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B Betschart

Publications and source records attributed to B Betschart.

At least 37 records · Page 2Linked to original sources
PubMed1994

The chromatin of trypanosomes.

The nuclear chromatin of trypanosomes is organised in the form of nucleosome filaments. When soluble chromatin is prepared under suitable conditions, a regular array of nucleosomes can be shown by electron microscopy. Chromatin of blood stream as well as procyclic culture forms of Trypanosoma brucei brucei and of T. cruzi shows limited compaction at salt concentrations increasing from 1 to 100 mM. No 30 nm fibres, typical for higher eukaryotes, are formed. Digestion of the nuclear chromatin with micrococcal nuclease and analysis of the histone proteins with various techniques reveal that the basic organisation of the trypanosome chromatin is similar but not identical as compared to that of higher eukaryotes. Distinct differences are present with respect to biochemical properties of the histones as well as to their interaction with the DNA. The primary structure of the histones also differs significantly from that found in other lower and higher eukaryotes. The function of the recently described H1-like proteins in trypanosomes is currently being investigated. The differences that have already been found in the structure and compaction of the trypanosome chromatin compared to that of higher eukaryotes lead us to expect differences of gene expression which, in turn, might offer targets for the control of trypanosomiasis.

Animals↗
PubMed1994

Heterogeneity in the properties of the trypanolytic factor in normal human serum.

Although it seems clear that the trypanolytic factor in human serum capable of killing Trypanosoma brucei brucei is high density lipoprotein (HDL), it nevertheless remains controversial as to whether the trypanolytic properties of HDL are confined to a specific subclass or whether all particles have activity. In the present study, we have compared the lytic activities of serum fractions from six normal individuals prepared by gradient ultracentrifugation and also, to avoid ultracentrifugally-induced loss of HDL apolipoproteins, by gel filtration using fast protein liquid chromatography (FPLC). All sera displayed trypanolytic activity in fractions corresponding to the general density (rho = 1.06-1.20 g ml-1) and size (59-440 kDa) limits conventionally used to describe bulk human HDL, the particles between rho = 1.18-1.20 g ml-1 and between 214-440 kDa being particularly lytic. But some sera additionally contained fractions with powerful activity outside these density (rho > 1.24 g ml-1) and size (> 1000 kDa) ranges. Nevertheless, such fractions were considered to contain material with HDL characteristics; apolipoprotein A-I, the major protein of HDL, was always present and the lytic activity of the sera could be completely neutralized by absorption with HDL antiserum. We conclude that all of the trypanolytic activity in human sera is associated with HDL particles and that it is a property of several HDL subpopulations with very different density and size characteristics. Presumably the well-recognized wide variation in trypanocidal activity of normal human sera reflects differences in the quantities of these HDL subpopulations rather than in the total amount of a single, uniquely lytic particle.

Adult↗
PubMed1993

Malaria vaccine: immunization of mice with a synthetic T cell helper epitope alone leads to protective immunity.

The immunogenicity of the non-repetitive sequences of the Plasmodium berghei circumsporozoite (CS) protein was studied using synthetic peptides. Two CS sequences (residues 20-39 and 57-70) exhibiting T cell helper activity were identified. Immunization of BALB/c mice with a branched peptide containing either the 20-39 or the 57-70 sequence and two repeats (B epitope) in a linear sequence induced high titers of anti-repeat and anti-sporozoite antibodies. Mice immunized with the T-B construct (high antibody titers) or with the 57-70 epitope alone (no serum anti-repeat or anti-peptide antibodies) were protected to a similar degree after challenge with infective sporozoites. No protection was obtained in mice immunized with the 20-39 epitope. These results indicate that BALB/c mice can be protected either by effector T cells or by high levels of anti-repeat antibodies. Thus, in the same strain, a double mechanism of protection can be obtained by a synthetic peptide vaccine.

Amino Acid Sequence↗
PubMed1993

Biochemical and functional characterization of histone H1-like proteins in procyclic Trypanosoma brucei brucei.

Four variants and/or posttranslational modifications of histone H1-like proteins of Trypanosoma brucei brucei procyclic culture forms were extracted with 0.25 N HCl from isolated nuclei and analyzed by two-dimensional gel electrophoresis. The amino acid composition of these proteins, their ability to space nucleosomes regularly and to induce salt-dependent condensation of the chromatin indicated their histone H1 nature. On the other hand, the histone H1-like proteins clearly differed from their higher-eukaryote counterparts by their weak interaction with DNA under low-salt conditions. As a consequence, intact nucleosome filaments were prepared according to a new preparation protocol especially adapted to the unstable chromatin of T. b. brucei. Our results indicate that the biochemical properties of the histone H1-like proteins contribute to the structural and functional differences between the chromatin of procyclic T. b. brucei and that of higher eukaryotes.

