Increased expression and possible roles of nicotinamide N-methyltransferase in pancreatic islets of STZ-induced diabetic monkeys

AIM: To verify and localize the expression of nicotinamide N-methyltransferase (NNMT) in pancreas of streptozotocin(STZ)-induced diabetic monkeys and understand its important role in β-cell destruction in the pathogenesis of diabetes. METHODS: Through an olig-microarray gene chip, NNMT was identified as the most obviously up-regulated genes in pancreas of STZ-induced diabetic monkeys versus controls. Semiquantitative RT-PCR and Western blotting were performed to verify the differential expression at mRNA and protein level respectively. Then the cellular localization of NNMT expression within pancreas was identified by immunohistochemical(IHC) staining.RESULTS: An obvious high expression of NNMT at both mRNA and protein levels was shown in pancreas of STZ-induced diabetic monkeys compared to that of controls. Further localization of the protein by IHC staining in pancreas specimens showed that its altered expression was restricted to central islets, most of which were β cells.CONCLUSION: Expression of NNMT is increased in islets of STZ- induced diabetic monkeys, which infers that NNMT might participate in the process of β cell damage in diabetes probably through the mechanism of energy metabolism disturbance.     

家蚕滞育卵中辅酶Ⅰ和辅酶Ⅱ的含量变化

利用苹果酸脱氢酶(MDH)和柠檬酸合酶(CS)串联反应以及葡萄糖-6-磷酸脱氢酶(G6PDH)催化葡萄糖-6-磷酸(G-6-P)脱氢反应,分别测定了家蚕滞育发动、即时浸酸和低温冷藏处理蚕卵辅酶Ⅰ(NAD+)和辅酶Ⅱ(NADP+)的含量。滞育发动时蚕卵NAD+含量显著下降,而即时浸酸和低温冷藏处理使蚕卵NAD+含量显著提高。蚕卵中的NADP+在滞育发动前大量积累,滞育发动时下降,即时浸酸处理后NADP+含量显著提高,在低温冷藏早期其含量进一步提高,低温冷藏处理后期又显著下降。分析上述结果认为,家蚕滞育卵中NAD+和NADP+含量的变化分别与呼吸耗氧量和糖元-山梨醇相互转化密切相关。     

Characterisation of the first wheat (Triticum aestivum L.) nucleotide pyrophosphatase/phosphodiesterase resembling mammalian counterparts

A formerly unknown nucleotide pyrophosphatase/phosphodiesterase (NPP) of wheat (Triticum aestivum L.) (TaNPP) was recombinantly produced in Pichia pastoris (TaNPPr) following assembly of the sequence out of wheat ESTs using sequences of known NPPs. Simultaneously, a phosphodiesterase was purified to homogeneity from wheat germs, characterised and identified as TaNPP. TaNPP contains the highly conserved catalytic substrate and metal binding residues and displays properties [high pH optimum, glycosylation, high thermostability and inhibition by EDTA and adenosine 5′-monophosphate (AMP)] similar to those of mammalian enzymes characterised earlier. The sequence of TaNPP includes that of a putative transmembrane domain, a characteristic of most NPPs. Both recombinant and native TaNPP partially occur as oligomeric proteins on SDS PAGE. Upon addition of DTT, both migrate as monomeric proteins. Part of the native wheat protein even occurs in its truncated form, thus lacking the transmembrane region. Out of a range of tested natural substrates, TaNPP had the highest affinity towards adenine containing nucleotides. While Michaelis-Menten kinetics were valid in the low substrate concentration range, at higher concentrations, they were no longer applicable. TaNPP shows no similarity to the recently characterised rice and barley enzymes and, is hence, one of the first characterised plant NPPs resembling mammalian NPPs.     

辣椒烟酰胺腺嘌呤二核苷酸磷酸基因(NADPH)在辣椒中的遗传转化及其抗病性鉴定

为了分析辣椒NADPH基因与辣椒疫病抗性之间的关系,明确其在辣椒抗疫病方面的作用,以辣椒NADPH基因为目的基因,利用载体pBI121构建了植物表达载体pBI121-NADPH,采用农杆菌介导法转化辣椒(Capsicum annuum L.)感病品种B12,获得了5个卡那霉素抗性转化株系。对卡那霉素抗性转化植株进行PCR和RT-PCR分子检测发现,这5个转化株系含有目的基因CanNADPH。应用辣椒疫病抗性离体叶片鉴定技术,鉴定转基因植株对疫霉菌(Phytophthora capsici)的抗病性,结果表明,转基因辣椒的发病率较非转基因植株降低了22.2%,其病情指数降低了46.5%,表明辣椒NADPH基因在辣椒抗疫病方面有重要作用。     

The first characterised wheat (Triticum aestivum L.) member of the nudix hydrolase family shows specificity for NAD(P)(H) and FAD

