Ginsenosides Rh1, Rg2, and Rg3 ameliorate dexamethasone-induced muscle atrophy in C2C12 myotubes.

High doses or prolonged use of the exogenous synthetic glucocorticoid dexamethasone (Dex) can lead to muscle atrophy. In this study, the anti-atrophic effects of ginsenosides Rh1, Rg2, and Rg3 on Dex-induced C2C12 myotube atrophy were assessed by XTT, myotube diameter, fusion index, and western blot analysis. The XTT assay results showed that treatment with Rh1, Rg2, and Rg3 enhanced cell viability in Dex-injured C2C12 myotubes. Compared with the control group, the myotube diameter and fusion index were both reduced in Dex-treated cells, but treatment with Rh1, Rg2, and Rg3 increased these parameters. Furthermore, Rh1, Rg2, and Rg3 significantly downregulated the protein expression of FoxO3a, MuRF1, and Fbx32, while also upregulating mitochondrial biogenesis through the SIRT1/PGC-1α pathway. It also prevents myotube atrophy by regulating the IGF-1/Akt/ mTOR signaling pathway. These findings indicate that Rh1, Rg2, and Rg3 have great potential as useful agents for the prevention and treatment of muscle atrophy.

Adenophora Stricta Root Extract Alleviates Airway Inflammation in Mice with Ovalbumin-Induced Allergic Asthma.

Adenophora stricta Miq. (Campanulaceae family) is a traditional herb used for relieving cough and phlegm in East Asia. This study explored the effects of A. stricta root extract (AsE) in ovalbumin (OVA)-induced allergic asthma and lipopolysaccharide (LPS)-stimulated macrophages. Administration of 100-400 mg/kg AsE dose-dependently decreased pulmonary congestion and suppressed the reduction of alveolar surface area in mice with OVA-mediated allergic asthma. Histopathological analysis of lung tissue and cytological analysis of bronchioalveolar lavage fluid showed that AsE administration significantly attenuated inflammatory cell infiltration into the lungs. In addition, AsE also alleviated OVA-specific immunoglobulin E, interleukin (IL)-4, and IL-5 production, which are essential for OVA-dependent activation of T helper 2 lymphocytes. In Raw264.7 macrophage cells, AsE significantly blocked nitric oxide, tumor necrosis factor-α, IL-1ß, IL-6, and monocyte chemoattractant factor-1 production in response to LPS. Results from an immunoblot assay revealed that AsE inhibited the phosphorylation of c-jun N-terminal kinase, inhibitory-κB kinase α/ß, and p65 in LPS-stimulated cells. Furthermore, 2-furoic acid, 5-hydroxymethylfurfural, and vanillic acid 4-ß-D-glucopyranoside in AsE were shown to inhibit the production of proinflammatory mediators by LPS. Taken together, the present results suggest that A. stricta root will be a useful herb for relieving allergic asthma through managing airway inflammation.

Adenophora Stricta Root Extract Protects Lung Injury from Exposure to Particulate Matter 2.5 in Mice.

Chronic exposure of particulate matter of less than 2.5 µm (PM2.5) has been considered as one of the major etiologies for various respiratory diseases. Adenophora stricta Miq. is a medicinal herb that has been used for treating respiratory diseases in East Asia. The present study investigated the effect of A. stricta root extract (AsE) on PM2.5-induced lung injury in mice. Oral administration of 100-400 mg/kg AsE for 10 days significantly reduced the PM2.5-mediated increase in relative lung weight, but there was no difference in body weight with AsE administration. In addition, AsE dose-dependently decreased congested region of the lung tissue, prevented apoptosis and matrix degradation, and alleviated mucus stasis induced by PM2.5. Moreover, cytological analysis of bronchioalveolar lavage fluid revealed that AsE significantly inhibited the infiltration of immune cells into the lungs. Consistently, AsE also decreased expression of proinflammatory cytokines and chemokines in lung tissue. Furthermore, AsE administration blocked reactive oxygen species production and lipid peroxidation through attenuating the PM2.5-dependent reduction of antioxidant defense system in the lungs. Therefore, A. stricta root would be a promising candidate for protecting lung tissue from air pollution such as PM2.5.

