Identification of a novel Sp1 splice variant as a strong transcriptional activator.

The transcription factor Sp1 regulates expression of numerous genes involved in many cellular processes. Different post-transcriptional modifications can influence the transcriptional control activity and stability of Sp1. In addition to these modifications, alternative splicing isoforms may also be the basis of its distinct functional activities. In this study, we identified a novel alternative splice isoform of Sp1 named Sp1c. This variant is generated by exclusion of a short domain, which we designate a, through alternative splice acceptor site usage in the exon 3. The existence of this new isoform was confirmed in vivo by Western blotting analysis. Although at very low levels, Sp1c is ubiquitously expressed, as seen in its fulllength Sp1. A preliminary characterization of Sp1c shows that: (a) Sp1c works as stronger activator of transcription than full-length Sp1; (b) percentage of HEK293 Sp1c-overexpressing cells is higher in G1 phase and lower in S phase than percentage of HEK293 Sp1-overexpressing cells

Impairment of methyl cycle affects mitochondrial methyl availability and glutathione level in Down's Sindrome

In Down's syndrome there is evidence that increased gene expression coding for specific cystathionine beta-synthase translates directly into biochemical aberrations, which result in a biochemical and metabolic imbalance of the methyl status. This event is destined to impact mitochondrial function since methylation is a necessary event in mitochondria and relies on the availability and uptake of the methyl donor S-adenosylmethionine. Indeed mitochondrial dysfunctions have been widely described in Down's syndrome, but they have never been correlated to a possible mitochondrial methyl unbalance. In the present study we find that the mitochondrial levels of S-adenosylmethionine are reduced in Down's syndrome compared to control cells demonstrating the effect of the methyl unbalance on mitochondria. The possible role of methylation in mitochondria is discussed and some preliminary results on a possible methylation target are presented

Impairment of methyl cycle affects mitochondrial methyl availability and glutathione level in Down's Sindrome

In Down's syndrome there is evidence that increased gene expression coding for specific cystathionine beta-synthase translates directly into biochemical aberrations, which result in a biochemical and metabolic imbalance of the methyl status. This event is destined to impact mitochondrial function since methylation is a necessary event in mitochondria and relies on the availability and uptake of the methyl donor S-adenosylmethionine. Indeed mitochondrial dysfunctions have been widely described in Down's syndrome, but they have never been correlated to a possible mitochondrial methyl unbalance. In the present study we find that the mitochondrial levels of S-adenosylmethionine are reduced in Down's syndrome compared to control cells demonstrating the effect of the methyl unbalance on mitochondria. The possible role of methylation in mitochondria is discussed and some preliminary results on a possible methylation target are presented

Transcriptional Regulation of the Mitochondrial Citrate and Carnitine/Acylcarnitine Transporters: Two Genes Involved in Fatty Acid Biosynthesis and E-oxidation

Transcriptional regulation of genes involved in fatty acid metabolism is considered the major long-term regulatory mechanism controlling lipid homeostasis. By means of this mechanism, transcription factors, nutrients, hormones and epigenetics control not only fatty acid metabolism, but also many metabolic pathways and cellular functions at the molecular level. The regulation of the expression of many genes at the level of their transcription has already been analyzed. This review focuses on the transcriptional control of two genes involved in fatty acid biosynthesis and oxidation: the citrate carrier (CIC) and the carnitine/ acylcarnitine/carrier (CAC), which are members of the mitochondrial carrier gene family, SLC25. The contribution of tissue-specific and less tissue-specific transcription factors in activating or repressing CIC and CAC gene expression is discussed. The interaction with drugs of some transcription factors, such as PPAR and FOXA1, and how this interaction can be an attractive therapeutic approach, has also been evaluated. Moreover, the mechanism by which the expression of the CIC and CAC genes is modulated by coordinated responses to hormonal and nutritional changes and to epigenetics is highlighte

The Human SLC25A33 and SLC25A36 Genes of Solute Carrier Family 25 Encode Two Mitochondrial Pyrimidine Nucleotide Transporters

The human genome encodes 53 members of the solute carrier family 25 (SLC25), also called the mitochondrial carrier family, many of which have been shown to transport inorganic anions, amino acids, carboxylates, nucleotides, and coenzymes across the inner mitochondrial membrane, thereby connecting cytosolic and matrix functions. Here two members of this family, SLC25A33 and SLC25A36, have been thoroughly characterized biochemically. These proteins were overexpressed in bacteria and reconstituted in phospholipid vesicles. Their transport properties and kinetic parameters demonstrate that SLC25A33 transports uracil, thymine, and cytosine (deoxy)nucleoside di- and triphosphates by an antiport mechanism and SLC25A36 cytosine and uracil (deoxy)nucleoside mono-, di-, and triphosphates by uniport and antiport. Both carriers also transported guanine but not adenine (deoxy)nucleotides. Transport catalyzed by both carriers was saturable and inhibited by mercurial compounds and other inhibitors of mitochondrial carriers to various degrees. In confirmation of their identity (i) SLC25A33 and SLC25A36 were found to be targeted to mitochondria and (ii) the phenotypes of Saccharomyces cerevisiae cells lacking RIM2, the gene encoding the well characterized yeast mitochondrial pyrimidine nucleotide carrier, were overcome by expressing SLC25A33 or SLC25A36 in these cells. The main physiological role of SLC25A33 and SLC25A36 is to import/export pyrimidine nucleotides into and from mitochondria, i.e. to accomplish transport steps essential for mitochondrial DNA and RNA synthesis and breakdown

