerted beneficial effects on the survival and weight gain of H1N1-challenged mice via its direct binding to PNP.To reveal for the first time,we found that PNP,activated by IAV,plays a hub role within H1N1-host interaction,simultaneously modulating viral replication and hyperinflammation through purine salvage.Our study sheds new light on a“two-for-one”strategy by targeting purine salvage in combating IAV-related pathology,suggesting PNP as a potential novel anti-influenza host target.展开更多
Objectives:Breast cancer(BC)is the leading cause of cancer-related mortality in women,largely due to metastasis.This study aims to explore the role of purine nucleoside phosphorylase(PNP),a key enzyme in purine metabo...Objectives:Breast cancer(BC)is the leading cause of cancer-related mortality in women,largely due to metastasis.This study aims to explore the role of purine nucleoside phosphorylase(PNP),a key enzyme in purine metabolism,in the aggressiveness and metastatic behavior of BC.Methods:A comprehensive analysis was performed using in silico transcriptomic data(n=2509 patients),immunohistochemical profiling of BC tissues(n=103),and validation through western blotting in multiple BC cell lines.Gene expression and survival analyses were conducted using Tumor Immune Estimation Resource(TIMER),Gene Expression Profiling Interactive Analysis 2(GEPIA2),and the cBioPortal for cancer genomics(cBioPortal)platforms.Correlations between PNP and key epithelial–mesenchymal transition(EMT)markers,molecular subtypes,tumor grades,and stages were examined.Results:PNP was significantly overexpressed in human epidermal growth factor receptor 2(HER-2)-positive and triple-negative BCs compared to luminal subtypes.High PNP levels were strongly associated with advanced BC stages,high-grade tumors,EMT phenotypes,and poor overall survival.Notably,HER-2 inhibition suppressed PNP expression,while PNP gene silencing induced HER-2 upregulation,revealing a reciprocal regulatory loop.Dual inhibition of PNP and HER-2 resulted in a significant reduction in cell viability compared to HER-2 inhibition alone.Conclusion:Collectively,PNP emerges as a promising biomarker of BC aggressiveness and progression.Its reciprocal interaction with HER-2 underscores its potential as a therapeutic target.Dual targeting of PNP and HER-2 may offer a novel strategy for improving outcomes in aggressive BC subtypes.展开更多
Background Pancreatic cancer is one of the most common tumors and has a 5-year survival for all stages of less than 5%. Most patients with pancreatic cancer are diagnosed at an advanced stage and therefore are not can...Background Pancreatic cancer is one of the most common tumors and has a 5-year survival for all stages of less than 5%. Most patients with pancreatic cancer are diagnosed at an advanced stage and therefore are not candidates for surgical resection. In recent years, investigation into alternative treatment strategies for this aggressive disease has led to advances in the field of gene therapy for pancreatic cancer. E. coil purine nucleoside phosphorylase/6-methylpurine deoxyribose （ePNP/MePdR） is a suicide gene/prodrug system where PNP enzyme cleaves nontoxic MePdR into cytotoxic membrane-permeable compounds 6-methylpurine （MeP） with high bystander activity, hTERT is expressed in cell lines and tissues for telomerase activity. In this study we examined the efficacy of ePNP under the control of hTERT promoter sequences and assessed the selective killing effects of the ePNP/prodrug MePdR system on pancreatic tumors. Methods Recombinant pET-PNP was established. The protein of E. coil PNPase was expressed and an antibody to E. coil PNPase was prepared. Transcriptional activities of hTERT promoter sequences were analyzed using a luciferase reporter gene. A recombinant phTERT-ePNP vector was constructed. The ePNP/MePdR system affects SW1990 human pancreatic cancer cell lines in vitro. Results The hTERT promoter had high transcriptional activity and conferred specificity on cancer cell lines. The antibody to E. coil PNPase was demonstrated to be specific for the ePNP protein. The MePdR treatment induced a high in vitro cytotoxicity on the sole hTERT-ePNP-producing cell lines and affected SW1990 cells in a dose-dependent manner. Conclusions The hTERT promoter control of the ePNP/MePdR system can provide a beneficial anti-tumor treatment in pancreatic cancer cell lines including a good bystander killing effect.展开更多
