Potential anti-inflammatory and antioxidant effects of Citrus aurantium essential oil against carbon tetrachloride-mediated hepatotoxicity: A biochemical, molecular and histopathological changes in adult rats.

The present study aimed (1) to investigate the chemical composition as well as the anti-inflammatory properties and in vitro antioxidant activity of Citrus aurantium peel essential oil (pEOCa) and (2) to evaluate its potential effect in vivo. The main results showed that the major components of pEOCa are Limonene and Linalool. Additionally, DPPH scavenging ability and ß-carotene bleaching inhibition tests confirmed the antioxidant capacity of pEOCa. Our oil reduced the production of NO by LPS-stimulated RAW264,7 macrophages in a concentration-dependent. This inhibition occurred at a transcriptional level. pEOCa in CCl4 treated rats alleviated hepatotoxicity as monitored by the improvement of hepatic oxidative stress biomarkers levels plasma biochemical parameters, and DNA molecule aspect. Furthermore, the mRNA gene expression of Cu-Zn SOD, CAT, and GPx increased under CCl4 + pEOCa exposure to reach the same value to the control. Similarly, antioxidant activities of these three enzymes changed in accordance with the mRNA levels. These results were confirmed by the histological results. It seems obvious that the treatment with pEOCa prevented liver damage induced by CCl4 , thus preventing the harmful effects of free radicals.

Apigenin isolated from A. americana encodes Human and Aspergillus oryzae S2 α-amylase inhibitions: credible approach for antifungal and antidiabetic therapies.

Agave americana extract was analyzed by reverse phase HPLC for characterization. Among phenolic compounds identified, apigenin was observed to be present. The finding showed an inhibitory effect of apigenin towards Human and Aspergillus oryzae S2 α-amylases. Apigenin inhibition towards Human and A. oryzae α-amylase activities was observed to be competitive. IC50 and  % inhibition of apigenin for A. oryzae α-amylase were 3.98 and 1.65 fold higher than for Human α-amylase. The inhibition of the described biocatalyst activity was significantly lowered when apigenin was pre-incubated with starch. In addition to the catalytic residues, 44 amino acid residues were involved on A. oryzae α-amylase-apigenin interactions while only 11 amino acid residues were exposed for Human α-amylase-apigenin complex. The binding site of apigenin showed 76 polar contacts for A. oryzae S2 α-amylase against 44 interactions for Human α-amylase. The docking studies confirmed the mode of action of apigenin and strongly suggested a higher inhibitory activity towards fungal amylase which was experimentally exhibited. These findings provided a rational reason to establish apigenin capability as a therapeutic target for postprandial hyperglycaemia modulation and antifungal therapy.

Trans-glycosylation capacity of a highly glycosylated multi-specific ß-glucosidase from Fusarium solani.

An extracellular ß-glucosidase from Fusaruim solani cultivated on wheat bran was purified by only two chromatographic steps. The purified enzyme exhibited optimal temperature and pH at 60 °C and pH 5, respectively. The purified ß-glucosidase behaves as a very large protein due to its high degree of glycosylation. More interestingly, the endoglycosidase H (Endo H) treatment led to 97.55% loss of its initial activity after 24 h of treatment. Besides, the addition of Tunicamycin (nucleoside antibiotic blocking the N-glycosylation first step) during the culture of the fungus affected seriously the glycosylation of the enzyme. Both treatments (endo H and Tunicamycin) strengthened the idea that the hyperglycosylation is involved in the ß-glucosidase activity and thermostability. This enzyme was also shown to belong to class III of ß-glucosidases (multi-specific) since it was able to act on either cellobiose, gentiobiose or sophorose which are disaccharide composed of two units of D-glucose connected by ß1-4, ß1-6 and ß1-2 linkage, respectively. The ß-glucosidase activity was strongly enhanced by ferrous ion (Fe2+) and high ionic strength (1 M KCl). The purified enzyme exhibited an efficient transglycosylation capacity allowing the synthesis of cellotriose and cellotetraose using cellobiose as donor.

Fast activated charcoal prepurification of Fusarium solani ß-glucosidase for an efficient oleuropein bioconversion.

