The Inhibitory Effect of KerraTM, KSTM, and MinozaTM on Human Papillomavirus Infection and Cervical Cancer.

Background and Objectives: Cervical cancer is one of the most common types of frequently found cancers in Thailand. One of the causative agents is the infection of the high-risk human papillomavirus (HPV) type 16 and 18. Traditional medicines are rich sources of bioactive compounds which are a valuable source for the development of novel cancer therapies. In this study, the therapeutic effects of 3 traditional medicines, KerraTM, KSTM, and MinozaTM, were studied on HeLa and CaSki cells. Materials and Methods: The effects of KerraTM, KSTM, and MinozaTM on cancer cells were evaluated through cytotoxicity and cell death assays. The infection assay using HPV-16 pseudovirus was also carried out. Results: All traditional medicines efficiently suppressed cell growths of HeLa and CaSki, with KerraTM being the most potent anticancer agent followed by KSTM and MinozaTM. KerraTM at 158 µg/mL and 261 µg/mL significantly increases the percentage inhibition of the HPV-16 pseudovirus infection in a pre-attachment step in a dose-dependent manner, while KSTM at 261 µg/mL efficiently inhibited viral infection in both pre-attachment and adsorption steps. However, KerraTM, KSTM, and MinozaTM at subtoxic concentrations could not reduce the viral E6 mRNA expressions of HPV-16 and HPV-18. Cell death assay by acridine orange/ethidium bromide showed that KerraTM increased population of dead cells in dose-dependent manner in both CaSki and HeLa. The percentage of secondary necrosis in KerraTM-treated CaSki was higher than that of HeLa cells, while the percentage of late apoptotic cells in HeLa was higher than that of CaSki, indicating that HeLa was more susceptible to KerraTM than CaSki. For KSTM and MinozaTM, these extracts at 250 µg/mL promoted autophagy over cell death. At 500 µg/mL, the percentage of dead cells in KerraTM was higher than that of KSTM and MinozaTM. Conclusions: KerraTM is a potent traditional medicine for promoting cancer cell death. KerraTM is possibly useful in the prevention and treatment of cervical cancer. Further investigation will be carried out to gain a better understanding of the biochemical mechanism and the pharmacological activity underlying this effect.

Biochemical and structural comparisons of non-nucleoside reverse transcriptase inhibitors against feline and human immunodeficiency viruses.

BACKGROUND: Feline immunodeficiency virus (FIV) causes an acquired immunodeficiency-like syndrome in cats. FIV is latent. No effective treatment has been developed for treatment the infected cats. The first and second generations non-nucleoside reverse transcriptase inhibitors (NNRTIs) for HIV treatment, nevirapine (NVP) and efavirenz (EFV), and rilpivirine (RPV), were used to investigate the potential of NNRTIs for treatment of FIV infection. OBJECTIVE: This study aims to use experimental and in silico approaches to investigate the potential of NNRTIs, NVP, EFV, and RPV, for inhibition of FIV reverse transcriptase (FIV-RT). METHODS: The FIV-RT and human immunodeficiency virus reverse transcriptase (HIV-RT) were expressed and purified using chromatography approaches. The purified proteins were used to determine the IC50 values with NVP, EFV, and RPV. Surface plasmon resonance (SPR) analysis was used to calculate the binding affinities of NNRTIs to HIV-RT and FIV-RT. The molecular docking and molecular dynamic simulations were used to demonstrate the mechanism of FIV-RT and HIV-RT with first and second generation NNRTI complexes. RESULTS: The IC50 values of NNRTIs NVP, EFV, and RPV against FIV-RT were in comparable ranges to HIV-RT. The SPR analysis showed that NVP, EFV, and RPV could bind to both enzymes. Computational calculation also supports that these NNRTIs can bind with both FIV-RT and HIV-RT. CONCLUSIONS: Our results suggest the first and second generation NNRTIs (NVP, EFV, and RPV) could inhibit both FIV-RT and HIV-RT.

Phytochemical and Pharmacological Properties of a Traditional Herb, Strobilanthes Cusia (Nees) Kuntze.

The present investigation aimed to determine the effectiveness of bioactive components extracted from Hom herbs (Strobilanthes cusia (Nees) Kuntze) using the solvent-free microwave-assisted extraction (MAE) method. The obtained bioactive components were analyzed for total phenolic content (TPC) and active ingredient content. The Hom extracts were examined for antioxidant, antibacterial, anti-inflammatory, cytotoxic, and anticancer activities. The comparative analysis of extraction methods MAE was studied by using different solvents such as ethanol (EtOH), 50% ethanol (50EtOH) and distilled water (DW). The results obtained by the MAE method with DW as solvent show the TPC of 104.41±1.36 mg GAE/g crude and tryptanthrin 0.1138±0.0014 mg/g crude and indigo 0.0622±0.0015 mg/g crude. Comparatively, values ​​detected in the 50% EtOH extract were not significantly different at the 95% confidence level. At the same time, levels of indirubin were detected at levels equivalent to that of ethanol extracts. The DW extract from MAE had an IC50 value against the DPPH scavenging assay of 0.1927±0.0756 mg/ml, comparable to the test results of extracts of ethanol and 50% ethanol. The bioactive extracted using the MAE with water as solvent had minimum inhibitory concentration (MIC) and could suppress infection at 10 mg/disc. It was also observed that the extracts from the conventional extraction technique using ethanol as the solvent continued to be highly effective against Bacillus cereus even after employing the EtOH or 50% EtOH. Hom extract's MIC value representing inhibiting B. cereus was 0.625 mg/disc. Still, EtOH-extracted Hom demonstrated the highest cytotoxicity against 16HBEo- by reducing cell survival rate by less than 50% while the others did not. Interestingly, Hom that had been extracted using 50EtOH and DW with MAE had an anticancer impact on A549 by reducing the survival rate in a dose-dependent manner.

