腺嘌呤葡萄糖苷的合成

以β-D-(+)-五乙酰基葡萄糖和HCl气体反应,得到1-氯-2,3,4,6-四-O-乙酰基葡萄糖,继而和硅醚化的6-氯嘌呤反应得到缩合物,缩合物在Na_2CO_3催化下脱除乙酰基,不经分离"一锅法"在饱和的NH_3/MeOH溶液中反应使6位氨解,最终以4步和80%的总收率得到腺嘌呤葡萄糖苷。考察了关键步骤缩合反应中催化剂、溶剂、投料比、反应时间、反应温度及反应规模对缩合物收率的影响,得到了较佳的反应条件:乙腈为溶剂,2%四氯化锡为催化剂,n(6-氯嘌呤)∶n[β-D-(+)-五乙酰基葡萄糖]=1∶1,60℃反应30 min,收率87%,不需要柱层析。缩合反应的反应规模可以扩大到200g,收率75%。

白细胞介素21在小鼠病毒性心肌炎中的表达及其与抗ANT抗体的相关性

目的:观察病毒性心肌炎(VMC)小鼠急性期不同时点白细胞介素21(IL-21)的表达及其与抗腺嘌呤核苷酸转位子(adenine nucleotide translocator,ANT)抗体的相关性,探究其在VMC发病中的作用及意义。方法:BALB/c小鼠腹腔注射柯萨奇病毒B3(CVB3)建立VMC模型,腹腔注射等量PBS的BALB/c小鼠作为对照组,在注射的第0、1、2、3、4和6周,应用荧光定量逆转录-聚合酶链反应(qRT-PCR)检测小鼠心肌组织中IL-21mRNA的表达、酶联免疫双夹心抗体法(ELISA)检测血清IL-21蛋白及抗ANT抗体的表达。结果:与对照组比较,VMC小鼠心肌组织中IL-21mRNA及血清中IL-21自1周开始高表达,2周时达峰值,并维持高表达至6周,各时点表达量均高于相应时点的对照组(P<0.05)。血清中抗ANT抗体在第1周时开始升高,2周时达峰值,维持高水平表达至第6周,除0周时点外,VMC小鼠与对照组小鼠比较差异均有统计学意义(P<0.05),Pearson相关分析显示,IL-21与抗ANT抗体存在相关性(r=0.88,P<0.01)。结论:IL-21在VMC小鼠心肌组织及外周血中高表达,并与血清抗ANT抗体呈正相关,提示IL-21参与VMC的发病过程。

Effects of hypobaric hypoxia on adenine nucleotide pools,adenine nucleotide transporter activity and protein expression in rat liver

AIM： To explore the effect of hypobaric hypoxia on mitochondrial energy metabolism in rat liver. METHODS： Adult male Wistar rats were exposed to a hypobadc chamber simulating 5000 m high altitude for 23 h every day for 0 （H0）, 1 （H1）, 5 （H5）, 15 （H15） and 30 d （H30） respectively. Rats were sacrificed by decapitation and liver was removed. Liver mitochondria were isolated by differential centrifugation program. The size of adenine nucleotide pool （ATP ADP, and AMP） in tissue and mitochondria was separated and measured by high performance liquid chromatography （HPLC）. The adenine nucleotide transporter （ANT） activity was determined by isotopic technique. The ANT total protein level was determined by Western blot. RESULTS： Compared with H0 group, intra-mitochondrial ATP content decreased in all hypoxia groups. However, the H5 group reached the lowest point （70.6%） （P〈 0.01） when compared to the control group. Intra-mitochondrial ADP and AMP level showed similar change in all hypoxia groups and were significantly lower than that in H0 group, In addition, extra-mitochondrial ATP and ADP content decreased significantly in all hypoxia groups. Furthermore, extra-mitochondrial AMP in groups H5, H15 and H30 was significantly lower than that in H0 group, whereas HI group had no marked change compared to the control situation. The activity of ANT in hypoxia groups decreased significantly, which was the lowest in H5 group （55.7%） （P〈0.01） when compared to H0 group. ANT activity in H30 group was higher than in H15 group, but still lower than that in H0 group. ANT protein level in H5, H15, H30 groups, compared with H0 group decreased significantly, which in H5 group was the lowest, being 27.1% of that in H0 group （P〈0.01）. ANT protein level in H30 group was higher than in H15 group, but still lower than in H0 group. CONCLUSION： Hypobaric hypoxia decreases the mitochondrial ATP content in rat liver, while mitochondrial ATP level recovers during long-term hypoxia exposure. The lower level of extra-mitochondrial ATP may be related to the decrease of ANT activity during hypoxia exposure.

