Partial characterization of a 36-kDa antigen of Entamoeba histolytica and its recognition by sera from patients with amoebiasis

A 36-kDa antigen of axenically grown pathogenic Entamoeba histolytica (HM1-IMSS) was eluted from the sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE)-resolved crude amoebic extract antigens. The immunoreactivity of this partially purified 36-kDa antigen with monoclonal antibody (MoAb) 3D(10) altered significantly (P<0.01) after heat and trypsin treatment but remained unaltered after treatment with sodium metaperiodate (P0.5), thereby indicating the protein nature of the epitope recognized by MoAb 3D(10). The epitope was found to be localized on the surface as well as in the cytoplasm of the E. histolytica trophozoites with the majority of it in the cytoplasm. In addition, this epitope was also found to be present on the cyst form of the parasite. The 36-kDa molecule was recognized by the sera from 29 (85%) of the 34 patients with amoebic liver abscess and five (83%) of the six patients with amoebic colitis. No serum samples from asymptomatic cyst passers, from patients with non-amoebic hepatic or intestinal disorders and apparently healthy subjects had antibodies that reacted with this 36-kDa molecule. The immune responses in man to this 36-kDa amoebic molecule indicate a potential specific role for this molecule in invasive amoebiasis.     

Circular and linear DNA molecules in the Entamoeba histolytica complex molecular karyotype

Entamoeba histolytica genome was analysed by pulsed field gel electrophoresis under conditions to separate linear chromosomes in the 170–1400 kb range. We identified linear DNA molecules of 227, 366, 631, 850, 1112 and 1361 kb (mean sizes obtained by three different methods) and we estimated their reorientation times and migration velocities at various experimental conditions. DNA shift mobility assays, using ethidium bromide, suggested that bands migrating at 227 and 631 kb contain linear and circular DNA, whereas a band at 436 kb has only circular DNA. We obtained a regression equation relating sizes of supercoiled DNA molecules with their migration velocities during a pulse at constant electric field and temperature. We also developed a computer program (EHPATTERNS) that predicts the migration per pulse and the resolution order of circular and linear E. histolytica DNA at different pulse times and constant driving and frictional forces. The simulation showed that linear DNA molecules frequently co-migrate with circular molecules, but circular molecules change when the pulse time varies. This molecular mixture generates broad bands and difficulties in the interpretation of the molecular karyotype of E. histolytica. Received: 19 January 1999 / Revised version: 3 November 1999 / Accepted: 22 November 1999     

Clinical performance of non-radioactive assays for HIV-1 DNA amplified by the polymerase chain reaction

A major factor preventing more widespread use of polymerase chain reaction in the clinical laboratory is the lack of convenient non-radioactive probe hybridization procedures which do not sacrifice sensitivity or specificity. In this report, we describe comparisons of probes labelled with biotin, digoxygenin, alkaline phosphatase, and³²P. We report the comparison of solution or liquid hybridization assay and Southern blotting with digoxygenin-labelled oligonucleotides on a total of 64 clinical specimens. Perfect diagnostic agreement between the³²P and digoxygenin probes was obtained. These data suggest that the non-radioactive assay as described is as sensitive and as specific as the assay with³²P-Iabelled probes.     

Detection of Glycogen-Debranching System in Trophozoites of Entamoeba histolytica

ABSTRACT. Homogenates of trophozoites of Entamoeba histolytica were shown to bring about the total degradation of glycogen while purified phosphorylase of the same source alone yielded a limit dextrin as end product. An enzyme system capable of debranching the limit dextrin was obtained from the 40,000 g pellet by extraction in aqueous medium, purified by gel filtration on Fractogel TSK HW-55(F), and separated from phosphorylase by chromatography on Blue Sepharose CL-6B and aminobutyl Agarose. The glycogen-debranching system was purified 540-fold to a state of homogeneity by criterion of disc-gel electrophoresis. The purified enzyme was able to degrade glycogen-limit dextrin in the presence of phosphorylase and exhibited activities of both amylo-1,6-glucosidase (EC 3.2.1.33) and 4- α -glucanotransferase (EC 2.4.1.25). Although amylo-1,6-glucosidase released glucose from a glycogen-phosphorylase limit dextrin, transferase activity moved single glucose residues from the limit dextrin to 4-nitrophenyl- α -glucoside yielding successively 4-nitrophenyl- α -maltoside and 4-nitrophenyl- α -maltotrioside that could be detected by HPLC. Native glycogen-debranching system exhibited a relative molecular mass of M_r= 180,000 ± 10% by gel filtration and gel electrophoresis in both denaturing and nondenaturating conditions.     

