Modulation of redox regulatory molecules and electron transport chain activity in muscle of air breathing fish Heteropneustes fossilis under air exposure stress

Responses of redox regulatory system to long-term survival (> 18 h) of the catfish Heteropneustes fossilis in air are not yet understood. Lipid and protein oxidation level, oxidant (H2O2) generation, antioxidative status (levels of superoxide dismutase, catalase, glutathione peroxidase and reductase, ascorbic acid and non-protein sulfhydryl) and activities of respiratory complexes (I, II, III and IV) in mitochondria were investigated in muscle of H. fossilis under air exposure condition (0, 3, 6, 12 and 18 h at 25 A degrees C). The increased levels of both H2O2 and tissue oxidation were observed due to the decreased activities of antioxidant enzymes in muscle under water deprivation condition. However, ascorbic acid and non-protein thiol groups were the highest at 18 h air exposure time. A linear increase in complex II activity with air exposure time and an increase up to 12 h followed by a decrease in activity of complex I at 18 h were observed. Negative correlation was observed for complex III and V activity with exposure time. Critical time to modulate the above parameters was found to be 3 h air exposure. Dehydration induced oxidative stress due to modulation of electron transport chain and redox metabolizing enzymes in muscle of H. fossilis was clearly observed. Possible contribution of redox regulatory system in muscle tissue of the fish for long-term survival in air is elucidated. Results of the present study may be useful to understand the redox metabolism in muscle of fishes those are exposed to air in general and air breathing fishes in particular.

FOLD TYPE II PYRIDOXAL 5 `-PHOSPHATE DEPENDENT ENZYMES: STRUCTURE, SUBSTRATE RECOGNITION AND CATALYSIS

Enzymes utilizing pyridoxal 5'-phosphate dependent mechanism for catalysis are observed in all cellular forms of living organisms. PLP-dependent enzymes catalyze a wide variety of reactions involving amino acid substrates and their analogs. Structurally, these ubiquitous enzymes have been classified into four major fold types. We have carried out investigations on the structure and function of fold type I enzymes serine hydroxymethyl transferase and acetylornithine amino transferase, fold type n enzymes catabolic threonine deaminase, D-serine deaminase, D-cysteine desulfhydrase and diaminopropionate ammonia lyase. This review summarizes the major findings of investigations on fold type II enzymes in the context of similar studies on other PLP-dependent enzymes. Fold type II enzymes participate in pathways of both degradation and synthesis of amino acids. Polypeptide folds of these enzymes, features of their active sites, nature of interactions between the cofactor and the polypeptide, oligomeric structure, catalytic activities with various ligands, origin of specificity and plausible regulation of activity are briefly described. Analysis of the available crystal structures of fold type II enzymes revealed five different classes. The dimeric interfaces found in these enzymes vary across the classes and probably have functional significance.

Food composition and digestive enzymes in the gut of the African electric catfish Malapterurus electricus (Gmelin 1789) (Malapteruridae)

Analyses of stomach contents of 330 Malapterurus electricus (standard length, 10.1-30.5 cm) in Mahin Lagoon (southwestern Nigeria) established it as a bottom feeder. There was a preponderance of insects accounting for >80% occurrence and >25% of total volume in stomachs of specimens, suggesting a stenophagous predatory habit. Qualitative and quantitative assays of digestive enzymes in the different regions of the gut (oesophagus, stomach, duodenum, ileum, rectum) were investigated. Carbohydrases (amylase, maltase), chitinase, proteases (pepsin, chymotrypsin, trypsin) and lipases were detected in different gut regions with different activity. The pattern of distribution and relative activity of the enzymes correlated with the predatory diet

Part I. Synthesis of L-amino acid oxidase by a serine- or glycine-requiring strain of neurospora. Part II. Studies concerning multiple electrophoretic forms of tyrosinase in neurospora

Part I: Synthesis of L-Amino Acid Oxidase by a Serine- or Glycine-Requiring Strain of Neurospora

Wild-type cultures of Neurospora crassa growing on minimal medium contain low levels of L-amino acid oxidase, tyrosinase, and nicotinarnide adenine dinucleotide glycohydrase (NADase). The enzymes are derepressed by starvation and by a number of other conditions which are inhibitory to growth. L-amino acid oxidase is, in addition, induced by growth on amino acids. A mutant which produces large quantities of both L-amino acid oxidase and NADase when growing on minimal medium was investigated. Constitutive synthesis of L-amino acid oxidase was shown to be inherited as a single gene, called P110, which is separable from constitutive synthesis of NADase. P110 maps near the centromere on linkage group IV.

