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Hymenolepis diminuta tapeworms of the WMS "strain", obtained at 1 month and at 2.5 months after the administration to rats of cysticercoids, showed a right-hand side position of the genital pores (PGP). In the 5th month of infection strobilas of variable PGP were also found to appear. These two types of strobilas (of right-hand side and variable PGP) appeared till the end of the observation period in the 23rd month after the infection of rats. It was established that the variability of PGP depends on the duration of the infection and not on the age of the final host. Apart from typical proglottids containing 1 testis on the poral side and 2 testes on the aporal side (lp2a) there were also proglottids of the following numbers and placement of testes (NDT): 0p0a, 0pla, 0p2a, 0p3a, 0p4a, 0p5a, 1p0a, 1p1a, 1p3a, 1p4a, 1p5a, 2p0a, 2p1a, 2p2a, 2p3a, 2p4a, 3p0a and 3p1a. Correlation was established between the frequency of occurrence of PGP changes and the number of proglottids with the following NDT: 1p2a, 2p1a, 3p0a, 3p1a and all deviations from 1p2a.
Crowded infrapopulations of H. diminuta obtained from the WMS i11 inbred line resembled those of the potentially non-inbred WMS strain in not showing either the abrupt reductions in the numbers of worms or the destrobilation described by other autors. Denser populations of both types of tapeworm have a greather abundance of 4 - testis proglottids of the lp3a type (having 1 testis on the poral side and three on the aporal), and fewer of type 0p3a. Changes in the positioning of genital ducts and pores show a marked positive correlation with the abundance of type 2pla proglottids. Strobilae of H. diminuta WMS i11 contain a relatively greather number of type 0p3a proglottids and fewer of type 1p3a, than those of the WMS „strain"; something which is probably linked with the respective selection of the maternal Tapeworms.
Variable position of genital ducts and pores (PGP) was found in 23.3% of five months old tapeworms obtained as a result of administering to rats a dose of 6 cysticercoids proceeding from the third generation of inbred Hymenolepis diminuta - WMS il1. In tapeworms coming from generations no 18 and 32 PGP changes were reported only in 7.5 month, with only 5.9% of strobilas in the 32 generation group being characterized by this feature. These results indicate a time shift with respect to the appearance of PGP changes in tapeworms proceeding from latter inbred generations to a further period of tapeworm invasion in rats. The number of PGP changes is positively correlated with the number of type 2p1a proglottids (two testes on the poral and one on the aporal side). The average number of type Op3a proglottids increases with the number of generations. In 5-month old tapeworms of unilateral PGP coming from generation no 3, 18 and 32 it amounts to 5.9%, 6.6% and 8.1 %, respectively.
The paper deals with methods facilitating the preparation of oncospheres of the cestode, Hymenolepis diminuta, for experimental studies. Described in detail are procedures for the infection of the definitive hosts with the oncospheres; collection and artificial hatching of oncospheres; purification of hexacanths; preparation of extracts from the hexacanths; and preparation of hexacanths for electronmicroscopic studies.
The investigations focused on tapeworms obtained as a result of the administration to individual rats a dose of 6 cysticercoids coming from the 39th and 40th generation of one-specimen inbred Hymenolepis diminuta - WMS il1. In 7.5-month old tapeworms the position of genital pores (PGP) was exclusively right-hand side with the genital ducts typical of this feature. Type 0p3a proglottids were the most common deviation from the typical number and topography of testes - 1p2a (one testis between the ovary and the genital pore marking the poral side and two testes on the aporal side) - in such tapeworms. Their mean incidence amounted to 8.9%, exceeding by at least 2.9% the mean quantitative value of this feature in tapeworms proceeding from non-inbred H. diminuta „strains" which have been subjected to similar studies. 19.5-month old tapeworms, exactly a year older than the previous ones, all had variable PGP. The number of PGP changes was here correlated positively with the number of type 2p1a proglottids and negatively with the number of type 1p2a and 0p3a proglottids.
In Hymenolepis diminuta WMS "strain" tapeworms which were obtained 25 months after the administration to rats of 2-month old larvae - cysticercoids, isolated from Tribolium destructor, only one-sided position of genital pores (PGP) developed. The extended 7-month duration of the invasion of H. diminuta larvae stimulates the development of variable PGP already in 14.1 % of 2.5-month tapeworms. In tapeworms with exclusively one-sided PGP in the first of the two groups mentioned, the number of type 1p2a proglottids (1 testis on the poral side and 2 testes on the aporal side) as well as type 0p3a, 1p3a and 2p1a amounted to 88.2%, 5.5%, 4.2% and 0.5%, respectively and similary in the second group - 88.3%, 5.6%, 4.5% and 0.6%, respectively. In tapeworms with variable PGP the mean number of the types of proglottids mentioned was diflerent Crom (86.0%, 4.1 %, 3.8% and 4.1%, respectively) the same figure in tapeworms with one-sided PGP. Particularly high correlation was found to occur between the number of PGP variations and type 2p1a proglottids (r= +0.928, P<0.01).
