Kinetics of the Anti-oxidant Response to Salinity in the Halophyte Cakile maritima

The effects of NaCl stress on the activity of anti-oxidant enzymes （superoxide dismutase, catalase （CAT）, peroxidase （POD）, ascorbate peroxidase （APX）, monodehydroascorbate reductase, dehydroascorbate reductase （DHAR）, and glutathione reductase （GR））, anti-oxidant molecules （ascorbate and glutathione）, and parameters of oxidative stress （malondialdehyde （MDA）, electrolyte leakage, and H2O2 concentrations） were investigated in Cakile maritima, a halophyte frequent along the Tunisian seashore. Seedlings were grown in the presence of salt （100, 200, and 400 mmol/L NaCl）. Plants were harvested periodically over 20 days. Growth was maximal in the presence of 0-100 mmol/L NaCl. At 400 mmol/L NaCl, growth decreased significantly. The salt tolerance of C. maritima, at moderate salinities, was associated with the lowest values of the parameters indicative of oxidative stress, namely the highest activities of POD, CAT, APX, DHAR, and GR and high tissue content of ascorbate and glutathione. However, prolonged exposure to high salinity resulted in a decrease in anti-oxidant activities and high MDA content, electrolyte leakage, and H2O2 concentrations. These results suggest that anti-oxidant systems participate in the tolerance of C. maritima to moderate salinities.     

Cytotoxicity and genotoxicity induced by aflatoxin B1, ochratoxin A,and their combination in cultured Vero cells

Aflatoxin B1 (AFB1) and ochratoxin A (OTA) are important food‐borne mycotoxins that have been implicated in human health. In this study, independent and combinative toxicities of AFB1 and OTA were tested in cultured monkey kidney Vero cells. The experiments reported here were conducted to evaluate the effect of these toxins on cell viability followed by the determination of cell death pathways, using the quantification of DNA fragmentation and the expression of p53 and bcl‐2 protein levels. Our results showed that AFB1 and OTA caused a marked decrease of cell viability in a dose‐dependent manner. Under the same conditions, these mycotoxins increased fragmented DNA levels. In addition, p53 was activated in response to DNA damage and the expression of the antiapoptotic factor bcl‐2 decreased significantly. According to these data, AFB1 and OTA seemed to be involved in an apoptotic process. Moreover, combined AFB1 and OTA induced all the toxicities observed with the mycotoxins separately. Therefore, this combination was classified as an additive response of the two mycotoxins. © 2010 Wiley Periodicals, Inc. J Biochem Mol Toxicol 24:42–50, 2010; Published online in Wiley InterScience ( www.interscience.wiley.com ). DOI 10.1002/jbt.20310     

Rendomab B4, a monoclonal antibody that discriminates the human endothelin B receptor of melanoma cells and inhibits their migration

Metastatic melanoma is an aggressive cancer with a poor prognostic, and the design of new targeted drugs to treat melanoma is a therapeutic challenge. A promising approach is to produce monoclonal antibodies (mAbs) against the endothelin B receptor (ETB), which is known to be overexpressed in melanoma and to contribute to proliferation, migration and vasculogenic mimicry associated with invasiveness of this cancer.

We previously described rendomab-B1, a mAb produced by DNA immunization. It is endowed with remarkable characteristics in term of affinity, specificity and antagonist properties against human ETB expressed by the endothelial cells, but, surprisingly, had poor affinity for ETB expressed by melanoma cells. This characteristic strongly suggested the existence of a tumor-specific ETB form. In the study reported here, we identified a new mAb, rendomab-B4, which, in contrast to rendomab-B1, binds ETB expressed on UACC-257, WM-266-4 and SLM8 melanoma cells. Moreover, after binding to UACC-257 cells, rendomab-B4 is internalized and colocalizes with the endosomal protein EEA-1. Interestingly, rendomab-B4, despite its inability to compete with endothelin binding, is able to inhibit phospholipase C pathway and migration induced by endothelin. By contrast, rendomab-B4 fails to decrease ERK1/2 phosphorylation induced by endothelin, suggesting a biased effect on ETB.

These particular properties make rendomab-B4 an interesting tool to analyze ETB-structure/function and a promising starting point for the development of new immunological tools in the field of melanoma therapeutics.     

