Transcriptome analysis of hybrid tilapia (Oreochromis spp.) with Streptococcus agalactiae infection identifies Toll-like receptor pathway-mediated induction of NADPH oxidase complex and piscidins as primary immune-related responses.

Streptococcus agalactiae infection is one of the most significant bacterial diseases in tilapia aquaculture. Identification of immune-related genes associated with Streptococcus agalactiae infection may provide a basis for breeding selection or therapeutics to augment disease resistance. Therefore, we utilized transcriptome profiling to study the host response in tilapia following Streptococcus agalactiae infection. Based on GO and KEGG enrichment analyses, we found that differentially expressed genes are widely involved in immune-related pathways, including the induction of antimicrobial peptides. Moreover, the main components of two immune-related pathways (Toll-like receptor signaling and leukocyte transendothelial migration) and four environmental information processing pathways (TNF, PI3K-Akt, Jak-STAT and MAPK) were identified. Finally, a time-course expression profile for several of the identified transcripts including tilapia piscidin 3 (TP3), tilapia piscidin 4 (TP4), TLR2, TLR5, MyD88, TRAF6, p38, and interleukin components was performed by qRT-PCR. Collectively, these results provide a starting point to study molecular mechanisms of tilapia immune response to Streptococcus agalactiae infection and may be applied as a basis for developing disease resistant strains by breeding selection.

Transgenic expression of omega-3 PUFA synthesis genes improves zebrafish survival during Vibrio vulnificus infection.

BACKGROUND: Highly desaturated n-3 polyunsaturated fatty acids (PUFAs), such as eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA), are synthesized by desaturases and elongase. They exert hepatoprotective effects to prevent alcoholic fatty liver syndrome or cholestatic liver injury. However, it is unclear how n-3 PUFAs improve immune function in liver. Vibrio vulnificus, a gram-negative bacterial pathogen, causes high mortality of aquaculture fishes upon infection. Humans can become infected with V. vulnificus through open wounds or by eating raw seafood, and such infections may result in systemic septicemia. Moreover, patients with liver diseases are vulnerable to infection, and are more likely than healthy persons to present with liver inflammation following infection. This study quantified n-3 PUFAs and their anti-bacterial effects in Fadsd6 and Elvol5a transgenic zebrafish. RESULTS: Two transgenic zebrafish strains with strong liver specific expression of Fadsd6 and Elvol5a (driven by the zebrafish Fabp10 promoter) were established using the Tol2 system. Synthesis of n-3 PUFAs in these strains were increased by 2.5-fold as compared to wild type (Wt) fish. The survival rate in 24 h following challenge with V. vulnificus was 20 % in Wt, but 70 % in the transgenic strains. In addition, the bacteria counts in transgenic fish strains were significantly decreased. The expression levels of pro-inflammatory genes, such as TNF-α, IL-1ß, and NF-κB, were suppressed between 9 and 12 h after challenge. This study confirms the anti-bacterial function of n-3 PUFAs in a transgenic zebrafish model. CONCLUSIONS: Fadsd6 and Elvol5a transgenic zebrafish are more resistant to V. vulnificus infection, and enhance survival by diminishing the attendant inflammatory response.

Modulation of nitrosative stress via glutathione-dependent formaldehyde dehydrogenase and S-nitrosoglutathione reductase.

Glutathione-dependent formaldehyde dehydrogenase (GFD) from Taiwanofungus camphorata plays important roles in formaldehyde detoxification and antioxidation. The enzyme is bifunctional. In addition to the GFD activity, it also functions as an effective S-nitrosoglutathione reductase (GSNOR) against nitrosative stress. We investigated the modulation of HEK (human embryonic kidney) 293T cells under nitrosative stress by transfecting a codon optimized GFD cDNA from Taiwanofungus camphorata (Tc-GFD-O) to these cells. The parental and transfected HEK 293T cells were then subjected to S-nitrosoglutathione treatment to induce nitrosative stress. The results showed that in Tc-GFD-O-transfected 293T cells, the expression and activity of GFD increased. Additionally, these cells under the nitrosative stress induced by S-nitrosoglutathione showed both higher viability and less apoptosis than the parental 293T cells. This finding suggests that the Tc-GFD-O in HEK 293T cells may provide a protective function under nitrosative stress.

