Enhanced protection against oxidative stress in an astaxanthin-overproduction Haematococcus mutant (Chlorophyceae)

Many unicellular green algae can become yellow or red in various natural habitats due to mass accumulation of a secondary carotenoid, such as lutein, or astaxanthin. The accumulation of secondary carotenoids is generally thought to be a survival strategy of the algae under photo-oxidative stress or other adverse environmental conditions. The physiological role of the carotenoids in stress response is less well understood at the subcellular or molecular level. In this study, a stable astaxanthin overproduction mutant (MT 2877) was isolated by chemical mutagenesis of a wild type (WT) of the green microalga Haematococcus pluvialis Flotow NIES-144. MT 2877 was identical to the WT with respect to morphology, pigment composition, and growth kinetics during the early vegetative stage of the life cycle. However, it had the ability to synthesize and accumulate about twice the astaxanthin content of the WT under high light, or under high light in the presence of excess amounts of ferrous sulphate and sodium acetate. Under stress, the mutant exhibited higher photosynthetic activities than the WT, based on considerably higher chlorophyll fluorescence induction, chlorophyll autofluorescence intensities, and oxygen evolution rates. Cell mortality caused by stress was reduced by half in the mutant culture compared with the WT. Enhanced protection of the mutant against stress is attributed to its accelerated carotenogenesis and accumulation of astaxanthin. Our results suggest that MT 2877, or other astaxanthin overproduction Haematococcus mutants, may offer dual benefits, as compared with the wild type, by increasing cellular astaxanthin content while reducing cell mortality during stress-induced carotenogenesis.

Construction and characterization of a live, attenuated esrB mutant of Edwardsiella tarda and its potential as a vaccine against the haemorrhagic septicaemia in turbot, Scophthamus maximus (L.)

The esrB gene of Edwardsiella tarda, which encodes a regulator protein of the type III secretion system, was mutated by the unmarked deletion method and reintroduced by allelic exchange into the chromosome of E. tarda LSE40 by means of the suicide vector pRE 112. The LSE40 esrB mutant was highly attenuated when inoculated intraperitoneally into turbot Scophthamus maximus L., showing a 50% lethal dose of 10(8.1) cfu/fish. The esrB mutants were not recoverable from the internal organs at 14 days post-inoculation. Vaccination with a single dose of 10(5)-10(7) cfu/fish of the esrB mutant elicited significant protection against the wildtype strain of E. tarda LSE40 (relative percentage survival > 50%). The protection correlated well with the antibody titres in the serum of vaccinated fish. (c) 2006 Elsevier Ltd. All rights reserved.

Identification and analysis of the immune effects of CpG motifs that protect Japanese flounder (Paralichthys olivaceus) against bacterial infection

CpG-containing oligodeoxynucleotides (ODNs) are known to be immunostimulatory in vertebrate systems and can activate both innate and adaptive immune responses. In this report, we described the selection, identification, and analysis of CpG motifs with immunoprotective effects in Japanese flounder. Sixteen CpG ODNs were synthesized and examined for the ability to inhibit bacterial dissemination in Japanese flounder blood. Four ODNs with the strongest inhibitory effects were selected and mixed to form ODNs 4M. In addition, a plasmid, pCN6, was constructed that contains the sequences of the four selected ODNs. When administered into Japanese flounder via intraperitoneal injection, both ODNs 4M and pCN6 could, in dose and time dependent manners, afford short-term protection against the infections of two different bacterial pathogens. Immunological analyses showed that ODNs 4M and, especially, pCN6 activated head kidney macrophages and enhanced serum bactericidal activity via probably the alternative pathway of complement activation. When used as a DNA vaccine to immunize Japanese flounder, pCN6 conferred apparent protections (42.9% and 52.6%, respectively, in terms of relative percent survival) against the challenges of two different fish pathogens at 4-week post-vaccination. Transcriptional analysis showed that vaccination with pCN6 upregulated the expression of the genes encoding NKEF, MHC II alpha, IL-1 beta, Mx, and MHC I alpha. These results demonstrate that ODNs 4M and pCN6 are immunostimulatory in Japanese flounder and can induce short- and long-term nonspecific protections against bacterial infections. (C) 2010 Elsevier Ltd. All rights reserved.

