Immunocytolocalization of human gastric lipase in chief cells of the fundic mucosa

The presence in human gastric juice of a lipase secreted by the gastric mucosae has been reported previously, but its exact cellular origin has not yet been established. Polyclonal antibodies specific to human gastric lipase (HGL) were prepared, and used by an immunofluorescence technique to label cells producing HGL. This immunocytolocalization was correlated with that of pepsin (chief cells) and parietal cells using specific polyclonal or monoclonal antibodies. Our results clearly establish that HGL is exclusively located in the chief cells of fundic mucosa; furthermore, it was found to be always co-located with pepsin. No HGL was observed in the parietal or mucus cells. HGL was always detected intracellularly, either in secretory granules of the apical region of the chief cells, or revealed by more diffuse cytoplasmic labelling.     

Newly synthesized dopamine ester derivatives and assessment of their antioxidant,antimicrobial and hemolytic activities

Preparation of dopamine derivatives was carried out as a response to the increasing demand for new lipophilized antioxidants in food, cosmetic and pharmaceutical industries. A large series of dopamine esters (DA-C₃ to DA-C_18:1) with increasing lipophilicity was synthesized using lipase from Candida antarctica (Novozyme 435) as a biocatalyst. The highest conversion yield (52.75%) was reached when caprylic acid (DA-C₈) was used as acyl donor. Synthesized compounds were purified and evaluated for their antioxidant activity using the DPPH and the ABTS tests. Results showed that esterification had little effect on radical-scavenging capacity. However, long chain fatty acid esters displayed higher protective effect of oil against oxidation at 70 °C as compared to the parent dopamine or to the BHT. The hemolytic activity of dopamine esters was studied. Middle chain length derivatives (DA-C₈ and DA-C₁₂) of dopamine and oleic acid derivative (DA-C_18:1) showed the highest hemolytic activity against human erythrocyte. The antimicrobial activities of dopamine esters were also evaluated using well diffusion and minimal inhibition concentration methods. Among all the tested compounds, DA-C₈ and DA-C₁₂ exhibited the highest antibacterial activities. These results open up potential applications by using dopamine derivatives as antioxidants and antimicrobial compounds in cosmetic, food and pharmaceutical industries.     

Biochemical and molecular characterisation of a thermoactive, alkaline and detergent-stable lipase from a newly isolated Staphylococcus aureus strain

A newly soil-isolated Staphylococcus aureus strain secretes a non-induced lipase in the culture medium. The extracellular lipase from S. aureus (SAL3) is purified to homogeneity. The purified enzyme is a tetrameric protein (180 kDa) corresponding to the association of four lipase molecules. The 15 N-terminal amino acid residues showed a high degree of homology with other staphylococcal lipase sequences. The part of the gene encoding the mature SAL3 is cloned and sequenced. The deduced polypeptide sequence, corresponding to the mature SAL3, was very similar to the mature Staphylococcus simulans lipase sequence with two additional amino acid residues (LK) at the N-terminus of SAL3. The lipase activity is maximal at pH 9.5 and 55 °C. The specific activity of about 4200 U/mg or 3500 U/mg was measured using tributyrin or olive oil emulsion as substrate, respectively, at pH 9.5 and 55 °C.In contrast to other staphylococcal lipases previously characterised, SAL3 is found to be stable between pH 5 and 12 after 24 h incubation. The enzyme retained 50% of its activity after 60 min incubation at 60 °C. This novel lipase is able to hydrolyse its substrate in presence of various oxidizing agents as well as some surfactants and some commercial detergents, then SAL3 can be considered as a good candidate for industrial and biotechnological applications.     

Cytoprotective Effects of the Red Marine Alga <Emphasis Type="Italic">Chondrus canaliculatus</Emphasis> Against Maneb-Induced Hematotoxicity and Bone Oxidative Damages in Adult Rats

The current study aimed at evaluating the ability of a mineral and antioxidant-rich extract from Chondrus canaliculatus to improve maneb (MB)-induced toxicity in adult rat. The animals were divided into four groups: group 1 used as a control group, group 2 received MB, group 3 received MB + C. canaliculatus extract, and group 4 received only the algal extract. MB, a Mn-containing ethylene-bis-dithiocarbamate fungicide, induced oxidative stress damages, mineral perturbations in the plasma, urine, and bone, and genotoxicity in rats. Hematological analysis revealed in the MB-treated group a disruption in the number of red blood cells, platelets, and white blood cells associated with a striking genotoxicity. Interestingly, a significant increase in malondialdehyde and advanced oxidation protein product levels in erythrocytes and bones were found. On the other hand, an impairment of the antioxidant status in both tissues was occurred. Along, our results revealed that MB injection caused a striking drop and disruption in bone’s mineral rates, especially calcium and phosphorus. These biochemical results were in accordance with the histological and molecular changes. However, co-treatment with C. canaliculatus extract showed, for the first time, that this alga was effective against MB-induced hematotoxicity, genotoxicity, and oxidative stress in the blood and bone and maintained osteomineral metabolism and bone histo-architecture. Such observations might be explained by the strong in vitro antioxidant and antibacterial activities exhibited by the alga, as well as by its high levels in several minerals: calcium, phosphorus, sodium, potassium, magnesium, iron, and zinc.     

