Effect of dietary selenium on plasma selenoprotein P, selenoprotein P1 and glutathione peroxidase in the rat

The purpose of this study was to determine the effect of dietary selenium on the abundance of selenium in plasma selenoprotein P, selenoprotein P1 and glutathione peroxidase. Weanling rats were provided water that contained 1.0, 0.1 or 0.01 ppm selenium and 75Se for 21 days. Gel filtration of denatured subunits was used to identify 75Se in the selenoproteins. Rats provided 1.0 ppm selenium accumulated 1.5 times more 75Se in liver cytosolic selenoprotein P1, but not in the two other selenoproteins, than did rats provided 0.1 ppm selenium. Most of the liver and blood selenium in rats provided 1.0 ppm selenium was insoluble and in an unknown chemical form. The tissue accumulation of unrecoverable selenium was apparently a response to the high dietary level of selenium. The proportion of selenium in plasma selenoprotein P, a putative selenium-transport protein, reflected the long-term selenium status of rats and varied from approximately 11-58% depending on the level of selenium supplementation. Turnover of selenium from this protein was affected by the dietary selenium of the rats. The results indicate that selenium incorporation into plasma selenoprotein P and selenoprotein P1 is affected by diet in ways that may reflect their importance to the rat.     

硒蛋白P的结构与功能预测

目的　预测硒蛋白P的结构和功能。方法　利用生物信息学方法分析硒蛋白P的氨基酸疏水性、跨膜螺旋、二硫键的形成、氨基酸残基相对可及性和二级结构的组成及外显子对应的肽序列 ,利用Smith Waterman法在EMBL蛋白数据库中对硒蛋白P进行同源性比对 ,ScanProsite程序在PROSITE数据库中搜索硒蛋白P中的基序 ,SMART服务器搜索结构域。结果　成熟的硒蛋白P有两个跨膜螺旋 ,由四个模块组成 :M1(Glu2 0 Ser6 7 Lys6 8)、M2 (Ser6 7 Lys6 8 Asp138 Arg139)、M3(Asp138 Arg139 Thr178) ,和M4 (Thr179 Asn381) ;在M2中有一螺旋的螺旋结构 ,其中的规则二级结构高于其他三个模块 ,在M1、M3、M4中环形 (loop)结构含量较高 ,M4含有富含组氨酸的序列 (His2 0 4 His2 5 7)且该序列位于硒蛋白P的表面 ;氨基酸溶剂可及性分析表明成熟的硒蛋白P不是球形结构。几种在脑的发育中起重要作用的蛋白HUNBSCAAL、ZP12BRARE、BR11BRARE ,和BRN1HUMAN与硒蛋白P有同源性 ,这种同源性可能是对脑的特异性 ;另外两个同源性蛋白是KNOBPLAFG和HPNHELPY ,KNOBPLAFG能锚定于内皮细胞受体与血小板反应蛋白形成的复合物上 ,而HPNHELPY对镍和锌有强烈的结合特性。结论　硒蛋白P有金属结合活性及与内皮细胞的重要关系 ,结果还显示硒蛋白P对脑的?     

Decreased selenoprotein expression alters the immune response during influenza virus infection in mice

Previous work from our laboratory demonstrated that host selenium (Se) deficiency results in greater lung pathology and altered immune function in mice infected with influenza virus. Because selenoproteins play a key role in determining the oxidant status of the host, we utilized a transgenic mouse line carrying a mutant selenocysteine (Sec) tRNA ([Ser]Sec) transgene (t-trspi(6)A(-)). The levels of selenoproteins are decreased in these mice in a protein- and tissue-specific manner. Male t-trspi(6)A(-) and wild-type (WT) mice were infected with influenza and killed at various time points postinfection (p.i.). Lung mRNA levels for innate and pro-inflammatory cytokines increased with infection but did not differ between groups. However, at d 2 p.i., chemokine levels were greater in the t-trspi(6)A(-) mice compared with WT mice. Additionally, IFN-gamma was higher at d 7 p.i. in the t-trspi(6)A(-) mice and viral clearance slower. Despite these immune system changes, lung pathology was similar in t-trspi(6)A(-) and WT mice. (75)Se labeling experiments demonstrated that glutathione peroxidase (GPX)-1 and thioredoxin reductase, although greatly diminished in the lungs of t-trspi(6)A(-) mice, were not altered as a result of infection. GPX-1 activity in the lungs of the t-trspi(6)A(-) mice was approximately 82% of the WT mice. In addition, the GPX-1 activity in the lungs of Se-deficient mice was 125% less than in the t-trspi(6)A(-) mice. These results suggest that although selenoproteins are important for immune function, there is a threshold of GPX-1 activity that can prevent an increase in lung pathology during influenza infection.     

