Localization of the proteinase inhibitor activity in the fish cestode Eubothrium rugosum.

The mechanisms enabling fish tapeworms to avoid proteolytic attacks by digestive enzymes of their fish host have been studied in less detail compared with mammalian cestodes. This study aimed to assess the inhibitory ability towards trypsin and chymotrypsin in Eubothrium rugosum, an intestinal parasite of burbot Lota lota, and establish its localization in the tapeworm. To this end, the worms were treated with Triton X-100 followed by differential centrifugation to isolate the tegumental brush border membrane. The protease inhibitory abilities of the worms were mostly determined by their excretory/secretory products released into the incubation medium. These inhibitory abilities proved to be linked mainly with the brush border fractions. Notably, the per cent inhibition of both studied digestive enzymes (trypsin and chymotrypsin) hardly depended on the duration of the parasite exposure in the incubation medium, probably due to intermittent glycocalyx renewal. Improved knowledge on functions of the excretory/secretory proteins produced by fish tapeworms may contribute to a better understanding of host-parasite relations and development of new approaches to the treatment and prevention of diseases caused by pathogenic helminths.

Activity of proteolytic enzymes in the intestine of bream Abramis brama infected with cestodes Caryophyllaeus laticeps (Cestoda, Caryophyllidea).

Adaptive mechanisms underlying the long-term existence of intestinal parasites in their enzymatically hostile environment are still poorly understood, particularly with regard to fish cestodes. The study describes the activity distribution of proteolytic enzymes along the gut of the bream Abramis brama infected with intestinal cestodes Caryophyllaeus laticeps and characterizes the capacity of these worms to inhibit host proteinase activity. Mucosal proteolytic activity was mainly presented by serine proteinases. The research revealed an insignificant increase in total proteolytic activity from anterior and middle to posterior part of the gut accompanied with changes in proportions of various proteinase subclasses along the intestine. The trypsin (but not chymotrypsin) activity in the posterior section was significantly higher than in the mid-section. Both the incubation medium of the worms and their extract had a significant inhibitory effect on mucosal proteolytic activity and commercial trypsin samples. In both instances, the effect was comparable with that of a synthetic serine protease inhibitor, PMSF. SDS-PAGE electrophoregrams of the incubation medium of C. laticeps and its extract revealed three common protein bands, with apparent molecular masses from 19 to 47 kDa, possibly responsible for the worms' inhibitory capacities. According to casein-zymography performed, the target host proteinases for a putative cestode inhibitor (inhibitors) have an approximate molecular weight of 28-53 kDa. A comparative test with the extracts from three other cestodes showed that each of them can suppress the proteolytic activity of the bream mucosa. The level of inhibitory activity was found to increase with protein content in the extracts of these tapeworms.

[Characteristics of the Effect of Cestodes Parasitizing the Fish Intestine on the Activity of the Host Proteinases].

The activity and spectrum of proteinases in the intestines of host fishes change upon infestation with cestodes. Serine proteinases are found to make a greater contribution to the total proteolytic activity. The reduction of proteolytic activity is associated with adsorption of the enzymes of the host on the surface of cestodes, and the increase in the activity is caused by the injury of the intestinal mucosa by the attachment apparatuses of cestodes. The inhibition of proteainase activity indicates the possible participation of microbiota enzymes in protein hydrolyses.

[Activity of digestive enzymes of thick-billed murre (Uria lomvia) and common murre (U. aalga) invaded by cestodes].

Activities of digestive enzymes (proteases, carbohydrases, acid and alkaline phosphatases) are determined in intestinal mucosa of the thick-billed and common murres Comparative analysis of the obtained results is performed for non-infected and for birds infested by cestodes. It has been established that at invasion by cestode Alcataenia armillaris (Cestoda: Tetrabothriidae), activities of carbohydrase and alkaline phosphatase in intestinal mucosa of the thick-billed murre decreases. Parasitizing of cestodes Tetrabothrius jaegerskieldi (Cestoda: Tetrabothriidae) in intestine of the common murre induces a decrease of saccharase activity. There is studied kinetics of desorption of enzymes from digestive-transport surfaces of the bird intestine. Peculiarities of firmness of enzyme fixation are established on the surface of intestinal mucosa of invaded murres. According to the obtained data, a decrease of the carbohydrase activities in intestine of infested murres is likely to be due to absorption of a part of enzymes hydrolyzing carbohydrates on the surface of cestodes.

