Aly Raies

Ca2+-ATPases in non-failing and failing heart: evidence for a novel cardiac sarco/endoplasmic reticulum Ca2+-ATPase 2 isoform (SERCA2c).

We recently documented the expression of a novel human mRNA variant encoding a yet uncharacterized SERCA [SR (sarcoplasmic reticulum)/ER (endoplasmic reticulum) Ca2+-ATPase] protein, SERCA2c [Gélébart, Martin, Enouf and Papp (2003) Biochem. Biophys. Res. Commun. 303, 676-684]. In the present study, we have analysed the expression and functional characteristics of SERCA2c relative to SERCA2a and SERCA2b isoforms upon their stable heterologous expression in HEK-293 cells (human embryonic kidney 293 cells). All SERCA2 proteins induced an increased Ca2+ content in the ER of intact transfected cells. In microsomes prepared from transfected cells, SERCA2c showed a lower apparent affinity for cytosolic Ca2+ than SERCA2a and a catalytic turnover rate similar to SERCA2b. We further demonstrated the expression of the endogenous SERCA2c protein in protein lysates isolated from heart left ventricles using a newly generated SERCA2c-specific antibody. Relative to the known uniform distribution of SERCA2a and SERCA2b in cardiomyocytes of the left ventricle tissue, SERCA2c was only detected in a confined area of cardiomyocytes, in close proximity to the sarcolemma. This finding led us to explore the expression of the presently known cardiac Ca2+-ATPase isoforms in heart failure. Comparative expression of SERCAs and PMCAs (plasma-membrane Ca2+-ATPases) was performed in four nonfailing hearts and five failing hearts displaying mixed cardiomyopathy and idiopathic dilated cardiomyopathies. Relative to normal subjects, cardiomyopathic patients express more PMCAs than SERCA2 proteins. Interestingly, SERCA2c expression was significantly increased (166+/-26%) in one patient. Taken together, these results demonstrate the expression of the novel SERCA2c isoform in the heart and may point to a still unrecognized role of PMCAs in cardiomyopathies.

Chemical Composition and Antimicrobial and Antioxidant Activities of Essential Oils and Various Extracts of Juniperus phoenicea L. (Cupressacees)

GC-FID and GC-MS analysis of essential oils of Juniperus phoenicea resulted in the identification of 30 compounds, representing more than 98% of the total composition. alpha-pinene (55.7% and 80.7%), delta-3-carene (10.7% and 4.5%), and gamma-cadinene (2.9% and 5.1%) were the main components, respectively, in leaves and berries essential oil. Extracts of J. phoenicea were obtained by different extraction solvents: methanol, ethanol, ethyl acetate, and dichloromethane and evaluated composition for polyphenols (gallic acid equivalent 52 to 217 g/kg), tannins (catechin equivalent 6.5 to 60.2 g/kg), antocyanins (cyanidin equivalent 84 to 373 mg/kg), and flavonoids (quercetin equivalent 6.4 to 29.3 g/kg). The samples (essential oils and extracts) were subjected to a screening for their antioxidant activity by using DPPH and ABTS assays; antimicrobial activity was tested with 6 bacteria (3 Gram-positive and 3 Gram-negative), 1 yeast, and 2 fungi. The strongest antioxidant activity was obtained by the methanolic extract (IC(50)= 6.5 +/- 0.3 mg/L). Flavonoids are likely to contribute to the antifungal activity against Saccharomyces cerevisiae. Correlations were studied between chemical composition and antioxidant and antimicrobial activities.

Compartmentalized expression of three novel sarco/endoplasmic reticulum Ca2+ATPase 3 isoforms including the switch to ER stress, SERCA3f, in non-failing and failing human heart

The human sarco/endoplasmic reticulum (ER) Ca(2+)ATPase 3 (SERCA3) gene gives rise to SERCA3a-3f isoforms, the latter inducing ER stress in vitro. Here, we first demonstrated the co-expression of SERCA3a, -3d and -3f proteins in the heart. Evidence for endogenous proteins was obtained by using isoform-specific antibodies including a new SERCA3d-specific antibody, and either Western blotting of protein lysates or immunoprecipitation of membrane proteins. An immunolocalization study of both left ventricle tissue and isolated cardiomyocytes showed a distinct compartmentalization of the SERCA3 isoforms, as a uniform distribution of SERCA3a was detected while -3d and -3f isoforms were observed around the nucleus and in close vicinity of plasma membrane, respectively. Second, we studied their expressions in failing hearts including mixed (MCM) (n=1) and idiopathic dilated (IDCM) cardiomyopathies (n=4). Compared with controls (n=5), similar expressions of SERCA3a and -3d mRNAs were observed in all patients. In contrast, SERCA3f mRNA was found to be up-regulated in failing hearts (125+/-7%). Remarkably, overexpression of SERCA3f paralleled an increase in ER stress markers including processing of X-box-binding protein-1 (XBP-1) mRNA (176+/-24%), and expression of XBP-1 protein and glucose-regulated protein (GRP)78 (232+/-21%). These findings revisit the human hearts Ca(2+)ATPase system and indicate that SERCA3f may account for the mechanism of ER stress in vivo in heart failure.

