16 results on '"Hseu RS"'
Search Results
2. Ganoderma microsporum immunomodulatory protein induces apoptosis and potentiates mitomycin C-induced apoptosis in urinary bladder urothelial carcinoma cells.
- Author
-
Huang SY, Chien CC, Hseu RS, Huang VYJ, Chiang SY, Huang CJ, Chen SK, Tsai RY, Lin HT, and Cheng YC
- Subjects
- Cell Line, Tumor, Gene Expression Regulation, Neoplastic drug effects, Humans, Neoplasm Proteins biosynthesis, Urinary Bladder Neoplasms metabolism, Urinary Bladder Neoplasms pathology, Apoptosis drug effects, Fungal Proteins pharmacology, Ganoderma chemistry, Immunologic Factors pharmacology, Mitomycin pharmacology, Urinary Bladder Neoplasms drug therapy
- Abstract
Current chemotherapy and immunotherapy treatments followed by transurethral resection for urinary bladder urothelial carcinoma (UC) usually suffer from poor prognosis and high recurrence rate. Design and modification of current formulation with the novel adjuvants are needed. A recombinant protein derived from Ganoderma microsporum named as Ganoderma microsporum immunomodulatory protein (GMIP) was used to treat UC cells. We found GMIP elicits a dose-dependent and time-dependent anti-UC cell proliferation effect, with a half-maximal inhibition concentration (IC
50 ) comparable to mitomycin C (MMC), a commonly used chemotherapy agent. After GMIP treatment, UC cells showed apoptotic phenomenon including cell cycle arrest in the G1 phase, elevated sub-G1 population, mitochondrial membrane potential loss, up-regulated p21 expression, p21 nuclear translocation, caspase activation, and PARP cleavage in a p53-independent but p21-mediated pathways. Unlike lung cancer cells, GMIP treated UC cells showed no autophagic scheme including Beclin-1, an autophagy to apoptosis switch marker, was not cleaved by caspase 3 and slight LC3B-II accumulation. Also, the classic autophagic inhibitor, chloroquine had no effect in GMIP-mediated cell death made us conclude that GMIP induced apoptosis through caspase activation but not autophagy in UC cells. Additionally, GMIP showed synergistic effects with MMC in killing UC cells and thus decreased the concentration of MMC usage to reach the comparable apoptotic effects. Our results delineate novel strategies for treatment of UC by GMIP alone or in combination with MMC application and provide a promising therapeutic cocktail for better treatment of urinary bladder urothelial carcinoma., (© 2017 Wiley Periodicals, Inc.)- Published
- 2018
- Full Text
- View/download PDF
3. Evidence for two types of nrDNA existing in Chinese medicinal fungus Ophiocordyceps sinensis .
- Author
-
Chen CS, Huang CT, and Hseu RS
- Abstract
Nuclear ribosomal DNA (nrDNA) sequences are widely used in the molecular classification of fungi. Previous phylogenetic studies of highly-valued traditional Chinese medicinal fungus Ophiocordyceps sinensis were mostly based on 18S and internal transcribed spacer (ITS) regions (ITS1, 5.8S and ITS2) of nrDNA. However, the disparity manifest in the low sequences identities between different O. sinensis isolates has led to argumentative hypotheses for this phenomenon, such as the "species complex" or "cryptic species" hypotheses. In the present study, four types of nrDNA (GC, AT-1, AT-2, and T) were identified using four primer pairs to amplify the nrDNA of six O. sinensis isolates. We demonstrate that each O. sinensis isolate contained two types of nrDNA, the omnipresent GC-type and a coexistent type alternating between the remaining three. This crucial discovery challenges the established notion of one type of nrDNA per species. We therefore propose that the composition of nrDNA types should be taken into consideration in studies of fungal genetics and classification., Competing Interests: Conflict of Interest: The authors have no conflict of interest to declare.
