A Dorsal homolog (FcDorsal) in the Chinese shrimp Fenneropenaeus chinensis is responsive to both bacteria and WSSV challenge

Rel/NF kappa B is a family of transcription factors. In the present study, a Rel/NF kappa B family member, Dorsal homolog (FcDorsal) was cloned from the Chinese shrimp Fenneropenaeus chinensis. The full length cDNA of FcDorsal consists of 1627 bp, revealed a 1071 bp open reading frame encoding 357 aa. The predicted molecular weight (MW)of the deduced amino acid sequence of FcDorsal was 39.78 kDa, and its theoretical pl was 8.85. Amino acid sequence analysis showed that FcDorsal contains a Rel homolog domain (RHD) and an IPT/TIG (Ig-like, plexins and transcriptions factors) domain. The signature sequence of dorsal protein existed in the deduced amino acid sequence. Spatial expression profiles showed that FcDorsal had the highest expression level in the hemocytes and lymphoid organ (Oka). The expression profiles in the hemocytes and lymphoid organ were apparently modulated when shrimp were stimulated by bacteria or WSSV. Both Gram-positive (G(+)) bacteria (Micrococcus lysodeikticus) and Gram-negative (G(-)) bacteria (Vibrio anguillarium) injection to shrimp caused the up-regulation of FcDorsal at the transcription level. DsRNA approach was used to study the function of FcDorsal and the data showed that FcDorsal was related to the transcription of Penaeidin 5 in shrimp. The present data provide clues that FcDorsal might play potential important roles in the innate immunity of shrimp. Through comparison of the expression profiles between FcDorsal and another identified Rel/NF kappa B member (FcRelish) in shrimp responsive to WSSV challenge, we speculate that FcDorsal and FcRelish might play different roles in shrimp immunity. (C) 2010 Elsevier Ltd. All rights reserved.

Culture of the terrestrial cyanobacterium, Nostoc flagelliforme (Cyanophyceae), under aquatic conditions

Both colonies and free-living cells of the terrestrial cyanobacterium, Nostoc flagelliforme (Berk. & Curtis) Bornet & Flahault, were cultured under aquatic conditions to develop the techniques for the cultivation and restoration of this endangered resource. The colonial filaments disintegrated with their sheaths ruptured in about 2 days without any desiccating treatments. Periodic desiccation played an important role in preventing the alga from decomposing, with greater delays to sheath rupture with a higher frequency of exposure to air. The bacterial numbers in the culture treated with seven periods of desiccation per day were about 50% less compared with the cultures without the desiccation treatment. When bacteria in the culture were controlled, the colonial filaments did not disintegrate and maintained the integrity of their sheath for about 20 days even without the desiccation treatments, indicating the importance of desiccation for N. flagelliforme to prevent them from being disintegrated by bacteria. On the other hand, when free-living cells obtained from crushed colonial filaments were cultured in liquid medium, they developed into single filaments with sheaths, within which multiple filaments were formed later on as a colony. Such colonial filaments were developed at 15, 25, and 30degreesC at either 20 or 60 mumol photons.m(-2).s(-1); colonies did not develop at 180 mumol photons.m(-2).s(-1), though this light level resulted in the most rapid growth of the cells. Conditions of 60 mumol photons.m(-2).s(-1) and 25degrees C appeared to result in the best colonial development and faster growth of the sheath-held colonies of N. flagelliforme when cultured indoor under aquatic conditions.

Enhanced affinochromism of polydiacetylene monolayer in response to bacteria by incorporating US nano-crystallites

By incorporating bio-specific receptors, such as p-10,12-pentacosadiyne-1-N-(3,6,9-trioxaundecylamide)-alpha-D-mannopyranoside (MPDA), into 10,12-pentacosadiyonic acid (PDA) monolayer, the MPDA/PDA monolayer underwent affinochromatic transition in response to the bacteria binding to the receptor. Here, we described a new method to study the membrane/macromolececular interaction between Escherichia coli (E coli) and mannose and its relative affinochromism by modifying MPDA/PDA with CdS nano-crystallites (MPDA/PDA-CdS). CdS not only triggered the strong tropism of the bacteria but also reduced the rigidity of the MPDA/PDA backbone, resulting in the enhanced affinochromism. This discovery might be of significance in basic biophysical studies of membrane/macromolececular and designing novel biosensor.