Geography, Plants, and Growing Systems Shape the Genetic Structure of Tunisian Botrytis cinerea Populations

Botrytis cinerea, considered for a long time as a generalist fungal pathogen of a multitude of plants, was recently shown to exhibit significant population structure in France according to the host, suggesting sympatric specialization. Recent models also showed that adaptation to new hosts may facilitate the process of sympatric speciation in fungal plant pathogens. The present work aimed at investigating if host plants, combined with geographic origin and growing systems, shape the diversity and structure of Tunisian populations of B. cinerea. We genotyped 153 isolates with 9 microsatellites. In all the investigated populations, the fungus reproduced mainly sexually. Gene flow was significantly reduced between greenhouses and open fields from strawberry but not from grapevine. Populations from tomatoes, sampled under greenhouses only, exhibited a low genotypic diversity. The effects of plant and geography from open fields were investigated on a sample of 74 isolates. Six populations were inferred, mainly structured according to a geographic barrier corresponding to the Grande Dorsale Mountain. However, this effect could not be separated from the host plant origin of isolates. The analysis of 63 isolates recovered from strawberries and faba beans in the Cap Bon and Centre regions did not reveal any significant effect of plant on pathogen population differentiation.

The influence of organ, season and drying method on chemical composition and antioxidant and antimicrobial activities of Juniperus phoenicea L. essential oils

BACKGROUND Juniperus phoenicea is an important medicinal plant. In the present study, essential oils (18 samples) from leaves and berries of Juniperus phoenicea L. (Cupressaceae), obtained by various drying methods and in different collection months, were analysed by gas chromatography-mass spectrometry and also evaluated for in vitro antimicrobial and antioxidant activities. Correlations were studied between antimicrobial activity and the chemical composition of essential oils. RESULTS Sixty-seven compounds were identified in essential oils, representing 97.7-100%. Essential oils were dominated by monoterpenes and sesquiterpenes, which presented 35.0-93.3% and 6.7-62.0%, respectively, depending of organ, season and drying method. Antimicrobial tests showed that essential oils strongly inhibited the growth of Gram-positive microorganisms and Mucor ramamnianus, but was inactive against Gram-negative strains. Antioxidant activity was tested using the ABTS radical-scavenging assay. Most samples showed good activity (the best IC(50) = 41.7 + or - 1.5 mg L(-1)). CONCLUSIONS It could be concluded that drying of leaves of J. phoenicea in the sun and berries in oven-drying was more suitable and was recommended for obtaining higher essential oil yield, but for a higher percentage of some special components such as alpha-pinene and delta-3-carene shade-drying was more suitable.

Platelet PMCA- and SERCA-type Ca2+-ATPase expression in diabetes: a novel signature of abnormal megakaryocytopoiesis

Summary.  Background: Previous studies have shown platelet Ca2+ abnormalities in diabetes mellitus and some reports suggest abnormal platelet production. Platelet Ca2+ homeostasis is controlled by a multi‐Ca2+‐ATPase system that includes two plasma membrane Ca2+‐ATPase (PMCA) and seven sarco/endoplasmic reticulum Ca2+‐ATPase (SERCA) isoforms. In addition, we recently found that the expression of PMCA4b and SERCA3 isoforms may serve as new markers of abnormal megakaryocytopoiesis [Nurden P et al. Impaired megakaryocytopoiesis in type 2B von Willebrand disease with severe thrombocytopenia. Blood 2006; 108: 2587–95]. Aim: To analyze the expression of major platelet Ca2+‐ATPases in 27 patients with type 1 or type 2 diabetes (T1D or T2D) compared with normal donors. Methods: Investigation of protein and mRNA expressions of PMCA1b and PMCA4b, and SERCA2b, SERCA3a and SERCA3b, using specific Western blotting and reverse transcriptase‐polymerase chain reaction, respectively. Results: Remarkably, all patients with T1D were found to present a higher expression of PMCA4b protein (212% ± 28%; n = 10) and PMCA4b mRNA (155% ± 16%; n = 17), coupled with a higher expression of SERCA3b mRNA (165% ± 9%) in some cases. Patients with T2D (n = 10) were also studied for protein expression and were found to present similar major upregulation of the expression of PMCA4b protein (180% ± 28%; n = 10). Lastly, five of 10 patients with T1D were studied for PMCA4b expression after insulin treatment, with four of five recovering normal expression (96% ± 15%; n = 5). Conclusions: Compared with the expression of PMCA4b upon platelet maturation, platelets from diabetic patients exhibit similarities with immature megakaryocytes. Thus, this study reinforces the idea that abnormal megakaryocytopoiesis can provide additional insights into diabetes and could represent a novel therapeutic target for antithrombotic drugs.

