• Journal of Microbiology and Biotechnology
• /
• v.22 no.8
• /
• pp.1141-1147
• /
• 2012

Polyhydroxyalkanoate (PHA) is a class of biodegradable plastics that have great potential applications in the near future. In this study, the micro-biodiversity and productivity of PHA-accumulating bacteria in activated sludge from a domestic wastewater treatment plant were investigated. A previously reported primer set and a self-designed primer set (phaCF1BO/phaCR2BO) were both used to amplify the PHA synthase (phaC) gene of isolated colonies. The new primers demonstrated higher sensitivity for phaC, and combining the PCR results of the two primer sets was able to widen the range of detected genera and raise the sensitivity to nearly 90%. Results showed that 85.3% of the identified bacteria were Gram-negative, with Ralstonia as the dominant genus, and 14.7% were Gram-positive. In addition, Zoogloea and Rhizobium contained the highest amounts of intracellular PHA. It is apparent that glucose was a better carbon source than pentone or tryptone for promoting PHA production in Micrococcus. Two different classes, class I and class II, of phaC were detected from alphaproteobacteria, betaproteobacteria, and gammaproteobacteria, indicating the wide diversity of PHA-accumulating bacteria in this particular sampling site. Simultaneous wastewater treatment and PHA production is promising by adopting the high PHA-accumulating bacteria isolated from activated sludge.

• Journal of Microbiology and Biotechnology
• /
• v.28 no.9
• /
• pp.1482-1492
• /
• 2018

Fusarium oxysporum trypsin (FOT) is a fungal serine protease similar to mammal trypsin. The FOT could be successfully expressed in Pichiapastoris by engineering the natural propeptide APQEIPN. In this study, we constructed two recombinant enzymes with engineered amino acid sequences added to the N-terminus of FOT and expressed in P. pastoris. The N-terminal residues had various effects on the structural and functional properties of trypsin. The FOT, and the recombinants TE (with peptide YVEF) and TS (with peptide YV) displayed the same optimum temperature ($40^{\circ}C$) and pH (8.0). However, the combinants TE and TS showed significantly increased thermal stability at $40^{\circ}C$ and $50^{\circ}C$. Moreover, the combinants TE and TS also showed enhanced tolerance of alkaline pH conditions. Compared with those of wild-type FOT, the intramolecular hydrogen bonds and the cation ${\pi}$-interactions of the recombinants TE and TS were significantly increased. The recombinants TE and TS also had significantly increased catalytic efficiencies (referring to the specificity constant, $k_{cat}/K_m$), 1.75-fold and 1.23-fold than wild-type FOT. In silico modeling analysis uncovered that the introduction of the peptides YVEF and YV resulted in shorter distances between the substrate binding pocket (D174, G198, and G208) and catalytic triad (His42, Asp102, and Ser180), which would improve the electron transfer rate and catalytic efficiency. In addition, N-terminal residues modification described here may be a useful approach for improving the catalytic efficiencies and characteristics of other target enzymes.

• Bulletin of the Korean Chemical Society
• /
• v.32 no.7
• /
• pp.2358-2368
• /
• 2011

Density functional theory (DFT) and time-dependent density functional theory (TDDFT) calculations, employing the B3LYP method and the LANL2DZ, 6-31G$^*$(LANL2DZ for Tc), 6-31G$^*$(cc-pVDZ-pp for Tc) and DGDZVP basis sets, have been performed to investigate the electronic structures and absorption spectra of the technetium-99m-labeled methylenediphosphonate ($^{99m}Tc$-MDP) complex of the simplest diphosphonate ligand. The bonding situations and natural bond orbital compositions were studied by the Mulliken population analysis (MPA) and natural bond orbital (NBO) analysis. The results indicate that the ${\sigma}$ and ${\pi}$ contributions to the Tc-O bonds are strongly polarized towards the oxygen atoms and the ionic contribution to the Tc-O bonding is larger than the covalent contribution. The electronic transitions investigated by TDDFT calculations and molecular orbital analyses show that the origin of all absorption bands is ascribed to the ligand-to-metal charge transfer (LMCT) character. The solvent effect on the electronic structures and absorption spectra has also been studied by performing DFT and TDDFT calculations at the B3LYP/6-31G$^*$(cc-pVDZ-pp for Tc) level with the integral equation formalism polarized continuum model (IEFPCM) in different media. It is found that the absorption spectra display blue shift in different extents with the increase of solvent polarity.

