Proteinases of hydrobionts--potential objects of fish feeding

The review analyzes data on activities and characteristics of proteinases of digestive system and of cathepsins of various tissues in potential preys of fish differing by the character of feeding. There are presented data on multiple forms and molecular mass of proteinases, species-related differences, dependence of proteinase activities on the life cycle stages, type of nutrition, and biochemical food composition as well as on temperature and pH optima. Role of cathepsin in evolution of enzymatic systems providing degradation of food protein components in fish is discussed.     

Activities of proteinases in invertebrate animals--potential objects of fish nutrition. Effects of temperature, pH, and heavy metals

Differences in the degree of separate and combined effects of temperature, pH, and heavy metals (zinc, copper) on the trypsin- and chymotrypsin-like proteinase activities have been established in the whole body of some invertebrate animals - potential objects of fish nutrition: pond snail Lymnaeae stagnalis, orb snail Planorbis purpura, zebra mussel Dreissena polymorpha, oligochaetae Tubifex sp. and Lumbriculus sp. in total, chironomid larvae Chironimus sp. and Ch. riparus, as well as crustacean zooplankton. It has been shown that enzymes of the potential victim at a low temperature can compensate low activity of intestinal proteinases of fish bentho- and planktophages.     

Purification of Yeast Proteinases

The presence of three types of proteinase A, B and C in the autolysate of baker’s yeast was demonstrated by the chromatography on DEAE-Sephadex A-50, and proteinase A and C were isolated and purified by a relatively simple procedure, which was mainly conducted by repeating the chromatography and alcohol fractionation. The final preparation of proteinase C was found to be homogeneous by various physical criteria and crystallized from alcohol solution. On the other hand, although the preparation of proteinase A also showed homogeneous in chromatographic and ultracentrifugal analyses, the result of electrophoresis disclosed the heterogeneity of the preparation. As the results of the chemical and physicochemical analyses, both enzymes showed large contents of carbohydrate, higher molecular weights and acidic isoelectric points, which seemed to be characteristic to the present proteinases. The properties of three types of proteinase from yeast are also discussed.     

Planktonic and symbiotic organisms in nutrition of coralobiont fish

Nutrition of coral reef fish closely associated with the colonies of scleractinian corals Acropora spp. (South China Sea, Gulf of Nha Trang) was investigated. Parabionts Dascyllus reticulatus and Chromis caeruleus (Pomacentridae) feed mainly on zooplankton in the water column above the hosts’ colony, and inbionts Gobiodon quinquestrgatus (Gobiidae) rarely leave a host colony and feed generally on coral reef invertebrates. A high part of secondary nutrition objects (on average 19–29%) that indicates deficiency in main food were noted in both categories of fish. Food of all three fish species contains parasites (from 19% in C. caeruleus and up to 25% in G. quinquestrigatus) whose significant part is represented by parasites of coral (approximately 7% in D. reticulatus and C. caeruleus and 15% in G. quinquestrigatus). In addition, larvae of parasites of Isopoda and Facetotecta that are fed by fish in the water column were found. Mucus from the surface of host coral which was recognized on the basis of a high number of nematocysts in fish stomachs was found in food of G. quinquestrigatus for the first time. Positive and negative effects of symbiosis of fish and host-coral were discussed. A commensal character of symbiosis was supposed.     

Electric images of two low resistance objects in weakly electric fish

Electroreceptive fish detect nearby objects by processing the information contained in the pattern of electric currents through their skin. In weakly electric fish, these currents arise from a self-generated field (the electric organ discharge), depending on the electrical properties of the surrounding medium. The electric image can be defined as the pattern of transepidermal voltage distributed over the receptive surface. To understand electrolocation it is necessary to know how electric image of objects are generated. In pulse mormyrids, the electric organ is localized at the tail, far from the receptors and fires a short biphasic pulse. Consequently, if all the elements in the environment are resistive, the stimulus at every point on the skin has the same waveform. Then, any measure of the amplitude (for example, the peak to peak amplitude) could be the unique parameter of the stimulus at any point of the skin. We have developed a model to calculate the image, corroborating that images are spread over the whole sensory surface and have an opposite center-surround, "Mexican-hat" shape. As a consequence, the images of different objects superimpose. We show theoretically and by simulation that the image of a pair of objects is not the simple addition of the individual images of these objects.     

