ZINGIPAIN,A CYSTEINE PROTEASE FROM Zingiber ottensii VALETON RHIZOMES WITH ANTIPROLIFERATIVE ACTIVITIES AGAINST FUNGI AND HUMAN MALIGNANT CELL LINES

The objective of this study was to investigate the activity of a protein identified as cysteine protease, purified from Zingiber ottensii Valeton rhizomes, in terms of antiproliferation against fungi, bacteria, and human malignant cell lines. By means of buffer extraction followed by (NH₄)₂SO₄ precipitation and ion-exchange chromatography, the obtained dominant protein (designated F50) was submitted to non-denaturing and reducing sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE), where a single band and three bands were revealed from eletrophoretic patterns, respectively. It could be concluded at this point that the F50 was potentially a heterotrimer or heterodimer composed of either two small (～13.8 and ～15.2 kD) subunits or these two together with a larger (～32.5 kD) one. In-gel digestion was carried out for the most intense band from reducing SDS-PAGE, and to the resulting material was applied liquid chromatography (LC)–mass spectroscopy (MS)/MS. The main F50 subunit was found to contain fragments with 100% similarity to zingipain-1, a cysteine protease first discovered in Zingiber officinale. The activity corresponding to the identified data, cysteine protease, was then confirmed in the F50 by azocasein assay and a positive result was obtained. The F50 then was further investigated for antiproliferation against three plant pathogenic fungi species by disk diffusion test, four bacterial species by direct exposure in liquid culture and dish diffusion tests, and five human malignant cell lines by tissue culture assay. It was found that a dose of 23.6 µg F50/0.3 cm² of paper disk exhibited the best inhibitory effect against Collectotrichum cassiicola, while lesser effects were found in Exserohilum turicicum and Fusarium oxysporum, respectively. No inhibitory effect against bacterial proliferation was detected in all studied bacterial strains. However, relatively strong antiproliferative effects were found against five human cell lines, with IC50 values ranging from 1.13 µg/mL (hepatoma cancer; HEP-G2) to 5.37 µg/mL (colon cancer; SW620). By periodic acid–Schiff's staining and phenol–sulfuric acid assay, the F50 was determined as a glycoprotein containing 26.30 ± 1.01% (by weight) of carbohydrate. Thus, a new glycoprotein with protease activity was successfully identified in Zingiber ottensii rhizome. The glycoprotein also contained antiproliferative activity against some plant pathogenic fungi and human cancer cell lines.     

Genome-wide location analysis reveals an important overlap between the targets of the yeast transcriptional regulators Rds2 and Adr1

A simple and rapid liquid chromatography tandem mass spectrometry (LC-MS/MS) method was developed for the quantification of tetrahydrobiopterin (BH4) and dopamine in rat brain using epsilon-acetamidocaproic acid (AACA) as an internal standard. Proteins in the samples were precipitated with acetonitrile and then the supernatants were separated by a Sepax Polar-Imidazole (2.1 × 100 mm, i.d., 3 μm) column using a mixture of 10mM ammonium formate in acetonitrile/water (75:25, v/v) as the mobile phase at a flow rate of 300 μl/min. Quantification was performed on a triple quadrupole mass spectrometer employing electrospray ionization with the operating conditions as multiple reaction monitoring (MRM) and positive ion mode from m/z 242.1 → 166.0 for BH4, m/z 154.1 → 90.0 for dopamine and m/z 174.1 → 114.0 for AACA (IS). The total chromatographic run time was for 5.5 min. The method was validated for the analysis of samples: the limit of detection was 10 ng/g. The calibration curve was linear between 10-2000 ng/g for BH4 (r(2)=0.995) and 10-5000 ng/g for dopamine (r(2)=0.997) in the rat brain. Thus, good correlated LC-ESI/MS/MS results were obtained and found to be a powerful tool for the quantitative analysis of BH4 and dopamine in the rat brain.     

Novel palmicolous taxa within Pleosporales: multigene phylogeny and taxonomic circumscription

Palm fungi are highly diverse in the tropical regions of Asia. Recent investigations on these palmicolous fungi have led to the collection of astrosphaeriella-like taxa from Calamus, Caryota, and Licuala species in Thailand (Chiang Rai and Narathiwat provinces) and southwest China (Yunnan Province). This study characterizes fungal taxa, which are new to science, based on morphological examination and concatenated DNA sequence data, to infer their familial relationships. Morphological comparisons reveal six new species, viz. Astrosphaeriellopsis caryotae, Fissuroma calami, F. caryotae, Neoastrosphaeriella sribooniensis, Pithomyces caryotae, and P. licualae.Their similarities and differences to other extant species are discussed. The phylogenetic results indicate that all of these new taxa belong to Aigialaceae and Astrosphaeriellaceae (Pleosporales) and support their establishment. Astrosphaeriellopsis is assigned to Astrosphaeriellaceae and the family is amended in order to accommodate both coelomycetous and hyphomycetous asexual morphs. A generic key is presented for Astrosphaeriellaceae to delimit Astrosphaeriella, Astrosphaeriellopsis, Pteridiospora, and Pithomyces. Asexual morph connections of Pithomyces caryotae and P. licualae are established from axenic cultures derived from single ascospores. DNA-based sequence data supports the establishment of our new species; however, the affinities of Astrosphaeriella tornata to other Astrosphaeriella and Pithomyces species are unclear and warrant further investigations with increased taxon sampling.     

