Effects of β-Glucan Supplementation on Repairing of Phenol-Induced Vaginal Mucosal Epithelium Damage in Rats

Objective: To investigate the effects of different concentrations of β-glucan on the repair of damaged vaginal mucosa, the expression of vascular endothelial growth factor (VEGF), and the inflammatory factor-6 (IL-6) in vaginal tissues. Methods: Thirty-six adult female specific pathogen free (SPF)-grade Wistar rats were randomly divided into 3 phase groups with 12 rats each. Vaginal inflammation rat models were established by injecting phenol gel into the vagina of each rat at a dose of 0.1 ml/100g body weight. After modeling, rats were divided into 4 groups based on different concentrations of the test agent. The control group was injected with 0.5 ml of saline, experimental group A was injected with 0.375 ml saline 0.125 ml β-glucan, experimental group B was injected with 0.25 ml saline 0.25 ml β-glucan, and experimental group C was injected with 0.50 ml β-glucan. The injection sites were selected at the 3 o’clock and 9 o’clock positions of the vagina. Rats were sacrificed at 7-, 14-, and 28-days post-injection, and tissue samples were collected from the injection sites and prepared for histological analysis. New blood vessels and fibroblast numbers in the tissues were observed after Hematoxylin-eosin (HE) staining. The expression levels of VEGF and IL-6 in the tissues were measured using quantificational reverse transcription polymerase chain reaction (qRT-PCR). Results: Histological examination of vaginal tissue specimens at 7-, 14-, and 28-days post-injection showed that on day 7, there were no significant changes in the experimental groups compared to the control group. However, on days 14 and 28, the experimental groups showed more new blood vessels, macrophages, and fibroblasts with increased activity compared to the control group. The expression levels of VEGF in vaginal tissues were elevated on days 14 and 28 in the experimental groups. The comparison of IL-6 levels in vaginal tissues on day 28 showed that serum IL-6 levels returned to normal, and there was no statistically significant difference between the experimental and control groups. Conclusion: In the 3 experimental phases, the increase in VEGF levels in vaginal tissues on day 14 post-injection was more pronounced with higher concentrations of β-glucan, and IL-6 levels returned to normal on day 28. β-Glucan can enhance VEGF levels in damaged vaginal tissues, promote the repair of damaged vaginal tissues, and higher concentrations of β-glucan have a better effect.

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.

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.

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 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.

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.

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.

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.

Mushroomβ-glucan and polyphenol formulations as natural immunity boosters and balancers:nature of the application

Mushrooms are experiencing a kind of renaissance as a part of the contemporary human diet.These valuable organisms are more than food,they fit in perfectly as a novel market group known as nutra-mycoceuticals.Immune-balancing mushroom dietary fibers and secondary metabolites such as polyphenols are the main focus of the healthcare industry.Wellness and cosmetic companies are increasingly using mushroom extracts rich in these ingredients.This review considers the basic molecular immunomodulatory mechanisms of action of the most commonly used mushroom dietary fibers,β-glucans.The literature data on their bioavailability,metabolic transformations,preclinical and human clinical research,and safety are discussed.Immunomodulatory mechanisms of polyphenol ingredients are also considered.These molecules present great potential in the design of the new immunity balancer formulations according to their widespread structural diversity.Finally,we draw attention to the perspectives of modern trends in mushroom nutraceutical and cosmeceutical formulations to strengthen and balance immunity.

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.

β-Glucans: Characterization, Extraction Methods, and Valorization

β-glucans are bioactive compounds with a wide range of biological properties, including anticancer, anti-inflammatory, antioxidant, and immune-modulating properties. Due to their specific physical properties, such as (in)solubility, viscosity, and gelation, β-glucans are increasingly being used in the food, pharmaceutical, and cosmetic industries. The purpose of this review is to provide an overview of the different types of β-glucans, their sources, especially Saccharomyces cerevisiae yeasts, and the methods of extraction, isolation, and purification of β-glucans, with the aim of optimizing these methods for the efficient production process. Moreover, the physico-chemical properties, modifications, current applications and future prospects of the use of β-glucans in food, medicines, cosmetics and other potential value-added products are summarized. The data presented indicate that β-glucans will play an increasingly important role in the sector of special-purpose food products as well as in other current and future areas.

嘌呤核苷磷酸化酶/嘧啶核苷磷酸化酶共表达及固定化催化合成阿糖核苷

将嘌呤核苷磷酸化酶(PNP)和尿苷磷酸化酶(UP)进行共表达和双酶固定化,提高生物法合成阿糖核苷过程中底物转化率和催化剂稳定性。首先,构建大肠杆菌内源(PNP)和(UP)过表达工程菌,考察工程菌催化不同阿糖核苷之间转化的效率。将PNP和UP双酶固定化,并对固定化双酶进行回收利用,考察重复使用过程中固定化双酶的催化效率。结果表明:工程菌催化阿糖尿苷和腺嘌呤生成阿糖腺苷的反应最高转化率可达93.6%。固定化双酶催化合成阿糖2,6-二氨基嘌呤核苷的最高转化率可达99.8%。重复回收使用固定化双酶19次后对底物的转化率达到60.6%,累计有效催化时长可达684 h。PNP和UP的双酶耦合体系能高效催化阿糖核苷之间的转化,固定化修饰有利于延长酶的使用寿命,为生物法生产核苷类似物提供重要科学参考。

