Thymidylate synthase and thymidine phosphorylase gene expression as predictive parameters for the efficacy of 5-fluorouraci-based adjuvant chemotherapy for gastric cancer

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.

Induction of nucleoside phosphorylase in Enterobacter aerogenes and enzymatic synthesis of adenine arabinoside

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.

Research development of the relationship between thymidine phosphorylase expression and colorectal carcinoma

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.

Relationship between the Expression of Thymidylate Synthase,Thymidine Phosphorylase and Dihydropyrimidine Dehydrogenase and Survival in Epithelial Ovarian Cancer

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.

Effects of thymidine phosphorylase on tumor aggressiveness and 5-fluorouracil sensitivity in cholangiocarcinoma

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.

Induction of Recombinant Uridine Phosphorylase and Its Application in Biosynthesis of Pyrimidine Nucleosides

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%.

Effects of Trinitrotoluene on Serum Phosphorylase A Activities and on Calcium Contents in Men and Rats

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.

Structural Insight into the Binding of Hypoxanthine with Purine Nucleoside Phosphorylase from Streptococcus mutants

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.

Methylthioadenosine (MTA) Rescues Methylthioadenosine Phosphorylase (MTAP)-Deficient Tumors from Purine Synthesis Inhibition <i>in Vivo</i>via Non-Autonomous Adenine Supply

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.

Case of purine nucleoside phosphorylase deficiency presented with hematuria

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.

Results of Combining Phosphorylase Kinase Inhibition with Removal of Precipitating Factors in Large Cohort of Psoriatic Patients: A Proof of Concept Study

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.

Immobilization of Starch Phosphorylase from Germinating Wheat Seeds

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.

Efficient synthesis of nigerose by a novel nigerose phosphorylase from Anaerosporobacter mobilis

Nigerose is a kind of rare disaccharide connected by anα-1,3 glucosidic bond,which is a potential probiotic due to its antidigestive properties and beneficial functions.This study identified and characterized a novel GH65 glycoside phosphorylase derived from Anaerosporobacter mobilis(AmNP).This new protein could specifically catalyze the phospholysis of nigerose to generate glucose and glucose-1-phosphate in the presence of phosphate,indicating it was a typical nigerose phosphorylase.Compared to the previously reported nigerose phosphorylases,AmNP exhibited lower affinity towards nigerose in phosphorolysis reaction and higher affinity towards glucose in reverse phosphorolysis reaction,which indicated that AmNP might be superior in the synthetic capability of disaccharide.Then AmNP was employed to synergize with maltose phosphorylase from Lactobacillus brevis(LbMP)to catalyze the synthesis of nigerose using maltose as the substrate.After optimization of reaction conditions,the highest nigerose yield reached 132.0 g/L with a 66.3%conversion rate,which was higher than ever reported cases using the same reaction pathway to our knowledge.These findings on AmNP in this work were expected to provide a new candidate for large-scale enzymatic synthesis of nigerose and have important theoretical significance for studying nigerose phosphorylase.

The role of glycogen phosphorylase in glycogen biogenesis in skeletal muscle after exercise

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.

hTERT-targeted E.coli purine nucleoside phosphorylase gene/ 6-methylpurine deoxyribose therapy for pancreatic cancer

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.

Starch phosphorylase 2 is essential for cellular carbohydrate partitioning in maize

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.

Disaccharide phosphorylases: Structure, catalytic mechanisms and directed evolution

Disaccharide phosphorylases(DSPs)are carbohydrate-active enzymes with outstanding potential for the biocatalytic conversion of common table sugar into products with attractive properties.They are modular enzymes that form active homo-oligomers.From a mechanistic as well as a structural point of view,they are similar to glycoside hydrolases or glycosyltransferases.As the majority of DSPs show strict stereo-and regiospecificities,these enzymes were used to synthesize specific disaccharides.Currently,protein engineering of DSPs is pursued in different laboratories to broaden the donor and acceptor substrate specificities or improve the industrial particularity of naturally existing enzymes,to eventually generate a toolbox of new catalysts for glycoside synthesis.Herein we review the characteristics and classifications of reported DSPs and the glycoside products that they have been used to synthesize.

Enhancing thermostability and activity of sucrose phosphorylase for high-level production of 2-O-α-d-glucosylglycerol

Sucrose phosphorylase(SPase)can transfer the glucosyl group of sucrose to different compounds and has been widely used in industry.To overcome the low thermostability of the sucrose phosphorylase from Leuconostoc mesenteroides ATCC 12291(LmSP),a method named PROSS was used to construct mutants with increased thermostability.All variants were screened by measuring their residual activities after heating at 50℃.Then,a single point mutant and a combined mutant with improved thermostability and activity were obtained.The half-lives of mutants at 50℃ were approximately twice as high as those of the wild type.In addition,2-O-α-d-glucosylglycerol(αGG)was synthesized by the wild type and the two improved variants,and the reaction conditions were optimized.Under the conditions of glycerol concentration of 3.2 mol/L,sucrose concentration of 1.2 mol/L,and enzyme concentration of 40 U/mL at 37℃ for 60 h,the yield ofαGG reached the maximum,and the sucrose conversion rate of the wild type,the mutant V23L and the combined mutant V23L/S424R were 62.3%,70.7%and 76.3%,respectively.In this study,SPase mutants with higher activity and stability were obtained,and achieved high-level production ofαGG.

Linc-RAM promotes muscle cell differentiation via regulating glycogen phosphorylase activity

Long non-coding RNAs(lncRNAs)are important regulators of diverse biological processes,especially skeletal muscle cell differentiation.Most of the lncRNAs identified to date are localized in the nucleus and play regulatory roles in gene expression.The cytoplasmic lncRNAs are less well understood.We previously identified a long intergenic non-coding RNA(linc-RNA)activator of myogenesis(Linc-RAM)that directly binds MyoD in the nucleus to enhance muscle cell differentiation.Here,we report that a substantial fraction of Linc-RAM is localized in the cytoplasm of muscle cells.To explore the molecular functions of cytoplasmic Linc-RAM,we sought to identify Linc-RAM-binding proteins.We report here that Linc-RAM physically interacts with glycogen phosphorylase(PYGM)in the cytoplasm.Knockdown of PYGM significantly attenuates the function of Linc-RAM in promoting muscle cell differentiation.Loss-of-function and gain-of function assays demonstrated that PYGM enhances muscle cell differentiation in an enzymatic activity-dependent manner.Finally,we show that the interaction between Linc-RAM and PYGM positively regulates the enzymatic activity of PYGM in muscle cells.Collectively,our findings unveil a molecular mechanism through which cytoplasmic Linc-RAM contributes to muscle cell differentiation by regulating PYGM activity.Our findings establish that there is crosstalk between lncRNAs and cellular metabolism during myogenic cell differentiation.

Gene expression and molecular characterization of a thermostable trehalose phosphorylase from Thermoanaerobacter tengcongensis

A gene encoding the trehalose phosphorylase (TreP), which reversibly catalyzes trehalose degradation and synthesis from α-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, 50 μg crude en- zyme of the TreP is able to catalyze the synthesis of trehalose up to 11.6 mmol/L from 25 mmol/L α-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.