A single pyrimidine nucleoside phosphorylase was found in the cytoplasmic extract from Hymenolepis diminuta. This enzyme preferentially cleaves uridine and, to a much lesser extent, thymidine. Its presence directly indicates the existence of pyrimidine nucleoside salvage pathway in this parasite. Detailed kinetic studies in the phosphorolytic and synthetic direction pointed to the sequential mechanism of these reactions. For phosphorolysis, Kurd = 33 ^M and Kp = 806 nM. For synthesis of uridine, KUra = 204 jiM and Ki.p.rib.= 50 pM. Over six times higher Km for uracil than for uridine indicates that phosphorolysis is the favoured reaction in this tapeworm. Well known inhibitors of mammalian uridine phosphorylase: 2,2'-anhydro-5- -ethyluridine and l-(l,3-dihydroxy-2-propoxymethyl)-5-benzyluracil (DHPBU), both with Ki = 0.07 nM were potent competitive inhibitors of the enzyme from H. diminuta. The newly synthesized 2,3'-anhydro-5-ethyluridine (K. Felczak,unpublished) showed only moderate inhibitory activity (Ki = 14 jiM) similarly as l-(l,3-dihydroxy-2- -propoxy-methyl)-5-benzyluracil. The same order of Ki values obtained for the investigated inhibitors vs uridine phosphorylase, irrespective whether the enzyme was isolated from rat intestinal mucosa (Drabikowska etal., 1987,Biochem. Pharmacol. 36,4125-4128) or H. diminuta may point to a great similarity between binding sites on the parasite and the host enzyme.
Acetylcholinesterase (AChE) sequentially extracted from mature specimens of Hymenolepis diminuta was shown to be a globular protein, the monomeric form of which (Ga₁) had molecular mass of 66 kDa as determined by SDS-PAGE. Amphiphilic character of the enzyme was revealed by Triton X-l14 phase partitioning. The cestode AChE preferred acetylthiocholine over propionyl- and butyrylthiocholine as substrate, split N-acetyl-ß-methylthiocholine and myristoylcholine but did not hydrolyze ß-carbonaphthoxycholine, a substrate for butyrylcholinesterases. It was sensitive to 10⁻⁵ M physostigmine and 10⁻⁵ M BW284C51 but not to 10⁻³ M iso-OMPA. No butyrylcholinesterase activity was detected in extracts from the parasite.
The distribution of acetylcholinesterase (AChE) in oncospheres and developing cysticercoids of Hymenolepis diminuta was examined. The enzyme was localized in the nervous system and in some non-nerve cells of these larvae. In oncospheres AChE was detected in hook muscles and in the binucleated medullar center that is known to enclose two neurons. At early developmental stages of the cysticercoids the enzyme was localized in the post-oncospheral hook muscles and in subtegumental muscle fibers of the cercomer. At medium and late stages of development the activity of AChE was detected in the developing nervous system and in two and, subsequently, in four populations of cells, which gradually spread over the whole internal wall of the cyst, thus forming a thin multilayer AChE-positive lining of the cyst cavity. Following withdrawal of the scolex the lining separates the parenchyma of the turned neck from the cyst tissues and remains AChE-positive during the whole life of the parasite, i.e. up to the death of the infected host. The role played by non-neural AChE associated with the cyst cavity lining is unknown, but seems to regulate both the transport of nutrients and minerals into the scolex and waste substances in the opposite direction.
9-months tapeworms obtained from 3- 6-specimen populations of the 42nd generation of Hymenolepis diminuta WMS „strain" were compared with tapeworms obtained from quantitatively similar populations deriving from one-specimen infections of H. diminuta WMS il1. In both groups of tapeworms the majority of strobilas had unilateral position of genital pares (PGP), while the remaining ones were characterized by variable PGP. The latter appeared in 41.4% of H. diminuta WMS „strain" tapeworms and in only 24.6% of tapeworms coming from H. diminuta WMS il1. In tapeworms with unilateral as well as with variable PGP deriving from H. diminuta WMS il1, the average number of type 0p3a proglottids (all three testes on the aporal side) was significantly higher than in H. diminuta WMS „strain". It results most probably from suitable selection of mother specimens for successive generations of H. diminuta WMS il1. In both compared groups the growth of the number of PGP changes was correlated positively with the number of type 2p1a proglottids and negatively with the numer of type 1p2a and 0p3a proglottids.
The accumulation of neutral lipids in the cells predetermined to evolve into the cyst cavity lining in Hymenolepis diminuta cysticercoids is presented. The differentiation of these cells from the mesenchyma cells and the accompanying systhesis of lipids began in very young, blastula-shaped larvae. Both the number of the cells and the amount of their lipids increased gradually during further development of the parasites. In the pre-encystment stage of the cysticercoids the cells concentrated around the cavity of the prospective cyst. Following encystment, the scolex and the inverted neck became surrounded by a compact mass of the cells, which started using the stored lipids as a source of energy indispensable for the formation of the cyst tissues and of the definitive multilayer lining of the cyst cavity. No neutral lipids were detected in the mature 23-day-old cysticercoids as well as in the experimentally excysted scoleces. Beside a low level of uniformly distributed phospholipids apparently associated with cell membranes, no local accumulations of these substances were observed at any step of the development of the larvae. The utilization of neutral lipids proves both the presence of a lipase-type enzyme(s) and an operative β-oxidation pathway in the cells of the cysticercoids, the latter feature being highly indicative of oxidative metabolism of these larvae.
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