Leptin and its receptor are overexpressed in breast cancer tissue of postmenopausal Mexican-Mestizo women with obesity.

The aim of this study was to investigate the expression of leptin (LEP) and its receptor (LEPR) in breast cancer tissue of postmenopausal women with different body mass indexes (BMI), as well as the relationship of this expression with the rate of recurrence free survival (RFS). Leptin and LEPR expression, determined by immunohistochemistry, were studied in breast cancer tissues of 154 patients. Qualitative and semi-quantitative analysis of protein expression was performed by the H-Score method, through the ImageJ's IHC Profiler software. Kaplan-Meier survival analysis and log-rank statistic were used to estimate RFS differences. Protein expression of LEP, was significantly higher in women with overweight or with obesity, when compared to women with normal BMI (P = 0.032 and P = 0.013, respectively). We also observed a significantly higher expression of LEPR in breast tumor cells of women with obesity (58.8%), when compared to women with normal BMI (32.7%) (P = 0.007). Five-year survival rate, regarding LEPR expression, was 82.4% when positive and 94% when negative (P = 0.024). In the Cox proportional-hazards regression model, LEPR expression represented a risk factor for disease recurrence after adjustment for confounding factors (HR = 4.67; 95% CI: 1.13-19.31; P = 0.033). In conclusion, postmenopausal women with obesity and breast cancer present higher LEP and LEPR expression in breast tumors, when compared to women with normal BMI. Independently from BMI, women with tumors LEPR positive have worst RFS, when compared to women with tumors LEPR negative.

Association of polymorphisms rs2303729, rs10880, and rs1131620 of LTBP4 with sarcopenia in elderly patients with type 2 diabetes mellitus.

BACKGROUND: Latent TGFß binding protein 4 (LTBP4) modifies skeletal muscle function, and polymorphisms in this gene have been associated with a longer ambulation time in patients with Duchenne muscular dystrophy. However, no studies associate these polymorphisms with an acquired muscle condition. AIM: The study aims to determine whether three functional variants within the LTBP4 were associated with sarcopenia in patients with type 2 diabetes mellitus (T2DM). SUBJECTS AND METHODS: We performed an analysis with 144 elderly individuals with T2DM, including 101 without sarcopenia and 43 with sarcopenia. Polymorphism frequency was determined by real-time PCR allelic discrimination TaqMan assay. RESULTS: Under different genetic models, the univariant analysis did not show a significant association of any polymorphism with sarcopenia. But the multivariate model analysis showed that variant rs1131620 (OR 7.852, 95% CI 1.854-33.257, p = 0.005) was significantly associated with sarcopenia under a dominant model. Under the same analysis, the variants rs2303729 and rs10880 had a more discrete association (OR 3.537 95% CI 1.078-11.607, p = 0.037; OR 5.008, 95% CI 1.120-22.399, p = 0.035, respectively). CONCLUSIONS: Our study highlights the importance of studying LTBP4 polymorphisms associated with sarcopenia. These findings suggest that the rs1131620 polymorphism of the LTBP4 may be part of the observed sarcopenia process in patients with T2DM.

Bone mineral density and fractures in postmenopausal women of Maya-Mestizo ethnic origin with different body mass indexes.

BACKGROUND: Obesity protects against bone loss, but it increases the risk of fragility fractures. AIM: To determine if bone mineral density (BMD) and the prevalence of fractures are different in postmenopausal Maya-Mestizo women grouped according to their body mass index (BMI). SUBJECTS AND METHODS: We studied 600 postmenopausal Maya-Mestizo women. A structured questionnaire for risk factors was applied. Body mass index was determined. BMD was assessed at the lumbar spine and total hip by dual-energy X-ray absorptiometry. History of low trauma fracture was determined from medical records. ANOVA was used to compare mean BMD between women with different BMI. To compare the frequency of fractures according to BMI group, we used χ2 test. RESULTS: According to WHO classification of BMI, 16.3% of women had normal BMI, 35.3% were overweight, and 48.4% had obesity. We found that women with obesity had a higher BMD versus women with normal BMI or overweight in all the anatomical sites analysed. The prevalence of history of fractures was 18.2%. We did not find differences between the women of different BMI; the wrist was the most frequent skeletal site of the fracture. CONCLUSION: Obesity in postmenopausal Maya-Mestizo women is not a risk factor for developing fragility fractures.

