RESUMO
Several density gradients were tested for the isolation of parasitic nematode, Angiostrongylus costaricensis, first-stage larvae from rodent feces. With a 45/72% Percoll gradient, 83-99% (89.56+/-6.57%) of the larvae were recovered in a clean preparation.
Assuntos
Angiostrongylus/isolamento & purificação , Fezes/parasitologia , Doenças dos Roedores/parasitologia , Infecções por Strongylida/veterinária , Animais , Centrifugação com Gradiente de Concentração , Coloides , Larva , Camundongos , Camundongos Endogâmicos C3H , Povidona , Dióxido de Silício , Infecções por Strongylida/parasitologiaRESUMO
Protozoan cysts were isolated according to a two-step sucrose gradient procedure. Pure cysts of Entamoeba histolytica, in fixed and native states, were injected into BALB/c mice intraperitoneally for immunization. The spleens of these animals were used for fusion with AG8 mouse myeloma cells. Hybridomas were obtained and tested for the recognition of E. histolytica, E. dispar, E. coli, E. hartmanni, Endolimax nana, Jodamoeba biitschlii, and Giardia lamblia. Three monoclonal antibodies were identified that reacted only with cysts and trophozoites of E. histolytica. These can be used for differentiation and identification of E. histolytica in feces.
Assuntos
Anticorpos Monoclonais/imunologia , Anticorpos Antiprotozoários/imunologia , Entamoeba histolytica/imunologia , Entamebíase/parasitologia , Fezes/parasitologia , Animais , Anticorpos Monoclonais/biossíntese , Anticorpos Antiprotozoários/biossíntese , Especificidade de Anticorpos , Western Blotting , Reações Cruzadas , Endolimax/imunologia , Endolimax/isolamento & purificação , Entamoeba/classificação , Entamoeba/imunologia , Entamoeba histolytica/isolamento & purificação , Entamebíase/diagnóstico , Técnica Indireta de Fluorescência para Anticorpo , Giardia lamblia/imunologia , Giardia lamblia/isolamento & purificação , Humanos , Hibridomas , Imunoglobulina G/biossíntese , Imunoglobulina G/classificação , Imunoglobulina G/imunologia , Camundongos , Camundongos Endogâmicos BALB CRESUMO
Blastocystis hominis isolates from asymptomatic carriers and symptomatic patients were cultured in vitro, purified from the co-cultivated bacterial flora and tested for cytopathic effects on monolayers of Chinese Hamster Ovary (CHO) cells and Adeno Carcinoma HT29 cells. In the case of the CHO cells, living B. hominis cells and B. hominis cell lysates were able to cause significant cytopathic effects, which were dependent on the concentration of cells employed. Destruction of the cell monolayers was observed to the same extent with patient isolates derived from healthy or symptomatic B. hominis carriers. HT29 cells were less susceptible: B. hominis cells and cell lysates caused only minor effects which were not statistically significant. Culture filtrates of B. hominis exhibited cytopathic potential on CHO and HT29 cells; however, the control which consisted of filtrates from Robinson's cultures in which B. hominis failed to grow showed similar effects, too. Therefore the culture supernatants could not be proven to produce a specific cytopathic effect on CHO and HT29 cells.
Assuntos
Blastocystis hominis/patogenicidade , Adenocarcinoma/parasitologia , Adenocarcinoma/patologia , Animais , Blastocystis hominis/crescimento & desenvolvimento , Blastocystis hominis/isolamento & purificação , Células CHO/parasitologia , Células CHO/patologia , Linhagem Celular Transformada , Sobrevivência Celular , Cricetinae , Cricetulus , Células HT29/parasitologia , Células HT29/patologia , HumanosRESUMO
DNA polymorphism of Blastocystis hominis isolates was examined by the amplification of a gene fragment coding for the 16S-like rDNA. Using identical primers, fragments of approximately 850 bp were amplified from 110 B. hominis isolates and fragments of 1.1 kbp were amplified from 48 isolates. Digestion of the amplification products with the restriction enzymes HinfI, RsaI, and AluI revealed different profiles for each fragment length. Subgroup I and II, resulting from digestion of the smaller 850 bp fragment, have identical HinfI and AluI restriction bands, subgroup III and IV have identical RsaI fragments after digestion of the 1.1 kbp DNA. Subgroup V resembles subgroup III in a few bands after the RsaI and AluI restriction, respectively. Ninety (54%) of the isolates studied were assigned to subgroup I, 20 (12%) to subgroup II, 35 (21%) to subgroup III, 12 (7%) to subgroup IV, and 1 (1%) to subgroup V. Five (3%) of the examined people were coinfected with B. hominis of the subgroup III, 3 (2%) carried B. hominis of the subgroup I and II. These results show that there are 5 B. hominis subgroups none of which was found to be significantly correlated with the reported disease.
