Studies on the carbohydrases in the digestive tract of the milkfish chanos chanos

Crude extracts from various regions of the digestive tract of pond grown milkfish were tested forttheir ability to catalyze the hydrolysis of various carbohydrates. The most active carbohydrases were those involved in the hydrolysis of -glucosidic bonds. Maltose, trehalose, dextrin, starch and glycogen were rapidly hydrolyzed in the presence of crude extracts from the intestines and the pyloric caeca. High amylase activity was observed in extracts from the intestines, pancreas, pyloric caeca and liver. The intestinal amylase had optimum activity at pH 6.2 and at a temperature of about 50°C. It was active at a chloride concentration of 10 to 40 ppt. The amylase activity in the intestines consistently peaked daily at about noon when the milkfish gut was full. In contrast, enzyme activity was significantly lower at 0030 hrs when the gut was empty. These results are consistent with earlier observations that the milkfish is a daytime feeder and suggest further that intestinal amylase secretion is in phase with the feeding activity of the milkfish. Although the fishes used in this study fed mostly on the naturally occurring algae in the ponds, no cellulase activity was detected in any region of the digestive tract. Less active carbohydrases that were detected include a -glucosidase and -galactosidase, both of which were of limited substrate specificity.SEAFDEC Contribution No. 71     

Genotoxic assessment of anthracene and benzo [a] pyrene to milkfish Chanos chanos

Mutagenic and genotoxic effects of polycyclic aromatic hydrocarbons, anthracene and benzo [a] pyrene (BaP), in milkfish Chanos chanos were determined using micronucleus (MN) test and comet assay (CA). Distinct mean frequencies of nuclear abnormalities such as MNs; binucleated micronuclei, nuclear bud, and fragmented apoptotic cells were measured. Significant increase in DNA damage with five classes of damage level was observed and expressed in terms of arbitrary unit (AU). Mean frequencies of total nuclear abnormalities were 0.5?±?0.25 cells in control; 0.67?±?0.33 cells in solvent control; 70?±?9.60 cells in 0.176?mg?L^?1 anthracene, and 91.83?±?6.25 cells in 0.031?mg?L^?1 BaP. The greatest DNA damage of 170AU was observed in 0.176?mg?L^?1 anthracene-exposed group and 182AU was observed in 0.031?mg?L^?1 BaP-treated fish. This study confirmed that the CA and MN assays are useful tools in determining potential genotoxicity of water pollutants and might be appropriate as a part of monitoring program.     

Effect of different diets on the ultrastructure of hepatocytes of Chanos chanos fry (Chanidae: Teleostei): an electron microscopic and morphometric analysis

The hepatocytes of milkfish fry offered different artificial diets (carbohydrate-, lipid-, protein-oriented) and live food (Artemia spp., Brachionus plicatilis) differ considerably both qualitatively and quantitatively as was shown by means of transmission electron microscopy and planimeter. Food deprivation, too, resulted in ultrastructural alterations of milkfish fry hepatocytes. Thus, this cell type might be used as an indicator of quality and quantity of food in teleosts.Contribution No. 131 of the SEAFDEC Aquaculture Department     

Tissular localization of the digestive proteases in the sedentary polychaetous annelid Sabellaria alveolata

The digestive tract of Sabellaria alveolata (L.) is partitioned into 4 main regions: oesophagus, gizzard, intestine and rectum. Histoenzymologic study of the repartition of the protease activities along the digestive tract has shown that the proteases are secreted in the anterior intestine, where numerous serous gland cells are present. These cells display many histochemical and cytological characteristics similar to those of the gland cells previously described in digestive organs of other polychaetes. The role of the other regions of the digestive tract is discussed.     

Population genetic structure of the milkfish,<Emphasis Type="Italic"> Chanos chanos</Emphasis>, based on PCR-RFLP analysis of the mitochondrial control region