We cloned the first nudix hydrolase of wheat (Triticum aestivum L.) (TaNUDT1) and expressed it using Escherichia coli as expression host. Properties of the purified His₆-tagged protein were studied. The sequence codes for a nudix hydrolase with a molecular mass of around 43x10³ and a predicted pI of 5.68. The characteristic residues of the nudix box were highly conserved in the wheat enzyme sequence, and TaNUDT1 is most probably targeted to the peroxisomes. In the presence of dithiothreitol and MnCl₂, the enzyme hydrolyses the nicotinamide coenzymes NAD(P)(H) as could be predicted by the presence of a conserved amino acid array C-terminal to the nudix box, and the enzyme has higher affinity towards the reduced forms of the coenzymes. In light of previous reports of nudix enzymes and the physiological concentration of their substrates, we found its K_m values towards some of the coenzymes to be relatively high. As NAD(P)(H) and FAD play crucial roles in cell metabolism, extensive hydrolysis could be detrimental to cell functioning. We speculate that a decreased enzyme affinity may point to a built-in restriction on coenzyme hydrolysis in vivo. Furthermore, by hydrolysing the cofactors of several redox enzymes, this enzyme may well affect several wheat based processes.     

Effect of plumbagin on levels of Nox4/ROS and α-SMA in human hepatic stellate cells

AIM: To observe the effect of plumbagin on the mRNA and protein expression of nicotinamide adenine dinucleotidephosphate oxidase 4 (Nox4), reactive oxygen species (ROS) level and protein expression of α-smooth muscle actin (α-SMA) in the HSC-LX2 cells stimulated with transforming growth factor β1 (TGF-β1) in vitro. METHODS: HSC-LX2 cells were cultured in vitro and divided into blank group, model group, high-, medium- and low-dose (2, 1.5 and 1 μmol/L) plumbagin groups. After incubated with each drug for 72 h, the mRNA expression of Nox4 was detected by RT-PCR. ROS levels were tested by in situ loading probe method. The protein contents of Nox4 and α-SMA were measured by Western blot. RESULTS: Compared with model group, after treated with plumbagin for 72 h, the mRNA expression of Nox4, ROS level and α-SMA protein were significantly decreased in high-and medium-dose plumbagin groups (P<0.01). CONCLUSION: Plumbagin inhibits the activation of HSC-LX2 cells via decreasing the expression of Nox4, thus decreasing ROS levels.     

烟酰胺对福建肉用番鸭性能影响的研究

用１日龄福建肉用雌性番鸭４００只进行试验,研究饲粮添加烟酰胺对肉用番鸭生长、血清生化指标和组织辅酶Ｉ（ＮＡＤ,下同）含量的影响。试鸭按体重随机分为５组,分别喂以不添加烟酰胺的玉米———豆粕型基础饲粮（烟酸含量：０～３周阶段４２．０ｍｇ／ｋｇ;４～１０周阶段１８．８ｍｇ／ｋｇ）和相同基础饲粮分别添加３０、６０、９０和１２０ｍｇ／ｋｇ烟酰胺的饲粮。添加烟酰胺对鸭前、后期的日增重、饲料转化效率（Ｆ／Ｇ,下同）、１０周末鸭血清中的总胆固醇、高密度脂蛋白胆固醇（ＨＤＬ—Ｃ,下同）、甘油三酯、白蛋白、球蛋白及尿酸浓度以及胸肌ＮＡＤ含量均无影响（Ｐ＞０．１０）,但降低了１０周末鸭血清甘油三酯浓度（Ｐ＜０．０２）,提高了３周末（Ｐ＜０．００１）与１０周末（Ｐ＜０．０９８）鸭肝组织ＮＡＤ含量。本研究结果表明,实用饲粮添加烟酰胺可能影响福建肉鸭的脂类代谢和肝中ＮＡＤ含量。     

烟酰胺对高产奶牛泌乳中期生产性能及血液指标的影响

本试验旨在研究烟酰胺对高产奶牛泌乳中期产奶性能、血液生化指标和经济效益的影响。选取40头泌乳中期的中国荷斯坦奶牛,随机分为4组,每组每头在基础日粮中分别添加0、6、10、14 g/d烟酰胺,检测采食量、产奶量、乳成分、血液生化指标。结果表明:各组奶牛干物质和粗蛋白质采食量差异不显著(P>0.05);烟酰胺的添加对乳脂率、乳蛋白率、乳糖率、乳脂产量、乳蛋白产量无显著影响(P>0.05);添加NAM后,显著降低了血清NEFA含量(P<0.05);而对血清葡萄糖、丙氨酸转氨酶、天门冬氨酸转氨酶、甘油三酯、碱性磷酸酶、尿素氮和胆固醇的影响差异不显著(P>0.05);添加10、14 g/d烟酰胺可比对照组显著增加产奶量0.86和1.34 kg/d(P<0.05)。在泌乳中期奶牛日粮中添加10~14 g烟酰胺,有助于奶牛生产潜力的发挥,经济效益显著。