Rapid identification of pathogenic bacteria by capillary electrophoretic analysis of rRNA genes

Molecular diagnosis is playing an increasingly important role in the rapid detection and identification of pathogenic organisms in clinical samples. The genetic variation of ribosomal genes in bacteria offers an alternative to culturing for the detection and identification of these organisms. Here 16S rRNA and 16S-23S rRNA spacer region genes were chosen as the amplified targets for single-strand conformation polymorphism (SSCP) and restriction fragment length polymorphism (RFLP) capillary electrophoresis analysis and bacterial identification. The multiple fluorescence based SSCP method for the 16S rRNA gene and the RFLP method for the 16S-23S rRNA spacer region gene were developed and applied to the identification of pathogenic bacteria in clinical samples, in which home-made short-chained linear polyacrylamide (LPA) was used as a sieving matrix; a higher sieving capability and shorter analysis time were achieved than with a commercial sieving matrix because of the simplified template preparation procedure. A set of 270 pathogenic bacteria representing 34 species in 14 genera were analyzed, and a total of 34 unique SSCP patterns representing 34 different pathogenic bacterial species were determined. Based on the use of machine code to represent peak patterns developed in this paper, the identification of bacterial species becomes much easier.

Rapid quantitative detection of Yersinia pestis by lateral-flow immunoassay and up-converting phosphor technology-based biosensor

Up-converting phosphor technology (UPT)-based lateral-flow immunoassay has been developed for quantitative detection of Yersinia pestis rapidly and specifically. In this assay, 400 nm up-converting phosphor particles were used as the reporter. A sandwich immumoassay was employed by using a polyclonal antibody against F1 antigen of Y. pestis immobilized on the nitrocellulose membrane and the same antibody conjugated to the UPT particles. The signal detection of the strips was performed by the UPT-based biosensor that could provide a 980 nm IR laser to excite the phosphor particles, then collect the visible luminescence emitted by the UPT particles and finally convert it to the voltage as a signal. V-T and V-c stand for the multiplied voltage units for the test and the control line, respectively, and the ratio V-T/V-C is directly proportional to the number of Y pestis in a sample. We observed a good linearity between the ratio and log CFU/ml of Y pestis above the detection limit, which was approximately 10(4) CFU/mI. The precision of the intra- and inter-assay was below 15% (coefficient of variation, CV). Cross-reactivity with related Gram-negative enteric bacteria was not found. The UPT-LF immunoassay system presented here takes less than 30 min to perform from the sample treatment to the data analysis. The current paper includes only preliminary data concerning the biomedical aspects of the assay, but is more concentrated on the technical details of establishing a rapid manual assay using a state-of-the-art label chemistry. (c) 2006 Elsevier B.V. All rights reserved.

Direct detection of Yersinia pestis from the infected animal specimens by a fiber optic biosensor

The FOB-3, anew type fiber optic biosensor, is designed to rapidly detect a variety of biological agents or analytes with better stability, sensitivity and specificity. In order to detect Y. Pestis, a sandwich immunoassay was developed by using the purified antibody against antigen FI immobilized on polystyrene probes as the capture antibody and the monoclonal antibody-Cy5 conjugate as the detector. After a series of optimization for the stability, sensitivity and specificity of the FOB-3, 50-1000 ng/ml of antigen FI and 6 x 10(1)-6 x 10(7) CFU/ml Y. pestis could be detected constantly in about 20 min, and Y pestis could be detected specifically from Y. pseudotuberculosis, Y. enterocolitica, B. anthracis and E. coli. Then, 39 blind samples, including 27 tissues of mice infected with Y pestis and 12 tissues of healthy mice as negative control, were detected with the FOB-3. 92.6% infected tissues were identified from the tissues of healthy mice and the tissues containing more than 100 CFU/ml bacteria could be detected by the biosensor. The results demonstrated the feasibility of the FOB-3 as an effective method to detect Y. pestis rapidly and directly from the infected animal specimens with the advantage of portability, simple-operation as well as high sensitivity and specificity. (c) 2006 Elsevier B.V. All rights reserved.

