Sequence analysis of cDNAs encoding the heavy and light chain variable regions of a WSSV-neutralizing monoclonal antibody.

Antibodies raised against individual viral envelope proteins have been used in white spot syndrome virus (WSSV) neutralization assays. We report here the sequence analysis of cDNAs encoding the variable regions of a novel monoclonal antibody that binds to the viral envelope protein and neutralizes WSSV infection. The heavy and light variable chains are most homologous to VH7183 germline gene (AF120472) and IgVk RF germline gene (AJ235936), respectively. Database searches using the derived sequences predicted residues comprising CDR loops. The 12 amino acid residue of the heavy chain CDR3 is rich in negatively charged aspartic acid (25%) and did not show significant homology to any murine V gene available on the database. This study provides insights on the paratope-epitope interaction and can be used to identify compounds with comparable properties as the paratope leading to future development of drugs and vaccines for WSSV infection.

Detection of White spot syndrome virus DNA in pond soil using a 2-step nested PCR.

White spot syndrome virus (WSSV) continues to be the most pathogenic among the penaeid shrimp viruses. In this study, WSSV DNA was detected in pond soil samples using a 2-step nested PCR. Primers described previously were used for the first round of amplification and based on the sequenced amplicon, an inner primer was designed for the 2nd round of amplification. Using plasmid DNA (pET 100) containing the 211 bp target WSSV sequence, analytical sensitivity showed that the 2-step nested PCR protocol was able to detect down to 0.015 fg of the plasmid DNA, or approximately 2 copies of the target DNA sequence. Persistence of WSSV DNA in pond soil samples after various time intervals was determined. WSSV-specific PCR product (161 bp) was still present in the soil samples even after 10 months of storage. The effect of soil heat treatment on the WSSV DNA was also examined. Soils were subjected to 25, 37, 50 and 70 degrees C for 1, 3 and 5 days. The results showed that PCR amplifiable WSSV DNA was still present even after 5 days at 70 degrees C. To our knowledge, this is the first report on the detection of WSSV DNA in soil samples. Based on these findings, it is concluded that the persistence of viral DNA in soil habitats may be an important aspect of WSSV ecology and may have an implication for viral transmissibility.

Changes in chlorinated organic pollutants and heavy metal content of sediments during pyrolysis.