Serum concentrations of apelin-17 isoform vary in accordance to blood pressure categories in individuals with obesity class 3.

Background: The aim of this study was to investigate if serum concentrations of apelin-36, apelin-17, apelin-13 or apelin-12 were different in obesity class 3 individuals with hypertension, when compared to those without hypertension (normal or high-normal).Subjects and Methods: Twenty six individuals with obesity class 3-related hypertension and thirty three individuals without hypertension, who were divided in individuals with normal (n = 23) or with high-normal (n = 10) blood pressure (BP) were analyzed. All individuals presented obesity class 3, without diabetes mellitus. Measurements of all apelin isoforms were performed using enzyme-linked immunosorbent assay kits. Analysis of differences between groups of Apelin isoform concentrations was performed by a One-way ANOVA, with a Tukey test post hoc.Results: The individuals of the hypertensive group presented a slightly lower serum concentration of all apelin isoforms, but these differences were not statistically significant. These results were more evident when the group of patients without hypertension were divided based in normal and high-normal BP, observing that apelin-17 isoform were higher in individuals with high-normal BP in comparison to subjects with normal BP (P = 0.018); concentrations were also higher when compared to subjects with hypertension (P = 0.004).Conclusions: To our knowledge, this is the first study regarding the differences of apelin-17 isoform concentrations in individuals pertaining to different categories of BP, who presented obesity class 3. The group of patients that presented hypertension showed a lower concentration of all isoforms. This observation could be due to the fact that these patients were taking antihypertensive medication.

Lack of Delta-Sarcoglycan (Sgcd) Results in Retinal Degeneration.

Age-related macular degeneration (AMD) is the leading cause of central vision loss and severe blindness among the elderly population. Recently, we reported on the association of the SGCD gene (encoding for δ-sarcoglycan) polymorphisms with AMD. However, the functional consequence of Sgcd alterations in retinal degeneration is not known. Herein, we characterized changes in the retina of the Sgcd knocked-out mouse (KO, Sgcd-/-). At baseline, we analyzed the retina structure of three-month-old wild-type (WT, Sgcd+/+) and Sgcd-/- mice by hematoxylin and eosin (H&E) staining, assessed the Sgcd-protein complex (α-, ß-, γ-, and ε-sarcoglycan, and sarcospan) by immunofluorescence (IF) and Western blot (WB), and performed electroretinography. Compared to the WT, Sgcd-/- mice are five times more likely to have retinal ruptures. Additionally, all the retinal layers are significantly thinner, more so in the inner plexiform layer (IPL). In addition, the number of nuclei in the KO versus the WT is ever so slightly increased. WT mice express Sgcd-protein partners in specific retinal layers, and as expected, KO mice have decreased or no protein expression, with a significant increase in the α subunit. At three months of age, there were no significant differences in the scotopic electroretinographic responses, regarding both a- and b-waves. According to our data, Sgcd-/- has a phenotype that is compatible with retinal degeneration.

Ursolic and Oleanolic Acids Induce Mitophagy in A549 Human Lung Cancer Cells.

Ursolic and oleanolic acids are natural isomeric triterpenes known for their anticancer activity. Here, we investigated the effect of triterpenes on the viability of A549 human lung cancer cells and the role of autophagy in their activity. The induction of autophagy, the mitochondrial changes and signaling pathway stimulated by triterpenes were systematically explored by confocal microscopy and western blotting. Ursolic and oleanolic acids induce autophagy in A549 cells. Ursolic acid activates AKT/mTOR pathways and oleanolic acid triggers a pathway independent on AKT. Both acids promote many mitochondrial changes, suggesting that mitochondria are targets of autophagy in a process known as mitophagy. The PINK1/Parkin axis is a pathway usually associated with mitophagy, however, the mitophagy induced by ursolic or oleanolic acid is just dependent on PINK1. Moreover, both acids induce an ROS production. The blockage of autophagy with wortmannin is responsible for a decrease of mitochondrial membrane potential (Δψ) and cell death. The wortmannin treatment causes an over-increase of p62 and Nrf2 proteins promote a detoxifying effect to rescue cells from the death conducted by ROS. In conclusion, the mitophagy and p62 protein play an important function as a survival mechanism in A549 cells and could be target to therapeutic control.