[Characteristics of protein hydrolysis on the digestive-transport surfaces of the intestine of the kittiwake Rissa tridactyla and Alcataenia larina (Cestoda, Dilepididae) parasitizing it].

Investigation of the activity of proteolytic enzymes functioning on the digestive-transport surfaces of the intestine of infected and noninfected kittiwakes Rissa tridactyla and cestodes Alcataenia larina (Cestoda, Dilepididae) parasitizing it was conducted. The protease activities depending on the pH values in the intestine of gulls and tapeworms were determined. The dynamics of desorption of enzymes of protein hydrolysis from the surface of the cestodes A. larina and the kittiwake intestine was studied. It was shown that the tapeworm invasion activates the processes of membrane and cavitary digestion of birds. It was determined that the degree of influence of the dilepidid infection on protease activity was defined mainly by the intensity of the invasion.

Fish hepatic glutathione-S-transferase activity is affected by the cestode parasites Schistocephalus solidus and Ligula intestinalis: evidence from field and laboratory studies.

The activity of hepatic glutathione-S-transferase (GST) was analysed in 3 different fish species with respect to fish sex and infection with parasites. In both sexes of laboratory bred three-spined sticklebacks (Gasterosteus aculeatus) experimentally infected with Schistocephalus solidus (Cestoda), a significantly lower GST-activity was found for infected fish compared to control. After field sampling of roach (Rutilus rutilus) from Lake Müggelsee (MS) and the Reservoir Listertalsperre (LTS), the GST-activity showed significantly lower values for males infected with Ligula intestinalis from MS (25%) and for infected females from LTS (55%). L. intestinalis-infected female chub (Leuciscus cephalus) from LTS also appeared to have a lower GST-activity. Thus, it could be shown that the presence of parasites significantly affects GST-activity in different fish species resulting in a decreased GST-activity due to infection. Our results therefore emphasize the need for more integrative approaches in environmental pollution research to clearly identify the possible effects of parasites in an effort to develop biomarkers for evaluating environmental health.

[Effect of Caryophyllaeus laticeps (Cestoda, Caryophyllidea) upon activity of digestive enzymes in bream].

The activities of the main digestive hydrolases were comparatively studied in bream infected and noninfected with cestodes Caryophyllaeus laticeps (Pallas, 1781). It was shown that enzyme activities are distributed in the fish intestine in an irregular manner; the gradient of protease and lipase activities along the gut is presented. Following the infection of bream by cestodes, the activities of the studied enzymes decreased and the percentages of activities of various proteinase subclasses changed. No relation between the distribution of worms along the intestine and the levels of activities of digestive hydrolases was revealed.

[Hydrolytic activity of enzymes produced by symbiotic microflora and its role in digestion processes of bream and its intestinal parasite Caryophyllaeus laticeps (Cestoda, Caryopyllidea)].

Bacteria capable to secrete enzymes hydrolyzing proteins and carbohydrates proved to be associated with the digestive-transport surfaces of the bream intestine and tegument of cestode Caryophyllaeus laticeps. Apparently, the contribution of symbiotic digestion to the hydrolysis of polymers increases during active feeding of fishes, while at low feeding rate, bacteria can compete with the host and its parasite for the available monomers. Secretion of bacterial enzymes hydrolyzing both complex carbohydrates and disaccharides reduces the metabolic expenditures of the macroorganisms for the synthesis of their own hydrolases. Glucose resulting from hydrolytic activity of bacterial enzymes can be used by all members of the established community. While bacteria colonizing the digestive-transport surfaces of the host and parasite were generally similar, some specific features were also observed probably due to the structural peculiarities of the intestinal mucosa and cestode tegument.

