Search PubMed⌕ Search

Biomedical subjects

D Mirelman

Publications and source records attributed to D Mirelman.

At least 55 records · Page 3Linked to original sources
PubMed1994

Characterization of two distinct gene transcripts for ribosomal protein L21 from pathogenic and nonpathogenic strains of Entamoeba histolytica.

A second gene (rp-L21) copy, clone g34, coding for ribosomal (r-) protein L21, was isolated from the pathogenic (P) strain HM-1:IMSS cl6 of the intestinal parasite Entamoeba histolytica (Eh). The gene was compared to the previously isolated copy, gLE3 [Petter et al., Mol. Biochem. Parasitol. 56 (1992) 329-334], with respect to its primary structure, mRNA levels and binding to the r-complex during translation. Unlike the gLE3 gene copy [Petter et al., Mol. Biochem. Parasitol. 56 (1992) 329-334], g34 was found not to be physically connected to an actin gene copy. Homologous copies of the two rp-L21 genes were also characterized from the nonpathogenic (NP) strain SAW1734R clAR, as well as from its P derivative. Sequence comparison of the coding regions of the two rp-L21 revealed almost full identity. Significant differences were found, however, within their 3' and 5' flanking regions. Using the 3' rapid amplification of cDNA ends (3' RACE) method [Frohman et al., Proc. Natl. Acad. Sci. USA 85 (1988) 8998-9002], as well as Northern and slot blot hybridizations, it was demonstrated that both rp-L21 mRNAs are found in similar amounts. However, as was shown by differential hybridization, the relative binding of each transcript to the r-complex varied somewhat between P and NP strains. This finding suggests that the control of expression of rp-L21 in Eh may involve regulation at the post-transcriptional level.

Amino Acid Sequence↗
PubMed1994

Alliin lyase (Alliinase) from garlic (Allium sativum). Biochemical characterization and cDNA cloning.

The garlic plant (Allium sativum) alliinase (EC 4.4.1.4), which catalyzes the synthesis of allicin, was purified to homogeneity from bulbs using various steps, including hydrophobic chromatography. Molecular and biochemical studies showed that the enzyme is a dimer of two subunits of MW 51.5 kDa each. Its Km using synthetic S-allylcysteine sulfoxide (+ isomer) as substrate was 1.1 mM, its pH optimum 6.5, and its isoelectric point 6.35. The enzyme is a glycoprotein containing 6% carbohydrate. N-terminal sequences of the intact polypeptide chain as well as of a number of peptides obtained after cyanogen bromide cleavage were obtained. Cloning of the cDNAs encoding alliinase was performed by a two-step strategy. In the first, a cDNA fragment (pAli-1-450 bp) was obtained by PCR using a mixed oligonucleotide primer synthesized according to a 6-amino acid segment near the N-terminal of the intact polypeptide. The second step involved screening of garlic lambda gt11 and lambda ZAPII cDNA libraries with pAli-1, which yielded two clones; one was nearly full length and the second was full length. These clones exhibited some degree of DNA sequence divergence, especially in their 3' noncoding regions, suggesting that they were encoded by separate genes. The nearly full length cDNA was fused in frame to a DNA encoding a signal peptide from alpha wheat gliadin, and expressed in Xenopus oocytes. This yielded a 50 kDa protein that interacted with the antibodies against natural bulb alliinase. Northern and Western blot analyses showed that the bulb alliinase was highly expressed in bulbs, whereas a lower expression level was found in leaves, and no expression was detected in roots. Strikingly, the roots exhibited an abundant alliinase activity, suggesting that this tissue expressed a distinct alliinase isozyme with very low homology to the bulb enzyme.

Amino Acid Sequence↗
PubMed1993

Interaction between trophozoites of Entamoeba histolytica and the human intestinal cell line HT-29 in the presence or absence of leukocytes.