Biomonitoring of selenoprotein P in human serum by fast affinity chromatography coupled to ICP-MS

Most of the Se in human serum is bound to selenoprotein P (SEPP1) in which Se is present in form of selenocysteine. The SEPP1 is a new possible biomarker for the Se status and for this reason we developed a fast, simple and reliable method for the quantitative determination of SEPP1 in serum by affinity chromatography coupled to ICP-MS. It is possible to separate SEPP1 from other selenoproteins in serum in only 5?min, which allows high sample throughput in clinical laboratories. Measured and certified concentrations of total Se and Se(SEPP1) are in good agreement for the reference material SRM 1950. The SEPP1 concentration was stable in serum samples of 3 persons for a minimum of 2 weeks. Further results of method validation were described including internal and external quality assurance.The analytical method was applied for a biomonitoring study of the SEPP1 and total Se concentration in human serum of 50 occupationally non-exposed persons living in northern Germany. Concentration ranges and mean concentrations for Se(SEPP1) are 31.1–59.7 and 46.2?μg/L, respectively. The corresponding values for total Se are 62–120 and 83.5?μg/L. The mean percentage of total Se in serum present as SEPP1 is 58%.     

Both selenoproteins and low molecular weight selenocompounds reduce colon cancer risk in mice with genetically impaired selenoprotein expression

Selenium has cancer protective effects in a variety of experimental systems. Currently, it is not known whether selenoproteins or low molecular weight selenocompounds are responsible for this activity. To evaluate the contribution of selenoproteins to the cancer protective effects of selenium, we used transgenic mice that carry a mutant selenocysteine transfer RNA gene, which causes reduced selenoprotein synthesis. Selenium homeostasis was characterized in liver and colon of wild-type and transgenic mice fed selenium-deficient diets supplemented with 0, 0.1, or 2.0 microg selenium (as selenite)/g diet. (75)Se-labeling, Western blot analysis, and enzymatic activities revealed that transgenic mice have reduced (P < 0.05) liver and colon glutathione peroxidase expression, but conserved thioredoxin reductase expression compared with wild-type mice, regardless of selenium status. Transgenic mice had more (P < 0.05) selenium in the nonprotein fraction of the liver and colon than wild-type mice, indicating a greater amount of low molecular weight selenocompounds. Compared with wild-type mice, transgenic mice had more (P < 0.05) azoxymethane-induced aberrant crypt formation (a preneoplastic lesion for colon cancer). Supplemental selenium decreased (P < 0.05) the number of aberrant crypts and aberrant crypt foci in both wild-type and transgenic mice. These results provide evidence that a lack of selenoprotein activity increases colon cancer susceptibility. Furthermore, low molecular weight selenocompounds reduced preneoplastic lesions independent of the selenoprotein genotype. These results are, to our knowledge, the first to provide evidence that both selenoproteins and low molecular weight selenocompounds are important for the cancer-protective effects of selenium.     

All regions of mouse brain are dependent on selenoprotein P for maintenance of selenium

The brain and testis retain selenium better than other tissues during selenium deficiency. Studies of mice with selenoprotein P (Sepp1) deleted (Sepp1(-/-) mice) showed that brain and testis selenium levels are largely dependent on Sepp1. Therefore, we examined tissue selenium in mice fed varying amounts of selenium and in Sepp1(-/-) mice to characterize better the role(s) of Sepp1. Mice were fed a selenium-deficient diet for 8 wk supplemented with selenium as selenite from none to 0.25 mg/kg diet and tissue selenium was measured. Brain and testis maintained their selenium better than did liver, kidney, and muscle when dietary selenium was limiting but testis selenium fell sharply in the group fed the deficient diet. Brain retained its selenium well, even in the group fed the deficient diet. After intravenous injection of (75)Se-Sepp1 into Sepp1(-/-) and Sepp1(+/+) mice, qualitative differences between brain and testis (75)Se uptake were noted, further suggesting differences in their uptake of selenium from Sepp1. Finally, selenium was measured in brain regions of Sepp1(-/-) and Sepp1(+/+) mice fed the diet supplemented with 1 mg selenium/kg and Sepp1(+/+) mice fed the deficient diet. Deletion of Sepp1 and selenium deficiency each lowered selenium a similar amount in cortex, midbrain, brainstem, and cerebellum. Selenium in the hippocampus was lowered by deletion of Sepp1 but not by selenium deficiency. These results suggest that Sepp1 is more important for maintaining selenium in the hippocampus than in other brain regions. They also confirm the position of the brain at the apex of the organ selenium hierarchy.     