Hyperhomocysteinemia: related genetic diseases and congenital defects, abnormal DNA methylation and newborn screening issues

Homocysteine, a sulfur-containing amino acid derived from the methionine metabolism, is located at the branch point of two pathways of the methionine cycle, i.e. remethylation and transsulfuration. Gene abnormalities in the enzymes catalyzing reactions in both pathways lead to hyperhomocysteinemia. Hyperhomocysteinemia is associated with increased risk for congenital disorders, including neural tube closure defects, heart defects, cleft lip/palate, Down syndrome, and multi-system abnormalities in adults. Since hyperhomocysteinemia is known to affect the extent of DNA methylation, it is likely that abnormal DNA methylation during embryogenesis, may be a pathogenic factor for these congenital disorders. In this review we highlight the importance of homocysteinemia by describing the genes encoding for enzymes of homocysteine metabolism relevant to the clinical practice, especially cystathionine-β-synthase and methylenetetrahydrofolate reductase mutations, and the impairment of related metabolites levels. Moreover, a possible correlation between hyperhomocysteine and congenital disorders through the involvement of abnormal DNA methylation during embryogenesis is discussed. Finally, the relevance of present and future diagnostic tools such as tandem mass spectrometry and next generation sequencing in newborn screening is highlighted

Hypoxia-Induced Molecular and Cellular Changes in the Congenitally Diseased Heart: Mechanisms and Strategies of Intervention

Tissue hypoxia plays a critical role in the pathobiology of congenital heart diseases, especially with regard to cyanotic patients. Here, we describe the cellular and molecular mechanisms induced by hypoxia in the diseased heart, with particular attention to the metabolic and functional changes that underlie the hypoxia-induced right ventricle remodelling. The role of reactive oxygen species in transcriptomic changes, DNA damage, contractile dysfunction and extracellular matrix remodelling will be addressed. Furthermore, the reoxygenation injury, which occurs when oxygen is reintroduced upon initiation of cardiopulmonary bypass, will be discussed. This allows a better understanding of the risks associated with the reoxygenation injury in children undergoing open-heart surgery and helps to improve strategies of intervention for myocardial protection

The sequences of human and bovine genes of the phosphate carrier from mitochondria contain evidence of alternatively spliced forms.

The sequences of the human and bovine genes for the phosphate carrier from the inner membranes of mitochondria have been determined. The genes have similar structures and each is divided into nine exons. In both genes, two exons, named IIIA and IIIB, are closely related, and they appear to the alternatively spliced. The human exon IIIB sequence is found in a published human heart cDNA sequence, and bovine exon IIIA forms part of a published bovine heart cDNA sequence. By further examination of the human heart cDNA library, sequences arising from both alternatively spliced forms of the phosphate carrier have been characterized. Both forms were also found in several bovine tissues, but the ratios of expression of the two forms varied. The form containing exon IIIA was expressed most highly in bovine heart and liver, less highly in brain and kidney, and only in low amounts in lung. The opposite hierarchy was found for the form containing exon IIIB; it was most highly expressed in lung and least in heart and liver. The alternative splicing mechanism affects amino acids 4-45 of the mature phosphate carrier protein, which is believed to form one of six transmembrane segments of the phosphate carrier and to emerge into a large extramembranous loop. The alternative splicing mechanism changes 13 and 11 amino acids in the human and bovine carrier proteins, respectively. As the function of this region of the phosphate carrier is not known, the effects of the changes on carrier function are not understood at present

The Intrinsic Virtues of EGCG, an Extremely Good Cell Guardian, on Prevention and Treatment of Diabesity Complications

The pandemic proportion of diabesity—a combination of obesity and diabetes—sets a worldwide health issue. Experimental and clinical studies have progressively reinforced the pioneering epidemiological observation of an inverse relationship between consumption of polyphenol-rich nutraceutical agents and mortality from cardiovascular and metabolic diseases. With chemical identification of epigallocatechin-3-gallate (EGCG) as the most abundant catechin of green tea, a number of cellular and molecular mechanisms underlying the activities of this unique catechin have been proposed. Favorable effects of EGCG have been initially attributed to its scavenging effects on free radicals, inhibition of ROS-generating mechanisms and upregulation of antioxidant enzymes. Biologic actions of EGCG are concentration-dependent and under certain conditions EGCG may exert pro-oxidant activities, including generation of free radicals. The discovery of 67-kDa laminin as potential EGCG membrane target has broaden the likelihood that EGCG may function not only because of its highly reactive nature, but also via receptor-mediated activation of multiple signaling pathways involved in cell proliferation, angiogenesis and apoptosis. Finally, by acting as epigenetic modulator of DNA methylation and chromatin remodeling, EGCG may alter gene expression and modify miRNA activities. Despite unceasing research providing detailed insights, ECGC composite activities are still not completely understood. This review summarizes the most recent evidence on molecular mechanisms by which EGCG may activate signal transduction pathways, regulate transcription factors or promote epigenetic changes that may contribute to prevent pathologic processes involved in diabesity and its cardiovascular complication