Yo-Chia Chen

Cucurbitane-type triterpenoids from the stems of momordica charantia.

Four new cucurbitane-type triterpenes, cucurbita-5,23(E)-diene-3beta,7beta,25-triol (1), 3beta-acetoxy-7beta-methoxycucurbita-5,23(E)-dien-25-ol (2), cucurbita-5(10),6,23(E)-triene-3beta,25-diol (5), and cucurbita-5,24-diene-3,7,23-trione (6), together with four known triterpenes, 3beta,25-dihydroxy-7beta-methoxycucurbita-5,23(E)-diene (3), 3beta-hydroxy-7beta,25-dimethoxycucurbita-5,23(E)-diene (4), 3beta,7beta,25-trihydroxycucurbita-5,23(E)-dien-19-al (7), and 25-methoxy-3beta,7beta-dihydroxycucurbita-5,23(E)-dien-19-al (8), were isolated from the methyl alcohol extract of the stems of Momordica charantia. The structures of the new compounds were elucidated by spectroscopic methods.

The identification, purification, and characterization of STXF10 expressed in Streptomyces thermonitrificans NTU-88

Multiple xylanolytic enzymes of Streptomyces thermonitrificans NTU-88 were induced by oat-spelt xylan and separated by two-dimensional polyacrylamide and zymogram gels. Nineteen clear spots differed in pI and molecular weight values were found on the zymogram, and only spot one was seen on the corresponding silver-stained gel. These results revealed that multiple xylanases were secreted when S. thermonitrificans NTU-88 was induced and the spot (STXF10), identified as being a glycosyl hydrolase family 10 xylanase, was the predominant one among xylanases. STXF10 showed a tolerance for high temperatures and broad pH ranges and high affinity and hydrolysis efficiency for xylans. Furthermore, it also featured the minor ability to degrade different lignocellulosic substrates. Although S. thermonitrificans NTU-88 possesses multiple xylanases, our results suggest that the major form of xylanase might be selectively and specifically induced depending on the type of substrate to which the microorganism is exposed.

The direct conversion of xylan to lactic acid by Lactobacillus brevis transformed with a xylanase gene.

A xylanase gene (xynR8), obtained from the DNA of a pool of uncultured rumen microbes, was introduced via a plasmid into Lactobacillus brevis. The recombinant xylanase, with an estimated molecular weight of 32 KDa, was expressed in the transformants and showed obvious xylanase activity (0.412 U ml−1) against oat-spelt xylan in broth when compared to the wild-type Lactobacillus brevisATCC367. The transformants all shared a similar ability to utilize and metabolize xylooligosaccharides. When a selected transformant was inoculated into modified MRS medium containing xylan as the main carbon source, the cell density reached 2.20 × 109 CFU ml−1 on day 4, while the wild-type strain without the plasmid containing the recombinant xylanase did not grow at all under the same conditions. After fermentation, 1.70 g l−1 of lactic acid and 0.44 g l−1 of ethanol were present in the culture supernatant of the strain containing the recombinant xylanase. These results indicate that Lactobacillus brevis containing the xylanase gene is capable of directly saccharifying and fermenting xylan to produce lactic acid in one step. This strain will enable the development of a feasible and economical approach to the production of lactic acid directly from xylan.

Structural modeling and further improvement in pH stability and activity of a highly-active xylanase from an uncultured rumen fungus

Rumen fungi are a rich source of enzymes degrading lignocelluloses. XynR8 is a glycosyl hydrolase family 11 xylanase previously cloned from unpurified rumen fungal cultures. Phylogenetic analysis suggested that xynR8 was obtained from a Neocallimastix species. Recombinant XynR8 expressed in Escherichia coli was highly active and stable between pH 3.0 and 11.0, and displayed a V(max) of 66,672μmolmin(-1)mg(-1), a k(cat) of 38,975s(-1), and a K(m) of 11.20mg/mL towards soluble oat spelt xylan. Based on molecular modeling, residues N41 and N58, important in stabilizing two loops and the structure of XynR8, were mutated to D. Both mutant enzymes showed higher tolerance to pH 2.0. The V(max), k(cat) and K(m) of the N41D and N58D mutant enzymes were 79,645μmolmin(-1)mg(-1), 46,493s(-1), 29.29mg/mL, and 96,689μmolmin(-1)mg(-1), 56,503s(-1), and 21.24mg/mL, respectively. Thus, they are good candidates for application, including biofuel production.