Whole-genome sequencing and identification of Morganella morganii KT pathogenicity-related genes.

BACKGROUND: The opportunistic enterobacterium, Morganella morganii, which can cause bacteraemia, is the ninth most prevalent cause of clinical infections in patients at Changhua Christian Hospital, Taiwan. The KT strain of M. morganii was isolated during postoperative care of a cancer patient with a gallbladder stone who developed sepsis caused by bacteraemia. M. morganii is sometimes encountered in nosocomial settings and has been causally linked to catheter-associated bacteriuria, complex infections of the urinary and/or hepatobiliary tracts, wound infection, and septicaemia. M. morganii infection is associated with a high mortality rate, although most patients respond well to appropriate antibiotic therapy. To obtain insights into the genome biology of M. morganii and the mechanisms underlying its pathogenicity, we used Illumina technology to sequence the genome of the KT strain and compared its sequence with the genome sequences of related bacteria. RESULTS: The 3,826,919-bp sequence contained in 58 contigs has a GC content of 51.15% and includes 3,565 protein-coding sequences, 72 tRNA genes, and 10 rRNA genes. The pathogenicity-related genes encode determinants of drug resistance, fimbrial adhesins, an IgA protease, haemolysins, ureases, and insecticidal and apoptotic toxins as well as proteins found in flagellae, the iron acquisition system, a type-3 secretion system (T3SS), and several two-component systems. Comparison with 14 genome sequences from other members of Enterobacteriaceae revealed different degrees of similarity to several systems found in M. morganii. The most striking similarities were found in the IS4 family of transposases, insecticidal toxins, T3SS components, and proteins required for ethanolamine use (eut operon) and cobalamin (vitamin B12) biosynthesis. The eut operon and the gene cluster for cobalamin biosynthesis are not present in the other Proteeae genomes analysed. Moreover, organisation of the 19 genes of the eut operon differs from that found in the other non-Proteeae enterobacterial genomes. CONCLUSIONS: This is the first genome sequence of M. morganii, which is a clinically relevant pathogen. Comparative genome analysis revealed several pathogenicity-related genes and novel genes not found in the genomes of other members of Proteeae. Thus, the genome sequence of M. morganii provides important information concerning virulence and determinants of fitness in this pathogen.

Dual expression of transgenic delta-5 and delta-6 desaturase in tilapia alters gut microbiota and enhances resistance to Vibrio vulnificus infection.

Omega-3 polyunsaturated fatty acids (n-3 PUFAs), such as eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA), exhibit antibacterial and anti-inflammatory activities. Furthermore, diets rich in n-3 PUFAs are known to improve disease resistance and limit pathogen infection in commercial aquaculture fishes. In this study, we examined the effects of transgenic overexpression of n-3 PUFA biosynthesis genes on the physiological response to bacterial infection in tilapia. We first established tilapia strains with single or dual expression of salmon delta-5 desaturase and/or delta-6 desaturase and then challenged the fish with Vibrio vulnificus infection. Interestingly, our data suggest that n-3 PUFA-mediated alterations in gut microbiota may be important in determining disease outcome via effects on immune response of the host. Both liver- and muscle-specific single and dual expression of delta-5 desaturase and delta-6 desaturase resulted in higher n-3 PUFA content in transgenic fish fed with a LO basal diet. The enrichment of n-3 PUFAs in dual-transgenic fish is likely responsible for their improved survival rate and comparatively reduced expression of inflammation- and immune-associated genes after V. vulnificus infection. Gut microbiome analysis further revealed that dual-transgenic tilapia had high gut microbiota diversity, with low levels of inflammation-associated microbiota (i.e., Prevotellaceae). Thus, our findings indicate that dual expression of transgenic delta-5 and delta-6 desaturase in tilapia enhances disease resistance, an effect that is associated with increased levels of n-3 PUFAs and altered gut microbiota composition.

