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The present study investigated the virulence and expression of innate immunity genes in isolates of infectious hematopoietic necrosis virus (IHNV) in Gangwon province, South Korea, by challenging rainbow trout, Atlantic salmon, and coho salmon. Eight IHNV isolates were used to infect RTG-2 cells for viral replication using plaque assays. Three isolates with the highest replication rates, the RtPc0314g and RtPc0314c isolates of the JRt-Shizuoka type and the RtPc0816g isolate of the JRt-Nagano type, were experimentally infected into the fish. In rainbow trout, both RtPc0314c and RtPc0314g isolates showed 100% cumulative mortality while the RtPc0816g isolate showed 60% cumulative mortality for 14 days. In contrast, all three isolates showed

The estrogen receptor (ER) and aryl hydrocarbon receptor (AhR) are ligand-activated transcription factors involved in estrogen or xenobiotic exposure, whereas the 90-kDa heat shock protein (HSP90), which is a ubiquitously expressed molecular chaperone, is involved in the signal transduction process. Although the interactions between these pathways have been under investigation, the mechanisms are unclear and the potential role of HSP90 in these interactions has not been reported. The results of goldfish primary hepatocytes showed that exposure to PCB77 and 17β-estradiol (E2) alone induced significant protein expression of cytochrome P450 1A (CYP1A) and vitellogenin (VTG), respectively. On the other hand, the combined exposure to PCB77 and E2 led to the reduction of CYP1A and VTG compared to the single treatments. Although the AhRs and ERs were naturally induced during the co-treatment, the total amount of HSP90 binding to the receptors was not changed. Furthermore, while the HSP90 chaperon activity was blocked by the specific inhibitor (geldanamycin), reciprocal inhibition between AhR and ER pathways was not observed. These findings indicate a potential role of HSP90 where competition between AhR and ER for binding to HSP90 can occur and cause reciprocal inhibition.

This study examined the influence of water temperature on the induction of vitellogenin (VTG) in walleye pollocks (Gadus chalcogrammus) in aquaculture. The full-length VTG-A (or VTG-I) gene of walleye pollock was cloned using RACE (Rapid amplification of cDNA ends)-PCR method from liver tissue and recombinant VTG protein (wpVTG) was expressed in E. coli. The wpVTG protein was used to construct polyclonal antibodies to specifically detect VTG in blood; the antibodies were verified using western blotting. The VTG in blood plasma was determined by ELISA. The relationship between GSI (gonadosomatic index), VTG and body length were all positively correlated, and the results indicated that VTG can be used as an indicator of sex and/or maturation of female G. chalcogrammus in aquaculture. When VTG was measured in by the aqua-cultured female pollocks raised in water temperature of 5 °C, 8 °C, 11 °C, and 14 °C, VTG was detected only at 5 °C and 8 °C. In the histological analysis of VTG-inducing individuals, vitellogenesis was found in ovaries. In those same samples, E2 (17beta-estradiol) was detected in the blood by LC-MS. On the other hand, there was no significant difference in level of VTG induction when E2 of same concentration was treated to hepatocyte culture in different water temperatures. These results seemed to indicate that VTG is not directly affected by water temperature. However, we considered that the water temperature indirectly affect VTG induction by affecting the E2 concentration level.

The Nackdong River is the longest river in South Korea and passes through major cities that have several industrial complexes, including chemical, electric, and petrochemical complexes, and municipal characteristics such as apartment complexes. Along the river, the Gumi region has an electric industrial complex and an apartment complex that may be possible point sources of xenoestrogens such as phenolic compounds. To identify the causative chemicals for estrogenic activity in the river water of this region, bioassay-directed chemical analysis was performed. All samples from six sampling sites (an upstream point: S1; hot spot points: S2-1, S2-2, and S2-3; and downstream points: S3, and S4) showed estrogenic activity in the E-screen assay, with bio-EEQs (17beta-E(2)-equivalent quantities) ranging from 25.35-677.15 pg/L. Samples from S2-2, the sampling point downstream of the junction of stream water, and domestic and industrial wastewater, contained the highest estrogenic activity. Since the bio-EEQ of the organic acid fraction (F2) of the S2-2 sample had the highest activity (823.25 pg-EEQ/L) and F2 may contain phenolic compounds, GC-MS analyses for phenolic xenoestrogens were conducted with the organic acid fractions of the river water samples. Six estrogenic phenolic chemicals, 4-NP, BPA, 4-t-OP, 4-t-BP, 4-n-OP, and 4-n-HTP, were detected, with the highest concentrations (I-EEQ) found in S2-2 (231.80 pg/L). Among these phenolic chemicals, 4-NP was the most potent estrogen (bio-EEF; 8.12 x1 0(-5)) and acted as a full agonist. Furthermore, 4-NP was present at levels (2.0 microg/L in S2-2) that can induce VTG induction in fish (1 microg/L). In addition, we confirmed that river water (S2-2) significantly increased serum VTG levels in crucian carp (Carassius auratus) in a fish exposure experiment under laboratory conditions. Therefore, phenolic xenoestrogens, especially 4-NP, may be the main causative compounds responsible for the estrogenic effect on the Nackdong River.

