Microarray analysis for gene expression that involves trace metals

Microarray plays a major role to identify the over- and under-expressed genes. It is a well-known fact that trace elements in our body play a major role in the metabolic processes of all living organisms. In this paper, the microarray studies related to major trace metals are reviewed. This review forms the basis for the converged effort to locate the genes that are either defective and destabilise the concentration of the trace metals or influenced by the changed concentration of the trace metals that are needed for proper functions of the human body, at different parts of the body.

Hybrid methods to select informative gene sets in microarray data classification

One of the key applications of microarray studies is to select and classify gene expression profiles of cancer and normal subjects. In this study, two hybrid approaches–genetic algorithm with decision tree (GADT) and genetic algorithm with neural network (GANN)–are utilized to select optimal gene sets which contribute to the highest classification accuracy. Two benchmark microarray datasets were tested, and the most significant disease related genes have been identified. Furthermore, the selected gene sets achieved comparably high sample classification accuracy (96.79% and 94.92% in colon cancer dataset, 98.67% and 98.05% in leukemia dataset) compared with those obtained by mRMR algorithm. The study results indicate that these two hybrid methods are able to select disease related genes and improve classification accuracy.

Genomic analyses and cloning of novel chicken natriuretic peptide genes reveal new insights into natriuretic peptide evolution

The natriuretic peptide (NP) family consists of multiple subtypes in teleosts, including atrial, B-type, ventricular, and C-type NPs (ANP, BNP, VNP, CNP-1–4, respectively), but only ANP, BNP, CNP-3, and CNP-4 have been identified in tetrapods. As part of understanding the molecular evolution of NPs in the tetrapod lineage, we identified NP genes in the chicken genome. Previously, only BNP and CNP-3 have been identified in birds, but we characterized two new chicken NP genes by cDNA cloning, synteny and phylogenetic analyses. One gene is an orthologue of CNP-1, which has only ever been reported in teleostei and bichir. The second gene could not be assigned to a particular NP subtype because of high sequence divergence and was named renal NP (RNP) due to its predominant expression in the kidney. CNP-1 mRNA was only detected in brain, while CNP-3 mRNA was expressed in kidney, heart, and brain. In the developing embryo, BNP and RNP transcripts were most abundant 24 h post-fertilization, while CNP mRNA increased in a stage-dependant manner. Synthetic chicken RNP stimulated an increase in cGMP production above basal level in chicken kidney membrane preparations and caused a potent dose-dependant vasodilation of pre-constricted dorsal aortic rings. From conserved chromosomal synteny, we propose that the CNP-4 and ANP genes have been lost in chicken, and that RNP may have evolved from a VNP-like gene. Furthermore, we have demonstrated for the first time that CNP-1 is retained in the tetrapod lineage.

Genetic variation in BEACON influences quantitative variation in metabolic syndrome-related phenotypes

The BEACON gene (also known as UBL5) was identified as differentially expressed between lean and obese Psammomys obesus, a polygenic animal model of obesity, type 2 diabetes, and dyslipidemia. The human homologue of BEACON is located on chromosome 19p, a region likely to contain genes affecting metabolic syndrome–related quantitative traits as established by linkage studies. To assess whether the human BEACON gene may be involved in influencing these traits, we exhaustively analyzed the complete gene for genetic variation in 40 unrelated individuals and identified four variants (three novel). The two more common variants were tested for association with a number of quantitative metabolic syndrome–related traits in two large cohorts of unrelated individuals. Significant associations were found between these variants and fat mass (P = 0.026), percentage of fat (P = 0.001), and waist-to-hip ratio (P = 0.031). The same variants were also associated with total cholesterol (P = 0.024), LDL cholesterol (P = 0.019), triglycerides (P = 0.006), and postglucose load insulin levels (P = 0.018). Multivariate analysis of these correlated phenotypes also yielded a highly significant association (P = 0.0004), suggesting that BEACON may influence phenotypic variation in metabolic syndrome–related traits.

Copper depletion down-regulates expression of the Alzheimer's Disease amyloid-ß precursor protein gene

Alzheimer's disease is characterized by the accumulation of amyloid-ß peptide, which is cleaved from the amyloid-ß precursor protein (APP). Reduction in levels of the potentially toxic amyloid-ß has emerged as one of the most important therapeutic goals in Alzheimer's disease. Key targets for this goal are factors that affect the regulation of the APP gene. Recent in vivo and in vitro studies have illustrated the importance of copper in Alzheimer's disease neuropathogenesis and suggested a role for APP and amyloid-ß in copper homeostasis. We hypothesized that metals and in particular copper might alter APP gene expression. To test the hypothesis, we utilized human fibroblasts overexpressing the Menkes protein (MNK), a major mammalian copper efflux protein. MNK deletion fibroblasts have high intracellular copper, whereas MNK overexpressing fibroblasts have severely depleted intracellular copper. We demonstrate that copper depletion significantly reduced APP protein levels and down-regulated APP gene expression. Furthermore, APP promoter deletion constructs identified the copper-regulatory region between -490 and +104 of the APP gene promoter in both basal MNK overexpressing cells and in copper-chelated MNK deletion cells. Overall these data support the hypothesis that copper can regulate APP expression and further support a role for APP to function in copper homeostasis. Copper-regulated APP expression may also provide a potential therapeutic target in Alzheimer's disease.

