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Biomedical subjects

E Tannich

Publications and source records attributed to E Tannich.

At least 55 records · Page 3Linked to original sources
PubMed1995

Spontaneous release of cysteine proteinases but not of pore-forming peptides by viable Entamoeba histolytica.

Invasive properties of pathogenic Entamoeba histolytica have been postulated to depend on the secretion or release of cysteine proteinases and pore-forming peptides (amoebapores) by trophozoites. To establish whether such toxic molecules are released by viable trophozoites or upon cellular disintegration, amoebae were maintained in various culture media, and activities in supernatants were monitored over time in correlation to cellular integrity. By measuring the release of the cytoplasmic marker enzyme NADP(+)-alcohol dehydrogenase, it became apparent that release of amoebapore was accompanied by cellular disintegration. In contrast, considerable quantities of cysteine proteinases were found to be present in culture supernatants also when amoebae remained intact. Treatment of amoebae with concanavalin A, bacterial lipopolysaccharides or the calcium ionophore A23187 did not result in amoebapore secretion suggesting that here target cell contact is required as an essential stimulus.

Animals↗
PubMed1995

Evaluation of three serological tests for the detection of antiamebic antibodies applied to sera of patients from an area endemic for amebiasis.

Two enzyme immuno assays based on a single recombinant Entamoeba histolytica antigen (P1-EIA) or soluble E. histolytica extract (SA-EIA) as well as a latex agglutination test using an E. histolytica membrane fraction (M-LA) were evaluated for its use to detect anti-amebic serum antibodies in patients from Durban, South Africa, an area endemic for amebiasis. In a previous study, all three test systems were found to be reliable in terms of sensitivity and specificity when applied to sera of European individuals. By analysing a total of 167 serum samples of patients from the Durban area, suffering from invasive amebiasis (n = 76) or miscellaneous diseases unrelated to E. histolytica infection (n = 91), the present study revealed sensitivity for the detection of anti-amebic antibodies of 97.4% for SA-EIA, 86.8% for P1-EIA and 96.1% for M-LA, respectively. Specificity was high for P1-EIA (96.7%) and M-LA (92.3%) but substantially lower for SA-EIA (62.6%). In addition, antibody responses to the recombinant P1 antigen were analysed in 16 patients with amebic liver abscess before and after anti-amebic treatment. The results indicated that most of the patients lost their specific antibody response within 7 month of follow up. Therefore, P1-EIA seems to be a valuable test for distinguishing between present and past E. histolytica infections.

Amebiasis↗
PubMed1994

Purification and molecular characterization of the NAD(+)-dependent acetaldehyde/alcohol dehydrogenase from Entamoeba histolytica.

A bifunctional 95 kDa polypeptide (EhADH2) harbouring acetaldehyde dehydrogenase and alcohol dehydrogenase activities was purified to homogeneity from trophozoite extracts of the protozoan parasite Entamoeba histolytica. Kinetic studies revealed that the enzyme utilizes NAD+ rather than NADP+ as cofactor. Km values for acetyl-CoA, acetaldehyde and ethanol were found to be 0.015, 0.15 and 80 mM respectively in the presence of 0.2 mM NAD+. The primary structure of EhADH2 as deduced from respective amoebic DNA sequences showed striking similarity to the trifunctional AdhE protein of Escherichia coli and the bifunctional AAD protein of Clostridium acetobutylicum. Alignment with a number of aldehyde dehydrogenases and alcohol dehydrogenases from various species suggested that the two catalytic functions of EhADH2 are located on separate parts of the molecule. By cross-linking experiments and electron-microscopic analysis, native EhADH2 was found to be organized in a homopolymeric fashion consisting of more than 20 associated promoters which form rods about 50-120 nm in length.

Alcohol Dehydrogenase↗
PubMed1994

HLA-D alleles associated with generalized disease, localized disease, and putative immunity in Onchocerca volvulus infection.

Human infections with the tissue nematode Onchocerca volvulus result in a variety of clinical conditions that possibly include protective immunity. In a West African area hyperendemic for human onchocerciasis, 120 residents were classified according to clinical and laboratory findings as presenting with generalized onchocerciasis, localized onchocerciasis, or as being putatively immune. The three groups differed in the distribution of HLA-D variants as determined by DNA typing. The most pronounced differences were found among alleles of the DQ loci. The haplotype DQA1*0501-DQB1*0301 was significantly more frequent among putatively immune individuals than among patients with generalized or localized disease. Conversely, DQA1*0101-DQB1*0501 and, independently, the allele DQB1*0201 were more frequent in generalized disease than in localized disease or putative immunity. In these correlations, the frequencies of allelic variants were in localized disease intermediate to those of the two other groups. The only distinct association found with localized disease was that of the DP allele DPB1*0402. The findings indicate that HLA-D variants influence the course of O. volvulus infection and help to define a state that may reflect protective immunity.