The milkfish, Chanos chanos (Forsskål, 1775) is a pelagic, monotypic gonorhynchiform widely distributed in the tropical Indo-Pacific. This study evaluates temporal variability of milkfish samples from the Philippine archipelago, and spatial variability at two geographic scales based on restriction fragment length polymorphism (RFLP) analysis of a portion of the mitochondrial control region. High levels of genetic diversity characterize the milkfish control region (mean h=0.908, =1.59%), with 74 haplotypes detected among the 367 fish analyzed. For temporal analysis of Philippine samples, milkfish were collected over 2 years from three sites (inter-annual variation), and sampled twice within a year during different seasons at four sites (intra-annual variation). No significant temporal variability was detected between or within years. Significant spatial differentiation among the Philippine samples was observed (F_ST=0.006, P<0.05), with two northeastern samples, Claveria and Dingalan, found to be genetically distinct. However, an hierarchical analysis of molecular variance (AMOVA), where samples were grouped into four geographic regions, revealed very low levels of genetic partitioning, with less than 1% of the total variation attributed to between-region differences, and lack of genetic structure. Nonetheless, the existence of putative northeastern Philippine populations is not discounted. Strong genetic structure across broad geographical scales was revealed by AMOVA, with 11% of the molecular variance based on haplotype frequencies allocated between three distinct groups: Indian Ocean, west Pacific (Philippines) and north central Pacific (Hawaii) The broad-scale genetic structure points to limited gene flow among disjunct Indo-Pacific populations.Communicated by T. Ikeda, Hakodate     

Studies on the digestive lipase of the surf clam Spisula solidissima

Crude molluscan lipase was prepared by homogenizing in tris buffer either fresh or lyophilized crystalline styles of Spisula solidissima. Both preparations showed specific activities less than 2% of standard hog pancreatic lipase, and pH and temperature optima of 8.0 and 20°C, respectively. When compared with mammalian hog pancreatic lipase, the clam lipase was found to be the more versatile enzyme on primary wax esters as well as methyl esters. Neither lipase was capable of hydrolyzing the oleate or palmitate esters of the secondary alcohols of glycerol, cholesterol or isopropyl alcohol; however, both showed activity on the oleate ester of isobutyl alcohol, a primary alcohol. S. solidissima lipase, hog pancreatic lipase, and a lipase isolated from the pancreas of the skate Raja erinacea, all demonstrated an inability to hydrolyze the methyl esters of eicosatetraenoic, eicosapentaenoic and docosahexaenoic acids. The lipase-resistant polyunsaturated fatty acid esters are discussed in terms of their role in the lipid biochemistry of marine organisms.     

Isolation,purification and partial characterization of four digestive proteases from the purple seastar Pisaster ochraceus

An investigation of enzymatic activity in the pyloric caeca of Pisaster ochraceus collected at Post Point, Bellingham, Washington, USA, in 1982, revealed the presence of four distinct proteases. These enzymes have been partially purified and characterized and display tryptic, chymotryptic and carboxypeptidase A-like activity. The two distinctive tryptic enzymes have molecular weights of 88 000 and 25 200 daltons, respectively, as determined by reducting SDS-PAGE electrophoresis. The chymotryptic and carboxypeptidase A proteases have molecular weights of 22 800 and 34 400 daltons, respectively. The tryptic and chymotryptic enzymes are inactivated by phenylmethylsulfonyl fluoride, indicating that all are serine proteases. Both trypsins are inhibited by soybean trypsin inhibitor; however, the high molecular weight trypsin was not inhibited by tosyl-L-lysine chloromethyl ketone. The chymotrypsin was inhibited by tosyl-L-phenylalanine chloromethyl ketone. Both reagents indicate the involvement of histidine in the active site. The low molecular weight trypsin and the chymotrypsin were able to activate bovine trypsinogen to trypsin, as determined in a modified PAGE electrophoresis with trypsinogen and casein copolymerized in the gel.     

Preliminary report on the digestive enzymes present in the digestive gland ofPerna viridis

Quantitative data on digestive enzymes of bivalves are very limited and so this study was performed to obtain such information, and to find out whether some of these enzymes are well adapted to conditions in the digestive gland. The green musselPerna viridis was obtained from Clementi West Market in 1985. The results show that various carbohydrases are present in the digestive gland. The activities of-amylase, cellulase and-glucosidase are higher than that of other carbohydrases, whilst the activities of both lipase and protease, especially the former, are low. The pH optima of the following enzymes are also given:-amylase = 5.8; laminarinase = 5.5;-glucosidase = 5.8; trehalase = 4.8;-glucosidase = 5.5. The only enzyme which was not well adapted to conditions in the digestive gland (pH 6.2) was trehalase. The wide spectrum of enzymes present in this bivalve indicates that it has the ability to utilize a wide range of nutrients efficiently.     