Immunomodulatory Effect of Polysaccharide from Fermented Morinda citrifolia L. (Noni) on RAW 264.7 Macrophage and Balb/c Mice.

This study aims to determine the immunomodulatory effects of a polysaccharide fraction from fermented M. citrifolia L. (FMP) in RAW 264.7 macrophages and Balb/c mice. M. citrifolia was fermented for 72 h using Lactobacillus brevis; polysaccharides were extracted using ethanol precipitation. The RAW 264.7 cells exposed to FMP (50, 100, and 200 µg/mL) for 24 h showed increased NO production, proinflammatory cytokine (IL-1ß, IL-6, and TNF-α) release, and COX-2 and iNOS protein expression. FMP (100, 200 mg/kg) and deacetylasperulosidic acid (DAA) (20 mg/kg) administered orally to Balb/c mice for 14 days upregulated NO production and NK cytotoxicity in abdominal cavity and spleen, respectively. Th1 and Th2 cytokines production and immune cell numbers increased in spleen, mesenteric lymph nodes (MLN), peritoneal exudate cells (PEC), Peyer's patches (PP), and peripheral blood mononuclear cells (PBMC). Therefore, FMP containing DAA can be used as materials for health functional foods to enhance immune responses.

Ultra-sensitive detection of kanamycin for food safety using a reduced graphene oxide-based fluorescent aptasensor.

Overuse of antibiotics has caused serious problems, such as appearance of super bacteria, whose accumulation in the human body through the food chain is a concern. Kanamycin is a common antibiotic used to treat diverse infections; however, residual kanamycin can cause many side effects in humans. Thus, development of an ultra-sensitive, precise, and simple detection system for residual kanamycin in food products is urgently needed for food safety. In this study, we identified kanamycin-binding aptamers via a new screening method, and truncated variants were analyzed for optimization of the minimal sequence required for target binding. We found various aptamers with high binding affinity from 34.7 to 669 nanomolar Kdapp values with good specificity against kanamycin. Furthermore, we developed a reduced graphene oxide (RGO)-based fluorescent aptasensor for kanamycin detection. In this system, kanamycin was detected at a concentration as low as 1 pM (582.6 fg/mL). In addition, this method could detect kanamycin accurately in kanamycin-spiked blood serum and milk samples. Consequently, this simple, rapid, and sensitive kanamycin detection system with newly structural and functional analysis aptamer exhibits outstanding detection compared to previous methods and provides a new possibility for point of care testing and food safety.

Mutational analysis of critical residues of FAD-independent catabolic acetolactate synthase from Enterococcus faecalis V583.

Catabolic acetolactate synthase (cALS) from Enterococcus faecalis is a FAD-independent enzyme, which catalyzes the condensation of two molecules of pyruvate to produce acetolactate. Mutational and kinetic analyses of variants suggested the importance of H111, Q112, and Q411 residues for catalysis in cALS. The wild-type and variants were expressed as equally soluble proteins and co-migrated to a size of 60 kDa on SDS-PAGE. Importantly, H111 in cALS, which is widely present as phenylalanine in many other ThDP-dependent enzymes, plays a crucial role in substrate binding. Interestingly, the H111 variants, H111R and H111F, demonstrated altered specific activity of H111 variants with 17- and 26-fold increases in Km, respectively, compared to wild-type cALS. Furthermore, Q112 variants, Q112E, Q112N, and Q112V, exhibited significantly lower specific activity with 70-, 15-, and 10-fold higher Ks for ThDP, respectively. In the case of Q411, the variant Q411E showed a 10-fold rise in Km and a 20-fold increase in Ks for ThDP. Further, the molecular docking results indicated that the binding mode of ThDP was slightly affected in the variants of cALS. Based on these results, we suggest that H111 plays a role in substrate binding, and further suggest that Q112 and Q411 might be involved in ThDP binding of cALS.

Characterization of recombinant FAD-independent catabolic acetolactate synthase from Enterococcus faecalis V583.

