Biomedical subjects

T Seebeck

Publications and source records attributed to T Seebeck.

At least 55 records · Page 3Linked to original sources

PubMed1988

Large microtubule-associated protein of T. brucei has tandemly repeated, near-identical sequences.

The parasitic protozoon Trypanosoma brucei contains a highly organized membrane skeleton, consisting of a dense array of parallel, singlet microtubules that are laterally interconnected and that are also in tight contact with the overlying cell membrane. A high molecular weight, heat-stable protein from this membrane skeleton was isolated that is localized along the microtubules. Protease digestion experiments and sequencing of a cloned gene segment showed that most of the protein is built up by more than 50 nearly identical tandem repeats with a periodicity of 38 amino acids.

Amino Acid Sequence↗

PubMed1988

A microtubule-binding protein of Trypanosoma brucei which contains covalently bound fatty acid.

Covalently linked fatty acids are increasingly recognized as an important type of post-translational protein modification. Many of such acylated proteins are found associated with cellular membranes. The membrane skeleton of the parasitic hemoflagellate Trypanosoma brucei consists of a regular array of microtubules which are tightly bound to the overlying cell membrane. A microtubule-binding protein (p41) has been identified within this structure which carries covalently bound fatty acid. The fatty acid linkage is sensitive to hydroxylamine treatment. After chemical transesterification, the released radioactivity co-migrates with fatty acid methyl esters in thin-layer chromatograms, establishing that the fatty acid was covalently bound to p41 via an ester (thioester) linkage. Upon detergent extraction of trypanosomes, p41 remains tightly bound to the cytoskeleton as long as Ca2+ ions are present. It can selectively be released from this structure by the addition of excess EGTA. Conversely, p41 binds to isolated cytoskeletons and to purified microtubules in vitro, the reaction again being entirely Ca2+-dependent.

Animals↗

PubMed1988

Production of a recombinant antigen of Echinococcus multilocularis with high immunodiagnostic sensitivity and specificity.

A cDNA library derived from mRNA of metacestodes from Echinococcus multilocularis was constructed in the Escherichia coli expression vector lambda gt11 and screened with human patients' sera. The recombinant proteins of 11 positive phages were further evaluated for their potential as immunodiagnostic reagent. One isolated clone (from lysogen II/3) synthesized a fusion protein which was rapidly degraded. This intracellular degradation process provided two distinct polypeptides with Mr of about 31,000 and 33,000, both showing strong binding activity with specific patients' antibodies. The diagnostic value of the II/3-antigen was evaluated by mini-Western-blot with sera from 41 patients infected with E. multilocularis and sera from 77 patients with other helminthic infections, resulting in a diagnostic sensitivity of 98% and an over-all specificity of 96%. These results suggest the usefulness of the recombinant II/3-antigen for immunodiagnosis of human alveolar echinococcosis.

Animals↗

PubMed1988

A 60-kDa cytoskeletal protein from Trypanosoma brucei brucei can interact with membranes and with microtubules.

The cytoskeleton of eukaryotic cells is a major determinant of cellular architecture and of many cellular functions. In addition to or in place of the transcellular cytoskeleton, many eukaryotic cells also contain membrane-associated cytoskeletal structures (membrane skeletons), which are important for cellular structure and function. The membrane skeleton of the parasitic hemoflagellate Trypanosoma brucei consists of a dense array of singlet microtubules (subpellicular microtubules), which are tightly associated to the overlying cell membrane. This study reports the identification of a microtubule-associated protein from Trypanosoma brucei that constitutes a component of the link between this microtubular array and the cell membrane. The protein can bind in vitro both to microtubules and to membrane vesicles or liposomes. Furthermore, it can crosslink microtubules and membrane vesicles, suggesting that it exerts a similar function in the membrane skeleton.

Animals↗

PubMed1988

Structure and transcription of the actin gene of Trypanosoma brucei.