Crystal structure and identification of amino acid residues for catalysis and binding of GH3 AnBX ß-xylosidase from Aspergillus niger.

ß-Xylosidases catalyze the hydrolysis of xylooligosaccharides to xylose in the final step of hemicellulose degradation. AnBX, which is a GH3 ß-xylosidase from Aspergillus niger, has a high catalytic efficiency toward xyloside substrates. In this study, we report the three-dimensional structure and the identification of catalytic and substrate binding residues of AnBX by performing site-directed mutagenesis, kinetic analysis, and NMR spectroscopy-associated analysis of the azide rescue reaction. The structure of the E88A mutant of AnBX, determined at 2.5-Å resolution, contains two molecules in the asymmetric unit, each of which is composed of three domains, namely an N-terminal (ß/α)8 TIM-barrel-like domain, an (α/ß)6 sandwich domain, and a C-terminal fibronectin type III domain. Asp288 and Glu500 of AnBX were experimentally confirmed to act as the catalytic nucleophile and acid/base catalyst, respectively. The crystal structure revealed that Trp86, Glu88 and Cys289, which formed a disulfide bond with Cys321, were located at subsite -1. Although the E88D and C289W mutations reduced catalytic efficiency toward all four substrates tested, the substitution of Trp86 with Ala, Asp and Ser increased the substrate preference for glucoside relative to xyloside substrates, indicating that Trp86 is responsible for the xyloside specificity of AnBX. The structural and biochemical information of AnBX obtained in this study provides invaluable insight into modulating the enzymatic properties for the hydrolysis of lignocellulosic biomass. KEY POINTS: • Asp288 and Glu500 of AnBX are the nucleophile and acid/base catalyst, respectively • Glu88 and the Cys289-Cys321 disulfide bond are crucial for the catalytic activity of AnBX • The W86A and W86S mutations in AnBX increased the preference for glucoside substrates.

Anti-HIV-1 reverse transcriptase property of some edible mushrooms in Asia.

Human immunodeficiency virus (HIV) causes acquired immunodeficiency syndrome (AIDS), which is a serious health threat worldwide. One of its core enzymes, reverse transcriptase (RT), is a target for HIV inhibition. A number of bioactive compounds have been successfully used for HIV treatment. However, HIV rapidly mutates, and long-term treatment can cause drug-resistant strains. Therefore, new inhibitors are required to overcome this problem. In this study, the aqueous, ethanolic and hexane crude extracts of 19 edible and medicinal mushrooms, which are widely grown and available commercially in Thailand, were screened against HIV-1 RT. The results showed that the water extracts of A. blazei and I. obliquus, the ethanol extracts of I. obliquus and P. igniarius and the hexane extract of I. obliquus exhibited strong anti-HIV-1 RT activity with IC50 values of 1.92 ± 0.15, 4.39 ± 0.79, 6.17 ± 0.76 and 7.75 ± 246 µg/ml, respectively. These mushrooms have the potential for HIV treatment, and further study on identification of the bioactive compounds against HIV-1 RT should be performed.

Anti-feline immunodeficiency virus reverse transcriptase properties of some medicinal and edible mushrooms.

BACKGROUND AND AIM: Feline immunodeficiency virus (FIV) causes AIDS-like symptoms in domestic and wild cats. Treatment of infected cats has been performed using human anti-HIV drugs, which showed some limitations. This study aimed to determine the anti-FIV potential of some mushrooms. MATERIALS AND METHODS: A total of 17 medicinal and edible mushrooms were screened to find their inhibitory effect against FIV reverse transcriptase (FIV-RT). Three solvents, water, ethanol, and hexane, were used to prepare crude mushroom extracts. Fluorescence spectroscopy was used to perform relative inhibition and 50% inhibitory concentrations (IC50) studies. RESULTS: The ethanol extract from dried fruiting bodies of Inonotus obliquus showed the strongest inhibition with an IC50 value of 0.80±0.16 µg/mL. The hexane extract from dried mycelium of I. obliquus and ethanol and water extracts from fresh fruit bodies of Phellinus igniarius also exhibited strong activities with the IC50 values of 1.22±0.20, 4.33±0.39, and 6.24±1.42 µg/mL, respectively. The ethanol extract from fresh fruiting bodies of Cordyceps sinensis, hexane extracts from dried mycelium of I. obliquus, ethanol extracts of Ganoderma lucidum, hexane extracts of fresh fruiting bodies of Morchella esculenta, and fresh fruiting bodies of C. sinensis showed moderate anti-FIV-RT activities with IC50 values of 29.73±12.39, 49.97±11.86, 65.37±14.14, 77.59±8.31, and 81.41±17.10 µg/mL, respectively. These mushroom extracts show anti-FIV potential. CONCLUSION: The extracts from I. obliquus, P. igniarius, C. sinensis, and M. esculenta showed potential anti-FIV activity.

