Iron Ion Binding Research Articles

THE EXPRESSION PROFILING OF INTESTINAL NUTRIENT TRANSPORTER GENES IN RATS WITH RENAL FAILURE

The understanding of intestinal function in chronic kidney disease (CKD) has been important elements in the clinical management of CKD with dietary and drug therapy. Numerous studies have indicated that CKD patients or model rats have enzymatic abnormalities and impairments of absorptive function in the small intestine. However, it has been still unclear how different of the intestinal function in CKD. In this study, we demonstrated the microarray analysis of global gene expression in intestine of adenine-induced CKD rat. DNA microarray analysis using Affymextrix rat gene chip revealed that CKD caused great changes in gene expression in the rat duodenum: about 400 genes exhibited more than a two-fold change in expression level. Gene ontology analysis showed that a global regulation of genes by CKD involved in iron ion binding, alcoholic, organic acid and lipid metabolism. Furthermore, we found markedly changes of a number of intestinal transporters gene expression related to iron metabolism. These results suggest that CKD may alter some nutrient metabolism in the small intestine by modifying the expression of specific genes. The intestinal transcriptome database of CKD might be useful to develop the novel drugs or functional foods for CKD patients.

Changes in the antimicrobial potential of egg albumen during the early stages of incubation and its impact on the growth and virulence response of Salmonella Enteritidis

The antimicrobial activity of egg albumen has been widely documented. However, the changes in the chemical nature and antimicrobial activity of egg albumen and the way it controls bacteria during the incubation period have not up to now been understood. The purpose of the present investigation was to analyze the antimicrobial potential of egg albumen during early incubation stages (0–5 days). The changes in ovotransferrin and lysozyme content in egg albumen were quantified via high performance liquid chromatography (HPLC). The results showed that the increase in concentration of ovotransferrin in egg albumen was closely related to the time of incubation, but that of lysozyme remained substantially unchanged and was very similar to that of the non-incubated eggs. The iron ion binding capacity and specific lysozyme activity of the egg albumen during early stages of incubation was also investigated. The changes occurring in egg albumen during early incubation could influence the growth of Salmonella Enteritidis. Very little difference was observed in the virulence response of Salmonella Enteritidis to egg albumen collected at different incubation periods by using the expression of hilA gene as an indicator. Since the first few days of incubation are likely to place heavy demands on the immune capacities of the egg, due to the defenceless immune system of the developing embryo, the changes in antimicrobial activity in egg albumen widen our understanding of the innate immune mechanism of the fertilized egg during its early embryonic development.

Genes and pathways affected by CAG-repeat RNA-based toxicity in Drosophila

Spinocerebellar ataxia type 3 is one of the polyglutamine (polyQ) diseases, which are caused by a CAG-repeat expansion within the coding region of the associated genes. The CAG repeat specifies glutamine, and the expanded polyQ domain mutation confers dominant toxicity on the protein. Traditionally, studies have focused on protein toxicity in polyQ disease mechanisms. Recent findings, however, demonstrate that the CAG-repeat RNA, which encodes the toxic polyQ protein, also contributes to the disease in Drosophila. To provide insights into the nature of the RNA toxicity, we extracted brain-enriched RNA from flies expressing a toxic CAG-repeat mRNA (CAG100) and a non-toxic interrupted CAA/G mRNA repeat (CAA/G105) for microarray analysis. This approach identified 160 genes that are differentially expressed specifically in CAG100 flies. Functional annotation clustering analysis revealed several broad ontologies enriched in the CAG100 gene list, including iron ion binding and nucleotide binding. Intriguingly, transcripts for the Hsp70 genes, a powerful suppressor of polyQ and other human neurodegenerative diseases, were also upregulated. We therefore tested and showed that upregulation of heat shock protein 70 mitigates CAG-repeat RNA toxicity. We then assessed whether other modifiers of the pathogenic, expanded Ataxin-3 polyQ protein could also modify the CAG-repeat RNA toxicity. This approach identified the co-chaperone Tpr2, the transcriptional regulator Dpld, and the RNA-binding protein Orb2 as modifiers of both polyQ protein toxicity and CAG-repeat RNA-based toxicity. These findings suggest an overlap in the mechanisms of RNA and protein-based toxicity, providing insights into the pathogenicity of the RNA in polyQ disease.