BACKGROUND: There has been an increasing concern about the treatment and disposal of contaminated sediment from dredged rivers, harbors or estuaries due to the accumulated toxic organics such as dioxins and inorganics, particularly heavy metals like Cr, Pb, Zn, Cu, Hg and Cd. However, considering the huge amount of materials and financial costs involved, any candidate technology must ultimately result in reusable, residual by-products. This can only be made possible if the toxic pollutants are removed or stabilized in the raw sediment and then fed back into the materials cycle. Currently, we are developing a pyrolysis process for the commercial-scale cleanup of dioxins and heavy metal-contaminated river sediment to yield reusable char for various economical applications. In this connection, this paper describes our preliminary investigation into the extent of dioxins and heavy metal volatilization from actual contaminated sediment. The stabilization of certain metallic species, particularly Cr ions, was studied. METHODS: Laboratory scale pyrolysis experiments were conducted using a special, horizontal lab-scale pyrolyzer. Sediment samples from Shanghai Suzhou Creek and Tagonoura harbor were pyrolyzed in the reactor under nitrogen gas at 800 degrees C and different retention times of 30, 60 and 90 min. A constant heating rate of 10 degrees C min(-1) was employed. The pyrolysis gas was first allowed to pass through a cold trap to condense the tar. Uncondensed gases were then channeled through a column containing an adsorbent (XAD-2 Resin) for dioxins. Heavy metal concentrations in the initial and final sediment residues were analyzed by ICP (Nippon Jarrel-Ash) following their acid and alkali (for Cr6+) digestion. Dioxin contents of the pyrolysis char, tar, and exhaust gases in the dioxin adsorbent were also determined. For comparative purpose, thermal treatment under air flow was conducted. RESULTS: The data for the removal of heavy metals from Suzhou Creek sediment showed very significant reductions in Pb, Zn and Cr6+ content of the sediment at this condition. Percentage removals were 42.4%, 60.8% and 42.2%, respectively. The disappearance of Cr6+ was due to reduction reactions rather than volatilization, since the total Cr content remained almost unchanged. Other heavy metals such as Cu, Fe and Ni showed very minimal reductions. Nonetheless, Toxicity Characteristics Leaching Procedure (TCLP) tests confirmed that these residual heavy metals were rather stable in the pyrolysis char. Reduction of toxic Cr6+ at 42.2% has also been achieved by pyrolysis (with N2) as opposed to the more than 580% increase in Cr6+ observed during thermal oxidation (with air). DISCUSSION: Pyrolysis also removes toxic organics, particularly dioxins, from the sediment. For the total dioxins, removal percentage of 99.9999% was achieved even at the lowest retention time of 30 min. Almost all polychlorinated dibenzo-p-dioxins (PCDDs) and polychlorinated dibenzo-furans (PCDFs) were removed at any retention time. The TEQs detected from the solid residues were mainly contributed by dioxin-like PCBs, yet these were present in relatively trace quantities. At the shortest retention time of 30 min, only 0.000085 pg-TEQ g(-1) of polychlorinated biphenyls (PCBs) was detected in the pyrolysis char. Furthermore, the residual PCBs have very low toxicity ratings and none of the highly toxic PCBs, which were initially present in the sediment such as 3,3',4,4',5-PeCB and 3,3',4,4'5,5'-HxCB, were detected in the char. Results further confirmed that most of the dioxins that were removed were transferred to the gas phase so that volatilization may be considered as the main mechanism for their removal. CONCLUSION: Some heavy metals, particularly Pb and Zn, can be volatilized under N2 pyrolysis at 800 degrees C. Pyrolysis also prevented the formation of more toxic Cr6+ ions and, at the same time, resulted in its reduction by around 42.2%, in contrast to the 580% increase during thermal oxidation. PCDDs and PCDFs have been removed and were not formed in the solid products over the retention time range of 30-90 min at 800 degrees C. Dioxin-like PCBs mostly remained and a retention time of 30 min was found to be sufficient for its maximum removal. Recommendations and Perspectives. With the above results, a temperature of 800 degrees C at a retention time of 30 min is sufficient for the removal of total dioxins and of some heavy metals by volatilization. It is, however, necessary to destroy the dioxins as well as recover heavy metals in the gas phase. Stability of remaining heavy metals in the char also needs to be confirmed by leaching tests. These are the major concerns, which we are currently evaluating in order to establish the feasibility of our proposed, large scale pyrolysis system for sediment treatment.

Immunostimulating effects of the polyphenol-rich fraction of sugar cane (Saccharum officinarum L.) extract in chickens.

The phagocytic activity of peripheral blood leukocytes (PBL) in chickens orally administered sugar cane extracts (SCE) or polyphenol-rich fraction (PRF) of SCE (500 mg/kg/day) for 3 consecutive days increased significantly, when compared with that of saline-administered control chickens. Chickens orally administered SCE or PRF (500 mg/kg/day) for 3 consecutive days showed significantly higher antibody responses against sheep red blood cells and Brucella abortus than control chickens. In addition, oral administration of SCE or PRF also resulted in a significant increase in the number of IgM- and IgG-plaque forming cell responses of PBL, intestinal leukocytes and splenocytes, when compared with those of control chickens. Furthermore, delayed type hypersensitivity responses to human gamma globulin significantly increased in chickens orally administered SCE or PRF, compared with those of control chickens when evaluated on the basis of net increased wattle thickness at 24, 48 and 72 h after challenge. These results suggest that PRF of SCE has an immunostimulating effect in chickens.