Identification and analysis of a CpG motif that protects turbot (Scophthalmus maximus) against bacterial challenge and enhances vaccine-induced specific immunity

Oligodeoxynucleotides (ODNs) containing unmethylated CpG motifs in certain contexts are known to be immunostimulatory in vertebrate systems. CpG ODNs with immune effects have been identified for many fish species but, to our knowledge, not for turbot. In this study, a turbot-effective CpG ODN, ODN 205, was identified and a plasmid, pCN5, was constructed which contains the CpG motif of ODN 205. When administered into turbot via intraperitoneal (i.p.) injection, both ODN 205 and pCN5 could (i) inhibit bacterial dissemination in blood in dose and time dependent manners, and (ii) protect against lethal bacterial challenge. Immunological analyses showed that in vitro treatment with ODN 205 stimulated peripheral blood leukocyte proliferation, while i.p. injection with ODN 205 enhanced the respiratory burst activity, chemiluminescence response, and acid phosphatase activity of turbot head kidney macrophages. pCN5 treatment-induced immune responses similar to those induced by ODN 205 treatment except that pCN5 could also enhance serum bactericidal activity in a calcium-independent manner. To examine whether ODN 205 and pCN5 had any effect on specific immunity, ODN 205 and pCN5 were co-administered into turbot with a Vibrio harveyi subunit vaccine, DegQ. The results showed that pCN5, but not ODN 205, significantly increased the immunoprotective efficacy of DegQ and enhanced the production of specific serum antibodies in the vaccinated fish. Further analysis indicated that vaccination with DegQ in the presence of pCN5 upregulated the expression of the genes encoding MHC class II alpha, IgM, Mx, and IL-8 receptor. Taken together, these results demonstrate that ODN 205 and pCN5 can stimulate the immune system of turbot and induce protection against bacterial challenge. In addition, pCN5 also possesses adjuvant property and can potentiate vaccine-induced specific immunity. (C) 2010 Elsevier Ltd. All rights reserved.

Effects of aniracetam on extracellular levels of transmitter amino acids in the hippocampus of the conscious gerbils: an intracranial microdialysis study

The effects of aniracetam on extracellular amino acid levels in the hippocampus of conscious gerbils, with or without transient cerebral ischemia/reperfusion, were measured by microdialysis and reverse phase-high performance liquid chromatography. Increased extracellular levels of aspartate and glutamate that were observed in the hippocampus of conscious gerbils during transient global forebrain ischemia were reversed by aniracetam. In contrast, the level of extracellular gamma-aminobutyric acid was increased, while taurine was maintained at a higher level than other amino acids by administration of aniracetam (100 mg/kg, p.o.) 60 min before ischemia. Further, in contrast to ischemic animals, administration of aniracetam (100 mg/kg, p.o.) enhanced the release of glutamate and aspartate in the normal gerbil hippocampus. The results suggest that these effects might be due to a partial calcium agonist activity of aniracetam, and that the effects of aniracetam on amino acid levels might be a mechanism of protection against delayed neuronal death in the ischemic hippocampus, thereby improving memory dysfunction induced by ischemia/reperfusion. (C) 2003 Elsevier Science Ireland Ltd. All rights reserved.

Genomic organization and expression analysis of Toll-like receptor 3 in grass carp (Ctenopharyngodon idella)

Toll-like receptor 3 (TLR3) participates in the innate immune response by recognizing viral pathogens. To investigate grass carp immune system responding to GCRV (grass carp reovirus) infection, the full-length cDNA sequence and genomic organization of grass carp TLR3 (CiTLR3) was identified and characterized. The full-length genome sequence of CiTLR3 is composed of 5668 nucleotides, including five exons and four introns. The full-length of CiTLR3 cDNA is 3681 bp in length and encodes a polypeptide of 904 amino acids with an estimated molecular mass of 102,765 Da and a predicted isoelectric point of 8.35. Analysis of the deduced amino acid sequence indicated that CiTLR3 has four main structural domains, including a signal peptide sequence, 14 LRR (leucine-rich repeat) motifs, a transmembrane region and a TIR (Toll/interleukin-1 receptor) domain. It is most similar to the crucian carp (Carassius auratus) TLR3 amino acid sequence with an identity of 99%. Quantitative RT-PCR analysis showed that CiTLR3 transcripts were significantly up-regulated starting at day 1 and continued through day 7 following GCRV infection (P < 0.05). These data implied that CiTLR3 is involved in antiviral defense, provide molecular and functional information for grass carp TLR3, and implicate their role in mediating immune protection against grass carp viral diseases. (C) 2009 Elsevier Ltd. All rights reserved.

