Evaluation of Oxidative Stress,Apoptosis, and Expression of MicroRNA-208a and MicroRNA-1 in Cardiovascular Patients

Background:MicroRNA expression signature and reactive oxygen species (ROS) production have been associated with the development of cardiovascular diseases (CVDs). This study aimed to evaluate oxidative stress, inflammation, apoptosis, and the expression of miRNA-208a and miRNA-1 in cardiovascular patients.Methods:The study population included four types of patients (acute coronary syndromes (ACS), myocardial infarction (MI), arrhythmia, and heart failure (HF)), with 10 people in each group, as well as a control group. Quantitative real-time PCR was performed to measure mir-208 and miR-1 expression, the mRNAs of inflammatory mediators (TNFα, iNOS/eNOS), and apoptotic factors (Bax and Bcl2). XOX, MDA, and antioxidant enzymes (CAT, SOD, and GPx) were measured by ZellBio GmbH kits by an ELISA Reader.Results:The results showed significant decreases in the activity of antioxidant enzymes (CAT, SOD, and Gpx) and a significant increase in the activity of the MDA and XOX in cardiovascular patients. Significant increases in IL-10, iNos, iNOS / eNOS, and TNF-α in cardiovascular patients were also observed. Also, a significant increase in the expression of miR-208 (HF> arrhythmia> ACS> MI) and a significant decrease in the expression of miR-1 (ACS> arrhythmia> HF> MI) were found in all four groups in cardiovascular patients.Conclusion:The results showed increases in oxidative stress, inflammation, apoptotic factors, and in the expression of miR-208a in a variety of cardiovascular patients (ACS, MI, arrhythmia, and HF). It is suggested that future studies determine the relationships that miR-1, miR-208, and oxidative stress indices have with inflammation and apoptosis.Key Words: Apoptosis, Cardiovascular diseases, Inflammation, microRNA-208a, microRNA-1, Oxidative stress     

Junctional Adhesion Molecule A Interacts with Afadin and PDZ-GEF2 to Activate Rap1A, Regulate β1 Integrin Levels, and Enhance Cell Migration

Junctional adhesion molecule-A (JAM-A) is a transmembrane tight junction protein that has been shown to regulate barrier function and cell migration through incompletely understood mechanisms. We have previously demonstrated that JAM-A regulates cell migration by dimerization of the membrane-distal immunoglobulin-like loop and a C-terminal postsynaptic density 95/disc-large/zona occludens (PDZ) binding motif. Disruption of dimerization resulted in decreased epithelial cell migration secondary to diminished levels of β1 integrin and active Rap1. Here, we report that JAM-A is physically and functionally associated with the PDZ domain-containing molecules Afadin and PDZ-guanine nucleotide exchange factor (GEF) 2, but not zonula occludens (ZO)-1, in epithelial cells, and these interactions mediate outside-in signaling events. Both Afadin and PDZ-GEF2 colocalized and coimmunoprecipitated with JAM-A. Furthermore, association of PDZ-GEF2 with Afadin was dependent on the expression of JAM-A. Loss of JAM-A, Afadin, or PDZ-GEF2, but not ZO-1 or PDZ-GEF1, similarly decreased cellular levels of activated Rap1, β1 integrin protein, and epithelial cell migration. The functional effects observed were secondary to decreased levels of Rap1A because knockdown of Rap1A, but not Rap1B, resulted in decreased β1 integrin levels and reduced cell migration. These findings suggest that JAM-A dimerization facilitates formation of a complex with Afadin and PDZ-GEF2 that activates Rap1A, which regulates β1 integrin levels and cell migration.     

Protein kinase C activation disrupts epithelial apical junctions via ROCK-II dependent stimulation of actomyosin contractility

Background  

Disruption of epithelial cell-cell adhesions represents an early and important stage in tumor metastasis. This process can be modeled in vitro by exposing cells to chemical tumor promoters, phorbol esters and octylindolactam-V (OI-V), known to activate protein kinase C (PKC). However, molecular events mediating PKC-dependent disruption of epithelial cell-cell contact remain poorly understood. In the present study we investigate mechanisms by which PKC activation induces disassembly of tight junctions (TJs) and adherens junctions (AJs) in a model pancreatic epithelium.     