WHOLE SEQUENCING OF THE MITOCHONDRIAL GENOME OF BREAST CANCER TISSUE IN MEXICAN-MESTIZO POSTMENOPAUSAL WOMEN WITH DIFFERENT BODY MASS INDEX.

BACKGROUND: Mitochondrial and oxidative stress has been related to obesity and breast cancer being this cancer more frequent and more aggressive in postmenopausal women with obesity. OBJECTIVE: The objective of this study was to investigate whether Mexican-Mestizo postmenopausal women with breast cancer and obesity present different somatic mutations in the mitochondrial DNA (mtDNA) when compared to women with normal body mass index (BMI). SUBJECTS AND METHODS: We included six Mexican-Mestizo postmenopausal women bearing breast cancer and who underwent mastectomy or breast-conserving surgery. BMI was determined in each case. Patients' genomic DNA was isolated from blood leukocytes and tumor tissue samples. Whole mtDNA sequence was determined by MitoChip v2.0 mitochondrial resequencing array, and data were analyzed using the GeneChip Sequence Analysis Software. Tumor mtDNA sequence was compared with matched leukocyte mtDNA sequence. RESULTS: Three women had a normal BMI and three presented obesity. Overall, we found 64 genetic variants: 53.1% were somatic mutations and 46.9% were polymorphisms; 44.1% were in the non-coding region and 55.9% were in genes that encode for mitochondrial proteins. Among the somatic mutations, 67.7% were in patients with normal BMI and 32.3% in patients with obesity. CONCLUSIONS: We did not find a higher frequency of mitochondrial somatic mutations in postmenopausal women with breast cancer and obesity compared to those with normal BMI. However, results could be due to the small number of women studied.

Duplication of SOX9 associated with 46,XX ovotesticular disorder of sex development.

RESEARCH QUESTION: The purpose of the present study was to investigate whether ten unrelated SRY-negative individuals with this sex differentiation disorder presented a double dose of SOX9 as the cause of their disease. DESIGN: Ten unrelated SRY-negative 46,XX ovotesticular disorder of sexual development (DSD) subjects were molecularly studied. Multiplex-ligation dependent probe amplification (MLPA) and quantitative real-time PCR analysis (qRT-PCR) for SOX9 were performed. RESULTS: The MLPA analysis demonstrated that one patient presented a heterozygous duplication of the entire SOX9 coding region (above 1.3 value of peak ratio), as well as at least a ~ 483 kb upstream duplication. Moreover, no duplication of other SOX9 probes was observed corresponding to the region between -1007 and -1500 kb upstream. A qRT-PCR analysis showed a duplication of at least -581 kb upstream and ~1.63 kb of the coding region that encompasses exon 3. The limits of the duplication were mapped approximately from ~71539762 to 72122741 of Chr17. No molecular abnormalities were found in the remaining nine patients. CONCLUSION: This study is thought to be the first report regarding a duplication of SOX9 that is associated with the presence of 46,XX ovotesticular DSD, encompassing at least -581 kb upstream, and the almost entire coding region of the gene.

Reconstructing Native American population history.

The peopling of the Americas has been the subject of extensive genetic, archaeological and linguistic research; however, central questions remain unresolved. One contentious issue is whether the settlement occurred by means of a single migration or multiple streams of migration from Siberia. The pattern of dispersals within the Americas is also poorly understood. To address these questions at a higher resolution than was previously possible, we assembled data from 52 Native American and 17 Siberian groups genotyped at 364,470 single nucleotide polymorphisms. Here we show that Native Americans descend from at least three streams of Asian gene flow. Most descend entirely from a single ancestral population that we call 'First American'. However, speakers of Eskimo-Aleut languages from the Arctic inherit almost half their ancestry from a second stream of Asian gene flow, and the Na-Dene-speaking Chipewyan from Canada inherit roughly one-tenth of their ancestry from a third stream. We show that the initial peopling followed a southward expansion facilitated by the coast, with sequential population splits and little gene flow after divergence, especially in South America. A major exception is in Chibchan speakers on both sides of the Panama isthmus, who have ancestry from both North and South America.