Characterization, molecular modelling and developmental expression of zebrafish manganese superoxide dismutase.

A 977 bp cDNA containing an open reading frame encoding 224 amino acid residues of manganese superoxide dismutase was cloned from zebrafish (zMn-SOD). The deduced amino acid sequence showed high identity with the sequences of Mn-SODs from human (85.1%) to nematode (61.6%). The 3-D structure model was superimposed on the relative domains of human Mn-SOD with the root mean square (rms) deviation of 0.0919 A. The recombinant mature zMn-SOD with enzyme activity was purified using His-tag technique. The half-life of the enzyme is approximately 48 min and its thermal inactivation rate constant k(d) is 0.0154 min(-1)at 70 degrees C. The enzyme was active under a broad pH (2.2-11.2) and in the presence of up to 4% SDS. Real-time RT-PCR assay was used to detect the zMn-SOD mRNA expression during the developmental stages following a challenge with paraquat. A high level expression of Mn-SOD mRNA was detected at the cleavage stage, but decreased significantly under paraquat treatment. The results indicated that Mn-SOD plays an important role during embryonic development.

Replacement of buried cysteine from zebrafish Cu/Zn superoxide dismutase and enhancement of its stability via site-directed mutagenesis.

Zebrafish Cu/Zn-superoxide dismutase (ZSOD1) has one free cysteine (Cys-7) in a first beta-strand with lower thermostability. We predicted the stability would be increased with single-point mutation at 70 degrees C via the I-Mutant 2.0 server, and generated a mutant SOD with replacement of the free Cys to Ala (ZSODC7A) by site-directed mutagenesis. The mutant was expressed and purified from the Escherichia coli strain AD494(DE3)pLysS and the yield was 2 mg from 0.4 L of culture. The ZSODC7A was heated at 90 degrees C. In a time-dependent assay, the time interval for 50% inactivation was 32 min, and its thermal inactivation rate constant K (d) was 2 x 10(-2) min(-1). The mutant was still activated in broad pH range (2.3-12), and had only a moderate effect under sodium dodecyl sulfate treatment. The calculated specific activity of the mutant was 3980 U/mg, twice that of wild-type ZSOD1. In addition, we soaked fish larva with equal enzyme units of either ZSOD1 or ZSODC7A for 2 h, and then stressed them with 100 ppm of paraquat to induce oxidative injury. The survival rate was significant.

Characterization of Fe/Mn-superoxide dismutase from diatom Thallassiosira weissflogii: cloning, expression, and property.

A cDNA clone of 1114 bp encoding a putative Mn-superoxide dismutase (Mn-SOD) from diatom Thallassiosira weissflogii was cloned by the PCR technique. Nucleotide sequence analysis of this cDNA clone revealed that it was translated into 201 amino acid residues. When the sequence was compared with Mn-SODs from Vibrio mimicus and Escherichia coli, as well as two Fe-SODs from E. coli and Photobacterium leiognathi, this SOD showed higher homology to Mn-SOD. The amino acid residues required to coordinate the single manganese ion were conserved in all reported Mn-SOD sequences. This cDNA was introduced in an expression vector, pET-20b(+), and transformed into E. coli BL21(DE3)pLysS. The expressed SOD protein was then purified by a His-tag column. The recombinant enzyme was heated at 55 degrees C with a time-dependent assay; the time interval for 50% inactivation was 23 min, and its thermal inactivation rate constant K(d) was 3.03 x 10(-)(2) min(-)(1). The enzyme was inactivated either in acidic pH (below 4.0) or in the presence of imidazole (above 1.6 M) and had only a moderate effect under SDS (above 4%), whereas it was not affected under an alkaline pH (above 9.0). The atomic absorption spectrometric assay showed that 0.6 atom of iron/manganese (3:1) was present in each subunit of SOD. Reconstitution study was suggested that diatom SOD was cambialistic (Fe/Mn)-SOD. The finding of this SOD cDNA could be used for a reference in comparing the differences among marine phytoplankton species and as a probe to detect the transcription level of this enzyme, which can be applied in cosmetics for skin protection or defending unesthetic effects caused by oxygen-containing free radicals.