Detection of Chlamydia psittaci using DNA probes and the polymerase chain reaction

Fewer than 10(5) elementary bodies of Chlamydia psittaci could be detected by using DNA hybridisation with a plasmid probe specific for avian chlamydial strains. PCR amplification of chlamydial DNA using primers specific for conserved regions of the major outer membrane protein gene enabled the detection of fewer than 10 elementary bodies. DNA could be amplified from 22 of the 24 chlamydial strains tested including avian, feline, ovine, caprine, koala and lymphogranuloma venereum strains.     

The plasma membrane of Entamoeba histolytica: structure and dynamics.

The plasma membrane components of the parasitic protozoan Entamoeba histolytica, the causative agent of human invasive amebiasis, have been biochemically and immunologically characterized during the last decade. In addition, genes coding for certain surface proteins have been cloned. In spite of these advances, a unified characterization of plasma membrane antigenic components of the parasite is still required for badly needed advancements in the design of useful diagnostic, epidemiologic, and immunoprophylactic tools. Here we review current knowledge on this issue and address the problem of the considerable variation in the electrophoretic profiles of plasma membrane proteins obtained by different groups. In addition, the differences in the degree of recognition of reported membrane antigens with human immune sera, and the diverse interpretations concerning the possible functions of the surface molecules characterized are discussed. A comparative analysis of plasma membrane proteins of E histolytica trophozoites using three different isolation methods revealed that it is possible to select for specific membrane proteins, depending on the lysis conditions. In our view, the method of Calderón and Avila preserves more proteins than other methods tested. Using sera from recent cases of invasive amebiasis studied by several laboratories in various geographical areas, a basic antigenic pattern of 11 principal proteins with molecular weights of 220, 170, 150, 125, 97, 80, 60, 45, 20 and 9 kDA was established for the pathogenic E histolytica strain HM1:IMSS, used by most research groups.     

Chip ligating human genomic DNA serves as storage material and template for polymerase chain reaction

A chip was developed to store DNA for medical research. The optional restriction site fixed on the chip can randomly ligate with whole human genomic DNA treated by the corresponding restriction enzyme. PCR can then use the chip as template DNA. Moreover, a chip fixing two restriction sites (e.g. EcoRI and HindIII) showed the amplification by PCR for any location of genomic DNA. Repetitive PCRs have confirmed that a DNA chip can be stored by at –4 °C for 2 years.     

Detection and identification ofCampylobacter coli andCampylobacter jejuni by two-step polymerase chain reaction

Flagellin gene was used as target sequence to detect and distinguishC. coli andC. jejuni by a “nested PCR” technique. The method shows a high level of sensitivity and specificity. Application of this rapid diagnostic tool could provide further information about epidemiological and pathogenetic implications of each of these two microorganisms.     

Detection of bovine kappa-casein genomic variants by the polymerase chain reaction method

Summary A method is described for identifying variants at the bovine κ-casein locus by combining polymerase chain reaction (PCR) amplification and restriction enzyme digests.     

Detection of Streptobacillus spp. in feral rats by specific polymerase chain reaction

Streptobacillus moniliformis is an etiological agent of rat-bite fever and Haverhill fever in human infection. As the currently available methods for identifying the causative bacteria are not satisfactory, we attempted to establish them by PCR using newly designed primers for the 16S rRNA gene of S. moniliformis. We then determined the prevalence of Streptobacillus spp. in two species of feral rats that inhabit an urban region in Japan, because information on the prevalence of the bacteria in feral rats is obscure. The use of PCR with newly designed primers showed that an extremely high proportion of R. norvegicus harbored the bacteria (61/66, 92%), whereas the prevalence was only 58% in R. rattus (30/52). The nucleotide sequence analysis of the 16S rRNA gene of Streptobacillus spp. isolated from oral swabs of feral rats showed at least two different types of bacteria among isolates from R. norvegicus and R. rattus.     

Microparticles from condensed DNA formed in the process of polymerase chain reaction

DNA microparticle formation in the course of a polymerase chain reaction (PCR) is reported. PCR with gene-specific and partially complementary primers and yeast genomic DNA as a template was shown to yield spherical DNA-composed microparticles as well as their aggregates and conglomerates, along with routine linear DNA. Microparticles were formed at late PCR stages and could be easily identified by the reaction with fluorescently labeled oligonucleotide primers or by staining of the PCR mixture with fluorescent dyes (acridine orange, propidium iodide or DAPI). According to the data of epifluorescent and electron microscopy, the microparticle size varied from 500 nm to 3–4 μm and the particles were multimeric star-shaped spheres or aggregates formed by several fused microspheres. Some properties of the microspheres were studied. It was found that the Mg⁺² cations comprising the PCR buffer played a key role in the formation of microparticles and the stabilization of their structures.     