Raet1e Polymorphisms Are Associated with Increased Risk of Developing Premature Coronary Artery Disease and with Some Cardiometabolic Parameters: The GEA Mexican Study.

In an animal model, new evidence has been reported supporting the role of raet1e as an atherosclerosis-associated gene. Our objective was to establish if raet1e polymorphisms are associated with the risk of developing premature coronary artery disease (CAD) or with the presence of cardiometabolic parameters. After an informatic analysis, five polymorphisms were chosen and determined in 1158 patients with premature CAD and 1104 controls using 5' exonuclease TaqMan genotyping assays. Standardized questionnaires were applied to all participants to obtain family medical history, demographic information, history of nutritional habits, physical activity, alcohol consumption, and pharmacological treatment. The functional effect of the rs7756850 polymorphism was analyzed by luciferase assays. Under different models, adjusted by age, gender, body mass index, current smoking, and type 2 diabetes mellitus, the rs6925151 (OR = 1.250, p heterozygote = 0.026; OR = 1.268, p codominant1 = 0.034), rs9371533 (OR = 1.255, p heterozygote = 0.024), rs7756850 (OR = 1.274, p heterozygote = 0.016; OR = 1.294, p codominant1 = 0.031), and rs9383921 (OR = 1.232, p heterozygote = 0.037) polymorphisms were associated with increased risk of premature CAD. When compared to the rs7756850 G allele, the C allele showed a decreased luciferase activity. In premature CAD patients, associations with low levels of adiponectin, with a high presence of hypertension, and with high levels of gamma-glutamyltransferase and total cholesterol were observed. In healthy controls, associations with a decrease in LDL pattern B, aspartate aminotransaminase, and hypo-α-lipoproteinemia were detected. An association of the raet1e polymorphisms with an increased risk of developing premature CAD and with cardiometabolic parameters has been shown for the first time. In addition, the functional effect of the rs7756850 polymorphism was defined.

Higher prepregnancy body mass index is a risk factor for developing preeclampsia in Maya-Mestizo women: a cohort study.

AIM: Preeclampsia and obesity are two closely related syndromes. The high maternal prepregnancy body mass index (BMI) is a risk factor for present preeclampsia, independently of the ethnic background of the studied population. The aim of this study was to analyse in a prospective cohort study the relation between prepregnancy BMI and development of preeclampsia in Maya-Mestizo women. DESIGN: This is a prospective cohort study of 642 pregnant women that were included in the first trimester of the pregnancy (gestational age ≤12 weeks at the first antenatal visit) and all of them were of Maya-Mestizo ethnic origin from the state of Yucatán, México. We assessed the potential risk factors for preeclampsia and documented the prepregnancy BMI (kg/m2) that was based on measured height and maternal self-report of prepregnancy weight at the initial visit. Besides, in the antenatal visit we documented if the pregnant women developed preeclampsia. RESULTS: Of the 642 pregnant Maya-Mestizo women, 49 developed preeclampsia, with an incidence of 7.6% (44.9% had severe and 55% mild). The prepregnancy BMI was higher in women with developed preeclampsia than in those with normal pregnancies. Women with overweight or obesity in comparison with normal weight presented a RR = 2.82 (95% CI: 1.32-6.03; P = 0.008) and RR= 4.22 (95% CI: 2.07-8.61; P = 0.001), respectively. CONCLUSIONS: Our findings expand the previous studies to show that the higher prepregnancy BMI is a strong, independent risk factor for preeclampsia.

Complete sequence of the ATP6 and ND3 mitochondrial genes in breast cancer tissue of postmenopausal women with different body mass indexes.

