日本沼虾MT基因克隆、组织差异性表达及与饲料铜、锌含量的相关性

为了更全面理解日本沼虾(Macrobrachium nipponense)的铜/锌营养生理作用,研究利用RACE技术从日本沼虾肝胰腺中克隆获得一金属硫蛋白基因cDNA全长(mn-MT),并对该基因分子特征、组织表达谱和饲料铜/锌水平对其表达的影响进行分析。结果显示:(1)mn-MT cDNA全长665 bp,含编码59个氨基酸的180 bp的开放阅读框,预测该多肽的理论分子量为6.085 kD,等电点为7.73。该蛋白中半胱氨酸含量最高(30.5%),其次是赖氨酸(16.95%)和丝氨酸(10.17%)。相似性分析显示mn-MT氨基酸序列与美洲海螯虾、斑节对虾和中华绒螯蟹MT的相似性分别达到78%、75%和75%。(2)qRT-PCR分析显示, mn-MT mRNA在肝胰腺、血细胞、鳃、胃、卵巢、肠和肌肉中都有表达,其中肝胰腺中表达量最高。(3)用4组铜添加量分别为0、20、40及160 mg/kg的饲料和3组锌添加水平分别为0、35和210 mg/kg的饲料饲喂初重为(0.1010.002) g日本沼虾56d后,分析各组虾肝胰腺的mn-MT mRNA表达。mn-MT mRNA表达随饲料铜水平的提高而升高,到40 mg/kg组达到最高(P0.05),而后开始下降;饲料中高锌(210 mg/kg)显著提高mn-MT表达(P0.05), 0和35 mg/kg组间差异不显著(P0.05)。结果表明饲料中铜/锌均可影响mn-MT表达,且呈现不同的剂量依赖效应。     

Regulation of zinc transporters by dietary zinc supplement in breast cancer

Zinc is essential for cell growth and is a co-factor for more than 300 enzymes, representing over 50 different enzyme classes. Two gene families have been identified involved in zinc homeostasis. ZnT transporters reduce intracellular zinc while ZIP transporters increase intracellular zinc. Previous studies have shown that zinc concentration in breast cancer tissues is higher than that in normal breast tissues. However, the mechanisms involved and the relations to zinc transporters are still unknown. A series of zinc transporters are characterized in this article and several of that are emphasized in view of their unique tissue-specific expressions. Established human breast cancer in a nude mice model is used. With a dietary zinc supplement treatment, ZnT-1 mRNA expression in established human breast cancer is raised by 24%, and is nearly 2 times of that in basal diet. ZIP1, ZIP2 and LIV-1 mRNA are the same between two treatment groups. Moreover, no significant changes of these zinc transporters expressions are found between differential breast cancer cell lines in the nude mice model. This is the first report, which detects the zinc transporters expressions in established human breast cancer in nude mice model. These results lead to the constitutive expression and response to zinc in different tissues. In addition to that, ZnT-1 seems to have played an important role in zinc homeostasis, even in breast cancer.     

Expression of metallothionein in the liver and kidney of rats is influenced by excess dietary histidine

It is well known that excess dietary histidine induces the metabolic changes in copper and zinc. Therefore, this study was carried out to clarify whether excess dietary histidine alters the gene expressions of metallothionein-1 and metallothionein-2 in the liver and kidney. Male rats were fed the control (ad libitum and pair-fed) or histidine-excess (50 g of L-histidine per kg of diet) diet for 0, 1 and 3 days. The levels of liver metallothionein-1 and metallothionein-2 mRNA were markedly lower in the rats fed the histidine-excess diet as compared to those of the control (ad libitum and pair-fed) diet, when fed for 1 or 3 days. The levels of renal metallothionein-1 and metallothionein-2 mRNA in the rats fed the histidine-excess diet were higher or tended to be higher as compared with the rats fed the control (ad libitum and pair-fed) diet when fed for 1 or 3 days, respectively. At the same time, hepatic copper content was decreased and renal zinc content was increased by dietary histidine. It thus appears, that such a response on the level of liver metallothionein mRNA might be related to the contents of liver copper, but of kidney metallothionein mRNA might be due to the content of zinc.     