Entamoeba histolytica: apoptosis induced in vitro by nitric oxide species

Apoptosis has been described in some parasites like Leishmania, Trypanosoma, and Trichomonas. This phenomenon has not been observed yet in Entamoeba histolytica. This work analyzed the in vitro effect of sodium nitroprusside, sodium nitrite and sodium nitrate (NOs) on E. histolytica apoptosis. Parasites incubated for 1h with NOs revealed apoptosis 6h later (95% viability), demonstrated by YOPRO-1, TUNEL, DNA fragmentation and low ATP levels. The caspase inhibitor Z-VAD-FMK inhibited total intracellular cysteine protease activity (CPA) but had no effect on apoptosis. When treated with NOs some amebic functions like complement resistance and hemolytic activity decreased but CPA and erythrophagocytosis remained unchanged. After treatment in vitro with NOs, parasite death was almost complete at 24h; but when injected into hamster livers they disappeared in less than 6h. These results show that apoptosis is induced in vitro by NOs in E. histolytica and renders them incapable of surviving in hamster's livers.     

Computing by polymerase chain reaction

A mathematical notation is introduced to represent, at a symbolic level, different mechanisms of DNA recombination, and a 'PCR lemma' is proven by analytically describing the combinatorial properties of the polymerase chain reaction process. This approach led to the discovery of novel techniques, based on a form of PCR which we called cross pairing PCR (briefly XPCR). They were mathematically analyzed and already experimentally proven in different contexts, such as DNA extraction and recombination. Thus, a mathematical analysis of standard methodologies may highlight novel mechanisms of DNA recombination and this can provide new technologies for DNA manipulation.     

The MAK16 Gene of Entamoeba histolytica and Its Identification in Isolates from Patients

To identify sequences of Entamoeba histolytica associated with the development of amebic liver abscess (ALA) in hamsters, subtractive hybridization of cDNA from E. histolytica HM-1:IMSS under 2 growth conditions was performed: 1) cultured in axenic medium and 2) isolated from experimental ALA in hamsters. For this procedure, 6 sequences were obtained. Of these sequences, the mak16 gene was selected for amplification in 29 cultures of E. histolytica isolated from the feces of 10 patients with intestinal symptoms and 19 asymptomatic patients. Only 5 of the 10 isolates obtained from symptomatic patients developed ALA and amplified the mak16 gene, whereas the 19 isolates from asymptomatic patients did not amplify the mak16 gene nor did they develop ALA. Based on the results of Fisher''s exact test (P<0.001), an association was inferred between the presence of the mak16 gene of E. histolytica and the ability to develop ALA in hamsters and with the patient''s symptoms (P=0.02). The amplification of the mak16 gene suggests that it is an important gene in E. histolytica because it was present in the isolates from hamsters that developed liver damage.     

Elucidation of the DNA Synthetic Cycle of Entamoeba Spp. Using Flow Cytometry and Mathematical Modeling

ABSTRACT. We developed a method to study the DNA synthetic cycles of Entamoeba histolytica and Entamoeba invadens by flow cytometry (FCM) based on a preparative procedure to reduce both high levels of natural fluorescence and non-specific adsorption of fluorochromes. We modeled G₁, S, and G₂ phases as a series of overlapping Gaussian curves. Both E. histolytica and E. invadens displayed G₁, S, and G₂ proportions that are consistent with eukaryotic cell populations in exponential or stationary growth phase. Exponential phase E. histolytica populations contained a hypodiploid subset with a mass of about 20% less than the diploid value which we estimate by FCM to be 24 × 10^-14 g DNA/cell. Exponential phase E. invadens populations contained a hypodiploid subset with a mass of about 6% less than the diploid value which we estimate by FCM to be 30 × 10^-14 g DNA/cell.     

Diagnosis of bovine freemartinism by the polymerase chain reaction method

Summary. Using sex chromosome specific primers, leucocyte chimaerism in heterosexual bovine female twins was identified by combining polymerase chain reaction (PCR) and restriction enzyme digests.     

Extraction of DNA from exfoliative cytology specimens and its suitability for analysis by the polymerase chain reaction

The extraction of DNA from archival exfoliative cytology samples would allow the molecular biological analysis of this readily available material using the polymerase chain reaction (PCR). We have quantitatively and qualitatively studied the extraction of DNA from a variety of cytological preparations. For both fresh and archival cervical smears, overnight incubation with proteinase K produces high yields of high molecular weight DNA, but simply boiling the samples produces DNA suitable for PCR amplification of a single copy gene. Increasing the proteinase K incubation to several days allows the extraction of DNA from fixed and stained archival cytology slides from a variety of sites. The extracted DNA was again suitable for PCR analysis. Fresh and archival cytological material can be utilized for molecular biological study of disease processes using PCR. Archival cytological material is probably the best source of DNA and RNA after stored frozen tissue.