Single Nucleotide Polymorphisms in Apolipoprotein B, Apolipoprotein E, and Methylenetetrahydrofolate Reductase Are Associated With Serum Lipid Levels in Northern Chilean Subjects. A Pilot Study.

Characterization of allelic variants is relevant to demonstrate associations among genetic background and susceptibility to develop cardiovascular diseases, which are the main cause of death in Chile. Association of APOB, APOE, and MTHFR polymorphisms with higher lipid levels and the risk of developing hypertension and cardiovascular diseases have been described. Thus, the aim of this study was to assess genotype distribution and relative allelic frequency of ApoB rs693, ApoE rs7412, ApoE rs429358, MTHFR rs1801131, and MTHFR rs1801133 allelic variants and their effects on lipid profile in young healthy men and women from Northern Chile. A group of 193 healthy subjects were enrolled for this study. Genotyping of rs693 (APOB), rs7412 and rs429358 (APOE), and rs1801131 and rs1801133 (MTHFR) polymorphisms were performed by real time PCR. In addition, lipid profiles were determined and associated to genetic data. The genotype distribution was APOB rs693 (CC = 37%, CT = 41%, and TT = 22%), APOE rs7412/rs429358 (E4 = 0.06, E3 = 0.91, and E2 = 0.03), MTHFR rs1801131 (AA = 57%, AC = 30%, and CC = 13%), and MTHFR rs1801133 (CC = 20%, CT = 47%, and TT = 33%). The association of the genetic variants with plasma lipid levels showed that women, but not men, carrying APOB mutated allele (T) and Apo E4 allele presented lower values of total cholesterol when compared with C/C homozygous genotype or E3 allele, respectively (p < 0.05). In addition, a subgroup analysis revealed that ApoB C/C homozygous women exhibited higher values of HDL-C when compared with men carrying identical genotype (p < 0.01). On the other hand, women carrying E4 allele exhibited lower values of triglycerides when compared with male carrying identical genotype (p < 0.05). Finally, women carrying mutate allele (C) for MTHFR rs1801131 showed lower levels of triglycerides when compared with A/A homozygous genotype (p < 0.05) and lower levels of LDL-C for MTHFR rs1801133 in females carrying (T) allele when compared with males carrying identical genotype (p < 0.05). In summary, the present data showed that APOB, APOE, and MTHFR single nucleotide polymorphisms are associated to lipid levels in a gender-dependent manner among healthy subjects from Northern Chile, especially in women.

Sulfate nutrition improves short-term Al3+-stress tolerance in roots of Lolium perenne L.

Trivalent aluminum ions (Al3+) in acidic soils are a major constraint for crop productivity inhibiting root elongation and promoting cell death. Al3+-toxicity has adverse biochemical and physiological effects on plant root growth. Sulfur is an essential macronutrient assimilated from the soil in the form of sulfate. However, the implication of sulfate nutritional status in the modulation of short-term Al3+-tolerance mechanisms in plant roots has not been previously reported. Here, we evaluated the effects of increased sulfate supply on short-term Al3+-toxicity in roots of Lolium perenne, measuring Al, Ca, Mg and S uptake, lipid peroxidation, total SOD activity, and transcriptional levels of Cu/Zn and Fe-SOD genes. First, the nitrogen sulfur ratio (N/S) in the TF nutrient solutions used in this study were computed to confirm that L. perenne plants were grown in sulfate deficiency (120 µM), optimal supply (240 µM), or overdoses conditions (360 µM), without affecting dry root biomass. Sulfate supplementation (>240 µM, and N/S ratio < 16) played a significant protection to Al3+-stress that prevents morphological changes in root tips, inhibits lipid peroxidation and differentially up-regulates total SOD activity, due changes in SOD gene expression. The results support the importance of sulfate nutritional status, on plant tissue homeostasis, enhancing the physiological tolerance mechanisms modulating lipid peroxidation damage induced by short-term Al3+-toxicity.

Characterization of LDLR rs5925 and PCSK9 rs505151 genetic variants frequencies in healthy subjects from northern Chile: Influence on plasma lipid levels.