• Asian Pacific Journal of Cancer Prevention
• /
• v.15 no.7
• /
• pp.3219-3226
• /
• 2014

Chemotherapy continues to be a mainstay of cancer treatment, although drug resistance is a major obstacle. Lipid metabolism plays a critical role in cancer pathology, with elevated ether lipid levels. Recently, alkylglyceronephosphate synthase (AGPS), an enzyme that catalyzes the critical step in ether lipid synthesis, was shown to be up-regulated in multiple types of cancer cells and primary tumors. Here, we demonstrated that silencing of AGPS in chemotherapy resistance glioma U87MG/DDP and hepatic carcinoma HepG2/ADM cell lines resulted in reduced cell proliferation, increased drug sensitivity, cell cycle arrest and cell apoptosis through reducing the intracellular concentration of lysophosphatidic acid (LPA), lysophosphatidic acid-ether (LPAe) and prostaglandin E2 (PGE2), resulting in reduction of LPA receptor and EP receptors mediated PI3K/AKT signaling pathways and the expression of several multi-drug resistance genes, like MDR1, MRP1 and ABCG2. ${\beta}$-catenin, caspase-3/8, Bcl-2 and survivin were also found to be involved. In summary, our studies indicate that AGPS plays a role in cancer chemotherapy resistance by mediating signaling lipid metabolism in cancer cells.

• Journal of Pharmaceutical Investigation
• /
• v.39 no.4
• /
• pp.257-261
• /
• 2009

Effective therapeutics for renal failures have not yet been developed until now. Recently, there was a report showing that Wen-pi-tang-Hab-Wu-ling-san (WHW) prescriptions had the potential to prevent renal failures through the increased expression of HSP-27 and HSP-72 after ischemia/reperfusion. Therefore, formulation studies by pH-specific released systems were carried out to exhibit the optimal activity of WHW prescriptions in this study. WHW prescriptions were separately extracted using water into two parts of stomach-released (SR) and intestine-released (IR) extracts. Subsequently, the double-layered tablet was prepared using the SR extracts and pharmaceutical additives and enteric-coated IR tablet. Dissolution studies were carried out to figure out the release of cinnamic acid and icarrin from SR tablet, IR tablet and double-layered tablet, respectively. The complete release of cinnamic acid from SR tablet showed 90min after dissolution in pH 1.2 and insignificant drug released from IR tablet. As well as, icarrin from IR tablet completely released in pH 6.8 and 7.4 as enteric-coating film dissolved.

• Journal of Ginseng Research
• /
• v.46 no.3
• /
• pp.418-425
• /
• 2022

Background: Sorafenib is effective in treating hepatoma, but most patients develop resistance to it. STAT3 signaling has been implicated in sorafenib resistance. Artesunate (ART) and 20(R)-ginsenoside Rg3 (Rg3) have anti-hepatoma effects and can inhibit STAT3 signaling in cancer cells. This study aimed to evaluate the effects of Rg3 in combination with ART (Rg3-plus-ART) in overcoming sorafenib resistance, and to examine the involvement of STAT3 signaling in these effects. Methods: Sorafenib-resistant HepG2 cells (HepG2-SR) were used to evaluate the in vitro anti-hepatoma effects of Rg3-plus-ART. A HepG2-SR hepatoma-bearing BALB/c-nu/nu mouse model was used to assess the in vivo anti-hepatoma effects of Rg3-plus-ART. CCK-8 assays and Annexin V-FITC/PI double staining were used to examine cell proliferation and apoptosis, respectively. Immunoblotting was employed to examine protein levels. ROS generation was examined by measuring DCF-DA fluorescence. Results: Rg3-plus-ART synergistically reduced viability of, and evoked apoptosis in HepG2-SR cells, and suppressed HepG2-SR tumor growth in mice. Mechanistic studies revealed that Rg3-plus-ART inhibited activation/phosphorylation of Src and STAT3 in HepG2-SR cultures and tumors. The combination also decreased the STAT3 nuclear level and induced ROS production in HepG2-SR cultures. Furthermore, overactivation of STAT3 or removal of ROS diminished the anti-proliferative effects of Rg3-plus-ART, and removal of ROS diminished Rg3-plus-ART's inhibitory effects on STAT3 activation in HepG2-SR cells. Conclusions: Rg3-plus-ART overcomes sorafenib resistance in experimental models, and inhibition of Src/STAT3 signaling and modulation of ROS/STAT3 signaling contribute to the underlying mechanisms. This study provides a pharmacological basis for developing Rg3-plus-ART into a novel modality for treating sorafenib-resistant hepatoma.