Fungal ß-glucosidases were extensively studied regarding their various potential biotechnology applications. Here, we report the selection of Fusarium solani strain producing high yield of ß-glucosidase activity. The effect of some factors on ß-glucosidase production was studied including: Initial pH, medium composition, concentration of carbon and nitrogen sources, and particle size of raw substrates. The optimal enzyme production was obtained with 4 units of pH. The highest ß-glucosidase activity was produced on 4% wheat bran (WB) as raw carbon sources, reaching 5 U/mL. A positive correlation between WB particle size and the ß-glucosidase production level was settled. The last one was enhanced to 13.60 U/mL in the presence of 0.5% (w/v) of ammonium sulfate. Interestingly, the activated charcoal was used as an inexpensive reagent enabling a rapid and efficient purification prior step that improved the enzyme-specific activity. Eventually, F. solani ß-glucosidase acts efficiently during the bioconversion process of oleuropein. Indeed, 82.5% of oleuropein was deglycosylated after 1 hr at 40°C. Altogether, our data showed that the ß-glucosidase of F. solani has a potential application to convert oleuropein to ameliorate food quality.

Durum wheat dehydrin (DHN-5) confers salinity tolerance to transgenic Arabidopsis plants through the regulation of proline metabolism and ROS scavenging system.

MAIN CONCLUSION: The wheat dehydrin (DHN-5) gives birth to salinity tolerance to transgenic Arabidopsis plants by the regulation of proline metabolism and the ROS scavenging system. Dehydrins (DHNs) are involved in plant abiotic stress tolerance. In this study, we reported that salt tolerance of transgenic Arabidopsis plants overexpressing durum wheat dehydrin (DHN-5) was closely related to the activation of the proline metabolism enzyme (P5CS) and some antioxidant biocatalysts. Indeed, DHN-5 improved P5CS activity in the transgenic plants generating a significant proline accumulation. Moreover, salt tolerance of Arabidopsis transgenic plants was accompanied by an excellent activation of antioxidant enzymes like catalase (CAT), superoxide dismutase (SOD) and peroxide dismutase (POD) and generation of a lower level of hydrogen peroxide (H2O2) in leaves compared to the wild-type plants. The enzyme activities were enhanced in these transgenic plants in the presence of exogenous proline. Nevertheless, proline accumulation was slightly reduced in transgenic plants promoting chlorophyll levels. All these results suggest the crucial role of DHN-5 in response to salt stress through the activation of enzymes implicated in proline metabolism and in ROS scavenging enzymes.

Biocatalysts: beautiful creatures.

The chemical industry has come under increasing pressure to make chemical production more eco-friendly and independent to fossil resources. The development of industrial processes based on micro-organisms can especially help to eliminate the use or the generation of hazardous substances and can support the transition from dependence on fossil resources towards real sustainable and eco-safety industrial processes. The biocatalysts are the best solution given by nature that can be used to improve some biotechnological applications. In this research review, we report some peculiar properties of biocatalysts, implicated in a range of metabolic pathways and biotechnological tools.

Cellobiose dehydrogenase influences the production of S. microspora ß-glucosidase.

BglG, a Stachybotrys microspora ß-glucosidase produced in the presence of glucose and cellobiose, was used individually as sole carbon source. The time course synthesis of BglG showed two aspects: (1) an exponential curve, observed in glucose Mandels medium, and (2) a cloche curve, observed in cellobiose containing cultures. A decrease was observed in bglG production at the 6th, 8th and 10th days during mycelium growth in cellobiose Mandels medium, which allowed for the assumption that the anabolism of a bglG inhibitor factor was produced with cellobiose but not with glucose. Cellobiose dehydrogenases (CDH) activity was, on another hand detected in cellobiose grown cultures but not in glucose containing ones. The aliquots, withdrawn at the time course of bglG production in the presence of cellobiose, gave rise to an inhibitory effect on bglG activity. This result was obtained with and without the heat treatment (5 min at 100°C) of the aliquots, which supported the non-proteinaceous nature of the inhibitor factor. Furthermore, sugar chromatographic analyses revealed the appearance of a secondary metabolite in the cellobiose Mandels medium and indicated that the factor behind the bglG activity cloche curve was a δ-gluconolactone. Seeing that the latter follows a strong inhibitory effect on bglG activity, it is speculated that the decrease in bglG activity during the time course of bglG synthesis in cellobiose Mandels medium is assigned to the release of δ-gluconolactone. This paper presents and validates an explanatory model for this hypothesis.