BACKGROUND: Identification and characterization of genetic variants and their effects on human health may allow to establish relationships between genetic background and susceptibility to developing cardiovascular diseases. LDLR and PCSK9 polymorphisms have been associated with higher lipid levels and risk of cardiovascular diseases. Thus, the main aim of this study was to evaluate genotype distribution and relative allelic frequency of LDLR rs5925 (1959C > T) and PCSK9 rs505151 (23968 A > G) genetic variants and their effects on lipid levels of healthy subjects from northern Chile. METHODS: A total of 178 healthy individuals were recruited for this study. The genotyping of rs5925 (LDLR) and rs505151 (PCSK9) polymorphisms was performed by PCR-RFLP and qPCR, respectively. In addition, glucose and lipid levels were determined and associated with the genetic data. RESULTS: Genotype distribution for LDLR rs5925 polymorphism was as follows: CC = 19%; CT = 53%; and TT = 28% (HWE: χ2  = 0.80; P = .37), and for PCSK9 rs505151 genetic variant was as follows: AA = 93%; AG = 7%; and GG = 0% (HWE: χ2  = 0.22; P = .64). The frequency of T (rs5925) and G (rs505151) mutated alleles was 0.55 and 0.03, respectively. Data showed that individuals carrying LDLR mutated allele (T) presented lower values of total cholesterol, triglycerides, and LDL-cholesterol when compared to CC homozygous genotype (P < .05). Subgroup analysis revealed that women carrying the PCSK9 mutated allele (G) exhibited higher values of total cholesterol, triglycerides, HDL-C, and LDL-C when compared to male group carrying the same genotype (P < .05). CONCLUSIONS: The effect of LDLR rs5925 and PCSK9 rs505151 gene polymorphisms on lipid levels is associated with gender among healthy subjects from northern Chile.

Protein kinase Czeta represses the interleukin-6 promoter and impairs tumorigenesis in vivo.

Gene alterations in tumor cells that confer the ability to grow under nutrient- and mitogen-deficient conditions constitute a competitive advantage that leads to more-aggressive forms of cancer. The atypical protein kinase C (PKC) isoform, PKCzeta, has been shown to interact with the signaling adapter p62, which is important for Ras-induced lung carcinogenesis. Here we show that PKCzeta-deficient mice display increased Ras-induced lung carcinogenesis, suggesting a new role for this kinase as a tumor suppressor in vivo. We also show that Ras-transformed PKCzeta-deficient lungs and embryo fibroblasts produced more interleukin-6 (IL-6), which we demonstrate here plays an essential role in the ability of Ras-transformed cells to grow under nutrient-deprived conditions in vitro and in a mouse xenograft system in vivo. We also show that PKCzeta represses histone acetylation at the C/EBPbeta element in the IL-6 promoter. Therefore, PKCzeta, by controlling the production of IL-6, is a critical signaling molecule in tumorigenesis.

The signaling adaptor p62 is an important NF-kappaB mediator in tumorigenesis.

The balance between cell death and survival, two critical aspects of oncogenic transformation, determines the outcome of tumorigenesis. Nuclear factor-kappaB (NF-kappaB) is a critical regulator of survival; it is induced by the oncogene Ras and, when inhibited, accounts for the cell death response of Ras-transformed cells. Here, we show that the signaling adaptor p62 is induced by Ras, its levels are increased in human tumors, and it is required for Ras-induced survival and transformation. p62-/- mice are resistant to Ras-induced lung adenocarcinomas. p62 is necessary for Ras to trigger IkappaB kinase (IKK) through the polyubiquitination of tumor necrosis factor (TNF) receptor-associated factor 6 (TRAF6), and its deficiency produces increased reactive oxygen species (ROS) levels, which account for the enhanced cell death and reduced tumorigenicity of Ras in the absence of p62.

A functional polymorphism of the Galphaq (GNAQ) gene is associated with accelerated mortality in African-American heart failure.