Adolescent↗
PubMed1994

Transfection and transient expression of chloramphenicol acetyltransferase gene in the protozoan parasite Entamoeba histolytica.

Hybrid plasmids were constructed and used for successful transfection and transient expression of the chloramphenicol acetyltransferase (CAT) gene in the protozoan parasite Entamoeba histolytica. Transfection was performed by electroporation of the amebae in a potassium phosphate-based buffer under conditions of 3000 V/cm and 25 microF, resulting in a time constant of 0.4 ms. Expression of CAT activity was achieved with constructs in which the CAT coding region was flanked by untranslated upstream and downstream sequences of E. histolytica genes. Highest activity was detected after culturing transfected cells for 48 hr. Activity was found to be proportional to the amount of DNA transfected.

Animals↗
PubMed1994

Induction of the iron-containing superoxide dismutase in Entamoeba histolytica by a superoxide anion-generating system or by iron chelation.

The regulation of superoxide dismutase (SOD) expression was studied in 4 Entamoeba histolytica isolates. In comparison to anaerobic conditions, cultivation of the amoebae in the presence of superoxide radical anions or a ferrous iron chelator revealed substantial increase of SOD expression. Under the different culture conditions, all SOD activity could be exclusively attributed to an iron-containing type (FeSOD). Northern blot analysis revealed that FeSOD expression was regulated on the transcriptional level. Within the 5'-flanking region of the amoebic FeSOD gene, a 19-bp fragment was found with 68% sequence identity to the consensus motif of the binding site for the ferric uptake regulation gene product of Escherichia coli. Electrophoretic mobility shift assays with this 19-bp fragment and with amoebic nuclear extracts revealed specific DNA/protein complex formation. The results indicate that the regulation of E. histolytica FeSOD expression is similar to that of the manganese-containing SOD (MnSOD) of E. coli.

Animals↗
PubMed1994

Amoebapores, a family of membranolytic peptides from cytoplasmic granules of Entamoeba histolytica: isolation, primary structure, and pore formation in bacterial cytoplasmic membranes.

Three peptides with pore-forming activity were isolated from the cytoplasmic granules of pathogenic Entamoeba histolytica by acidic extraction, gel filtration and reversed-phase high-performance liquid chromatography. Partial amino acid sequence analysis of the three active peptides revealed that the most abundant of them was amoebapore and the other two were isoforms thereof. Cloning and sequencing of genomic DNA resolved the amino acid sequence of the two newly recognized peptides. The three peptides designated amoebapores A, B and C were found to have the same molecular size but to differ markedly in their primary structure, although all six cysteine residues are conserved. Despite sequence divergence, structural implications predict for the three peptides a similar amphipathic alpha-helical conformation stabilized by disulphide bonds. All three isoforms exhibit pore-forming activity toward lipid vesicles, but they differ in their kinetics. They also are capable of perturbing the integrity of bacterial cytoplasmic membranes and thereby kill Gram-positive bacteria. The amoebapores represent a distinct family of membrane-active peptides that may function intracellularly as antimicrobial agents but may also confer cytolytic activity on the parasite.

Amino Acid Sequence↗
PubMed1993

A family of transcripts (K2) of Entamoeba histolytica contains polymorphic repetitive regions with highly conserved elements.

Sera from patients with invasive amebiasis were used to identify a cDNA clone (K2p-1) encoding a commonly recognized, repeat-containing antigen of the pathogenic Entamoeba histolytica HM-1:IMSS. K2p-1 was used to isolate 3 cDNA clones (K2 clones); one K2p-1 related clone from the same pathogenic E. histolytica strain and 2 from the nonpathogenic E. histolytica strain SAW-142. The nucleotide sequence and predicted amino acid sequence revealed a closely related family of transcripts differing mainly in the extent and arrangement of an internal region consisting of tandemly arranged repetitive elements. The repetitive units encoding either 12 or 8 amino acids were found to be highly conserved in all the K2 clones analyzed so far, suggesting that the repeat motifs perform functions common to both pathogenic and nonpathogenic E. histolytica. The genomic organization of the K2 genes was different when compared in pathogenic and nonpathogenic E. histolytica and may therefore be used to discriminate between pathogenic and nonpathogenic E. histolytica strains.