- Published
- 2017
- Full Text
- View/download PDF
4. Potent In Vitro Protection Against PM₂.₅-Caused ROS Generation and Vascular Permeability by Long-Term Pretreatment with Ganoderma tsugae.
- Author
-
Tseng CY, Chung MC, Wang JS, Chang YJ, Chang JF, Lin CH, Hseu RS, and Chao MW
- Subjects
- Glutathione metabolism, Human Umbilical Vein Endothelial Cells, Humans, Intercellular Junctions metabolism, Myocardial Infarction chemically induced, Oxidation-Reduction drug effects, Oxidative Stress drug effects, Particulate Matter metabolism, Time Factors, Vascular Endothelial Growth Factor A metabolism, Capillary Permeability drug effects, Ganoderma chemistry, Particulate Matter adverse effects, Plant Extracts administration & dosage, Plant Extracts pharmacology, Reactive Oxygen Species metabolism
- Abstract
Epidemiological studies show increased particulate matter (PM[Formula: see text]) particles in ambient air are correlated with increased myocardial infarctions. Given the close association of capillaries and alveoli, the dysfunction is caused when inhaled PM[Formula: see text] particles come in close proximity to capillary endothelial cells. We previously suggested that the inhalation of PM[Formula: see text] diesel exhaust particles (DEP) induces oxidative stress and upregulates the Nrf2/HO-1 pathway, inducing vascular permeability factor VEGFA secretion, which results in cell-cell adherens junction disruption and PM[Formula: see text] transmigratation into circulation. Here, we minimized the level that PM[Formula: see text] traveled in the bloodstream by pre-supplementing with a traditional Chinese medicine (TCM) Ganoderma tsugae DMSO extract (GTDE) prior to PM[Formula: see text] exposure. Our results show that PM[Formula: see text] caused alterations in enzyme activities and cellular anti-oxidant balance. We found decreased glutathione levels, a reduced cellular redox ratio, increased ROS generation and cytotoxicity in the cellular fractions. The oxidative stress caused DNA damage and apoptosis, likely causing downstream molecular events that trigger vasculature permeabilization and, eventually, cardiovascular disorders. Our results show long-term GTDE treatment increased endogenous glutathione level, while PM[Formula: see text]-reduced glutathione levels and the cellular redox ratio. GTDE was protective against the genotoxic and apoptotic effects initiated by PM[Formula: see text] oxidative stress. Vascular permeability revealed that PM[Formula: see text] only accumulated on the surface of cells after GTDE treatment; no penetration was detected. After two weeks of GTDE treatment, VEGFA secretion was significantly reduced in human umbilical vein endothelial cells (HUVEC) and endothelial cell migration was blocked. Our results suggest GTDE prevents PM[Formula: see text] transmigration into the bloodstream, and the resultant dysfunction, by inhibiting oxidative stress production and endothelial permeability.
- Published
- 2016
- Full Text
- View/download PDF
5. Purification and characterization of a cellulolytic multienzyme complex produced by Neocallimastix patriciarum J11.
- Author
-
Wang HC, Chen YC, and Hseu RS
- Subjects
- Animals, Blotting, Western, Buffaloes microbiology, Cellulases genetics, Cellulases metabolism, Chromatography, Gel, Fungal Proteins genetics, Fungal Proteins metabolism, Multienzyme Complexes genetics, Multienzyme Complexes metabolism, Neocallimastix isolation & purification, Protein Subunits, Recombinant Proteins chemistry, Recombinant Proteins genetics, Recombinant Proteins metabolism, Rumen microbiology, Tandem Mass Spectrometry, Cellulases isolation & purification, Fungal Proteins isolation & purification, Multienzyme Complexes isolation & purification, Neocallimastix enzymology
- Abstract
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., (Copyright © 2014 Elsevier Inc. All rights reserved.)