Wheat dehydrin DHN-5 exerts a heat-protective effect on beta-glucosidase and glucose oxidase activities.

Group-2 late embryogenesis abundant (LEA) proteins, also known as dehydrins, are claimed to stabilize macromolecules against damage caused by freezing, dehydration, ionic or osmotic stresses. However, their precise function remains unknown. Here, we investigated the effect of wheat dehydrin (DHN-5) protein on the activity and thermostability of two distinct enzymes, beta-glucosidase (bglG) and glucose oxidase/peroxidase (GOD/POD) in vitro. The purified DHN-5 protein had the capacity to preserve and stabilize the activity of bglG subjected to heat treatment. In addition, DHN-5 stabilized oxidizing enzymes, as it improved reliability in measuring glucose concentrations with a glucose oxidase/peroxidase (GOD/POD) kit while the temperature increased from 37 to 70 degrees C. All together the data presented provide evidence that DHN-5 is a dehydrin able to preserve enzyme activities in vitro from adverse effects induced by heating.

Antioxidant and hepato-preventive effect of Citrus aurantium extract against carbon tetrachloride-induced hepatotoxicity in rats and characterisation of its bioactive compounds by HPLC-MS.

The objective of this study is to explore the preventive effects of ethyl acetate fraction from Citrus aurantium leaf extract (EACA), associated with its phytochemical content, against the toxic impacts of acute exposure to carbon tetrachloride (CCl4) in the liver of adult rats. HPLC analysis of ethyl acetate fraction from extract C. aurantium revealed eight compounds. Administration of a single dose of CCl4 caused hepatoxicity as monitored by an increase in lipid peroxidation (thiobarbituric acid reactive substances) and in protein carbonyl level but a decrease in antioxidant markers in the liver tissue. The pre-treatment with EACA; significantly prevented the increased plasma levels of hepatic markers and lipid levels induced by CCl4 in rats. Furthermore, this fraction ameliorated biochemical and histological parameters as compared to CCl4-treated group. Our results suggest that C. aurantium contains promising substances to counteract the CCl4 intoxication and can be efficient in the prevention of hepatotoxicity complications.

Effect of Agave americana L. on the human, and Aspergillus oryzae S2 α-amylase inhibitions.

Among phenolic compounds, Agave americana L. extract contained puerarin (38.4%) and p-coumaric acid (12.29%) (pCa). From the Lineweaver-Burk plots, pCa and puerarin demonstrated a competitive and a non competitive inhibitions towards human α-amylase activity, respectively. PCa exhibited a higher human inhibitory activity with an IC50 of 98.8 µM which was about 2.3 times than acarbose. Puerarin (IC50 = 3.87 µM) and pCa (IC50 = 10.16 µM) also showed an excellent inhibition for Aspergillus oryzae S2 α-amylase activity. The inhibitions of the described biocatalysts compounds towards both amylases were significantly decreased when they were pre-incubated with starch. The binding modes of these compounds were evaluated in silico. The binding efficiency order of these molecules in terms of polar contact numbers for both enzymes was in agreement with the in vitro studies. These findings provided a rational reason to establish the isolated compounds capability as therapeutic target for hyperglycaemia modulation and antifungal therapy.

Causal Enzymology and Physiological Aspects May Be Accountable to Membrane Integrity in Response to Salt Stress in Arabidopsis thaliana Lines.