Salinity effects on polyphenol content and antioxidant activities in leaves of the halophyte Cakile maritima.

Cakile maritima is a local oilseed halophyte exhibiting potential for secondary metabolite production. In the present study, plant growth, leaf polyphenol content and antioxidant activity were comparatively analyzed in two C. maritima Tunisian accessions (Jerba and Tabarka, respectively sampled from arid and humid bioclimatic stages) under salt constraint. Three-week-old plants were subjected to 0, 100, and 400 mM NaCl for 28 days under glasshouse conditions. A significant variability in salt response was found between both accessions: while Tabarka growth (shoot biomass, leaf expansion) was significantly restricted at 100 and 400 mM NaCl, compared to the control, Jerba growth increased at 100mM before declining at 400 mM NaCl. The better behaviour of Jerba salt-challenged plants, compared to those of Tabarka, may be related to their higher polyphenol content (1.56- and 1.3-fold the control, at 100 and 400 mM NaCl respectively) and antioxidant activity (smaller IC(50) values for both 1,1-diphenyl-2-picrylhydrazyl and superoxide scavenging), associated with lower leaf MDA accumulation (ca. -66% of the control at 100mM NaCl). Taken together, our findings suggest that halophytes may be interesting for production of antioxidant compounds, and that the accession-dependent capacity to induce antioxidative mechanisms in response to salt, may result in a corresponding variability for growth sustainability.     

Identification and characterization of solvent-filled channels in human ferrochelatase

Ferrochelatase catalyzes the formation of protoheme from two potentially cytotoxic products, iron and protoporphyrin IX. While much is known from structural and kinetic studies on human ferrochelatase of the dynamic nature of the enzyme during catalysis and the binding of protoporphyrin IX and heme, little is known about how metal is delivered to the active site and how chelation occurs. Analysis of all ferrochelatase structures available to date reveals the existence of several solvent-filled channels that originate at the protein surface and continue to the active site. These channels have been proposed to provide a route for substrate entry, water entry, and proton exit during the catalytic cycle. To begin to understand the functions of these channels, we investigated in vitro and in vivo a number of variants that line these solvent-filled channels. Data presented herein support the role of one of these channels, which originates at the surface residue H240, in the delivery of iron to the active site. Structural studies of the arginyl variant of the conserved residue F337, which resides at the back of the active site pocket, suggest that it not only regulates the opening and closing of active site channels but also plays a role in regulating the enzyme mechanism. These data provide insight into the movement of the substrate and water into and out of the active site and how this movement is coordinated with the reaction mechanism.     

Influence of biological, environmental and technical factors on phenolic content and antioxidant activities of Tunisian halophytes

Halophyte ability to withstand salt-triggered oxidative stress is governed by multiple biochemical mechanisms that facilitate retention and/or acquisition of water, protect chloroplast functioning, and maintain ion homeostasis. Most essential traits include the synthesis of osmolytes, specific proteins, and antioxidant molecules. This might explain the utilization of some halophytes as traditional medicinal and dietary plants. The present study aimed at assessing the phenolic content and antioxidant activities of some Tunisian halophytes (Cakile maritima, Limoniastrum monopetalum, Mesembryanthemum crystallinum, M. edule, Salsola kali, and Tamarix gallica), depending on biological (species, organ and developmental stage), environmental, and technical (extraction solvent) factors. The total polyphenol contents and antioxidant activities (DPPH and superoxide radicals scavenging activities, and iron chelating and reducing powers) were strongly affected by the above-cited factors. Such variability might be of great importance in terms of valorising these halophytes as a source of naturally secondary metabolites, and the methods for phenolic and antioxidant production.     

Anti-cariogenic and anti-biofilms activity of Tunisian propolis extract and its potential protective effect against cancer cells proliferation

