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D Mirelman

Publications and source records attributed to D Mirelman.

At least 73 records · Page 4Linked to original sources

Repetitive DNA elements characteristic of pathogenic Entamoeba histolytica strains can also be detected after polymerase chain reaction in a cloned nonpathogenic strain.

Strains of Entamoeba histolytica which were isolated from symptomatic patients and which possess a characteristic pathogenic isoenzyme pattern (zymodeme) have extrachromosomal circular DNA molecules containing RNA genes and clusters of tandemly reiterated PvuI elements. The nucleotide sequence of comparable reiterated BamHI elements present in amebae with nonpathogenic zymodemes differs from that found in pathogenic ones. By using the polymerase chain reaction, it was demonstrated that the cloned, nonpathogenic E. histolytica strain SAW 1734R clAR also contains one or few of the tandemly repeated DNA PvuI elements characteristic of the pathogenic amebae. Sequences were detected by hybridization with the P-145 probe after in vitro amplification. Because of technical difficulties, it was impossible to resolve whether single copies of the nonpathogenic BamHI repetitive elements are present in pathogenic amebae. Our findings suggest that in the nonpathogenic amebae, the signal to start amplifying the PvuI-type elements may be induced during the process of elimination of bacterial associates from their growth environment.

Animals↗

Pathogenic and nonpathogenic strains of Entamoeba histolytica can be differentiated by monoclonal antibodies to the galactose-specific adherence lectin.

Entamoeba histolytica infection results in either asymptomatic colonization or invasive colitis and liver abscess. E. histolytica isolates from patients with invasive disease have characteristic isoenzyme profiles (pathogenic zymodemes), suggesting a role for parasite factors in determining the severity of infection. A galactose-specific cell surface lectin from a pathogenic zymodeme was shown to mediate in vitro adherence to human colonic mucins and contact-dependent killing of target cells. Six nonoverlapping antigenic determinants were identified on the 170-kilodalton heavy subunit of the pathogenic lectin. Anti-lectin monoclonal antibodies (MAb) directed against epitopes 1 and 2 enhanced adherence whereas MAb to epitopes 3 through 6 either inhibited or had no effect on adherence. We tested 50 pathogenic and nonpathogenic strains for reactivity to these anti-lectin MAb by radioimmunoassay. MAb to epitopes 1 through 6 reacted in the radioimmunoassay with all 16 pathogenic zymodeme strains tested. In contrast, only MAb to epitopes 1 and 2 bound to the lectin from nonpathogenic strains. Western immunoblots with anti-lectin antibodies showed that the 170-kilodalton heavy subunit was present in the nonpathogenic amebae. Adherence of the nonpathogenic SAW 760 strain to human erythrocytes was enhanced by MAb to epitope 1 and blocked by galactose, confirming the presence of a functionally active lectin. A lectin radioimmunoassay based on MAb to epitopes 1 and 3 proved to be a simple and rapid method to distinguish pathogenic from nonpathogenic amebae in culture. Further exploration of the functional consequences of the antigenic differences demonstrated for the lectin may lead to a better understanding of its role in pathogenesis.

Animals↗

Differentiation of clinical isolates of Entamoeba histolytica by using specific DNA probes.

Most individuals infected with Entamoeba histolytica are reported to be clinically asymptomatic. On the basis of the electrophoretic migration of hexokinase and phosphoglucomutase isoenzymes, two groups of E. histolytica isolates have been classified. Those derived from symptomatic cases were found to have fast-migrating hexokinase bands and were labeled pathogenic. The others, isolated from cyst passers, had (in most cases) slow-migrating bands and were called nonpathogenic. Differences between these two groups of E. histolytica were found recently at the DNA level. Two sets of different DNA probes derived from tandemly repeated sequences present in extrachromosomal circular DNA elements in each group of E. histolytica were characterized. Using these probes with procedures for direct hybridization of trophozoites on nylon membranes, we could correctly correlate hexokinase electromobility with the DNA hybridization signal of 81 different isolates of E. histolytica. The advantages of using DNA probes lie in their sensitivity (fewer than 200 trophozoites can be detected) and specificity. The probes hybridized only with amebae from the E. histolytica species and not with other enteric protozoa and can be useful as a diagnostic tool.