[A comparative biochemical research in the Schistocephalus solidus (Cestoda)--three-spined stickleback Gasterosteus aculeatus L. system]. / Sravnitel'no-biokhimicheskie issledovaniia v sisteme Schistocephalus solidus (Cestoda)--koliushka trekhiglaia Gasterosteus aculeatus L.

The activity of five lysosomal hydrolases (acid phosphatase, DNAase, RNAase, beta-glucosidase, beta-galactosidase), alkaline phosphatase and aldolase have been examined in tissues of the cestode Schistocephalus solidus (Müller, 1776) and the three-spined stickleback Gasterosteus aculeatus (L.) forming a stable parasite-host system. As a rule, the activity of enzymes was higher in a cestode body than in fish tissues. The acid and alkaline phosphatases were the exception. The activity and variation of lysosomal nucleases and aldolase in the parasite differed notably from those in both infested and healthy hosts. The paper discusses the role of lysosomal and cytoplasmic enzymes in a cestode adaptation to parasitism, as well as in the mechanisms of the host's chemical and immunological response to infection.

Suppression of anti-CD3-induced interleukin-4 and interleukin-5 release from splenocytes of Mesocestoides corti-infected BALB/c mice by phosphodiesterase 4 inhibitors.

We investigated the suppressive effects of rolipram, RP 73401 (piclamilast), and other structurally diverse inhibitors of adenosine 3'5'-cyclic monophosphate (cAMP)-specific phosphodiesterase (PDE4) on anti-CD3-stimulated interleukin (IL)-4 and IL-5 generation by splenocytes from BALB/c mice infected with Mesocestoides (M) corti. RP 73401 (IC40: 0.011 +/- 0.004 microM) was a very potent inhibitor of anti-CD3-induced IL-4 release, being approximately 40-fold more potent than (+/-)-rolipram (IC40: 0.43 +/- 0.09 microM). A maximal inhibition of 60-70% of the response was achieved at the top concentrations of RP 73401 (1 microM) and rolipram (100 microM). These PDE inhibitors also suppressed IL-5 generation over the same concentration ranges, but the maximal suppression achieved was only 30-40%. R-(-)-rolipram (IC40: 0.39 +/- 0.09 microM) was approximately 6-fold more potent than S-(+)- rolipram (IC40: 2.6 +/- 0.95 microM) in inhibiting IL-4 release. A close correlation (r2 = 0.82) was observed between suppression of IL-4 release by PDE inhibitors and inhibition of CTLL cell PDE4, a form against which R-(-)-rolipram displayed relatively weak inhibitory potency. A poorer correlation (r2 = 0.26) was observed between suppression of IL-4 release and affinities of cAMP PDE inhibitors for the high-affinity rolipram binding site in mouse brain membranes. The cGMP-inhibited PDE (PDE3) inhibitor, siguazodan, had little or no effect (IC40 > 100 microM) on anti-CD3-stimulated release of either IL-4 or IL-5 and did not significantly enhance the inhibitory action of RP 73401 on the release of either of these cytokines. Finally, RP 73401 (IC50: 0.41 +/- 0.19 nM) inhibited anti-CD3-stimulated DNA synthesis in splenocyte preparations from M. corti-infected mice and siguazodan (10 microM) had no effect on this response, either alone or in combination with the PDE4 inhibitor. The results show that PDE4 inhibitors suppress the release of Th2 cytokines from anti-CD3-stimulated murine spenocytes and that this effect is correlated with inhibition of a low-affinity PDE4 form.

Proteinase activity in the plerocercoid of Proteocephalus ambloplitis (Cestoda).

