EFFECTS OF IRON ON PHYSIOLOGICAL AND BIOCHEMICAL CHARACTERISTICS OF Microcystis wesenbergii (Kom.) Kom. (Cyanobacterium)

Iron is an essential trace element for biological requirements of phytoplankton. Effects of iron on physiological and biochemical characteristics of Microcystis wesenbergii were conducted in this study. Results showed that 0.01 mu M [Fe3+] seriously inhibited growth and chlorophyll synthesis of M. wesenbergii, and induced temporary increase of ATPase activities, however, NR. ACP and ALP activities were restrained by iron limitation. Interestingly, iron addition on day 8 resulted in the gradual restoration of structures and functions of above enzymes and resisted a variety of stresses from iron limitation. M. wesenbergii in 10 mu M [Fe3+] treatment group grew normally. enzymes maintained normal levels, and residual phosphate contents in cultures first sharply decreased, then smoothly as M. wesenbergii has a characteristic of luxury consumption of phosphorus. Above parameters in 100 mu M [Fe3+] treatment group were almost same with those in 10 mu M [Fe3+] treatment group except for NR, ACP and ALP activities. In 100 mu M [Fe3+] treatment group, activities of ACP and ALP had temporary increase because phosphate and ferric iron could form insoluble compound - ferric phosphate (Fe3PO4) through adsorption effect. resulting in lack of bioavailable phosphate in culture media. The experiment suggested that too low or too high iron can affect obviously physiological and biochemical characteristics of M. wesenbergii.

First Identification of the Hepatotoxic Microcystins in the Serum of a Chronically Exposed Human Population Together with Indication of Hepatocellular Damage

Hepatotoxic microcystins (MCs) are the most commonly reported cyanotoxins in eutrophic freshwaters. In 1996, human intoxications by MCs caused deaths of 76 patients at Caruaru dialysis centers in Brazil. So far, there have been no direct evidences of MC occurrence in human tissue in consequence of exposure to MC. In this study, we improved cleanup procedures for detecting MCs in serum sample using liquid chromatographymass spectrometry, and confirmed for the first time the presence of MCs in serum samples (average 0.39 ng/ml, which amounts to ca. 1/87 of the concentrations found in tissue samples of the Caruaru victims) of fishermen at Lake Chaohu. Daily intake by the fishermen was estimated to be in the range of 2.2-3.9 mu g MC-LReq, whereas the provisional World Health Organization tolerable daily intake (TDI) for daily lifetime exposure is 0.04 mu g/kg or 2-3 mu g per person. Moreover, statistical analysis showed closer positive relationships between MC serum concentrations and concentrations of alanine aminotransferase, aspartate aminotransferase, alkaline phosphatase, and lactate dehydrogenase than between the MC concentrations and other biochemical indicators. Thus, the data raise the question whether extended exposure in the range of the TDI or up to a factor of 10 above it may already lead to indication of liver damage. The results also demonstrate a risk of health effects from chronic exposure to MCs at least for populations with high levels of exposure, like these fishermen.

Mouse Toxicity of Anabaena flos-aquae from Lake Dianchi, China

Some species of the genera Anabaena can produce various kinds of cyanotoxins, which may pose risks to environment and human health. Anabaena has frequently been observed in eutrophic freshwater of China in recent years, but its toxicity has been reported only in a few studies. In the present study, the toxicity of an Anabaena flos-aquae strain isolated from Lake Dianchi was investigated. Acute toxicity testing was performed by mouse bioassay using crude extracts from the lyophilized cultures. The mice exposed to crude extracts showed visible symptoms of toxicity and died within 10-24 h of the injection. Serum biochemical parameters were evaluated by the use of commercial diagnostic kits. Significant alterations were found in the serum biochemical parameters: alkaline phosphatase (AKP), gamma-glutamyl transpepticlase (gamma-GT), aspartate amino transferase (AST), alanine amino transferase (ALT), AST/ALT ratio, total protein content, albumin content, albumin/globulin (A/G) ratio, blood urea nitrogen (BUN), serum creatinine (Ssr), and total antioxidative capacity (T-AOC). Histopathological observations were carried out with hematoxylin and eosin (HE) stain under light microscope. Severe lesions were seen in the livers, kidneys, and lungs of the mice injected with crude extracts. The alterations of biochemical parameters were in a dose-dependent manner, and the severities of histological lesions were in the same manner. Based on biochemical and histological studies, this research firstly shows the presence of toxin-producing Anabaena species in Lake Dianchi and the toxic effects of its crude extracts on mammals. (C) 2008 Wiley Periodicals, Inc. Environ Toxicol 24: 10-18, 2009.