Due to the fact that mitochondrial defects and oxidative stress have been related with obesity and breast cancer is more aggressive in women with obesity, we investigated if postmenopausal Mexican-Mestizo women with breast cancer presented somatic mutations in the sequence of the ATP6 and/or ND3 genes. Twenty one postmenopausal Mexican-Mestizo women with breast cancer who underwent mastectomy or breast conserving surgery were studied. Height and weight were used to calculate body mass index. DNA from tumor tissue samples and blood leukocytes was amplified by polymerase chain reaction and sequenced the ATP6 and ND3 mitochondrial genes. Ages ranged from 46 to 82. According to World Health Organization criteria among the 21 women, 7 had a normal BMI, 7 were overweight and 7 had obesity. In regard to the molecular study, after sequencing the coding region of ATP6 and ND3 genes of the DNA obtained from both leukocytes and tumor tissue, we did not find somatic mutations. All of the changes that we found in both genes were polymorphisms: in ATP6, we identified in ten patients 3 non-synonymous nucleotide changes and in ND3 we observed that six patients presented polymorphisms, three of them were synonymous and two non-synonymous. To our knowledge, this constitutes the first report where the complete sequence of the ATP6 and ND3 genes has been analyzed in postmenopausal Mexican-Mestizo women with breast cancer and diverse BMI. Our results differ with those reported in Caucasian and Asian populations, possibly due to ethnic differences.

HDL-Mediated Lipid Influx to Endothelial Cells Contributes to Regulating Intercellular Adhesion Molecule (ICAM)-1 Expression and eNOS Phosphorylation.

Reverse cholesterol transport (RCT) is considered as the most important antiatherogenic role of high-density lipoproteins (HDL), but interventions based on RCT have failed to reduce the risk of coronary heart disease. In contrast to RCT, important evidence suggests that HDL deliver lipids to peripheral cells. Therefore, in this paper, we investigated whether HDL could improve endothelial function by delivering lipids to the cells. Internalization kinetics using cholesterol and apolipoprotein (apo) AI fluorescent double-labeled reconstituted HDL (rHDL), and human dermal microvascular endothelial cells-1 (HMEC-1) showed a fast cholesterol influx (10 min) and a slower HDL protein internalization as determined by confocal microscopy and flow cytometry. Sphingomyelin kinetics overlapped that of apo AI, indicating that only cholesterol became dissociated from rHDL during internalization. rHDL apo AI internalization was scavenger receptor class B type I (SR-BI)-dependent, whereas HDL cholesterol influx was independent of SR-BI and was not completely inhibited by the presence of low-density lipoproteins (LDL). HDL sphingomyelin was fundamental for intercellular adhesion molecule-1 (ICAM-1) downregulation in HMEC-1. However, vascular cell adhesion protein-1 (VCAM-1) was not inhibited by rHDL, suggesting that components such as apolipoproteins other than apo AI participate in HDL's regulation of this adhesion molecule. rHDL also induced endothelial nitric oxide synthase eNOS S1177 phosphorylation in HMEC-1 but only when the particle contained sphingomyelin. In conclusion, the internalization of HDL implies the dissociation of lipoprotein components and a SR-BI-independent fast delivery of cholesterol to endothelial cells. HDL internalization had functional implications that were mainly dependent on sphingomyelin. These results suggest a new role of HDL as lipid vectors to the cells, which could be congruent with the antiatherogenic properties of these lipoproteins.

Association of CYP1A1 and CYP1B1 polymorphisms with bone mineral density variations in postmenopausal Mexican-Mestizo women.

Herein, we investigated potential associations between polymorphisms of genes related to estrogen metabolism and bone mineral density (BMD) in postmenopausal women. This was a cross-sectional study, in which two hundred and ninety postmenopausal Mexican-Mestizo women were studied. The BMD of the lumbar spine (LS), total hip (TH), and femoral neck (FN) was measured. The distribution of the genetic polymorphisms, including rs1799814 and rs1048943 at CYP1A1 as well as rs1056836 at CYP1B1, were analyzed by polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP), single-stranded conformational polymorphism (SSCP), and DNA sequencing. Deviations from Hardy-Weinberg equilibrium (HWE) were tested, and linkage disequilibrium (LD) was calculated by direct correlation (r2). Moreover, haplotype analysis was performed. All polymorphisms were in HWE. The genotype and allele distributions of the three single nucleotide polymorphisms (SNPs) studied showed no significant differences. However, statistical significance was reached when constructing haplotypes. The CG haplotype in CYP1A1 was associated with variations in LS and FN BMD after adjustment for covariates (p = 0.021 and 0.045, respectively), but the association with TH BMD was not significant. These results suggested that the CG haplotype in CYP1A1 may play an important role in the mechanism of osteoporosis and may be useful as a genetic marker.