Purification and characterization of a cellulolytic multienzyme complex produced by Neocallimastix patriciarum J11

Understanding the roles of the components of the multienzyme complex of the anaerobial cellulase system, acting on complex substrates, is crucial to the development of efficient cellulase systems for industrial applications such as converting lignocellulose to sugars for bioethanol production. In this study, we purified the multienzyme complex of Neocallimastix patriciarum J11 from a broth through cellulose affinity purification. The multienzyme complex is composed of at least 12 comprised proteins, based on sodium dodecyl sulfate polyacrylamide gel electrophoresis. Eight of these constituents have demonstrated β-glucanase activity on zymogram analysis. The multienzyme complex contained scaffoldings that respond to the gathering of the cellulolytic components. The levels and subunit ratio of the multienzyme complex from N. patriciarum J11 might have been affected by their utilized carbon sources, whereas the components of the complexes were consistent. The trypsin-digested peptides of six proteins were matched to the sequences of cellulases originating from rumen fungi, based on identification through liquid chromatography/mass spectrometry, revealing that at least three types of cellulase, including one endoglucanase and two exoglucanases, could be found in the multienzyme complex of N. patriciarum J11. The cellulolytic subunits could hydrolyze synergistically on both the internal bonds and the reducing and nonreducing ends of cellulose. Based on our research, our findings are the first to depict the composition of the multienzyme complex produced by N. patriciarum J11, and this complex is composed of scaffoldin and three types of cellulase.

Characterization of the activities of p21Cip1/Waf1 promoter-driven reporter systems during camptothecin-induced senescence-like state of BHK-21 cells

AbstractIt was attempted in this work to establish a cell line in which senescent cells can be readily and directly identified in situ in live culture. Transcriptional activation of p21Cip1/Waf1 gene is known to be one of the key steps in the development of cellular senescence, whereas the elements within the p21Cip1/Waf1 promoter that regulate the transcriptional activation of p21Cip1/Waf1 during cellular senescence have not been clearly defined. Thus, several reporter plasmids were constructed in each of which the gene of green fluorescent protein was placed under the control of a selected fragment of p21Cip1/Waf1 promoter, and stably transfected into BHK-21 cells. The transfected cells were induced to become senescence-like by camptothecin and assayed for fluorescence intensity. It was shown that the reporter system constructed with bases −2504 to +406 of the p21Cip1/Waf1 promoter was very efficient in reflecting the senescence of BHK-21 cells by increased cytosolic fluorescence, and the fluorescence intensity of senescent cells was easily distinguished from that of quiescent cells.

Cloning and characterization of a thermostable and pH-stable cellobiohydrolase from Neocallimastix patriciarum J11.

An 1888-bp cDNA designated celA, isolated from a cDNA library of Neocallimastix patriciarum J11 was cloned. The celA had an open reading frame of 1530 bp encoding J11 CelA of 510 amino acids. The primary structure analysis of J11 CelA revealed a complete cellulose-binding domain at the N-terminal, followed by an Asn, Ala, Gly, Gln and Pro-rich linker and ending with a C-terminal glycosyl hydrolase family 6 catalytic domain. The mature J11 CelA was overexpressed in Escherichia coli and purified to homogeneity. This enzyme had high specific activities towards barley β-glucan and lichenan, low toward carboxymethyl cellulose (CMC), Avicel, and H3PO4-swollen Avicel (PSA). The product of Avicel hydrolysis was cellobiose indicating that J11 CelA is a typical cellobiohydrolase. The recombinant J11 CelA had an optimal pH of 6.0 and was stable over a wide range of pH (5.2-11.3). The enzyme showed an optimal temperature of 50°C and was still maintained approximately 50% of the maximum activity in response to the treatment at 70°C for 1h. Cobalt and Fe(3+) at 1 mM greatly activated the enzyme activity. As a thermostable and pH stable enzyme with crystalline cellulose-degrading activity, J11 CelA is a potential candidate for the bioethanol industry.

