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E Orozco

Publications and source records attributed to E Orozco.

At least 91 records · Page 5Linked to original sources
PubMed1990

[Antigens specific to pre-cysts and in vivo chitin synthetase activity in Entamoeba invadens].

In this paper we studied the transformation of a trophozoite into a cyst in Entamoeba, using E. invadens as a model. We had the following objectives: a) identification of precyst-specific proteins (P), by a monoclonal antibody against E. invadens and heterologous polyclonal antibodies against cellular (165) fractions of Mucar rouxii, which are chitin synthetase activity rich; and b) in vivo determination of the time required for the expression of activity of the chitin synthetase during encystment. We found P markers which are not found in either trophozoites nor cysts. Monoclonal F507 antibody recognized a 33 kDa protein in P and the polyclonal anti-16S antibodies reacted with a 90 kDa protein to P. Even though the 33 and 90 kDa proteins have a different molecular weight from the chitin synthetase described in fungi (57 kDa and 65 kDa) and yeasts (63 kDa), we conclude that these proteins are specific to P and that the 90 kDa one shares epitopes with chitosomal fractions of M. rouxii. Also, the mayor accumulation of alkali-resistant material, sensitive to chitinase, occurred during the formation of P, between 40 and 50 hours post incubation, during encystment. One may conclude that chitin polymers are synthesized during the P phase.

Animals↗
PubMed1990

Emetine resistant mutants of Entamoeba histolytica overexpress mRNAs for multidrug resistance.

Although drug therapy is critical for control of amoebiasis, little is known about mechanisms of drug resistance by E. histolytica parasites. Here we tested the hypothesis that multidrug resistant (mdr) amoeba mutants, similar to mdr tumor cells, are drug resistant based upon overexpression of a P-glycoprotein pump that effluxes drugs from the cells. Using primers to conserved regions of the human P-glycoprotein and the polymerase chain reaction (PCR), we identified multiple 344 base par segments of amoeba DNA similar to the mammalian P glycoprotein. The amino acid sequences of amoeba mdr-like PCR products were from 53 to 97 identical with each other, 55 to identical to human mdr1 sequences, and 41-44% identical with P. falciparum mdr-like sequences. On northern blots, the mdr-like PCR products identified amoeba mRNAs 4.5-5 kilobases long, similar to the 5 kilobase mRNAs reported for the mammalian mdr gene. These mRNAs were increased at least seven times in emetine resistant mutant clone C2 amoebae versus wild-type clone A parasites. Further, the expression of the mdr-like mRNAs was increased three to four times when clone C2 mutants were grown under drug pressure versus the same parasites grown without emetine. In contrast, the number of genomic copies of the mdr-like DNA segments was not increased in the mutant clone C2 versus the wild-type clone A amoebae, and no rearrangements of the mdr-like DNA segments by the mutant were identified on Southern blots. In conclusion there appears to be a family of mdr-like genes in E. histolytica, which may be involved in drug resistance by the parasite.

ATP Binding Cassette Transporter, Subfamily B, Mem↗
PubMed1990

[Immunochemical differences in the surface polysaccharides obtained from Entamoeba histolytica strain HM1:IMSS and its virulent (C-A) and non-virulent (L-6) clones].

There probably exist many different surface molecular polysaccharides with different compositions of their constituent sugars, in the different zymodemes. This fact might have a repercussion upon the resistance to the lysis of the complement. Seeking to prove this hypothesis, the ideal would be to obtain polysaccharide molecules of type LPFG of pathogenic and non-pathogenic zymodemes, demonstrating that there is a difference in the chemical composition of their polysaccharides. If this were true, a serologic classification, of the different zymodemes, would be possible in a fashion similar to gram-negative bacteria. Our results suggest the existence of structural differences between the polysaccharides of a virulent clone (C-A) and a non-virulent one (L-6).

Animals↗
PubMed1990

[Identification of Entamoeba histolytica chromosomes by pulsed field gradient electrophoresis].

