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E Tannich

Publications and source records attributed to E Tannich.

At least 19 recordsLinked to original sources
PubMed1992

Ubiquitin of Entamoeba histolytica deviates in six amino acid residues from the consensus of all other known ubiquitins.

The amino acid sequence of ubiquitin from Entamoeba histolytica, as deduced from a cDNA nucleotide sequence, deviated at six positions from the consensus of all other known ubiquitins (ranging from Trypanosoma cruzi to Homo sapiens). The corresponding residues were scattered over the primary sequence, but came close together on the surface of the folded protein structure. We conclude that (i) E. histolytica branched off very early from the main eukaryotic line, and (ii) this organism may yield clues as to the evolutionary development of the ubiquitin system.

Amino Acid Sequence↗
PubMed1992

Primary and secondary structure of the pore-forming peptide of pathogenic Entamoeba histolytica.

A pore-forming peptide is implicated in the potent cytolytic activity of pathogenic Entamoeba histolytica. Using NH2-terminal sequence information of this peptide, the corresponding cDNA was isolated. The cDNA-deduced amino acid sequence revealed a putative signal peptide and a mature peptide of 77 amino acids including six cysteine residues. Computer-aided secondary structure analysis predicted that the peptide would be composed of four adjacent alpha-helices, and CD spectroscopy indicated an all alpha-helical conformation. The tertiary structure appears to be stabilized by three disulfide bonds; the pore-forming activity was not sensitive to heat but was lost in the presence of reducing agents. Sequence homology was found to the saposins and to surfactant-associated protein B, both mammalian polypeptides of similar size and secondary structure but of non-lytic function. In particular, the six cysteine residues were found to be conserved, suggesting a common motif for stabilizing a favourable tertiary structure. Compared with previously characterized toxic peptides also containing three disulfide bonds, the amoeba peptide may represent a distinct class of biologically active peptides.

Amino Acid Sequence↗
PubMed1992

Sensitive and specific serodiagnosis of invasive amebiasis by using a recombinant surface protein of pathogenic Entamoeba histolytica.

A recombinantly expressed protein, recEh-P1, representing part of an immunodominant surface antigen of pathogenic Entamoeba histolytica, was used for serodiagnosis of invasive amebiasis. Expression was performed under the control of a T7-RNA promoter by using a modified procaryotic expression vector, designated pHisT7. This vector allowed high-yield expression of recEh-P1 fused to a stretch of sequence containing eight histidine residues, which facilitated purification by metal chelate affinity chromatography on Ni2+ columns under highly denatured conditions. Purified recEh-P1 was found to be water soluble after prolonged dialysis and was used as the antigen for the detection of antiamebic serum antibodies by immunoblotting and enzyme-linked immunosorbent assay. In both tests all sera of patients with invasive amebiasis reacted to recEh-P1 whereas none of those collected from healthy controls, including individuals with noninvasive amebiasis, or from patients suffering from bacterial or protozoan infections unrelated to E. histolytica did so.

Amino Acid Sequence↗
PubMed1992

Host tissue destruction by Entamoeba histolytica: molecules mediating adhesion, cytolysis, and proteolysis.

Entamoeba histolytica, the protozoan parasite causing human amoebiasis, has recently been found to comprise two genetically distinct forms, potentially pathogenic and constitutively nonpathogenic ones. Host tissue destruction by pathogenic forms is believed to result from cell functions mediated by a lectin-type adherence receptor, a pore-forming peptide involved in host cell lysis, and abundant expression of cysteine proteinase(s). Isolation and molecular cloning of these amoeba products have provided the tools for structural analyses and manipulations of cell functions including comparisons between pathogenic and nonpathogenic forms.

Animals↗
PubMed1992

Recent progress in the molecular biology of Entamoeba histolytica.

Entamoeba histolytica, a protozoan parasite causing human amoebiasis, has recently been found to comprise two genetically distinct forms, potentially pathogenic and constitutively nonpathogenic ones. Host tissue destruction by pathogenic forms is believed to result from cell functions mediated by a lectin-type adherence receptor, a pore-forming peptide involved in host cell lysis, and abundant expression of cysteine proteinases. Comparisons of these products from pathogenic and nonpathogenic E. histolytica suggest that they have evolved to serve functions in free-living or commensal behaviour. Isolation of the corresponding genes have provided the tools for detailed structural studies and manipulations of amoeba cell functions.