L-amino acid oxidase produced constitutively by P110 was partially purified and compared to partially purified L-amino acid oxidase produced by derepressed wild-type cultures. The enzymes are identical with respect to thermostability and molecular weight as judged by gel filtration.

The mutant P110 was shown to be an incompletely blocked auxotroph which requires serine or glycine. None of the enzymes involved in the synthesis of serine from 3-phosphoglyceric acid or glyceric acid was found to be deficient in the mutant, however. An investigation of the free intracellular amino acid pools of P110 indicated that the mutant is deficient in serine, glycine, and alanine, and accumulates threonine and homoserine.

The relationship between the amino acid requirement of P110 and its synthesis of L-amino acid oxidase is discussed.

Part II: Studies Concerning Multiple Electrophoretic Forms of Tyrosinase in Neurospora

Supernumerary bands shown by some crude tyrosinase preparations in paper electrophoresis were investigated. Genetic analysis indicated that the location of the extra bands is determined by the particular T allele present. The presence of supernumerary bands varies with the method used to derepress tyrosinase production, and with the duration of derepression. The extra bands are unstable and may convert to the major electrophoretic band, suggesting that they result from modification of a single protein. Attempts to isolate the supernumerary bands by continuous flow paper electrophoresis or density gradient zonal electrophoresis were unsuccessful.

Feeding stimulatory effects of Cyperus rotundus tuber on Cirrhinus mrigala

Traditionally tubers of cyperus (Cyperus rotundus) and its extracts have been used for alluring fish during harvesting in India. An experiment was conducted to evaluate its feeding stimulatory activity and effect on the growth of a commercially important freshwater fish, Cirrhinus mrigala. Three isonitrogenous and isocaloric formulated diets viz. plant ingredient based control and control supplemented with cyperus tuber (CS) at 1% and 5% levels were fed to the fingerlings of mrigal, C. mrigala (2.68+0.20 g) for a period of 45 days. The growth performance and the activity of metabolic enzymes, aspartate aminotransferase (AST) and alanine aminotransferase (ALT), in liver, gill and muscle tissues of mrigal were studied during every 15 days interval. Highest relative growth (72.28%) was obtained in the mrigal fed with the diet containing 5% cyperus (5% CS), while the relative growths were 66.18% and 43.40% for the fish fed with the 1% CS diet and control respectively. The activities of AST and ALT were significantly higher (p<0.01) in both 1% and 5% CS diets as compared to the control in all the tissues studied. Higher aminotransferase activities were observed in the tissues of 5% CS group than in those of 1% CS group throughout the experimental period. The observed higher enzymatic activity was concomitant with the higher growth rate in fish. The results suggested that cyperus tuber supplementation increased feed palatability and growth.

Time-Dependent Oxidative Stress Responses of Crucian Carp (Carassius auratus) to Intraperitoneal Injection of Extracted Microcystins

This study was conducted to investigate time-dependent changes in oxidative enzymes in liver of crucian carp after intraperitoneally injection with extracted microcystins 600 and 150 mu g kg(-1) body weight. The results showed that activities of antioxidant enzymes, including superoxide dismutase, catalase, glutathione peroxidase and glutathione reductase generally exhibited a rapid increase in early phase (1-3 h post injection), but gradually decreased afterwards (12-48 h) compared with the control, with an evident time-dependent effect. These zigzag changes over time contributed a better understanding on oxidative stress caused by microcystins in fish.

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.