Plasma biochemical responses of the omnivorous crucian carp (Carassius auratus) to crude cyanobacterial extracts

Healthy crucian carp (Carassius auratus) were treated by intraperitoneal (i.p.) injection of crude cyanobacterial extracts at two doses, 50 and 200 mu g MC-LR equiv kg(-1) BW. High mortality (100%) was observed within 60 h post injection in the high-dose group. In the treated fish, activities of four plasma enzymes, alanine aminotransferase (ALT), alkaline phosphatase (ALP), aspartate aminotransferase (AST), and lactate dehydrogenase (LDH), all showed substantial increases, with both dose and time-dependent effects. These increases of enzyme activity indicate severe impairment occurred in the liver of crucian carp over time. Plasma concentrations of energy-related biomolecules including glucose (GLU), cholesterol (CHO), triglyceride (TG), and total protein (TP) showed marked changes in the high-dose group, possibly a nutritional imbalance correlated with the liver injury caused by intraperitoneal exposure to crude cyanobacterial extracts.

Kinetic study of the 2-methyl-3-methoxy-4-phenylbutanoic acid produced by oxidation of microcystin in aqueous solutions

Microcystins (MCs) are a family of related cyclic hepatotoxic heptapeptides, of which more than 70 types have been identified. The chemically unique nature of the C20 beta-amino acid, (2S, 3S, 8S, 9S)-3-amino-9-methoxy-2,6,8-trimethyl-10-phenyldeca4,6-dienoic acid (Adda), portion of the MCs has been exploited to develop a strategy to analyze the entirety. Oxidation of MCs causes the cleavage of MC Adda to form 2-methyl-3-methoxy-4-phenylbutanoic acid (MMPB). In the present study, we investigated the kinetics of MMPB produced by oxidation of the most-often-studied MC variant, MC-LR (L = leucine, R = arginine), with permanganate-periodate. This investigation allowed insight regarding the influence of the reaction conditions (concentration of the reactants, temperature, and pH) on the conversion rate. The results indicated that the reaction was second order overall and first order with respect to both permanganate and MC-LR. The second-order rate constant ranged from 0.66 to 1.35 M/s at temperatures from 10 to 30 degrees C, and the activation energy was 24.44 kJ/mol. The rates of MMPB production can be accelerated through increasing reaction temperature and oxidant concentration, and sufficient periodate is necessary for the formation of MMPB. The initial reaction rate under alkaline and neutral conditions is higher than that under acidic conditions, but the former decreases faster than the latter except under weakly acidic conditions. These results provided new insight concerning selection of the permanganate-periodate concentration, pH, and temperature needed for the oxidation of MCs with a high and stable yield of MMPB.

Isolation, characterization and genome sequence of a birnavirus strain from flounder Paralichthys olivaceus in China

A birnavirus strain, Paralichthys olivaceus birnavirus (POBV), was isolated and characterized from cultured flounder in China, and its complete genomic sequence was subsequently determined. The virus could induce cytopathic effects (CPE) in four of seven fish cell lines and was resistant to chloroform, 5-iodo-2'-deoxyuridine, acid and alkaline pH, and heat treatment. Purified virus particles had a typical icosahedral shape, with a diameter of approximately 55-60 nm. The genomic segments A and B of POBV were 3,091 and 2,780 bp in length and shared many of the features of the members of the family Birnaviridae. Segment A contained two partially overlapping ORFs encoding a polyprotein, pVP2-VP4-VP3, and a nonstructural protein, VP5, while segment B had only one ORF encoding for the VP1, a viral RNA-dependent RNA polymerase (RdRp). This is the first report about a birnavirus strain from a new non-salmonid host in China and its complete genome sequence.

