Exploration of antimicrobial and anticancer activities of L-amino acid oxidase from Egyptian Naja haje venom.

Hepatocellular carcinoma and bacterial resistance are major health burdens nowadays. Thus, providing new therapies that overcome that resistance is of great interest, particularly those derived from nature rather than chemotherapeutics to avoid cytotoxicity on normal cells. Venomous animals are among the natural sources that assisted in the discovery of novel therapeutic regimens. L-amino acid oxidase Nh-LAAO (140 kDa), purified from Egyptian Naja haje venom by a successive two-step chromatography protocol, has an optimal pH and temperature of 8 and 37 °C. Under standard assay conditions, Nh-LAAO exhibited the highest specificity toward L-Arg, L-Met and L-Leu, with Km and Vmax values of 3.5 mM and 10.4 µmol/min/ml, respectively. Among the metal ions, Ca+2, Na+, and K+ ions are activators, whereas Fe+2 inhibited LAAO activity. PMSF and EDTA slightly inhibited the Nh-LAAO activity. In addition, Nh-LAAO showed antibacterial and antifungal activities, particularly against Gentamicin-resistant P. aeruginosa and E. coli strains with MIC of 18 ± 2 µg/ml, as well as F. proliferatum and A. parasiticus among the selected human pathogenic strains. Furthermore, Nh-LAAO exhibited anti-proliferative activity against cancer HepG2 and Huh7 cells with IC50 of 79.37 and 60.11 µg/ml, respectively, with no detectable effect on normal WI-38 cells. Consequently, the apoptosis % of the HepG2 and Huh7 cells were 12 ± 1 and 34.5 ± 2.5 %, respectively, upon Nh-LAAO treatment. Further, the Nh-LAAO arrested the HepG2 and Huh7 cell cycles in the G0/G1 phase. Thus, the powerful selective cytotoxicity of L-amino acid oxidase opens up the possibility as a good candidate for clinical cancer therapy.

Purification and characterization of cysteine protease of Sarcocystis fusiformis from infected Egyptian water buffaloes.

Sarcocystis spp. infects water buffaloes (Bubalus bubalis) causing sarcocystosis. In the present study, Sarcocystis fusiformis was recognized in Egyptian water buffaloes based on histological observation and molecular analysis of internal transcribed spacer 1 (ITS1), 18S ribosomal RNA (18S rRNA) and cytochrome c oxidase subunit I (COX-1) gene fragments. Chemotherapy and vaccines against Sarcocystis spp. could potentially target proteases because they may play a crucial role in the infection. Cysteine proteases are multifunctional enzymes involved in vital metabolic processes. However, the involvement of proteases in S. fusiform infection has not yet been characterized. Here, the purification and study on some biochemical properties of protease isolated from cysts of S. fusiform were carried out. Protease with a molecular weight of 100 kDa was purified. LC-MS/MS analyzed the protein sequence of purified protease and the data suggested that the enzyme might be related to the cysteine protease. The purified protease exhibited maximum activity at pH 6 and a temperature of 50 °C. The Michaelis-Menten constant (Km), the maximum velocity (Vmax), and the turnover number (Kcat) were determined. The complete inhibition effect of cysteine inhibitors indicated that the purified enzyme is a cysteine protease. The results suggested that S. fusiform proteolytic enzyme may be necessary for parasite survival in water buffaloes by digesting host tissues. Therefore, cysteine protease could be a suitable target for vaccinations.

Egyptian chia seeds (Salvia hispanica L.) during germination: Upgrading of phenolic profile, antioxidant, antibacterial properties and relevant enzymes activities.

Little studies on chia sprouts were not deeply address the polyphenols profiles and their functional properties during long period of germination. This study aims to evaluate the impact of germination process on the phenolic profile, antioxidant and antibacterial properties and relevant enzymes activities of Egyptian chia seeds. The total phenolic and flavonoid contents of chia sprouts increased several times during ten days of germination and maximized on 7-day sprouts (6.4 and 11.5 folds, respectively). In HPLC analysis, seventeen phenolic compounds were detected on 7-day sprouts compared to fifteen in dry seeds, where two new phenolic compounds (p-coumaric acid and kaempferol) were detected. The concentrations of all the identified phenolic compounds increased several folds (1.8-27) on 7-day sprouts. The total antioxidant activity increased 10, 17, and 29 folds on 7-day sprouts using DPPH, ABTS and PMC antioxidant methods, respectively compared to the dry seeds. Both antioxidant and carbohydrate-cleaving enzymes increased in chia sprouts and correlated with their phenolic content and antioxidant activity. The phenolic content of 7-day sprouts showed a potent antibacterial activity against some human enteric pathogenic bacteria including Escherichia coli O157-H7, Salmonella typhi, Pseudomonas aeruginosa and Staphylococcus aureus with lower MIC values compared to the raw seeds.