VNTR polymorphism of the insulin gene and childhood overweight in a general population

Objective: The VNTR polymorphism 5' of the insulin gene has been related to obesity in a previous study on children with early onset of severe obesity. Our purpose was to analyze the association between this polymorphism and adiposity variability in an unselected population of children and adolescents in northern France.

Research Methods and Procedures: In 293 nuclear families from the Fleurbaix Laventie Ville Santé study, we genotyped the INS VNTR polymorphism in 431 children and adolescents (8 to 18 years of age) and their parents. Overweight was defined according to the international definition in both children and adults. A transmission disequilibrium test in families with an overweight offspring was performed. The prevalence of overweight was compared according to genotype. The effect of the genotype on BMI and waist circumference was tested with a linear regression model, adjusting for age, gender, and Tanner stage.

Results: There was an undertransmission of class III alleles from heterozygous parents to their overweight offspring (p < 0.002). Overweight was associated with class I alleles in children and adolescents (12% I/I, I/III vs. 3% III/III; p < 0.08). Those with a class III/III genotype had a 1 kg/m2 lower mean BMI (p = 0.04) and 3 cm lower waist circumference (p = 0.02) than those bearing one or two class I alleles. No association of adiposity or obesity with class I alleles was found in parents.

Discussion: INS VNTR polymorphism seems to contribute to differences in adiposity level in the general population of children and adolescents.

Mice lacking angiotensin-converting enzyme have increased energy expenditure, with reduced fat mass and improved glucose clearance

In addition to its role in the storage of fat, adipose tissue acts as an endocrine organ, and it contains a functional renin-angiotensin system (RAS). Angiotensin-converting enzyme (ACE) plays a key role in the RAS by converting angiotensin I to the bioactive peptide angiotensin II (Ang II). In the present study, the effect of targeting the RAS in body energy homeostasis and glucose tolerance was determined in homozygous mice in which the gene for ACE had been deleted (ACE^-/-) and compared with wild-type littermates. Compared with wild-type littermates, ACE^-/- mice had lower body weight and a lower proportion of body fat, especially in the abdomen. ACE^-/- mice had greater fed-state total energy expenditure (TEE) and resting energy expenditure (REE) than wild-type littermates. There were pronounced increases in gene expression of enzymes related to lipolysis and fatty acid oxidation (lipoprotein lipase, carnitine palmitoyl transferase, long-chain acetyl CoA dehydrogenase) in the liver of ACE^-/- mice and also lower plasma leptin. In contrast, no differences were detected in daily food intake, activity, fed-state plasma lipids, or proportion of fat excrete in fecal matter. In conclusion, the reduction in ACE activity is associated with a decreased accumulation of body fat, especially in abdominal fat depots. The decreased body fat in ACE^-/- mice is independent of food intake and appears to be due to a high energy expenditure related to increased metabolism of fatty acids in the liver, with the additional effect of increased glucose tolerance.

Expression of natriuretic peptide receptor mRNA and functional response to atrial natriuretic peptide and C-type natriuretic peptide in rainbow trout (Oncorhynchus mykiss) head kidney leucocytes

The stimulatory effect of vasomodulatory natriuretic peptide hormones on macrophages and peripheral blood leucocytes in mammals is well-established. However, the relationship in lower vertebrates has not been characterised. Expression of atrial natriuretic peptide, ventricular natriuretic peptide and C-type natriuretic peptide-1, and the guanylyl cyclase-linked (GC) natriuretic peptide receptor-A and -B-type receptors (NPR-A and NPR-B, respectively) was determined by PCR from the mRNA of rainbow trout head kidney leucocytes yielding gene fragments with 100% homology to the same respective natriuretic peptide and NPR-A and -B sequences obtained from other rainbow trout tissues. A mixed population of isolated rainbow trout head kidney leucocytes was stimulated in vitro with trout atrial natriuretic peptide (specific NPR-A agonist) and trout C-type natriuretic peptide (NPR-A and -B agonist) as well as the cGMP agonist 8-bromo-cGMP or the GC inhibitor 8-bromo-phenyl-eutheno-cGMP. Respiratory burst was stimulated by trout atrial natriuretic peptide, trout C-type natriuretic peptide-1 and 8-bromo-cGMP in a dose dependant manner with the highest activity as a result of stimulation with trout C-type natriuretic peptide-1 in excess of that achieved by phorbol myristate acetate (PMA). Equimolar concentrations of the inhibitor, inhibited the respiratory burst caused by the natriuretic peptides and 8-bromo-cGMP. The natriuretic peptide receptors on rainbow trout head kidney leucocytes appear to have a stimulatory function with regard to respiratory burst that is activated through a cGMP second messenger pathway and the natriuretic peptides expressed in the head kidney leucocytes may well act in a paracrine/autocrine manner.