Carbohydrate partitioning is essential for plant growth and development,and its hindrance will result in excess accumulation of carbohydrates in source tissues.Most of the related mutants in maize(Zea mays L.)display ...Carbohydrate partitioning is essential for plant growth and development,and its hindrance will result in excess accumulation of carbohydrates in source tissues.Most of the related mutants in maize(Zea mays L.)display impaired whole-plant sucrose transport,but other mechanisms affecting carbohydrate partitioning have seldom been reported.Here,we characterized chlorotic leaf3(chl3),a recessive mutation causing leaf chlorosis with starch accumulation excessively in bundle sheath chloroplasts,suggesting that chl3 is defective in carbohydrate partitioning.Positional cloning revealed that the chl3 phenotype results from a frameshift mutation in ZmPHOH,which encodes starch phosphorylase 2.Two mutants in ZmPHOH exhibited the same phenotype as chl3,and both alleles failed to complement the chl3 mutant phenotype in an allelism test.Inactivation of Zm PHOH in chl3 leaves reduced the efficiency of transitory starch conversion,resulting in increased leaf starch contents and altered carbohydrate metabolism patterns.RNA-seq revealed the transcriptional downregulation of genes related to photosynthesis and carbohydrate metabolism in chl3 leaves compared to the wild type.Our results demonstrate that transitory starch remobilization is very important for cellular carbohydrate partitioning in maize,in which Zm PHOH plays an indispensable role.展开更多
文摘AIM： To investigate the prognostic role of thymidylate synthase （TS） and thymidine phosphorylase （TP） mRNA levels in T3 or T4 gastric cancer treated with 5-fluorouraci-based adjuvant chemotherapy. METHODS： Fifty-one patients with T3 or T4 gastric cancer received systemic 5-fluorouraci-based adjuvant chemotherapy, and intratumoral expression of TS and TP in 51 gastric cancer tissue samples was tested by realtime quantitative PCR.RESULTS： The median disease-free survival （DFS） time was 10.2 mo in the patients. There were no significant differences in DFS between the groups with high and low levels of TP. However, the group with low level of TS had a longer DFS （14.4 mo vs 8.3 mo, P = 0.017）. The median overall survival （OS） time was 18.5 mo, and there were significant differences in OS between the groups with high and low levels of TS or TP （for TS, 17.0 mo vs 21.3 mo, P = 0.010; for TP, 16.6 mo vs 22.5 too, P = 0.009）. Moreover, the coupled low expression of these two genes was strongly associated with a longer survival time of patients as compared with that of a single gene.CONCLUSION： Expression of TS and TP mRNA is a useful predictive parameter for the survival of postoperative gastric cancer patients after 5-fluorouracilbased adjuvant chemotherapy.
基金Project (No. 07C26213101283) supported by the Innovation Fundfor Technology Based Firms from the Ministry of Science andTechnology of China
文摘Nucleoside phosphorylases (NPases) were found to be induced in Enterobacter aerogenes DGO-04, and cytidine and cytidine 5′-monophosphate (CMP) were the best inducers. Five mmol/L to fifteen mmol/L cytidine or CMP could distinctly increase the activities of purine nucleoside phosphorylase (PNPase), uridine phosphorylase (UPase) and thymidine phosphorylase (TPase) when they were added into medium from 0 to 8 h. In the process of enzymatic synthesis of adenine arabinoside from adenine and uracil arabinoside with wet cells of Enterobacter aerogenes DGO-04 induced by cytidine or CMP, the reaction time could be shortened from 36 to 6 h. After enzymatic reaction the activity of NPase in the cells induced remained higher than that in the cells uninduced.