ZnO–CuxO/polypyrrole nanocomposite modified electrode for simultaneous determination of ascorbic acid,dopamine, and uric acid

Novel zinc oxide (ZnO) nanosheets and copper oxide (Cu_xO, CuO, and Cu₂O) decorated polypyrrole (PPy) nanofibers (ZnO–Cu_xO–PPy) have been successfully fabricated for the simultaneous determination of ascorbic acid (AA), dopamine (DA), and uric acid (UA). The morphology and structure of ZnO–Cu_xO–PPy nanocomposites were characterized by scanning electron microscopy (SEM), X-ray diffraction (XRD), and Raman spectroscopy. Compared with the bare glassy carbon electrode (GCE), PPy/GCE, Cu_xO–PPy/GCE, and ZnO–PPy/GCE, ZnO–Cu_xO–PPy/GCE exhibits much higher electrocatalytic activities toward the oxidation of AA, DA, and UA with increasing peak currents and decreasing oxidation overpotentials. Cyclic voltammetry (CV) results show that AA, DA, and UA could be detected selectively and sensitively at ZnO–Cu_xO–PPy/GCE with peak-to-peak separation of 150 and 154 mV for AA–DA and DA–UA, respectively. The calibration curves for AA, DA, and UA were obtained in the ranges of 0.2 to 1.0 mM, 0.1 to 130.0 μM, and 0.5 to 70.0 μM, respectively. The lowest detection limits (signal/noise = 3) were 25.0, 0.04, and 0.2 μM for AA, DA, and UA, respectively. With good selectivity and sensitivity, the current method was applied to the determination of DA in injectable medicine and UA in urine samples.     

Zinc-induced upregulation of metallothionein (MT)-2A is predicted by gene expression of zinc transporters in healthy adults

The usefulness of zinc transporter and metallothionein (MT) gene expressions to detect changes in zinc intake remains unclear. This pilot study aimed to determine the effects of zinc supplementation on zinc transporter and MT gene expressions in humans. Healthy adults (n = 39) were randomised to zinc treatment (ZT), receiving 22 mg Zn/day (n = 19), or no treatment (NT) (n = 20). Blood samples were collected on Days 0, 2, 7, 14, and 21. Plasma zinc and serum C-reactive protein concentrations were analysed. Gene expression of zinc transporters and MT in peripheral blood mononuclear cells was analysed using real-time PCR. Using repeated-measures ANOVA, MT-2A gene expression and fold change were found to be higher in the ZT group (P = 0.025 and P = 0.016, respectively) compared to the NT group, specifically at Day 2 (40 ± 18 % increase from baseline, P = 0.011), despite no significant increase in plasma zinc concentration. In a multiple regression model exploring the changes in gene expressions between Days 0 and 21, the change in MT-2A gene expression was correlated with changes in all zinc transporter expressions (r² = 0.54, P = 0.029); the change in ZIP1 expression emerged as a univariate predictor (P = 0.003). Dietary zinc intake was predictive of zinc transporter and MT expressions (P = 0.030). Physical activity level was positively correlated with baseline ZIP7 expression (r = 0.36, P = 0.029). The present study shows that MT-2A expression is related to changing expression of zinc transporter genes, specifically ZIP1, in response to zinc supplementation. The current report adds to our understanding of MT in the coordinated nature of cellular zinc homeostasis.

Electronic supplementary material

The online version of this article (doi:10.1007/s12263-015-0494-y) contains supplementary material, which is available to authorized users.     