Depressed serum selenoprotein P: possible new predicator of increased risk for cerebrovascular events

Selenium protection against cellular damage by oxygen radicals is accomplished through selenoproteins. Thus, selenium protection during the development of stroke, an oxidative stress–related disease, may not be appropriately reflected in the total serum selenium concentration. Therefore, we hypothesized that serum selenoproteins should also be measured to understand the relationship between selenium status and oxidative stress. To establish whether stroke is associated with changes in serum selenoprotein levels, a population-based, nested case-control study was performed. The subjects were recruited from 1632 residents older than 40 years who had completed health examinations in 1992. Blood samples collected from 30 controls and 30 initial stroke victims between 1992 and 1994 were analyzed for total serum selenium and selenium-containing protein distribution. Selenium-containing proteins were separated using 2 high-performance liquid chromatography columns in tandem and detected by inductively coupled plasma–mass spectrometry. The mean serum selenium concentration was lower in the patients who had a stroke than in the controls (105.2 vs 116.5 μg/L). Selenium contents in glutathione peroxidase and albumin did not show any significant difference; however, selenoprotein P was significantly lower in the stroke cases than in the controls (54.5 vs 63.0 μg/L, P = .006). Results from multivariate logistic regression analysis showed that reduced serum level of selenoprotein P was associated with a higher risk of stroke (odds ratio = 0.28; 95% confidence interval, 0.10-0.85).     

硒蛋白对糖尿病小鼠血糖、Ca2+转运以及NO系统影响的实验研究

目的研究硒蛋白对糖尿病小鼠血糖、Ca2+转运及NO系统的调控作用.方法体重(20.3±1.7)g昆明种雄性小鼠,腹腔注射200mg/kgbw,2%的四氧嘧啶造糖尿病(DM)模型.实验分6组正常对照组(Ⅰ)、正常+硒蛋白组(Se 100μg/kgbw)(Ⅱ)、糖尿病对照组(Ⅲ)、DM+硒蛋白低剂量组(Se 100μg/kgbw)(Ⅳ)、DM+硒蛋白高剂量组(Se 300μg/kgbw)(Ⅴ)、DM+亚硒酸钠组(Se 100μg/kgbw)(Ⅵ).结果Ⅴ组血糖(20.4±6.3)mmol/L明显低于Ⅲ组(45.3±3.3)mmoi/L,P＜0.05;肾脏三磷酸腺苷酶(Ca2+-ATPase)活性,Ⅴ组0.90±0.5明显高于Ⅲ组(0.35±0.1)μmol/(h·mg prot),P＜0.05;一氧化氮合酶(NOS)活性,Ⅴ组(25.0±4.3)U/ml明显低于Ⅲ组(35.2±4.4)U/ml,P＜0.05.结论补硒剂量为Se 300μg/kgbw的硒蛋白能够显著的降低糖尿病小鼠血糖、提高肾脏Ca2+-ATPase活性和降低血浆NOS活性.     

Aging deteriorates quality of sperm produced by male mice with partial Yq deletion

The aim of the study was to assess the cumulative effects of aging and Y-chromosome long arm deletion on sperm quality parameters. Motility, mitochondrial activity, and head morphology were evaluated for sperm of 3- and 12-month-old males from B10.BR-Y^del and B10.BR congenic mouse strains. The study revealed that quality and fertilizing potential of sperm produced by younger and older B10.BR males persist on similar levels, but worsen significantly with age of B10.BR-Y^del males. The findings imply that partial Yq deletions might be more harmful for spermiogenesis in advancing age and may be applicable to other species including humans.

Abbreviations: AZF: azoospermia factor; MSYq: male-specific region of the Y-chromosome long arm     

奇异变形菌ppk1基因缺失株的构建与鉴定

目的构建奇异变形菌(PMI)聚磷酸盐激酶1(polyphosphate kinase 1,PPK1)基因缺失株,为研究ppk1基因在PMI致尿路感染中的作用及其机制奠定基础。方法通过融合PCR将目的基因上游及下游同源臂连接成一个片段,并克隆入自杀质粒pCVD442,将重组质粒转化入大肠埃希菌SM10λpir中,再与PMI受体菌进行接合试验,通过氨苄西林、蔗糖筛选得到ppk1基因缺失株(△pk1),进行PCR和DNA测序鉴定;体外比较野生株和缺失株在低营养条件下的生存能力。结果利用融合PCR将目的基因上、下游片段连接成一个重组片段,并连接至pCVD442自杀质粒;重组自杀质粒进入PMI后出现了同源重组事件,同源片段发生替换,ppk1基因被敲除,通过PCR及测序分析证实基因组目的基因已缺失;经体外测定MOPS培养基中的生长曲线,发现敲除株与野生株相比,在低营养环境中生存能力明显降低。结论自杀质粒同源重组方法可用于PMI基因敲除株的构建,是研究PMI基因功能的重要手段,ppk1基因与PMI在低营养环境中的生存能力有关。     