Galphaq, encoded by the human GNAQ gene, is an effector subunit of the Gq heterotrimeric G-protein and the convergence point for signaling of multiple Gq-coupled neurohormonal receptors. To identify naturally occurring mutations that could modify GNAQ transcription, we examined genomic DNA isolated from 355 normal subjects for genetic variants in transcription factor binding motifs. Of seven variants identified, the most common was a GC to TT dinucleotide substitution at -694/-695 (allele frequency of 0.467 in Caucasians and 0.329 in African Americans) within a GC-rich domain containing consensus binding sites for Sp-1, c-rel and EGR-1. In promoter-reporter analyses, the TT substitution increased promoter activity in cultured neonatal rat cardiac myocytes and human HEK fibroblasts by approximately 30% at baseline and after stimulation with phorbol ester. Two other relatively common polymorphisms, -173G/A and -168G/A, did not affect promoter activity. Since altered expression/activity of Galphaq is implicated in heart disease, we re-sequenced the GNAQ promoter in 1052 prospectively followed heart failure patients. The TT variant was not increased in heart failure, but was associated with decreased survival time among African Americans, with an adjusted RR of death/cardiac transplant of 1.95 (95% CI = 1.21-3.13) for heterozygotes and 2.4 (95% CI = 1.36-4.26) for homozygotes. Gel mobility shift assays showed that this GC/TT substitution eliminated Sp-1 binding without affecting c-rel or EGR-1 binding to this promoter fragment. Thus, the GNAQ -694/-695 promoter polymorphism alters transcription factor binding, increases promoter activity and adversely affects outcome in human heart failure.

Cardiomyocyte degeneration with calpain deficiency reveals a critical role in protein homeostasis.

Regulating the balance between synthesis and proteasomal degradation of cellular proteins is essential for tissue growth and maintenance, but the critical pathways regulating protein ubiquitination and degradation are incompletely defined. Although participation of calpain calcium-activated proteases in post-necrotic myocardial autolysis is well characterized, their importance in homeostatic turnover of normal cardiac tissue is controversial. Hence, we evaluated the consequences of physiologic calpain (calcium-activated protease) activity in cultured cardiomyocytes and unstressed mouse hearts. Comparison of in vitro proteolytic activities of cardiac-expressed calpains 1 and 2 revealed calpain 1, but not calpain 2, activity at physiological calcium concentrations. Physiological calpain 1 activation was evident in adenoviral transfected cultured cardiomyocytes as proteolysis of specific substrates, generally increased protein ubiquitination, and accelerated protein turnover, that were each inhibited by coexpression of the inhibitor protein calpastatin. Conditional forced expression of calpain 1, but not calpain 2, in mouse hearts demonstrated substrate-specific proteolytic activity under basal conditions, with hyperubiquitination of cardiac proteins and increased 26S proteasome activity. Loss of myocardial calpain activity by forced expression of calpastatin diminished ubiquitination of 1 or more specific myocardial proteins, without affecting overall ubiquitination or proteasome activity, and resulted in a progressive dilated cardiomyopathy characterized by accumulation of intracellular protein aggregates, formation of autophagosomes, and degeneration of sarcomeres. Thus, calpain 1 is upstream of, and necessary for, ubiquitination and proteasomal degradation of a subset of myocardial proteins whose abnormal accumulation produces autophagosomes and degeneration of cardiomyocytes with functional decompensation.

Distinct pathways regulate proapoptotic Nix and BNip3 in cardiac stress.