- Published
- 2014
- Full Text
- View/download PDF
6. Hemodynamic and neuropathological analysis in rats with aluminum trichloride-induced Alzheimer's disease.
- Author
-
Chen SM, Fan CC, Chiue MS, Chou C, Chen JH, and Hseu RS
- Subjects
- Aluminum Chloride, Aluminum Compounds, Alzheimer Disease physiopathology, Amyloid beta-Peptides metabolism, Animals, Blood Viscosity, Brain blood supply, Catalase metabolism, Cerebral Cortex enzymology, Cerebral Cortex pathology, Cerebrovascular Circulation, Chlorides, Environment, Escape Reaction, Glutathione Peroxidase metabolism, Hippocampus enzymology, Hippocampus pathology, Immunohistochemistry, Magnetic Resonance Angiography, Male, Maze Learning, Memory, Neurons metabolism, Neurons pathology, Oxidative Stress, Rats, Wistar, Superoxide Dismutase metabolism, Task Performance and Analysis, Alzheimer Disease blood, Alzheimer Disease pathology, Brain pathology, Brain physiopathology, Hemodynamics
- Abstract
Background and Aims: Hemodynamic normality is crucial to maintaining the integrity of cerebral vessels and, therefore, preserving the cognitive functions of Alzheimer's disease patients. This study investigates the implications of the hemodynamic changes and the neuropathological diversifications of AlCl3-induced AD., Methods: The experimental animals were 8- to 12-wk-old male Wistar rats. The rats were randomly divided into 2 groups: a control group and a (+)control group. Food intake, water intake, and weight changes were recorded daily for 22 wk. Synchronously, the regional cerebral blood flow (rCBF) of the rats with AlCl3-induced AD were measured using magnetic resonance imaging (MRI). The hemorheological parameters were analyzed using a computerized auto-rotational rheometer. The brain tissue of the subjects was analyzed using immunohistological chemical (IHC) staining to determine the beta-amyloid (Aβ) levels., Results: The results of hemodynamic analysis revealed that the whole blood viscosity (WBV), fibrinogen, plasma viscosity and RBC aggregation index (RAI) in (+)control were significantly higher than that of control group, while erythrocyte electrophoresis (EI) of whole blood in (+)control were significantly lower than that of control group. The results of acetylcholinesterase-RBC (AChE-RBC)in the (+)control group was significantly higher than that of the control group. The results also show that the reduction of rCBF in rats with AlCl3-induced AD was approximately 50% to 60% that of normal rats. IHC stain results show that significantly more Aβ plaques accumulated in the hippocampus and cortex of the (+)control than in the control group., Conclusion: The results accentuate the importance of hemorheology and reinforce the specific association between hemodynamic and neuropathological changes in rats with AlCl3-induced AD. Hemorheological parameters, such as WBV and fibrinogen, and AChE-RBC were ultimately proven to be useful biomarkers of the severity and progression of AD patients. In addition, the parameters can be substituted for invasive inspection in therapeutic intervention.
- Published
- 2013
- Full Text
- View/download PDF
7. Cloning and characterization of a thermostable and pH-stable cellobiohydrolase from Neocallimastix patriciarum J11.
- Author
-
Wang HC, Chen YC, Huang CT, and Hseu RS
- Subjects
- Amino Acid Sequence, Carboxymethylcellulose Sodium chemistry, Carboxymethylcellulose Sodium metabolism, Cellulose 1,4-beta-Cellobiosidase metabolism, Cloning, Molecular, DNA, Complementary chemistry, DNA, Complementary metabolism, Enzyme Stability, Escherichia coli genetics, Fungal Proteins chemistry, Fungal Proteins metabolism, Hot Temperature, Hydrogen-Ion Concentration, Molecular Sequence Data, Recombinant Proteins chemistry, Recombinant Proteins genetics, Recombinant Proteins metabolism, Cellulose 1,4-beta-Cellobiosidase chemistry, Cellulose 1,4-beta-Cellobiosidase genetics, Fungal Proteins genetics, Neocallimastix enzymology
- Abstract
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., (Copyright © 2013 Elsevier Inc. All rights reserved.)