In Trypanosoma brucei, the actin gene is present in a cluster of two, three, or four tandemly linked copies, depending on the strain. Each cluster seems to exist in two allelic versions, as suggested by the polymorphism of both gene number and restriction fragment length in the DNA from cloned trypanosomes. The amplification of the gene copy number probably occurs through unequal sister chromatid exchange. The chromosomes harboring the actin genes belong to the large size class. The coding sequence was 1,128 nucleotides long and showed 60 to 70% homology to other eucaryotic actin genes. Surprisingly, this homology seemed weaker with Trypanosoma congolense, Trypanosoma cruzi, Trypanosoma vivax, Trypanosoma mega, or Leishmania actin-specific sequences. The mRNA was around 1.6 kilobases long and was synthesized at the same level in bloodstream and procyclic forms of the parasite. Large RNA precursors, up to 7.7 kilobases, were found in a pattern identical in strains containing either two or three gene copies. Probing of the flanking regions of the gene with either steady-state or in vitro transcripts, as well as S1 nuclease protection and primer extension experiments, allowed mapping of the 3' splice site of the actin mRNA, 38 nucleotides upstream from the translation initiation codon. A variably sized poly(dT) tract was found about 30 base pairs ahead of the splice site. The largest detected actin mRNA precursor seemed to give rise to at least two additional stable mRNAs. The RNA polymerase transcribing the actin gene exhibited the same sensitivity to inhibition by alpha-amanitin as that transcribing both the spliced leader and the bulk of polyadenylated mRNAs.

Actins↗

PubMed1988

Spectrin-like proteins in the paraflagellar rod structure of Trypanosoma brucei.

A polyclonal, monospecific rabbit antibody to human erythrocyte spectrins cross-reacted with two sets of proteins (a doublet of 180/200K and a triplet of 67-66-65K; K = 10(3) Mr) in the parasitic protozoon Trypanosoma brucei brucei. Except for the 66K protein, the cross-reacting proteins are localized in the flagellum, on the basis of evidence from cell fractionation and immunofluorescence microscopy. Immunogold labelling and electron micrographs further revealed that the spectrin-like proteins are confined to the paraflagellar rod structure. The spectrin-like proteins with apparent molecular weights of 180 and 200 share homology with spectrin band 1, since V8-protease from Staphylococcus aureus generated similarly sized, antigenic peptides from these proteins. The results indicate homology between the cross-reacting proteins and human red cell spectrin.

Animals↗

PubMed1987

Transcription of the intergenic regions of the tubulin gene cluster of Trypanosoma brucei: evidence for a polycistronic transcription unit in a eukaryote.

The tubulin genes of T. brucei are clustered in a tightly packed array of alternating alpha- and beta-genes. The steady state mRNA contains one abundant mRNA species each for alpha- and beta-tubulin, both carrying the identical 35 nt mini-exon sequence at their 5'-ends. We have used in vitro run-on transcription assays to investigate the mechanism of tubulin gene transcription in T. brucei. Our results show that the regions between the individual tubulin genes are transcribed at the same rate as are the genes themselves. On the other hand, transcripts containing the intergenic regions could not be detected by Northern analysis or in vivo labelling experiments. We conclude that putative transcripts from the intergenic regions have a half-life of less than one minute. These results suggest that the tubulin gene cluster is transcribed as a single contiguous transcription unit yielding a primary transcript which is rapidly processed into individual mRNAs by the polyadenylation and mini-exon trans splicing machineries.

Animals↗

PubMed1987

Subpellicular and flagellar microtubules of Trypanosoma brucei brucei contain the same alpha-tubulin isoforms.

The cytoskeleton of the parasitic hemoflagellate Trypanosoma brucei brucei essentially consists of two microtubule-based structures: a subpellicular layer of singlet microtubules, which are in close contact with the cell membrane, and the flagellar axoneme. In addition, the cells contain a small pool of soluble tubulin. Two-dimensional gel electrophoretic analysis of the tubulins present in these subcellular compartments revealed two distinct electrophoretic isoforms of alpha-tubulin, termed alpha 1 and alpha 3. alpha 1-Tubulin most likely represents the primary translation product, while alpha 3-tubulin is a posttranslationally acetylated derivative of alpha 1-tubulin. In the pool of soluble cytoplasmic tubulin, alpha 1 is the predominant species, while the very stable flagellar microtubules contain almost exclusively the alpha 3-tubulin isoform. The subpellicular microtubules contain both isoforms. Neither of the two alpha-tubulin isoforms is organelle specific, but the alpha 3 isoform is predominantly located in stable microtubules.

Acetates↗

PubMed1987

Distinct localization and cell cycle dependence of COOH terminally tyrosinolated alpha-tubulin in the microtubules of Trypanosoma brucei brucei.

alpha-Tubulin can be posttranslationally modified in that its COOH-terminal amino acid residue, tyrosine, can be selectively removed and replaced again. This reaction cycle involves two enzymes, tubulin carboxypeptidase and tubulin tyrosine ligase. The functional significance of this unusual modification is unclear. The present study demonstrates that posttranslational tyrosinolation of alpha-tubulin does occur in the parasitic hemoflagellate Trypanosoma brucei brucei and that posttranslational tyrosinolation can be detected in both alpha-tubulin isoforms found in this organism. Trypanosomes contain a number of microtubular structures: the flagellar axoneme; the subpellicular layer of singlet microtubules which are closely associated with the cell membrane; the basal bodies; and a cytoplasmic pool of soluble tubulin. Tyrosinolated alpha-tubulin is present in all these populations. However, immunofluorescence studies demonstrate a distinct localization of tyrosinolated alpha-tubulin within individual microtubules and organelles. This localization is subject to a temporal modulation that correlates strongly with progress of a cell through the cell cycle. Our results indicate that the presence of tyrosinolated alpha-tubulin is a marker for newly formed microtubules.