Assuntos
Infecções por Blastocystis/parasitologia , Blastocystis hominis/genética , DNA de Protozoário/análise , RNA Ribossômico 16S/genética , Animais , Sequência de Bases , Blastocystis hominis/classificação , Clonagem Molecular , Fezes/parasitologia , Genes de Protozoários , Humanos , Epidemiologia Molecular , Dados de Sequência Molecular , Reação em Cadeia da Polimerase , Polimorfismo de Fragmento de Restrição , Alinhamento de Sequência , Análise de Sequência de DNARESUMO
Entamoeba histolytica and E. dispar detected in fecal samples of German travelers and residents of tropical and subtropical countries were cultured and differentiated by the polymerase chain reaction (PCR) and hexokinase isoenzyme typing. Twenty-one of 60 isolates were identified as E. histolytica. It was shown that 13 of 18 German short-term travelers were infected with E. histolytica, whereas only three of 22 of the examined inhabitants of tropical countries and five of 20 German long-term travelers harbored E. histolytica. It appears that short-term travelers represent a high risk group for infection with the pathogen E. histolytica.
Assuntos
Doenças Endêmicas , Entamoeba histolytica/classificação , Entamoeba/classificação , Entamebíase/epidemiologia , Entamebíase/parasitologia , Viagem , Animais , Anticorpos Antiprotozoários/sangue , Entamoeba histolytica/imunologia , Ensaio de Imunoadsorção Enzimática , Fezes/parasitologia , Alemanha , Hexoquinase/análise , Humanos , Isoenzimas/análise , Reação em Cadeia da PolimeraseRESUMO
Twenty-one Entamoeba histolytica and 56 Entamoeba dispar patient isolates were investigated for their sensitivity to the classical and alternative pathway of human complement, E. histolytica and E. dispar patient isolates were differentiated by polymerase chain reaction and hexokinase isoenzyme typing. It was found that 90.3% (+/- 12.0%) of the trophozoites of E. histolytica were lysed after 30 min by the alternative pathway of complement in the presence of 50% human serum (19 isolates showed lysis rates higher than 80%), whereas E. dispar cells were less susceptible to the alternative pathway as 68.8% (+/- 28.2%) of lysis occurred. However, 23 of the E. dispar isolates were lysed between 100 and 80% (90.9% +/- 9.1%), demonstrating that about half of the tested E. dispar isolates were highly sensitive to complement lysis. Only 11 of the E. dispar isolates were proven to be ¿resistant' to the alternative pathway of complement and were lysed less than 40%. These results are in conflict to earlier publications, describing resistance of E. dispar to complement lysis (Hamelmann et al. 1992, 1993).
Assuntos
Proteínas do Sistema Complemento/imunologia , Entamoeba histolytica/imunologia , Entamoeba/imunologia , Entamebíase/imunologia , Animais , Entamoeba/classificação , Entamoeba/genética , Entamoeba/isolamento & purificação , Entamoeba histolytica/classificação , Entamoeba histolytica/genética , Entamoeba histolytica/isolamento & purificação , Entamebíase/parasitologia , HumanosRESUMO
Isolates of Blastocystis hominis derived from patients with intestinal symptoms and from healthy carriers were cultured in vitro and isoenzyme patterns of hexokinase (E.C. 2.7.1.1), phosphoglucomutase (E.C. 2.7.5.1) and glucose phosphate isomerase (E.C. 5.3.1.9) were investigated to find evidence for pathogenic and non-pathogenic subspecies of B. hominis. For this purpose we examined 2000 patients of the out-patient department of the Institute for Tropical Medicine in Tübingen. From these, we obtained 232 B. hominis patient isolates; 119 isolates could be tested for the three isoenzymes. Blastocystis hominis possesses 5 patterns for hexokinase, 11 for phosphoglucomutase and 35 for glucose phosphate isomerase, showing that B. hominis is highly polymorphic. However, there was no correlation between isoenzyme patterns and disease of patients.