Effects of solar UV radiation on morphology and photosynthesis of filamentous cyanobacterium Arthrospira platensis

To study the impact of solar UV radiation (UVR) (280 to 400 nm) on the filamentous cyanobacterium Arthrospira (Spirulina) platensis, we examined the morphological changes and photosynthetic performance using an indoor-grown strain (which had not been exposed to sunlight for decades) and an outdoor-grown strain (which had been grown under sunlight for decades) while they were cultured with three solar radiation treatments: PAB (photosynthetically active radiation [PAR] plus UVR; 280 to 700 nm), PA (PAR plus UV-A; 320 to 700 nm), and P (PAR only; 400 to 700 nm). Solar UVR broke the spiral filaments of A. platensis exposed to full solar radiation in short-term low-cell-density cultures. This breakage was observed after 2 h for the indoor strain but after 4 to 6 h for the outdoor strain. Filament breakage also occurred in the cultures exposed to PAR alone; however, the extent of breakage was less than that observed for filaments exposed to full solar radiation. The spiral filaments broke and compressed when high-cell-density cultures were exposed to full solar radiation during long-term experiments. When UV-B was screened off, the filaments initially broke, but they elongated and became loosely arranged later (i.e., there were fewer spirals per unit of filament length). When UVR was filtered out, the spiral structure hardly broke or became looser. Photosynthetic 0, evolution in the presence of UVR was significantly suppressed in the indoor strain compared to the outdoor strain. UVR-induced inhibition increased with exposure time, and it was significantly lower in the outdoor strain. The concentration of UV-absorbing compounds was low in both strains, and there was no significant change in the amount regardless of the radiation treatment, suggesting that these compounds were not effectively used as protection against solar UVR. Self-shading, on the other hand, produced by compression of the spirals over adaptive time scales, seems to play an important role in protecting this species against deleterious UVR. Our findings suggest that the increase in UV-B irradiance due to ozone depletion not only might affect photosynthesis but also might alter the morphological development of filamentous cyanobacteria during acclimation or over adaptive time scales.

Antibody response of carp, Cyprinus carpio to the cestode, Bothriocephalus acheilognathi

The humoral antibody response and the number of pronephric antibody-secreting cells were examined in naturally Bothriocephalus acheilognathi-infected carp. Cyprinus carpio, and in those injected intraperitoneally with an extract of the cestode. In the extract-injected fish, specific antibody was detected 3 weeks after a second injection given 2 weeks after the primary injection, and antibody levels persisted for more than 200 days. A third injection also enhanced the antibody level in the extract-injected carp. The numbers of antibody-secreting cells were significantly higher in carp injected 3 times with the extract than in the control. In naturally-infected fish, the serum antibody levels and the number of pronephric antibody-secreting cells were higher in infected fish than in uninfected individuals although this difference was not statistically significant. The relevance of these results to immune protection against infection is discussed.

The direct electrochemistry of cryo-hydrogel immobilized myoglobin at a glassy carbon electrode

A cryo-hydrogel membrane (CHM) immobilized at a glassy carbon (GC) electrode is reported for the direct electron transfer of redox proteins. The most attractive characteristics of this CHM were its hydrophilic micro-environment for incorporated proteins to retain their activities, its high ability for protection against interference of denatured and adsorbed proteins at the electrode, its potential applications for various proteins or enzymes, as well as its high mechanical strength and thermal stability. A clear well developed and stable redox wave was obtained for commercially available horse heart myoglobin without further purification, giving a peak to peak separation Delta E(p) = 93 mV at 5 mV s(-1) and the formal electrode potential E(0)' = -0.158 V (vs. Ag/AgCl). The formal heterogeneous electron transfer rate constant was calculated as k(0)' = 5.7 X 10(-4) cm s(-1) at pH 6.5, showing rapid electron transfer was achieved. The pH controlled conformational equilibria, acid state --> natural state --> basic I state --> basic II state, of myoglobin at the CHM GC electrode in the pH range 0-13.8 were also observed and are discussed in detail.