Amino Acid Sequence↗
PubMed1993

Comparison of pore-forming peptides from pathogenic and nonpathogenic Entamoeba histolytica.

Similar to the findings obtained with pathogenic Entamoeba histolytica, nonpathogenic isolates were found to kill mammalian cells in vitro, and cell extract caused pore formation in liposome membranes. A pore-forming peptide termed APnp was isolated from a nonpathogenic isolate using the schedule developed for the purification of APp or amoebapore, the homologous peptide of the pathogenic isolate HM-1:IMSS. Compared to APp, the specific activity of APnp in pore formation was 60% lower. cDNA sequencing indicated 95% identity of the primary structures of APnp and APp, and secondary structure predictions revealed a high degree of similarity. Notably, a glutamic acid residue at position 2 of APp is in APnp replaced by proline, which shortens one of the two amphipathic alpha-helices considered crucial for the pore-forming function. This structural divergence of the two peptides might explain the difference in their pore-forming activities.

Amino Acid Sequence↗
PubMed1993

Unusual gene organization in the protozoan parasite Entamoeba histolytica.

We have analyzed three independent genomic loci of the protozoan parasite Entamoeba histolytica that contain coding regions for the iron-containing superoxide dismutase, the pore-forming peptide, and the galactose-inhibitable lectin. All of the three structural genes were found to be closely linked unidirectionally to other coding sequences. The intergenic regions did not exceed 1,350 nucleotides. Nuclear run-on data demonstrated that at least the galactose-inhibitable lectin gene is transcribed in a monocistronic fashion. Comparison of the genomic sequences described here with several others reported previously for E. histolytica revealed a number of invariable peculiarities for the gene organization of this parasite: (i) Coding sequences are not interrupted by introns; (ii) 5' untranslated regions are rather short and transcription starts at the consensus sequences ATTCA or ATCA; (iii) an unusual TATA-motif is located about 30 nucleotides upstream of the start of transcription and comprises the sequence TATTTAAA, which reveals protein binding activity as determined by gel retardation assays; (iv) the conserved pentanucleotide motif TAA/TTT is found within the relatively short 3' untranslated regions and functions putatively as the transcription termination signal; and (v) a stretch of up to 12 pyrmidine residues is located at the end of transcribed sequences.

Animals↗
PubMed1993

Crude or recombinant proteins applied to latex agglutination, complement fixation and enzyme-linked immunosorbent assays for the serodiagnosis of invasive amebiasis.

Three different agglutination tests were developed for the detection of serum antibodies against Entamoeba histolytica. These tests are based on carboxylated polystyrene beads loaded either with a purified recombinant E. histolytica protein, designated recEh-P1, or a soluble fraction, or a membrane fraction (M-LA) both prepared from E. histolytica trophozoites. The three agglutination tests were compared with an enzyme-linked immunosorbent assay and a complement fixation test based on crude soluble E. histolytica antigens as well as with an enzyme-linked immunosorbent assay using recEh-P1 as antigen (P1-EIA). Serum samples from patients with invasive amebiasis (n = 30), or infectious diseases unrelated to E. histolytica (n = 57), as well as sera of apparently healthy individuals (n = 25) including some with noninvasive amebiasis (n = 5) were analysed by all six methods. Depending on the assay used, the results obtained, revealed sensitivities ranging from 83% to 100% and specificities ranging from 93% to 100%. P1-EIA and M-LA exhibited best results, both with a sensitivity of 100% and a specificity of 98%.

Animals↗
PubMed1993

Analysis of the genomic sequence encoding the 29-kDa cysteine-rich protein of Entamoeba histolytica.

By analyzing cDNA and genomic clones coding for the 29-kDa cysteine-rich protein of Entamoeba histolytica, substantial sequence differences were found to the 5'-end of a previously described full-length cDNA coding for the same protein (Reed et al., 1992), which was reported to contain an untranslated 5'-sequence of at least 171 nucleotides, unusual for E. histolytica cDNAs. We found evidence that the cDNA published by Reed et al. (1992) represents a hybridclone composed of two unrelated sequences and that the gene coding for the 29-kDa molecule comprises all of the features typical for E. histolytica genes. A data base analysis revealed substantial sequence homology of the 29-kDa protein to a class of polypeptides found in prokaryotic organisms that may be involved in the inactivation of hydrogen peroxide.

Amino Acid Sequence↗
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↗