The use of pulsed field gradient electrophoresis (PFG) has simplified the study of chromosomic organization in protozoan parasites whose chromosomes do not condense during division. In this paper we used PCG to analyzed chromosome organization Entamoeba histolytica's. In the conditions which we used (200 volts, 24 hours, 3 minute pulses and the anode at 7.5 cm. from the end of the cathode) 6 and 9 band of amebic DNA were resolved. By means of a ribosomal DNA probe from Trypanosoma brucei amebic DNA was detected in the gels representing chromosomal sizes, in accord with the reproducibility of our results and the hybridization pattern we obtained. The numbers of 6 to 9 obtained for PFG is in accord with the results which we have previously reported. The suggest that Entamoeba histolytica has between 12 and 16 structures similar to chromosomes, if Entamoeba histolytica were diploid, and therefore each band would consist of two chromosomes. Nevertheless small condensations of DNA similar to minichromosomes of kinetoplasas cannot be ruled out, and this would considerably increase the number of chromosomes estimated in this paper.

Animals↗
PubMed1990

[Identification and localization of molecules which intervene in the interaction between Entamoeba histolytica and epithelial cells].

During the initial stages of host-parasite interaction between Entamoeba histolytica and its target cell, superficial molecules of both cells participate. The purpose of this study has been the study of the parasite's proteins which are involved in the process of adhesion, as well as localize the superficial sites of the host which bind with the amebic proteins. Antibodies directed against adhesin 112 kDa, and other amebic adhesins, specifically inhibited the destruction of the monolayers of the MDCK epithelial cell line. Soluble proteins of MDCK cells recognized was separated by electrophoresis and transferred to nitrocellulose paper, the amebic proteins of 112, 90 and 45 to 50 kDa. In a similar fashion, membrane proteins of MDCK cells marked with 125[I] specifically recognized amebic proteins of 112, 90, 45 and 32 kDa, as well as others of higher molecular wight. The localization of the binding sites of the E. histolytica molecules on semi-confluent monolayers of MDCK cells, was brought about by immunofluorescence using antibodies against the parasite's total proteins or against adhesins. For electron microscopy antibodies labelled with peroxidase were used. The amebic proteins were mainly found on the periphery of free cells, land in the intercellular cell junctions of monolayers.

Animals↗
PubMed1989

Entamoeba histolytica: molecules involved in the target cell-parasite relationship.

To detect molecules of Entamoeba histolytica involved in the trophozoite-target cell interaction, three different antisera were generated: (a) two rabbit antisera, one against total amebic proteins and another directed specifically to the 112-kDa adhesin; and (b) a mouse antiserum against amebic molecules adhering to the red blood cell (RBC) surface after incubation of RBCs with total soluble protein from trophozoites (anti-adhesion serum). All three antisera recognized the 112-kDa adhesion. Adhesion of this molecule to the RBC surface was temperature-dependent. More of the 112-kDa adhesion was found on the surface of RBCs incubated with trophozoites at 37 degrees C than on RBCs incubated at room temperature or at 0 degree C. Experiments using both anti-adhesin and anti-total ambebic protein sera revealed the presence of 210, 160, 112, 90, 70, 50, and 24-kDa proteins on RBC incubated with trophozoites. Surface proteins obtained from iodinated MDCK cells recognized amebic proteins of 112, 90, and 48-50 kDa. Virulence-deficient mutants presented a similar amount of the 112-kDa adhesin to the wild-type strain. However, in mutants, the adhesion was not functional, since they did not adhere to RBCs. 90- and 24-kDa proteins were also found to be altered in mutants.

Animals↗
PubMed1988

Entamoeba histolytica: cell cycle and nuclear division.