Characterization of fish Cu/Zn-superoxide dismutase and its protection from oxidative stress.

Copper/zinc superoxide dismutase was cloned from the zebrafish ( Danio rerio). The full coding region of the zebrafish superoxide dismutase (ZSOD) complementary DNA was ligated with pET-20b(+) and successfully expressed in Escherichia coli strain AD494(DE3)pLysS. The active enzyme was purified by His tagging. The ZSOD yield was 6 mg from 0.2 L of E. coli culture, and the specific activity was 2000 U/mg as assayed using a RANSOD kit. The enzyme stability was characterized by reaction to temperature, pH, and detergent treatment. The results showed enzyme activity was still active after heat treatment at 70 degrees C for 10 minutes, resistant to pH treatment from 2.3 to 12, and resistant to treatment with sodium dodecyl sulfate (SDS) under 4%. In addition, the recombinant ZSOD was used to protect fish from 100 ppm of paraquat-induced oxidative injury by soaking fish larva in 55 micro g/ml SOD enzyme. The results were significant.

Copper/zinc-superoxide dismutase from Epinephelus malabaricus cDNA and enzyme property.

A full-length cDNA of 803 base pairs encoding a putative copper/zinc-superoxide dismutase (Cu/Zn-SOD) from Epinephelus malabaricus was cloned by the polymerase chain reaction approach. Nucleotide sequence analysis of this cDNA clone revealed that it comprises a complete open reading frame coding for 154 amino acid residues. The deduced amino acid sequence showed high similarity (65-91%) with the sequences of the Cu/Zn-SOD from other species. Computer analysis of the residues required for coordinating copper (His-49, -64, and -121) and zinc (His-64, -72, and -81 and Asp-84), as well as the two cysteines (58 and 147) that form a single disulfide bond, was well-conserved among all reported Cu/Zn-SOD sequences. To further characterize the E. malabaricus Cu/Zn-SOD, the coding region was subcloned into an expression vector, pET-20b(+) and transformed into Escherichia coli BL21(DE3)pLysS. The expression of the Cu/Zn-SOD was confirmed by enzyme activity stained on a native gel and purified by Ni(2+)-nitrilotriacetic acid Sepharose. The enzyme activity was inhibited under basic pH (higher than 10.0). The enzyme retained 65% activity after heating at 60 degrees C for 10 min. The inactivation rate constant (k(d)) was 6.64 x 10(-2) min(-1) at 60 degrees C. The enzyme activity was only some decrease under 3% sodium dodecyl sulfate. The enzyme was resistant to proteolysis by trypsin and chymotrypsin. The finding of Cu/Zn-SOD cDNA could be used as a probe to detect the transcription level of this enzyme, which can be used as an early biomarker of environmental pollution. The property of this enzyme could provide a reference as compared to the oxidized forms or new isoforms, which could be induced under the experiments of pollution.

Characterization of copper/zinc-superoxide dismutase from Pagrus major cDNA and enzyme stability.