Hepatic Gene Expression Profiling Reveals Key Pathways Involved in Leptin-Mediated Weight Loss in ob/ob Mice

BackgroundLeptin, a cytokine-like protein, plays an important role in the regulation of body weight through inhibition of food intake and stimulation of energy expenditure. Leptin circulates in blood and acts on the brain, which sends downstream signals to regulate body weight. Leptin therapy has been successful in treating leptin deficient obese patients. However, high levels of leptin have been observed in more common forms of obesity indicating a state of leptin resistance which limits the application of leptin in the treatment of obesity. If the central effect of leptin could be by-passed and genes which respond to leptin treatment could be regulated directly, new therapeutic targets for the treatment of obesity may be possible. The purpose of this study was to identify genes and subsequent pathways correlated with leptin-mediated weight loss.Methodology/Principal FindingsWe utilized microarray technology to compare hepatic gene expression changes after two types of leptin administration: one involving a direct stimulatory effect when administered peripherally (subcutaneous: SQ) and another that is indirect, involving a hypothalamic relay that suppresses food intake when leptin is administered centrally (intracerebroventricular: ICV). We identified 214 genes that correlate with leptin mediated weight loss. Several biological processes such as mitochondrial metabolic pathways, lipid metabolic and catabolic processes, lipid biosynthetic processes, carboxylic acid metabolic processes, iron ion binding and glutathione S-transferases were downregulated after leptin administration. In contrast, genes involved in the immune system inflammatory response and lysosomal activity were found to be upregulated. Among the cellular compartments mitochondrion (32 genes), endoplasmic reticulum (22 genes) and vacuole (8 genes) were significantly over represented.Conclusions/SignificanceIn this study we have identified key molecular pathways and downstream genes which respond to leptin treatment and are involved in leptin-mediated weight loss. Many of these genes have previously been shown to be associated with obesity; however, we have also identified a number of other novel target genes. Further investigation will be required to assess the possible use of these genes and their associated protein products as therapeutic targets for the treatment of obesity.

Perfusion of the Human Placenta with Red Blood Cells and Xanthine Oxidase Mimics Preeclampsiain-vitro

Preeclampsia is a major obstetric problem of unknown etiology. The fact that removal of the placenta is the only cure for preeclampsia, has led to the well-established hypothesis, that the placenta is central in the etiology. Gene profiling and proteomics studies have suggested oxidative stress caused by reperfusion and free oxygen radicals as a potential pathophysiological mechanism in preeclampsia. In this study, the dual placental perfusion model was used in order to evaluate the damaging effects of oxidative stress induced by xanthine/xanthine oxides and free hemoglobin. The dual placenta perfusion model is a well-established in vitro model for functional placental studies. Placentas were perfused with medium containing either xanthine/xanthine oxidase or erythrocytes as a source of free hemoglobin. Concentration of free hemoglobin in the medium was measured by means of ELISA. Whole genome microarray technique and bioinformatics were used to evaluate the gene expression profile in the two groups. Substantial levels of free adult hemoglobin were detected in the perfusions. A total of 58 genes showed altered gene expression, the most altered were hemoglobin alpha, beta and gamma, tissue factor pathway inhibitor 2 and superoxide dismutase 2. Bioinformatics revealed that biological processes related to oxidative stress, anti-apoptosis and iron ion binding were significantly altered. The results suggest that perfusion with xanthine/xanthine oxidase and free hemoglobin induce changes in gene expression similar to what has been described for the preeclamptic placenta.

Gene expression profiles in testis of pigs with extreme high and low levels of androstenone

Background:Boar taint is a major obstacle when using uncastrated male pigs for swine production. One of the main compounds causing this taint is androstenone, a pheromone produced in porcine testis. Here we use microarrays to study the expression of thousands of genes simultaneously in testis of high and low androstenone boars. The study allows identification of genes and pathways associated with elevated androstenone levels, which is essential for recognising potential molecular markers for breeding purposes.Results:Testicular tissue was collected from 60 boars, 30 with extreme high and 30 with extreme low levels of androstenone, from each of the two breeds Duroc and Norwegian Landrace. The samples were hybridised to porcine arrays containing 26,877 cDNA clones, detecting 563 and 160 genes that were differentially expressed (p < 0.01) in Duroc and Norwegian Landrace, respectively. Of these significantly up- and down-regulated clones, 72 were found to be common for the two breeds, suggesting the possibility of both general and breed specific mechanisms in regulation of, or response to androstenone levels in boars. Ten genes were chosen for verification of expression patterns by quantitative real competitive PCR and real-time PCR. As expected, our results point towards steroid hormone metabolism and biosynthesis as important biological processes for the androstenone levels, but other potential pathways were identified as well. Among these were oxidoreductase activity, ferric iron binding, iron ion binding and electron transport activities. Genes belonging to the cytochrome P450 and hydroxysteroid dehydrogenase families were highly up-regulated, in addition to several genes encoding different families of conjugation enzymes. Furthermore, a number of genes encoding transcription factors were found both up- and down-regulated. The high number of clones belonging to ferric iron and iron ion binding suggests an importance of these genes, and the association between these pathways and androstenone levels is not previously described.Conclusion:This study contributes to the understanding of the complex genetic system controlling and responding to androstenone levels in pig testis. The identification of new pathways and genes involved in the biosynthesis and metabolism of androstenone is an important first step towards finding molecular markers to reduce boar taint.