Studies on digestion and the fine structure of digestive caeca in Eurydice pulchra (Crustacea: Isopoda)

In the carnivorous isopod Eurydice pulchra Leach the mid-gut caeca have a pH of 5.6 and produce a strong acid proteinase, a lipase and a carbohydrase. Electron microscope studies of the digestive caeca of starved and fed animals show that secretion is merocrine and may originate from a complex involving the basal cell membrane and associated mitochondria, producing lysosomes. Absorbed materials, in fed animals, appear to take the form of neutral lipid bodies, glycogen, and proteinaceous crystals, the latter forming within the golgi complex. Digestion of both lipid and protein inclusions was observed to occur intra-cellularly by fusion with free lysosomes. Secretion and absorption may occur in the same cell, and the scheme for the sequence of epithelial cell stages is proposed.     

Accumulation and elimination of cadmium and lead in juvenile milkfish during sublethal exposure

Pollution of aquatic environments by trace metals is a worldwide environmental problem. Metal pollutants are increasingly being released into the environment as a result of industrialization. In this study, the bioaccumulation of cadmium and lead in young juvenile milkfish liver (Chanos chanos) was investigated after exposure to three sublethal concentration of each pollutants (1/20, 1/10, and 1/5 LC₅₀ of 96-h LC₅₀) for acute time 12, 24, and 96 h and subchronically for 7, 14, and 21 days. Cadmium and lead accumulation in liver increased with the exposure period and concentrations of pollutant. Compared to controls, the uptake of cadmium is much higher than that of lead. Accumulation factors showed an increase with exposure time and for lead an inverse relationship between accumulation factor and exposure concentration. The elimination of the two pollutants during the 30 days depuration was investigated after 30 days depuration time. During this phase, cadmium and lead concentrations decreased.     

Histoenzymological study on the toxicity of copper sulphate in the digestive glands of Lymnaea luteola

During 24 and 48 hr of exposure, the digestive glands of Lymnaea treated with a lethal concentration of 0.038 mgl(-1) CuSO4 revealed intense activity of acid phosphatase in perilobular margin. On the other hand, same area of the gland showed moderate activity of ATPase during 24 and 48 hr of exposure. However, alkaline phosphatase showed average activity in perialveolar region and perilobular margin during 24 and 48, and 72 hr of exposure respectively The changes in the activity of these enzymes were nonsignificant in alveolar margin and perialveolar region of the gland. It is interesting to note moderate activity of acid phosphatase in perialveolar region during 24 hr of exposure.     

Chitinolytic enzymes in the digestive system of marine fishes

Chitinase, exo-N-acetyl--D-glycosaminidase (NAGase) and lysozyme activities were assayed in the digestive tract of 6 species of marine fishes: Myxine glutinosa (cyclostome), Chimaera monstrosa (holocephalan), Squalus acanthias, Etmopterus spinax, Raja radiata (elasmobranchs) and Coryphaenoides rupestris (teleost). Strong chitinase activity was found in the gastric mucosa of the elasmobranchs (S. acanthias, E. spinax and R. radiata) and the teleost (Coryphaenoides rupestris). A remarkably high chitinase activity occurred in the pancreas of the stomachless holocephalan fish Chimaera monstrosa. NAGase activity was strong in the digestive tract of all species. It could be concluded that marine fishes with diets consisting largely of chitinous invertebrates may display high chitinase and NAGase activities in their digestive system; however, only low chitinase activity was found in the intestine of the cyclostome Myxine glutinosa. Coryphaenoides rupestris gastric mucosa chitinase had one optimum activity at pH 1.25, whereas S. acanthias chitinase had two optima, at pH 1.6 and 3.6. The NAGase pH-activity curves from S. acanthias and R. radiata gastric mucosa displayed similar optima, at pH 4.5 and 4.25 respectively. Chimaera monstrosa pancreatic chitinase had a very strong optimum around pH 8 to 10, and one less strong at pH 3. These enzyme activities could not be separated by gel filtration or isoelectric focusing. The pI (isoelectric point) was approximately 4.9 for both enzymes. The molecular weight of the C. monstrosa pancreatic chitinase was estimated to be approximately 43 000. Lysozyme activity was absent or extremely weak in the material studied.     

