Jack bean (Canavalia ensiformis) seeds contain several biologically important proteins among which α-mannosidase (EC 3.2.1.24) has been purified, its biochemical properties studied and widely used in glycan analysis. In the present study, we have used the purified enzyme and derived its amino acid sequence covering both the known subunits (molecular mass of ∼66,000 and ∼44,000 Da) hitherto not known in its entirety. Peptide de novo sequencing and structural elucidation of N-glycopeptides obtained either directly from proteolytic digestion or after zwitterionic hydrophilic interaction liquid chromatography solid phase extraction-based separation were performed by use of nanoelectrospray ionization quadrupole time-of-flight mass spectrometry and low-energy collision-induced dissociation experiments. De novo sequencing provided new insights into the disulfide linkage organization, intersection of subunits and complete N-glycan structures along with site specificities. The primary sequence suggests that the enzyme belongs to glycosyl hydrolase family 38 and the N-glycan sequence analysis revealed high-mannose oligosaccharides, which were found to be heterogeneous with varying number of hexoses viz, Man8-9GlcNAc2 and Glc1Man9GlcNAc2 in an evolutionarily conserved N-glycosylation site. This site with two proximal cysteines is present in all the acidic α-mannosidases reported so far in eukaryotes. Further, a truncated paucimannose type was identified to be lacking terminal two mannose, Man1(Xyl)GlcNAc2 (Fuc).
Polygalacturonase-inhibitor proteins (PGIPs) are important plant defense proteins which modulate the activity of microbial polygalacturonases (PGs) leading to elicitor accumulation. Very few studies have been carried out towards understanding the role of PGIPs in monocot host defense. Hence, present study was taken up to characterize a native PGIP from pearl millet and understand its role in resistance against downy mildew. A native glycosylated PGIP (PglPGIP1) of ~43 kDa and pI 5.9 was immunopurified from pearl millet. Comparative inhibition studies involving PglPGIP1 and its non-glycosylated form (rPglPGIP1; recombinant pearl millet PGIP produced in Escherichia coli) against two PGs, PG-II isoform from Aspergillus niger (AnPGII) and PG-III isoform from Fusarium moniliforme, showed both PGIPs to inhibit only AnPGII. The protein glycosylation was found to impact only the pH and temperature stability of PGIP, with the native form showing relatively higher stability to pH and temperature changes. Temporal accumulation of both PglPGIP1 protein (western blot and ELISA) and transcripts (real time PCR) in resistant and susceptible pearl millet cultivars showed significant Sclerospora graminicola-induced accumulation only in the incompatible interaction. Further, confocal PGIP immunolocalization results showed a very intense immuno-decoration with highest fluorescent intensities observed at the outer epidermal layer and vascular bundles in resistant cultivar only. This is the first native PGIP isolated from millets and the results indicate a role for PglPGIP1 in host defense. This could further be exploited in devising pearl millet cultivars with better pathogen resistance.
Alcohol abuse causes severe metabolic abnormalities inducing hepatic damage and malnutrition. Since higher Fischer ratio proteins have therapeutic value in liver disease conditions, an investigation was undertaken to study the ameliorative effect of the enhanced Fischer ratio flaxseed protein hydrolysate (EFR-FPH) alone and in combination with antioxidant micronutrients on ethanol-induced hepatotoxicity in a rat model. The EFR-FPH was prepared by dual enzymatic hydrolysis and charcoal treatment of flaxseed protein. The ratio of the branched-chain to aromatic amino acids (Fischer ratio) was found to be 7.08. The EFR-FPH characterized using LC-MS/MS, showed the abundance of free leucine and isoleucine compared to phenylalanine and tyrosine. The MALDI-TOF MS analysis revealed the larger peptides present in EFR-FPH with mass 2.3 kDa. The EFR-FPH improved the nutritional status, liver function and antioxidant enzymes activity in the ethanol hepatotoxicity induced rat model. The hepatoprotective effect of EFR-FPH was significantly enhanced when combined with selenium or vitamin E. Ethanol-induced changes in the histology of liver tissue were effectively suppressed in the group receiving EFR-FPH. Flaxseed-based hepatoprotective dietary supplement was formulated incorporating an optimum level of EFR-FPH (10%) based on sensory acceptability and was fortified with selenium and vitamin E. The hepatoprotective formulation significantly lowered AST, ALT, ALP, and bilirubin by 47, 61, 55, and 78%, respectively, and improved the antioxidant defense system in the ethanol hepatotoxicity induced rat model. The current investigation suggests that EFR-FPH in synergy with antioxidant micronutrients is potent in ameliorating ethanol-induced hepatotoxicity and has a potential to form a hepatoprotective dietary supplement.
BACKGROUND:Ruta graveolens is one of the most used phytomedicines. To date, there is no report of determining the bioactivity of R. graveolens against cariogenic causing bacteria (Streptococcus mutans and Streptococcus sobrinus).OBJECTIVE:The objective of the present study was to determine the antibacterial activity and metabolite profile of R. graveolens against S. mutans and S. sobrinus.MATERIALS AND METHODS:R. graveolens plant material was collected and processed in the month of February. The plant material was extracted by Soxhlet apparatus using methanol solvent. Two strains of S. mutans and two strains of S. sobrinus were isolated from dental caries-active participants and cultured on mitis salivarius-bacitracin agar. The antibacterial susceptibility testing of methanolic extract of R. graveolens was performed by disc diffusion method. The metabolite profile of the plant extract was determined using electrospray ionization-tandem mass spectrometry.RESULTS:The methanolic extract of R. graveolens showed a promising antibacterial activity against S. mutans and S. sobrinus. Two compounds named γ-fagarine and kokusaginine were identified from the methanolic extract of R. graveolens.CONCLUSION:The study concluded that R. graveolens contains significant antibacterial activity. However, further investigations are suggested to understand the anticaries properties of these pure compounds.
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