Animals↗

PubMed1986

Processing in the external transcribed spacer of ribosomal RNA from Physarum polycephalum.

The rDNA of the myxomycete Physarum polycephalum is transcribed to give a 13.3 kb precursor of ribosomal RNA. At 1.7 kb downstream of the primary initiation site there is a processing site or a second initiation site. This site was studied by S1-mapping, DNA sequencing and electron microscopy. None of these methods could conclusively distinguish between the two formal possibilities. However, capping experiments indicate that rapid processing is taking place at this site rather than reinitiation. In addition, primary transcripts and processed molecules were assayed throughout the synchronous mitotic cycle. During all interphase stages newly initiated transcripts of rDNA and products of the first processing step are present in similar amounts, indicating control of initiation and not of maturation as being the main regulatory step for the accumulation of mature rRNAs. During the brief period of mitosis the level of newly initiated rRNA precursors is lowered.

Base Sequence↗

PubMed1986

Tubulin mRNAs of Trypanosoma brucei.

The tubulin genes of Trypanosoma brucei are located in a single, tightly packed cluster of ten tandemly arranged alternating alpha and beta-genes. No tubulin genes are detected outside the clustered array. Therefore, the cluster can be assumed to be the locus of tubulin gene expression. Single bands of alpha and beta-tubulin mRNAs are observed in cultured procyclic as well as in bloodstream trypanosomes. Both alpha and beta-tubulin mRNAs have distinct 5' termini, which carry a 35-nucleotide mini-exon sequence. The 3' termini of both mRNA populations are heterogeneous.

Animals↗

PubMed1986

Monoclonal antibodies against a 60 kDa phenothiazine-binding protein from Trypanosoma brucei can discriminate between different trypanosome species.

The dominant structure of the cytoskeleton of the Trypanosomatidae consists of a tight array of singlet pellicular microtubules, which surround the entire cell body. These microtubules are in close and stable contact with the cellular membrane. These contacts can be selectively disrupted by the action of phenothiazine drugs, which are potent trypanocides in vitro. Phenothiazine-affinity chromatography of detergent solubilized proteins from Trypanosoma brucei has resulted in the isolation of a protein of an apparent molecular weight of 60 000. Polyclonal antibodies raised against this protein (p60) have been used to investigate the presence of similar proteins in other protozoa. No such crossreacting proteins have been observed outside the family Trypanosomatidae. Within this family, a strong crossreactivity was observed with Crithidia fasciculata, while only a marginal reaction was seen with two species of Leishmania and, quite unexpectedly, also with the stercorarian trypanosomes T. cruzi and T. rangeli. Different monoclonal antibodies against p60 are able to clearly distinguish different subgenera of salivarian trypanosomes, and most notably to differentiate between various isolates of T. congolense. Therefore, these antibodies may prove valuable for diagnostic and epidemiological applications.

Animals↗

PubMed1986

Tubulin isotypes: generation of diversity in cells and microtubular organelles.

Diversity of tubulin isotypes is illustrated by consideration of the beta-tubulin isotypes of higher plants and the eukaryotic microbe, Physarum polycephalum, and by the alpha-tubulin isotypes of the protozoan, Trypanosoma brucei. The carrot plant expresses six, well-defined beta-tubulin isotypes that possess characteristic two-dimensional gel coordinates. These six beta-tubulin isotypes are differentially expressed during development of the flowering plant. In a similar manner, Physarum expresses three separate beta-tubuli isotypes during its life cycle; of the two beta 1 isotypes, one is expressed solely in the myxamoeba whilst the other is expressed both in the myxamoeba and in the plasmodium. A further beta-tubulin isotype, beta 2, is expressed only in the plasmodium. In carrot and in Physarum the generation of beta-tubulin diversity appears, in the main, to be generated by the differential expression of a beta-tubulin multi-gene family. However, tubulin isotypes can also be generated by post-translational modifications and T. brucei utilizes two different modifications within one cell. First, the primary translation product, the alpha 1-tubulin isotype, can be acetylated to produce the alpha 3 isotype. Second, both the alpha 1 and alpha 3 isotypes appear to exist in both tyrosinated and detyrosinated forms. The generation of these alpha-tubulin isotypes within the same cell and their presence in particular cellular domains, modulated throughout the cell cycle, reveals a complex relationship between alpha-tubulin isotypes produced by post-translational modifications and the dynamics of microtubule construction.