文摘Thymidine phosphorylase (TP) is a key enzyme that contributes to the composition and decomposition of pyrimidine nucleotides. TP seems homologous to platelet-derived endothelial cell growth factor, and its effects on inducing vascularization and anti-apoptosis are closely related to growth and metastasis of colorectal carcinoma. In addition, TP is a key enzyme that catalyzes the transformation from 5-fluorouracil (FU) prodrugs of 5′-deoxy-5-fluorouridine (5′- DFUR) to 5-FU. The activity of TP is closely related to the sensitivity of colorectal carcinoma cells to fluorouracil drugs and targeted therapy. Given the important functions of TP in growth, metastasis, tumor treatment, and prognosis, determining its expression mechanism is significant. This article summarizes the research development of TP expression in colorectal carcinoma, tumor neovascularization, cytotoxicity activation of 5′-DFUR, and colorectal carcinoma therapy.
基金supported by a grant from the National Natural Sciences Foundation of China (No. 30973184)
文摘The mRNA and protein expression of thymidylate synthase (TS), thymidine phosphorylase (TP) and dihydropyrimidine dehydrogenase (DPD) and their relationship with prognosis were investigated. Real-time quantitative RT-PCR (Taqman) was used to detect the mRNA expression of TS, TP and DPD in formalin-fixed and paraffin-embedded 106 samples of epithelial ovarian cancer and 29 normal ovaries. A TATA box-binding protein (TBP) was used as an endogenous reference gene. A relationship between TS, TP, DPD expression and clinicopathologic features was investigated. The protein location and expression of TS, TP and DPD was examined in the same patients by an avidin-biotin-peroxidase immunohistochemistry. TS and TP mRNA expression levels were significantly higher in tumor group than in normal controls, with the average value of TS and TP mRNA being 6.14±0.62 and 0.59±0.06 in tumor tissue, and 0.71±0.14 and 0.16±0.04 in normal tissue, respectively. DPD mRNA expression levels were significantly lower in tumor group (0.11±0.02) than in normal controls (0.38±0.05). There was statistically significant difference in TS and TP mRNA expression levels among different pathological grades and clinical stages (P<0.05), but histological subtype was not significantly associated with TS and TP mRNA expression. DPD gene expression was not significantly associated with any clinicopathological parameters. Immunohistochemistry revealed that TP protein was mainly distributed in nucleus, and TS and DPD mainly in cytoplasm. The protein expression intensity of TS, TP and DPD was coincided with the mRNA expression levels. It was concluded that TS, TP mRNA and protein expression levels were significantly higher in epithelial ovarian cancer, and DPD mRNA and protein expression levels were significantly lower. The expression levels of TS and DPD were related to the patients’ prognosis and survival. Combined gene expression levels of TS, TP and DPD represent a new variable to predict the clinical outcome in ovarian cancer. The association of TS, TP and DPD expression levels with survival suggests an importance of these genes for tumor occurrence and progression.
基金Supported by The Thailand Research Fund through The Royal Golden Jubilee PhD Program Grant No. PHD/0037/2544 for Thanasai J and Limpaiboon T and grants-in-aid from the Centre for Research and Development of Medical Diagnostic Laboratories, Faculty of Associated Medical Sciences, Khon Kaen University, Thailand, and from the Ministry of Education, Sports, Science, Culture and Technology, Japan
文摘AIM: To evaluate the role of thymidine phosphorylase (TP) in cholangiocarcinoma using small interfering RNA (siRNA). METHODS: A human cholangiocarcinoma-derived cell line KKU-M139, which has a naturally high level of endogenous TP, had TP expression transiently knocked down using siRNA. Cell growth, migration, in vitro angiogenesis, apoptosis, and cytotoxicity were assayed in TP knockdown and wild-type cell lines. RESULTS: TP mRNA and protein expression were decreased by 87.1% ± 0.49% and 72.5% ± 3.2%, respectively, compared with control cells. Inhibition of TP significantly decreased migration of KKU-M139, and suppressed migration and tube formation of human umbilical vein endothelial cells. siRNA also reduced the ability of TP to resist hypoxia-induced apoptosis, while suppression of TP reduced the sensitivity of KKU-M139 to 5-fluorouracil. CONCLUSION: Inhibition of TP may be beneficial in decreasing angiogenesis-dependent growth and migration of cholangiocarcinoma but may diminish the response to 5-fluorouracil chemotherapy.