Animals↗

Entamoeba histolytica ribosomal RNA genes are carried on palindromic circular DNA molecules.

Highly abundant DNA fragments obtained after restriction enzyme digests of nuclear DNA of Entamoeba histolytica strain HM-1:IMSS have been cloned and characterized. Northern blot hybridization to E. histolytica rRNA and sequence analysis identified the abundant DNAs as ribosomal DNA containing species. Several overlapping clones containing these abundant DNAs were isolated from 4 different genomic libraries of E. histolytica. Alignment of the restriction maps was consistent with a circular molecule, about 24.6 kilobase pairs (kb) in size. Nuclease BA131 digestion provided additional evidence for the circular nature of this DNA. The ribosomal DNA molecule contains two large inverted repeat-regions, each at least 5.2 kb in length. Sequence analysis of clone R715 revealed homology to the large rRNA units of various eukaryotic organisms. This clone was located in both inverted repeats, suggesting two rRNA cistrons per molecule. The inverted repeats are flanked by stretches of DNA which contain tandemly reiterated sequences. Southern blot analysis of E. histolytica nuclear DNA revealed the presence of two populations of molecules. These molecules have identical arrangements of restriction sites, but differ in size (0.7 kb) in a fragment containing tandemly reiterated sequences. Analysis of E. histolytica nuclear DNA by electron microscopy also revealed circular molecules. These molecules are about 26.6 kb +/- 0.5 kb in size and contain structural features predicted by the restriction map of the extrachromosomal ribosomal DNA of E. histolytica.

Animals↗

Effects of covalently bound silica-nitroimidazole drug particles on Entamoeba histolytica.

Most commonly used antiamoebic drugs are effective in invasive amebiasis, but their response against trophozoites of Entamoeba histolytica, present in the lumen of the human colon, is inadequate. We report the development of an antiamoebic drug carrier that may be effective against luminal infections. Our preparation consists of small silica particles (5-10 microns in diameter) covalently linked to a potent antiamoebic drug, 2-(4-aminophenoxymethyl)-5-nitro-1-methyl imidazole. Silica-drug particles were injected into mice, hamsters, and guinea pigs. We found that trophozoites phagocytosed the particles in vivo and in vitro, followed by rapid cell death due to the released drug. Analysis of mouse serum revealed that no drug was absorbed from the intestine after placement of the drug-containing particles in the intestine. The antiamoebic activity of particles recovered from the intestine was almost fully retained. This novel antiamoebic concept may be useful for luminal therapy for asymptomatic amebiasis and may minimize side effects and frequency of administration.

Amebicides↗

DNA probes specific for Entamoeba histolytica possessing pathogenic and nonpathogenic zymodemes.

A number of DNA probes which hybridize to highly abundant DNA sequences of Entamoeba histolytica were developed. Variations in the hybridization patterns of different E. histolytica strains were detected with selected probes. Four types of restriction fragment length patterns were obtained. Of these, the first class belonged to E. invadens and E. histolytica-like var. Laredo. The next two classes consisted of various strains of E. histolytica which were originally isolated from symptomatic patients and possessed pathogenic patterns of isoenzymes (zymodemes), whereas the fourth group contained E. histolytica strains with nonpathogenic zymodemes obtained from asymptomatic carriers. DNA probes, based on DNA sequences specific to E. histolytica isolates with pathogenic and nonpathogenic zymodemes were isolated, and their nucleotide sequences were determined. These probes (P145 and B133) hybridized selectively to DNA of isolates possessing either pathogenic or nonpathogenic isoenzyme patterns. The newly developed probes could be useful for diagnostic purposes and could serve as tools to investigate the molecular basis of pathogenicity and the genetic mechanisms which regulate the variable aggressive behavior of the parasite.

