Interethnic differences of CYP2C9 alleles in healthy Hungarian and Roma population samples: Relationship to worldwide allelic frequencies

CYP2C9 gene polymorphisms are widely studied in several ethnic groups, however they are less known in the Roma population. The aim of this work was to study the ethnic differences of the CYP2C9 allele distribution in a healthy Roma population in order to compare them with a healthy Hungarian population. A total of 535 Hungarian and 465 Roma volunteers were genotyped for the CYP2C92 (Arg144Cys) and CYP2C93 (Ile359Leu) allelic variants by PCR-RFLP assay. The frequencies of the CYP2C91, 2 and 3 alleles in the Hungarian population were 0.787, 0.125, and 0.088 and in Roma 0.727, 0.118, and 0.155, respectively. We found a significant difference in CYP2C93 prevalence between the Hungarian and Roma populations, which have therapeutic consequences (p < 0.005). The distribution of 1/1, 1/2, 1/3, 2/2, 2/3, and 3/3 genotypes in Hungarians were 0.620, 0.195, 0.139, 0.021, 0.015, and 0.011, while in Roma were 0.533, 0.168, 0.219, 0.011, 0.047, and 0.022, respectively. A significant difference was found between the Hungarian and Roma populations regarding the 1/1, 1/3 and the 2/3 (p < 0.005) genotypes. This is the first study to investigate the polymorphisms of CYP2C9 gene in the two largest populations in Hungary, healthy Hungarians and Roma. The prevalence of variant CYP2C9 alleles in the Hungarian population is similar to that observed in other European populations. In contrast, the Roma population differs from Hungarians, from most of other Caucasian groups, and from Indians in the incidence of CYP2C9 common variants. The difference in allele distribution patterns between the two populations studied has therapeutic implications as it influences the optimization of therapies.     

Distribution of CYP1A1*2A polymorphism in adult patients with acute lymphoblastic leukemia in a Mexican population

The effects of the CYP1A12A genotype on susceptibility to leukemia have received particular attention in recent years because this enzyme plays a central role in the activation of carcinogens. Several polymorphisms at the CYP1A1 locus have been identified and their genotypes appear to exhibit population frequencies that depend on ethnicity. We evaluated the role of the CYP1A12A genotype in adults with acute lymphoblastic leukemia (ALL) by genotyping 210 patients and 228 healthy controls from the Mexican population. The frequency of the CC genotype was 8% (18/228) in the control group and 42% (88/210) in ALL patients; the frequency of the CT genotype was 39% (89/228) and 29.5% (62/210), respectively; and that of the TT genotype was 53% (121/228) and 28.5% (60/210), respectively. The odds ratio was 8.4 (95% CI, 4.7–15.5; P < 0.001). These data indicate that the CYP1A12A genotype contributes significantly to susceptibility to adult ALL in a sample of the Mexican population.     

Netrin-1 prevents ischemia/reperfusion-induced myocardial infarction via a DCC/ERK1/2/eNOSs1177/NO/DCC feed-forward mechanism

We have recently shown that a novel endothelial mitogen netrin-1 potently stimulates nitric oxide (NO) production via a DCC-ERK1/2 dependent mechanism. In view of the well-established cardioprotective role of NO, the present study investigated whether netrin-1 is cardioprotective via NO signaling in the heart. Netrin-1 receptor DCC was abundantly expressed in the C57BL/6J mouse hearts. Perfusion of heart with netrin-1 (100 ng/mL) using a Langendorff system significantly increased NO production. Under ischemia/reperfusion (I/R), netrin-1 induced a substantial reduction in infarct size (21.8 ± 4.9% from 42.5 ± 3.6% in the controls), which was accompanied by an augmented production of NO. Pre-perfusion with DCC-antibody, U0126 (MEK1/2 inhibitor), L-NAME or PTIO (NO scavenger) attenuated protective effects of netrin-1 on infarct size and NO production, indicating upstream roles of DCC and ERK1/2 in NO production, as well as an essential role of NO in cardioprotection. Netrin-1 induced reduction in infarct size was significantly attenuated in DCC+/− mice, confirming an intermediate role of DCC. In additional experiments we found netrin-1 increased ERK1/2 and eNOS_s1177 phosphorylation, and DCC protein expression, which was diminished by I/R. Furthermore, netrin-1-induced DCC upregulation was NO and ERK1/2-dependent, implicating a feed-forward mechanism. DAF-AM staining revealed enhanced NO production in both cardiac endothelial cells (ECs) and myocytes. In primarily isolated cardiomyocytes, netrin-1 also increased NO production, DCC abundance and ERK1/2 phosphorylation. Of note, cardiac apoptosis was significantly attenuated by netrin-1, which was reversed by DCC-antibody, U0126, L-NAME or PTIO. In summary, our data clearly demonstrate that netrin-1 potently protects the heart from I/R injury by stimulating NO production from cardiac ECs and myocytes. This potent effect is mediated by a DCC/ERK1/2/eNOS_s1177/NO/DCC feed-forward mechanism in both cell types.     