长链非编码RNA在痛风性关节炎研究中的新进展

随着基因技术的发展,长链非编码RNA(lncRNA)已成为当前研究的热点。痛风性关节炎是最常见的炎性关节炎,是尿酸以尿酸钠晶体的形式沉积在关节诱发的炎症反应。痛风发病率逐年升高,已严重影响人类的健康。目前已知痛风的免疫学发病机制主要包括核因子κB信号通路、NOD样受体蛋白3炎性小体信号通路和腺苷三磷酸-嘌呤能配体门控离子通道7信号通路。因此lncRNA与人类多种疾病尤其是免疫相关性疾病的发生密切相关。

8-Hydroxydeoxyguanosine:Not mere biomarker for oxidative stress,but remedy for oxidative stress-implicated gastrointestinal diseases

Reactive oxygen species （ROS） attack guanine bases in DNA easily and form 8-hydroxydeoxyguanosine （8-OHdG）, which can bind to thymidine rather than cytosine, based on which, the level of 8-OHdG is gen- erally regarded as a biomarker of mutagenesis conse- quent to oxidative stress. For example, higher levels of 8-OHdG are noted in Helicobacter pylori-associated chronic atrophic gastritis as well as gastric cancer. However, we have found that exogenous 8-OHdG can paradoxically reduce ROS production, attenuate the nuclear factor-KB signaling pathway, and ameliorate the expression of proinflammatory mediators such as interleukin （IL）-I, IL-6, cyclo-oxygenase-2, and induc- ible nitric oxide synthase in addition to expression of nicotinamide adenine dinucleotide phosphate oxidase （NOX）-I, NOX organizer-1 and NOX activator-1 in vari- ous conditions of inflammation-based gastrointestinal （GI） diseases including gastritis, inflammatory bowel disease, pancreatitis, and even colitis-associated carci- nogenesis. Our recent finding that exogenous 8-OHdG was very effective in either inflammation-based or oxidative-stress-associated diseases of stress-related mucosal damage has inspired the hope that synthetic 8-OHdG can be a potential candidate for the treatment of inflammation-based GI diseases, as well as the pre- vention of inflammation-associated GI cancer. In this editorial review, the novel fact that exogenous 8-OHdG can be a functional molecule regulating oxidative- stress-induced gastritis through either antagonizing Rac-guanosine triphosphate binding or blocking the signals responsible for gastric inflammatory cascade is introduced.

Rebamipide suppresses diclofenac-induced intestinal permeability via mitochondrial protection in mice

AIM: To investigate the protective effect and mechanism of rebamipide on small intestinal permeability induced by diclofenac in mice. METHODS: Diclofenac (2.5 mg/kg) was administered once daily for 3 d orally. A control group received the vehicle by gavage. Rebamipide (100 mg/kg, 200 mg/kg, 400 mg/kg) was administered intragastrically once a day for 3 d 4 h after diclofenac administration. Intestinal permeability was evaluated by Evans blue and the FITC-dextran method. The ultrastructure of the mucosal barrier was evaluated by transmission electron microscopy (TEM). Mitochondrial function including mitochondrial swelling, mitochondrial membrane potential, mitochondrial nicotinamide adenine dinucleotide-reduced (NADH) levels, succinate dehydrogenase (SDH) and ATPase activities were measured. Small intestinal mucosa was collected for assessment of malondialdehyde (MDA) content and myeloperoxidase (MPO) activity. RESULTS: Compared with the control group, intestinal permeability was significantly increased in the diclofenac group, which was accompanied by broken tight junctions, and significant increases in MDA content and MPO activity. Rebamipide significantly reduced intestinal permeability, improved inter-cellular tight junctions, and was associated with decreases in intestinal MDA content and MPO activity. At the mitochondrial level, rebamipide increased SDH and ATPase activities, NADH level and decreased mitochondrial swelling. CONCLUSION: Increased intestinal permeability induced by diclofenac can be attenuated by rebamipide, which partially contributed to the protection of mitochondrial function.

Histone modifications and alcohol-induced liver disease:Are altered nutrients the missing link?

Alcoholism is a major health problem in the United States and worldwide,and alcohol remains the single most significant cause of liver-related diseases and deaths.Alcohol is known to influence nutritional status at many levels including nutrient intake,absorption,utilization,and excretion,and can lead to many nutritional disturbances and deficiencies.Nutrients can dramatically affect gene expression and alcohol-induced nutrient imbalance may be a major contributor to pathogenic gene expression in alcohol-induced liver disease(ALD).There is growing interest regarding epigenetic changes,including histone modifications that regulate gene expression during disease pathogenesis.Notably,modifications of core histones in the nucleosome regulate chromatin structure and DNA methylation,and control gene transcription.This review highlights the role of nutrient disturbances brought about during alcohol metabolism and their impact on epigenetic histone modifications that may contribute to ALD.The review is focused on four critical metabolites,namely,acetate,S-adenosylmethionine,nicotinamide adenine dinucleotide and zinc that are particularly relevant to alcohol metabolism and ALD.