Amino Acids↗
PubMed1993

Trypanosoma brucei brucei: differences in the nuclear chromatin of bloodstream forms and procyclic culture forms.

Nucleosome filaments of two stages of the life-cycle of Trypanosoma brucei brucei, namely bloodstream forms and procyclic culture forms, were investigated by electron microscopy. Chromatin of bloodstream forms showed a salt-dependent condensation. The level of condensation was higher than that shown by chromatin from procyclic culture forms, but 30 nm fibres as formed in rat liver chromatin preparations were not found. Analysis of histones provided new evidence for the existence of H1-like proteins, which comigrated in the region of the core histones in SDS-PAGE and in front of the core histones in Triton acid urea gels. Differences were found between the H1-like proteins of the two trypanosome stages as well as between the core histones in their amount, number of bands and banding pattern. It can be concluded that T. b. brucei contains a full set of histones, including H1-like proteins, and that the poor condensation of its chromatin is not due to the absence of H1, but most probably due to histone-DNA interaction being weak. It is obvious that structural and functional differences of the chromatin exist not only between T. b. brucei and higher eukaryotes, but also between various stages of the life-cycle of the parasite. It is therefore not adequate to investigate the chromatin only of the procyclic culture forms as a model for all stages of the life-cycle of T. b. brucei.

Animals↗
PubMed1992

Histone-DNA interactions in the chromatin of procyclic Trypanosoma brucei brucei.

The dissociation of histone proteins a-d from the chromatin of Trypanosoma brucei brucei procyclic culture forms was investigated by removing the proteins from the DNA by centrifugation of soluble chromatin through isokinetic sucrose gradients in the presence of NaCl. The dissociation of the T. b. brucei histones was compared with that of their higher-eukaryote counterparts H3, H2A, H2B and H4. All four histones of T. b. brucei remained bound to the DNA at 500 mM NaCl, were partially released at 750 mM NaCl and were completely dissociated from the DNA at 1 M NaCl. These interactions of histones a-d with the DNA were comparable with those of the H2 histones in the chromatin of higher eukaryotes, and histones a and d interacted with the DNA more weakly than did their higher-eukaryote counterparts H3 and H4. Substoichiometric amounts of an additional protein were recovered in the top fractions of the gradients under all dissociation conditions. This protein migrated in the H1 region of rat-liver chromatin in various gel systems. Its early release from the DNA also indicated a resemblance to histone H1. The presence of only small amounts of this protein and the relatively weak interactions of histones a and d with the DNA suggest that the mechanisms involved in chromatin compaction in T. b. brucei are different from those in higher eukaryotes.

Animals↗
PubMed1992

Structural differences between the chromatin of procyclic Trypanosoma brucei brucei and of higher eukaryotes as probed by immobilized trypsin.

Soluble chromatin of Trypanosoma brucei brucei procyclic culture forms was submitted to digestion with free or immobilized trypsin. Digestion with trypsin in salt solutions of low and high ionic strengths generated characteristic sets of limit histone peptides. After incubation of chromatin with immobilized trypsin in a solution of low ionic strength, histones were not degraded, whereas a selective proteolysis occurred at 50 mM NaCl. Histones a and d, which correspond to H3 and H4 of higher eukaryotes, were rapidly attacked. Histones b and c, the counterparts of H2A and H2B, were more resistant. The results indicated that probably the basic N-terminal tails of the proteins a and d are located on the surface of the core particle. The location of d on the surface differs from the internal one proposed for histone H4. The salt-induced increase of susceptibility of histones to proteolysis reflects structural changes of T.b. brucei chromatin, which may result in partial chromatin compaction.

Animals↗
PubMed1992

Sequence differences between histones of procyclic Trypanosoma brucei brucei and higher eukaryotes.

Four histones, a, b, c, d from procyclic Trypanosoma brucei brucei, which show similarities with the amino acid composition of the core histones H3, H2A, H2B and H4, were isolated and cleaved with Endoproteinase Glu-C. The fragments were separated by FPLC reversed phase chromatography and a subset of the fragments (a5, a9, b6, c8, d3, d9, d11) was subjected to sequence analysis. A 54-71% identity was found in the sequences of the fragment c8 and the C-terminal half of H2B and of three fragments of protein d covering the N-terminal half as well as the C-terminal region of H4. The amino acid sequence of the fragment a9 showed a 57 and 54% identity with H3 sequences of Saccharomyces cerevisiae and Xenopus laevis. Neither the a5 nor the b6 sequence could be aligned with histone sequences of other eukaryotes. The significant differences of 21-48% between the T.b. brucei histone sequences and those of calf thymus histones, which are more pronounced than the differences of Tetrahymena pyriformis and the higher eukaryote, resulted partially from replacements of amino acids with different properties and indicate specific patterns of histone-histone and/or histone-DNA contact sites in the nucleosome of T.b. brucei. These differences, together with the lack of a functional histone H1, may be sufficient to explain the lack of a salt-dependent formation of the nucleosome filament into the 30 nm fibre, which reflects alternative methods of organizing and processing the genetic information in the nucleus of the protozoan parasite and which may be of chemotherapeutic significance.