Reduced lipopolysaccharide (LPS)-induced nitric oxide production in peritoneal macrophages and inhibited LPS-induced lethal shock in mice by a sugar cane (Saccharum officinarum L.) extract.

A sugar cane extract (SCE) has been found to have an immunostimulating effect in several animals. Lipopolysaccharide (LPS) is known to induce endotoxin shock via the production of inflammatory modulators such as tumor necrosis factor (TNF)-alpha and nitric oxide (NO). We examined in the present study the effects of SCE on the TNF-alpha and NO production in LPS-stimulated mice peritoneal cells and the endotoxin shock in mice. The supplementation of SCE to peritoneal macrophages cultured with LPS resulted in a significant decrease in NO production. All the mice injected intraperitoneally with LPS and D-galactosamine (LPS+GalN) died within 24 h. However, a peritoneal injection, but no intravenous or oral administration, of SCE (500-1,000 mg/kg) at 3 to 48 h before the LPS+GalN-challenge resulted in a significantly improved survival rate. These results suggest that SCE had a protective effect on LPS-induced endotoxin shock via one of possible mechanisms involving the suppression of NO production in the mouse peritoneal cavity.

Comparison of Vibrio harveyi strains isolated from shrimp farms and from culture collection in terms of toxicity and antibiotic resistance.

Vibrio harveyi strains isolated from shrimp farms (wild strains) were compared with those from culture collections in terms of minimum inhibitory concentration (MIC) and toxicity. Wild strains had higher MIC values for four antibiotics (kanamycin, carbenicillin, oxytetracycline and ampicillin) and also showed higher toxicity compared with culture collection strains. Vibrio harveyi with the lowest antibacterial resistance was chosen to test if a gradual increase in antibiotic concentration and frequent subculture would enhance its antibiotic resistance. Results showed that V. harveyi was able to develop resistance to oxytetracycline. The MIC value was 250 times higher compared with the MIC before subculturing. Moreover, the V. harveyi strain developed slightly higher toxicity. Therefore, it is possible that there is a relationship between antibiotic resistance and toxicity in V. harveyi.

Removal of PCDD/Fs and PCBS from sediment by oxygen free pyrolysis.

Abstract: Few studies have dealt on the evaluation of volatilization and decomposition reactions of dioxins from sediment by oxygen free pyrolysis. In this study, the performance of pyrolysis on the removal of dioxins from sediment was investigated. Dioxin concentrations of the raw sediment and the solid residues after pyrolysis were analyzed at different conditions. Results showed a removal efficiency of 99.9999% for total dioxins at 800 degrees C and retention time of 30 min. All the polychlorinated dibenzo-furans (PCDFs) have been removed and were not formed in the solid residues at the retention time range of 30-90 min at 800 degrees C. Close to 100% removal of polychlorinated dibenzo-p-dioxins (PCDDs) was also achieved. Only trace PCDDs were detected in the solid yields at a retention time of 60 min. The highest removal efficiency of polychlorinated biphenyls (PCBs) was more than 99.9994% at a retention time of 30 min. During cooling period following pyrolysis, however, the concentration of total dioxins in solid residues increased 130 times as compared to that of the raw sediment under air atmosphere. This confirmed that some complex reactions do occur to form PCDD/Fs and PCBs from 800 to 400 degrees C in the presence of oxygen. Oxygen-free atmosphere therefore can prevent formation of dioxin during thermal process thus generating clean solid residues.

Screening of novel cellulose-degrading bacterium and its application to denitrification of groundwater.

To establish an environmentally friendly groundwater bioremediation process using a cellulose carrier combined with cellulose-utilizing, denitrifying microorganisms, a novel psychrophilic bacterium, designated CL-5, which can degrade a commercial-based cellulose carrier as the sole carbon source, was screened. Since the denitrification capability of CL-5 is low, complex microbial systems were constructed together with other denitrifying bacteria designated NR-1 and NR-2 that were also isolated from soil. The nitrate-reducing activities of mixed cultures were much higher than those of the pure cultures of CL-5, NR-1 and NR-2. The highest N(2)O and N(2) formation activities were observed in the mixed culture of CL-5+NR-2.