慢性心房颤动患者P-选择素水平及内皮功能障碍

目的　研究慢性心房颤动 (房颤 )患者血小板活化和内皮功能障碍的有关标志物 ,探讨其临床意义。方法　采用酶联免疫双抗体夹心法测定心脏病患者房颤组 (30例 )、无房颤组 (2 0例 )和正常对照组 (17例 )的血浆P -选择素及血管性血友病因子 (vWF)的水平。结果　房颤组血浆P -选择素及vWF水平显著高于无房颤组及正常对照组 (P均 <0 .0 1) ,无房颤组与正常对照组比较上述指标亦增高 (P <0 .0 1)。结论　慢性房颤组患者存在血小板激活和内皮功能障碍 ,这些异常可能参与了房颤患者血栓的形成。     

硒蛋白对氧化还原信号的调控作用

在生命过程中,机体通过特定的机制抑制以ROS为传感器和信号的还原氧造成的氧化损伤.Cys和Met的氧化修饰对蛋白质活性、结构、稳定性及亚细胞的定位均有深远影响.硒是人体不可缺少的微量元素,是红细胞抗氧化剂- GSH-Px的重要成份,充足的硒可使GSH-Px有效地将H2O2转变为水.硒的大部分生物活性取决于硒蛋白的Sec...     

硒蛋白对糖尿病小鼠血糖、Ca~(2 )转运以及NO系统影响的实验研究

目的研究硒蛋白对糖尿病小鼠血糖、Ca2 转运及NO系统的调控作用。方法体重(203±17)g昆明种雄性小鼠,腹腔注射200mgkgbw,2%的四氧嘧啶造糖尿病(DM)模型。实验分6组正常对照组(Ⅰ)、正常 硒蛋白组(Se100μgkgbw)(Ⅱ)、糖尿病对照组(Ⅲ)、DM 硒蛋白低剂量组(Se100μgkgbw)(Ⅳ)、DM 硒蛋白高剂量组(Se300μgkgbw)(Ⅴ)、DM 亚硒酸钠组(Se100μgkgbw)(Ⅵ)。结果Ⅴ组血糖(204±63)mmolL明显低于Ⅲ组(453±33)mmolL,P<005;肾脏三磷酸腺苷酶(Ca2 ATPase)活性,Ⅴ组090±05明显高于Ⅲ组(035±01)μmol(h·mgprot),P<005;一氧化氮合酶(NOS)活性,Ⅴ组(250±43)Uml明显低于Ⅲ组(352±44)Uml,P<005。结论补硒剂量为Se300μgkgbw的硒蛋白能够显著的降低糖尿病小鼠血糖、提高肾脏Ca2 ATPase活性和降低血浆NOS活性。     

Association of CYP2A6 gene deletion with cigarette smoking status in Japanese adults

BACKGROUND: Genetic variation of CYP2A6 is shown to alter nicotine metabolism. This study was developed to investigate the genetic influence of the whole deletion-allele of CYP2A6 on active and passive smoking behavior. METHODS: Two hundred and forty Japanese adults, who visited Aichi Cancer Center as outpatients, were genotyped for the wild-type (CYP2A6*1A, CYP2A6*1B) and the whole deletion-type (CYP2A6*4C) polymorphism of CYP2A6. Information about active and passive smoking status was obtained by a self-administered questionnaire. Genetic influence of CYP2A6 polymorphism on smoking behavior was evaluated using the Mantel extension test. RESULTS: The frequency of the deletion allele was 18%. All 8 subjects carrying two deletion alleles had no smoking habit, and the homozygous deletion genotype showed a tendency to correlate with active smoking status after adjustment for sex and age (p = 0.054). However, the proportion of never smokers among heterozygous subjects was almost the same as among subjects carrying no deletion allele (54% and 58%, respectively). Furthermore, CYP2A6 genotypes were correlated neither with the number of cigarettes smoked per day nor with the age at starting smoking (p = 0.364 and 0.880, respectively). Among never smokers, CYP2A6 genotypes were not correlated with exposure to passive smoking at home or in the workplace (p = 0.623 and 0.484, respectively). CONCLUSION: Despite the possible protection against active smoking behavior in subjects homozygous for the deletion allele, the CYP2A6 polymorphism has only a limited impact on public health because no protective effect was found in heterozygous subjects.     

