Delta-aminolevulinic acid dehydratase activity in weanling and adult rats exposed to lead acetate.
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Administration, Oral↗
Biomedical subjects
M E Pereira
Publications and source records attributed to M E Pereira.
Plasmodium falciparum lacks sialidase and trans-sialidase activity.
Sialic acid on the red cell surface plays a major role in invasion by the malaria parasite Plasmodium falciparum. The NeuAc(alpha 2,3) Gal motif on the O-linked tetrasaccharides of the red cell glycophorins is a recognition site for the parasite erythrocyte-binding antigen (EBA-175). Consequently, the interaction of P. falciparum and the red cell might share homology with that of the influenza virus. The cellular interactions of P. falciparum were examined for their sensitivity to 4-guanidino-2,3-didehydro-D-N-acetyl neuraminic acid (4-guanidino Neu5Ac2en), a potent inhibitor of influenza virus sialidase. Parasite invasion and subsequent development was unaffected by the sialidase inhibitor. The inhibitor did not affect rosette formation of parasite-infected erythrocytes with uninfected cells nor their cytoadherence to C32 melanoma cells. Furthermore, we were unable to confirm the presence of a previously reported parasite sialidase using sensitive fluorometric or haemagglutination assays, neither was any malarial trans-sialidase identified. We conclude that P. falciparum possesses neither sialidase nor trans-sialidase activity and that an inhibitor of influenza virus sialidase has no effect on important cellular interactions of this parasite.
Animals↗
Trypanosome invasion of mammalian cells requires activation of the TGF beta signaling pathway.
Trypanosoma cruzi invades most nucleated mammalian cells by as yet unknown mechanisms. We report here that while T. cruzi attaches to epithelial cells lacking signaling transforming growth factor beta (TGF beta) receptor I or II, the adherent parasites cannot penetrate and replicate inside the mutant cells, as they do in parental cells. Invasion of the mutants is restored by transfection with the TGF beta receptor genes, as are biological responses to TGF beta. Similar rescue of both TGF beta antiproliferative response and T. cruzi invasion was demonstrated in a hybrid of TGF beta-resistant bladder and colon carcinoma cells. In addition, T. cruzi did not efficiently invade epithelial cells with dysfunction of the intracellular signaling cascade caused by the constitutive expression of the cyclin-dependent kinase cdk4 or of the oncogene H-ras. Treatment with TGF beta, but not with other antiproliferative agents of non-phagocytic cells, greatly enhances T. cruzi invasion. Moreover, infective, but not noninfective, trypanosomes strongly induce a TGF beta-responsive reporter gene in TGF beta-sensitive, but not in TGF beta-insensitive, cell lines. Thus, T. cruzi itself may directly trigger activation of the TGF beta signaling pathway required for parasite entry into the mammalian cells.
Animals↗
Effect of treatment with mercury chloride and lead acetate during the second stage of rapid postnatal brain growth on delta-aminolevulinic acid dehydratase (ALA-D) activity in brain, liver, kidney and blood of suckling rats.
The sensitivity of developing rodents to toxic metals differs considerably from that of adults. In the present study, we investigated the in vivo and in vitro effects of inorganic mercury and lead on delta-aminolevulinic acid dehydratase (ALA-D) from brain, liver, kidney and blood of young rats. Eight day-old rats were injected with one or five doses of lead acetate (0, 3.5, or 7.0 mg/kg) or HgCl2 (0, 2.5, or 5.0 mg/kg). In vitro, the IC50 for mercury inhibition of cerebral, renal and hepatic ALA-D was in the 124 to 160 microM range, while values for lead acetate was in the 7 to 12 microM range. The IC50 of blood enzyme for lead (0.8 microM) and mercury (6.5 microM) was significantly lower than that observed for the other tissues. A single dose of lead did not affect the enzyme activity, but a single dose of HgCl2 (5 mg/kg) caused a significant inhibition of ALA-D from kidney (40%, P < 0.01) and liver (25%, P < 0.05). Five doses of lead acetate (3.5 or 7 mg/kg) caused an inhibition of about 25 and 40%, respectively (P < 0.01), of hepatic ALA-D, and an increase of 1.4-fold (P < 0.05) and 2.6-fold (P < 0.01) of blood enzyme, respectively. Treatment with five doses of HgCl2 (5 mg/kg) caused an inhibition of about 25, 60, 50, and 80% of ALA-D from brain, blood, liver and kidney, respectively (all P < 0.05). Five doses of 2.5 mg/kg HgCl2 caused an inhibition of ALA-D from liver (40%, P < 0.01) and kidney (45%, P < 0.01). These results demonstrate that ALA-D from young rat tissues show different sensitivities to mercury and lead. The enzyme was more affected by mercury than by lead in vivo, while in vitro lead was more potent that mercury as an ALA-D inhibitor.