Dietary phosphorus requirement of juvenile black seabream, Sparus macrocephalus

A growth trial was conducted to estimate the optimum requirement of dietary available phosphorus (P) for black seabream (Sparus macrocephalus) in indoor net cages (1.5x1.0x1.0 m). Triplicate groups of black seabream (11.45 +/- 0.02 g) were fed diets containing graded levels (0.18, 0.36, 0.54, 0.72, 0.89 and 1.07%) of available P to satiation for 8 weeks. The basal diet (diet 1), containing 0.18% available P, was supplemented with graded levels of monosodium phosphate (NaH2PO4 2H(2)O) to formulate five experimental diets. The fish were fed twice daily (08:00 h and 16:00 h) and reared in seawater (salinity, 26-29 g l(-1)) at a temperature of 28 +/- 1 degrees C. Dissolved oxygen during the experiment was above 5 mg l(-1). The specific growth rate (SGR), weight gain (WG), feed efficiency (FE) and protein efficiency ratio (PER) were all significantly improved by dietary phosphorus up to 0.54% (P<0.05) and then leveled off beyond this level. Hepatosomatic index (HSI) was inversely correlated with dietary phosphorus levels (P< 0.05). Efficiency of P utilization stabled in fish fed diets containing 0.18%-0.54% available P and then decreased dramatically with further supplementation of dietary phosphorus. Body composition analysis showed that the whole-body lipid, ash, calcium and phosphorus contents were all significantly affected by dietary available P concentration (P<0.05), however, no significance were found in whole-body calcium/phosphorus (Ca/P) ratios among all the treatments (P>0.05). Dietary phosphorus levels also affected the mineralization of vertebrae, skin and scale (P<0.05). Ca/P ratios in vertebrae and scale were not influenced by dietary P supplementation, while skin Ca/P ratio increased statistically with dietary available P levels (quadratic effect, P<0.001). The blood chemistry analysis showed that dietary available P had distinct effects on enzyme activities of alkaline phosphatase (ALP) and plasma lysozyme (LSZ), as well as contents of triacyglycerol (TG) and total cholesterol (T-CHO) (P<0.05). Broken-line analysis showed maximum weight gain (WG) was obtained at dietary available P concentrations of 0.55%. Quadratic analysis based on P contents in whole fish, vertebrae or scale indicated that the requirements were 0.81, 0.87 and 0.88%, respectively. Signs of phosphorus deficiency were characterized by poor growth, slightly reduced mineralization and an increase in body lipid content. (C) 2008 Published by Elsevier B.V.

Application of methyl parathion hydrolase (MPH) as a labeling enzyme

Methyl parathion hydrolase (MPH) is an enzyme that catalyzes the degradation of methyl parathion, generating a yellow product with specific absorption at 405 nm. The application of MPH as a new labeling enzyme was illustrated in this study. The key advantages of using MPH as a labeling enzyme are as follows: (1) unlike alkaline phosphatase (AP), horseradish peroxidase (HRP), and glucose oxidase (GOD), MPH is rarely found in animal cells, and it therefore produces less background noise; (2) its active form in solution is the monomer, with a molecular weight of 37 kDa; (3) its turnover number is 114.70 +/- 13.19 s(-1), which is sufficiently high to yield a significant signal for sensitive detection; and (4) its 3D structure is known and its C-terminal that is exposed to the surface can be easily subjected to the construction of genetic engineering monocloning antibody-enzyme fusion for enzyme-linked immunosorbent assay (ELISA). To demonstrate its utility, MPH was ligated to an single-chain variable fragment (scFv), known as A1E, against a white spot syndrome virus (WSSV) with the insertion of a [-(Gly-Ser)(5)-] linker peptide. The resulting fusion protein MPH-A1E possessed both the binding specificity of the scFv segment and the catalytic activity of the MPH segment. When MPH-A1E was used as an ELISA reagent, 25 ng purified WSSV was detected; this was similar to the detection sensitivity obtained using A1E scFv and the HRP/Anti-E Tag Conjugate protocol. The fusion protein also recognized the WSSV in 1 mu L hemolymph from an infected shrimp and differentiated it from a healthy shrimp.

Comparative studies on physiological responses to phosphorus in two phenotypes of bloom-forming Microcystis

Toxic Microcystis blooms frequently occur in eutrophic water bodies and exist in the form of colonial and unicellular cells. In order to understand the mechanism of Microcystis dominance in freshwater bodies, the physiological and biochemical responses of unicellular ( 4 strains) and colonial ( 4 strains) Microcystis strains to phosphorus ( P) were comparatively studied. The two phenotype strains exhibit physiological differences mainly in terms of their response to low P concentrations. The growth of four unicellular and one small colonial Microcystis strain was significantly inhibited at a P concentration of 0.2 mg l - 1; however, that of the large colonial Microcystis strains was not inhibited. The results of phosphate uptake experiments conducted using P- starved cells indicated that the colonial strains had a higher affinity for low levels of P. The unicellular strains consumed more P than the colonial strains. Alkaline phosphatase activity in the unicellular strains was significantly induced by low P concentrations. Under P- limited conditions, the oxygen evolution rate, Fv/ Fm, and ETRmax were lower in unicellular strains than in colonial strains. These findings may shed light on the mechanism by which colonial Microcystis strains have an advantage with regard to dominance and persistence in fluctuating P conditions.