Animals↗
PubMed1992

Recombinant expression, purification and biochemical characterization of a superoxide dismutase from Entamoeba histolytica.

A recombinant iron-containing superoxide dismutase (recFeSOD) of Entamoeba histolytica was produced in a prokaryotic expression system. Purified recFeSOD was found to be enzymatically active as determined by (i) inhibition of ferri-cytochrome c reduction, (ii) dismutation of superoxide anions generated by human neutrophils and (iii) inhibition of nitroblue tetrazolium reduction. The enzymatic properties of recFeSOD were similar to those of the native protein in trophozoite extracts. In an ELISA using recFeSOD as antigen, 96% of sera from patients having invasive amebiasis were reactive whereas none of the healthy controls or of patients suffering from malaria, bacterial or viral infections were reactive. Only sera of Toxoplasma-, Leishmania- or Trypanosoma-infected individuals exhibited partial cross-reactivity to recFeSOD.

Amino Acid Sequence↗
PubMed1992

[Recent findings on the pathogenicity of Entamoeba histolytica].

Entamoeba histolytica is the causative agent of human amoebiasis. During recent years, research in amoebiasis has concentrated on two subjects: 1. the dual manifestation of the infection as harmless colonization of the intestinal cavity or pathogenic tissue invasion and 2. the molecular analysis of functions of E. histolytica that are considered essential for pathogenicity. Besides epidemiological studies and isoenzyme analyses, molecular genetics have revealed additional evidence that two genetically distinct forms of E. histolytica do exist, named "pathogenic" and "nonpathogenic" forms, respectively. Both can infect humans but only the "pathogenic" form is able to invade the tissue and cause disease whereas the "nonpathogenic" is not. Questions remain open about the mechanism that triggers "pathogenic" E. histolytica to become invasive and about the molecules that are involved. Current data indicate that at least three functions of the amoebae are considered essential for pathogenic tissue invasion. Pathogenicity is viewed as a result of 1. adherence of the amoeba to host cells, predominantly mediated by a galactose- and N-acetylgalactosamine-inhibitable lectin, 2. killing of host cells by a pore-forming peptide known as amoebapore, and 3. proteolysis of the host's extracellular matrix mediated by cysteine proteinases. Structural detailed molecular analysis including cloning of the corresponding genes have led to a better understanding of the function of these proteins.

Animals↗
PubMed1991

Direct sequencing of variable HLA gene segments after in vitro amplification and allele separation by temperature-gradient gel electrophoresis.

Previously unrecognized variants of human leukocyte antigens (HLA) are currently being analyzed by in vitro amplification and sequencing of the variable gene segments. In heterozygous individuals, molecular cloning is required to separate the two concomitantly amplified haplotypic gene segments. A method is presented which facilitates the procedure of separating the two haplotypic gene segments by using a temperature-gradient gel electrophoresis (TGGE). The procedure comprises PCR amplification of the variable HLA gene segments, allele separation by TGGE, re-amplification of each of the separated allelic segments, and direct DNA sequencing using the PCR primers.

Amino Acid Sequence↗
PubMed1991

Homologous cysteine proteinases of pathogenic and nonpathogenic Entamoeba histolytica. Differences in structure and expression.

A cDNA clone derived from the gene encoding a cysteine proteinase of pathogenic Entamoeba histolytica was isolated using an antiserum to the purified enzyme. This clone was used to identify the homologous clone in a cDNA library from nonpathogenic E. histolytica. Sequence analysis and comparison of the predicted amino acid sequences revealed a sequence divergence of 16%. Southern blot analyses indicated that (i) pathogenic isolates may contain more genes coding for these or related enzymes than nonpathogenic isolates, (ii) the structure and organization of these genes are conserved within each group of amoebae, and (iii) none of the genes is found in both pathogenic and nonpathogenic E. histolytica, underlining the notion that the two groups are genetically distinct. Northern blot analyses suggested that the cysteine proteinase is expressed by pathogenic isolates in substantially higher amounts than by nonpathogenic isolates. Overexpression of this enzyme may be an important factor in the pathogenicity of E. histolytica.

Amino Acid Sequence↗
PubMed1991

Primary structure of the 170-kDa surface lectin of pathogenic Entamoeba histolytica.