The involvement of the antioxidant system in protection of desert cyanobacterium Nostoc sp against UV-B radiation and the effects of exogenous antioxidants

In this study, we found that UV-B radiation decreased photosynthetic activity and boosted lipid peroxidation of desert Nostoc sp., and exogenous chemicals (ascorbate acid (ASC), N-acetylcysteine (NAC), and sodium nitroprusside (SNP)) had obvious protective effects on photosynthesis and membranes under UV-B radiation. High-concentration SNP boosted the activities of antioxidant enzymes, but low-concentration SNP reduced the activities of antioxidant enzymes. Both NAC and ASC treatments of cells decreased activities of antioxidant enzymes. The results suggested that those chemicals possibly had different mechanisms of protection of algae cells against UV-B radiation. SNP might play double roles as a signal molecule in the formation of algae cell protection of Photosystem 11 under UV-B radiation and as a (reactive oxygen species) scavenger, while NAC and ASC might function as antioxidant reagents or precursors of other antioxidant molecules, which could protect cells directly against ROS initiated by UV-B radiation. (c) 2006 Elsevier Inc. All rights reserved.

Effects of microcystin-RR on the antioxidant system of Bacillus subtilis

Microcystins are a kind of cyclic hepatotoxins produced by many cyanobacterial species. Many works have been done concerning, the toxic effects of microcystins on animals and plants. However, the reports about their effects on microbial cells are very limited. In the present paper, Bacillus subtilis (B. subtilis) was used to determine the dose- and time-effect of microcystin-RR, and the results showed that the activity of antioxidant enzymes including superoxide dismutase (SOD) and catalase (CAT) was significantly increased to that of control, when exposed to 5 or 10 mu g/ml microcystin-RR for 1 h. The contents of thiobarbituric acid-reactive sub-stances (TBARS) and glutathione (GSH) as well as glu-tathione reductase (GR) activity were obviously increased only when exposed to 10 mu g/ml microcystin-RR. For the time-effect of microcystin-RR on B. subtilis, the activities of antioxidant enzymes including SOD and CAT as well as GR activity and TBARS, GSH contents in B. subtilis were at first significantly increased, and then subsequently de-creased. These results suggested that microcystin-RR could induce the oxidative stress of B. subtilis for a short period. The antioxidant system protects B. subtilis from oxidative damage.

Responses of antioxidant systems in the hepatocytes of common carp (Cyprinus carpio L.) to the toxicity of microcystin-LR

The freshwater, bloom-forming cyanobacterium (blue-green alga) Microcystis aeruginosa produces a peptide hepatotoxin, which causes the damage of animal liver. Recently, toxic Microcystis blooms frequently occur in the eutrophic Dianchi Lake (300 km(2) and located in the South-Westem of China). Microcystin-LR from Microcystis in Dianchi was isolated and purified by high performance liquid chromatography (HPLC) and its toxicity to mouse and fish liver was studied (Li et al., 2001). In this study, six biochemical parameters (reactive oxygen species, glutathione, superoxide dismutase, catalase, glutathione peroxide and glutathione S-transferase) were determined in common carp hepatocytes when the cells were exposed to 10 mug microcystin-LR per litre. The results showed that reactive oxygen species (ROS) contents increased by more than one-time compared with the control after 6 h exposure to the toxin. In contrast, glutathione (GSH) levels in the hepatocytes exposed to microcystin-LR decreased by 47% compared with the control. The activities of superoxide dismutase (SOD), catalase (CAT) and glutathione peroxide (GSH-Px) increased significantly after 6 h exposure to microcystin-LR, but glutathione S-transferase (GST) activity showed no difference from the control. These results suggested that the toxicity of microcystin-LR caused the increase of ROS contents and the depletion of GSH in hepatocytes exposed to the toxin and these changes led to oxidant shock in hepatocytes. Increases of SOD, CAT and GSH-Px activities revealed that these three kinds of antioxidant enzymes might play important roles in eliminating the excessive ROS. This paper also examined the possible toxicity mechanism of microcystin-LR on the fish hepatocytes and the results were similar to those with mouse hepatocytes. (C) 2003 Elsevier Science Ltd. All rights reserved.

Enzymatic reaction of the immobilized enzyme on porous silicon studied by matrix-assisted laser desorption/ionization-time of flight-mass spectrometry

Desorption/ionization on silicon mass spectrometry (DIOS-MS) is a matrix-free technique that allows for the direct desorption/ionization of low-molecular-weight compounds with little or no fragmentation of analytes. This technique has a relatively high tolerance for contaminants commonly found in biological samples. DIOS-MS has been applied to determine the activity of immobilized enzymes on the porous silicon surface. Enzyme activities were also monitored with the addition of a competitive inhibitor in the substrate solution. It is demonstrated that this method can be applied to the screening of enzyme inhibitors. Furthermore, a method for peptide mapping analysis by in situ digestion of proteins on the porous silicon surface modified by trypsin, combined with matrix-assisted laser desorption/ionization-time of flight-MS has been developed.