Effects of zinc deficiency and zinc supplementation on homocysteine levels and related enzyme expression in rats

Methionine synthase (MS) and betaine-homocysteine methyltransferase (BHMT) are both zinc (Zn)-dependent methyltransferases and involved in the methylation of homocysteine. The objective of this study was to investigate the effects of dietary Zn supply on homocysteine levels and expression of the two enzymes in growing rats. Male weanling Sprague-Dawley rats were assigned randomly to four dietary groups (n = 8/group) for 3 weeks: Zn deficient (ZD; <1 mg Zn/kg); Zn control (ZC; 30 mg Zn/kg); Zn supplemented (ZS; 300 mg Zn/kg); pair fed (PF; 30 mg Zn/kg) to the ZD group. Serum and femur Zn concentrations were 83% and 58% lower in ZD, and 49% and 62% higher in ZS compared to ZC (P < 0.001), respectively. The ZD rats had lower feed intake (37%), body weight gains (45%), liver (43%) and kidney (31%) weights than those of ZC (P < 0.001), but these parameters in ZD were not significantly different from the PF controls. Serum homocysteine concentrations were 65% higher in ZD compared to PF (P < 0.05), and there was no significant difference in serum folate levels between ZD and PF groups. The mRNA expression of liver and kidney MS was 57% and 38% lower in ZD than PF (P < 0.001), respectively. Hepatic and renal BHMT mRNA levels were not altered in ZD compared to controls. The aforementioned measurements were not significantly different between ZS and ZC groups, except Zn levels. These results demonstrated that homocysteine homeostasis appeared to be disturbed by Zn deficiency but not Zn supplementation, and elevated serum homocysteine might be due to reduced expression of MS during Zn deficiency.     

Fungicidal synergistic effect of biogenically synthesized zinc oxide and copper oxide nanoparticles against Alternaria citri causing citrus black rot disease

Citrus black rot disease being caused by Alternaria citri is a major disease of citrus plants with 30–35% economic loss annually. Fungicides had not been effective in the control of this disease during last few decades. In the present study, antifungal role of green synthesized zinc oxide (ZnO) and copper oxide (CuO) nanoparticles (NPs) were studied against Alternaria citri. Alternaria citri was isolated from disease fruits samples and was identified by staining with lacto phenol cotton blue. Furthermore, CuO and ZnO NPs were synthesized by utilizing the lemon peels extract as the reducing and capping agent. Nanoparticles were characterized by X-ray diffraction (XRD) and scanning electron microscopy (SEM) techniques. From the XRD data, the calculated size of CuO NPs was to be 18 nm and ZnO NPs was16.8 nm using Scherrer equation. The SEM analyses revealed the surface morphology of all the metal oxide NPs synthesized were rounded, elongated and or spherical in the shape. The zone of inhibition was observed to be 50 ± 0.5 mm by CuO NPs, followed by 51.5 ± 0.5 mm by ZnO NPs and maximum zone of antifungal inhibition was observed to be 53 ± 0.6 mm by mix metal oxide NPs. The results of minimum inhibitory concentration (MIC) and minimum fungicidal concentration (MFC) of the synthesized nanoparticles showed that at the certain concentrations (80 mg ml^?1), these NPs were capable of inhibiting the fungal growth, whereas above that specified concentrations (100 mg ml^?1), NPs completely inhibited the fungal growth. Based on these findings, the green synthesized NPs can be used as alternative to fungicide in order to control the citrus black rot disease.     

Resveratrol: Is There Any Effect on Healthy Subject?