A highly active beta-glucanase from a new strain of rumen fungus Orpinomyces sp.Y102 exhibits cellobiohydrolase and cellotriohydrolase activities

A new strain of rumen fungus was isolated from Bos taurus, identified and designated Orpinomyces sp.Y102. A clone, celC7, isolated from the cDNA library of Orpinomyces sp.Y102, was predicted to encode a protein containing a signal peptide (Residues 1-17), an N-terminal dockerin-containing domain, and a C-terminal cellobiohydrolase catalytic domain of glycoside hydrolase family 6. CelC7 was insoluble when expressed in Escherichia coli. Deletion of 17 or 105 residues from the N-terminus significantly improved its solubility. The resulting enzymes, CelC7(-17) and CelC7(-105), were highly active to β-glucan substrates and were stable between pH 5.0 and 11.0. CelC7(-105) worked as an exocellulase releasing cellobiose and cellotriose from acid-swollen Avicel and cellooligosaccharides, and displayed a Vmax of 6321.64μmole/min/mg and a Km of 2.18mg/ml to barley β-glucan. Further, the crude extract of CelC7(-105) facilitated ethanol fermentation from cellulose. Thus, CelC7(-105) is a good candidate for industrial applications such as biofuel production.

Avian reovirus S1133-induced apoptosis is associated with Bip/GRP79-mediated Bim translocation to the endoplasmic reticulum

In this study the mechanism of avian reovirus (ARV) S1133-induced pathogenesis was investigated, with a focus on the contribution of ER stress to apoptosis. Our results showed that upregulation of the ER stress response protein, as well as caspase-3 activation, occurred in ARV S1133-infected cultured cells and in SPF White Leghorn chicks organs. Upon infection, Bim was translocated specifically to the ER, but not mitochondria, in the middle to late infectious stages. In addition, ARV S1133 induced JNK phosphorylation and promoted JNK–Bim complex formation, which correlated with the Bim translocation and apoptosis induction that was observed at the same time point. Knockdown of BiP/GRP78 by siRNA and inhibition of BiP/GRP78 using EGCG both abolished the formation of the JNK–Bim complex, caspase-3 activation, and subsequent apoptosis induction by ARV S1133 efficiently. These results suggest that BiP/GRP78 played critical roles and works upstream of JNK–Bim in response to the ARV S1133-mediated apoptosis process.

RhoA/ROCK1 regulates Avian Reovirus S1133-induced switch from autophagy to apoptosis

BackgroundAutophagy is an essential process in the control of cellular homeostasis. It enables cells under certain stress conditions to survive by removing toxic cellular components, and may protect cells from apoptosis. In the present study, the signaling pathways involved in ARV S1133 regulated switch from autophagy to apoptosis were investigated.ResultsARV S1133 infection caused autophagy in the early to middle infectious stages in Vero and DF1 cells, and apoptosis in the middle to late stages. Conversion of the autophagy marker LC3-I to LC3-II occurred earlier than cleavage of the apoptotic marker caspase-3. ARV S1133 also activated the Beclin-1 promoter in the early to middle stages of infection. Levels of RhoA-GTP and ROCK1 activity were elevated upon ARV S1133 infection, while inhibition of RhoA and ROCK1 reduced autophagy and subsequent apoptosis. Conversely, inhibition of caspase-3 did not affect the level of autophagy. Beclin-1 knockdown and treatment with autophagy inhibitors, 3-MA and Bafilomycin A1, suppressed ARV S1133-induced autophagy and apoptosis simultaneously, suggesting the shift from autophagy to apoptosis. A co-immunoprecipitation assay demonstrated that the formation of a RhoA, ROCK1 and Beclin-1 complex coincided with the induction of autophagy.ConclusionOur results demonstrate that RhoA/ROCK1 signaling play critical roles in the transition of cell activity from autophagy to apoptosis in ARV S1133-infected cells.