The catabolic acetolactate synthase (cALS) of Enterococcus faecalis V583 was cloned, expressed in Escherichia coli, and purified to homogeneity. The purified protein had a molecular weight of 60 kDa. The cALS of E. faecalis is highly homologous with other cALSs, while sharing low homology with its anabolic counterparts. The cALS of E. faecalis exhibits optimum activity at a temperature of 37°C and pH 6.8. Based on the enzyme characterization, the apparent K(m) for pyruvate was calculated to be 1.37 mM, while the K(c) for thiamin diphosphate (ThDP) and Mg(2+) were found to be 0.031 µM and 1.27 mM, respectively. Negligible absorbance at 450 nm and lack of activity enhancement upon addition of flavin adenine dinucleotide (FAD) to the assay buffer suggest that the cALS of E. faecalis is not FAD-dependent. The enzyme showed extreme stability against the organic solvent dimethyl sulfoxide (DMSO), whereas the activity decreased to less than 50% in the presence of acetone and ethanol.

Cloning, characterization and evaluation of potent inhibitors of Shigella sonnei acetohydroxyacid synthase catalytic subunit.

Acetohydroxyacid synthase (AHAS) is a thiamin diphosphate (ThDP)- and flavin adenine dinucleotide (FAD)-dependent plant and microbial enzyme that catalyzes the first common step in the biosynthesis of essential amino acids such as leucine, isoleucine and valine. To identify strong potent inhibitors against Shigella sonnei (S. sonnei) AHAS, we cloned and characterized the catalytic subunit of S. sonnei AHAS and found two potent chemicals (KHG20612, KHG25240) that inhibit 87-93% S. sonnei AHAS activity at an inhibitor concentration of 100uM. The purified S. sonnei AHAS had a size of 65kDa on SDS-PAGE. The enzyme kinetics revealed that the enzyme has a K(m) of 8.01mM and a specific activity of 0.117U/mg. The cofactor activation constant (K(s)) for ThDP and (K(c)) for Mg(++) were 0.01mM and 0.18mM, respectively. The dissociation constant (K(d)) for ThDP was found to be 0.14mM by tryptophan fluorescence quenching. The inhibition kinetics of inhibitor KHG20612 revealed an un-competitive inhibition mode with a K(ii) of 2.65mM and an IC(50) of 9.3µM, whereas KHG25240 was a non-competitive inhibitor with a K(ii of) 5.2mM, K(is) of 1.62mM and an IC(50) of 12.1µM. Based on the S. sonnei AHAS homology model structure, the docking of inhibitor KHG20612 is predicted to occur through hydrogen bonding with Met 257 at a 1.7Å distance with a low negative binding energy of -9.8kcal/mol. This current study provides an impetus for the development of a novel strong antibacterial agent targeting AHAS based on these potent inhibitor scaffolds.

Evaluation of substituted triazol-1-yl-pyrimidines as inhibitors of Bacillus anthracis acetohydroxyacid synthase.

Acetohydroxyacid synthase (AHAS), a potential target for antimicrobial agents, catalyzes the first common step in the biosynthesis of the branched-chain amino acids. The genes of both catalytic and regulatory subunits of AHAS from Bacillus anthracis (Bantx), a causative agent of anthrax, were cloned, overexpressed in Escherichia coli, and purified to homogeneity. To develop novel anti-anthracis drugs that inhibit AHAS, a chemical library was screened, and four chemicals, AVS2087, AVS2093, AVS2387, and AVS2236, were identified as potent inhibitors of catalytic subunit with IC(50) values of 1.0 +/- 0.02, 1.0 +/- 0.04, 2.1 +/- 0.12, and 2.0 +/- 0.08 microM, respectively. Further, these four chemicals also showed strong inhibition against reconstituted AHAS with IC(50) values of 0.05 +/- 0.002, 0.153 +/- 0.004, 1.30 +/- 0.10, and 1.29 +/- 0.40 microM, respectively. The basic scaffold of the AVS group consists of 1-pyrimidine-2-yl-1H-[1,2,4]triazole-3-sulfonamide. The potent inhibitor, AVS2093 showed the lowest binding energy, -8.52 kcal/mol and formed a single hydrogen bond with a distance of 1.973 A. As the need for novel antibiotic classes to combat bacterial drug resistance increases, the screening of new compounds that act against Bantx-AHAS shows that AHAS is a good target for new anti-anthracis drugs.