This preliminary study was planned to investigate the effects of resveratrol on oxidative–nitrosative stress markers and on trace element concentrations in blood and on circulatory system parameters in rats. Twenty-five Sprague–Dawley male rats, 10–12 weeks old, with mean body weight of 295 g were used in the study. Administration of resveratrol (0.5 ml/day) was performed in experimental group in 10 days. In control (n = 10) and in experimental groups (n = 15), after 1 week training period, systolic arterial blood pressures and heart rates were recorded daily. At the end of the tenth day, blood samples of control and experimental groups were drawn. Total nitrite, nitrite, nitrate, malondialdehyde, copper, zinc concentrations in plasma, superoxide dismutase, and catalase activities and copper, zinc concentrations in red cell were determined both in control and experimental groups. Alterations in oxidative and nitrosative stress markers, trace element concentrations, and circulatory system parameters in experimental group compared to controls were observed. The results of this study were discussed according to the effect of resveratrol. This study was presented at “The 5th International Congress of Pathophysiology (ISP2006)” June 28–July 1, 2006 Beijing, China.     

苦荞锌指蛋白基因FtLOL1的克隆及其对非生物胁迫的应答

根据苦荞(Fagopyrum tataricum)花期转录组数据,采用RT-PCR技术和PCR技术克隆得到一个C2C2型锌指蛋白基因Ft LOL1(Ft LSD-one-like 1,Gen Bank登录号:KP260662),获得c DNA和DNA全长序列,采用实时荧光定量PCR技术研究Ft LOL1在3种非生物处理条件下的表达模式。结果显示,苦荞Ft LOL1基因DNA全长1 720bp,由5个外显子和4个内含子组成,符合GT-AG剪切原则;c DNA包含一个444 bp的开放阅读框,编码147个氨基酸,具有LSD1家族的典型特征;2mmol/L水杨酸、UV-B照射和4℃冷处理均能导致Ft LOL1基因表达量下降,其中水杨酸和UV-B处理均在12h达到最小值,分别为对照组(0h)的11.9%和33.8%,而4℃冷处理条件下,Ft LOL1表达量12h开始下降,至24h达到最小值,为对照组(0h)的50.7%,36h后表达量有所回升,稳定在对照组(0h)的60%左右。推测该基因可能参与苦荞抗高浓度水杨酸、UV-B照射和冷胁迫等非生物胁迫的应答反应,为探究苦荞抗逆性提供了新的视角。     

Use of dual section mRNA in situ hybridisation/immunohistochemistry to clarify gene expression patterns during the early stages of nephron development in the embryo and in the mature nephron of the adult mouse kidney

The kidney is the most complex organ within the urogenital system. The adult mouse kidney contains in excess of 8,000 mature nephrons, each of which can be subdivided into a renal corpuscle and 14 distinct tubular segments. The histological complexity of this organ can make the clarification of the site of gene expression by in situ hybridisation difficult. We have defined a panel of seven antibodies capable of identifying the six stages of early nephron development, the tubular nephron segments and the components of the renal corpuscle within the embryonic and adult mouse kidney. We have analysed in detail the protein expression of Wt1, Calb1 Aqp1, Aqp2 and Umod using these antibodies. We have then coupled immunohistochemistry with RNA in situ hybridisation in order to precisely identify the expression pattern of different genes, including Wnt4, Umod and Spp1. This technique will be invaluable for examining at high resolution, the structure of both the developing and mature nephron where standard in situ hybridisation and histological techniques are insufficient. The use of this technique will enhance the expression analyses of genes which may be involved in nephron formation and the function of the mature nephron in the mouse.     