Oxidative inactivation of purified plasma membrane Ca2+-ATPase by hydrogen peroxide and protection by calmodulin

Calmodulin (CaM)-regulated plasma membrane Ca(2+)-ATPase (PMCA) is critical for the regulation of free intracellular Ca(2+) levels. PMCA activity and levels in neuronal membranes are decreased with aging, possibly due to oxidation-induced inactivation. In the present studies, inhibition of PMCA by H(2)O(2) was characterized in enzyme purified from human erythrocyte membranes. Basal and CaM-stimulated PMCA activities were inhibited by exposure to H(2)O(2) (25-100 microM). However, neither the concentration-dependent enhancement of PMCA activity by CaM nor the binding of CaM to H(2)O(2)-exposed PMCA was disrupted by treatment with H(2)O(2). Rates of inactivation by H(2)O(2) of basal and CaM-stimulated PMCA were nearly identical. The addition of CaM after exposure to H(2)O(2) did not protect enzyme activity, although the binding of CaM to PMCA before exposure to H(2)O(2) protected the enzyme completely, indicating a CaM-induced conformational state resistant to oxidation. H(2)O(2) quenched Trp fluorescence in PMCA, an index of conformational changes, with a rate similar to that observed for enzyme inactivation. H(2)O(2) enhanced the solvent accessibility of Trp residues in PMCA, whereas accessibility of the only Trp residue in the CaM-binding domain peptide was unaltered. Exposure of PMCA to H(2)O(2) led to aggregate formation partially reversible by dithiothreitol (DTT) but not to recovery of activity. Amino acid analysis indicated Cys modification following H(2)O(2) exposure but no Cys oxyacids. Because DTT did not reverse inactivation by H(2)O(2), it appears that the disulfide bond formation led to conformational changes that were not fully reversed when the bonds were reduced. Preincubation of PMCA with CaM protected the enzyme from undergoing this conformational change.     

Vitamin D in healthy Tunisian population: Preliminary results

BackgroundVitamin D deficiency is one of the most common medical conditions worldwide. In Tunisia, several studies evaluated Vitamin D status, but this was concerning specific populations (pregnant women, obese or diabetic patients and children with asthma). The only study that evaluated Vitamin D status in a healthy Tunisian population was conducted by Meddeb and associeties in 2002. The update of data available, based on the currently recommended limits, is necessary. This study aimed to estimate the prevalence of hypovitaminosis D in a healthy Tunisian population, and correlate the values with potential risk factors.MethodsIt was conducted on 209 Tunisian healthy subjects. Data collected included clinical characteristics and dietary intakes. We measured 25-hydroxyvitamin D (25(OH)D), parathyroid hormone (PTH), glycemia, creatinine, calcium, phosphorus, and alkaline phosphatase concentrations. Hypovitaminosis D was retained for 25(OH)D concentrations <75 nmol/L. Vitamin D deficiency was defined by 25(OH)D concentrations <25 nmol/L.ResultsThe prevalence of hypovitaminosis D and vitamin D deficiency were respectively 92.3% and 47.6%. The main factors that were significantly associated with low vitamin D levels in our multivariate analysis were veiling, living in rural areas and sunscreen use. However, sex, age, socioeconomic level, phototype, solar exposure score, smoking and bone mass index, were not statistically associated with hypovitaminosis D. The study of relationship between vitamin D status and serum PTH levels showed a significative and negative correlation (P < 0.005).ConclusionsGiven the high prevalence of vitamin D, an adapted health policy is essential. A widespread vitamin D supplementation and food fortification seems to be necessary in Tunisia.     

Assembly and stability of Salmonella enterica ser. Typhi TolC protein in POPE and DMPE

In this work we assessed the suitability of two different lipid membranes for the simulation of a TolC protein from Salmonella enterica serovar Typhi. The TolC protein family is found in many pathogenic Gram-negative bacteria including Vibrio cholera and Pseudomonas aeruginosa and acts as an outer membrane channel for expulsion of drug and toxin from the cell. In S. typhi, the causative agent for typhoid fever, the TolC outer membrane protein is an antigen for the pathogen. The lipid environment is an important modulator of membrane protein structure and function. We evaluated the conformation of the TolC protein in the presence of DMPE and POPE bilayers using molecular dynamics simulation. The S. typhi TolC protein exhibited similar conformational dynamics to TolC and its homologues. Conformational flexibility of the protein is seen in the C-terminal, extracellular loops, and α-helical region. Despite differences in the two lipids, significant similarities in the motion of the protein in POPE and DMPE were observed, including the rotational motion of the C-terminal residues and the partially open extracellular loops. However, analysis of the trajectories demonstrated effects of hydrophobic matching of the TolC protein in the membrane, particularly in the lengthening of the lipids and subtle movements of the protein’s β-barrel towards the lower leaflet in DMPE. The study exhibited the use of molecular dynamics simulation in revealing the differential effect of membrane proteins and lipids on each other. In this study, POPE is potentially a more suitable model for future simulation of the S. typhi TolC protein.     