Assuntos
Infecções por Blastocystis/parasitologia , Blastocystis hominis/enzimologia , Portador Sadio/parasitologia , Isoenzimas/isolamento & purificação , Animais , Blastocystis hominis/isolamento & purificação , Blastocystis hominis/patogenicidade , Fezes/parasitologia , Alemanha , Glucose-6-Fosfato Isomerase/isolamento & purificação , Hexoquinase/isolamento & purificação , Humanos , Fosfoglucomutase/isolamento & purificação , Viagem , VirulênciaRESUMO
A new method for the purification of protozoan cysts from feces was established, allowing to isolation of native cysts. The procedure consists of two sucrose-density gradients and enzymatic digestion of cellulose particles by cellulase and can be accomplished in a few hours. The cyst fractions were differentiated into Entamoeba histolytica and E. dispar using the DNA probes P145 and B133 and a dot-blot test.
Assuntos
Entamoeba histolytica/isolamento & purificação , Entamoeba/isolamento & purificação , Fezes/parasitologia , Animais , Celulose/análise , Centrifugação com Gradiente de Concentração , Cistos , Sondas de DNA , DNA de Protozoário/análise , Entamoeba/classificação , Entamoeba histolytica/classificação , Entamebíase/parasitologia , Humanos , MétodosRESUMO
One of the three cysteine proteinase genes, ACP1 (or CP 3), has been reported to be missing in non-pathogenic strains of Entamoeba histolytica (or Entamoeba dispar as recently labeled). Unexpectedly, a gene fragment very similar in its sequence (95% homology) to ACP1 of pathogenic strains was obtained by use of the polymerase chain reaction from genomic DNA and cDNA of various cloned non-pathogenic strains as well as in 23 clinical isolates from asymptomatic carriers. The finding of the ACP1 homologue in non-pathogenic or E. dispar strains rules out the proposed use of its absence for diagnostic purposes.
Assuntos
Cisteína Endopeptidases/genética , Entamoeba histolytica/enzimologia , Entamoeba histolytica/genética , Entamoeba/enzimologia , Entamoeba/genética , Genes de Protozoários , Sequência de Aminoácidos , Animais , Sequência de Bases , Primers do DNA/genética , DNA de Protozoário/genética , Entamoeba/patogenicidade , Entamoeba histolytica/patogenicidade , Dados de Sequência Molecular , Reação em Cadeia da Polimerase , Homologia de Sequência de Aminoácidos , Homologia de Sequência do Ácido Nucleico , Especificidade da EspécieRESUMO
The Rz lysis gene of bacteriophage lambda was cloned into the expression vectors, pT7-3 and pT7-7. The recombinant plasmids expressed either a protein of an unexpected 6.5-kDa size (pT7-3H and pSB54) or two proteins of 6.5 and 17.2 kDa (pBH21). The 6.5-kDa protein alone did not complement the lysis defect of the lambda Rz mutant; hence, this protein was not the Rz gene product. Complementation observed as a result of pBH21 expression thus can be ascribed to the 17.2-kDa protein, which agrees with the size based on the nucleotide sequence of Rz. The 6.5 kDa is a product of an open reading frame entirely encompassed within the Rz sequence and denoted by us Rz1. Both proteins were detectable only by autoradiography, which may mean that the genes are expressed at low rates. Polyclonal anti-Rz antibodies (Ab) were obtained by rabbit immunization with a synthetic polypeptide corresponding to an antigenic determinant of Rz defined by a computer program. The Ab reacted with the 17.2-kDa protein resulting from pBH21 expression, as well as with the 17.2-kDa protein present in the induced Escherichia coli W3350(lambda cI857Sam7) lysate.
Assuntos
Bacteriófago lambda/genética , Genes Virais , Proteínas Virais/genética , Proteínas Estruturais Virais/genética , Sequência de Aminoácidos , Sequência de Bases , Clonagem Molecular , Códon , Escherichia coli/genética , Expressão Gênica , Teste de Complementação Genética , Lisogenia , Dados de Sequência Molecular , Peso Molecular , Fases de Leitura Aberta , Proteínas Virais/imunologiaRESUMO
The localization of the major autolytic enzyme, the soluble lytic transglycosylase, in the different cell compartments of Escherichia coli was investigated by immunoelectron microscopy. Ultrathin sections were labeled with a specific antiserum against purified soluble lytic transglycosylase, and the antibody-enzyme complexes were visualized with colloidal protein A-gold. A preferential localization of the lytic transglycosylase in the envelope was observed, with only 20 to 30% of the enzyme left in the cytoplasm. Most of the enzyme associated with the cell wall was tightly bound to the murein sacculus. Sacculi prepared by boiling of cells in 4% sodium dodecyl sulfate could be immunolabeled with the specific antiserum, indicating a surprisingly strong interaction of the lytic transglycosylase with murein. The enzyme-substrate complex could be reconstituted in vitro by incubating pronase-treated, protein-free murein sacculi with purified lytic transglycosylase at 0 degrees C. Titration of sacculi with increasing amounts of enzyme indicated a limiting number of binding sites for about 1,000 molecules of enzyme per sacculus. Ruptured murein sacculi obtained after penicillin treatment revealed that the enzyme is exclusively bound to the outer surface of the sacculus. This finding is discussed in the light of recent evidence suggesting that the murein of E. coli might be a structure of more than one layer expanding by inside-to-outside growth of patches of murein.