Apart from their significance in the protection against stress conditions, the plant cell membranes are essential for proper development of the diverse surface structures formed on aerial plant organs. In addition, we signal that membrane remodeling and integrity are function of some of causal physiological and enzymological aspects such as the MDA, the ion leakage and also the monitoring of some phytozymes involved in lipid and cellulose metabolisms. Those last ones are related to the membrane structure (lipases and cellulases), that were assessed in durum wheat dehydrin transgenic context (YS, K1-K2, DH2, and DH4), proline metabolic mutant (P5CS1-4) per comparison with the wild-type plant (Wt). We report also the docking data reinforcing the fact that the membrane integrity seems to be function of causal enzymological behaviors, through the molecular dynamic investigation resulting from the dehydrin-phytozyme interactions and also from the inhibition effect of the durum wheat LTP4 on the lipase activity.

A stress-associated protein, LmSAP, from the halophyte Lobularia maritima provides tolerance to heavy metals in tobacco through increased ROS scavenging and metal detoxification processes.

Agricultural soil pollution by heavy metals is a severe global ecological problem. We recently showed that overexpression of LmSAP, a member of the stress-associated protein (SAP) gene family isolated from Lobularia maritima, in transgenic tobacco led to enhanced tolerance to abiotic stress. In this study, we characterised the response of LmSAP transgenic tobacco plants to metal stresses (cadmium (Cd), copper (Cu), manganese (Mn), and zinc (Zn)). In L. maritima, LmSAP expression increased after 12 h of treatment with these metals, suggesting its involvement in the plant response to heavy metal stress. LmSAP transgenic tobacco plants subjected to these stress conditions were healthy, experienced higher seedling survival rates, and had longer roots than non-transgenic plants (NT). However, they exhibited higher tolerance towards cadmium and manganese than towards copper and zinc. LmSAP-overexpressing tobacco seedlings accumulated more cadmium, copper, and manganese compared with NT plants, but displayed markedly decreased hydrogen peroxide (H2O2) and lipid peroxidation levels after metal treatment. Activities of the antioxidant enzymes superoxide dismutase (SOD), catalase (CAT), and peroxidase (POD) were significantly higher in transgenic plants than in NT plants after exposure to metal stress. LmSAP overexpression also enhanced the transcription of several genes encoding metallothioneins (Met1, Met2, Met3, Met4, and Met5), a copper transport protein CCH, a Cys and His-rich domain-containing protein RAR1 (Rar1), and a ubiquitin-like protein 5 (PUB1), which are involved in metal tolerance in tobacco. Our findings indicate that LmSAP overexpression in tobacco enhanced tolerance to heavy metal stress by protecting the plant cells against oxidative stress, scavenging reactive oxygen species (ROS), and decreasing the intracellular concentration of free heavy metals through its effect on metal-binding proteins in the cytosol.

The LmSAP gene isolated from the halotolerant Lobularia maritima improves salt and ionic tolerance in transgenic tobacco lines.

The A20/AN1 zinc-finger domain-containing proteins of the stress-associated proteins (SAPs) family are fast emerging as potential candidates for biotechnological approaches to improve abiotic stress tolerance in plants. We identified LmSAP, one of the SAPs genes in Lobularia maritima (L.) Desv., a halophyte brassicaceae, through its transcript accumulation in response to salinity and ionic stresses. Sequence homology analysis revealed that LmSAP contains two conserved zinc-finger domains A20 and AN1. Phylogeny analyses showed that LmSAP exhibited high amino acid sequence identity to other plant SAPs. Heterologous expression of LmSAP in yeast increased cell tolerance to salt and osmotic stress. In addition, the overexpression of LmSAP conferred high salt and ionic tolerance to transgenic tobacco plants. Transgenic tobacco seedlings showed higher survival rates and antioxidant activities under salt and ionic stresses. Enhanced antioxidant activities paralleled lower malondialdehyde and superoxide anion O2- levels in the LmSAP transgenic seedlings. Overall, our results suggest that overexpression of LmSAP enhanced salt tolerance by maintaining ionic balance and limiting oxidative and osmotic stresses.

Role of the durum wheat dehydrin in the function of proteases conferring salinity tolerance in Arabidopsis thaliana transgenic lines.