A full-length cDNA of 794 bp encoding a putative copper/zinc-superoxide dismutase (Cu/Zn-SOD) from Pagrus major was cloned by the PCR approach. Nucleotide sequence analysis of this cDNA clone revealed that it comprises a complete open reading frame coding for 154 amino acid residues. The deduced amino acid sequence showed high similarity (53-91%) with the sequences of Cu/Zn-SOD from other species. Computer analysis of the residues required for coordinating copper (His-47, 49, 64, and 121) and zinc (His-64, 72, 81, and Asp-84), as well as the two cysteines (58 and 147) that form a single disulfide bond, were well conserved among all reported Cu/Zn-SOD sequences. To further characterize the Pagrus major Cu/Zn-SOD, the coding region was subcloned into an expression vector, pET-20b(+), and transformed into Escherichia coli BL21(DE3). The expression of the Cu/Zn-SOD was confirmed by enzyme activity stained on a native-gel and purified by Ni(2+)-nitrilotriacetic acid Sepharose superflow. Dimer was the major form of the enzyme in equilibrium. The dimerization of the enzyme was inhibited under acidic pH (below 4.0 or higher than 10.0). The half-life was 8.6 min and the inactivation rate constant (k(d)) was 9.69 x 10(-2) min(-1) at 70 degrees C. The enzyme activity was not significantly affected under 4% SDS or 0.5 M imidazole. The enzyme was resistant to proteolysis by both trypsin and chymotrypsin.

Biochemical characterization of a functional recombinant aryl-alcohol dehydrogenase from Taiwanofungus camphorata.

BACKGROUND: Aryl-alcohol dehydrogenases (AADs) have been known to involve in the metabolism of aromatic compounds. RESULTS: One TcAAD cDNA (GenBank HQ453361) encoding a putative aryl-alcohol dehydrogenase (AAD) was cloned from Taiwanofungus camphorata. The deduced amino acid sequence is conserved among the reported AADs. A 3-D structural model of the TcAAD has been created based on the known structure of voltage-dependent potassium channels subunit beta-2 (PDB code: 3EAU). To characterize the TcAAD, the coding region was subcloned into an expression vector and transformed into Saccharomyces cerevisiae. The recombinant His6-tagged TcAAD was overexpressed and purified by Ni affinity chromatography. The purified enzyme showed a band of approximately 39 kDa on a 12% SDS-PAGE. The molecular mass determined by MALDI-TOF is 40.58 kDa which suggests that the purified enzyme is a monomeric enzyme. Using veratraldehyde as a substrate, the KM, Vmax of TcADD was determined at pH 6.0. Using benzyl alcohol derivatives as substrates, the oxidizing power of TcADD via NAD+ at pH 9.6 was studied. CONCLUSIONS: The coding sequence of the TcAAD cDNA was introduced into an S. cerevisiae expression system and the active enzyme purified and characterized. Understanding the properties of this TcAAD will be beneficial for its potential in xenobiotic detoxification or production of natural flavors.

Real time in vivo investigation of superoxide dynamics in zebrafish liver using a single-fiber fluorescent probe.

Superoxide anion is the key radical that causes intracellular oxidative stress. The lack of a method to directly monitor superoxide concentration in vivo in real time has severely hindered our understanding on its pathophysiology. We made transgenic zebrafish to specifically express yellow fluorescent proteins, a reversible superoxide-specific indicator, in the liver and used a fiber-optic fluorescent probe to noninvasively monitor the superoxide concentration in real time. Several superoxide-inducing and scavenging reagents were administrated onto the fish to alter superoxide concentrations. The distinct biochemical pathways of the reagents can be discerned from the transient behaviors of fluorescence time courses. These results demonstrate the feasibility of this method for analyzing superoxide dynamics and its potential as an in vivo pharmaceutical screening platform.

Taiwanofungus camphorata nitroreductase: cDNA cloning and biochemical characterisation.