基金Supported by"Production,Education&Research"item of Shanghai Baoshan(08-H-4)
文摘Recombinant Escherichia coli pUDP,which overexpressed uridine phosphorylase（UPase）,was constructed.0.5 mmol·L 1lactose had a similar induction effect as the commonly used inducer IPTG during 2.5-5.5 h of cell growth.The lactose-induced UPase was stable at 50°C.Wet cells of pUDP was used as catalyst to biosynthesize 5-fluorouridine from 30 mmol·L 1uridine and 5-fluorouracil in phosphate buffer（pH 7.0）catalyzed at 50°C for 1.5 h and the yield of 5-fluorouridine was higher than 68%.Under the optimum reaction conditions for production of 5-fluorouridine,5-methyluridine and azauridine were synthesized from uridine by pUDP,the yield was 61.7%and 47.2%respectively.Deoxynucleosides were also synthesized by pUDP,but the yield was only about 20%.
文摘Wistar rats were exposed to trinitrotoluene (TNT) for 6 weeks. After initiation of TNT exposure, serum phosphorylase A activities and calcium contents were assayed for every 2 weeks. Both of these 2 parameters increased in rats treated with 50 and 100 mg TNT/kg b.w. at 3 intervals. Serum phosphorylase A activities and calcium contents of TNT exposure worker increased too.
文摘Nucleoside phosphorylase is an important enzyme involved in the biosynthesis of nucleosides. In this study, purine nucleoside phosphorylase and pyrimidine nucleoside phosphorylase were co-expressed in Escherichia coli and the intact cells were used as a catalyst for the biosynthesis of nucleosides. For protein induction, lactose was used in place of isopropyl β-D-l-thiogalactopyranoside （IPTG）. When the concentration of lactose was above 0.5 mmol/L, the ability to induce protein expression was similar to that of IPTG. We determined that the reaction conditions of four bacterial strains co-expressing these genes （TUD, TAD, DUD, and DAD） were similar for the biosyntheses of 2,6-diaminopurine nucleoside and 2,6-diaminopurine deoxynucleoside. When the substrate concentration was 30 mmol/L and 0.5% of the recombinant bacterial cell volume was used as the catalyst （pH 7.5）, a greater than 90% conversion yield was reached after a 2-h incubation at 50℃. In addition, several other nucleosides and nucleoside derivatives were efficiently synthesized using bacterial strains co-expressing these recombinant enzymes.
基金Supported by the National Natural Science Foundation of China (30530190 to XDS and 30700115 to NY)
文摘Purine nucleoside phosphorylase is a key enzyme in the purine-salvage pathway and an attractive target for drug design. The crystal structure of Streptococcus mutants purine nucleoside phosphorylase（Smu PNP） has been solved by molecular replacement at 1.80 resolution and refined to R factors of 19.9%/23.7%（Rcryst/Rfree） . Sequence alignment and structural comparison show that Smu PNP has more similarity with PNPs isolated from human and malarial sources than the bacterial PNPs. The structure complexed with hypoxanthine（HPA） and sulfate ion was solved at 2.24A resolution and refined to R factors of 21.6%/24.1%（Rcryst/Rfree） . It is interesting to note that the resulting electron density indicated the product,HPA,presents in the active site although inosine was included in the crystallization mixture with Smu PNP. Asn233 and Glu191 are the important residues for ligand binding and recognition. Comparison with PNPs from different species gives detailed information about binding of small molecules on the active site,which is important for the studies of enzymatic mechanism and rational design of specific inhibitors for PNPs.