Proteinases in fungal morphogenesis

Fungal morphogenesis is a regulated series of events, leading to changes from one state to another, in which proteolysis could be regarded as one of the controlling functions. Proteinases are essential for the supply of amino acids, selective inactivation of specific growth phase proteins not required during development and for the activation and modification of the enzymes involved in cell wall synthesis. A critical evaluation of the role of proteinases as a biochemical correlate in fungal morphogenesis is discussed.     

Auditory evoked potential audiometry in fish

A recent survey lists more than 100 papers utilizing the auditory evoked potential (AEP) recording technique for studying hearing in fishes. More than 95 % of these AEP-studies were published after Kenyon et al. introduced a non-invasive electrophysiological approach in 1998 allowing rapid evaluation of hearing and repeated testing of animals. First, our review compares AEP hearing thresholds to behaviorally gained thresholds. Second, baseline hearing abilities are described and compared in 111 fish species out of 51 families. Following this, studies investigating the functional significance of various accessory hearing structures (Weberian ossicles, swim bladder, otic bladders) by eliminating these morphological structures in various ways are dealt with. Furthermore, studies on the ontogenetic development of hearing are summarized. The AEP-technique was frequently used to study the effects of high sound/noise levels on hearing in particular by measuring the temporary threshold shifts after exposure to various noise types (white noise, pure tones and anthropogenic noises). In addition, the hearing thresholds were determined in the presence of noise (white, ambient, ship noise) in several studies, a phenomenon termed masking. Various ecological (e.g., temperature, cave dwelling), genetic (e.g., albinism), methodical (e.g., ototoxic drugs, threshold criteria, speaker choice) and behavioral (e.g., dominance, reproductive status) factors potentially influencing hearing were investigated. Finally, the technique was successfully utilized to study acoustic communication by comparing hearing curves with sound spectra either under quiet conditions or in the presence of noise, by analyzing the temporal resolution ability of the auditory system and the detection of temporal, spectral and amplitude characteristics of conspecific vocalizations.     

The application of radiography to the study of fish nutrition

The measurement of individual food consumption rates of fish held in groups using radiography has enabled the development of a new approach to fish nutrition trials. In order to compare diets, groups of individually numbered fish are fed different experimental diets over extended periods of time (similar to standard nutrition trials) and food consumption rates are measured regularly over the course of the experiment. Analysis of covariance is then used to compare regression coefficients, obtained from mean consumption-growth relationships, from each diet. The advantages of the approach are several: (1) differences in appetite between fish fed different diets are monitored; (2) fewer fish are needed to establish consumption-growth curves over a large range of consumption rates; (3) measured food consumption rates, not ration levels, are used to calculate 'true' growth efficiencies; and (4) other factors, such as absorption efficiency, trypsin activity, the concentration of free amino acids in tissues and protein turnover can be measured for individual fish and related to differences in food consumption between fish in the same group. The approach has been used successfully with a variety of species to compare the growth response of groups fed two or more diets.     