Studies on the interaction between trophozoites of Entamoeba histolytica of pathogenic or non-pathogenic origin and epithelial cells of the human intestine can contribute to the understanding of the pathogenesis of invasive amoebiasis. We have examined the interaction of virulent E. histolytica with the human colonic carcinoma cell line HT-29. Differentiated HT-29 cells are comparable to the mucosa cells to which E. histolytica attaches physiologically. Adherence between E. histolytica trophozoites and HT-29 cells was effectively inhibited by glycoconjugates containing galactose, indicating the importance of the 170-kDa lectin of E. histolytica in binding to intestinal cells. Adherence was not significantly inhibited by glycoconjugates containing N-acetyl-glucosamine, indicating that the 220-kDa lectin of E. histolytica is not involved in binding to HT-29 cells. The destruction of HT-29 cells by pathogenic E. histolytica was dependent on adherence. The destruction was enhanced when polymorphonuclear granulocytes were added to the E. histolytica trophozoites.

Adenocarcinoma↗
PubMed1993

Shigella flexneri transformants expressing type 1 (mannose-specific) fimbriae bind to, activate, and are killed by phagocytic cells.

Shigella flexneri M90T (invasive) and BS176 (noninvasive) are typical nonfimbriated organisms that do not bind to or activate phagocytic cells. We demonstrate that S. flexneri M90Tp and BS176p, obtained by transformation of the strains named above with the cluster of genes encoding type 1 (mannose-specific) fimbriae of Escherichia coli, express the functional fimbriae, as shown by electron microscopy, by binding of antifimbria antibodies and by yeast cell aggregation. The transformants, but not the parental strains, bound to human granulocytes and mouse peritoneal macrophages. This binding was inhibited by methyl alpha-D-mannoside but not by methyl alpha-D-galactoside. The bound bacteria induced oxidative burst activation and degranulation of the granulocytes in vitro. With mouse peritoneal macrophages, the binding of the fimbriated bacteria induced degranulation in vitro. Injection of the bacteria into mouse peritoneum also induced degranulation of the macrophages in vivo; no such effect was observed with the nonfimbriated strains. The bound fimbriated transformants were effectively killed by the human granulocytes in vitro in the absence of opsonins or after opsonization with human anti-S. flexneri antiserum. The nonfimbriated strains were killed only after opsonization. These results provide further evidence for the role of type 1 fimbriae in lectin-mediated nonopsonic phagocytosis.

Animals↗
PubMed1993

Electrophoretic karyotype and chromosome assignments for a pathogenic and a nonpathogenic strain of Entamoeba histolytica.

The electrophoretic karyotypes of a pathogenic and a nonpathogenic strain of Entamoeba histolytica were determined by pulsed-field gel electrophoresis. A number of previously isolated genes were assigned to specific chromosomal bands. Significant differences between the chromosomal patterns of these strains as well as in the assignment of most genes were found.

Animals↗
PubMed1992

A model of interaction between Entamoeba histolytica and Shigella flexneri.

The establishment of a relationship between Entamoeba histolytica and certain bacteria may contribute to the expression and/or enhancement of the pathogenicity of this parasite. Recent experiments have shown that bacteria expressing mannose-binding lectins on their surface could attach to mannose-containing molecules on the surface of amoebae. In this study, we established a model of interaction between. E. histolytica and Shigella flexneri. Using well-characterized mutants of S. flexneri, we studied the role of type I pili expression and the invasive phenotype of S. flexneri in the interaction between amoebae and the bacteria. Type I pili expression allowed attachment and subsequent internalization of S. flexneri by amoebae, these events were not observed in isogenic strains that did not express type I pili. Invasive as well as non-invasive variants of S. flexneri expressing type I pili were slowly digested by amoebae following internalization. Morphological studies showed that the specific features of the interaction depend on the dynamics of the distribution of mannose residues on the amoebic membrane during the interaction.

Animals↗
PubMed1992

Cloning and partial characterization of an antigen detected on membrane surfaces of non-pathogenic strains of Entamoeba histolytica.

A novel and unique mAb (318-28) which specifically interacts with a 60 kDa antigen that is found only on the surfaces of non-pathogenic (NP) strains of E. histolytica has been recently characterized. The antigen appears to be present also on (NP) cyst forms of amebas but was not detected on any of the various (P)-strains tested. It was also not found on other Entamoeba species such as Moshkovskii, Laredo, Huff, Coli, Gingivalis, or Invadens. Clinical trails for the differentiation of (P) and (NP) amebas directly from stools using this mAb are in progress. Cloning of the gene encoding for the (NP)-specific antigen was achieved after screening with mAb 318-28 a lambda gt11 expression library of NP strain SAW 1734R. No sequence homology to any known protein was found in data bases.