文摘Methylthioadenosine phosphorylase, (MTAP) is a key enzyme in the adenine and methionine salvage pathways. MTAP is encoded on human chromosome 9p21 in close proximity to the p16INK4a and p14ARF tumor suppressor genes and is frequently co-deleted with p16INK4a in many cancers. Deletion of MTAP has been reported to create a reliance of MTAP–/– tumors on de novo purine synthesis to maintain adequate pools of AMP, leading to increased sensitivity to purine synthesis inhibitors, such as L-alanosine. The ‘Achilles heel’ created by the loss of MTAP in cancer cells provides a unique therapeutic opportunity whereby MTAP–/– tumors could be selectively targeted with purine synthesis inhibitors and normal tissues could be preferentially rescued with MTAP substrates, such as MTA. We demonstrate that, in contrast to published literature, MTAP–/– cells are not more sensitive to inhibition of de novo purine synthesis than MTAP+/+ cells. Although MTA can preferentially rescue MTAP+/+ cells from purine-synthesis inhibitor toxicity in vitro, MTA protects cells of both genotypes from L-alanosine equivalently in vivo. Our data demonstrate that in vivo, adenine salvaged from plasma and adjacent tissues is sufficient to protect MTAP–/– tumors from the effects of purine synthesis inhibitors. These results suggest targeting MTAP–/– tumors with de novo purine synthesis inhibitors is unlikely to provide significant benefit over other therapeutic strategies and may explain, at least in part, the lack of efficacy of L-alanosine in clinical trials.
文摘Purine Nucleoside Phosphorylase (PNP) deficiency is a rare autosomal recessive metabolic disorder. In PNP-deficiency disorder, the deficient enzyme leads to accumulation of toxic metabolites, especially in lymphocytes and the metabolites exert toxic effect on T-cell generation. Purine nucleoside phosphorylase deficiency causes decreased numbers of T cells and lymphopenia. The patients suffering from PNP-deficiency may be admitted with recurrent infections, as well as neurological and autoimmune findings. We hereby presented a case admitted with the symptom of hematuria in which we established the diagnosis of PNP-deficiency early on the basis of detection of lymphopenia and low level of uric acid.
文摘Background: Phosphorylase kinase (PhK) activity is induced by injurious stimuli, which is known to precipitate psoriasis. We had previously reported that elevated PhK activity in psoriatic epidermis correlated with increased psoriatic activity, and that suppression of PhK activity by its inhibitor, curcumin gel, correlated with disease resolution. Objective: We evaluated the efficacy of a strategy of combining PhK inhibition by topical curcumin with elimination of PhK-generating precipitating factors from various injurious stimuli in producing improvement of psoriatic activity, aiming at complete resolution. Patients and Methods: We studied a cohort of 647 consecutive patients with mild to severe psoriasis in a single center. Our therapeutic regimen consisted of curcumin gel, topical steroids, strict avoidance of contact allergens, avoidance of dairy products in lactose-intolerant patients, and treatment of infections to eliminate bacterial superantigens. Results: PASI scores at 0 wk was 24.7 +/– 17.1 (SD), n = 647. PASI scores improved significantly at 4 weeks to 11.5 +/– 8.1 (n = 638;p < 0.0001), at 8 weeks to 4.5 +/– 4.2 (n = 636, p < 0.0001), and at 16 weeks to 0.9 +/– 2.5 (n = 641, p < 0.0001). At 16 weeks, 72.2% of patients were completely clear of psoriatic activity (PASI = 0). Conclusion: Our results indicate that a regimen of PhK inhibition by topical curcumin with elimination of PhK-generating factors is effective in producing significant reduction of psoriatic activity at 16 weeks, with complete clearance of psoriasis in 72.2% of patients.