Assuntos
Escherichia coli/enzimologia , Glicosiltransferases , Transferases/metabolismo , Compartimento Celular , Escherichia coli/ultraestrutura , Imuno-Histoquímica , Peptidoglicano/metabolismo , Solubilidade , Transferases/imunologiaRESUMO
Specific localization of the lysis (L) protein of bacteriophage MS2 in the cell wall of Escherichia coli was determined by immunoelectron microscopy. After induction of the cloned lysis gene, the cells were plasmolyzed, fixed, and embedded in either Epon or Lowicryl K4M. Polyclonal L-protein-specific antiserum was purified by preabsorption to membranes from cells harboring a control plasmid. Protein A-gold was used to label the protein-antibody complexes. Between 42.8% (Lowicryl) and 33.8% (Epon) of the label was found in inner and outer membranes, but 30.3% (Lowicryl) and 32.8% (Epon) was present mostly in clusters in the adhesion sites visible after plasmolysis. The remaining label (26.9 and 33.4%, respectively) appeared to be present in the periplasmic space but may also have been part of membrane junctions not visible because of poor contrast of the specimen. In contrast, a quite different distribution of the L protein was found in cells grown under conditions of penicillin tolerance, i.e., at pH 5, a condition that had previously been shown to protect cells from L-protein-induced lysis. At tolerant conditions, only 21.0% of the L protein was in the adhesion sites; most of the protein (68.2%) was found in inner and outer membranes. It is concluded that lysis of the host, E. coli, was a result of the formation of specific L-protein-mediated membrane adhesion sites.
Assuntos
Colífagos/fisiologia , Escherichia coli/ultraestrutura , Proteínas Virais/metabolismo , Membrana Celular/ultraestrutura , Parede Celular/metabolismo , Imuno-Histoquímica , Microscopia EletrônicaRESUMO
Bacterial lysis induced by the expression of the cloned lysis gene of the RNA bacteriophage MS2 in Escherichia coli was shown to be under the same regulatory control mechanisms as penicillin-induced lysis. It was controlled by the stringent response and showed the phenomenon of tolerance when E. coli was grown at pH 5. Changes in the fine structure of the murein were found to be the earliest physiological changes in the cell, taking place 10 min before the onset of cellular lysis and inhibition of murein synthesis. Both the average length of the glycan strands and, with a time lag, the degree of cross-linkage were altered, indicating that a lytic transglycosylase and a DD-endopeptidase had been triggered. After extensive separation of the membranes by isopycnic sucrose gradient centrifugation, the lysis protein was present predominantly in the cytoplasmic membrane and in a fraction of intermediate density and, to a lesser degree, in the outer membrane, irrespective of the conditions of growth. However, only under lysis-permissive conditions could a 17% increase in the number of adhesion sites between the inner and outer membranes be observed. Thus, a casual relationship between lysis and the formation of lysis protein-induced adhesion sites seems to exist.
Assuntos
Colífagos/genética , Escherichia coli/genética , Genes Virais , Genes , Proteínas Virais/genética , Bacteriólise , Membrana Celular/metabolismo , Membrana Celular/ultraestrutura , Clonagem Molecular , Escherichia coli/ultraestrutura , L-Lactato Desidrogenase/análise , Microscopia Eletrônica , Peptidoglicano/análiseRESUMO
Expression of the cloned lysis protein of phage MS2, which is sufficient to lyse wild type Escherichia coli, does not cause lysis of mutants lacking the osmoregulatory membrane-derived oligosaccharides (MDO). The lysis gene product normally found in the membrane fraction was not stably inserted into the membranes of a mdoA mutant; rather degradation and release from the membrane occurred. Gentle plasmolysis of the MDO-lacking mutant clearly showed an increased periplasmic space as compared to wild type cells. It is concluded that the MDOs play an important role in maintaining a proper arrangement of inner and outer membrane, a prerequisite for a functional insertion of the MS2 lysis protein.