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Peptidases of monogeneans of the family Diplozoidae
Jedličková, Lucie ; Mikeš, Libor (advisor) ; Dvořák, Jan (referee)
The blood processing mechanisms in monogeneans of the subclass Polyopisthocotylea are known from ultrastructural and histochemical analyses only. In contrast to other blood- feeding parasites, just few biochemical and molecular analyses have been done on digestive enzymes in monogeneans. Therefore, we focused on the biochemical and molecular characterization of hydrolytic enzymes (peptidases) in the hematophagous species Paradiplozoon bliccae and Eudiplozoon nipponicum. The presence of the cysteine class peptidases, mainly cathepsin L, in excretory- secretory products and soluble protein extracts of P. bliccae and E. nipponicum we found. Detection was carried out using fluorogenic substrates, specific inhibitors and the labelled probe DCG-04. On the gels / membranes after electrophoresis / blotting we detected bands of approximately size of 35 kDa in the case of both species and 24 kDa for E. nipponicum. Soluble protein extracts of worms were separated by 2D gel electrophoresis and relevant spots around 35 kDa (P. bliccae) and around 25 ˗ 35 kDa (E. nipponicum) were confirmed by mass spectrometry as cathepsins L. Using degenerate primers based on the conserved motifs of cysteine class peptidases, a partial sequence of cathepsin L gene from E. nipponicum was obtained. Furthermore, 3'RACE PCR method...
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Acid peptidases of schistosomes and haematophagous monogeneans
Dvořáková, Hana ; Mikeš, Libor (advisor) ; Horn, Martin (referee) ; Sojka, Daniel (referee)
Blood is a complex nutrient-rich mixture. No wonder that haematophagy has been adopted as a feeding strategy by many invertebrates, including many parasitic helminths. In general, processing of haemoglobin (and other blood proteins) in blood-feeding helminths relies on an evolutionary conserved network of cysteine and aspartic peptidases (e.g., cathepsins L, B and D). However, some helminth taxa have been neglected from this point of view - very little information has been available about the occurrence of these enzymes in haematophagous monogeneans. Therefore, the presented thesis focuses on the molecular and biochemical characteristics of peptidases that maybe potentially involved in blood processing by the monogenean Eudiplozoon nipponicum (Heteronchoinea, Diplozoidae), an ectoparasite inhabiting gills of common carp. We show that the most abundant haemoglobinolytic endopeptidase activities in soluble protein extracts and excretory/secretory products of E. nipponicum belong to the cysteine and aspartic classes, with cathepsin L-like activity predominating over cathepsin B-like activity and supplemented with cathepsin D-like activity (paper 1). Additionally, we found that E. nipponicum adults express a variety of cathepsins L with different structural characteristics and probably different...
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Peptidases of monogeneans of the family Diplozoidae
Jedličková, Lucie ; Mikeš, Libor (advisor) ; Dvořák, Jan (referee)
The blood processing mechanisms in monogeneans of the subclass Polyopisthocotylea are known from ultrastructural and histochemical analyses only. In contrast to other blood- feeding parasites, just few biochemical and molecular analyses have been done on digestive enzymes in monogeneans. Therefore, we focused on the biochemical and molecular characterization of hydrolytic enzymes (peptidases) in the hematophagous species Paradiplozoon bliccae and Eudiplozoon nipponicum. The presence of the cysteine class peptidases, mainly cathepsin L, in excretory- secretory products and soluble protein extracts of P. bliccae and E. nipponicum we found. Detection was carried out using fluorogenic substrates, specific inhibitors and the labelled probe DCG-04. On the gels / membranes after electrophoresis / blotting we detected bands of approximately size of 35 kDa in the case of both species and 24 kDa for E. nipponicum. Soluble protein extracts of worms were separated by 2D gel electrophoresis and relevant spots around 35 kDa (P. bliccae) and around 25 ˗ 35 kDa (E. nipponicum) were confirmed by mass spectrometry as cathepsins L. Using degenerate primers based on the conserved motifs of cysteine class peptidases, a partial sequence of cathepsin L gene from E. nipponicum was obtained. Furthermore, 3'RACE PCR method...
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Monogenean parasites of fishes of economic importance
Gelnar, M. ; Blažek, R. ; Matějusová, I. ; Ondračková, Markéta ; Šimková, A.
To date , altogether 165 species of monogenean parasites have been recorded on the territory of Czech and Slovak Republics. In comparison with the status of monogenean organismal diversity described in monograph published by Ergens and Lom (1970): "Causative Agents of Fish Diseases", there is a significant increase in regional parasite species richness. During last 30 years, 47 species of monogeneans have been newly recorded in the mentioned territory. Following three reasons trying to explain the situation: 20 monogenean species have been recognized like species nova for science after 1970; another 14 parasite species have represented a new zoogeographical records in central Europe and finally altogether 11 monogenean species have been introduced to our fauna from exotic regions.
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