文摘Initially it was believed that phosphorylase was responsible for both glycogen breakdown and synthesis in the living cell.The discovery of glycogen synthase and McArdle's disease(lack of phosphorylase activity),together with the high Pi/glucose 1-P ratio in skeletal muscle,demonstrated that glycogen synthesis could not be attributed to reversal of the phosphorylase reaction.Rather,glycogen synthesis was attributable solely to the activity of glycogen synthase,subsequent to the transport of glucose into the cell.However,the well-established observation that phosphorylase was inactivated(i.e.,dephosphorylated)during the initial recovery period after prior exercise,when the rate of glycogen accumulation is highest and independent of insulin,suggested that phosphorylase could play an active role in glycogen accumulation.But the quantitative contribution of phosphorylase inactivation was not established until recently,when studying isolated murine muscle preparations during recovery from repeated contractions at temperatures ranging from 25 to 35C.Thus,in both slow-twitch,oxidative and fast-twitch,glycolytic muscles,inactivation of phosphorylase accounted for 45%–75%of glycogen accumulation during the initial hours of recovery following repeated contractions.Such data indicate that phosphorylase inactivation may be the most important mechanism for glycogen accumulation under defined conditions.These results support the initial belief that phosphorylase plays a quantitative role in glycogen formation in the living cell.However,the mechanism is not via activation of phosphorylase,but rather via inactivation of the enzyme.
文摘Starch phosphorylase is an industrially important enzyme used in the production of glucose-l-phosphate. Here, we extracted the enzyme from the germinating wheat seeds and partially purified using ammonium sulfate fractionation. The partially purified enzyme showed maximum enzyme activity at pH 6.2 and pH 7.2 in the polysaccharide and glucose-l-phosphate formation directions, respectively. The enzyme showed maximum enzyme activity at 37 ℃ temperature with 50% of the enzyme activity at 32 ℃ and 42 ℃. The desalted ammonium sulfate fractionated enzyme has been immobilized on brick dust with nearly 60% enzyme activity retention. The specific activity of the immobilized starch phosphorylase increased from 0.410 to 0.925. There was a slight alkaline shift in the optimum pH when assayed in both the directions. The immobilized starch phosphorylase also displayed increased optimum temperature and thermo-stability and could be reused many times. The desalted ammonium sulfate fractionated enzyme exhibited a half life of 4 h at 30 ℃ and 30 rain at 50 ℃ whereas brick dust immobilized enzyme exhibited a half life of 7 h at 30 ℃ and 45 min at 50 ℃. The immobilized enzyme may be exploited for glucose-l-phosphate production.
基金This work was supported by the Chinese Acad-emy of Sciences(Grant No.KSCX2-SW-112)the National Natural Science Foundation of China(Grant No.30170015).
文摘A gene encoding the trehalose phosphorylase(TreP),which reversibly catalyzes trehalose degradation and synthesis fromα-glucose-1-phosphate(α-Glc-1-P)and glucose,was cloned from Thermoanaerobacter tengcongensis and successfully expressed in Escherichia coli.The overexpressed TreP,with a molecular mass of approximately 90 kDa,was determined by SDS-PAGE.It catalyzes trehalose synthesis and degradation optimally at 70℃(for 30 min),with the optimum pHs at 6.0 and 7.0,respectively.It is highly thermostable,with a 77%residual ac-tivity after incubation at 50℃for 7 h.Under the optimum reaction conditions,50μg crude en-zyme of the TreP is able to catalyze the synthesis of trehalose up to 11.6 mmol/L from 25 mmol/Lα-Glc-1-P and 125 mmol/L glucose within 30 min,while only 1.5 mmol/L out of 250 mmol/L tre-halose is degraded within the same time period.Dot blotting revealed that the treP gene in T.tengcongensis was upregulated in response to salt stress but downregulated when trehalose was supplied.Both results indicate that the dominant function of the T.tengcongensis TreP is catalyzing trehalose synthesis but not degradation.Thus it might provide a novel route for indus-trial production of trehalose.