Animals↗

Entamoeba histolytica: virulence potential and sensitivity to metronidazole and emetine of four isolates possessing nonpathogenic zymodemes.

The pathogenic potential of four Entamoeba histolytica isolates obtained from asymptomatic carriers and possessing nonpathogenic zymodemes was compared to four E. histolytica strains obtained from invasive cases of amebiasis and having pathogenic zymodemes. Both xenic and axenic cultures of a number of strains were tested. Determinations of cytopathogenicity were done in vitro by measuring the rates of destruction of tissue cultured monolayers of baby hamster kidney cells by intact amebae or by its cell-free extracts. The in vivo virulence was tested by assessing their capacity to form hepatic abscesses in hamsters or cecal ulcerations in rats. The results obtained show that two of the isolates from asymptomatic carriers (strains SAW 1734R clAR and WI:0385:191) were as virulent as three of the invasive ones (HM-1:IMSS, 200:NIH, and SAW 408). Two other isolates from asymptomatic carriers and one from a dysentery case were avirulent. All the E. histolytica isolates tested were similarly sensitive to metronidazole and emetine (IC50 1-10 micrograms/ml). The results indicate that the pathogenic potential of E. histolytica varies between isolates and can be affected by culture conditions and by the presence or absence of bacterial cells. These findings suggest that virulence does not necessarily correlate with a pathogenic zymodeme.

Amebiasis↗

Nucleotide sequence analysis of an Entamoeba histolytica ferredoxin gene.

A cDNA clone (subclone B) previously isolated from the human parasite Entamoeba histolytica was characterized. DNA sequence analysis of subclone B identified the DNA as that encoding apoferredoxin. E. histolytica ferredoxin cDNA contains unusually short 5' and 3' noncoding regions of 9 and 25 nucleotides, respectively. A genomic ferredoxin clone was isolated from E. histolytica DNA, and comparison of genomic and cDNA sequences revealed that the ferredoxin gene is unspliced. The deduced amino acid sequence of E. histolytica ferredoxin resembles clostridial type of ferredoxins, and shows an arrangement of cysteines characteristic for the coordination of 2[4Fe-4S] centres. Of interest is the absence of an aromatic amino acid in the N-terminal region of the protein, a feature which is conserved in clostridial ferredoxins. Southern blot analysis of three different E. histolytica strains (200:NIH, Rahman and HM-1:IMSS) demonstrated the presence of a family of at least two ferredoxin genes. One of these genes is marked by restriction length polymorphisms in different strains of E. histolytica.

Amino Acid Sequence↗

Appearance of sialoglycoproteins in encysting cells of Entamoeba histolytica.

Amoeba-bacterium cultures of Entamoeba histolytica transferred to a hypoosmotic medium depleted of nutrients changed morphologically and biochemically. The cells ejected grains of rice starch, rounded up, and formed a distinct cell wall that was resistant to detergent, bound the sialic acid-specific lectin from Limulus polyphemus, and became fluorescent with Calcofluor M2R. A subpopulation of these cells displayed more than one nucleus. All these signs are characteristic of encysting cells and were also observed in cysts obtained from a human patient. The morphological changes were accompanied by the appearance of two new glycoproteins with apparent molecular sizes of 100 and 150 kilodaltons which contained sialic acid. Sialic acid has been reported to be absent from trophozoites of Entamoeba species. The presence of this sugar residue on cyst-specific proteins parallels recently reported findings during the encystation of the related reptilian parasite Entamoeba invadens. This may indicate a basic role for sialic acid in the encystation of Entamoeba parasites.

Animals↗

Isolation and partial characterization of the hexokinase isoenzymes from pathogenic and non-pathogenic strains of Entamoeba histolytica.