Amino Acid Sequence↗
PubMed1992

Effect of hamster liver passage on the isoenzyme patterns of Entamoeba histolytica.

The effect of hamster liver passage on the isoenzyme patterns of isolates of Entamoeba histolytica was investigated. Three isolates, F, G and T were originally obtained from patients with acute amebic dysentery and another strain, C, was obtained from an asymptomatic carrier. All these strains were maintained for over two years in axenic culture. The isoenzyme pattern (zymodeme) of hexokinase (HK), phosphoglucomutase (PGM) and glucose phosphoisomerase (GPI) of these strains was found to belong to non-pathogenic group X, but the isoenzyme pattern of GPI resembled less pathogenic zymodeme XX and might be an intermediate type. Following inoculation of trophozoites into hamster livers and recovery after abscess formation, their isoenzyme pattern changed and revealed that they belonged to pathogenic type XIV. Liver passage caused an enhancement in amebic virulence as evidenced by their increased ability to destroy leukocytes. The results indicate that isoenzyme pattern is not a stable property of E. histolytica.

Animals↗
PubMed1992

Interaction of various Entamoeba histolytica strains with human intestinal cell lines.

The interaction of four pathogenic and three nonpathogenic E. histolytica strains with two human intestinal cell lines (Caco-2 and HT-29) was examined. The adherence of pathogenic and nonpathogenic E. histolytica to these cells was similar, indicating that defective adherence to intestinal cells is not a common feature of nonpathogenic strains. The addition of different carbohydrates confirmed the importance of the galactose-binding lectin of E. histolytica in binding to these intestinal cells. On the other hand, only virulent E. histolytica strains damaged monolayers of intestinal cells. The results indicate that Caco-2 cells and differentiated HT-29 cells are useful models for research of intestinal amebiasis.

Animals↗
PubMed1992

Chromosome walk in Entamoeba histolytica: the gene encoding for ribosomal protein L21 neighbors one of the actin genes.

Actin is one of the most abundant proteins in the motile intestinal protozoan parasite E. histolytica. A number of actin gene copies have been detected. The cDNA and genomic sequences of two of the actin genes have been independently reported (1,2). Almost complete homology was detected between the coding regions of the two genes; however, significant differences were observed in the sequences of their 5' untranslated regions. Using the coding region of actin as the focal point, we performed a chromosome walk to identify the neighboring genes and the intergenic regulatory domains. A genomic library containing large fragments of DNA was screened with the coding and non-coding regions of the actin gene. An insert of 8.5 kb reacted on Northern blots with actin and two additional transcripts. The large (approximately 2.5 kb) transcript has not yet been identified, but the smaller one (600 bp), was shown to encode for the ribosomal protein L21. Both the cDNA and genomic sequences of this gene were determined. The RP-L21 gene was found to be physically connected to the actin gene by a 2.1 kb intergenic stretch. The actin gene on this DNA fragment contained a 5' untranslated region that was identical to the sequence described by Edman et al. The actin gene isolated by Huber et al. was located by PFGE on another chromosomal band that did not contain the RP-L21 gene.

Actins↗
PubMed1991

Cloning and characterization of an unusual elongation factor-1 alpha cDNA from Entamoeba histolytica.

The coding sequence deduced from two overlapping cDNA inserts obtained from a pathogenic strain of Entamoeba histolytica revealed a striking homology (greater than 85%) with elongation factor EF-1 alpha from Saccharomyces cerevisiae and Artemia salina. The deduced amino acid sequence predicted a size of 49 kDa, and antibodies raised against the S. cerevisiae EF-1 alpha cross-reacted with an amoebic protein of similar size (45-47 kDa). Sequence analysis of the cDNA revealed that the 5' untranslated region contained a stretch of 190 nucleotides which was perfectly complementary to a segment of the 3' terminal coding region situated 1015 bases downstream of the methionine initiation codon. Electron microscopy of self-renatured cDNA confirmed the potential of such molecules to form a stem-loop secondary structure. The presence of the complementary sequences was confirmed at the genomic level by sequence analysis of polymerase chain reaction-amplified segments which span both the 3' and 5' terminal complementary regions. Comparison of the deduced amino acid sequence of E. histolytica EF-1 alpha with Ef-Tu from Escherichia coli and EF-1 alpha from different sources, suggested that the major functional domains of the protein are located within the loop structure.