基质金属蛋白酶ADAMTS的研究进展

ADAMTS是含有TSP基序的分泌性多结构域蛋白水解酶。ADAMTS基因的突变或过表达与多种生理学和病理学过程相关。ADAMTS基因的过表达促进了细胞外基质成分的降解,加速了关节炎和动脉粥样硬化的疾病进程。ADAMTS基因的突变则与肿瘤的生长和侵袭以及遗传性发育紊乱等密切相关。该文将结合作者的研究工作,重点对ADAMTS-7的当前研究概况进行综述,讨论其结构、功能、调节及其在相关炎性疾病中的作用等。     

Expanding the moral circle: farmed fish as objects of moral concern

Until recently fish welfare attracted little attention, but international and national legislation and standards of fish welfare are now emerging and an overview of these developments is presented in this study. Whereas animal welfare legislation is based on public morality, animal ethics does not automatically accept public morality as normative and elaborates arguments regarding the way humans should treat animals (referred to as moral standards). In this study we present the most common animal ethics theories. For most of these, sentience is considered a demarcation line for moral concern: if an animal is sentient, then it should be included in the moral circle, i.e. receive moral consideration in its own right and some basic welfare should be ensured. As for fish, research has revealed that the sensory system of teleosts can detect noxious stimuli, and that some kind of phenomenal consciousness, allowing the fish to feel pain, seems to be present. This raises the ethical question as to how much evidence we need in order to act on such indications of fish sentience. A simple risk analysis shows that the probability that fishes can feel pain is not negligible and that if they do indeed experience pain the consequences in terms of the number of suffering individuals are great. We conclude that farmed fish should be given the benefit of the doubt and we should make efforts that their welfare needs are met as well as possible. Finally, the way forward is briefly discussed: efforts must be made to understand what fish welfare means in practical fish farming. This will involve the development of research and education, greater accountability and transparency, compliance with and control of policies, and quality assurance schemes.     

Dietary self-selection in fish: a new approach to studying fish nutrition and feeding behavior

The principles of modern aquaculture encourage the development of fish feeds containing low fish meal content and several types of plant ingredients plus nutrients to avoid depleting global fish stocks and to reduce costs. However, food constituents can affect animal nutrition and feeding behavior, so the effect of different diets on fish behavior and growth needs to be understood to optimize the use of nutrients and to improve fish welfare. The development of multiple-choice self-feeding systems led to a new perspective for investigating these issues in aquaculture species. Our purpose with this review is to summarize the information that has been published to date on this topic and to identify gaps in knowledge where research is needed. Key subjects are assessed under the following major headings: How do we study dietary selection in fish? What food signals do fish use to choose the right diet? and How do fish respond to food challenges? The present review will provide a picture of the main results obtained to date in these studies in aquaculture fish species, as well as perspectives for future research in the field.     

Activation of Intracellular Proteinases of Yeast

The existence of the inactive precursors of yeast proteinases B and C was confirmed in the autolysate of baker’s yeast and they were named as pro-proteinases B and C, respectively. The active and inactive forms of proteinase C were two distinct proteins, separable by chromatographical procedures. The two precursors were markedly activated by incubation at pH 5 or by treatment with denaturing agents, e.g. urea, dioxane, acetone and certain alcohols.

These activations were also observed with extracts from acetone-dried cells and from mechanically destructed cells, but the activation of proteinase A was not demonstrated under any conditions tested. Therefore, it was assumed that most of proteinases B and C exist in vivo as inactive precursors, whereas proteinase A originally exists in an active form.

Pro-proteinase C, the latent form of yeast proteinase C, was partially purified from the autolysate of baker’s yeast. It was strongly activated by incubation at pH 5 or by treatment with urea or dioxane. The former activation was prevented by treatment to inactivate yeast proteinase A, which co-existed with the pro-enzyme in the present preparation, but was promoted by addition of purified proteinase A. Thus, it was confirmed that A could activate pro-proteinase C. Furthermore, it was found that activation could be caused by extremes in pH or by heating to 55~60°C, accompanied by the simultaneous destruction of the enzyme produced. Pro-proteinase C was stable over a range of pH 5 to 8 after 60 min incubation at 50°C.     