- Published
- 2013
- Full Text
- View/download PDF
8. Gene expression profiling of dendritic cells in different physiological stages under Cordyceps sinensis treatment.
- Author
-
Li CY, Chiang CS, Cheng WC, Wang SC, Cheng HT, Chen CR, Shu WY, Tsai ML, Hseu RS, Chang CW, Huang CY, Fang SH, and Hsu IC
- Subjects
- Cells, Cultured, Humans, Lipopolysaccharides pharmacology, Oligonucleotide Array Sequence Analysis, Cordyceps chemistry, Dendritic Cells drug effects, Dendritic Cells metabolism, Drugs, Chinese Herbal pharmacology, Gene Expression Profiling methods
- Abstract
Cordyceps sinensis (CS) has been commonly used as herbal medicine and a health supplement in China for over two thousand years. Although previous studies have demonstrated that CS has benefits in immunoregulation and anti-inflammation, the precise mechanism by which CS affects immunomodulation is still unclear. In this study, we exploited duplicate sets of loop-design microarray experiments to examine two different batches of CS and analyze the effects of CS on dendritic cells (DCs), in different physiology stages: naïve stage and inflammatory stage. Immature DCs were treated with CS, lipopolysaccharide (LPS), or LPS plus CS (LPS/CS) for two days, and the gene expression profiles were examined using cDNA microarrays. The results of two loop-design microarray experiments showed good intersection rates. The expression level of common genes found in both loop-design microarray experiments was consistent, and the correlation coefficients (Rs), were higher than 0.96. Through intersection analysis of microarray results, we identified 295 intersecting significantly differentially expressed (SDE) genes of the three different treatments (CS, LPS, and LPS/CS), which participated mainly in the adjustment of immune response and the regulation of cell proliferation and death. Genes regulated uniquely by CS treatment were significantly involved in the regulation of focal adhesion pathway, ECM-receptor interaction pathway, and hematopoietic cell lineage pathway. Unique LPS regulated genes were significantly involved in the regulation of Toll-like receptor signaling pathway, systemic lupus erythematosus pathway, and complement and coagulation cascades pathway. Unique LPS/CS regulated genes were significantly involved in the regulation of oxidative phosphorylation pathway. These results could provide useful information in further study of the pharmacological mechanisms of CS. This study also demonstrates that with a rigorous experimental design, the biological effects of a complex compound can be reliably studied by a complex system like cDNA microarray.
- Published
- 2012
- Full Text
- View/download PDF
9. An immunomodulatory protein, Ling Zhi-8, induced activation and maturation of human monocyte-derived dendritic cells by the NF-kappaB and MAPK pathways.