Comparative analysis of bacterial diversity in freshwater sediment of a shallow eutrophic lake by molecular and improved cultivation-based techniques.

Comparative analysis of bacterial diversity in freshwater sediment collected from a shallow eutrophic lake was performed by using 16S rRNA gene clone library and improved cultivation-based techniques. Our study demonstrated that the use of gellan gum as a gelling reagent instead of agar was more effective at increasing culturability, cultivating a diverse array of novel microbes, and reducing the gaps of the results between molecular and cultivation-based analyses.

Distribution characteristics of ammonia-oxidizing bacteria in the Typha latifolia constructed wetlands using fluorescent in situ hybridization (FISH).

A molecular biology method, fluorescent in situ hybridization (FISH), in which the pre-treatment was improved in allusion to the media of the constructed wetlands (CW), e.g. the soil and the grit, was used to investigate the vertical distribution characteristics of ammonia-oxidizing bacteria (AOB) quantity and the relation with oxidation-reduction potential (ORP) in the Typha latifolia constructed wetlands under three different loadings in summer from May to September. Results showed that the quantity of the AOB decreased in the Typha latifolia CW with the increase of vertical depth. However, the AOB quantity was 2-4 times the quantity of the control in the root area. Additionally, ORP in the rhizosphere was found to be higher than other areas, which showed that Typha latifolia CW was in an aerobic state in summer when using simulated non-point sewage at the rural area of Taihu Lake in China and small town combined sewage.

Detection of telomerase activity in tissues and primary cultured lymphoid cells of Penaeus japonicus.

Telomerase is a ribonucleoprotein enzyme that can elongate telomeric DNA, which is thought to be required for the development of cellular immortality and oncogenesis in mammals. We examined telomerase activity in tissues and primary cultured lymphoid cells of adult penaeid shrimps. Using the telomeric repeat amplification protocol (TRAP), we studied the characteristics of a putative novel telomerase in Penaeus japonicus. This telomerase could be inactivated by heating or treatment with RNase A or proteinase K. At elongation, this telomerase required dATP, dGTP, and dTTP, but not dCTP, as substrates. Sequence analysis of the TRAP product revealed that this telomerase synthesized (TTAGG)(n) repeated sequences. The activity of this telomerase was decreased but still readily detectable in 100 ng of protein extract from lymphoid tissue. The telomerase activity was detected in all examined tissues including testis, ovary, lymphoid, heart, hepatopancreas, and muscle. The highest telomerase activity was in the extract of ovarian tissues. In primary cultured lymphoid cells, the telomerase activity was retained. Thus, primary cultured lymphoid cells of Penaeus japonicus possess one of the factors necessary for cell line establishment.

Electrochemical reduction of organohalogen compound by noble metal sintered electrode.

1,2,3-Trichlorobenzene (1,2,3-TCB) was used as a model sample of persistent organic pollutants (POPs) which was dechlorinated by a closed electrochemical reduction system under an inert gas atmosphere. The effect of the electrode material was examined in the reaction. Dechlorination yields in different type of cathode electrodes using sintered RuO2 (major)/Pt/PdO, sintered Pt(major)/IrO2/RuO2, sintered RuO2, sintered PdO, sintered Pt, sintered PdO/Pt, sintered Pd/Pt and plain Pd plate were 91%, 81%, 59%, 96%, 53%, 97%, 82% and 70% respectively, at reaction times of 60 or 120 min. The reaction was exothermic after initially starting at room temperature. This electrochemical reduction system was friendly technology for environment using cation exchange membrane, supplying sodium ions from sodium hydroxide solution as anolyte. Trace amounts of dichlorobenzene, as products of stepwise dechlorination, were observed with different pathways, depending on the electrode material. Electrodes with Ru and Pd were selective mainly for meta-position dechlorination, while those with Pt groups selective mainly for ortho-position (o-position) dechlorination. A PdO sintered electrode had an especially high selectivity for meta-position (m-position) dechlorination. The results suggest that dechlorination is an electrocatalytic reduction in this cation supply system.

