Backgroundα–glucosidase (HBGase) plays a key role in hydrolyzing α-glucosidic linkages. In Apis mellifera, three isoforms of HBGase (I, II and III) have been reported, which differ in their nucleotide composition, encoding amino acid sequences and enzyme kinetics. Recombinant (r)HBGase II from A. cerana indica (rAciHBGase II) was focused upon here due to the fact it is a native and economic honeybee species in Thailand. The data is compared to the two other isoforms, AciHBGase I and III from the same bee species and to the three isoforms (HBGase I, II and III) in different bee species where available.ResultsThe highest transcript expression level of AciHBGase II was found in larvae and pupae, with lower levels in the eggs of A. cerana indica but it was not found in foragers. The full-length AciHBGase II cDNA, and the predicted amino acid sequence it encodes were 1,740 bp and 579 residues, respectively. The cDNA sequence was 90% identical to that from the HBGase II from the closely related A. cerana japonica (GenBank accession # NM_FJ752630.1). The full length cDNA was directionally cloned into the pPICZαA expression vector in frame with a (His)6 encoding C terminal tag using EcoRI and KpnI compatible ends, and transformed into Pichia pastoris. Maximal expression of the rAciHBGase II–(His)6 protein was induced by 0.5% (v/v) methanol for 96 h and secreted into the culture media. The partially purified enzyme was found to have optimal α-glucosidase activity at pH 3.5 and 45°C, with > 80% activity between pH 3.5–5.0 and 40–55°C, and was stabile (> 80% activity) at pH 4–8 and at < 25–65°C. The optimal substrate was sucrose.ConclusionsLike in A. mellifera, there are three isoforms of AciHBGase (I, II and III) that differ in their transcript expression pattern, nucleotide sequences and optimal enzyme conditions and kinetics.
A BSTRACT Aim: This in vitro study aimed to investigate the effect of propolis extracts from two different solvents on human submandibular salivary gland (HSG) tumor cell line. Materials and Methods: Propolis was extracted by dichloromethane (DCM) and hexane (HEX). Crude extracts were prepared from 6.25 to 200 µg/mL in Dulbecco’s modified eagle medium without serum. Flavonoid and total phenolic contents of crude extracts were measured using a modified colorimetric method. The cytotoxicity was evaluated by 3-[4, 5-dimethylthiazol-2-yl]-2,5 diphenyl-tetrazolium (MTT) assay and lactate dehydrogenase (LDH) release assay. The statistics were analyzed by independent sample t-test. Results: Propolis extracts obtained using DCM and HEX exhibited comparable % yield (38.58 and 38.25) and physical characteristics and different amounts of flavonoid (0.439 ± 0.02 and 0.250 ± 0.01 mg catechin/g sample) and total phenolic compounds (3.759 ± 0.03 and 1.618 ± 0.03 mg gallic acid equivalents/g sample). The DCM group at 25, 50, 100, and 200 µg/mL as well as the HEX group at 50, 100, and 200 µg/mL significantly displayed a decrease in % cell viability and an increase in % cytotoxicity, compared with the untreated control group ( P < 0.05). The DCM group showed the half-maximal inhibitory concentration (IC50) of MTT (42.93 ± 2.70) and LDH (34.94 ± 0.22). The HEX group showed the IC50 of MTT (61.30 ± 5.39) and LDH (42.32 ± 1.00). Propolis extracts obtained using both DCM and HEX are effective to inhibit HSG viability. Conclusion: Regarding to the cell morphological observation, MTT and LDH assays, propolis extracts obtained using DCM and HEX exhibited the cytotoxic effect on HSG tumor cell line. Based on our knowledge, this research demonstrates the first preliminary result suggesting propolis as a natural product of choice for salivary gland cancer prevention and therapy.
Red kwao krua, Butea superba Roxb., is a herbal leguminous plant endemic in Thailand whose tuberous roots are used for male rejuvenation and the prevention of erectile dysfunction. Different populations from different provinces vary in their bioactivities, raising the need to evaluate the causes prior to optimal population selection and growth. Morphometric analysis of leaves collected from 34 populations from 24 provinces throughout Thailand using nine parameters for factor and cluster analyses were correlated with latitude and longitude revealing a clinal pattern. From the North to the South, leaf length increased in size in factor 1 but decreased in size in factor 2. Genetic analysis on the same samples, using DNA sequence analysis from the chloroplast rbcL, trnLF-cd, and trnLF-cf regions and maximum parsimony and neighbourjoining phylogenetic analysis, revealed essentially no genetic variation in the rbcL region, as expected. However, some between-population genetic variation was revealed by trnLF-cd and trnLF-cf sequences, suggesting potentially considerable genetic polymorphism. This was supported by preliminary RAPD analyses using five primers which indicated high genetic variation within and between populations.
Recombinant -glucosidase III (rHBGase III) from Apis cerana indica Fabricus (rAciHBGase III) was expressed in the yeast Pichia pastoris GS115, enriched and characterized. The full length cDNA of AciHbgase III (1.8 kb) was amplified by RT-PCR, cloned into the pPICZA expression vector and used to transform P. pastoris GS115. The maximum secreted expression level of rAciHBGase III [as an N terminal (His) 6 tagged chimera] was found 144 h after induction by 1% (v/v) methanol. Enrichment of the enzyme using histrap affinity purification revealed a single active glucosidase band with a molecular mass of ~68 kDa. The optimal pH and temperature for glucosidase activity of the enriched rAciHBGase III were pH 5.0 and 37°C, respectively, whilst the enzyme showed a good pH stability between pH 5.0 to 7.5, but not below pH 5.0, and a poor thermotolerance with < 10% and 0% residual activity at 40 and >50°C, respectively. The rAciHBGase showed a relatively high substrate specificity for maltose (K m of 4.5 mM) and p-nitrophenyl -D-glucoside (K m of 4.4 mM) compared to other reported HBGase enzymes.
Objective This study aimed to examine the effect of periodontal ligament stem cell conditioned medium (PDLSC-CM) on human gingival fibroblast (HGF) migration and collagen synthesis. Materials and Methods To assess cell viability, we extracted PDLSC-CM, and the total derived protein concentration was adjusted to 12.5 to 200 µg/mL, followed by treatment with HGFs. The viability of HGFs was observed for 24 hours using the MTT assay. Cell migration was monitored for 24 to 48 hours by wound healing and Boyden chamber assays. Collagen synthesis from HGFs was examined by picrosirius red dye and real-time polymerase chain reaction (PCR) to measure collagen type I and III gene expression for 7 to 10 days. A comparison among the groups was assessed using a one-way analysis of variance (ANOVA) and Bonferroni post hoc test, with the exception of the cell viability assay, which was subjected to Welch's test and Dunnett's T3 post hoc test. Results HGF viability was significantly enhanced by 12.5, 25, and 50 µg/mL PDLSC-CM. The HGFs treated with 50 µg/mL PDLSC-CM promoted cell migration as shown by wound healing and Boyden chamber assays. At this concentration, collagen synthesis increased at 10 days. Collagen type I gene expression increased by 1.6-fold (p < 0.001) and 4.96-fold (p < 0.001) at 7 and 10 days, respectively. Collagen type III gene expression showed an increase of 1.76-fold (p < 0.001) and 6.67-fold (p < 0.001) at the same time points. Conclusion Our study suggested that a low concentration of PDLSC-CM at 50 µg/mL has given an amelioration of HGFs providing for periodontal wound healing and periodontal regeneration, particularly migration and collagen synthesis.
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