A biosensor based on cytochrome c 3 (cyt c 3 ) has been introduced to detect and quantify superoxide radical (O 2 − ). Cyt c 3 , isolated from the sulfate-reducing bacterium ( Desulfovibrio vulgaris Miyazaki F. strain), and its mutant were immobilized onto a conducting polymer coated electrodes by the covalent bonding with carbodiimide chemistry. The immobilization of cyt c 3 was investigated with quartz crystal microbalance, electrochemical impedance spectroscopy, and cyclic voltammetric studies. The CVs recorded for cyt c 3 and a mutant modified-electrodes showed a quasi-reversible behavior having the formal potential of about −471 and −476 mV ( versus AgǀAgCl), respectively, in a 0.1 M phosphate buffer solution (pH 7.0). The modified electrodes showed the surface controlled process and the electron transfer rate constants ( k s ) were evaluated to be 0.47 and 0.51 s −1 for cyt c 3 and mutant modified electrodes, respectively. A potential application of the cyt c 3 modified electrode was evaluated by monitoring the bioelectrocatalytic response towards the O 2 − . The hydrodynamic range of 0.2–2.7 μmole L −1 and the detection limit of 0.05 μmole L −1 were obtained.

Bisphenol A (BPA), generally known as bisphenols, has been identified as a potential estrogenic substance. BPA must be conjugated to carrier protein and BSA was commonly used. 4,4-Bis(4-hydroxyphenyl) valeric acid (BHPVA) has a bisphenolic structure and a long carbon chain with a reactive carboxyl group on the end. In this study, BHPVA-BSA was used to produce polyclonal antibody against bisphenolic structure, and a modified competitive ELISA method for quantification of BPA was developed. This system was based on BHPVA-BSA for polyclonal antibody production against bisphenolic structure, and BHPVA-HRP for determination of BPA substituting detection antibody in competitive reaction. Recovery was assessed at 10 different concentrations (2-1000 ng/ml) of BHPVA, and the recovery range was from 96.3% to 107.2%. The variation was from 6.2% to 9.8% for intra assay and from 10.1% to 12.6% for inter assay. The quadratic was used to establish the curve regression. The range was found to be between 2 and 1000 ng/ml. This modified competitive ELISA method has proven to be a very useful tool for quantification of BPA without the unexpected interaction of BSA and anti-BSA polyclonal antibody.

A sandwich enzyme-linked immunosorbant assay (ELISA) was developed to quantitatively detect Bullfrog (Rana catesbeiana) vitellogenin (VTG) levels. This procedure involved a sandwich ELISA using monoclonal antibodies of BVmA1 and BVmA2 against Bullfrog-VTG, and BVmA1 conjugated to horseradish peroxidase as the detection antibody. The assay range was between 9.4 ng/ml and 1200 ng/ml and the recovery of the VTG added to Bullfrog control male serum was 92.0-108.8%. Male Bullfrog was induced by injection 17beta-estradiol (E2) for four weeks and Bullfrog-VTG levels were measured each week. Histological analysis was performed for investigating the correlation of the effect to male reproduction and Bullfrog-VTG level variation depending on E2 dose. After two weeks of E2 exposure, the induced Bullfrog-VTG level was significantly higher than Bullfrog control female (p0.05). After four weeks of E2 exposure, the rupture and fusion of seminiferous tubule in the testes of male Bullfrog were shown and provided direct evidence that the reproduction of male Bullfrog was affected by estrogenic compounds. Bullfrog-VTG bioassay, using the sandwich ELISA, could be a sensitive and useful tool for quantification of estrogenic principles in aquatic environments.