Mechanistic insights into folic acid-dependent vascular protection: Dihydrofolate reductase (DHFR)-mediated reduction in oxidant stress in endothelial cells and angiotensin II-infused mice: A novel HPLC-based fluorescent assay for DHFR activity

Folate supplementation improves endothelial function in patients with hyperhomocysteinemia. Mechanistic insights into potential benefits of folate on vascular function in general population however, remain mysterious. Expression of dihydrofolate reductase (DHFR) was markedly increased by folic acid (FA, 50 μmol/L, 24 h) treatment in endothelial cells. Tetrahydrofolate (THF) is formed after incubation of purified DHFR or cellular extracts with 50 μmol/L of substrate dihydrofolic acid. THF could then be detected and quantified by high performance liquid chromatography (HPLC) with a fluorescent detector (295/365 nm). Using this novel and sensitive assay, we found that DHFR activity was significantly increased by FA. Furthermore, FA improved redox status of Ang II treated cells by increasing H₄B and NO bioavailability while decreasing superoxide (O₂⁻) production. It however failed to restore NO levels in DHFR siRNA-transfected or methotrexate pre-treated cells, implicating a specific and intermediate role of DHFR. In mice orally administrated with FA (15 mg/kg/day, 16 days), endothelial upregulation of DHFR expression and activity occurred in correspondence to improved NO and H₄B bioavailability, and this was highly effective in reducing Ang II infusion (0.7 mg/kg/day, 14 days)-stimulated aortic O₂⁻ production. 5′-methyltetrahydrofolate (5′-MTHF) levels, GTPCH1 expression and activity remained unchanged in response to FA or Ang II treatment in vitro and in vivo. FA supplementation improves endothelial NO bioavailability via upregulation of DHFR expression and activity, and protects endothelial cells from Ang II-provoked oxidant stress both in vitro and in vivo. These observations likely represent a novel mechanism (intermediate role of DHFR) whereby FA induces vascular protection.     

Effects of mild calorie restriction and high-intensity interval walking in middle-aged and older overweight Japanese

We investigated whether a combination of mild calorie restriction (MCR) and high-intensity interval walking (HIW) improved physical fitness more than HIW alone in middle-aged and older overweight Japanese (40–69 years old, BMI  23.6 kg/m²). Forty-seven women and 16 men were divided into MCR + HIW and HIW groups. All subjects performed HIW: 5 sets of 3-min low-intensity walking (40% peak aerobic capacity for walking, VO_2peak) and 3-min high-intensity walking (70% VO_2peak) per day, 4 days per week, for 16 weeks while energy expenditure was monitored with a tri-axial accelerometer. The MCR + HIW group consumed meal replacement formula (240 kcal): a mixture of low-carbohydrates and -fat and high-protein, for either lunch or dinner everyday and therefore, had 87% of the energy intake of the HIW group during the intervention period. Although the HIW group showed improvements in BMI, blood pressure, and several blood chemicals, the MCR + HIW group had greater improvement. Moreover, the medical expenditure for the 6 months including the intervention period was 59% lower in the MCR + HIW group than in the HIW group. Our strategy of a short-term combination of MCR and HIW may thus prevent lifestyle-associated diseases and improve health in middle-aged and older overweight Japanese.     