Evolution of phenylalanyl-tRNA synthetase by domain losing

The gene duplication, fusion and horizontal transfer are the frequent events during evolution of many proteins, including the aminoacyl-tRNA synthetases (AARSs). However, in this work, it was shown that the main event during evolution of phenylalanyl-tRNA synthetase (PheRS) is a domain loss, and the function/activity of PheRS is not affected by domain losing. Generally, the size of genome and number of genes are increased during evolution from bacteria to eukaryote, but the interesting thing is that the type and number of PheRS domains in eukaryotae are obviously less than those in bacteria. The evolution of PheRS by domain losing seems to be related to the functional evolution of some AARSs from the multiple specificities to the single specificity.

Seasonal Variations in Chlorophyll a Concentrations in Relation to Potentials of Sediment Phosphate Release by Different Mechanisms in a Large Chinese Shallow Eutrophic Lake (Lake Taihu)

The relationship between chlorophyll a and fractionation of sediment phosphorus, inorganic phosphate-solubilizing bacteria (IPB), and organic phosphate-mineralizing bacteria (OPB) was evaluated in a large Chinese shallow eutrophic lake (Lake Taihu) and its embayment (Wuli Bay). At the three study sites, the increase of chlorophyll a concentrations in April paralleled those of the iron bound phosphate accounting for major portion of sediment inorganic phosphate, and in June significantly higher OPB and IPB numbers (especially OPB) in sediment were main contributors to the peaks of chlorophyll a concentration. Even though IPB peaked from February to June, it should serve as an unimportant P source due to the irrelevancy with chlorophyll a and soluble reactive phosphorus (SRP). By contrast, at the other site in the embayment, the calcium-bound phosphate was predominant and solid, which was difficult to be released, and neither IPB nor OPB were detectable in the sediment, indicating weak potential for phosphorus release from the sediment, which was reflected in the small seasonal variation in SRP concentration in water column. Hence, the extents to which the three general mechanisms behind phosphate release from sediment (desorption of iron bound phosphate, solubilization by IPB and enzymatic hydrolysis by OPB) operated were different depending on seasons and sites in Lake Taihu, they may jointly drive phosphate release and accelerate the eutrophication processes.

Toll-like receptor 4 signaling pathway can be triggered by grass carp reovirus and Aeromonas hydrophila infection in rare minnow Gobiocypris rarus

Toll-like receptor 4 (TLR4) is critical for LPS recognition and cellular responses. It also recognizes some viral envelope proteins. Detection mostly results in the inflammation rather than specific antiviral responses. However, it's unclear in fish. In this report, a TLR4 gene (named as GrTLR4b) was cloned and characterized from rare minnow Gobiocypris rarus. The full length of GrTLR4b cDNA consists of 2766 nucleotides and encodes a polypeptide of 818 amino acids with an estimated molecular mass of 94,518 Da and a predicted isoelectric point of 8.41. The predicted amino acid sequence comprises a signal peptide, six leucine-rich repeat (LRR) motifs, one leucine-rich repeat C-terminal (LRRCT) motif, followed by a transmembrane segment of 23 amino acids, and a cytoplasmic region of 167 amino acids containing one Toll - interleukin 1 - receptor (TIR) motif. It's closely similar to the zebrafish (Danio rerio) TLR4b amino acid sequence with an identity of 77%. Quantitative RT-PCR analysis showed GrTLR4b mRNA was constitutive expression in gill, heart, intestine, kidney, liver, muscle and spleen tissues in healthy animals and up-regulated by viruses and bacteria. After being infected by grass carp reovirus or Aeromonas hydrophila, GrTLR4b expressions were up-regulated from 24 h post-injection and lasted until the fish became moribund (P < 0.05). These data implied that TLR4 signaling pathway could be activated by both viral and bacterial infection in rare minnow. (C) 2009 Elsevier Ltd. All rights reserved.

Aerobic biodegradation of hexachlorobenzene by an acclimated microbial community

The aerobic degradation of hexachlorobenzene (HCB) by an acclimated microbial community which isolated from a contaminated site and acclimated in our laboratory was investigated. The enriched microbial community was capable of biodegrading HCB when cultivated in minimal salts medium and supplied HCB as the sole carbon source. The efficiencies of microbial community in the degradation of HCB under different pH and temperatures were examined. The phylogenetic analysis for the nearly complete sequences of 16S rDNA demonstrated that the bacteria assemblage in the microbial community was dominated by Azospirillum and Alcaligenes groups.