Up-regulation of myocardial Nix and BNip3 is associated with apoptosis in cardiac hypertrophy and ischemia, respectively. To identify mechanisms of gene regulation for these critical cardiac apoptosis effectors, the determinants of Nix and BNip3 promoter activation were elucidated by luciferase reporter gene expression in neonatal rat cardiac myocytes. BNip3 transcription was increased by hypoxia but not by phenylephrine (10 microM), angiotensin II (100 nM), or isoproterenol (10 microM). In contrast, Nix transcription was increased by phenylephrine but not by isoproterenol, angiotensin II, or hypoxia. Since phenylephrine stimulates cardiomyocyte hypertrophy via protein kinase C (PKC), the effects of phorbol myristate acetate (PMA, 10 nM for 24 h) and adenoviral PKC expression were assessed. PMA and PKC alpha, but not PKC epsilon or dominant negative PKC alpha, increased Nix transcription. Multiple Nix promoter GC boxes bound transcription factor Sp-1, and basal and PMA- or PKC alpha-stimulated Nix promoter activity was suppressed by mithramycin inhibition of Sp1-DNA interactions. In vivo determinants of Nix expression were evaluated in Nix promoter-luciferase (NixP) transgenic mice that underwent ischemia-reperfusion (1 h/24 h), transverse aortic coarctation (TAC), or cross-breeding with the G(q) overexpression model of hypertrophy. Luciferase activity increased in G alpha(q)-NixP hearts 3.2 +/- 0.4-fold and in TAC hearts 2.8 +/- 0.4-fold but did not increase with infarction-reperfusion. NixP activity was proportional to the extent of TAC hypertrophy and was inhibited by mithramycin. These studies revealed distinct mechanisms of transcriptional regulation for cardiac Nix and BNip3. BNip3 is hypoxia-inducible, whereas Nix expression was induced by G alpha(q)-mediated hypertrophic stimuli. PKC alpha, a G(q) effector, transduced Nix transcriptional induction via Sp1.

Perindopril regulates beta-agonist-induced cardiac apoptosis.

Administration of the beta-adrenergic agonist isoproterenol results in cardiac apoptosis. The effect of short-term beta-adrenergic stimulation by isoproterenol on the activity of plasma, lung, and left ventricular (LV) angiotensin I-converting enzyme (ACE) activity and its association with the development of cardiac apoptosis was investigated. beta-Adrenergic stimulation for 24 hours produced an early increase only in the proapoptotic proteins bax and bcl-XS without changes in the levels of the antiapoptotic protein bcl-XL. The ratio between these bcl family proteins was indicative of apoptosis and correlated with an early and significant increase (300%) in DNA laddering. However, after 5 days of the beta-adrenergic stimulation, the ratio changed in favor of antiapoptotic proteins and correlated with the absence of DNA fragmentation. In addition, LV and plasma ACE activities increased markedly with isoproterenol over the study period up to 5 days. ACE activity also regulated expression of the antiapoptotic gene bcl-XL. The administration of perindopril (an ACE inhibitor) prevented the observed increase in bax and bcl-XS levels and attenuated (50% decrease, P<0.05) the effect of isoproterenol on DNA fragmentation. Thus, early and transient cardiac apoptosis triggered by the beta-adrenergic agonist isoproterenol is reversed in the presence of perindopril.

Aldose reductase induced by hyperosmotic stress mediates cardiomyocyte apoptosis: differential effects of sorbitol and mannitol.

Cells adapt to hyperosmotic conditions by several mechanisms, including accumulation of sorbitol via induction of the polyol pathway. Failure to adapt to osmotic stress can result in apoptotic cell death. In the present study, we assessed the role of aldose reductase, the key enzyme of the polyol pathway, in cardiac myocyte apoptosis. Hyperosmotic stress, elicited by exposure of cultured rat cardiac myocytes to the nonpermeant solutes sorbitol and mannitol, caused identical cell shrinkage and adaptive hexose uptake stimulation. In contrast, only sorbitol induced the polyol pathway and triggered stress pathways as well as apoptosis-related signaling events. Sorbitol resulted in activation of the extracellular signal-regulated kinase (ERK), p54 c-Jun N-terminal kinase (JNK), and protein kinase B. Furthermore, sorbitol treatment resulting in induction and activation of aldose reductase, decreased expression of the antiapoptotic protein Bcl-xL, increased DNA fragmentation, and glutathione depletion. Apoptosis was attenuated by aldose reductase inhibition with zopolrestat and also by glutathione replenishment with N-acetylcysteine. In conclusion, our data show that hypertonic shrinkage of cardiac myocytes alone is not sufficient to induce cardiac myocyte apoptosis. Hyperosmolarity-induced cell death is sensitive to the nature of the osmolyte and requires induction of aldose reductase as well as a decrease in intracellular glutathione levels.