Phenolic contents and antioxidant activity of ethanolic extract of Capparis spinosa

Caper plant (Capparis spinosa) extracts have been associated with diverse biological activities including anti-oxidant properties. In this work, we characterized the hydro-ethanolic extract obtained from C. spinosa leaves [hydroethanolic extract of C. spinosa (HECS)] by analyzing the content in anti-oxidant compounds such as polyphenols, flavonoids and anthocyanins. Further, we evaluated HECS antioxidant activities in vitro using bleaching of 1,1-diphenyl-2-picrylhydrazyl radical and ABTS test as well as by pretreatment of HeLa cells exposed to Fe²⁺ or H₂O₂. Our findings indicate that HECS contains high amount of total phenolic compounds and high levels of flavonoids and anthocyanins. Furthermore, HECS exhibited antioxidant activity in both chemical and biological tests. Specially, pretreatment of HeLa cells with different concentrations of the extract conferred protection against lipid peroxidation and modulated activities of two antioxidant enzymes, SOD and catalase. These results revealed HECS antioxidant effects and suggest that C. spinosa leaves are a potential source of natural antioxidant molecules with possible applications in industry and medicine.     

A novel high molecular weight thermo-acidoactive β-glucosidase from <Emphasis Type="Italic">Beauveria bassiana</Emphasis>

An extracellular high molecular weight β-glucosidase was secreted by a local strain P1 of Beauveria bassiana. The enzyme was produced in the presence of various carbon sources, namely glucose, maltose, lactose, glycerol, starch, wheat bran and gruel. The highest level of β-glucosidase activity was produced with wheat bran at the concentration of 3%. Glucose caused a repressor effect on the β-glucosidase expression in a dose-dependent manner. The highest enzyme production level was obtained at initial pH of 6.0 and 7.0 in the culture medium. The zymography analysis revealed that B. bassiana secreted a β-glucosidase with high molecular weight between 400 and 600 kDa. The enzymatic preparation was characterized and showed temperature and pH optima of 55°C and 5.0, respectively. The enzyme was stable at 40 and 50°C but its stability declined at 60°C. Interestingly, this β-glucosidase had high stability at acid and basic pH saving its initial activity after 24 h incubation at pH from 3.0 to 11.0. It was stable also in presence of monovalent Na⁺ and K⁺ ions saving 60% of its initial activity at 2 M salts. Bivalent metal ions preserved totally or partially the enzymatic activity; in addition, Ba²⁺ was revealed as an activator. This is the first report that focuses on the production and the biochemical characterization of a β-glucosidase from the entomopathogenic fungus, B. bassiana.     

Purification and biochemical characterization of a transglucosilating β-glucosidase of <Emphasis Type="Italic">Stachybotrys</Emphasis> strain

The filamentous fungus Stachybotrys sp has been shown to possess a rich β-glucosidase system composed of five β-glucosidases. One of them was already purified to homogeneity and characterized. In this work, a second β-glucosidase was purified and characterized. The filamentous fungal A19 strain was fed-batch cultivated on cellulose, and its extracellular cellulases (mainly β-glucosidases) were analyzed. The purified enzyme is a monomeric protein of 78 kDa molecular weight and exhibits optimal activity at pH 6.0 and at 50°C. The kinetic parameters, K _m and V _max, on para-nitro-phenyl-β-d-glucopyranosid (p-NPG) as a substrate were, respectively, 1.846 ± 0.11 mM and 211 ± 0.08 μmol min⁻¹ ml⁻¹. One interesting feature of this enzyme is its high stability in a wide range of pH from 4 to 10. Besides its aryl β-glucosidase activity towards salicin, methylumbellypheryl-β-d-glucoside (MU-Glc), and p-NPG, it showed a true β-glucosidase activity because it splits cellobiose into two glucose monomers. This enzyme has the capacity to synthesize short oligosaccharides from cellobiose as the substrate concentration reaches 30% with a recovery of 40%. We give evidences for the involvement of a transglucosylation to synthesize cellotetraose by a sequential addition of glucose to cellotriose.