肠炎沙门菌pagC基因缺失株构建及其相关生物学特性研究

目的研究肠炎沙门菌外膜蛋白PagC的生物学功能以及在肠炎沙门菌致病过程中的作用。方法利用λ-Red同源重组系统构建了肠炎沙门菌(C50336)pagC基因缺失突变(C50336Δhtra),在此基础上构建基因回补菌株。测定肠炎沙门菌各菌株在培养基中的生长曲线;利用生化鉴定系统测定各菌株的生化特性;分析肠炎沙门菌各菌株在酸性、碱性和高渗环境下的存活能力;利用K-B纸片法测定肠炎沙门菌各菌株的耐药性。结果成功构建了肠炎沙门菌pagC基因缺失株C50336ΔpagC。生长曲线显示,与野生型菌株和回补菌株相比,C50336ΔpagC缺失株生长速度、生化特性无明显差异,表明pagC不影响肠炎沙门菌的生长特性。应激实验结果显示,pagC基因缺失后,肠炎沙门菌应对酸碱应激和高盐应激能力显著下降,表明pagC影响肠炎沙门菌应激条件下生存。药物敏感性试验结果显示,pagC基因缺失后,肠炎沙门菌对多种抗菌药物敏感性增高,表明pagC参与肠炎沙门菌耐药过程。结论 PagC蛋白与细菌抗环境应激和耐药性相关,为进一步阐释肠炎沙门菌致病机制和耐药机理奠定基础。     

Multiple variant mRNAs with different length tandem repeats of (CAYYCC)n produced from bovine selenoprotein P-like protein gene

In contrast to selenoprotein Ps (SeIPs) from other animal species, bovine selenoprotein P-like-protein (SeIPLP) was found to contain a tandem repeat of (CAYYCC)₁₁. During an investigation into whether SeIPLP was a bovine substitute for SeIP or uniquely bovine, its mRNA was found to consist of multiple variants with different length tandem repeat, namely p(0) with (CAYYCC)₁₁, p(−4) lacking (CAYYCC)₄, p(−8) lacking (CAYYCC)₈, and p(−9) lacking (CAYYCC)₉. Although they were encoded on a single gene locus, neither classicalGT-AG nor minor classAT-AC donator-acceptor sequences for alternative splicing were identified. A subsequent S1 protection assay using oligonucleotides, whose sequence may occur as variants, performed against bovine poly(A)⁺RNA identified a total of nine variants. Judging from the sequence of these variants and the branch point mapping, the consensus sequence for recognition of the donator was CACCCCCAC and of the acceptor and the branch point A nucleotide,ACCCCCAT orACCCCCATCCCCAT. Furthermore, when the p(0) insert mRNA was expressed in COS-7 cells derived from an African green monkey kidney, cDNAs corresponding to p(−8) and p(−9) could be isolated. Therefore, the bovine SeIPLP mRNAs consisted of multiple variants probably due to a novel splicing mechanism which was not bovine-specific but common to other mammals.     

Bioproduction of conjugated linoleic acid by probiotic bacteria occurs in vitro and in vivo in mice

Probiotics have been shown to reduce the incidence of colon cancer in animal models. The mechanisms responsible for this activity are poorly defined. Conjugated linoleic acids (CLA) are a group of isomers of linoleic acid (LA) possessing anti-inflammatory and anticarcinogenic properties, which can be produced from LA by certain bacterial strains. In this study, the ability of probiotic bacteria to exert anticarcinogenic effects through the production of CLA was assessed. Incubation of probiotic bacteria (VSL3, Lactobacillus acidophilus, L. bulgaricus, L. casei, L. plantarum, Bifidobacterium breve, B. infantis, B. longum, and Streptococcus thermophilus) in the presence of LA yielded CLA production as measured by gas chromatography. Conditioned medium, containing probiotic-produced CLA, reduced viability and induced apoptosis of HT-29 and Caco-2 cells, as assessed by MTT assay and DNA laddering, respectively. Western blotting demonstrated an increased expression of PPARgamma in cells treated with conditioned medium compared with LA alone. Incubation of murine feces with LA after administering VSL3 yielded 100-fold more CLA than feces collected prior to VSL3 feeding. This study supports a role for supplemental probiotics as a strategy both for attenuating inflammation and for preventing colon cancer.