Ruey-Shyang Hseu

Gene phylogeny of the Ganoderma lucidum complex based on ribosomal DNA sequences. Comparison with traditional taxonomic characters

A gene phylogeny of 29 isolates of the G. lucidum complex collected in temperate and subtropical areas was produced by parsimony analysis from nucleotide sequence data of the internal transcribed spacer region of the ribosomal gene and from divergent domain D2 of the large ribosomal subunit gene, and serves as hypothesis of natural relationships between taxa. Results were compared with morphological, ecological, cultural and mating data. They show that extensive convergence or parallelism of morphological characters has occurred during Ganoderma evolution, but also that remarkable morphological difference may occur with little divergence time. Monophyletic groups correlate fairly well with geographical origin of the taxa and/or host relationships. Phylogenetically related isolates have similar culture characteristics, but they may share these characteristics with distant taxa. Therefore, culture characters are less polymorphic than morphological characters between recently diverged taxa, but are useless in recognizing monophyletic groups. Isolates belonging to the same biological species were monophyletic with one exception. A species concept based on monophyly and potential evidence of genetic isolation is proposed, and taxonomy of the G. lucidum complex is revised. Collections named G. lucidum in North America and in Asia are not conspecific with European G. lucidum. The sister group of European G. lucidum is an Argentinian taxon labelled G. oerstedii. North American G. lucidum is related to a Formosan isolate identified as G. boninense. G. tsugae is absent from Taiwan and probably also from Japan and China, although it was commonly reported there. G. tsugae belongs to a lineage restricted to coniferous forests in the more Northern latitudes, of which the taxonomy remains unresolved (the G. valesiacum complex). Correct naming and distribution of several taxa are still to be investigated. From observation of distribution of taxa within monophyletic groups it is speculated that laccate Ganoderma may have originated in the tropics.

Reishi immuno-modulation protein induces interleukin-2 expression via protein kinase-dependent signaling pathways within human T cells†

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. J. Cell. Physiol. 215: 15–26, 2008.

PURIFICATION AND CHARACTERIZATION OF MANGANESE SUPEROXIDE DISMUTASE FROM GANODERMA MICROSPORUM

Manganese superoxide dismutase (Mn‐SOD) in the mycelium of Ganoderma microsporum was purified to homogeneity by heat treatment at 70°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 isoeleetric focusing. Comparisons of 17 amino acids from the N‐terminus ofMn‐SOD subunit with the derived amino acid sequences from the reported Mn‐SOD eDNA 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.

Two-sided effect of Cordyceps sinensis on dendritic cells in different physiological stages

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.

The genetic similarity of different generations of Neocallimastix frontalis SK.

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.

Partial purification and characterization of a 1,3-β-d-glucanase fromGanoderma tsugae

SummaryThe crude broth from a submerged culture ofGanoderma tsugae was assayed for activity against laminarin, and enzyme staining indicated the presence of 1,3-β-d-glucanases. 1,3-β-d-glucanase activity increased rapidly after the stationary growth phase ofGanoderma tsugae. At the same time the glucose was almost depleted and the amount of extracellular polysaccharide decreased in the broth medium. There were two 1,3-β-d-glucanase isozymes in the culture broth ofGanoderma tsugae. After gel filtration, the first peak showed a high activity for laminarin; it was designated as 1,3-β-d-glucanase (I). The second peak exhibited much less activity; it was designated as 1,3-β-d-glucanase (II). Since 1,3-β-d-glucanase (II) activity was very low, and there were probably some inhibitors present in the 1,3-β-d-glucanase (II) fractions, we collected only the fractions that contained 1,3-β-d-glucanase (I) activity. 1,3-β-d-glucanase (I) was specific for laminarin and was found to hydrolyze extracellular polysaccharide in the culture broth ofGanoderma tsugae. The maximum activity of 1,3-β-d-glucanase (I) was at about 50 °C and pH 5.0. The enzyme was stable at pH 5.0 and at temperatures below 40 °C.

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.

Hemodynamic and Neuropathological Analysis in Rats with Aluminum Trichloride-Induced Alzheimer's Disease

Background and Aims Hemodynamic normality is crucial to maintaining the integrity of cerebral vessels and, therefore, preserving the cognitive functions of Alzheimers 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.

Piromyces polycephalus (Neocallimastigaceae), a new rumen fungus

A new anaerobic gut fungus was isolated from the rumen fluid of water buffalo. This fungus appears to be an undescribed species of Piromyces, possessing uniflagellate zoospores and monocentric thalli. It has a distinctive spherical to oval basal body, connected to the sporangia by minute spicules. The fungus strongly resembles Piromyces communis but differs by the basal body and multisporangiate sporangia. It is described as P. polycephalus because of its characters of sporangial development. The species of Piromyces are keyed out.

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.