Analysis of the rs13306560 functional variant in the promoter region of the MTHFR gene in sporadic Parkinson´s disease.

OBJECTIVE: Among susceptibility genes for Sporadic Parkinson´s Disease (SPD), the MTHFR gene has been suggested as candidate. The A allele of the functional variant rs13306560 in its promoter region has been liked to decreased transactivation capacity. Therefore, we sought to determine a possible association of the rs13306560 and SPD. METHODS: In total, 237 individuals were genotyped, 113 patients with SPD diagnosed according to the Queen Square Brain Bank criteria and 124 neurologically healthy controls. Genotyping was performed using TaqMan probes for the rs13306560 and real-time PCR. RESULTS: The A allelle was associated to protection in SPD, under the dominant model, (OR=0.22, C.I.=[0.048-1.080], p=0.04), nevertheless, after logistic regression analysis with adjustment for gender, resulted only in a trend (Exp (ß)=0.211, [I.C. 95.0%, 0.042-1.057], p=0.058). CONCLUSION: Although further studies are needed, our data suggest an important role of the MTHFR gene variants in the fine-tuning regulation of one-carbon metabolism in the brain.

Dysferlin quantification in monocytes for rapid screening for dysferlinopathies.

INTRODUCTION: In this study, we determined normal levels of dysferlin expression in CD14+ monocytes by flow cytometry (FC) as a screening tool for dysferlinopathies. METHODS: Monocytes from 183 healthy individuals and 29 patients were immunolabeled, run on an FACScalibur flow cytometer, and analyzed by FlowJo software. RESULTS: The relative quantity of dysferlin was expressed as mean fluorescence intensity (MFI). Performance of this diagnostic test was assessed by calculating likelihood ratios at different MFI cut-off points, which allowed definition of 4 disease classification groups in a simplified algorithm. CONCLUSION: The MFI value may differentiate patients with dysferlinopathy from healthy individuals; it may be a useful marker for screening purposes. Muscle Nerve 54: 1064-1071, 2016.

Polymorphisms of APLN-APLNR system are associated with essential hypertension in Mexican-Mestizo individuals.

The aim of this study was to evaluate if polymorphisms of APLN and APLNR genes may play a role as susceptibility markers for hypertension in a group of Mexican-Mestizo patients. A case-control study was carried out including normotensive and hypertensive individuals. For these, two polymorphisms of APLN (rs3761581 and rs56204867) and two of APLNR () genes were genotyped by 5' exonuclease TaqMan assay in 400 normotensive individuals and 383 patients. The results showed that, under an additive model adjusted by BMI, HDL, triglycerides, glucose and family history of essential hypertension, the rs7119375 and rs10501367 polymorphisms of APLNR gene were associated significantly with a decreased risk of essential hypertension (P=0.039 and P=0.029, respectively). Besides, the haplotypes analysis of these polymorphisms showed that H1 haplotype was associated with an increased risk of essential hypertension (P=0.026), while the H2 haplotype was associated with a decreased risk (P=0.032). Contrary, the rs3761581 and rs56204867 polymorphisms of APLN gene were not associated with essential hypertension (P=0.1707 and P=0.0769, respectively). The data suggest that APLNR rs7119375 and rs10501367 are associated with a decreased risk of essential hypertension in our Mexican-Mestizo studied group, but further studies are warranted.

Serum Levels of MicroRNA-206 and Novel Mini-STR Assays for Carrier Detection in Duchenne Muscular Dystrophy.