- Author
-
Lin YL, Liang YC, Tseng YS, Huang HY, Chou SY, Hseu RS, Huang CT, and Chiang BL
- Subjects
- Animals, Antigens, CD biosynthesis, Cytokines biosynthesis, Cytokines immunology, Dendritic Cells cytology, Dendritic Cells immunology, Fungal Proteins chemistry, Gene Expression Regulation drug effects, Gene Expression Regulation immunology, HLA-DR Antigens biosynthesis, HLA-DR Antigens immunology, Humans, Immunoglobulin G immunology, Immunoglobulin G pharmacology, Immunologic Factors chemistry, MAP Kinase Signaling System physiology, Mice, Mice, Inbred BALB C, Monocytes cytology, Monocytes immunology, NF-kappa B immunology, Reishi chemistry, Th1 Cells cytology, Th1 Cells immunology, Th1 Cells metabolism, Toll-Like Receptor 4 antagonists & inhibitors, Toll-Like Receptor 4 immunology, Toll-Like Receptor 4 metabolism, Dendritic Cells metabolism, Fungal Proteins pharmacology, Immunologic Factors pharmacology, MAP Kinase Signaling System drug effects, Monocytes metabolism, NF-kappa B metabolism
- Abstract
Ganoderma lucidum, an oriental medicinal mushroom, has been widely used in Asia to promote health and longevity. LZ-8 is a protein derived from the fungus G. lucidum and has immunomodulatory capacities. In this study, we investigated the immune modulatory effects of rLZ-8 on human monocyte-derived DCs. Treatment of DC with rLZ-8 resulted in the enhanced cell-surface expression of CD80, CD86, CD83, and HLA-DR, as well as the enhanced production of IL-12 p40, IL-10, and IL-23, and the capacity for endocytosis was suppressed in DCs. In addition, treatment of DCs with rLZ-8 resulted in an enhanced, naïve T cell-stimulatory capacity and increased, naïve T cell secretion of IFN-gamma and IL-10. Neutralization with antibodies against TLR4 inhibited the rLZ-8-induced production of IL-12 p40 and IL-10 in DCs. rLZ-8 can stimulate TLR4 or TLR4/MD2-transfected HEK293 cells to produce IL-8. These results suggested an important role for TLR4 in signaling DCs upon incubation with rLZ-8. Further study showed that rLZ-8 was able to augment IKK, NF-kappaB activity, and also IkappaBalpha and MAPK phosphorylation. Further, inhibition of NF-kappaB by helenalin prevented the effects of rLZ-8 in the expression of CD80, CD86, CD83, and HLA-DR and production of IL-12 p40 and IL-10 in various degrees. To confirm the in vitro data, we investigated the effect of rLZ-8 further on antigen-specific antibody and cytokine production in BALB/c mice. Immunization with OVA/rLZ-8 showed that the anti-OVA IgG2a, IFN-gamma, and IL-2 were increased significantly compared with OVA alone in BALB/c mice. In conclusion, our experiments demonstrated that rLZ-8 can effectively promote the activation and maturation of immature DCs, preferring a Th1 response, suggesting that rLZ-8 may possess a potential effect in regulating immune responses.
- Published
- 2009
- Full Text
- View/download PDF
10. Two-sided effect of Cordyceps sinensis on dendritic cells in different physiological stages.
- Author
-
Li CY, Chiang CS, Tsai ML, Hseu RS, Shu WY, Chuang CY, Sun YC, Chang YS, Lin JG, Chen CS, Huang CL, and Hsu IC
- Subjects
- Cell Differentiation drug effects, Cell Proliferation drug effects, Chromatography, High Pressure Liquid, Dendritic Cells drug effects, Dendritic Cells immunology, Humans, Interleukin-12 biosynthesis, Interleukin-1beta biosynthesis, Interleukin-6 biosynthesis, Lipopolysaccharides pharmacology, Phenotype, Th1 Cells cytology, Th1 Cells drug effects, Th1 Cells immunology, Th2 Cells cytology, Th2 Cells drug effects, Th2 Cells immunology, Tumor Necrosis Factor-alpha biosynthesis, Cordyceps metabolism, Dendritic Cells cytology
- Abstract
Cordyceps sinensis (CS), a Chinese tonifying herb, has been widely used for centuries in Asian countries as a medicine and a health supplement. Although ample evidence indicates that CS can modulate immune responses, the functional effect of CS on dendritic cells (DCs) is still unclear. This study examines how CS affects human monocyte-derived DCs in two physiological states: naïve and LPS-induced inflammatory. Our experimental results demonstrate that CS acts as an activator and maturation inducer of immature DCs by stimulating the expression of costimulatory molecules and proinflammatory cytokines by DCs, enhancing the DC-induced, allogeneic T cell proliferation, and reducing the endocytic ability of DCs. In contrast, CS suppresses the LPS-induced, inflammatory response by decreasing the LPS-induced expression of costimulatory molecules and proinflammatory cytokines by DCs. CS also suppresses the LPS-induced, DC-elicited, allogeneic T cell proliferation and shifts the LPS-activated, DC-driven Th1 response toward a Th2 response. These results demonstrate that CS differentially regulates the DC activities according to the presence or absence of the inflammatory signs. Restated, with the lack of an ongoing inflammatory environment, CS primes DCs toward a Th1-type immunity, whereas in a potential inflammatory reaction, CS balances the over-reactivity of elicited Th1 immunity. This investigation illustrates the Yin-Yang balancing effects of CS as a medicine and a health supplement.