The adherence of Entamoeba histolytica to colonic mucins and to host cells appears to be predominantly mediated by a 170-kDa surface lectin of the amoebae. By using an antiserum to the purified lectin, the corresponding cDNA was isolated from an expression library of the pathogenic E. histolytica isolate HM-1:IMSS. Northern blot analysis indicated a transcript of approximately 4 kilobases, and Southern blot analyses suggested that multiple genes may encode the lectin or closely related proteins in HM-1:IMSS trophozoites. The cDNA-deduced amino acid sequence revealed an N-terminal signal peptide and a mature protein of 1270 amino acids corresponding to a molecular mass of 143 kDa, which comprises a short C-terminal cytoplasmic domain with potential phosphorylation sites, a transmembrane region, and a large extracellular portion with nine potential asparagine-linked glycosylation sites. The extracellular portion may be separated into a cysteine-poor domain and a cysteine-rich domain, the latter of which shows in part repetitive structural elements with a low degree of sequence homology to wheat germ agglutinin and to pDd63, a developmentally expressed protein of Dictyostelium discoideum.

Amino Acid Sequence↗
PubMed1991

Pathogenic and nonpathogenic Entamoeba histolytica: identification and molecular cloning of an iron-containing superoxide dismutase.

Superoxide dismutase (SOD) activity was determined in the cell lysate of the axenically cultured Entamoeba histolytica isolate HM-1:IMSS. Under anaerobic culture conditions, 18.7 (+/- 4.9) units SOD activity (mg protein)-1 were found. By inhibition studies the activity was attributed to an iron-containing type of SOD (FeSOD). Using degenerate oligonucleotide primers derived from regions highly conserved in prokaryotic FeSOD sequences, a genomic DNA fragment was amplified by the polymerase chain reaction. The fragment was used to isolate FeSOD specific cDNA clones from a pathogenic and a nonpathogenic E. histolytica isolate. A comparison of the 2 sequences revealed 5% nucleotide differences resulting in a single amino acid exchange. The primary structure showed the characteristics of an iron-containing type of SOD with a homology of approximately 55% with other FeSOD sequences. The enzyme was found to be encoded by single copy genes in both the pathogenic and the nonpathogenic E. histolytica, but restriction fragment lengths differed between the 2 groups. In 5 isolates studied, no correlation was found between pathogenic behavior of the amebae and the expression of FeSOD-related mRNA.

Amino Acid Sequence↗
PubMed1991

Differentiation of pathogenic from nonpathogenic Entamoeba histolytica by restriction fragment analysis of a single gene amplified in vitro.

We previously reported the identification of homologous cDNA clones derived from a pathogenic isolate and a nonpathogenic isolate of Entamoeba histolytica, which had been designated cEh-P1 and cEh-NP1, respectively. Sequence analysis of both clones had revealed 10% nucleic acid substitutions, which were dispersed over the entire sequence. This genetic difference had been found to be conserved between all four pathogenic and all five nonpathogenic laboratory strains of E. histolytica tested. On the basis of nucleic acid substitutions, we have now developed a sensitive assay to distinguish pathogenic from nonpathogenic forms of E. histolytica by using fresh clinical isolates. Comparing the sequence of cEh-P1 and cEh-NP1, we identified a 482-bp segment that contained identical 5' and 3' ends but differed in internal cleavage sites for restriction endonucleases. By using oligonucleotide primers corresponding to the 5' and 3' ends of this segment, the corresponding gene was amplified by the polymerase chain reaction. Endonuclease digestion of the amplified DNA yielded restriction fragments that are characteristic for pathogenic and nonpathogenic forms. This assay allows the detection and classification of fewer than 10 amoebae within a few hours. The differentiation of 48 isolates into pathogenic and nonpathogenic strains by using this method corresponded to the clinical status of the infected individuals and to the classification obtained by isoenzyme determination. The results further support the concept that pathogenic and nonpathogenic strains of E. histolytica constitute distinct subspecies.

Animals↗
PubMed1990

Deletion mutagenesis in M13 by polymerase chain reaction using universal sequencing primers.

A simple procedure is described for the efficient deletion of large DNA sequences. The method involves a combination of oligonucleotide-directed mutagenesis in bacteriophage M13 and amplification of the mutagenized product by polymerase chain reaction. In contrast to other protocols employing polymerase chain reaction, synthesis of only one specific primer is required. The efficiency of heteroduplex formation between mutagenic primers directing large deletions and single-stranded template is discussed.

Bacteriophages↗