Physical modeling of localized scour for the Yangtze Estuary Waterway Improvement Project, Phase I

In order to examine the effectiveness of engineering protection against localized scour in front of the south groin-group of the Yangtze Estuary Waterway Improvement Project, Phase I, an undistorted physical model on a geometric scale of 1:250 is built in this study, covering two groins and their adacent estuarine areas. By use of rinsing fix-bed model as well as localized mobile-bed model the experiment is undertaken under bi-directional steady flow. According to the experimental results, waterway dredging leads to the increase in steram velocity, the increase being larger during the ebb than during the flood. Construction of the upstream groin has some influence on the flow patterns near the downstream groin. Localized scour in front of the groin-heads is controlled mainly by ebb flow. In the case of a riverbed composed entirely of silt, the depths of localized scour in front of the two groin-heads are 27 m and 29 m, respectively. In reality, the underneath sediment of the prototype riverbed is clay whose threshold velocity is much higher than the stream velocity in the Yangtze Estuary; therefore, the depths of localized scour will not be much larger than the thickness of the silt layer, i. e. 7.4 m and 4.7 m, respectively. The designed aprons covering the riverbed in fron of the groin-heads are very effective in scour control. Aprons of slightly smaller size can also fulfill the task of protection, but the area of localized scour increases significantly.

Construction and evaluation of DNA vaccines encoding Edwardsiella tarda antigens

Edwardsiella tarda is an opportunistic pathogen that can infect humans, animal, and fish. Two E. tarda antigens, Eta6 and FliC, which are homologues to an ecotin precursor and the FliC flagellin, respectively, were identified by in vivo-induced antigen technology from a pathogenic E. tarda strain isolated from diseased fish. When used as a subunit vaccine, purified recombinant Eta6 was moderately protective against lethal challenge of E. tarda in a Japanese flounder model, whereas purified recombinant FliC showed no apparent immunciprotectivity. Similarly, DNA vaccines based on eta6 and fliC in the form of plasmids pEta6 and pFliC induced, respectively, moderate and marginal protection against E. tarda infection. To improve the vaccine efficacy of eta6, a chimeric DNA vaccine, pCE6, was constructed, which encodes Eta6 fused in-frame to FliC. pCE6 was found to induce significantly higher level of protection than pEta6. Likewise, another chimeric DNA vaccine, pCE18, which expresses FliC fused to a previously identified E. tarda antigen Et18, elicited significantly stronger protective immunity than the DNA vaccine based on et18 alone. Fish immunized with pEta6 and pCE6 produced specific serum antibodies and exhibited significantly enhanced expression of the genes encoding elements that are involved in both innate and adaptive immune responses. Furthermore, the induction magnitudes of most of these genes were significantly higher in pCE6-vaccinated fish than in pEta6-vaccinated fish. (C) 2009 Elsevier Ltd. All rights reserved.

Identification of an Edwardsiella tarda surface antigen and analysis of its immunoprotective potential as a purified recombinant subunit vaccine and a surface-anchored subunit vaccine expressed by a fish commensal strain

Edwardsiella tarda is the etiological agent of edwardsiellosis, a systematic disease that affects a wide range of marine and freshwater fish cultured worldwide. In order to identify E. tarda antigens with vaccine potential, we in this study conducted a systematic search for E. tarda proteins with secretion capacity. One of the proteins thus identified was Esa1, which contains 795 amino acid residues and shares extensive overall sequence identities with the D15-like surface antigens of several bacterial species. In silico analyses indicated that Esa1 localizes to outer membrane and possesses domain structures that are conserved among bacterial surface antigens. The vaccine potential of purified recombinant Esa1 was examined in a Japanese flounder (Paralichthys olivaceus) model, which showed that fish vaccinated with Esa1 exhibited a high level of survival and produced specific serum antibodies. Passive immunization of naive fish with antisera raised against Esa1 resulted in significant protection against E. tarda challenge. Taking advantage of the secretion capacity of Esa1 and the natural gut-colonization ability of a fish commensal strain, we constructed an Esa1-expressing recombinant strain, FP3/pJsa1. Western immunoblot and agglutination analyses showed that FP3/pJsa1 produces outer membrane-localized Esa1 and forms aggregates in the presence of anti-Esa1 antibodies. Vaccination analyses showed that FP3/pJsa1 as an intraperitoneal injection vaccine and an oral vaccine embedded in alginate microspheres produced relative percent survival rates of 79% and 52%, respectively, under severe challenging conditions that resulted in 92-96% mortality in control fish. Further analyses showed that following oral vaccination, FP3/pJsa1 was able to colonize in the gut but unable to disseminate into other tissues. Together these results indicate that Esa1 is a protective immunogen and an effective oral vaccine when delivered by FP3/pJsa1 as a surface-anchored antigen. (c) 2010 Elsevier Ltd. All rights reserved.