Multiple mutations in the aglycone binding pocket to convert the substrate specificity of dalcochinase to linamarase.

Dalcochinase from Dalbergia cochinchinensis Pierre and linamarase from Manihot esculenta Crantz are ß-glucosidases which share 47% sequence identity, but show distinct substrate specificities in hydrolysis and transglucosylation. Previously, three amino acid residues of dalcochinase, namely I185, N189 and V255, were identified as being important for determining substrate specificity. In this study, kinetic analysis of the ensuing double and triple mutants of dalcochinase showed that only those containing the 185A mutation could appreciably hydrolyze linamarin as well as transfer glucose to 2-methyl-2-propanol. So, the space provided by the I185A mutation appeared to be a prerequisite for accommodation of the aglycone moiety containing three substituents at the carbinol carbon. However, quantitative analysis of the energy parameters revealed mostly antagonistic interactions between these mutations. In addition, the N189F mutant showed a potential for use in enzymatic synthesis of alkyl glucosides via transglucosylation and reverse hydrolysis reactions. Thus, substitution of only 2-3 key residues in the aglycone binding pocket of dalcochinase could convert its specificities to that of linamarase, as well as to be suitable for any chosen hydrolytic or synthetic applications.

Cloning, expression and characterization of ß-xylosidase from Aspergillus niger ASKU28.

ß-Xylosidases catalyze the breakdown of ß-1,4-xylooligosaccharides, which are produced from degradation of xylan by xylanases, to fermentable xylose. Due to their important role in xylan degradation, there is an interest in using these enzymes in biofuel production from lignocellulosic biomass. In this study, the coding sequence of a glycoside hydrolase family 3 ß-xylosidase from Aspergillus niger ASKU28 (AnBX) was cloned and expressed in Pichia pastoris as an N-terminal fusion protein with the α-mating factor signal sequence (α-MF) and a poly-histidine tag. The expression level was increased to 5.7 g/l in a fermenter system as a result of optimization of only five codons near the 5' end of the α-MF sequence. The recombinant AnBX was purified to homogeneity through a single-step Phenyl Sepharose chromatography. The enzyme exhibited an optimal activity at 70°C and at pH 4.0-4.5, and a very high kinetic efficiency toward a xyloside substrate. AnBX demonstrated an exo-type activity with retention of the ß-configuration, and a synergistic action with xylanase in hydrolysis of beechwood xylan. This study provides comprehensive data on characterization of a glycoside hydrolase family 3 ß-xylosidase that have not been determined in any prior investigations. Our results suggested that AnBX may be useful for degradation of lignocellulosic biomass in bioethanol production, pulp bleaching process and beverage industry.

Mutational analysis in the glycone binding pocket of Dalbergia cochinchinensis ß-glucosidase to increase catalytic efficiency toward mannosides.

Dalcochinase and Abg are glycoside hydrolase family 1 ß-glucosidases from Dalbergia cochinchinensis Pierre and Agrobacterium sp., respectively, with 35% sequence identity. However, Abg shows much higher catalytic efficiencies toward a broad range of glycone substrates than dalcochinase does, possibly due to the difference in amino acid residues around their glycone binding pockets. Site-directed mutagenesis was used to replace the amino acid residues of dalcochinase with the corresponding residues of Abg, generating three single mutants, F196H, S251V, and M369E, as well as the corresponding three double mutants and one triple mutant. Among these, the F196H mutant showed increases in catalytic efficiency toward almost all glycoside substrates tested, with the most improved catalytic efficiency being a 3-fold increase for hydrolysis of p-nitrophenyl ß-D-mannoside, suggesting a preferred polar residue at this position and consistent with the presence of histidine at this position in two other GH1 glycosidases from barley and rice that prefer ß-mannosides. In addition, the M369E mutation resulted in a small increase in catalytic efficiency for cleavage of p-nitrophenyl ß-D-galactoside. By contrast, the multiple mutants were up to 8-fold less efficient than the recombinant wild-type dalcochinase, and displayed primarily antagonistic interactions between these residues. Thus, differences in catalytic efficiency between dalcochinase and Abg are therefore not primarily due to differences in the residues that directly contact the substrate, but derive largely from contributions from more remote residues and the overall architecture of the active site.