Effects of copper on the latency of lysosomal hexosaminidase in the digestive cells of Mytilus edulis

Mytilus edulis collected from Tomales Bay, California, USA, during mid-winter 1979 were exposed to increased concentrations of dissolved copper under controlled laboratory conditions. A dose-dependent reduction in the latency of lysosomal hexosaminidase activity in digestive cells was induced after a 30 d exposure to copper. The half-time of the hexosaminidase staining reaction in sections of digestive gland from control mussels was 15.5 min; for mussels exposed to 25, 50, and 75 g Cu l^-1 it was 11.8, 8.5 and 5.5 min, respectively. In addition, the dyecoupled reaction product was seen earlier in sections from individuals exposed to 50 and 75 g Cu l^-1 (after 30 s) and 25 g Cu l^-1 (1 min) than in sections from control individuals (2.5 min). Copper accumulations were demonstrated histochemically to have the same distribution as the hexosaminidase reaction product, indicating that copper is sequestered in lysosomes. Copper concentrations in digestive gland tissue, were related to the concentrations of copper in the water to which the mussels were exposed.     

Physiology of the digestive tract of the sedentary polychaete Terebellides stroemi

The digestive tract of the sedentary polychaete Terebellides stroemi, collected from 88 m depth on the French Catalan Coast, was studied using histological, biochemical, cytological and spectrographic techniques. The ciliated oesophagus possesses a ventral pharyngeal pouch with a muscle bulb. The stomach is divided into two parts, the fore stomach is ciliated, but the hind stomach takes the place of a gizzard with a thick peritrophic membrane and a thick muscle mantle. The digestive gland enters the digestive tract at the boundary of the two stomachal areas; it is comprised of epithelial lamellae with a glandular epithelium. The gland cells contain a spherocrystal secretion with a high calcium and phosphorus content. The cells are also absorbant due to their microvillar brush border and to the storage of glycogen and lipids in the basal epithelium. A biochemical study using the Apizym System confirmed the secretion of enzymes such as phosphatases, esterases and various carbohydrases. Tryptic and chymotryptic enzymes were visualized by biochemical hydrolysis of synthetic substrates. The epithelium of the fore intestine is similar in appearance to the epithelium of the digestive gland, but consists of a single epithelial layer. Hind intestine and rectum are only excretory systems. The presence of this digestive gland does not seem to have been described elsewhere in polychaetes but is similar to the Morren's gland of some oligochaetes. The study was carried out during 1982 and 1983.     

Quantitative alterations in the structure of the digestive cell of Littorina littorea on exposure to cadmium

The effects of sublethal concentrations of cadmium (Cd) on the morphology of the digestive gland of the periwinkle Littorina littorea have been investigated using a planimetric procedure. Five planimetric parameters were recorded (mean epithelial thickness: MET; mean diverticular radius: MDR; mean luminal radius: MLR; MLR/MET and MET/MDR) and tested by diverse statistical methods. The results indicate that MET is depressed after long exposures at high Cd concentrations whilst MLR increases. Variations in MDR have been attributed to different conditions in the tissues surrounding digestive tubules, and are not-significant in relation to the treatment. However, certain increases might be significant due to the presence of parasites or hemocyte proliferation. The ratios MLR/MET and MET/MDR are highly sensitive, and can be expressed as statistical functions of metal concentration and exposure time. Combination of these five planimetric parameters is reliably indicative of the structural changes occurring in the digestive gland of molluscs under conditions of environmental stress.     

Antioxidant enzymes in the digestive gland of the common musselMytilus edulis

Antioxidant enzymes function to remove deleterious reactive oxygen species, including the superoxide anion radical and H₂O₂. Subcellular distributions and optimal and other properties of catalase (EC. 1.11.1.6), superoxide dismutase (SOD; EC. 1.15.1.1), selenium-dependent glutathione peroxidase (Se-GPX; EC. 1.11.1.9) and total glutathione peroxidase (GPX) activities were determined in the digestive gland of the common musselMytilus edulis L. by spectrophotometric and cytochemical/electron microscopic (catalase) techniques. Assay conditions for Se-GPX and total GPX activities were determined which optimized the difference between the non-enzymic and enzymic rates of reaction. General peroxidase activity (guaiacol as substrate) (EC. 1.11.1.7) was not detectable in any subcellular fraction. Catalase was largely, if not totally, peroxisomal, whereas SOD and GPX activities were mainly cytosolic. Distinct mitochondrial (Mn-SOD) and cytosolic (CuZn-SOD) SOD forms were indicated. Catalase properties were consistent with a catalase, rather than a catalase-peroxidase. The pH-dependence and temperature-dependence of GPX activity were different with H₂O₂ or CHP as substrate, and these and other observations indicate the existence of a distinct Se-GPX. Under saturating or optimal (GPX) assay conditions, the apparent Michaelis constantsK _m (mM) were: catalase, 48 to 68 (substrate, H₂O₂); Se-GPX, 0.11 (H₂O₂) and 2.0 (glutathione); and total GPX, 2.2 (eumene hydroperoxide) and 1.2 (glutathione). Calculated catalase activity was 2 to 4 orders of magnitude greater than Se-GPX activity over an [H₂O₂] of 1 to 1000 M. The results are discussed in relation to theoretical calculations of in vivo oxyradical production and phylogenetic differences in antioxidant enzyme activities.     