Kinetic evidence for modulation by glycophorin A of a conformational equilibrium between two states of band 3 (SLC4A1) bound reversibly by the competitive inhibitor DIDS

Recent evidence has suggested that erythrocytes naturally deficient in glycophorin A (GPA) have a reduced V_max for monovalent anion exchange. Unanswered is whether miss-folding of band 3 during biosynthesis, or the absence of GPA modulation of properly folded band 3 is responsible. Here, I determine the effect of selective depletion of GPA on the kinetics of reversible binding of the competitive transport inhibitor DIDS (4,4′-diisothiocyanato-2,2′-stilbenedisulfonate) to properly folded band 3. Reversible binding of DIDS follows biphasic kinetics: a fast phase {DIDS + band 3  (DIDS − band 3), k₁, k_− 1} and a slower phase {(DIDS − band 3)  (DIDS − band 3), k₂, k_− 2}. Selective depletion of GPA was accomplished by pretreating membranes with Triton X-100, over a range where erythrocyte hemolysis is inhibited by the detergent (0% to 0.03%, v/v). Pretreatment with sublytic Triton X-100: (a) virtually completely depleted GPA, (b) did not deplete membrane-bound band 3, and (c) slowed the overall rate of reversible binding of DIDS to band 3. Data analysis and model simulation studies indicated that the decrease in the rate of binding of DIDS was due exclusively to a decrease in k_− 2, with no change in the initial rate of binding. Thus, depletion of GPA does not alter the native conformation of band 3 at the DIDS binding site, but rather modulates a conformational equilibrium between two states of the binary complex formed by the competitive inhibitor DIDS, reversibly bound to properly folded band 3.     

Direct effect of cocaine on epigenetic regulation of PKC gene repression in the fetal rat heart

Maternal cocaine administration during gestation caused a down-regulation of PKC expression in the heart of adult offspring resulting in an increased sensitivity to ischemia and reperfusion injury. The present study investigated the direct effect of cocaine in epigenetic modification of PKC gene repression in the fetal heart. Hearts were isolated from gestational day 17 fetal rats and treated with cocaine in an ex vivo organ culture system. Cocaine treatment for 48 h resulted in significant decreases in PKC protein and mRNA abundance and increases in CpG methylation at two SP1 binding sites in the PKC promoter region (− 346 and − 268). Electrophoretic mobility shift assays demonstrated that CpG methylation of both SP1 sites inhibited SP1 binding. Consistently, chromatin immunoprecipitation assays showed that cocaine treatment significantly decreased binding of SP1 to the SP1 sites in the intact fetal heart. Reporter gene assays revealed that site-directed mutations of CpG methylation at both SP1 sites significantly reduced the PKC promoter activity while methylation of a single site at either − 346 or − 268 did not have a significant effect. The causal effect of increased methylation in the cocaine-induced down-regulation of PKC was demonstrated with the use of DNA methylation inhibitors. The presence of either 5-aza-2′-deoxycytodine or procainamide blocked the cocaine-induced increase in SP1 sites methylation and decrease in PKC mRNA. The results demonstrate a direct effect of cocaine in epigenetic modification of DNA methylation and programming of cardiac PKC gene repression linking prenatal cocaine exposure and pathophysiological consequences in the heart of adult offspring.     

Prevalence of iodine deficiency in Europe in 2010

The adverse effects of iodine deficiency (ID)  intellectual impairment, damaged reproduction, goiter and hypo- and hyperthyroidism  are well known and easily corrected with salt iodization, but they continue to impair health and socioeconomic development, with more than two billion people at risk worldwide. During the major global expansion of salt iodization over the past four decades, much of Europe has remained iodine deficient. Although every European country endorsed the goal of eliminating iodine deficiency at the 1992 World Health Assembly, control of iodine deficiency has received low priority in much of Europe. However, there has been recent progress in the region and the number of children with low iodine intakes has decreased by ca. 30% since 2003. This paper presents an estimate of the prevalence of iodine deficiency in Europe in 2010, based on a systematic review to update the WHO Vitamin and Mineral Nutrition Information System (VMNIS) database.     