The cell cycle of Entamoeba histolytica, the duration of its phases, and the details of the nuclear division stages are described in this paper. Trophozoites from clone L-6, strain HM1:IMSS, were synchronized by colchicine. Synchrony was observed immediately after treatment and cultures remained synchronous for at least three replicative cycles with synchrony indexes between 13 and 15 hr. The stages of nuclear division were studied by light and electron microscopy. Four stages of the nuclear division were defined: prophase, early anaphase, late anaphase, and telophase. No metaphase stage was observed by light or electron microscopy. One of the first events in the nuclear division was the presence of a bud close to the juxtanuclear body, which grew to a daughter nucleus. The karyosome and the nuclear membrane remained throughout the mitotic process. Bundles of intranuclear microtubules were observed forming a "V" from the center of the nucleus to one of the poles, and associated with them, 12 to 16 chromosomes-like structures appeared. The results of these studies strongly suggest that division of E. histolytica involved a pleuromitotic process which is carried out in about 120 min.

Anaphase↗
PubMed1988

Binding and internalization of rat colonic mucins by the galactose/N-acetyl-D-galactosamine adherence lectin of Entamoeba histolytica.

Purified rat colonic mucins inhibit Entamoeba histolytica in vitro adherence to and lysis of colonic epithelial cells by binding to the amoebic galactose/N-acetyl-D-galactosamine (Gal/GalNAc)-inhibitable adherence lectin. We found that 125I-labeled mucins demonstrated saturable Gal-specific binding to E. histolytica trophozoites (strain HM1:IMSS), with 2.8 x 10(3) binding sites per amoeba and a dissociation constant of 8.20 x 10(-11) M-1. Inhibition of parasite protein synthesis completely abrogated mucin binding; elevation of amoebic vesicle pH with ammonium chloride (10 mM) had no effect. Surface-bound 125I-labeled mucins were rapidly internalized and released from amoebae without evidence of proteolytic degradation. Three avirulent HM1 clones contained immunoreactive Gal/GalNAc lectin molecules with high-affinity binding of 125I-labeled mucins but exhibited markedly reduced rates of uptake and exocytosis of bound mucins. High-affinity binding by the Gal/GalNAc adherence lectin was followed by rapid internalization and eventual release of the colonic mucins. Additionally, defects in lectin surface expression and endocytosis were found in the avirulent clones.

Animals↗
PubMed1988

Phorbol esters specifically enhance the cytolytic activity of Entamoeba histolytica.

Entamoeba histolytica causes invasive amebiasis by lysis of host tissue and inflammatory cells. The in vitro cytolysis of target Chinese hamster ovary (CHO) cells by axenic E. histolytica trophozoites (strain HM1:IMSS) is a calcium- and phospholipase A-dependent event initiated by the binding to the target cell of the galactose-inhibitable surface lectin of the parasite. We utilized phorbol esters as a probe to determine whether an amebic protein kinase C has a role in the cytolytic event. The addition of phorbol 12-myristate 13-acetate (PMA) at 10(-6) or 10(-7) M resulted in a greater than twofold enhancement of amebic killing of target CHO cells over 30 min (P less than 0.01). Prior exposure of only the amebae, but not the CHO cells, to PMA produced a similar effect (P less than 0.01). The inactive analog 4-alpha-phorbol had no effect on amebic killing of CHO cells. The PMA-mediated enhancement of amebic cytolysis persisted for up to 60 min after a 5-min exposure; however, after a 30-min exposure to PMA (10(-6) M) there was no augmentation of amebic killing of CHO cells. PMA (10(-6) M) did not promote adherence of parasites to CHO cells but did enhance amebic cytolysis of previously adherent target cells (P less than 0.01). Sphingosine, a specific inhibitor of protein kinase C, abolished both the PMA-stimulated and the basal cytolytic activity of E. histolytica. PMA enhanced CHO cell cytolysis by the less virulent wild-type strain H-303:NIH (P less than or equal to 0.02) but did not augment the activity of the less virulent strain H-200:NIH or two avirulent clones of HM1 (L6 and C919). In summary, these experiments with the phorbol esters and sphingosine as probes to modulate the activity of protein kinase C indicate participation of a parasite protein kinase C in the cytolytic activity of virulent, axenic E. histolytica trophozoites and thus in the pathogenesis of amebiasis.

Adhesiveness↗