Simplified and efficient DNA extraction protocol for Meliolaceae specimens

Meliolaceae is an obligate biotrophic fungal family which cannot be cultured in artificial media. This has resulted in a lack of DNA sequence data in public databases to better resolve species taxonomy. The main criterion for specific classification, therefore, relied heavily on host association. Fresh collections from living leaves of Croton persimilis and Tamarindus indica with black colonies in Mae Fah Luang University, Thailand yielded a new species, Irenopsis crotonicola sp. nov., and a new record of Meliola tamarindi. These taxa are described and phenotypic comparisons are made with known species. To better classify the putative novel species that cannot be cultivated, we outline the protocol to extract DNA from fruiting bodies and generate new phylogenetic data. The results indicate that this direct DNA extraction method is suitable to yield quality DNA sufficient for polymerase chain reaction (PCR) amplifications of several commonly used DNA regions in fungal systematics. The 28S rDNA phylogram generated confirms the position of our taxa within Meliolaceae and indicate a close relationship of I. crotonicola sp. nov. to I. walsurae. Sequences of tef, β-tubulin, and GPDH regions of Meliolaceae are provided as well.     

Characterization and high-quality draft genome sequence of Herbivorax saccincola A7, an anaerobic,alkaliphilic, thermophilic,cellulolytic, and xylanolytic bacterium

An anaerobic, cellulolytic-xylanolytic bacterium, designated strain A7, was isolated from a cellulose-degrading bacterial community inhabiting bovine manure compost on Ishigaki Island, Japan, by enrichment culture using unpretreated corn stover as the sole carbon source. The strain was Gram-positive, non-endospore forming, non-motile, and formed orange colonies on solid medium. Strain A7 was identified as Herbivorax saccincola by DNA-DNA hybridization, and phylogenetic analysis based on 16S rRNA gene sequences showed that it was closely related to H. saccincola GGR1 (= DSM 101079^T). H. saccincola A7 (= JCM 31827 = DSM 104321) had quite similar phenotypic characteristics to those of strain GGR1. However, the optimum growth of A7 was at alkaline pH (9.0) and 55 °C, compared to pH 7.0 at 60 °C for GGR1, and the fatty acid profile of A7 contained 1.7-times more C_17:0 iso than GGR1. The draft genome sequence revealed that H. saccincola A7 possessed a cellulosome-like extracellular macromolecular complex, which has also been found for Clostridium thermocellum and C. clariflavum. H. saccincola A7 contained more glycoside hydrolases (GHs) belonging to GH families-11 and -2, and more diversity of xylanolytic enzymes, than C. thermocellum and C. clariflavum. H. saccincola A7 could grow on xylan because it encoded essential genes for xylose metabolism, such as a xylose transporter, xylose isomerase, xylulokinase, and ribulose-phosphate 3-epimerase, which are absent from C. thermocellum. These results indicated that H. saccincola A7 has great potential as a microorganism that can effectively degrade lignocellulosic biomass.     

Isolation and characterization of a bluegill-degrading microorganism, and analysis of the root hair-promoting effect of the degraded products

Bluegill-degrading bacteria were isolated from various environmental sources. Brevibacillus sp. BGM1 degraded bluegill efficiently at 50 degrees C, and its culture supernatant showed the highest peptide and amino acid concentrations as trichloroacetic acid (TCA) soluble fraction (ASF) (10.7 mg/ml) of all supernatants obtained with bluegill as a substrate. Strain BGM1 secreted a protease(s) into the medium, and the concentration of peptides and amino acids gradually increased. The fertile effect of the degraded bluegill products (DGP) on Brassica rapa was also investigated. The root hair density of B. rapa grown with DGP at a concentration of 30 mug peptides and amino acids/ml was about 1.7 times higher than when grown with the same concentration of undegraded bluegill. DGP was shown to increase root hair numbers and adventitious root formation. The results of this study suggest that a specific peptide(s) for promotion of root hair is produced from the order Perciformes with a protease(s) from BGM1.     