Attenuated expression of SECIS binding protein 2 causes loss of telomeric reserve without affecting telomerase

The family of selenoproteins have a broad range of functions, including protection against oxidative damage. Previous studies have shown that elevated levels of oxidative damage can induce accelerated loss of telomeric DNA during proliferation of mammalian cells. The incorporation of selenocysteine (Sec) into proteins in mammalian cells requires the Sec insertion sequence (SECIS) binding protein 2 (SBP2). Thus in the present study we have assessed the effect of knocking down the expression of SBP2 on telomere length. Following knock-down of SBP2 expression in two different human cell lines, the MSTO mesothelioma cell line (5 Kb average telomere length) and SY5Y neuroblastoma cell line (4.2 Kb average telomere length), we observed a significant reduction (−0.6 to −1.1 Kb; P  0.01) in telomere length as compared to control cells. This reduction in telomere length was independent of affects on telomerase, since both telomerase activity levels and Tert mRNA expression levels were not altered by knock-down of SBP2 expression. Furthermore, telomeres were particularly sensitive to S1 nuclease digestion following SBP2 knock-down, indicating an increased frequency of oxidative damage-induced lesions in the telomeric DNA in these cells. Together, these observations imply that selenoproteins may help protect telomeric reserve in mammalian cells.     

Role of reactive oxygen species (ROS) in angiotensin II-induced stimulation of the cardiac Na/HCO3 cotransport

The sarcolemmal Na⁺/HCO₃⁻ cotransporter (NBC) plays an important role in intracellular pH (pH_i) regulation in the heart. In the present work we studied, in isolated cat ventricular myocytes, the role of Angiotensin II (Ang II) and reactive oxygen species (ROS) production as potential activators of the NBC. pH_i was measured in single cells in a medium with HCO₃⁻ using the fluorescent pH indicator BCECF. The NH₄⁺ pulse method was used to induce an intracellular acid load and the acid efflux (J_H) in the presence of the Na⁺/H⁺ exchanger blocker HOE642 (10 μM) was calculated as indicator of NBC activity. The following J_H data are presented at pH_i of 6.8 ( and ^# indicate p < 0.05 after ANOVA vs. control and Ang II, respectively). The basal J_H (1.03 ± 0.12 mM/min, n = 11) was significantly increased in the presence of 100 nM Ang II (1.70 ± 0.15 mM/min, n = 8). This effect of Ang II was abolished when we added to the extracellular solution 2 mM MPG (ROS scavenger; 0.80 ± 0.08 mM/min, n = 11^#), 300 μM apocynin (NADPH oxidase blocker; 0.80 ± 0.13 mM/min, n = 6^#), 500 μM 5-hydroxidecanoate (mitochondrial ATP dependent K⁺ channel, mK_ATP, blocker; 0.97 ± 0.21 mM/min, n = 9^#), or the inhibitor of the MAP kinase ERK pathway U0126 (10 μM; 0.56 ± 0.18 mM/min, n = 6^#). We also determined the phosphorylation of ERK during the first min of acidosis and we detected that Ang II significantly enhanced the ERK phosphorylation levels, an effect that was cancelled by scavenging ROS with MPG. In conclusion, we propose that Ang II enhances the production of ROS through the activation of the NADPH oxidase, which in turn triggers mK_ATP opening and mitochondrial ROS production (“ROS-induced ROS-release mechanism”). Finally, these mitochondrial ROS stimulate the ERK pathway, leading to the activation of the NBC.     

Regular moderate exercise reduces advanced glycation and ameliorates early diabetic nephropathy in obese Zucker rats

Advanced glycation end products (AGEs) play a key role in the pathogenesis of diabetes and its complications, including the diabetic nephropathy. The renoprotective effects of exercise are well known; however, the mechanisms remain elusive. Here we examined whether a regular moderate exercise in obese Zucker rats (OZR), a model of diabetes- and obesity-associated nephropathy, will affect the development of early renal injury in OZR possibly via alteration of AGEs formation. The OZR were left without exercise (sedentary) or subjected to 10 weeks intermittent treadmill running of moderate intensity. Compared with sedentary OZR, kidneys of running OZR had significantly less glomerular mesangial expansion and tubulointerstitial fibrosis. Running OZR had significantly lower plasma AGEs-associated fluorescence and N-carboxymethyllysine. Correspondingly, renal AGEs and N-carboxymethyllysine content were lower in running OZR. Systemically, exercise increased aerobic metabolism, as apparent from urinary metabolite profiling. No differences in plasma glucose, insulin, or lipid profile were found between the 2 groups. Apart from lower advanced oxidation protein products (a marker of myeloperoxidase activity), no other marker of inflammation was altered by exercise, either systemically or locally in kidneys. No indication of changed oxidative status was revealed between the groups. Exercise in OZR decreased advanced glycation. This might represent the early event of exercise-induced renoprotection in diabetic nephropathy in OZR. If confirmed in clinical studies, regular moderate exercise could represent an easy and effective nonpharmacologic approach to reduce advanced glycation.     