Antimicrobial activity-specific to Gram-negative bacteria and immune modulation-mediated NF-kappa B and Sp1 of a medaka beta-defensin

Defensins are a group of cationic antimicrobial peptides which play an important role in the innate immune system by exerting their antimicrobial activity against pathogens. In this study, we cloned a novel beta-defensin cDNA from medaka (Oryzias latipes) by rapid amplification of cDNA ends (RACE) technique. The full-length cDNA consists of 480 bp, and the open reading frame (CRF) of 189 bp encodes a polypeptide of 63 amino acids (aa) with a predicted molecular weight of 7.44 kDa. Its genomic organization was analyzed, and Southern blot detection confirmed that only one copy of beta-defensin exists in the medaka HNI strain. RT-PCR, Western blot and immunohistochemistry detections showed that the beta-defensin transcript and protein could be detected in eyes, liver, kidney, blood, spleen and gill, and obviously prevalent expression was found in eyes. Antimicrobial activity of the medaka beta-defensin was evaluated, and the antibacterial activity-specific to Gram-negative bacteria was revealed. Furthermore, the lipopolysaccharide (LPS), a major component of the outer membrane of Gram-negative bacteria, was demonstrated to be able to induce about 13-fol up-regulation of the beta-defensin within first 12 h. In addition, promoter and promoter mutagenesis analysis were performed in the medaka beta-defensin. A proximal 100 base pair(bp) sequence (+26 to -73)and the next 1700 bp sequence (-73 to -1755) were demonstrated to be responsible for the basal promoter activity and for the transcription regulation. Three nuclear factor kappa B (NF-kappa B) cis-elements and a Sp1 cis-element were revealed by mutagenesis analysis to exist in the 5' flanking sequence, and they were confirmed to be responsible for the up-regulation of medaka beta-defensin stimulated by LPS. And, the Sp1 cis-element was further revealed to be related to the basal promoter activity, and transcriptional factor II D (TFIID) was found to be in charge of the gene transcription initiation. All the obtained data suggested that the novel medaka beta-defensin should have antimicrobial activity-specific to Gram-negative bacteria, and the antibacterial immune function should be modulated by NF-kappa B and Sp1. (C) 2008 Elsevier Ltd. All rights reserved.

Plankton community composition in the Three Gorges Reservoir Region revealed by PCR-DGGE and its relationships with environmental factors

To explore the relationships between community composition and the environment in a reservoir ecosystem, plankton communities from the Three Gorges Reservoir Region were studied by PCR-denaturing gradient gel electrophoresis fingerprinting. Bacterial and eukaryotic operational taxonomic units (OTUs), generated by DGGE analysis of the PCR-amplified 16S and 18S rRNA genes, were used as surrogates for the dominant "biodiversity units". OTU composition among the sites was heterogeneous; 46.7% of the total bacteria] OTUs (45) and 64.1% of the eukaryotic OTUs (39) were identified in less than half of the sampling sites. Unweighted pair group method with arithmetic averages (UPGMA) clustering of the OTUs suggested that the plankton communities in the Xiangxi Rive sites were not always significantly different from those from the Yangtze River sites, despite clear differences in their environmental characterizations. Canonical correspondence analysis (CCA) was applied to further investigate the relationships between OTU composition and the environmental factors. The first two CCA ordination axes suggested that the bacterial community composition was primarily correlated with the variables of NO3--N, dissolved oxygen (DO), and SiO32--Si, whereas, the eukaryotic community was mainly correlated with the concentrations of DO, PO43--P, and SiO32--Si.

Biodegradation of dibenzofuran by Janibacter terrae strain XJ-1

The dibenzofuran (DF)-degrading bacterium, Janibacter terrae strain XJ-1, was isolated from sediment from East Lake in Wuhan, China. This strain grows aerobically on DF as the sole source of carbon and energy; it has a doubling time of 12 hours at 30 degrees C; and it almost completely degraded 100 mg/L-1 DF in 5 days, producing 2,2',3-trihydroxybiphenyl, salicylic acid, gentisic acid, and other metabolites. The dbdA (DF dioxygenase) gene cluster in the strain is almost identical to that on a large plasmid in Terrabacter sp. YK3. Unlike Janibacter sp. strain YY-1, XJ-1 accumulates gentisic acid rather than catechol as a final product of DF degradation.