Dehydrins are claimed to stabilize macromolecules against freezing damage, dehydration, ionic or osmotic stresses, thermal stress and re-folding yield. However, their precise function remains unknown. In this context, we report the behavior of protease activities in dehydrin transgenic Arabidopsis lines against the wild type plant under salt stress (100mM NaCl). Indeed, proteases play key roles in plants, maintaining strict protein quality control and degrading specific sets of proteins in response to diverse environmental and developmental stimuli. We proved that durum wheat DHN-5 modulates the activity of some proteases, summarized on the promotion of the Cysteinyl protease and the decrease of the Aspartyl protease activity. This fact is also upgraded in salt stress conditions. We conclude that the dehydrin transgenic context encodes salinity tolerance in transgenic lines through the modulation of the interaction not only at transcriptional level but also at protein level and also with the impact of salt stress as an endogenous and exogenous effector on some biocatalysts like proteases.

Bridging Between Proline Structure, Functions, Metabolism, and Involvement in Organism Physiology.

Much is now known about proline multifunctionality and metabolism; some aspects of its biological functions are still unclear. Here, we discuss some cases in the proline, structure, definition, metabolism, compartmentalization, accumulation, plausible functions and also its implication in homeostasis and organism physiology. Indeed, we report the role of proline in cellular homeostasis, including redox balance and energy status and their implication as biocatalyst for aldolase activity. Proline can act as a signaling molecule to modulate mitochondrial functions, influence cell proliferation or cell death, and trigger specific gene expression, which can be essential for plant recovery from stresses. Although, the regulation and the function of proline accumulation, during abiotic stresses, are not yet completely understood. The engineering of proline metabolism could lead to new opportunities to improve plant tolerance against environmental stresses. This atypical amino acid has a potential role in the toxicity during growth of some microorganism, vegetal, and mammalian species. Furthermore, we note that the purpose through the work is to provide a rich, concise, and mostly cohesive source on proline, considered as a platform and an anchor between several disciplines and biological functions.

Comparative functional analysis of two wheat Na(+)/H (+) antiporter SOS1 promoters in Arabidopsis thaliana under various stress conditions.

The bread wheat TaSOS1 has been previously shown to be induced by salt stress treatment. To further investigate the regulation of the TaSOS1 gene, the two genomic fragments Pr SOS1-AB and Pr SOS1-D have been isolated and sequenced. Pr SOS1-AB and Pr SOS1-D are the promoter regions of SOS1 alleles, which are localised on genomes A and/or B, and on genome D, respectively. Sequence analysis of these two promoters revealed the presence of cis-regulatory elements which could be required for abiotic stress and abscisic acid (ABA) responsiveness. Histochemical assays of stably transformed Arabidopsis T3 plants showed that Pr SOS1-AB and Pr SOS1-D are active in this heterologous system, and their activities were almost the same at early developmental stages (4-, 8- and 12-day-old transgenic Arabidopsis seedlings). Nevertheless, ß-glucuronidase (GUS) activity was detected only in plants carrying the Pr SOS1-AB -gusA construct grown for 20 or 30 days. Furthermore, in these plants, the application of abiotic stress produced an accumulation in gusA transcripts. Taken together, these results show that, in this heterologous dicot system and under normal growth conditions, Pr SOS1-AB and Pr SOS1-D are age-dependent and organ-specific promoters. However, in the presence of different stress conditions, the activities of these two promoters became different and only Pr SOS1-AB is an abiotic stress-inducible promoter at different developmental stages. Thus, Pr SOS1-AB can be used for the development of abiotic stress-tolerant transgenic plants.

Wheat dehydrin K-segments ensure bacterial stress tolerance, antiaggregation and antimicrobial effects.

Dehydrins are a group of plant proteins that have been shown to be involved in the tolerance of various abiotic stresses such as dehydration, salinity, and low temperature. We have previously shown that the K-segments of the wheat dehydrin DHN-5 are essential for the protection of enzyme activities in vitro. In this study, we further investigate the role of the K-segments in the growth of Escherichia coli under various stresses, and we tested their antibacterial and antifungal activities. Our results showed that the truncated forms of DHN-5 containing the two K-segments enhanced tolerance of E. coli against diverse stresses by protecting proteins against aggregation. In addition, we demonstrated that the K-segments have antibacterial and antifungal activities against Gram-positive and Gram-negative bacteria and fungi. Based on these results, we propose that the K-segments may play a protective role in plants not only under abiotic stress conditions but also most likely during defense mechanisms.