Propeptide-Mediated Inhibition of Cognate Gingipain Proteinases

Porphyromonas gingivalis is a major pathogen associated with chronic periodontitis. The organism’s cell-surface cysteine proteinases, the Arg-specific proteinases (RgpA, RgpB) and the Lys-specific proteinase (Kgp), which are known as gingipains have been implicated as major virulence factors. All three gingipain precursors contain a propeptide of around 200 amino acids in length that is removed during maturation. The aim of this study was to characterize the inhibitory potential of the Kgp and RgpB propeptides against the mature cognate enzymes. Mature Kgp was obtained from P. gingivalis mutant ECR368, which produces a recombinant Kgp with an ABM1 motif deleted from the catalytic domain (rKgp) that enables the otherwise membrane bound enzyme to dissociate from adhesins and be released. Mature RgpB was obtained from P. gingivalis HG66. Recombinant propeptides of Kgp and RgpB were produced in Escherichia coli and purified using nickel-affinity chromatography. The Kgp and RgpB propeptides displayed non-competitive inhibition kinetics with K_i values of 2.04 µM and 12 nM, respectively. Both propeptides exhibited selectivity towards their cognate proteinase. The specificity of both propeptides was demonstrated by their inability to inhibit caspase-3, a closely related cysteine protease, and papain that also has a relatively long propeptide. Both propeptides at 100 mg/L caused a 50% reduction of P. gingivalis growth in a protein-based medium. In summary, this study demonstrates that gingipain propeptides are capable of inhibiting their mature cognate proteinases.     

Cationic Inhibitors of Serine Proteinases from Buckwheat Seeds: Study of Their Interaction with Exogenous Proteinases

The inhibition of exogenous serine proteinases of different origin by cationic protease inhibitors BWI-1c, -2c, -3c, and -4c from buckwheat (Fagopyrum esculentum Moench) seeds has been studied. High efficiency of the inhibitors in binding bovine trypsin and chymotrypsin as well as their broad antiprotease effect, including inhibition of proteinases secreted by fungi and bacteria, has been demonstrated. According to the data obtained, it is proposed that cationic inhibitors from buckwheat seeds may participate in the defense of plants against fungal and bacterial infection.     

Discrimination of objects through electrolocation in the weakly electric fish,Gnathonemus petersii

Summary Three weakly electric fish (Gnathonemus petersii) were force-choice trained in a two-alternative procedure to discriminate between objects differing in their electrical characteristics. The objects were carbon dipoles in plexiglass tubing (length 2.5 cm, diameter 0.6 cm). Their electrical characteristics could be changed by varying the impedance of an external circuit to which they were connected (Fig. 1). In one (the capacitance dipole) the resistance was very low(< 3 ) and the capcitance variable. In the other (the resistance dipole) the resistance was variable and the capacitance low (<50 pF).Capacitances from several hundred pF (lower thresholds, Fig. 2) to several hundred nF (upper thresholds, Fig. 3) could be discriminated from both insulators and good conductors. In all cases the reward-negative stimulus was the capacitance dipole, which was avoided by all fish spontaneously. Thresholds were defined at 70% correct choices.The fish were then tested for their ability to discriminate between one object with a given capacitance and another with resistances varying from 3 to 200 k. The capacitance dipole continued to be the negative stimulus throughout. All 3 fish avoided it in at least 80% of the trials at each stimulus combination (Fig. 4). This result suggests that Gnathonemus perceives the capacitance and the resistance of objects differentially.The effect of the dipole-objects as well as some natural objects on the local EOD was recorded differentially very close to the fish's skin (Fig. 5). The amplitude of the local EODs was affected by all types of objects as they approached the skin. However, the waveform was changed only by capacitance dipoles and some natural objects (Figs. 6 and 7). It appears that the fish perceive not only intensity changes in the local EOD but wave-form deformations as well and can thus distinguish objects of different complex impedances.Abbreviations EOD electric organ discharge - f _max maximal spectral frequency - GP Gnathonemus petersii - LFS local filtered signal - PMA probing motor act - S+ positive stimulus - S negative stimulus     