Some properties of trypsin-like proteases extracted from the seaweedCodium fragile and their purification

Two enzymes were purified from the green algaCodium fragile, collected in 1988 at Fukuoka, Japan, by batch-wise ion-exchange extraction, affinity chromatography and ion-exchange high-performance liquid chromatography (HPLC) using Boc-Ala-Ala-Pro-Arg-pNA as a substrate. The molecular weights of the enzymes were estimated as 38 000 and 39 000, using gel filtration HPLC. The enzymes had the same optimal pH range of 7 to 9 for both activities, and an exclusively hydrolyzed peptide bond on the carboxyl-terminal side of theL-arginine of peptidep-nitroanilides. The ratios of the enzymatic activity for X-Arg-pNA to X-Lys-pNA were larger than 100. The enzymes exhibited 30 times higher activity toward Boc-Ala-Ala-Pro-Arg-pNA when compared with trypsin. The activities were strongly inhibited by diisopropyl phosphofluoridate (DFP), and partially inhibited by phenylmethylsulfonyl fluoride (PMSF), benzamidine, leupeptin and antipain. The isolated enzymes were presumed to be trypsin-like serine protease from their primary substrate specificities and inactivation behavior.     

Effects of algal diet on digestive enzyme activity in Calanus helgolandicus

Adult female Calanus helgolandicus were transferred, immediately after collection in the English Channel in June 1984, to two unialgal diets one of which, the flagellate Cryptomonas maculata, was rich in starch, while the other, the diatom Thalassiosira weissflogii, contained no starch. The activity of the digestive enzymes amylase, trypsin and laminarinase was measured in these two populations under foodsaturating conditions over an acclimation period of eight days. Ingestion rates were measured on a daily basis and the results confirmed, together with a constant level of body protein, that the experimental conditions were above the incipient limiting concentration. In the long-term (4 to 8 d), the activities of all three enzymes were significantly elevated in the C. maculata-fed copepods, whereas ingestion rates were lower than those on T. weissflogii. These results observed under food-saturating conditions indicate a compensatory mechanism between digestive enzymes and the substrate ingested. They are consistent with previous work on Artemia sp. suggesting that the rate of assimilation is a function of the digestive enzyme activity and ingestion rate. Enzyme activity exhibited differing shortterm responses (<48 h) on transfer to the two algal diets, which are interpreted in relation to the in situ activity of the field population.Communicated by J. Mauchline, Oban     

氧化石墨烯遗传毒性的实验研究

研究氧化石墨烯(GO)的遗传毒性,考察其致突变作用,为GO在生物领域的安全应用提供依据。采用Ames试验、体外CHL细胞染色体畸变试验和小鼠体内染色体畸变试验,分别在细菌水平、细胞水平及整体动物水平研究GO的遗传毒性。GO各剂量组的Ames试验结果为阴性。CHL试验中,CHL细胞染色体畸变率随GO浓度的增加而升高,其中1.000 mg·mL~(-1)剂量组(+S_9)和0.500 mg·mL~(-1)剂量组(-S_9)畸变率显著升高(P 0.05)。小鼠骨髓细胞染色体畸变试验中,骨髓细胞染色体畸变率同样随GO浓度的增加而升高,1.000和0.500 mg·kg~(-1)剂量组的畸变率显著提高(P 0.05)。虽然Ames试验结果没有反映出GO的遗传毒性,但在体外及体内染色体畸变试验中,GO均表现出对哺乳动物细胞染色体有潜在的遗传毒性。     

土壤汞污染研究进展

对国内外学者近些年来对土壤汞污染研究的工作进展作了系统的综述。主要包括土壤汞污染的途径、迁移方式和治理办法。其中重点介绍了大气汞的干湿沉降、土壤汞的释放及生物修复治理土壤汞污染。最后,还介绍了土壤汞形态的分析方法以及土壤挥发性汞释放通量的测量方法。