Propolis is a multifunctional substance used by bees to maintain the safety of their hives. It is worldwide used for its potential therapeutic effects. In this study, Tunisian propolis ethanol extract (EEP) was tested for their anti-cariogenic, anti-biofilms and antiproliferative effects of many cell lines. The Tunisian EEP was evaluated in vitro against 33 oral pathogens including streptococci and enterococci using broth microdilution method. The anti-biofilms activity of EEP was assessed via Crystal Violet staining and MTT assays. The Tunisian EEP antiproliferative effect was evaluated on normal (MRC-5) and cancer cell lines (HT-29, A549, Hep-2, raw 264.7, Vero) by the ability of the cells to metabolically reduce MTT to a formazan dye. Our results revealed that Tunisian EEP possessed excellent protective effects against cariogenic and biofilms activity of oral streptococci. Furthermore, EEP showed a strong antiproliferative potencies against all studied cancer cell lines as judged by IC50 and its value ranges from 15.7 ± 3.4 to 200 ± 22.2 μg mL?1. These results suggest that EEP is able to inhibit cancer cell proliferation, cariogenic bacteria and oral biofilms formation. It could have a promising role in the future medicine and nutrition when used as antibiotic or food additive.     

Ultrastructure of Aeluropus littoralis leaf salt glands under NaCl stress

The effects of salt uptake on the morphology and ultrastructure of leaf salt glands were investigated in Aeluropus littoralis plants grown for two months in the presence of 400 mM NaCl. The salt gland is composed of two linked cells, as observed in some other studied Poaceae species. The cap cell, which protrudes from the leaf surface, is smaller than the basal cell, which is embedded in the leaf mesophyll tissues and bears the former. The cuticle over the cap cell is frequently separated from the cell wall to form a cavity where salts accumulate prior to excretion. The basal cell cytoplasm contains an extensive intricate or partitioning membrane system that is probably involved in the excretion process, which is absent from the cap cell. The intricate membrane system seems to be elongated and heavily loaded with salt. The presence of 400 mM NaCl induced the disappearance of the collecting chamber over the glands and an increase in the number of vacuoles and their size in both gland cells. In the basal cell, salt greatly increased both the density and size of the intricate membrane system. The electron density of both gland cells observed under salt treatment reflects a high activity. All these changes probably constitute special adaptations for dealing with salt accumulation in the leaves. Despite the high salt concentration used, no serious damage occurred in A. littoralis salt gland ultrastructure, which consolidates the assumption that they are naturally designated for this purpose.     

The effect of calcium on the antioxidant systems in the halophyte <Emphasis Type="Italic">Cakile maritima</Emphasis> under salt stress

The purpose of the current investigation was to study the effect of Ca²⁺ (0, 3.5 and 20 mM concentrations) on the antioxidant systems in the halophyte Cakile maritima under NaCl stress (0, 100, 200 and 400 mM NaCl). Plants treated with both moderate calcium (3.5 mM) and salt levels (100 mM) showed the maximum growth, and the addition of 20 mM calcium to the nutrient media did not significantly reduce the growth under the moderate salt treatment. The absence of calcium associated with high salt concentration induced a strong reduction of biomass production. The tolerance of C. maritima at moderate salinity and calcium was related with the lowest values of the parameters indicative of oxidative stress (malondialdehyde, electrolyte leakage and hydrogen peroxide concentration). This was accompanied with a higher peroxidase, superoxide dismutase, ascorbate peroxidase, monodehydroascorbate reductase, dehydroascorbate reductase and glutathione reductase activities. In contrast, in the absence of calcium, those enzymes showed the lowest activities under all salt treatments. As a whole, it can be noticed that salt tolerance was improved by moderate calcium concentration; however, the absence of calcium has a drastic effect on C. maritima.     

Salt impact on photosynthesis and leaf ultrastructure of <Emphasis Type="Italic">Aeluropus littoralis</Emphasis>

The effects of salinity (400 mM NaCl) on growth, biomass partitioning, photosynthesis, and leaf ultrastructure were studied in hydroponically grown plants of Aeluropus littoralis (Willd) Parl. NaCl produced a significant inhibition of the main growth parameters and a reduction in leaf gas exchange (e.g. decreased rates of photosynthesis and stomatal conductance). However, NaCl salinity affected neither the composition of photosynthesis pigments nor leaf water content. The reduction in leaf gas exchange seemed to correlate with a decrease in mesophyll thickness as well as a severe disorganisation of chloroplast structure, with misshapen chloroplasts and dilated thylakoid membranes. Conspicuously, mesophyll chloroplasts were more sensitive to salt treatment than those of bundle sheath cells. The effects of NaCl toxicity on leaf structure and ultrastructure and the associated physiological implications are discussed in relation to the degree of salt resistance of A. littoralis.