Green tea, black tea, and epigallocatechin modify body composition, improve glucose tolerance, and differentially alter metabolic gene expression in rats fed a high-fat diet

The mechanisms of how tea and epigallocatechin-3-gallate (EGCG) lower body fat are not completely understood. This study investigated long-term administration of green tea (GT), black tea (BT), or isolated EGCG (1 mg/kg per day) on body composition, glucose tolerance, and gene expression related to energy metabolism and lipid homeostasis; it was hypothesized that all treatments would improve the indicators of metabolic syndrome. Rats were fed a 15% fat diet for 6 months from 4 weeks of age and were supplied GT, BT, EGCG, or water. GT and BT reduced body fat, whereas GT and EGCG increased lean mass. At 16 weeks GT, BT, and EGCG improved glucose tolerance. In the liver, GT and BT increased the expression of genes involved in fatty acid synthesis (SREBP-1c, FAS, MCD, ACC) and oxidation (PPAR-α, CPT-1, ACO); however, EGCG had no effect. In perirenal fat, genes that mediate adipocyte differentiation were suppressed by GT (Pref-1, C/EBP-β, and PPAR-γ) and BT (C/EBP-β), while decreasing LPL, HSL, and UCP-2 expression; EGCG increased expression of UCP-2 and PPAR-γ genes. Liver triacylglycerol content was unchanged. The results suggest that GT and BT suppressed adipocyte differentiation and fatty acid uptake into adipose tissue, while increasing fat synthesis and oxidation by the liver, without inducing hepatic fat accumulation. In contrast, EGCG increased markers of thermogenesis and differentiation in adipose tissue, while having no effect on liver or muscle tissues at this dose. These results show novel and separate mechanisms by which tea and EGCG may improve glucose tolerance and support a role for these compounds in obesity prevention.

Identification and characterisation of novel genes involved in skeletal muscle hypertrophy

There is mounting evidence in support of the view that skeletal muscle hypertrophy results from the complex and coordinated interaction of numerous signalling pathways. Well characterised components integral to skeletal muscle adaptation include the transcriptional activity of the members of the myogenic regulatory factors, numerous secreted peptide growth factors, and the regenerative potential of satellite cells. Whilst studies investigating isolated components or pathways have enhanced our current understanding of skeletal muscle hypertrophy, our knowledge of how all of these components react in concert to a common stimulus remains limited. The broad aim of this thesis was to identify and characterise novel genes involved in skeletal muscle hypertrophy. We have created a customised human skeletal muscle specific microarray which contains ∼11,000 cDNA clones derived from a normalised human skeletal muscle cDNA library as well as 270 genes with known functional roles in human skeletal muscle. The first aspect of this thesis describes the production of the microarray and evaluates the robustness and reproducibility of this analytical technique. Study one aimed to use this microarray in the identification of genes that are differentially expressed during the forced differentiation of human rhabdomyosarcoma cells, an in vitro model of skeletal muscle development. Firstly using this unique model of aberrant myogenic differentiation we aimed to identify genes with previously unidentified roles in myogenesis. Secondly, the data from this study permitted the examination of the performance of the microarray in detecting differential gene expression in a biological system. We identified several new genes with potential roles in the myogenic arrest of rhabdomyosarcoma and further characterised the expression of muscle specific genes in rhabdomyosarcoma differentiation. In study two, the molecular responses of cell cycle regulators, muscle regulatory factors, and atrophy related genes were mapped in response to a single bout of resistance exercise in human skeletal muscle. We demonstrated an increased expression of MyoD, myogenin and p21, whilst the expression of myostatin was decreased. The results of this study contribute to the existing body of knowledge on the molecular regulation skeletal muscle to a hypertrophic stimulus. In study three, the muscle samples collected in study two were analysed using the human skeletal muscle specific microarray for the identification of novel genes with potential roles in the hypertrophic process. The analysis uncovered four interesting genes (TXNIP, MLP, ASB5, FLJ 38973) that have not previously been examined in human skeletal muscle in response to resistance exercise. The functions of these genes and their potential roles in skeletal muscle are discussed. In study four, the four genes identified in study three were examined in human primary skeletal muscle cell cultures during myogenic differentiation. Human primary skeletal muscle cells were derived from the vastus lateralis muscle of 8 healthy volunteers (6 males and 2 females). Cell cultures were differentiated using serum withdrawal and serum withdrawal combined with IGF-1 supplementation. Markers of the cell proliferation, cell cycle arrest and myogenic differentiation were examined to assess the effectiveness of the differentiation stimulus. Additionally, the expressions of TXNIP, MLP, ASB5 and FLJ 38973 measured in an attempt to characterise further their roles in skeletal muscle. The expression of TXNIP changed markedly in response to both differentiation stimuli, whilst the expression of the remaining genes were not altered. Therefore it was suggested that expression of these genes might be responsive to the mechanical strain or contraction induced by the resistance exercise. In order to examine whether these novel genes responded specifically to resistance type exercise, their expression was examined following a single bout of endurance exercise. The expression of TXNIP, MLP, and FLJ 38973 remained unchanged whilst ASB5 increased 30 min following the cessation of the exercise.