The second anti-lipopolysaccharide factor (EsALF-2) with antimicrobial activity from Eriocheir sinensis

The anti-lipopolysaccharide factor CALF) is a small basic protein that can bind and neutralize lipopolysaccharide (LPS), mediating degranulation and activation of an intracellular coagulation cascade. In the present study, cDNA of the second Eriocheir sinensis ALF (designated as EsALF-2) was cloned and the full-length cDNA of EsALF-2 was of 724 bp, consisting of an open reading frame (ORF) of 363 bp encoding a polypeptide of 120 amino acids. The deduced amino acid of EsALF-2 shared 82% similarity with EsALF-1 from E. sinensis and about 53-65% similarity with ALFs from other crustaceans. The potential tertiary structures of EsALF-1 and EsALF-2 contained two highly conserved-cysteine residues to define the LPS binding site, but the N-terminal of EsALF-1 formed a single additional alpha-helix compared to EsALF-2, implying that EsALF-1 and EsALF-2 might represent different biological functions in E. sinensis. The mRNA transcript of EsALF-2 was detected in all examined tissues of healthy crabs, including haemocytes, hepatopancreas, gill, muscle, heart and gonad, which suggested that EsALF-2 could be a multifunctional molecule for the host immune defense responses and thereby provided systemic protection against pathogens. The mRNA expression of EsALF-2 was up-regulated after Listonelln anguillarum and Pichia pastoris challenge and the recombinant protein of EsALF-2 showed antimicrobial activity against L. anguillarum and P. pastoris. indicating that EsALF-2 was involved in the immune defense responses in Chinese mitten crab against L. anguillarum and P. pastoris. These results together indicated that there were abundant and diverse ALFs in E. sinensis with various biological functions and these ALFs would provide candidate promising therapeutic or prophylactic agents in health management and diseases control of crab aquaculture. (C) 2010 Elsevier Ltd. All rights reserved.

Identification and analysis of a Scophthalmus maximus ferritin that is regulated at transcription level by oxidative stress and bacterial infection

Ferritins are conserved Iron storage proteins that exist in most living organisms and play an essential role in Iron homeostasis. In this study, we reported the identification and analysis a ferritin M subunit, SmFerM, from turbot Scophthalmus maximus. The full length cDNA of SmFerM contains a 5'-untranslated region (UTR) of 232 bp, an open reading frame (ORF) of 531 bp, and a 3'-UTR of 196 bp The ORF encodes a putative protein of 176 amino acids, which shares extensive sequence identities with the M terrains of several fish species. In silico analysis identified in SmFerM both the ferroxidase center of mammalian H ferritins and the iron nucleation site of mammalian L ferritins. Quantitative real time reverse transcriptase-PCR analysis indicated that SmFerM expression was highest in muscle and lowest in heart and responded positively to experimental challenges with bacterial pathogens and poly(I center dot C) Exposure of cultured turbot hepatocytes to treatment of stress inducers (iron, copper, and H2O2) significantly upregulated the expression of SmFerM in a dose dependent manner. Iron chelating analysis showed that recombinant SmFerM purified from Escherichia coli exhibited apparent iron binding activity. These results suggest that SmFerM is a functional M ferritin and is likely to play a role in iron sequestration and protection against oxidative stress and microbial infection (C) 2010 Elsevier Inc All rights reserved