Cytosolic Neutral Proteinases of Paracoccidioides brasiliensis

Cytosolic proteinases were assayed in both morphological phases of Paracoccidioides brasiliensis. Preparations from the mycelial phase were more active in vitro than those from the yeast cells. Optimal proteinase activities for both phases occurred at pH's between 6.0 and 9.0, and at 45°C. Gelatin-SDS-PAGE electrophoresis separated several bands (58–112 kDa) in mycelial preparations; a single band (70 kDa) was seen in yeast preparations. Enzymatic activities were inhibited by antipain, phenyl methyl sulfonyl fluoride (PMSF), and chymostatin, suggestive of serine proteinases. Partial inhibition of the mycelial enzymes by ethylene diamine tetraacetic acid (EDTA), 1,10-phenanthroline, and iodoacetamide, also suggested the presence of cysteine- and metallo-proteinases. The enzymatic activity increased in preparations extracted from yeast cells transforming to mycelia, and decreased in preparations obtained from the reverse process. Received: 29 September 1997 / Accepted: 19 February 1998     

The potential of fish production based on periphyton

Periphyton is composed of attached plant andanimal organisms embedded in amucopolysaccharide matrix. This reviewsummarizes research on periphyton-based fishproduction and on periphyton productivity andingestion by fish, and explores the potentialof developing periphyton-based aquaculture.Important systems with periphyton arebrush-parks in lagoon areas and freshwaterponds with maximum extrapolated fish productionof 8 t ha^–1 y^–1 and 7 t ha^–1y^–1, respectively. Experiments with avariety of substrates and fish species havebeen done, sometimes with supplemental feeding.In most experiments, fish production wasgreater with additional substrates compared tocontrols without substrates. Colonization ofsubstrates starts with the deposition oforganic substances and attraction of bacteria,followed by algae and invertebrates. Afterinitial colonization, biomass density increasesto a maximum when competition for light andnutrients prevents a further increase. Often,more than 50% of the periphyton ash-free drymatter is of non-algal origin. Highest biomass(dm) in natural systems ranges from 0 to 700g m^–2 and in aquaculture experiments wasaround 100 g m^–2. Highest productivity wasfound on bamboo in brush-parks (7.9 gC m^–2 d^–1) and on coral reefs (3 gC m^–2 d^–1). Inorganic and organicnutrients stimulate periphyton production.Grazing is the main factor determiningperiphyton density, while substrate type alsoaffects productivity and biomass. Better growthwas observed on natural (tree branches andbamboo) than on artifical materials (plasticand PVC). Many herbivorous and omnivorous fishcan utilize periphyton. Estimates of periphytoningestion by fish range from 0.24 to 112 mg dm(g fish)^–1 d^–1. Ingestion rates areinfluenced by temperature, fish size, fishspecies and the nutritional quality of theperiphyton. Periphyton composition is generallysimilar to that of natural feeds in fishponds,with a higher ash content due to the entrapmentof sand particles and formation of carbonates.Protein/Metabolizable Energy (P/ME) ratios ofperiphyton vary from 10 to 40 kJ g^–1.Overall assimilation efficiency of fish growingon periphyton was 20–50%. The limited work onfeed conversion ratios resulted in valuesbetween 2 and 3. A simple simulation model ofperiphyton-based fish production estimates fishproduction at approximately 2.8 t ha^–1y^–1. Together with other food resources infishponds, total fish production with thecurrent technology level is estimated at about5 t ha^–1 y^–1. Because grazingpressure is determined by fish stocking rates,productivity of periphyton is currently themain factor limiting fish production. Weconclude that periphyton can increase theproductivity and efficiency of aquaculturesystems, but more research is needed foroptimization. Areas for attention include theimplementation and control of periphytonproduction (nutrient levels, substate types andconformations), the ratio of fish to periphytonbiomass, options for utilizing periphyton inintensive aquaculture systems and with marinefish, and possibilities for periphyton-basedshrimp culture.