Duchenne Muscular Dystrophy (DMD) is an X-linked neuromuscular disorder in which the detection of female carriers is of the utmost importance for genetic counseling. Haplotyping with polymorphic markers and quantitation of creatine kinase levels (CK) allow tracking of the at-risk haplotype and evidence muscle damage, respectively. Such approaches are useful for carrier detection in cases of unknown mutations. The lack of informative markers and the inaccuracy of CK affect carrier detection. Therefore, herein we designed novel mini-STR (Short Tandem Repeats) assays to amplify 10 loci within the DMD gene and estimated allele frequencies and the polymorphism information content among other parameters in 337 unrelated individuals from three Mexican populations. In addition, we tested the utility of the assays for carrier detection in three families. Moreover, given that serum levels of miR-206 discern between DMD patients and controls with a high area under the curve (AUC), the potential applicability for carrier detection was assessed. The serum levels of miR-206 of non-carriers (n = 24) and carriers (n = 23) were compared by relative quantitation using real-time PCR (p < 0.05), which resulted in an AUC = 0.80 in the Receiver Operating Characteristic curve analysis. In conclusion, miR-206 has potential as a "liquid biopsy" for carrier detection and genetic counseling in DMD.

Description of a utrophin associated protein complex in lipid raft domains of human artery smooth muscle cells.

The dystrophin-associated protein complex (DAPC) is a multimeric complex that links the extracellular matrix to the actin cytoskeleton, and in some cases dystrophin can be substituted by its autosomal homologue utrophin to form the utrophin-associated protein complex (UAPC). Both complexes maintain the stability of plasma membrane during contraction process and play an important role in transmembrane signaling. Mutations in members of the DAPC are associated with muscular dystrophy and dilated cardiomyopathy. In a previous study with human umbilical cord vessels, we observed that utrophin colocalize with caveolin-1 (Cav-1) which proposed the presence of UAPC in the plasma membrane of vascular smooth muscle (VSM). In the current study, we demonstrated by immunofluorescence analysis, co-immunoprecipitation assays, and subcellular fractionation by sucrose gradients, the existence of an UAPC in lipid raft domains of human umbilical artery smooth muscle cells (HUASMC). This complex is constituted by utrophin, ß-DG, ε-SG, α-smooth muscle actin, Cav-1, endothelial nitric oxide synthase (eNOS) and cavin-1. It was also observed the presence of dystrophin, utrophin Dp71, ß-SG, δ-SG, δ-SG3 and sarcospan in non-lipid raft fractions. Furthermore, the knockdown of α/ß-DG was associated with the decrease in both the synthesis of nitric oxide (NO) and the presence of the phosphorylated (active) form of eNOS; and with a reduction in the downstream activation of some cGMP signaling transduction pathway components. Together these results show the presence of an UAPC complex in HUASMC that may participate in the activity regulation of eNOS and in the vascular function.

Reduction of no-reflow and reperfusion injury with the synthetic 17ß-aminoestrogen compound Prolame is associated with PI3K/Akt/eNOS signaling cascade.

A high proportion of primary percutaneous coronary interventions performed in the setting of acute myocardial infarction, concur with inadequate myocardial perfusion at the microvascular level. This phenomenon, known as "no-reflow" contributes to reperfusion injury, poor prognosis and to unfavorable clinical outcome. In this study, we evaluated the hypothesis that the synthetic 17ß-aminoestrogen Prolame, may confer cardioprotection and prevent against no-reflow. In an open-chest model of 30-min ischemia and 90-min reperfusion, male Wistar rats were randomly assigned to different groups: Control, Prolame, Prolame followed by the nitric oxide synthase inhibitor (L-NAME), and 17ß-estradiol. Areas of risk, infarct size and no-reflow were determined by planimetry with triphenyltetrazolium chloride and thioflavin-S stains. Structural damage of the vasculature was measured as capillary compression in clarified tissue after intra-atrial injection of Microfil. Hemodynamic function was obtained at the end of stabilization, ischemia and reperfusion; nitric oxide (NO·) content was determined indirectly using the Griess reaction. Activation of the eNOS signaling cascade was determined by western blot. Prolame reduced the infarcted area, decreased the zones of no-reflow and capillary compression by activating the PI3K/Akt/eNOS signaling pathway in correlation with NO· increase. Prolame also activated endothelial cells augmenting NO· production, which was inhibited by ICI182780 (a selective estrogen receptor down-regulator), supporting the notion that the cardioprotective effect of Prolame involves the preservation of endothelium through the activation of estrogen receptor downstream signaling. Our results provide evidence that Prolame has potential therapeutic application in patients with AMI, as it prevents from both vascular and cardiac tissue damage.