A wheat lipid transfer protein (TdLTP4) promotes tolerance to abiotic and biotic stress in Arabidopsis thaliana.

Lipid transfer proteins (LTPs) are members of the family of pathogenesis-related proteins (PR-14) that are believed to be involved in plant defense responses. In this study, we report the isolation and characterization of a novel gene TdLTP4 encoding an LTP protein from durum wheat [Triticum turgidum L. subsp. Durum Desf.]. Molecular Phylogeny analyses of wheat TdLTP4 gene showed a high identity to other plant LTPs. Predicted three-dimensional structural model revealed the presence of six helices and nine loop turns. Expression analysis in two local durum wheat varieties with marked differences in salt and drought tolerance, revealed a higher transcript accumulation of TdLTP4 under different stress conditions in the tolerant variety, compared to the sensitive one. The overexpression of TdLTP4 in Arabidopsis resulted in a promoted plant growth under various stress conditions including NaCl, ABA, JA and H2O2 treatments. Moreover, the LTP-overexpressing lines exhibit less sensitivity to jasmonate than wild-type plants. Furthermore, detached leaves from transgenic Arabidopsis expressing TdLTP4 gene showed enhanced fungal resistance against Alternaria solani and Botrytis cinerea. Together, these data provide the evidence for the involvement of TdLTP4 gene in the tolerance to both abiotic and biotic stresses in crop plants.

Medium initial pH and carbon source stimulate differential alkaline cellulase time course production in Stachybotrys microspora.

The production profile of cellulases of the mutant strain A19 from the filamentous fungus Stachybotrys microspora was studied in the presence of various carbon sources (glucose, lactose, cellulose, carboxymethylcellulose (CMC), and wheat bran) and a range of medium initial pH (5, 7, and 8). Two extracellular cellulases from the Stachybotrys strain (endoglucanases and ß-glucosidases) were monitored by enzymatic assay, sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and zymogram analysis. Glucose and lactose repressed CMCase time course production while they permitted a strong ß-glucosidase one. On Avicel cellulose, CMC, and wheat bran, both activities were highly produced. Wheat bran (WB) is the best carbon source with an optimum of production at days 5 and 6. The production kinetics of both activities were shown to depend on the medium initial pH, with a preference for neutral or alkaline pH in the majority of conditions. The exception concerned the ß-glucosidase which was much more produced at acidic pH, on glucose and cellulose. Most interestingly, a constitutive and conditional expression of an alkaline endoglucanase was revealed on the glucose-based medium at an initial pH of 8 units. The zymogram analysis confirmed such conclusions and highlighted that carbon sources and the pH of the culture medium directed a differential induction of various endoglucanases and ß-glucosidases.

Improvement of cellulase and xylanase production by solid-state fermentation of Stachybotrys microspora.

The current study investigated the production of cellulases and xylanases from the rare fungus Stachybotrys microspora under solid-state fermentation (SSF) on wheat bran (WB). A comparison of both activities was first performed in submerged cultures using various concentrations of WB, glucose, and cellulose as substrates. The maximal activity of ß-glucosidases and xylanases was obtained with 2% and 4% WB, respectively, whereas cellulose yielded the highest endoglucanase production. The SSF conditions were therefore consequently optimized. A moisture content of 70% gave the most significant levels of enzyme production. Inoculation by spores led to better results than by preculture, with 10(5) spores per gram of dried matter as the best inoculum dose for all activities tested. Interestingly, the WB-based medium need not to be supplemented by an exogeneous nitrogen source. Considering the richness of S. microspora secreted proteins as lytic hydrolases, the crude extracellular enzyme extracts were successfully tested in two different biotechnological fields: protoplasting of fungi and subsequent extraction of their DNA, paper pulp hydrolysis to produce fermentable sugars.