- Published
- 2009
- Full Text
- View/download PDF
11. Reishi immuno-modulation protein induces interleukin-2 expression via protein kinase-dependent signaling pathways within human T cells.
- Author
-
Hsu HY, Hua KF, Wu WC, Hsu J, Weng ST, Lin TL, Liu CY, Hseu RS, and Huang CT
- Subjects
- CD3 Complex metabolism, Calcium Signaling drug effects, Cells, Cultured, Fungal Proteins immunology, Fungal Proteins pharmacology, Humans, Immunologic Factors immunology, Interleukin-2 metabolism, Isoenzymes metabolism, Jurkat Cells, Lymphocyte Activation drug effects, Mitogen-Activated Protein Kinases metabolism, Phosphorylation drug effects, Reactive Oxygen Species metabolism, Receptors, Antigen, T-Cell metabolism, Recombinant Proteins pharmacology, Reishi chemistry, T-Lymphocytes enzymology, T-Lymphocytes metabolism, Time Factors, Type C Phospholipases metabolism, src-Family Kinases metabolism, Gene Expression Regulation drug effects, Immunologic Factors pharmacology, Interleukin-2 genetics, Protein Kinase C metabolism, Reishi immunology, Signal Transduction drug effects, T-Lymphocytes drug effects
- Abstract
Ganoderma lucidum, a medicinal fungus is thought to possess and enhance a variety of human immune functions. An immuno-modulatory protein, Ling Zhi-8 (LZ-8) isolated from G. lucidum exhibited potent mitogenic effects upon human peripheral blood lymphocytes (PBL). However, LZ-8-mediated signal transduction in the regulation of interleukin-2 (IL-2) gene expression within human T cells is largely unknown. Here we cloned the LZ-8 gene of G. lucidum, and expressed the recombinant LZ-8 protein (rLZ-8) by means of a yeast Pichia pastoris protein expression system. We found that rLZ-8 induces IL-2 gene expression via the Src-family protein tyrosine kinase (PTK), via reactive oxygen species (ROS), and differential protein kinase-dependent pathways within human primary T cells and cultured Jurkat T cells. In essence, we have established the nature of the rLZ-8-mediated signal-transduction pathways, such as PTK/protein kinase C (PKC)/ROS, PTK/PLC/PKCalpha/ERK1/2, and PTK/PLC/PKCalpha/p38 pathways in the regulation of IL-2 gene expression within human T cells. Our current results of analyzing rLZ-8-mediated signal transduction in T cells might provide a potential application for rLZ-8 as a pharmacological immune-modulating agent., ((c) 2008 Wiley-Liss, Inc.)