Diabetic complications and oxidative stress: The role of phenolic-rich extracts of saw palmetto and date palm seeds.

Recently, we reported that the date palm seed (DP) and saw palmetto seed (SP) extracts possessed a great amount of phenolic contents with potent antioxidant, antimicrobial, and anti-inflammatory activities. Therefore, this study aimed to assess the role of DP and SP phenolic-rich extracts in modulating diabetic complications and oxidative stress in the STZ- diabetic rat. DP and SP extracts significantly inhibited both microbial and pancreatic α-amylases. The STZ-induced diabetic rat groups treated with DP and SP extracts exhibited reversed hyperglycemia (40% and 54%, p < .001-.01) and body weight (70%, p < .001) alteration close to normal. Moreover, DP and SP extracts modulated serious damages in the structures of the pancreas, kidney, and liver of diabetic rats. DP and SP extracts improved the decline of the activities of antioxidant enzymes: Catalase, glutathione-S-transferase, glutathione reductase, and glutathione peroxidase in liver, kidney, and pancreas of the diabetic rats. PRACTICAL APPLICATIONS: Generally, date seed is a rich source of dietary fibers, polyphenols, and antioxidants and used in foods and pharmaceuticals. Our study reported that date palm seed (DP) and saw palmetto seed (SP) phenolic-rich extracts attenuated diabetes and its complications, probably tissue regeneration and normalizing the oxidative stress in the STZ-induced diabetic rat.

l-Amino acid oxidase from Cerastes vipera snake venom: Isolation, characterization and biological effects on bacteria and tumor cell lines.

A homodimeric l-amino acid oxidase enzyme (Cv-LAAOI) was isolated from the venom of Cerastes vipera (Egyptian Sand viper) using gel filtration followed by anion exchange chromatography. The molecular mass of Cv-LAAO is 120 kDa in its native form and 60 kDa in its monomeric form. The optimum enzyme activity was achieved on l-Leucine as a substrate in 50 mM buffer pH 7.5 at 50 °C. The Cv-LAAOI activity was significantly reduced by increasing the temperature over 40 °C, lost 75% of its activity at 60 °C and inhibited completely at 80 °C. The Cv-LAAOI attains the highest substrate specificity towards L-Met. The results have also indicated that Mn2+ enhances the enzyme activity by 10%, while Cu2+, Hg2+, Ni2+, Co2+ have suppressive effects on the Cv-LAAOI activity. On the other hand, EDTA has no significant effect on the enzyme activity. The kinetic parameters of Cv-LAAOI activity (Km, Kcat and Vmax) estimated on l-Leucine at pH 8 and 37 °C were found to be 2 mM, 12 S-1 and 16.7 µmol/min/ml, respectively. In addition, the results have shown that Cv-LAAOI exhibits a significant bactericidal activity against gram-positive and gram-negative bacteria, particularly Staphylococcus aureus and Escherichia coli with MIC values of 20 µg/ml. Moreover, Cv-LAAOI has exhibited a considerable cytotoxic activity against breast cancer cell line (MCF-7) with IC50 value 2.75 ±â€¯0.38 µg/ml compared with different tumor cell lines (liver HepG2, lung A549, colon HCT116 and prostate PC3). Furthermore, Cv-LAAOI has triggered antiproliferative activity via extensive H2O2 generation as indicated by the increase in H2O2 and TBARS levels accompanied by the depletion in the catalase activity (CAT) in MCF-7 treated cells compared to the untreated ones. Thus, these findings clearly indicate that Cv-LAAOI has a selective cytotoxic effect on breast cancer cell line, demonstrating a great prospective for future use in cancer therapy.