Convergent and construct validity and test–retest reliability of the Caen Chronotype Questionnaire in six languages

ABSTRACT

Chronotype questionnaires provide a simple and time-effective approach to assessing individual differences in circadian variations. Chronotype questionnaires traditionally focused on one dimension of chronotype, namely its orientation along a continuum of morningness and eveningness. The Caen Chronotype Questionnaire (CCQ) was developed to assess an additional dimension of chronotype that captures the extent to which individual functioning varies during the day (amplitude). The aim of this study was to provide a multilanguage validation of the CCQ in six world regions (Arabic, Dutch, German, Italian, Portuguese and Spanish). At Time 1, a total of 2788 participants agreed to take part in the study (Arabic, n = 731; Dutch, n = 538; German, n = 329; Italian, n = 473; Portuguese, n = 361; Spanish, n = 356). Participants completed an assessment of the CCQ together with the Morningness-Eveningness Questionnaire (MEQ; Horne & Ostberg 1976) as well as questions related to factors theoretically related to chronotype (age, shift work, physical activity, sleep parameters and coffee consumption). One month later, participants again completed the CCQ. Results showed that the two-factor structure (morningness-eveningness and amplitude) of the CCQ could be replicated in all six languages. However, measurement invariance could not be assumed regarding the factor loadings across languages, meaning that items loaded more on their factors in some translations than in others. Test–retest reliability of the CCQ ranged from unacceptable (German version) to excellent (Dutch, Portuguese). Convergent validity was established through small–medium effect size correlations between the morningness-eveningness dimension of the CCQ and the MEQ. Taken together, our findings generally support the use of the translated versions of the CCQ. Further validation work on the CCQ is required including convergent validation against physiological markers of sleep, health and well-being.     

An angiotensin I-converting enzyme insertion/deletion polymorphism is associated with Pakistani asthmatic cases and controls

Asthma is a chronic disease due to inflammation of the airways of lungs that is clinically characterized by variable symptoms including wheezing, coughing and shortness of breath. Angiotensin I-converting enzyme (ACE) plays a major role in fibrous tissue formation and is highly expressed in lungs. The main aim of this research work was to study the role of ACE insertion/deletion (I/D) polymorphism, rs4646994, in asthma in Pakistani patients. A total of 854 subjects, including 333 asthma patients and 521 ethnically matched controls, were studied. The ACE (I/D) polymorphism was genotyped using polymerase chain reaction (PCR). Chi-square, Fisher’s exact and Hardy–Weinberg equilibrium tests were used to compare groups. Homozygous insertion genotype II (p<0.0001, OR=3.38) and insertion allele (I) was significantly more frequent in Pakistani asthmatics than in healthy controls (p=0.0007, OR=1.40). The ID genotype (p<0.0001, OR=0.43) and the deletion allele (D) were associated with protection of disease in Pakistani patients (p=0.0007, OR=0.71). These data suggest the involvement of ACE I/D polymorphism in asthma risk in the Pakistani population. This marker may be an important indication in the molecular mechanism of asthma and can become a useful tool in risk assessment and help in designing strategy to combat disease.     

Melanocyte differentiation antigen RAB38/NY-MEL-1 induces frequent antibody responses exclusively in melanoma patients

Expression pattern and immunogenicity are critical issues that define tumor antigens as diagnostic markers and potential targets for immunotherapy. The development of SEREX (serological analysis of recombinant expression libraries) has provided substantial progress in the identification of tumor antigens eliciting both cellular and humoral immune responses in cancer patients. By SEREX, we have previously identified RAB38/NY-MEL-1 as a melanocyte differentiation antigen that is highly expressed in normal melanocytes and melanoma tissues but not in other normal tissues or cancer types. In this study, we further demonstrate that RAB38/NY-MEL-1 is strongly immunogenic, leading to spontaneous antibody responses in a significant proportion of melanoma patients. The immune response occurs solely in malignant melanoma patients and was not detected in patients with other diseases, such as vitiligo, affecting melanocytes. Fine analysis of the spontaneous anti-RAB38/NY-MEL-1 antibody response reveals a polyclonal B cell recognition targeting various epitopes, although a dominant immunogenic region was preferentially recognized. Interestingly, our data indicate that this recognition is not rigid in the course of a patient’s response, as the dominant epitope changes during the disease evolution. Implications for the understanding of spontaneous humoral immune responses are discussed.Alfred Zippelius and Asma Gati contributed equally to this work.     

<Emphasis Type="Italic">Paracoccidioides brasiliensis</Emphasis> Infection Mimicking Recurrent Hodgkin Lymphoma: A Case Report and Review of the Literature

Paracoccidioides infection is a rare entity in the USA. This dimorphic fungus is found in Central and South America and is thought to be acquired by inhalation through the soil. We report a case of Paracoccidioides brasiliensis infection presenting as a clavicular bone lesion, peripancreatic mass, and various skin lesions. A 35-year-old man with a history significant for Hodgkin lymphoma presented with a left clavicular mass that was suspected clinically and radiologically as recurrent Hodgkin lymphoma. He was not experiencing any associated symptoms and was undergoing chemotherapy treatment for his known Hodgkin disease. On CT imaging, the mass was seen as a lytic bone lesion with an overlying soft tissue mass. This was biopsied and histologically diagnosed as a Paracoccidioides brasiliensis infection with associated necrotizing granulomatous inflammation. Also found on the CT scan was an enlarging peripancreatic mass which on endoscopic biopsy had similar histologic findings. In conclusion, this report presents a rare case of Paracoccidioides brasiliensis infection mimicking recurrent Hodgkin lymphoma.