Nitroreductases (Nrs) play important roles in redox system via NADPH or NADH as a reductant. A TcNr cDNA encoding a putative Nr was cloned from Taiwanofungus camphorata. A 3-D structural model of the TcNr has been created based on the known structure of BcNr (Bacillus cereus). To characterise the TcNr, the coding region was subcloned into an expression vector and transformed into Escherichia coli. The recombinant His(6)-tagged TcNr was purified by Ni affinity chromatography. The purified enzyme showed a single band at molecular mass of approximately 25 kDa on 12% sodium dodecyl sulphate-polyacrylamide gel electrophoresis. The enzyme exhibited Nr activity via ferricyanide assay. The Michaelis constant (K(M)) value for ferricyanide was 0.86 mM. The enzyme(')s half-life of deactivation at 45°C was 12.3 min. The enzyme was most active at pH 6. The enzyme's preferred substrate is 1-chloro-2, 4-dinitrobenzene.

Monothiol glutaredoxin cDNA from Taiwanofungus camphorata: a novel CGFS-type glutaredoxin possessing glutathione reductase activity.

Glutaredoxins (Grxs) play important roles in the redox system via reduced glutathione as a reductant. A TcmonoGrx cDNA (1039 bp, EU158772) encoding a putative monothiol Grx was cloned from Taiwanofungus camphorata (formerly named Antrodia camphorata). The deduced amino acid sequence is conserved among the reported monothiol Grxs. Two 3-D homology structures of the TcmonoGrx based on known structures of human Grx3 (pdb: 2DIY_A) and Mus musculus Grx3 (pdb: 1WIK_A) have been created. To characterize the TcmonoGrx protein, the coding region was subcloned into an expression vector pET-20b(+) and transformed into E. coli C41(DE3). The recombinant His6-tagged TcmonoGrx was overexpressed and purified by Ni(2+)-nitrilotriacetic acid Sepharose. The purified enzyme showed a predominant band on 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). The enzyme exhibited glutathione reductase (GR) activity via dithionitrobenzoate (DTNB) assay. The Michaelis constant (K(M)) values for GSSG and NADPH were 0.064 and 0.041 mM, respectively. The enzyme's half-life of deactivation at 60 °C was 10.5 min, and its thermal inactivation rate constant (k(d)) was 5.37 × 10(-2) min(-1). The enzyme was active under a broad pH range from 6 to 8. The enzyme retained 50% activity after trypsin digestion at 37 °C for 40 min. Both mutants C(40)→S(40) and C(165)→S(165) lost 40-50% GR activity, whereas the mutant S(168)→C(168) showed a 20% increase in its GR activity.

Betanodavirus induces oxidative stress-mediated cell death that prevented by anti-oxidants and zfcatalase in fish cells.

The role of oxidative stress in the pathogenesis of RNA nervous necrosis virus infection is still unknown. Red-spotted grouper nervous necrosis virus (RGNNV) induced free radical species (ROS) production at 12-24 h post-infection (pi; early replication stage) in fish GF-1 cells, and then at middle replication stage (24-48 h pi), this ROS signal may upregulate some expressions of the anti-oxidant enzymes Cu/Zn SOD and catalase, and eventually expression of the transcription factor Nrf2. Furthermore, both antioxidants diphenyliodonium and N-acetylcysteine or overexpression of zebrafish catalase in GF-1 cells also reduced ROS production and protected cells for enhancing host survival rate due to RGNNV infection. Furthermore, localization of ROS production using esterase activity and Mitotracker staining assays found that the ROS generated can affect mitochondrial morphology changes and causes ΔΨ loss, both of which can be reversed by antioxidant treatment. Taken together, our data suggest that RGNNV induced oxidative stress response for playing dual role that can initiate the host oxidative stress defense system to upregulate expression of antioxidant enzymes and induces cell death via disrupting the mitochondrial morphology and inducing ΔΨ loss, which can be reversed by anti-oxidants and zfcatalase, which provide new insight into betanodavirus-induced ROS-mediated pathogenesis.

Cloning, expression, and characterization of a thioredoxin reductase cDNA from Taiwanofungus camphorata.