Functional group analysis during ozonation of sunflower oil methyl esters by FT-IR and NMR.

Ozonation of neat sunflower oil (SFO) methyl esters was monitored by FT-IR and 1H and 13C NMR spectroscopy. During the early stage of ozonation, ozone absorption was essentially quantitative. This was accompanied by the formation of 1,2,4-trioxolane. IR and NMR spectra of ozonated samples showed that scission of ozonide to give aldehyde were minimal. 1H NMR analysis revealed that the amount of ozonide relative to aldehyde was more than 90% regardless of the extent of ozonation. Complete ozonation was attained after supplying around 0.20 g O3/ml methyl ester after which ozone absorption suddenly dropped to around 25%. At the latter part of ozonation, ozonide and aldehyde reacted with excess ozone to give carboxylic acid. Reaction products were identified according to Criegee mechanism.

Flavobacterium limicola sp. nov., a psychrophilic, organic-polymer-degrading bacterium isolated from freshwater sediments.

Three novel strains of cold-adapted bacteria, ST-82T, ST-10 and ST-92, were isolated from freshwater sediments. These three isolates were very similar to each other in phenotypic and chemotaxonomic traits, as well as in 16S rDNA sequence. The strains were Gram-negative, elongated filament-like rods that formed bright yellow colonies. They showed neither flexirubin pigments nor gliding motility. The strains were able to hydrolyse casein, gelatin, starch, agar, aesculin, urea, uric acid and tyrosine. They also lysed cells of Escherichia coil and Pseudomonas putida. The temperature range for growth was 0-25 degrees C, with optimum growth occurring at 15-20 degrees C. For all isolates, protease secretion increased as temperature decreased. Sodium chloride inhibited their growth, although the strains tolerated up to 1.5% (w/v) NaCl. Menaquinone-6 was the major respiratory quinone. The major cellular fatty acids were C15 : 0, iso-C15 : 0, anteiso-C15 : 0, C15:1, iso-C15:1, C16 : 1omega7cis, iso-C16 : 1, iso-C17 : 1, iso-C15 : 3-OH and iso-C16 : 0 3-OH. The DNA G + C content was 34.0-34.8 mol%. Phylogenetic analysis based on 16S rDNA sequences suggested that the strains belonged to the genus Flavobacterium and were closely related to Flavobacterium xanthum and Flavobacterium frigidarium, with sequence similarities of 96.9 and 96.3%, respectively. In physiological and biochemical analyses, the isolates were differentiated from all known members of the genus Flavobacterium. The name Flavobacterium limicola is proposed for these novel strains, and the type strain is ST-82T (=JCM 11473T =DSM 15094T).

A novel cyanobacteriolytic bacterium, Bacillus cereus, isolated from a Eutrophic Lake.

Isolation and screening of cyanobacteriolytic bacteria were carried out. Fifteen strains of cyano-bacteriolytic bacteria were isolated by the double layer method using the cyanobacterium, Microcystis, as a sole nutrient. The isolate, N-14, showing the highest cyanobacteriolytic activity was identified as Bacillus cereus based on the 16S rRNA sequence. Components among the extracellular products in the culture supernatant of B. cereus were responsible for the cyanobacteriolytic activity. Lytic assay tests of culture supernatants indicated that the major substances for lytic activity could be non-proteinaceous, and hydrophilic, heat stable, and with a molecular weight of less than 2 kDa. The highest lytic activity was obtained under alkaline conditions, indicating an advantage for the practical application of water bloom control in eutrophic lakes where the pH is usually in the alkaline region. The lytic substance of B. cereus N-14 were compared with enterotoxins and an emetic toxin produced by a pathogenic strain of B. cereus, and also with a known algicide produced by Bacillus brevis, gramicidin. From these results, the lytic substance seemed to be a novel algicide.