Host invasion and tissue migration of several helminths have been linked to expression and release of parasite-derived proteinases. The plerocercoid of the cestode Proteocephalus ambloplitis can migrate into the visceral organs or, in the case of bass, from them into the intestinal tract of the same individual fish. It does this within a few hours, aided by secretion of a substance from its apical gland. Proteinase activity in this plerocercoid, obtained from the host liver, was defined by pH optimum, by substrate and inhibitor specificity, and by electrophoretic and chromatographic techniques. Homogenates of plerocercoid contained a metalloproteinase exhibiting a molecular weight of 30,000 determined by gelatin substrate gel electrophoresis. Peak activity of this proteolytic enzyme in gel filtration fractions when azocoll was used as substrate then corresponded to a molecular weight of 31,500. The proteinase showed collagenolytic, haemoglobinolytic and slight elastinolytic activity, and it had a pH optimum at 9.0. Enzyme activity could be inhibited by various chelating agents. The metalloproteinase identified in this study constitutes the only enzyme class present in this larval stage of P. ambloplitis. We suggest that the plerocercoid's metalloproteinase is the substance secreted from the apical organ, necessary for the previously recognized tissue migration phase. This enzyme might also have a nutritional function.

[Lactate dehydrogenase activity and changes in its isoenzyme spectrum in the liver of the bream Abramis brama parasitized by pleurocercoids of the cestode Digramma interrupta]. / Aktivnost' laktatdegidrogenazy i izmenenie spektra ee izofermentov v pecheni leshcha Abramis brama pri parazitirovanii plerotserkoidov tsestody Digramma interrupta.

Changes of lactate-dehydrogenase (KF 1.1.1.27) activity have been studied in the liver of the bream been infected with pleurocercoids of the cestode Digramma interrupta. It is showed, that an increase of enzyme activity depends upon a slowly migrating from LDH-6. Problems of interaction at the level of substrate within a system parasite-host is considered.

[An enzyme study of purine nucleotide biosynthesis in the plerocercoids of cestodes in the fam. Ligulidae]. / Izuchenie fermentov biosinteza purinovykh nukleotidov u plevrotserkoidov tsestod sem. Ligulidae.

By means of spectrophotometric method there was determined the activity of three enzymes of biosynthesis of purine nucleotides: amino imidazole ribonucleotide-carboxylase (AIR-carboxylase, EC 4.1.1.21), an enzyme of biosynthesis of purine nucleotides de novo in plerocercoids of Schistocephalus pungitii and Digramma interrupta; inosine monophosphate-dehydrogenase (IMPh-dehydrogenase, EC 1.2.1.14), an enzyme of salvage path, and adenylosuccinate lyase (EC 4.3.2.2), an enzyme taking part both in biosynthesis de novo and salvage in plerocercoids of Schistocephalus pungitii. The activity of AIR-carboxylase was not determined. Specific activities of adenylosuccinate lyase and IMPh-dehydrogenase amount to (1.3 +/- 0.3) x 10(-3) and (1.2 +/- 0.4) x 10(-3) mumole/min.mg protein, respectively. The activity of the three enzymes was determined in the liver of ten-spined stickleback, a host of S. pungitii plerocercoids. The question of metabolic dependence of Ligulidae plerocercoids on hosts to provide for purine bases is discussed.

[The dynamics of carbohydrase desorption from the surface of the intestines in fish and in their parasitizing cestodes]. / Dinamika desorbtsii karbogidraz s poverkhnosti kishechnika ryb i parazitiruiushchikh v nikh tsestod.

Data are obtained on the fixation strength of carbohydrases on the structures of digestive-absorptive surfaces of cestodes and intestines of their fish hosts. A dependence of the parasite's digestive activity on the activity of the host's enzymes has been established. General regularities of desorption dynamics of carbohydrases in studied animals and their specific peculiarities are noted.

[The spatial structure and interrelation of plerocercoids of Digramma interrupta (Cestoda, Ligulidae) and the bream (Abramis brama) in the Kuibyshev reservoir]. / Izuchenie prostranstvennoi struktury i vzaimootnoshenii mezhdu plerotserkoidami Digramma interrupta (Cestoda, Ligulidae) i leshchom (Abramis brama) Kuibyshevskogo vodokhranilishcha.

The character of the distribution of the plerocercoid D. interrupta in the bream Abramis brama from the Kuibyshev water reservoir was studied. The spread of the parasites in A. brama population is of complex character and changes with the increase of fish body length. The factors affecting the maintenance and regulation of the relationships in the host-parasite system (size composition of fishes, peculiarities of the host's genotype, values of occurrence of different numbers of the parasites, variability of plerocercoids) are discussed.