Amino Acid Sequence↗
PubMed1991

Biochemical properties of histone-like proteins of procyclic Trypanosoma brucei brucei.

Four histone-like proteins a, b, c, d were extracted with 0.2 M H2SO4 from soluble nuclear chromatin of Trypanosoma brucei brucei procyclic culture forms and purified by FPLC reversed phase chromatography. The amino acid composition of these proteins and their electrophoretic mobilities in three different gel systems strongly indicated their core histone nature. Similarities were found between a, b, c and d with the core histones H3, H2A, H2B and H4 of higher eukaryotes, respectively. On the other hand, these proteins also showed differences as compared to higher eukaryotes; proteins a and d clearly differed from their counterparts H3 and H4 on the basis of their hydrophobic properties. The results indicate the occurrence of core histone variants in T.b. brucei which may influence DNA-histone and histone-histone interactions as well as the chromatin compaction in the nucleus of this protozoan parasite.

Amino Acids↗
PubMed1990

Analysis of the cuticular collagens of Ascaris suum.

The nematode cuticle is an extracellular structure composed mainly of collagens, with an insoluble epicuticle on the surface. The extracted collagens from adult Ascaris suum can be separated by SDS-PAGE into three major groups of polypeptides with apparent molecular masses of 34, 60-70 and 120-140 kDa. Densitometric evaluation of the polypeptide bands indicated that the three groups are present in the ratio of 1:2:6. Rotary shadowing of reduced, extracted molecules showed fibers 45 nm in length. This length is in excellent agreement with the calculated total length of amino acids in (Gly-X-Y) regions deduced from the collagen gene sequence of Caenorhabditis elegans and A. suum. It is proposed that the three groups of collagen polypeptides found in SDS-PAGE correspond to collagen monomers, dimers and trimers, and that the molecules in the dimeric and trimeric forms are cross-linked via non-reducible bonds.

Animals↗
PubMed1990

Antibodies against the cuticlin of Ascaris suum cross-react with epicuticular structures of filarial parasites.

The insoluble cuticlin from the cortical zone of the cuticle of adult Ascaris suum was purified and used to raise antibodies in C57/bl mice. The specificity of the antibodies for the external cortical layer was shown in the indirect immunofluorescence antibody test and by immunoelectronmicroscopy. A very high specificity for the external cortical layer was found. Some cross-reactions with cuticular collagens occurred, and increased after booster immunizations. The anti-cuticlin antibody cross-reacted with the electron-dense layers of the cortical zones of adult Acanthocheilonema viteae and Brugia pahangi. A very weak reaction was found in the cortical zone of adult Onchocerca volvulus. In the cuticles of third stage larvae of all three species mainly epitopes in the cortical zones were labelled. In no case did the anti-cuticlin antibody interact with the outermost surface of the cuticle.

Animals↗
PubMed1990

Interaction of monoclonal antibodies with cuticular antigens of filarial parasites, Brugia malayi and Wuchereria bancrofti.

Monoclonal antibodies (mAbs) have been prepared against excretory-secretory-metabolic (ESM) antigens of microfilariae (mf) of Wuchereria bancrofti (WbmfESM) and against third stage larvae (L3) of Brugia malayi (BmL3), and purified from ascites fluids with ammonium sulphate. Both antibodies were of the IgM type and did not react with phosphorycholine. The mAb against BmL3 (F46) reacted in ELISA with antigens of L3 of B. malayi, B. pahangi and W. bancrofti and of adults of B. malayi. The mAb raised against wbmfESM (F32) resembled F46 in this respect, though with a lower titer towards the antigens, and in addition reacted with the ESM-antigens of mf and of L3 of W. bancrofti. F46 was able to detect L3 antigens of filarial parasites in spiked serum samples with a detection limit of 8-16 ng in absolute amount. The antibody was found to label the cuticular portion of L3 and adults of the lymphatic parasites, and not the epicuticular surface, in immunoelectron microscopic studies. The antibody recognized a 36 kDa component of the beta-mercaptoethanol extracts of B. pahangi-adults in Western blot analysis.

Animals↗
PubMed1990

Plasmodium berghei subunit vaccine: repeat synthetic peptide of circumsporozoite protein comprising T- and B-cell epitopes fails to confer immunity.