Performance and mechanism of phosphorus removal in an integrated vertical flow constructed wetland treating eutrophic lake water

Phosphorus removal performance and a possible mechanism for the phosphorus removal from an eutrophic lake water were investigated using a medium-scale integrated vertical constructed wetland (combined vertical and reverse-vertical systems) from April, 11, 2001 to September, 28, 2004. Environmental factors affecting phosphorus removal and release profiles were monitored simultaneously under hydraulic loads from 400 to 2000 mm per day. The phosphorus removal rate varied with the environmental conditions. The removal rate for acidic influent water was superior to that for alkaline influent water. The substrate in the wetland chamber acted as a buffer to regulate the pH value of the water sample. As regards the water temperature, no significant differences were observed for the removal rate of total phosphorus (TP) and soluble reactive phosphorus (SRP) between low (lower than 15 degrees C) medium (16-25 degrees C) and high temperature (higher than 26 degrees C) conditions. Under a hydraulic load of 400 mm per day, the removal rate reached over 70%, the highest value achieved in this work. In addition, the highest hydraulic load of 2000 mm/d did not result in the lowest removal rate, as had been expected. After a two-year high hydraulic load test, the removal rate decreased significantly. Phosphorous release from the substrate was examined using a spatial sampling method. Depth profiles of total phosphorus and different states of phosphorus present in the substrate were recorded. This further study demonstrated that binding of phosphorus by iron and calcium might be another major factor in the removal and release of TP and SRP in this wetland system. The distribution of the speciated phosphorus showed that the amount of phosphorus captured in the substrate of the down-flow chamber was significantly higher than that captured in the up-flow chamber, suggesting that the up-flow chamber was the main source of phosphorus release in this constructed wetland.

Identification and characterization of a cathepsin L-like cysteine protease from Taenia solium metacestode

Taenia solium metacestode, a larval pork tapeworm, is a causative agent of neurocysticercosis, one of the most common parasitic diseases in the human central nervous system. In this study, we identified a cDNA encoding for a cathepsin L-like cysteine protease from the T solium metacestode (TsCL-1) and characterized the biochemical properties of the recombinant enzyme. The cloned cDNA of 1216 bp encoded 339 amino acids with an approximate molecular weight of 37.6 kDa which containing a typical signal peptide sequence (17 amino acids), a pro-domain (106 amino acids), and a mature domain (216 amino acids). Sequence alignments of TsCL-1 showed low sequence similarity of 27.3-44.6 to cathepsin L-like cysteine proteases from other helminth parasites, but the similarity was increased to 35.9-55.0 when compared to mature domains. The bacterially expressed recombinant protein (rTsCL-1) did not show enzyme activity; however, the rTsCL-1 expressed in Pichia pastoris showed typical biochemical characteristics of cysteine proteases. It degraded human immunoglobulin G (IgG) and bovine serum albumin (BSA), but not collagen. Western blot analysis of the rTsCL-1 showed antigenicity against the sera from patients with cysticercosis, sparganosis or fascioliasis, but weak or no antigenicity against the sera from patients with paragonimiasis or clonorchiasis. (c) 2006 Published by Elsevier B.V.

Meat and bone meal replacement in diets for juvenile gibel carp (Carassius auratus gibelio): effects on growth performance, phosphorus and nitrogen loading