A cDNA encoding putative thioredoxin reductase (TR) was identified from a medicinal mushroom, Taiwanofungus camphorata (T. camphorata). Alignment of the deduced amino acid sequence with TRs from other organisms showed high levels of identity (59-74%). A three-dimensional (3-D) homology structure was created for this TR. Functional T. camphorata TR (TcTR) was overexpressed in yeast and purified. The purified enzyme showed a monomic form on a 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). The enzyme's half-life of deactivation at 60 degrees C was 12.9 min, and its thermal inactivation rate constant K(d) was 5.37 x 10(-2) min(-1). The optimal pH for the enzyme was pH 8 and retained about 76% activity in the presence of 0.1 M imidazole. The enzyme showed 50% activity after 10 min of incubation at 37 degrees C with chymotrypsin. The Michaelis constant (K(m)) value for dithionitrobenzoate (DTNB) was 1.59 mM.

Cloning, expression, and characterization of an enzyme possessing both glutaredoxin and dehydroascorbate reductase activity from Taiwanofungus camphorata.

Glutaredoxins (Grxs) play important roles in the reduction of disulfides via reduced glutathione as a reductant. A cDNA (503 bp, EU193660) encoding a putative Grx was cloned from Taiwanofugus camphorata (Tc). The deduced amino acid sequence is conserved among the reported dithiol Grxs. A 3D homology structure was created for this TcGrx. To characterize the TcGrx enzyme, the coding region was subcloned into an expression vector pET-20b(+) and transformed into Escherichia coli . Functional TcGrx was expressed and purified by Ni(2+)-nitrilotriacetic acid Sepharose. The purified enzyme showed bands of approximately 15 kDa on 15% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). The TcGrx encodes a protein possessing both Grx and dehydroascorbate reductase (DHAR) activity. The Michaelis constant (K(m)) values for beta-hydroxyethyl disulfide (HED) and dehydroascorbate (DHA) were 0.57 and 1.85 mM, respectively. The half-life of deactivation of the protein at 100 degrees C was 8.5 min, and its thermal inactivation rate constant K(d) was 6.52 x 10(-2) min(-1). The enzyme was active under a broad pH range from 6.0 to 10.0 and in the presence of imidazole up to 0.4 M. The enzyme was susceptible to SDS denaturation and protease degradation/inactivation.

Biochemical characterization of a novel 2-Cys peroxiredoxin from Antrodia camphorata.

Peroxiredoxins (Prxs) play important roles in antioxidation and cell signaling. A gene encoding a novel 2-Cys Prx was identified based on sequence homology in an expressed sequence tag database of the Antrodia camphorata, a medicinal mushroom found only in Taiwan. The 2-Cys Prx cDNA (940 bp) encodes a protein of 188 amino acid residues with calculated molecular mass of 20,965 Da and a pI of 5.89. The coding region was subcloned into pAVD10, transformed into Escherichia coli, and expressed as a His-tagged fusion protein. The purified enzyme was characterized under various conditions. The Prx retained 68% activity after being heated at 60 degrees C for 2 min. It was stable under a broad pH range from 5 to 11. The enzyme activity was slightly decreased in the presence of 1% sodium dodecyl sulfate. The enzyme was somewhat susceptible to chymotrypsin treatment but resistant to digestion by trypsin.

Curcumin affects development of zebrafish embryo.

Embryotoxic and teratogenic effects of curcumin on the development of zebrafish embryo were investi-gated in this study. The LD(50) values of curcumin (24-h incubation) were estimated at 7.5 microM and 5 microM for embryos and larvae, respectively. The developmental defects caused by curcumin treatments include bent or hook-like tails, spinal column curving, edema in pericardial sac, retarded yolk sac resorption, and shorter body length. In curcumin-treated larvae, fluorescence signals of curcumin were found in edamae sac and some skin cells. Together, these results indicate that zebrafish are suitable model organisms to study the toxic effects of curcumin.