- Published
- 2008
- Full Text
- View/download PDF
12. Effects of dockerin domains on Neocallimastix frontalis xylanases.
- Author
-
Huang YH, Huang CT, and Hseu RS
- Subjects
- Amino Acid Sequence, Circular Dichroism, Endo-1,4-beta Xylanases genetics, Endo-1,4-beta Xylanases metabolism, Hydrogen-Ion Concentration, Molecular Sequence Data, Recombinant Proteins genetics, Recombinant Proteins metabolism, Sequence Deletion, Substrate Specificity, Temperature, Xylosidases genetics, Xylosidases metabolism, beta-Glucosidase genetics, beta-Glucosidase metabolism, Endo-1,4-beta Xylanases chemistry, Gene Expression Regulation, Fungal, Neocallimastix enzymology, Xylans metabolism, Xylosidases chemistry, beta-Glucosidase chemistry
- Abstract
Two xylanase genes were cloned from the anaerobic fungus Neocallimastix frontalis. Xyn11A had a modular structure of two catalytic domains and two dockerin domains, while Xyn11B had one catalytic domain and two dockerin domains. The characteristics of the xylanases with and without dockerin domains were investigated. The deletion of dockerin domains had little influence on the optimal pH of xylanases, while it significantly affected the optimal temperatures. The optimal temperatures increased from 55 to 60 degrees C for Xyn11A and 60 to 65 degrees C for Xyn11B after the deletion of dockerin domains. The increase of optimal temperatures was attributed to the lower stability of the second structure in full length xylanase than that in the truncated one as evidenced by the circular dichroism spectroscopy. The specific activity of Xyn11A and Xyn11B increased about 64% and 330%, respectively, after the deletion of the dockerin domains. The removal of dockerin domains appeared to increase the overall efficiency of Xyn11A' (1.2-) and Xyn11B' (2.9-) fold with oat spelts xylan as reflected by the values of k(cat)/K(m). The results suggest that the dockerin domain might play an important role in the characteristics of xylanases from anaerobic fungi.
- Published
- 2005
- Full Text
- View/download PDF
13. The genetic similarity of different generations of Neocallimastix frontalis SK.
- Author
-
Chen YC, Hseu RS, and Cheng KJ
- Subjects
- Amino Acid Sequence, DNA, Fungal analysis, DNA, Ribosomal Spacer analysis, Molecular Sequence Data, Neocallimastix classification, Random Amplified Polymorphic DNA Technique, DNA, Ribosomal genetics, DNA, Ribosomal Spacer genetics, Genes, rRNA, Genetic Variation, Neocallimastix genetics
- Abstract
The genetic similarity of different generations of Neocallimastix frontalis SK was examined by random amplified polymorphic DNA (RAPD) profiling and internal transcribed spacer 1 (ITS1) sequence analysis. N. frontalis SK was subcultured every 2-4 days, and SK-1, SK-3M, and SK-1Y represented N. frontalis SK cultures after one subculture, 50 subcultures, and 150 subcultures. The DNA polymorphisms of the different N. frontalis SK generations were compared by RAPD profiling. The RAPD results gave the same patterns for SK-1, SK-3M and SK-1Y using 12 selected random primers. The partial 18S rDNA, 5.8S rDNA, and ITS1 regions of different generations of N. frontalis SK were amplified and sequenced. The results of alignment and pairwise similarity indicated that the analyzed rRNA sequences of SK-1, SK-3M and SK-1Y were totally identical. This study thus demonstrated genetically identical DNA polymorphisms by RAPD profiling and an unvaried ITS1 region for N. frontalis SK when the strain is subcultured frequently. This suggests that this strain is homokaryotic and grows via an asexual life cycle in vitro.
- Published
- 2003
- Full Text
- View/download PDF
14. Purification and characterization of manganese superoxide dismutase from Ganoderma microsporum.
- Author
-
Pan SM, Ye JS, and Hseu RS
- Subjects
- Amino Acid Sequence, Electrophoresis, Polyacrylamide Gel, Molecular Sequence Data, Sequence Alignment, Superoxide Dismutase genetics, Superoxide Dismutase metabolism, Fungi enzymology, Superoxide Dismutase isolation & purification
- Abstract
Manganese superoxide dismutase (Mn-SOD) in the mycelium of Ganoderma microsporum was purified to homogeneity by heat treatment at 70 degrees C, ammonium sulfate fractionation, DEAE-52 anion-exchange chromatography, and Sephacryl SH-200 chromatography. The molecular mass of its native form was estimated to be 98 kD by size-exclusion chromatography. This enzyme is tetrameric composed of four subunits of equal size of 25 kD. The pI of this purified Mn-SOD was located at pH 6.34 and 5.06 by isoelectric focusing. Comparisons of 17 amino acids from the N-terminus of Mn-SOD subunit with the derived amino acid sequences from the reported Mn-SOD cDNA clones of other sources indicated a high degree of homology among the Ganoderma genus but the Mn-SOD from G. microsporum showed a high variation when compared with other organisms.