Isoenzyme electrophoretic patterns (zymodemes) are increasingly used to distinguish between pathogenic and non-pathogenic strains of Entamoeba histolytica. Isolates of E. histolytica from asymptomatic and symptomatic cases have been shown to differ in the electrophoretic mobility of their hexokinase and phosphoglucomutase isoenzymes. The hexokinase isoenzymes from a non-pathogenic strain and from a pathogenic strain of E. histolytica were purified by fast protein liquid chromatography in several steps, which included a separation by size, chromatofocusing, and anion exchange chromatography. The isoenzymes differed in their isoelectric points, which ranged from pH 4.8-5.4, but had very similar kinetic properties and almost identical apparent molecular weights (48,000) in sodium dodecyl sulfate polyacrylamide gels, as well as on gel filtration columns. Comparison of tryptic peptide analysis of each of the isoenzymes indicated considerable homology between the non-pathogenic and pathogenic forms. Antibodies produced against each of the two pathogenic hexokinase isoenzymes inhibited their enzymatic activity. The antibodies also inhibited the activity of the isoenzymes of the non-pathogenic strain. Our findings suggest that the isoenzymes have structural similarities, and that the pathogenic ones differ from the non-pathogenic ones in their electromobility due to post-translational modifications.

Animals↗

A stage-specific sialoglycoprotein in encysting cells of Entamoeba invadens.

A novel sialoglycoprotein with an apparent molecular mass of approximately 250 kDa was detected on the surface of cysts of Entamoeba invadens. Sialic acid was identified in this glycoprotein by gas chromatography after methanolysis; N-acetyl- and N-glycolyl neuraminic acid were identified by thin layer chromatography in hydrolysates of partially purified preparations of the 250 kDa glycoprotein as well as in whole cysts. The sialoglycoprotein is stage-specific and could be detected by binding of wheat germ agglutinin and a specific monoclonal antibody (JAM3) only to precysts and mature cysts but not to trophozoites. A 250 kDa protein could be metabolically labeled with [35S]methionine. This, together with the absence of such a glycoprotein in the encystation medium, suggests that the 250 kDa sialoglycoprotein is not an adsorbed serum glycoprotein. Indirect evidence suggests that the parasite may utilize serum components as a source for sialic acid.

Animals↗

Entamoeba histolytica: cloning and characterization of actin cDNA.

In order to study gene expression in the human parasite Entamoeba histolytica, a cDNA library of E. histolytica strain 200:NIH was constructed using the phage vector lambda gt10. Three cDNA clones (A, B and C) were selected for further analysis. Each of the three clones hybridized to a distinct mRNA. Two of these mRNAs were translated in vitro after hybrid selection, and yielded distinct translation products. One of these mRNAs, selected by hybridization to clone A, encodes the most abundantly expressed protein in E. histolytica. DNA sequence analysis of this cDNA clone identified the DNA as that encoding actin. The deduced amino acid sequence of E. histolytica actin resembles both cytoplasmic and muscle actins and has an unusual N-terminal glycine residue. We have shown that a family of actin genes is present in E. histolytica. Six different E. histolytica actin clones were obtained from a lambda gt10 genomic library using subcloned cDNA probes. Southern analysis of three different E. histolytica strains (200:NIH, Rhaman, and HM-1:IMSS) revealed at least four different actin genes. Strain HM-1:IMSS, however, differs by the presence of an additional actin gene.

Actins↗

Aromatic alpha-glycosides of mannose are powerful inhibitors of the adherence of type 1 fimbriated Escherichia coli to yeast and intestinal epithelial cells.