Cytochrome c3 (cyt c3) can mediate electron transport across phosphatidylcholine (PC)/cardiolipin (CL) and PC/phosphatidylglycerol (PG) membranes. A two-molecule process is involved in the electron transport across PC/CL membranes in the liquid-crystalline state. In contrast, a single-molecule process dominates the electron transport across PC/CL membranes in the gel state and PC/PG membranes in the liquid-crystalline and gel states. Namely, the electron transport mechanism differs with the phospholipid composition and membrane fluidity. The rate-limiting step of the two-molecule process was lateral diffusion of cyt c3 in membranes. The rate constants for the three single-molecule process cases were similar to each other. To elucidate these reaction processes, interactions between cyt c3 and phosphate groups and between cyt c3 and the glycerol backbones of phospholipid bilayers were investigated by means of 31P and 2H solid-state NMR, respectively, for CL and PC/CL membranes. The results showed that the polar headgroups of both phosphatidylcholine and CL are involved in the binding of cyt c3. Also, cyt c3 penetrates into membranes, which would induce distortion of the lipid bilayer. The molecular mechanisms underlying the single- and two-molecule processes are discussed in terms of membrane structure.

A simple and direct immunosensor for the determination of carp ( Carassius auratus ) vitellogenin (Vtg), a female-specific protein, has been proposed based on an antibody-captured conducting polymer-coated electrode. The monoclonal antibody specific to carp ( C. auratus ) Vtg was immobilized by covalent coupling to the carboxylic acid group on the polymer. The antibody immobilization and antibody–antigen interaction have been demonstrated by means of quartz crystal microbalance and impedance spectroscopic techniques. The impedance change occurred at the sensor surface due to the specific immuno-interaction was utilized to determine Vtg. The sensor showed high selectivity and sensitive response to Vtg in a buffered medium without redox probe. Vtg was determined in the linear range from 1.0 to 8.0 μg/l with the standard deviation of ±0.13 ( n =3) and the detection limit was determined to be 0.42 μg/l. This method was applied to the determination of Vtg in real male and female carp ( C. auratus ) serum samples.

The interaction of cytochrome c (cyt c) with phospholipids was investigated using electrochemical, in-situ UV–visible, and FTIR spectrophotometric methods, which showed that the electrostatic interaction between cyt c and cardiolipin (CL) gave a rapid direct electron transfer. This was studied for the effect of charges of lipids on the redox reaction of cyt c and investigated for electrochemical behavior according to the quantity of CL, the accumulation time, pH, temperature, and the stability of the CL layer. The spectroelectrochemical results showed that only the absorption band appearing at 550 nm, which is one of the Q bands, was directly related to the redox reaction of iron ions in cyt c. The kinetic parameters, DO and ko for the electron transfer reaction of cyt c on the lipid layer were determined to be 3.09 (±0.02)×10−7 and 2.06 (±0.04)×10−3 cm s−1.

Eukaryotic replication protein A (RPA) is a single-stranded DNA-binding protein with multiple functions in DNA replication, repair, and genetic recombination. RPA contains an evolutionarily conserved 4-cysteine-type zinc finger motif (X(3)CX(2-4)CX(12-15)CX(2)C) that has a potential role in regulation of DNA replication and repair (Dong, J., Park, J-S., and Lee, S-H. (1999) Biochem. J. 337, 311-317 and Lin, Y.-L., Shivji, M. K. K., Chen, C., Kolodner, R., Wood, R. D., and Dutta, A. (1998) J. Biol. Chem. 273, 1453-1461), even though the zinc finger itself is not essential for its DNA binding activity (Kim, D. K., Stigger, E., and Lee, S.-H. (1996) J. Biol. Chem. 271, 15124-15129). Here, we show that RPA single-stranded DNA (ssDNA) binding activity is regulated by reduction-oxidation (redox) through its zinc finger domain. RPA-ssDNA interaction was stimulated 10-fold by the reducing agent, dithiothreitol (DTT), whereas treatment of RPA with oxidizing agent, diazene dicarboxylic acid bis[N,N-dimethylamide] (diamide), significantly reduced this interaction. The effect of diamide was reversed by the addition of excess DTT, suggesting that RPA ssDNA binding activity is regulated by redox. Redox regulation of RPA-ssDNA interaction was more effective in the presence of 0.2 M NaCl or higher. Cellular redox factor, thioredoxin, was able to replace DTT in stimulation of RPA DNA binding activity, suggesting that redox protein may be involved in RPA modulation in vivo. In contrast to wild-type RPA, zinc finger mutant (cysteine to alanine mutation at amino acid 486) did not require DTT for its ssDNA binding activity and is not affected by redox. Together, these results suggest a novel function for a putative zinc finger in the regulation of RPA DNA binding activity through cellular redox.