- Published
- 1997
- Full Text
- View/download PDF
15. [Identification of Candida albicans by specific primers of polymerase chain reaction and DNA probes].
- Author
-
Huang CC, Tsai WC, Hseu RS, and Wang HH
- Subjects
- Base Sequence, Molecular Sequence Data, Sensitivity and Specificity, Candida albicans isolation & purification, DNA Probes, Polymerase Chain Reaction
- Abstract
Candida albicans is a pathogenic yeast. Two sets of universal primers were used for specific identification of Candida albicans with PCR-amplified ribosomal DNA internal transcribed spacers (ITS). Among the species of Candida, the amplified ITSI and ITSII of DNA fragments were similar in size. The PCR product was purified and labeled with digoxigenin and used as DNA probe in the detection with target DNA of Candida albicans by hybridization. Two sets of specific primers (CA1 and CA2 to amplify ITSI, CA3 and CA4 to amplify ITSII) were designed by alignment of ribosomal ITS sequence of pathogenic Candida albicans with other species to detect C. albicans by PCR. The sensitivity of PCR using the specific primers to detect pure culture of C. albicans was 0.1 ng (about 10(3)-10(4) cells). If the yeast cells were mixed with two other strains, there was a 10-fold decrease in sensitivity (1 ng or 10(4)-10(5) cells) under the same PCR conditions.
- Published
- 1997
16. Differentiation and grouping of isolates of the Ganoderma lucidum complex by random amplified polymorphic DNA-PCR compared with grouping on the basis of internal transcribed spacer sequences.
- Author
-
Hseu RS, Wang HH, Wang HF, and Moncalvo JM
- Subjects
- Base Sequence, DNA Fingerprinting, DNA Primers genetics, Evaluation Studies as Topic, Molecular Sequence Data, Mycology methods, Phylogeny, Polymorphism, Genetic, Basidiomycota classification, Basidiomycota genetics, DNA, Fungal genetics, DNA, Ribosomal genetics, Random Amplified Polymorphic DNA Technique
- Abstract
Laccate polypores of the Ganoderma lucidum species complex are widespread white rot fungi of economic importance, but isolates cannot be identified by traditional taxonomic methods. Parsimony analysis of nucleotide sequences from the internal transcribed spacers (ITS) of the ribosomal gene (rDNA) distinguished six lineages in this species complex. Each ITS lineage may represent one or more putative species. While some isolates have identical ITS sequences, all of them could be clearly differentiated by genetic fingerprinting using random amplified polymorphic DNA (RAPD). To investigate the suitability of RAPD markers for taxonomic identification and grouping of isolates of the G. lucidum complex, RAPD fragments (RAPDs) were used as phenotypic characters in numerical and parsimony analyses. Results show that data from RAPDS do not distinguish the same clades as ITS data do. Groupings based on analysis of RAPD data were very sensitive to the choice of the grouping method used, and no consistent grouping of isolates could be proposed. However, analysis with RAPDs did resolve several robust terminal clades containing putatively conspecific isolates, suggesting that RAPDs might be helpful for systematics at the lower taxonomic levels that are unresolved by ITS sequence data. The limitations of RAPDs for systematics are briefly discussed. The conclusion of this study is that ITS sequences can be used to identify isolates of the G. lucidum complex, whereas RAPDs can be used to differentiate between isolates having identical ITS sequences. The practical implications of these results are briefly illustrated.
- Published
- 1996
- Full Text
- View/download PDF
Catalog
Discovery Service for Jio Institute Digital Library
For full access to our library's resources, please sign in.