Adherence of bacteria via their surface lectins to host epithelial cells is considered an important initial event in bacterial pathogenesis. Mannose-specific (type 1) fimbriae are among the most commonly found lectins in enterobacteria. We studied the effect of aromatic alpha-glycosides of mannose on the agglutination of mannan-containing yeasts by different strains of Escherichia coli and on the adherence of the bacteria to guinea pig ileal epithelial cells. In both systems these compounds were considerably more effective inhibitors than methyl alpha-mannoside, with 4-methylumbelliferyl alpha-mannoside and p-nitro-o-chlorophenyl alpha-mannoside being the strongest inhibitors. Both compounds were approximately 400-times stronger inhibitors of yeast agglutination by E. coli O128 than was methyl alpha-mannoside and 1,000- and 470-fold stronger, respectively, than was methyl alpha-mannoside in inhibiting the adherence of the bacteria to ileal epithelial cells. 4-Methylumbelliferyl alpha-mannoside was 540 to 1,000 times more effective in inhibiting yeast agglutination by four additional strains of mannose-specific E. coli. It was also more efficient than methyl alpha-mannoside in removing adherent E. coli O128 from ileal epithelial cells. Our results provide further evidence that type 1 fimbriae of E. coli possess a hydrophobic region next to the mannose-binding site. The results suggest that 4-methylumbelliferyl alpha-mannoside and p-nitro-o-chlorophenyl alpha-mannoside are good candidates for the design of therapeutic agents that may prevent adherence in vivo and infection by E. coli strains that express type 1 fimbriae.

Agglutination↗

Nonimmunoglobulin fraction of human milk inhibits the adherence of certain enterotoxigenic Escherichia coli strains to guinea pig intestinal tract.

The protecting effect of human milk against intestinal infections has been well documented, but its mechanism not completely understood. We have examined the effect of the nonimmunoglobulin fraction (NIgF) of human milk and colostrum on bacterial adherence to the intestinal tract. The NIgF was prepared by passing the milk through an immunosorbent column containing rabbit antihuman gamma-globulin (IgG and IgA). The effluent fraction did not contain gamma-globulins as shown by immunodiffusion on agarose and by using rabbit antihuman Ig, that was then detected with fluorescently-labeled goat antirabbit Ig. The effect of the NIgF of human milk on the adherence of enterotoxigenic Escherichia coli strains to guinea pig intestinal tract was quantitatively determined using radiolabeled bacteria which were incubated with suspensions of viable intestinal cells. Thirteen to 17 bacteria adhered per intestinal cell. NIgF of human milk and colostrum (300 microliter, 6.7 mg) caused about 50% inhibition of the adherence of enterotoxigenic E. coli strains whose attachment was mediated by colonization factor antigen I and II. No inhibition was noted on the adherence of enterotoxigenic E. coli strains containing type I pili. The inhibitory activity resisted boiling and proteolytic digestion with trypsin, but was completely abolished by periodate treatment, indicating that carbohydrate residues were probably involved. Examination of the effect of NIgF of human milk on bacterial adherence to intact intestinal surfaces revealed comparable results. Observations with scanning electron microscopy confirmed, morphologically, the attachment of the bacteria and the inhibitory effect of human milk.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗

Entamoeba histolytica: effect of growth conditions and bacterial associates on isoenzyme patterns and virulence.

In xenic culture, isolates of Entamoeba histolytica from asymptomatic carriers are characterized, with rare exception, by possession of a nonpathogenic zymodeme. During the process of axenizing such an isolate, strain CDC:0784:4, a change in the pattern of the isoenzymes from nonpathogenic zymodeme I to pathogenic zymodeme II was observed 40 days after the amebae had been transferred from a medium for xenic cultivation to one used for axenic cultivation, but before axenization of the amebae had actually occurred. Axenization was accomplished by feeding the amebae lethally irradiated bacteria while suppressing and finally eradicating with antibiotics the bacterial flora accompanying the amebae in the original xenic culture. The change in zymodeme was accompanied by a change in virulence as evidenced by the ability of the amebae to produce hepatic abscesses in hamsters and to destroy monolayers of tissue culture cells. Two explanations are offered for the observed changes in zymodeme and virulence: a zymodeme is not a stable inherent property of the ameba. Alternatively, the original isolate consisted of two zymodeme populations and the conditions of growth selected for one or the other of the populations. In either case, our results suggest that the finding of a particular zymodeme in a culture of E. histolytica isolated from an asymptomatic carrier of the parasite cannot be used to predict a clinical condition or serve as a basis for the recommendation of therapy.

Amebiasis↗