The macroscopic and microscopic redox potentials of cytochrome c 3 from Desulfovibrio vulgaris Hildenborough ( Dν H) were estimated by the combined use of 1 H NMR and differential pulse polarography at two ionic strengths. The redox potentials obtained were compared with those of Desulfovibrio vulgaris Miyazaki F ( Dν MF) to see the effect of amino acid substitution in the 13 residues Although the general tendencies are similar to each other, there are distinct differences. The average redox potentials of Dν H cytochrome c 3 were lower than those of Dν MF by about 34 mV. The difference in the microscopic redox potentials was significant for hemes 1 and 3. The cooperative interaction between hemes 1 and 2 was larger in Dν H cytochrome c 3 than in Dν MF cytochrome c 3 and the former was ionic strength dependent. The microscopic redox potentials of Dν H cytochrome c 3 increased with an increase of ionic strength.

The assignment of 1 H-NMR signals of the heme methyl and propionate groups of cytochrome c 3 of D. vulgaris Miyazaki F was performed. The heme assignment was revised for hemes 2 and 3 (sequential heme numbering). Namely, heme 4 is mainly reduced at first with hemes 1, 2 and 3 following it in this order. The p 2 H titration of heme methyl signals in four macroscopic oxidation states was performed in the p 2 H range of 5.2 to 9.0. While the heme methyl resonances in the fully oxidized state showed just small changes with p 2 H most resonances in the intermediate oxidation states revealed clear p 2 H dependence. In particular, the methyl resonances of heme 1 shifted significantly in the acidic region. Then, the chemical shifts of β-CH 2 (next to the carboxyl group) of all propionate groups in the fully oxidized state were observed at various p 2 H in the range of 4.5 to 9.0. Only the propionate group at C-13 (IUPAC-IUB nomenclature) of heme 1 showed a clear change in this p 2 H range, its titration curve being similar to those of the methyl resonances of heme 1 in the intermediate oxidation states. pK a of the propionate group was 5.95 ± 0.05. Analysis of the microscopic formal redox potentials was carried out for the observations at p 2 H 5.2, 7.1 and 9.0. The redox potentials of heme 1 showed the most remarkable p 2 H dependence, resulting in the change of the order of the redox potentials of four berries. A significant change was also found in the interacting potential between hemes 1 and 2. In the light of the p 2 H-titration experiments, the propionate at C-13 of heme 1 was identified as the most plausible ionizable group responsible for the p 2 H dependence of microscopic redox potentials of heme 1 in the acidic region.

Conditions for the specific labelling of the tetrahaeme protein cytochrome c 3 of Desulfovibrio vulgaris Miyazaki F during culture of this sulphate-reducing bacterium in a minimal medium were established. Phenylalanine and tyrosine residues were specifically deuterated at more than 85% efficiency. Cytochrome c 3 has nine histidine, three tyrosine and two phenylalamine residues. Eight histidine imidazoles are ligated to four haeme groups. Using the deuterated cytochrome c 3 , aromatic proton signals of phenylalanine and tyrosine residues in the fully oxidized state were identified. However, the signals of one phenylalanine residue were missing and this was tentatively assigned to Phe20. The aromatic proton signals of His67 were also assigned p 2 H titration. Its p k 1 was much higher than that for the free histidine residue. No tyrosine residue was ionized up to p 2 H 12.