Multiple Strategies for Metabolic Engineering of Escherichia coli for Efficient Production of Coenzyme Q_（10）

Escherichia coli BW25113 was metabolically engineered for CoQ10 production by replacing ispB with ddsA from Gluconobacter suboxydans.Effects of precursor balance and reduced nicotinamide-adenine dinucleotide phosphate （NADPH） availability on CoQ10 production in E.coli were investigated.The knockout of pykFA along with pck overexpression could maintain a balance between glyceraldehyde 3-phosphate and pyruvate,increasing CoQ10 production.Replacement of native NAD-dependent gapA with NADP-dependent gapC from Clostridium acetobutylicum,together with the overexpression of gapC,could increase NADPH availability and then enhanced CoQ10 production.Three effects,overexpressions of various genes in CoQ biosynthesis and central metabolism,different vectors and culture conditions on CoQ10 production in E.coli,were all investigated.The investigation of different vectors indicated that low copy number vector may be more beneficial for CoQ10 production in E.coli.The recombinant E.coli （△ispB：：ddsA,△pykFA and △gapA：：gapC）,harboring the two plasmids encoding pck,dxs,idi and ubiCA genes under the control of PT5 on pQE30,ispA,ddsA from Gluconobacter suboxydans and gapC from Clostridium acetobutylicum under the control of PBAD on pBAD33,could produce CoQ10 up to 3.24 mg·g-1 dry cell mass simply by changing medium from M9YG to SOB with phosphate salt and initial culture pH from 7.0 to 5.5.The yield is unprecedented and 1.33 times of the highest production so far in E.coli.

Leptin influences estrogen metabolism and increases DNA adduct formation in breast cancer cells

Objective: The elevated incidence of obesity has been paralleled with higher risks of breast cancer. High adiposity increases leptin secretion from adipose tissue, which in turn increases cancer cell proliferation. The interplay between leptin and estrogen is one of the mechanisms through which leptin influences breast carcinogenesis. An unbalanced estrogen metabolism increases the formations of catechol estrogen quinones, DNA adducts, and cancer mutations. This study aims to investigate the effect of leptin on some estrogen metabolic enzymes and DNA adduction in breast cancer cells.Methods: High performance liquid chromatography(HPLC) was performed to analyze the DNA adducts 4-OHE1[E2]-1-N3 adenine and 4-OHE1[E2]-1-N7 guanine. Reporter gene assay, real time reverse transcription polymerase chain reaction(real time RT-PCR), and Western blot were used to assess the expression of estrogen metabolizing genes and enzymes: Cytochrome P-4501B1(CYP1B1), Nicotinamide adenine dinucleotide phosphate-quinone oxidoreductase1(NQO1), and Catechol-O-methyl transferase(COMT).Results: Leptin significantly increased the DNA adducts 4-OHE1[E2]-1-N3 adenine and 4-OHE1[E2]-1-N7 guanine.Furthermore, leptin significantly upregulated CYP1B1 promoter activity and protein expression. The luciferase promoter activities of NQO1 and m RNA levels were significantly reduced. Moreover, leptin greatly reduced the reporter activities of the COMT-P1 and COMT-P2 promoters and diminished the protein expression of COMT.Conclusions: Leptin increases DNA adduct levels in breast cancer cells partly by affecting key genes and enzymes involved in estrogen metabolism. Thus, increased focus should be directed toward leptin and its effects on the estrogen metabolic pathway as an effective approach against breast cancer.

Morphological effects of autoclaved diet on the myenteric neurons of rats

AIM:To evaluate the effect of autoclaved diet on the jejunum neurons of the myenteric plexus of rats during their growth.METHODS:The experimental groups were made up of rats going through weaning whose mothers received either an autoclaved or a non-autoclaved diet during gestation and lactation,and rats that were fed the same diet as their mothers during the post-weaning period.In order to measure the neurons'body profile and to quantify the number of neurons per area,preparations were stained by the nicotinamide adenine dinucleotide-diaphorase method.RESULTS:No significant changes were observed in rats'body weight or in the number of neurons regardless of the diet used(P>0.05) .There was a decrease in the jejunum-ileum length in rats treated with an autoclaved diet(P<0.05) .An increase in the neuronal cross-sectional area was seen in rats that had received the autoclaved diet,an effect that was significant for animals undergoing weaning.In addition,all observed factors showed significant differences when related to the age of the animals.CONCLUSION:The autoclaved diet did not alter the quantity of neurons,but increased their cell body area,suggesting changes similar to those observed in protein deficiency.