Evolutionary relationships among bluetongue and related orbiviruses

Polymerase chain reaction (PCR) sequencing of specific viral gene segments was used to investigate the phylogenetic relationships among the orbiviruses. Sequence comparisons of the bluetongue virus (BTV) RNA3 from different regions of the world (North America, South Africa, India, Indonesian, Malaysia, Australia and the Caribbean region) showed that geographic separation had resulted in significant divergence, consistent with the evolution of distinct viral populations. There were at least 3 topotypes (Gould, 1987); the Australasian, African - American and another topotype represented by BTV 15 isolated in Australia in 1986. The topotypes of BTV had RNA3 nucleotide sequences that differed by approximately 20 per cent. Analysis of BTV-specific gene segments from animal and insect specimens showed that bluetongue viruses had entered northern Australia from South East Asia, possibly by wind-borne vectors. Nucleotide sequence comparisons were used to show the close genetic relationship between BTV 2 (Ona-A strain) from Florida and BTV 12 from Jamaica, and to investigate the reassortment of BTV genome segments in nature. The mutation rates of the BTV RNA2 and RNA3 segments were estimated to be of the order of 10(-4) nucleotide changes/site/year, similar in magnitude to that reported for other RNA viruses.

Effects of vegetarianism and exercise on muscle creatine loading and gene expression

The results of this thesis indicate that vegetarianism and prior exercise can increase skeletal muscle creatine loading. The mechanisms by which these phenomena occur remain unknown but may be related to changes in the gene expression of a cellular pump within the muscle known as the "creatine transporter".

Dehydration and the natriuretic peptide system in the toad, Bufo marinus

The natriuretic peptide system is a family of related proteins that in humans function to reduce blood pressure and blood volume. However, in amphibians the function is less well understood. This thesis demonstrates that the amphibian, Bufo marinus, possesses a well-developed natriuretic peptide system that is capable of responding to changes induced by dehydration.

The Ikaros gene family

Ikaros-related transcription factors are essential for adaptive immunity. Evolutionary analysis suggested derivation from a precursor similar to the 'atypical' Pegasus protein, and possibly related to invertebrate Hunchback. Pegasus was shown to regulate neural, gut and blood cell development in zebrafish, with Ikaros identified as one of several potential gene targets.

The effect of folic acid supplementation on dna biomarkers of colorectal cancer risk (uracil misincorporation, global and gene-specific dna hypomethylation) : a randomised intervention study

Background - Alterations in folate status are associated with colorectal carcinogenesis. Folate’s role has been postulated to be either via prevention of changes in DNA methylation or uracil misincorporation.
Objective - To investigate the effect of folic acid supplementation on colonocyte folate status and DNA biomarkers.
Design - Twenty individuals harbouring colonic adenomas were randomised to receive folic acid (600 g daily) or placebo for 6 months post polypectomy. Systemic and colonocyte folate status was determined at baseline and following the intervention. Modified Comet assays were used to determine uracil misincorporation and global DNA hypomethylation at the site adjacent to the polyp and a site distal to the polyp.
Outcomes - Supplementation resulted in increased colonocyte folate, which approached significance, at the site adjacent to the polyp (P= 0.06) but not distal to the polyp (P= 0.36); correspondingly there was a reduction in uracil misincorporation at the site adjacent to the polyp (P = 0.02) and the distal site showed no such trend (P= 0.39). There were no significant changes in global DNA hypomethylation at either site post-intervention.
Conclusions - Folic acid supplementation resulted in increased colonocyte folate and decreased uracil misincorporation at the site of the adenoma but not distal to the adenoma. This supports the hypothesis that localised areas of folate deficiency may exist in human colonic mucosa which respond to folic acid supplementation through increasing colonocyte folate and improving folate-related DNA biomarkers of cancer risk.