The application of matrix-assisted laser desorption jonization mass spectrometry in enzymes of Agkistrodon blomhoffii Ussurensis venom analysis

The matrix-assisted laser desorption ionization mass spectrometry (MALDI-TOF-MS) spectra of four enzymes (PLA, AEase, Fibrolase, L-a.a. oxidase) in Agkistrodon blomhoffii Ussurensis venom, were given and interpreted. The experiment data showed that MALDI-TOF-MS can be used directly in enzyme analysis with high sensitivity and rapidity. In addition, the results were better than those from sodium dodecyl sulfate-polyacrylamide.

Matrix-assisted laser desorption/ionization time-of-flight mass spectrometric analysis of phospholipase A(2) and fibrinolytic enzyme, two enzymes obtained from Chinese Agkistrodon blomhoffii Ussurensis venom

Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOFMS) was used to analyze two enzymes, phospholipase AZ and fibrinolytic enzyme isolated from Chinese Agkistrodon blomhoffii Ussurensis venom. Using sinapinic acid as the matrix, positive ion mass spectra of the enzymes were obtained, In addition to the dominant protein [M+H](+) ions, multimeric and multiply charged ions were also observed in the mass spectra, The higher the concentration of the enzymes, the more multiply charged polymer and multimeric ions were detected, Our results indicate that MALDI-TOFMS can provide a rapid and accurate method for molecular weight determination of snake venom enzymes, Mass accuracies of 0.1 and 0.3 % were achieved by analysis of highly dialyzed phospholipase A2 and fibrinolytic enzyme, and these results are much better than those obtained using sodium dodecyl sulfate-palyacrylamide gel electrophoresis. MALDI-TOFMS thus provides a reliable method to determine the purity and molecular weight of these enzymes, which are of potential use as therapeutants, Copyright (C) 1999 John Wiley & Sons, Ltd.

Investigations of the characteristics and mode of action of an algalytic bacterium isolated from Tai Lake

An algalytic bacterium provisionally designated as TL1 was isolated from Tai Lake, a large freshwater lake in the Yangtze Delta plain on the border of the Jiangsu and Zhejiang provinces and close to Wuxi city in the People's Republic of China. Strain TL1 was identified as Achromobacter sp. based on its biophysical and biochemical properties and the analysis of its 16S rRNA sequence. Microcystis aeruginosa, which is the most common toxic cyanobacterium in eutrophic freshwater, could be decomposed by strain TL1. The results showed that after inoculation with the algalytic bacterium, the content of chlorophyll-a, maximum PSII quantum yield, and maximum electron transport rates of the alga decreased sharply. At first, the algal cells enhanced the activities of some antioxidative enzymes, but subsequently, the activities of antioxidative enzymes fell sharply once damage of the algal cells was achieved. The filtrate from strain TL1 culture suspension, after autoclaving and treatments with proteinase K, strongly inhibited algal growth, indicating that the lytic metabolites were extracellular and thermostable, not a protein.

In vivo antioxidant activity of polysaccharide fraction from Porphyra haitanesis (Rhodephyta) in aging mice

Sulfated polysaccharide fraction F2 from Porphyra haitanesis (Rhodephyta) showed inhibitory effect on the in vitro lipid peroxidation. In the present study, the age-related changes in the antioxidant enzyme activity, lipid peroxidation, and total antioxidant capacity (TAOC) in different organs in mice were investigated and the in vivo antioxidant effect of F2 in aging mice was checked. Increased endogenous lipid peroxidation and decreased TAOC were observed in aging mice. Intraperitoneal administration of F2 significantly decreased the lipid peroxidation in a dose-dependent manner. F2 treatment increased TAOC and the activity of superoxide dismutase (SOD) and glutathione peroxidase (GSH-Px) in all the organs tested in aging mice. It is concluded that the sulfated polysaccharide fraction F2 can be used in compensating the decline in TAOC and the activities of antioxidant enzymes and thereby reduces the risks of lipid peroxidation. (C) 2003 Elsevier Science Ltd. All rights reserved.