Synthesis of NAD analogs to develop bioorthogonal redox system

Three new nicotinamide adenine dinucleotide(NAD) analogs were synthesized,and their characteristics as cofactors for Escherichia coli malic enzyme(ME) and its double mutant ME L310R/Q401C were analyzed.Each pair of the NAD analog and the double mutant showed good orthogonality to the natural pair of NAD and ME in terms of catalyzing oxidative decarboxylation of L-malic acid.Results indicated that molecular interactions between redox enzyme and cofactor could be further explored to generate new bioorthogonal redox systems.

Preparation, Characterization, and Electrocatalytic Performance of Graphene-Methylene Blue Thin Films

We report a general method to graft aromatic molecules onto graphene thin film electrodes through a simple immersion process. Large-area electroactive graphene thin films grafted with methylene blue （MB） have been developed as electrocatalytic electrodes for the oxidation of β-nicotinamide adenine dinucleotide （NADH）. The oxidation of NADH starts from -0.08 V （vs. Ag/AgC1） at the graphene-MB thin film electrodes, showing a decrease of 530 mV in overpotential compared to a Ti metal electrode. The graphene-MB thin films have promising applications in biosensors and biofuel cells due to their ability to promote NADH electron transfer reaction.

Sirtuin 5:a review of structure,known inhibitors and clues for developing new inhibitors

Sirtuins(SIRTs) are nicotinamide adenine dinucleotide(NAD^+)-dependent protein deacetylases,which regulate important biological processes ranging from apoptosis,age-associated pathophysiologies,adipocyte and muscle differentiation,and energy expenditure to gluconeogenesis.Very recently,sirtuin 5(SIRT5) has received considerable attention due to that it was found to have weak deacetylase activity but strong desuccinylase,demalonylase and deglutarylase activities,and it was also found to be associated with several human diseases such as cancer,Alzheimer's disease,and Parkinson's disease.In this review,we for the first time summarized the structure characteristics,known peptide and small-molecule inhibitors of SIRT5,extracted some clues from current available information and introduced some feasible,practical in silico methods,which might be useful in further efforts to develop new SIRT5 inhibitors.

Electrochemistry of carboxylated nanodiamond films

The electrochemical behavior of nanodiamond （ND） film functionalized with carboxylic acid groups was studied systemati- cally on a glassy carbon （GC） electrode. One stable redox couple corresponding to the carboxylic acid group was observed. At the scan rate of 0.1 V/s, the cathodic and anodic peak potentials were -0.093 V and 0.088 V （vs. Ag/AgCI）, respectively. The carboxylic acid groups on the ND surface were reduced to CH2OH via a four electron redox process. The ND film modified electrode showed favorable electrocatalytic behavior toward the oxidation as well as the reduction of biomolecules, such as tryptophan and nicotinamide adenine dinucleotide.

Protective effect of dihydropteridine reductase against oxidative stress is abolished with A278C mutation

Objective: To evaluate the antioxidation of dihydrobiopterin reductase and to explore the effect of A278C mutation of the quinoid dihydropteridine reductase(QDPR) gene on its antioxidant activity. Methods: First, plasmids with different genes(wild and mutant QDPR) were constructed. After gene sequencing, they were transfected into human kidney cells(HEK293T). Then, the intracellular production of reactive oxygen species(ROS) and tetrahydrobiopterin(BH4) was detected after cells were harvested. Activations of nicotinamide adenine dinucleotide phosphate oxidase 4(NOX4), glutathione peroxidase 3(GPX3), and superoxide dismutase 1(SOD1) were analyzed to observe the oxidative stress after transfection. The expression of the neuronal nitric oxide synthase(n NOS) gene was analyzed by semiquantitative reverse-transcription polymerase chain reaction(RT-PCR). We also detected the activation of transforming growth factor β1(TGF-β1) by enzyme-linked immunosorbent assay(ELISA) to observe the connection of TGF-β1 and oxidative stress. Results: The exogenous wild-type QDPR significantly decreased the expression of n NOS, NOX4, and TGF-β1 and induced the expression of SOD1 and GPX3, but the mutated QDPR lost this function and resulted in excessive ROS production. Our data also suggested that the influence on the level of BH4 had no significant difference between mutated and the wild-type QDPR transfection. Conclusions: Wild-type QDPR played an important role in protecting against oxidative stress, but mutant QDPR failed to have these beneficial effects.