Proteomic analysis identifies candidate proteins associated with distant recurrences in breast cancer after adjuvant chemotherapy

Breast cancer is a heterogenous disease and it is of importance to select patients with regard to different prognosis and treatment sensitivity to individualize treatment regimes. In this study we successfully adapted a protein extraction protocol from mRNA extracted tumor samples enabling two-dimensional gel electrophoresis (2-DE) analysis of samples previously analyzed by cDNA microarray. The aim was to find candidate proteins that distinguish breast cancer patients with or without recurrences after adjuvant CMF (cyclophosphamide, methotrexate and 5-FU) treatment within four years to follow-up. We identified several proteins distinguishing the recurrence group from the non-recurrence group, especially in the ER and PgR positive subgroup ( n = 7). The induced proteins were involved in translation/folding, iron ion binding, and protease inhibition, whereas proteins involved in signaling, ubiquitination, and splicing were decreased in expression. These results show that it is possible to use 2-DE to separate high abundant proteins in breast cancer tissue and to find discriminating proteins to identify patients with different prognosis after adjuvant CMF treatment.

Calcium Indicator Dye Quin2 Inhibits Hydrogen Peroxide-Induced DNA Strand Break Formation via Chelation of Iron

Exposure of HeLa cells to H2O2 at 4°C caused DNA strand breakage which was prevented by the metal chelator 1,10-phenanthroline, but not by neocuproine. This is believed to indicate the participation of iron, rather than copper, in the formation of reactive hydroxyl radicals (· OH) from H2O2. The calcium indicator dye Quin2 also prevented H2O2-induced DNA fragmentation. The inhibition of oxidant-induced DNA fragmentation at 37°C by Quin2 is often presented as evidence for the involvement of Ca2+-dependent endonucleases in damage. However, our finding that Quin2 inhibits DNA fragmentation at 4°C led us to investigate the possibility that Quin2 may also inhibit DNA damage via its effects on metal-dependent · OH formation. Using ESR spin trapping techniques and in vitro DNA oxidation measurements, we found that the binding of Fe by Ouin2 does not prevent · OH formation, but inhibits DNA damage. This is believed to reflect the prevention of iron ion binding to DNA by Quin2, directing · OH generation to the bulk solution, thereby preventing damage. The Cu-Quina complex was found to be a poor catalyst of both · OH formation and in vitro DNA damage. These findings suggest that the frequently reported protective effect of Quina toward DNA in cells exposed to oxidants may be due to the chelation of metal ions rather than the prevention of Ca2+-dependent endonuclease activation.

Effect of various detergents on kinetic characteristics of lipid peroxidation systems in rat liver microsomes

The vari-directional and biphasic actions of detergents on the lipid peroxidation (LP) in microsome systems have been demonstrated in our experiments. Anionic detergents deoxycholate (DOC) and sodium dodecyl sulfate (SDS), when applied in relatively small concentrations, activate the ascorbate-dependent LP and lower the K m for iron ions in ascorbate-dependent and in NADPH-dependent LP. Besides, DOC weakens the inhibiting influence of promethazine and EDTA on both LP systems. The nonionic detergent Triton X-100 does not affect the investigated characteristics of peroxidation in microsomal lipids. The cationic detergent cetyltrimethylammonium bromide inhibits both LP microsome systems and raises the K m for iron ions in these reactions. Comparatively high concentrations of DOC, SDS, and Triton X-100 suppress both the NADPH- and ascorbate-dependent LP reactions in the microsomes. A reasonable hypothesis is that involvement of anionic detergents in the microsome membrane is attended by an increase in iron ion binding, whereas inclusion of cationic detergents results in a reverse effect. Under discussion is the interconnection between the amount of radical-forming centers, essential components of which are iron ions, and LP activity in the microsomes.