Animals↗

PubMed1985

Two simple media for biochemical experimentation with cultured procyclic Trypanosoma brucei.

Two simple media are presented which are particularly well suited for biochemical experimentation with procyclic culture forms of Trypanosoma brucei. ME-83 is a simple semi-defined medium which supports active cell growth, and from which individual components can conveniently be deleted or replaced. HHP-84 is a fully defined minimal medium, which allows vigorous cell motility over extended times, but in which cell proliferation is not occurring.

Animals↗

PubMed1984

Partial purification and characterization of microtubular protein from Trypanosoma brucei.

The tubulin proteins of the parasitic hemoflagellate Trypanosoma brucei brucei were purified and characterized. Cytoskeletal microtubules of trypanosomes do not disrupt under conditions used to solubilize brain tubulins. Trypanosomal tubulins, solubilized by extensive sonication, were partially purified from the crude cell extracts by taxol-mediated polymerization. Taxolinduced microtubules were identified by electron microscopy and analyzed biochemically. They consist predominantly of two proteins of about 52,000 and 56,000 Da. Their mobilities on sodium dodecyl sulfate gels differ slightly from those of bovine brain tubulins. Immunological cross-reactivity with antibodies raised against bovine brain tubulins confirmed the nature of the trypanosomal proteins. Peptide mapping of bovine and trypanosomal alpha- and beta-tubulins was performed by enzymatic digestion with staphylococcal protease V8 and chemical cleavage with N-chlorosuccinimide. In both cases, the peptide patterns generated from the trypanosomal alpha- and beta-tubulins were closely related to each other. This suggests that the trypanosomal alpha- and beta-tubulins may have remained more conserved during evolution than the tubulins from higher eukaryotes. The trypanosomal alpha-tubulin is post-translationally modified in vivo by the reversible addition of a tyrosine residue at its COOH terminus. As in higher eukaryotes, this reaction is completely specific for the alpha-polypeptide chain. Our observation represents the first documentation of the occurrence of COOH-terminal tyrosinolation of alpha-tubulin in an eukaryotic microorganism.

Animals↗

PubMed1984

Isolation and purification of transcriptionally active ribosomal chromatin from the slime mould, Physarum polycephalum.

In the acellular slime mold Physarum polycephalum the ribosomal genes are all located on linear, extrachromosomal DNA molecules which are clustered in the nucleolus. This report describes the isolation and purification of these ribosomal genes as functionally active chromatin particles. Nucleolar lysates are fractionated by gel filtration to remove ribosomal precursors and other soluble material. The ribosomal chromatin is subsequently separated from contaminating nuclear chromatin by a sucrose gradient centrifugation step. This procedure allows the isolation of the ribosomal genes as intact nucleoprotein particles, which are now amenable to a biochemical analysis of their structural and functional properties.

Cell Fractionation↗

PubMed1983

Localization and DNA sequence around the initiation site of ribosomal RNA transcription in Physarum polycephalum.

We have used S1 nuclease to map the initiation site of ribosomal RNA transcription in the acellular slime mold Physarum polycephalum, and we have determined the sequence of 1011 nucleotides surrounding the start site. Consistent with others' observations, there is little homology with the comparable region of other species. As predicted by previous restriction mapping, direct repeats roughly 30 base pairs in length are present upstream of the initiation site and a 148 base pair duplication occurs in the external transcribed spacer. The results also suggest the presence of a processing site within the external transcribed spacer of the ribosomal transcription unit.

Base Composition↗

PubMed1983

Trypanocidal action of neuroleptic phenothiazines in Trypanosoma brucei.

This paper reports the effects of a series of phenothiazine derivatives on trypanosomes in vitro. Our results demonstrate that trypanosomes are very sensitive to some of these compounds at micromolar concentrations. Electron microscopic analysis of drug-treated cells shows that the pellicular layer of microtubules is selectively disintegrated by phenothiazine treatment, while the flagellar axoneme is entirely resistant to the drug. These observations suggest that phenothiazines might constitute a promising candidate group for the development of new trypanocidal drugs. Furthermore, they indicate that the mechanism of drug action is mediated through a specific interaction of phenothiazines with the pellicular, but not with the flagellar, microtubules.

Animals↗