A 11-week growth trial was conducted in a flow-through system with juvenile gibel carp Carassius auratus gibelio to evaluate the effects of gradual replacement of fish meal (FM) by meat and bone meal (MBM) on growth performance, phosphorus (P) and nitrogen (N) loading. Six isonitrogenous (crude protein: 410 g kg(-1)) and isoenergetic (gross energy: 18 kJ g(-1)) diets were formulated. FM was used as the control protein. In the other five diets, 20, 40, 60, 80 and 100% FM protein was substituted with MBM20, MBM40, MBM60, MBM80, MBM100, respectively. Total P content in the diets ranged from 16.0 to 28.3 g kg(-1) and the available P was 5.0-6.6 g kg(-1). The results showed that the best growth was achieved with fish fed on the control diet and MBM20. Final body weight, weight gain, feed efficiency, protein retention efficiency and energy retention efficiency decreased with increased dietary MBM. No significant differences were found in the feeding rate and hepatosomatic index between the groups. Apparent digestibility coefficient (ADC) of dry matter, protein and P decreased with increase in dietary MBM, while there were no significant differences in the ADC of energy. P and N retention decreased linearly while P and N loading increased linearly with the increased dietary MBM levels. No significant differences were observed in the activity of alkaline phosphatase, aspartate aminotransferase and alanine aminotransferase, as well as pyruvate kinase in liver or in serum. Total superoxide dismutase activity in MBM20 was significantly higher than that of MBM100.

Kinetics of the oxidation of MCRR by potassium permanganate

The occurrence of the microcystins in the water bodies, especially in drinking water resources, has received considerable attentions. In situ chemical oxidation is a promising cost-effective treatment method to remove MC from water body. This research investigated the reaction kinetics of the oxidation of MCRR by permanganate. Experimental results indicate that the reaction is second order overall and first order with respect to both permanganate and MCRR, and has an activation energy of 18.9 kJ/mol. The second-order rate constant ranges from 0.154 to 0.225 l/mg/min at temperature from 15 to 30 degrees C. The MCRR degradation rates can be accelerated through increasing reaction temperature and oxidant concentration. The reaction under acid conditions was slightly faster than under alkaline conditions. The half-life of the reaction was less than 1 min, and more than 99.5% of MCRR was degraded within 10 min. (c) 2005 Elsevier Ltd. All rights reserved.

Characterization of two membrane-associated protease genes obtained from screening out-membrane protein genes of Flavobacterium columnare G(4)

In order to identify genes encoding the outer membrane proteins (OMPs) of the myxobacter Flavobacterium columnare G(4), the expression library of the bacterium was screened by using rabbit antisera developed against its OMPs. Positive colonies of Escherichia coli M15 containing fragments encoding the bacterial OMPs were selected for cloning the relevant genes by genomic walking methods. Two genes encoding a membrane-associated zinc metalloprotease and prolyl oligopeptidase are reported in this paper. The membrane-associated zinc metalloprotease gene (map) is 1800 bp in length, coding for 449 amino acids (aa). Despite the presence of a conserved motif HEXXH for all metalloproteases, the special HEXXH similar to 32 aa similar to E motif of the F. columnare G(4) Map and its low level of identity with other reported zinc-containing metalloproteases may imply that the membrane-associated zinc metalloprotease of F. columnare G(4) represents a new family of zincins. The gene encoding prolyl oligopeptidase (Pop), a serine proteinase, is 2352 bp in length, coding for 649 aa. Sequence homology analysis revealed that the Pop is also novel as it has <50% identity with other reported prolyl oligopeptidase family proteins. The present study represents the first to employ anti-fish bacterial OMP sera to screen genes of membrane-associated proteases of fish pathogenic bacteria, and to provide necessary information for the examination of the role of the two genes in the infection and pathogenesis of F. columnare.

Identification and characterization of Bacillus anthracis by multiplex PCR on DNA chip

Bacillus anthracis can be identified by detecting virulence factor genes located on two plasmids, pXO1 and pXO2. Combining multiplex PCR with arrayed anchored primer PCR and biotin-avidin alkaline phosphatase indicator system, we developed a qualitative DNA chip method for characterization of B. anthracis, and simultaneous confirmation of the species identity independent of plasmid contents. The assay amplifies pag gene (in pXO1), cap gene (in pXO2) and Ba813 gene (a B. anthracis specific chromosomal marker), and the results were indicated by an easy-to-read profile based on the color reaction of alkaline phosphatase. About 1 pg of specific DNA fragments on the chip wells could be detected after PCR. With the proposed method, the avirulent (pXO1(+)/2(-), pXO1(-)/2(+) and pXO1(-)/2(-)) strains of B. anthracis and distinguished 'anthrax-like' strains from other B. cereus group bacteria were unambiguously identified, while the genera other than Bacillus gave no positive signal. (C) 2004 Elsevier B.V. All rights reserved.