High-affinity binding proteins for N-acetylchitooligosaccharide elicitor in the plasma membranes from wheat, barley and carrot cells: conserved presence and correlation with the responsiveness to the elicitor.

Binding experiments as well as affinity labeling with an (125)I-labeled 2-(4-aminophenyl)ethylamino derivative of N-acetylchitooctaose revealed the presence of high-affinity binding sites/proteins for N-acetylchitooligosaccharide elicitor in the plasma membrane preparation from suspension-cultured carrot cells, barley cells and wheat leaves. Their binding specificity corresponded with the elicitor activity of N-acetylchitooligosaccharides and related sugars in these plant cells/tissues, and was similar to that reported for the binding site/protein previously reported for suspension-cultured rice cells. The molecular size of the binding proteins identified in carrot, barley and wheat was slightly smaller than that of rice. These plant cells were shown to respond to N-acetylchitooligosaccharides and generate reactive oxygen species, induced medium alkalinization, or previously shown to initiate lignification (wheat leaves, Barber et al. (1989) Physiol. Mol. Plant Pathol. 34: 3). No elicitor-binding protein nor the elicitor-induced cellular responses was detected for a cell line of tobacco BY-2 (BY-2T). On the other hand, another cell line of tobacco BY-2 (BY-2N) showed the presence of elicitor-binding protein and also elicitor-induced medium alkalinization. Thus, there was a good correlation between the existence of high-affinity binding proteins for the elicitor and elicitor-induced cellular responses among tested plant cells. These results indicated the wide distribution of N-acetylchitooligosaccharide elicitor-binding protein among various plants and added further support for the function of these plasma membrane proteins in the perception of the elicitor signal.

Penaeid (Penaeus japonicus) lymphoid cells replicate by cell division in vitro.

Penaeid cell culture has gained much attention as a potential model to facilitate researches on the characterization of the virus and to develop more sophisticated and improved diagnostic procedures for use in the aquaculture industry. However, to date, cell division processes of cultured penaeid cells have not been found, which is suggested as one of the reasons that block the establishment of the continuous penaeid cell lines. We reported here the cell division processes of cultured lymphoid cells of Penaeus japonicus. The culture medium used was based on M199 and was modified by supplementing saline components. Cultures were incubated at 25 degrees C, and 5% CO2 was supplemented. In primary cultured lymphoid cells, dividing cells in different shapes were found. Cell division processes of 12 dividing lymphoid cells were tracked. After cell division, their daughter cells turned into fibroblast-like or epithelioid cells. These results proved that the culture conditions used were suitable for lymphoid cells of I japonicus to proliferate in vitro and that cultured lymphoid cells still had the ability to carry out cell division. These findings would give light to the establishment of continuous penaeid cell lines and would also provide us with the knowledge of cell division processes of the penaeid.

On-site nitrate removal of groundwater by an immobilized psychrophilic denitrifier using soluble starch as a carbon source.

On-site denitrification of nitrate-contaminated groundwater was conducted at 15 degrees C using a facultative psychrophilic denitrifier (strain 47) immobilized on macro-porous cellulose carriers and utilizing soluble starch as a non-toxic carbon source. A C/N ratio of 2.5 to 3.0 and a P/N ratio of 0.05 to 0.10 were found to allow complete denitrification of the groundwater used in this study. Under these conditions, the long-term performance of the system (4 months) was examined by decreasing the HRT (hydraulic retention time) from 4 h to 0.25 h. The process was stable and 95 to 100% of the influent nitrogen (NO3-N ranging from 13.0 to 16.5 mgl(-1) was removed until an HRT of 0.75 h was reached. The maximum NO3-N removal rate was 0.46 kg-Nm(-3)d(-1) at an HRT of 0.75 h. Nitrogen removal efficiency of 99.5% at an HRT of 1 h was obtained with a C/N ratio 2.58, corresponding to 4.3 g of soluble starch per 1 g of NO3-N.