In the murine malaria model induced by Plasmodium berghei, we studied the immunogenicity of the repeat region of the circumsporozoite (CS) protein, which is the main target of the antibody response in infected animals. We immunized several strains with a synthetic peptide--Y(DPPPPNPN)3--corresponding to one of the two P. berghei repeat sequences in complete Freund's adjuvant. Only C57BL/6 immune sera reacted with the synthetic peptide in ELISA and with the native CS protein on P. berghei sporozoites, as detected by immunofluorescence. From lymph node cells of immunized C57BL/6 we isolated two repeat-specific T-cell lines which proliferated in the presence of the synthetic peptide or the recombinant CS protein. We analysed the protective role of this repeat-specific response by injecting infectious sporozoites into mice immunized with irradiated sporozoites or with the repeat peptide. The percentage of mice developing parasitaemia was 80-90% in the peptide-immunized group and only 10-20% in the group immunized with irradiated sporozoites. Anti-repeat antibody titres were comparable in the two groups. On the basis of these results, we can conclude that the T- and B-cell response to the CS repeat obtained with this synthetic peptide immunization is not sufficient for a protective immunity.

Amino Acid Sequence↗
PubMed1989

Characterization of West African Trypanosoma (Trypanozoon) brucei isolates from man and animals using isoenzyme analysis and DNA hybridization.

A total of 18 West African Trypanosoma (Trypanozoon) brucei stocks isolated from man and animals were characterized using isoenzyme analysis with isoelectric focusing (IEF) and DNA hybridization. They were compared with four T. (T.) brucei isolates from East and West Africa that had previously been analysed and well defined. All experiments were carried out with cell lysates of procyclic trypanosomes produced in vitro. The different stocks could be separated into two distinct groups according to their isoenzyme and DNA patterns. The homogeneous group of T. b. gambiense was characterized by zymodeme A and highly specific DNA-banding patterns (type G) always associated with stable human serum resistance. The non-gambiense group (consisting of T.b. rhodesiense and T.b. brucei) was determined by a great variation in these markers. Our results clearly indicate the existence, of T.b. rhodesiense-like parasites in West African patients. Due to their lack of human serum resistance, the four characterized animal isolates can be referred to as T. b. brucei.

Africa, Western↗
PubMed1989

Instability of the nuclear chromatin of procyclic Trypanosoma brucei brucei.

Digestion of nuclear chromatin of Trypanosoma brucei brucei procyclic culture forms with micrococcal nuclease yielded DNA fragments which formed DNA ladders in agarose gels, similar to those of rat liver. However, the chromatin of trypanosomes was digested more rapidly. The digestion of T. b. brucei chromatin yielded a large amount of DNA fragments of core-particle size. The numbers of base pairs per nucleosomal and linker DNA were identical in both species, if the digestion conditions were reduced in the case of T. b. brucei. Psoralen cross-linking of soluble chromatin of trypanosomes at 5 mM salt at pH 7 or pH 10 resulted in an irregular array of single-stranded (ss) bubbles separated by variable stretches of double-stranded (ds) DNA. The proportion of ss DNA was low compared with the ratio of ss/ds stretches in rat liver chromatin, which also showed regularly arranged nucleosomal DNA. Soluble chromatin of T. b. brucei, pre-treated with 500 mM NaCl to remove a potential H1 and psoralen cross-linked at 5 mM salt at pH 7 or pH 10 was to a great extent ds in both situations. The true nucleosome filament organization of T. b. brucei chromatin could only be shown by psoralen cross-linking the DNA in whole nuclei under physiological conditions. The results indicate that the chromatin of procyclic T. b. brucei differs significantly in its compaction pattern from rat liver chromatin; a typical histone H1 is not found, and the DNA-protein interactions are also less stable and can more easily be destabilized by experimental conditions.

Animals↗
PubMed1989

Liver disease in rural Tanzania--a diagnostic approach.

In a prospective study on the aetiology of liver disease and its diagnostic approach in a District hospital in rural Tanzania, 48 consecutive patients with evidence of liver disorders were investigated by physical examination, biochemical tests, laparoscopy and histology. Liver cirrhosis (posthepatic, alcoholic) was found in 31%; non cirrhotic alcoholic liver disease in 15%; viral, bacterial and protozoal liver disorders in 33%, and neoplastic liver changes in 21% of all patients. Clinical impression alone coincided with the final diagnosis in 40% of all cases. This figure was increased to 46%, when haematological and biochemical results were included, and to 71%, when laparoscopy (without histology) was used in addition. Laparoscopy was particularly decisive in the diagnosis and further management of cirrhosis, liver abscess and neoplastic liver disorders. The additional information obtained from histology led to the final diagnosis. Histology was specially useful for the diagnosis of alcoholic liver disease, tropical splenomegaly syndrome and non specific reactive hepatitis. The usefulness of laparoscopy as a diagnostic tool in a district hospital is discussed.

Adolescent↗