Thai Acanthamoeba isolate (T4) induced apoptotic death in neuroblastoma cells via the Bax-mediated pathway

A Thai Acanthamoeba isolate named AS recovered from a corneal scraping of a keratitis patient was genotypically determined as T4. AS trophozoites were used for studying Acanthamoeba-induced apoptosis in mouse neuroblastoma NA cells during in vitro co-cultivation. The Acanthamoeba-exposed NA cells showed signs of apoptosis including cell shrinkage, nuclear condensation and DNA fragmentation. The effect was confirmed by DNA laddering electrophoresis. Involvement of caspase enzymes and mitochondrial pro- and anti-apoptotic proteins (Bax and Bcl-2) in AS-induced apoptosis was determined. The use of Z-VAD-FMK, a pan-caspase inhibitor, significantly reduced the apoptotic effect, while Bax/Bcl-2 ratio analysis showed a significant increase in the expression of apoptotic proteins in AS-exposed NA cells. These results strongly indicated that apoptosis induced by AS trophozoites is caspase-dependent and is mediated by over-expression of pro-apoptotic proteins in the mitochondrial pathway. This is the first report on the role of Bax in mediating apoptosis induced by Acanthamoeba.     

The gene mpn310 (hmw2) from Mycoplasma pneumoniae encodes two proteins, HMW2 and HMW2-s, which differ in size but use the same reading frame

The gene mpn310 from Mycoplasma pneumoniae encodes the proteins HMW2 with a molecular weight of 215 621 and the smaller P28, here called HMW2-s. Because HMW2-s is not well defined, it was isolated from protein extracts of M. pneumoniae cells and its N-terminal end was determined by MS. HMW2-s starts with the methionine at the amino acid position 1620 of HMW2 and its residual sequence is identical to the last 198 amino acids of HMW2, predicting a molecular weight of 23 204. These results were confirmed by the comparative MS analysis of HMW2-s that had been synthesized in Escherichia coli . A precursor–product relationship between HMW2 and HMW2-s could be excluded, because HMW2-s can be translated from a specific mRNA starting within mpn310 . The conservation of an HMW2-s like protein in M. pneumoniae and Mycoplasma genitalium emphasizes its possible functional importance.     

Zingipain, A cysteine protease from Zingiber ottensii Valeton rhizomes with antiproliferative activities against fungi and human malignant cell lines

The objective of this study was to investigate the activity of a protein identified as cysteine protease, purified from Zingiber ottensii Valeton rhizomes, in terms of antiproliferation against fungi, bacteria, and human malignant cell lines. By means of buffer extraction followed by (NH(4))(2)SO(4) precipitation and ion-exchange chromatography, the obtained dominant protein (designated F50) was submitted to non-denaturing and reducing sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE), where a single band and three bands were revealed from eletrophoretic patterns, respectively. It could be concluded at this point that the F50 was potentially a heterotrimer or heterodimer composed of either two small (～13.8 and ～15.2?kD) subunits or these two together with a larger (～32.5?kD) one. In-gel digestion was carried out for the most intense band from reducing SDS-PAGE, and to the resulting material was applied liquid chromatography (LC)-mass spectroscopy (MS)/MS. The main F50 subunit was found to contain fragments with 100% similarity to zingipain-1, a cysteine protease first discovered in Zingiber officinale. The activity corresponding to the identified data, cysteine protease, was then confirmed in the F50 by azocasein assay and a positive result was obtained. The F50 then was further investigated for antiproliferation against three plant pathogenic fungi species by disk diffusion test, four bacterial species by direct exposure in liquid culture and dish diffusion tests, and five human malignant cell lines by tissue culture assay. It was found that a dose of 23.6?μg F50/0.3?cm(2) of paper disk exhibited the best inhibitory effect against Collectotrichum cassiicola, while lesser effects were found in Exserohilum turicicum and Fusarium oxysporum, respectively. No inhibitory effect against bacterial proliferation was detected in all studied bacterial strains. However, relatively strong antiproliferative effects were found against five human cell lines, with IC50 values ranging from 1.13?μg/mL (hepatoma cancer; HEP-G2) to 5.37?μg/mL (colon cancer; SW620). By periodic acid-Schiff's staining and phenol-sulfuric acid assay, the F50 was determined as a glycoprotein containing 26.30?±?1.01% (by weight) of carbohydrate. Thus, a new glycoprotein with protease activity was successfully identified in Zingiber ottensii rhizome. The glycoprotein also contained antiproliferative activity against some plant pathogenic fungi and human cancer cell lines.     

Structural and biochemical characterization of the inhibitor complexes of xenotropic murine leukemia virus-related virus protease

Interactions between the protease (PR) encoded by the xenotropic murine leukemia virus-related virus and a number of potential inhibitors have been investigated by biochemical and structural techniques. It was observed that several inhibitors used clinically against HIV PR exhibit nanomolar or even subnanomolar values of K(i) , depending on the exact experimental conditions. Both TL-3, a universal inhibitor of retroviral PRs, and some inhibitors originally shown to inhibit plasmepsins were also quite potent, whereas inhibition by pepstatin A was considerably weaker. Crystal structures of the complexes of xenotropic murine leukemia virus-related virus PR with TL-3, amprenavir and pepstatin A were solved at high resolution and compared with the structures of complexes of these inhibitors with other retropepsins. Whereas TL-3 and amprenavir bound in a predictable manner, spanning the substrate-binding site of the enzyme, two molecules of pepstatin A bound simultaneously in an unprecedented manner, leaving the catalytic water molecule in place.