基金Supported by National High Technology(863)Program(Grant No.2006AA02Z312)the State Key Laboratory of Chemo/Biosensing and Chemometrics Foundation(Grant No.0501201)Chongqing University Innovation Fund(Grant No.030506)
文摘Canceling grids accommodating probes in comparative molecular field analysis(CoMFA),the idea of flexibleness is introduced into the CoMFA,and in combination with swarm intelligent algorithm which attempts to optimize distributions of diverse probes around drug molecules,a new 3D-QSAR method is proposed in this context as flexible comparative molecular field analysis(FCoMFA).In preliminary at-tempts to performing QSAR studies on 47 glycogen phosphorylase inhibitors,FCoMFA is employed and confirmed to be potent to exploring ligand-receptor interaction manners at active positions and thus to generating stable and predictable models.Simultaneously by an intuitive graphics regarding probe distribution patterns,impacts of different substituted groups on activities is also given an insight into.
基金supported by the National Natural Science Foundation of China(81830101 and 82301991)。
文摘Influenza A virus(IAV)poses a significant threat to human health.The outcome of IAV results from the viral-host interaction,with the underlying molecular mechanisms largely unknown.By integrating the plasma proteomics data of the IAV-infected patients into the viral-inflammation protein-protein interaction(VI-PPI)network created in this study,purine nucleoside phosphorylase(PNP),the critical enzyme in purine salvage,was identified as a potential hub gene that connected the different stages of IAV infection.Extended survival rates and reduced pulmonary inflammatory lesions were observed in alveolar epithelial cell(AEC)-specific PNP conditional knockout mice upon H1N1 infection.Mechanistically,PB1-F2 of IAV was revealed as a novel viral transcriptional factor to bind to the TATA box of PNP promoter,leading to enhanced purine salvage in H1N1-challenged AECs.The activation of PNP-mediated purine salvage was verified in IAV-infected patients and A549 cells.PNP knockdown elicited a purine metabolic shift from augmented salvage pathway to de novo synthesis,constraining both viral infection and pro-inflammatory signaling through APRT-AICAR-AMPK activation.Moreover,durdihydroartemisinin(DHA),predicted by VI-PPI as a novel PNP inhibitor,exerted beneficial effects on the survival and weight gain of H1N1-challenged mice via its direct binding to PNP.To reveal for the first time,we found that PNP,activated by IAV,plays a hub role within H1N1-host interaction,simultaneously modulating viral replication and hyperinflammation through purine salvage.Our study sheds new light on a“two-for-one”strategy by targeting purine salvage in combating IAV-related pathology,suggesting PNP as a potential novel anti-influenza host target.
基金funded by Al Jalila Foundation-Research Grant(AJF2023-078)to SSMS.
文摘Objectives:Breast cancer(BC)is the leading cause of cancer-related mortality in women,largely due to metastasis.This study aims to explore the role of purine nucleoside phosphorylase(PNP),a key enzyme in purine metabolism,in the aggressiveness and metastatic behavior of BC.Methods:A comprehensive analysis was performed using in silico transcriptomic data(n=2509 patients),immunohistochemical profiling of BC tissues(n=103),and validation through western blotting in multiple BC cell lines.Gene expression and survival analyses were conducted using Tumor Immune Estimation Resource(TIMER),Gene Expression Profiling Interactive Analysis 2(GEPIA2),and the cBioPortal for cancer genomics(cBioPortal)platforms.Correlations between PNP and key epithelial–mesenchymal transition(EMT)markers,molecular subtypes,tumor grades,and stages were examined.Results:PNP was significantly overexpressed in human epidermal growth factor receptor 2(HER-2)-positive and triple-negative BCs compared to luminal subtypes.High PNP levels were strongly associated with advanced BC stages,high-grade tumors,EMT phenotypes,and poor overall survival.Notably,HER-2 inhibition suppressed PNP expression,while PNP gene silencing induced HER-2 upregulation,revealing a reciprocal regulatory loop.Dual inhibition of PNP and HER-2 resulted in a significant reduction in cell viability compared to HER-2 inhibition alone.Conclusion:Collectively,PNP emerges as a promising biomarker of BC aggressiveness and progression.Its reciprocal interaction with HER-2 underscores its potential as a therapeutic target.Dual targeting of PNP and HER-2 may offer a novel strategy for improving outcomes in aggressive BC subtypes.