蔗糖磷酸化酶的异源表达及合成蜜二糖条件优化

为挖掘蔗糖磷酸化酶(sucrose phosphorylase,SPase)合成蜜二糖的潜力、降低蜜二糖制备的工业成本,该研究利用大肠杆菌对SPase基因进行异源表达,通过镍柱纯化后表征其酶学性质,采用响应面试验优化合成蜜二糖的最佳反应条件,成功将芒果源肠膜明串珠菌(Leuconostoc mesenteroides)的SPase基因在大肠杆菌BL21(DE3)中表达,获得可溶性SPase。酶学性质研究结果表明其比酶活为78 U/mg,最适反应温度和pH值分别为40℃和6.0。以此为基础利用SPase进行蜜二糖的合成优化,结果表明,在40℃条件下,添加200 g/L蔗糖、400 g/L D-半乳糖和390 U/L的SPase,反应24 h最高可获得浓度为(34.20±0.92)g/L的蜜二糖。

长双歧杆菌蔗糖磷酸化酶的异源表达及其酶学性质

为挖掘来源于长双歧杆菌的蔗糖磷酸化酶(Bifidobacterium longum sucrose phosphorylase,BloSPase)合成2⁃O⁃α⁃D⁃吡喃葡糖基⁃L⁃抗坏血酸(2⁃O⁃α⁃D⁃glucopyranosyl⁃L⁃ascorbic acid,AA⁃2G)的潜力,该研究将BloSPase基因在大肠杆菌中异源表达,以BloSPase的水解酶活力为指标,通过单因素试验和响应面法对其表达条件进行优化;并通过镍柱纯化后对其转糖基化活力进行研究。结果表明,BloSPase的最佳表达条件为诱导时间10 h、异丙基⁃β⁃D⁃硫代半乳糖苷(isopropylthio⁃β⁃D⁃galactoside,IPTG)浓度0.47 mmol/L、诱导温度23℃。在该条件下,水解酶活力可达到(730.35±0.58)U/mL,比酶活达到(95.22±2.45)U/mg。酶学性质研究显示,BloSPase转糖基化活力的最适反应温度为50℃,最适反应pH值为5.2,在40、50℃时表现出较强的热稳定性。其动力学参数Km值为(266.80±23.76)mmol/L,Vmax值为(0.2350±0.0099)mmol/(L·min)。

C端非催化结构域对嗜热α-葡聚糖磷酸化酶TsGP酶学性质的影响

为获得异源表达水平与酶学性质良好的α-葡聚糖磷酸化酶并考察结构域对酶的影响,研究通过数据库挖掘和序列分析,获得了来源于超级嗜热古菌Thermococcus sp.EP1的新型α-葡聚糖磷酸化酶TsGP。利用AlphaFold2预测三维结构确定TsGP的C端为非催化结构域后,构建了C端截短的突变体ΔTsGP。在E.coli BL21(DE3)中对TsGP与ΔTsGP进行异源重组表达,并对其酶学性质进行了表征。结果表明,ΔTsGP在大肠杆菌中的表达水平较TsGP提高了3.76倍。在最适反应温度为70℃时,ΔTsGP的比酶活为23.87 U/mg,较TsGP提高1.3倍。在50~65℃的工业酶催化条件下,ΔTsGP的热稳定性与TsGP相当,且酶活力更高。此外,ΔTsGP与TsGP具有相同的底物特异性,最小底物为麦芽三糖,且随着底物链长的增加,比酶活逐渐提升。以麦芽七糖为底物,ΔTsGP的kcat值为37.09 s^(−1),比TsGP提高了1.21倍,但底物亲和力(K_(m)=3.30 mmol/L)降低了2.77倍。研究通过结构分析与非催化结构域截短策略成功提高了TsGP在大肠杆菌中的表达水平和工业酶催化条件下的比酶活。这为αGP酶蛋白的高效表达与应用提供了借鉴,并为进一步通过工程改造优化其性能奠定了基础。

MTAP在前列腺癌中的表达及对免疫细胞的影响初探

目的:探究甲硫腺苷磷酸化酶(methylthioadenosine phosphorylase,MTAP)在前列腺癌不同临床阶段的表达差异及其对免疫细胞的影响。方法:从TCGA和GTEx数据库中获取MTAP的表达谱数据,利用生物信息学方法进行泛癌分析。免疫组化验证上述结论并探究MTAP与临床病理指标之间的关联。RT-qPCR和Western blot检测MTAP在不同前列腺癌细胞系中的表达水平,分析MTAP与肿瘤恶性程度之间的关联。生物信息学方法分析前列腺癌中MTAP与免疫细胞的关联并进行免疫组化验证。结果:在前列腺癌中,MTAP的表达在肿瘤发生早期升高后逐渐降低,与T分期和Gleason评分呈负相关(rs=-0.576,P=0.000,rs=-0.284,P=0.020)。MTAP与多种免疫细胞存在相关关系,其中MTAP与CD4+T细胞呈正相关(r=0.643,P=0.000),与NK CD56bright细胞呈负相关(r=-0.570,P=0.000)。结论:前列腺癌中,MTAP的表达呈现早期升高后逐渐下降的趋势,与T分期和Gleason评分呈负相关。MTAP对免疫细胞的浸润和细胞功能的发挥有调节作用,是一种潜在的生物学标志物和免疫治疗靶点。