Amino Acid Sequence↗
PubMed1990

Autonomous replication sequences in an extrachromosomal element of a pathogenic Entamoeba histolytica.

Entamoeba histolytica possesses a 24.5 kilobase plasmid-like molecule which encodes for the organism's ribosomal RNAs. Sequence analysis of this extrachromosomal element revealed the presence of AT rich sequences which show homology to the origin of replication of other lower eucaryotes. An 802 bp fragment containing these sequences was cloned into a yeast shuttle vector lacking the origin of replication and the construct tested for its ability to replicate autonomously in yeast. Mitotic stability tests as well as evidence for plasmid maintenance indicate that the transformed cells contained self-replicating episomes and not stably integrated molecules. The nucleotide sequence of this ARS-containing fragment is presented.

Animals↗
PubMed1990

Identification of protein kinase C and its potential substrate in Entamoeba histolytica.

1. Protein kinase C (PKC) activity has been identified in various strains of the human parasite, Entamoeba histolytica. 2. An amoebic protein of mol. wt 78,000 was recognized by polyclonal antibodies raised against the 82,000 mol. wt rat brain protein kinase C. 3. A partially purified PKC preparation from E. histolytica phosphorylated histone I in the presence of calcium, phospholipids and diacylglycerol, and specifically bound tritiated phorbol ester at an apparent KD of 9 nM. 4. A relocalization of the amoebic PKC activity from the cytosol to the membrane fraction was observed when trophozoites were actively phagocytising bacteria. Under these conditions, a labelled phosphoprotein of mol. wt 68,000 was identified. 5. Similar to what was found during macrophage activation, a myristoylated mol. wt 68,000 protein was detected in amoebae grown in the absence of bacteria, but not in amoebae which were active in phagocytosis.

Animals↗
PubMed1990

Specific labeling of cysteine proteinases in pathogenic and nonpathogenic Entamoeba histolytica.

Growth of Entamoeba histolytica trophozoites was inhibited by 50% at low concentrations (2.0 micrograms/ml) of the diazopeptidyl inhibitor benzyloxycarbonyl-leucyl-L-tyrosyldiazomethane (Z-L-Leu-L-Tyr-CHN2). Iodination of the tyrosine residue lowered the growth inhibitory efficacy of the diazopeptidyl inhibitor (50% inhibition, approximately 10 micrograms/ml). However, even at this concentration, practically all of the cysteine proteinase activity of the cells was irreversibly inactivated as shown by fluorescence microscopy with the dipeptide substrate L-Arg-L-Arg-4-methoxy-beta-napthylamide or colorimetrically with azocasein as the substrate. Growth of trophozoites of E. histolytica from various strains, including both pathogenic and nonpathogenic zymodemes, was similarly inhibited. The concentration of inhibitor required to inactivate the proteinase activity of nonpathogenic cells was lower. Lysates from trophozoites grown in the presence of sublethal concentrations of 125I-labeled protease inhibitor (10 micrograms/ml) showed as many as eight radioactive bands by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (molecular sizes, 73, 68, 56, 40, 39, 35, 29, and 27 kilodaltons). Two of these bands (molecular sizes, 29 and 27 kilodaltons) could be seen in gels of the cytoplasmic fraction, whereas the high-molecular-size bands were mostly associated with the membrane fraction. The radioactive bands in pathogenic and nonpathogenic strains were very similar with only minor differences. The results obtained show that E. histolytica cells, irrespective of their pathogenicity, possess a number of cysteine proteinases of similar molecular sizes which are vital for cell growth.

Affinity Labels↗