Atrial fibrillation with left bundle branch block and intermittent right axis deviation during acute myocardial infarction

A survey of the litterature was made to precise the characteristics of infectious endocarditis (IE) in the elderly as defined by the diagnostic criteria of Von Reyn. The IE presents at times with a fallacious symptomatology where fever and cardiac bruit are often lacking. With regard to the cardiac disease, there is an increase in the incidence of calcifying degenerative valvulopathy responsible for IE. On the bacteriological point of view, streptococcal infections are still predominent in etiologgy for endocarditis but opportunistic infections dues to other bacterial species are increasing in frequency. Lastly, on the therapeutical side, there is a new trend to reduce the proeminence of curative treatment with aminoglycoside agents in favor of the antibiotic prophylaxis.

Changes of intra-mitochondrial Ca in adult ventricular cardiomyocytes examined using a novel fluorescent Ca indicator targeted to mitochondria

In this study a Ca²⁺ sensitive protein was targeted to the mitochondria of adult rabbit ventricular cardiomyocytes using an adenovirus transfection technique. The probe (Mitycam) was a Ca²⁺-sensitive inverse pericam fused to subunit VIII of human cytochrome c oxidase. Mitycam expression pattern and Ca²⁺ sensitivity was characterized in HeLa cells and isolated adult rabbit cardiomyocytes. Cardiomyocytes expressing Mitycam were voltage-clamped and depolarized at regular intervals to elicit a Ca²⁺ transient. Cytoplasmic (Fura-2) and mitochondrial Ca²⁺ (Mitycam) fluorescence were measured simultaneously under a range of cellular Ca²⁺ loads. After 48 h post-adenoviral transfection, Mitycam expression showed a characteristic localization pattern in HeLa cells and cardiomyocytes. The Ca²⁺ sensitive component of Mitycam fluorescence was 12% of total fluorescence in HeLa cells with a K_d of  220 nM. In cardiomyocytes, basal and beat-to-beat changes in Mitycam fluorescence were detected on initiation of a train of depolarizations. Time to peak of the mitochondrial Ca²⁺ transient was slower, but the rate of decay was faster than the cytoplasmic signal. During spontaneous Ca²⁺ release the relative amplitude and the time course of the mitochondrial and cytoplasmic signals were comparable. Inhibition of mitochondrial respiration decreased the mitochondrial transient amplitude by  65% and increased the time to 50% decay, whilst cytosolic Ca²⁺ transients were unchanged. The mitochondrial Ca²⁺ uniporter (mCU) inhibitor Ru360 prevented both the basal and transient components of the rise in mitochondrial Ca²⁺. The mitochondrial-targeted Ca²⁺ probe indicates sustained and transient phases of mitochondrial Ca²⁺ signal, which are dependent on cytoplasmic Ca²⁺ levels and require a functional mCU.     

Cardioprotective signaling to mitochondria

Mitochondria are central players in the pathophysiology of ischemia–reperfusion. Activation of plasma membrane G-coupled receptors or the Na,K-ATPase triggers cytosolic signaling pathways that result in cardioprotection. Our working hypothesis is that the occupied receptors migrate to caveolae, where signaling enzymes are scaffolded into signalosomes that bud off the plasma membrane and migrate to mitochondria. The signalosome–mitochondria interaction then initiates intramitochondrial signaling by opening the mitochondrial ATP-sensitive K⁺ channel (mitoK_ATP). MitoK_ATP opening causes an increase in ROS production, which activates mitochondrial protein kinase C epsilon (PKC), which inhibits the mitochondrial permeability transition (MPT), thus decreasing cell death. We review the experimental findings that bear on these hypotheses and other modes of protection involving mitochondria.