Human platelet Ca2+-ATPases: New markers of cell differentiation as illustrated in idiopathic scoliosis

The aetiology of adolescent idiopathic scoliosis (AIS), the most common form of scoliosis, is unclear. Previous studies showed controversial platelet abnormalities including intracellular calcium. Platelet Ca2+ homeostasis is controlled by a multi-Ca2+-ATPase system including SERCA (sarco/endoplasmic reticulum Ca2+-ATPase) and PMCA (plasma membrane Ca2+-ATPase) isoforms. Here, we first investigated the expression of PMCA4b, SERCA3a and SERCA2b isoforms in platelets of 17 patients with AIS. Patients presenting thoracic curves were found to present a higher PMCA4b expression coupled to a lower SERCA3a one in agreement with an abnormality in platelet maturation. Indeed, using PMA-treated MEG 01 cells, an in vitro model of megakaryocytopoiesis, we found an increase in SERCA3a expression, associated to a caspase-3 mediated C terminal proteolysis of PMCA4b. To look whether platelets reflect a basic defect in cell differentiation, we next identified osteoblast Ca2+-ATPases and studied their expressions in AIS. Major expressions of PMCA4b and SERCA2b were found in normal osteoblasts. Comparing platelets and osteoblasts in two additional patients with AIS, we found opposite and concerted regulations of the expressions of PMCA4b and caspase-3 substrate, PARP in both cell types. A systemic defect in cell differentiation involving caspase-3 can be proposed as a novel mechanism in the etiopathogenesis of the most frequent type of AIS.

Plant assemblage composition and soil P concentration differentially affect communities of AM and total fungi in a semi-arid grassland

Adding inorganic P- and N-fixing legumes to semi-arid grasslands can increase forage yield, but soil nutrient concentrations and plant cover may also interact to modify soil fungal populations, impacting short- and long-term forage production. We tested the effect of plant assemblage (seven native grasses, seven native grasses + the domesticated N-fixing legume Medicago sativa, seven native grasses + the native N-fixing legume Dalea purpurea or the introduced grass Bromus biebersteinii + M. sativa) and soil P concentration (addition of 0 or 200 P2O5 kg ha(-1) at sowing) on the diversity and community structure of arbuscular mycorrhizal (AM) fungi and total fungi over two consecutive years, using 454-pyrosequencing of 18S rDNA and ITS amplicons. Treatment effects were stronger in the wet year (2008) than the dry year (2009). The presence of an N-fixing legume with native grasses generally increased AM fungal diversity, while the interaction between soil P concentration and plant assemblage modified total fungal community structure in 2008. Excluding interannual variations, which are likely driven by moisture and plant productivity, AM fungal communities in semi-arid grasslands appear to be primarily affected by plant assemblage composition, while the composition of other fungi is more closely linked to soil P.

Influence of the process, season, and origin on volatile composition and antioxidant activity of Juniperus phoenicea L. leaves essential oils.

Essential oils of Juniperus phoenicea L. leaves cultivated in 3 regions, Korbos, Matmata, and Tabarka of Tunisia were obtained by hydrodistillation (HD), steam distillation (SD), and Soxhlet (SH) extraction methods. The essential oils were analyzed and quantified by capillary gas chromatography using flame ionization detection (GC-FID) and mass spectrometry (GC-MS). The highest yield was observed in HD process (1.12%). Tabarka essential oil provided the best yield 0.79% compared to other regions. December month SD essential oil was the highest in oxygenated monoterpenes (52.7%). Nevertheless, SH essential oil showed a higher content in sesquitepenes hydrocarbons (64.5%). α-Terpinol (25.5%) was the main oxygenated component in Matmata juniper essential oil, extracted by SD. Moreover, the antioxidant activity of essential oils was evaluated using ABTS assays. The strongest antioxidant activity (IC(50) = 22.6 ± 0.7 mg/L) was obtained by the Matmata (October 2007) SD essential oil.

Increased expression of plasma membrane Ca2+ATPase 4b in platelets from hypertensives: A new sign of abnormal thrombopoiesis?

Platelet Ca2+ homeostasis is controlled by a multi-Ca2+ATPase system including two PMCA (plasma membrane Ca2+ATPase) and seven SERCA (sarco/endoplasmic reticulum Ca2+ATPase) isoforms. Previous studies have shown similar platelet Ca2+ abnormalities in diabetic and hypertensive patients, including an increase in intracellular [Ca2+]I, a possible modulation of PMCA activity and increased PMCA tyrosine phosphorylation. Very recently, we found that platelets from diabetic patients also exhibited increased PMCA4b expression. In the present study we looked for further similarities between diabetic and hypertensive patients. We first confirmed a decrease in Ca2+ATPase activity (mean 55 + 7%) in mixed platelet membranes isolated from 10 patients with hypertension compared with those from 10 healthy controls. In addition, the decreased Ca2+ATPase activity correlated with the DBP of the different patients, as expected for PMCA activity. Second, we performed a pilot study of six hypertensives to examine their expressions of PMCA and SERCA mRNA and proteins. Like the diabetic patients, 100% of hypertensives were found to present a major increase in PMCA4b expression (mean value of 218 ± 21%). We thus determined that platelets from diabetic and hypertensive patients showed similar increased PMCA4b isoform. Since increased PMCA4b expression was recently found to be associated with a perturbation of megakaryocytopoiesis, these findings may also point to an abnormality in platelet maturation in hypertension.