Serum Zinc and Copper Levels in Ischemic Cardiomyopathy

Changes in the copper (Cu) and zinc (Zn) concentrations have been reported previously in ischemic cardiomyopathy (ISCMP). Due to controversial results, the aims of this study were to compare levels of Cu, Zn, and Zn/Cu ratio of ISCMP patients with healthy volunteers and also to investigate the possible relationship between trace elements status in ISCMP patients with the severity of clinical disease based on the New York Heart Association (NYHA) classification. The subjects of this study consisted of 30 ISCMP and 27 healthy volunteers. ISCMP was diagnosed with a history of previous myocardial infarction and also coronary artery disease was confirmed by coronary angiography. Exclusion criteria were renal or hepatic insufficiency, alcohol usage, and intake of supplements containing Cu or Zn within 1 week. Cu and Zn levels have been assayed with atomic absorption spectrophotometry. Statistical analysis was performed with the SPSS 10 software using independent sample t test for comparing the levels of Cu and Zn between ISCMP and normal subjects. The mean Cu level of the ISCMP group (1.54 ± 0.52 mg/L) was significantly more than the Cu levels of the healthy volunteers (1.31 ± 0.24 mg/L; p = 0.048). The mean Zn levels of the ISCMP and healthy volunteers were 1.05 ± 0.28 and 1.12 ± 0.42, respectively, without any significant difference between groups. There was a trend for higher Cu level, lower Zn level, and lower Zn/Cu ratio in NYHA III patients in comparison with NYHA II group. Considering the results of this study, Cu may have a role in the development of ISCMP. Interventions such as administration of Cu chelators to relieve the symptoms or to decrease the progression of ISCMP is needed to be examined in large clinical trials. In this study, the Zn level of ISCMP patients was not significantly different in comparison with the healthy volunteers.     

Zinc transporter gene expression and glycemic control in post-menopausal women with Type 2 diabetes mellitus

Type 2 diabetes mellitus (DM) is associated often with underlying zinc deficiency and nutritional supplements such as zinc may be of therapeutic benefit in the disease. In a randomized, double-blind, placebo-controlled, 12-week trial in postmenopausal women (n = 48) with Type 2 DM we investigated the effects of supplementation with zinc (40 mg/d) and flaxseed oil (FSO; 2 g/d) on the gene expression of zinc transporters (ZnT1, ZnT5, ZnT6, ZnT7, ZnT8, Zip1, Zip3, Zip7, and Zip10) and metallothionein (MT-1A, and MT-2A), and markers of glycemic control (glucose, insulin, glycosylated hemoglobin [HbA1c]). The homeostasis model assessment of insulin resistance (HOMA-IR) was calculated. No significant effects of zinc or FSO supplementation were observed on glycemic marker concentrations, HOMA-IR or fold change over 12 weeks in zinc transporter and metallothionein gene expression. In multivariate analysis, the change over 12 weeks in serum glucose concentrations (P = 0.001) and HOMA-IR (P = 0.001) predicted the fold change in Zip10. In secondary analysis, marginal statistical significance was observed with the change in both serum glucose concentrations (P = 0.003) and HOMA-IR (P = 0.007) being predictive of the fold change in ZnT6. ZnT8 mRNA expression was variable; HbA1c levels were higher (P = 0.006) in participants who exhibited ZnT8 expression compared to those who did not. The significant predictive relationships between Zip10, ZnT6, serum glucose and HOMA-IR are preliminary, as is the relationship between HbA1c and ZnT8; nevertheless the observations support an association between Type 2 DM and zinc homeostasis that requires further exploration.     

Towards therapeutic applications of engineered zinc finger proteins

It has long been the goal of molecular biologists to design DNA-binding proteins for the specific control of gene expression. The zinc finger design is ideally suited for such purposes, discriminating between closely related sequences both in vitro and in vivo. Whereas other DNA-binding proteins generally make use of the 2-fold symmetry of the double helix, zinc fingers do not and so can be linked linearly in tandem to recognize DNA sequences of different lengths, with high fidelity. This modular design offers a large number of combinatorial possibilities for the specific recognition of DNA. By fusing zinc finger peptides to repression or activation domains, genes can be selectively targeted and switched off and on. Several recent applications of such engineered zinc finger proteins (ZFPs) are described, including the activation of vascular endothelial growth factor (VEGF) in a human cell line and an animal model. Clinical trials have recently begun on using VEGF-activating ZFPs to treat human peripheral arterial disease, by stimulating vascular growth. Also in progress are pre-clinical studies using ZFPs to target the defective genes in two monogenic disorders, SCID and SCA. The aim is to replace them in each case by a correct copy from an extrachromosomal DNA donor by means of homologous recombination. Promising results are reported.