基金the 135 Project and Science-Technology Fund of Health Department of Jiangsu Province(Z200618)the Science-Technology Fund of Southeast University(XJ0690219)
文摘Background Pancreatic cancer is one of the most common tumors and has a 5-year survival for all stages of less than 5%. Most patients with pancreatic cancer are diagnosed at an advanced stage and therefore are not candidates for surgical resection. In recent years, investigation into alternative treatment strategies for this aggressive disease has led to advances in the field of gene therapy for pancreatic cancer. E. coil purine nucleoside phosphorylase/6-methylpurine deoxyribose （ePNP/MePdR） is a suicide gene/prodrug system where PNP enzyme cleaves nontoxic MePdR into cytotoxic membrane-permeable compounds 6-methylpurine （MeP） with high bystander activity, hTERT is expressed in cell lines and tissues for telomerase activity. In this study we examined the efficacy of ePNP under the control of hTERT promoter sequences and assessed the selective killing effects of the ePNP/prodrug MePdR system on pancreatic tumors. Methods Recombinant pET-PNP was established. The protein of E. coil PNPase was expressed and an antibody to E. coil PNPase was prepared. Transcriptional activities of hTERT promoter sequences were analyzed using a luciferase reporter gene. A recombinant phTERT-ePNP vector was constructed. The ePNP/MePdR system affects SW1990 human pancreatic cancer cell lines in vitro. Results The hTERT promoter had high transcriptional activity and conferred specificity on cancer cell lines. The antibody to E. coil PNPase was demonstrated to be specific for the ePNP protein. The MePdR treatment induced a high in vitro cytotoxicity on the sole hTERT-ePNP-producing cell lines and affected SW1990 cells in a dose-dependent manner. Conclusions The hTERT promoter control of the ePNP/MePdR system can provide a beneficial anti-tumor treatment in pancreatic cancer cell lines including a good bystander killing effect.
基金supported by the Science and Technology Major Program of Hubei Province(2021ABA005)the National Natural Science Foundation of China(31971951 and 31771796)。
文摘Carbohydrate partitioning is essential for plant growth and development,and its hindrance will result in excess accumulation of carbohydrates in source tissues.Most of the related mutants in maize(Zea mays L.)display impaired whole-plant sucrose transport,but other mechanisms affecting carbohydrate partitioning have seldom been reported.Here,we characterized chlorotic leaf3(chl3),a recessive mutation causing leaf chlorosis with starch accumulation excessively in bundle sheath chloroplasts,suggesting that chl3 is defective in carbohydrate partitioning.Positional cloning revealed that the chl3 phenotype results from a frameshift mutation in ZmPHOH,which encodes starch phosphorylase 2.Two mutants in ZmPHOH exhibited the same phenotype as chl3,and both alleles failed to complement the chl3 mutant phenotype in an allelism test.Inactivation of Zm PHOH in chl3 leaves reduced the efficiency of transitory starch conversion,resulting in increased leaf starch contents and altered carbohydrate metabolism patterns.RNA-seq revealed the transcriptional downregulation of genes related to photosynthesis and carbohydrate metabolism in chl3 leaves compared to the wild type.Our results demonstrate that transitory starch remobilization is very important for cellular carbohydrate partitioning in maize,in which Zm PHOH plays an indispensable role.