Analysis of Variance↗
Trypanosoma cruzi trans-sialidase: enhancement of virulence in a murine model of Chagas' disease.
Trypanosoma cruzi, the etiological agent of Chagas' disease, expresses a trans-sialidase at highest levels in infective trypomastigotes, where it attaches to the plasma membrane by a glycophosphoinositol linkage. Bound enzyme sheds into the extracellular milieu in a soluble form. Experiments performed in vitro suggest that the trans-sialidase participates in several parameters of T. cruzi-host interactions, like cell adhesion and complement resistance. However, the role that membrane-bound and soluble trans-sialidase plays in the infection of mammals is not understood. To begin to study the role the enzyme may play in vivo, T. cruzi trypomastigotes were inoculated subcutaneously into mice that had been sensitized for various times with the purified protein. A single dose of either endogenous or recombinant trans-sialidase injected into the connective tissues of BALB/c mice greatly enhanced parasitemia and mortality. Maximum enhancement was achieved with 1-2-h priming. Injection of the enzyme after the parasites had been established in the inoculation site had little, if any, consequence in modifying virulence. The enhancement did not seem to be through a direct effect of the enzyme on trypomastigote-host cell interactions because it occurred when the sites of trans-sialidase sensitization and parasite inoculation were physically separate. Rather, virulence enhancement seemed to depend on inflammatory cells, since priming with trans-sialidase had no significant effect in severe combined immunodeficiency mice, which lack functional T and B lymphocytes. However, antibody response to T. cruzi in the trans-sialidase-primed BALB/c mice was the same as in the control animals. Virulence enhancement was specific for the trans-sialidase because it did not occur in mice primed with Newcastle virus sialidase, which has the same substrate specificity as the T. cruzi enzyme, or with the sialidase from the bacterium Vibrio cholerae, whose substrate specificity is broader than the trypanosome sialidase. Furthermore, no enhancement of virulence occurred after sensitization with another adhesion protein (penetrin) purified from T. cruzi trypomastigotes and engineered bacteria, nor with bacterial lipopolysaccharide. The virulence-promoting activity of soluble trans-sialidase in the mouse model may be physiologically relevant because it was achieved with tiny doses, approximately 1-2 microgram/kg, raising the possibility that neutralization of the enzyme with specific probes could impair the development of Chagas' disease. In fact, a monoclonal antibody specific for the tandem repeat in the trans-sialidase COOH terminus enhanced infection of BALB/c mice, in agreement with earlier experiments in vitro, whereas antibodies against an amino acid sequence in the Cys region had the opposite effect.
Amino Acid Sequence↗
Atropine reverses antinociception induced by 2,5-hexanedione in rats.
2,5-Hexanedione is a n-hexane metabolite with neurotoxic properties. We have previously demonstrated that acute administration of 2,5-hexanedione causes analgesia in the tail-flick test in rats. In the present investigation, we examined the possible involvement of a cholinergic component in the 2,5-hexanedione-induced antinociception, since literature data indicate that this hexacarbon compound may act as a competitive inhibitor of acetylcholinesterase and that cholinesterase inhibitors are analgesic to rodents. Rats were treated with saline or with 5 or 25 mg/kg atropine (intraperitoneally) 10 min. before the injection of vehicle or 800 mg/kg 2,5-hexanedione (intraperitoneally). 2,5-Hexanedione caused a significant increase in tail-flick latencies at 10, 30, 60 and 90 min. after hexacarbon injection. Atropine (5 or 25 mg/kg) partially reversed the analgesia caused by 2,5-hexanedione at 60 and 90 min. When effects of 2,5-hexanedione on brain acetylcholinesterase was assessed in vitro, the results demonstrated that a competitive component is involved in enzyme inhibition. Taken together, these data support the involvement of a cholinergic (muscarinic) component in 2,5-hexanedione-induced analgesia.
Analgesia↗
Flow cytometry, a new approach to detect anti-live trypomastigote antibodies and monitor the efficacy of specific treatment in human Chagas' disease.
Sera from patients chronically infected with Trypanosoma cruzi display antibodies that bind to epitopes of living trypomastigotes, known as lytic antibodies (LA), and are detected by a complement-mediated lysis test. Conventional serology antibodies (CSA) are also present in sera from patients with chronic infections but, in contrast to LA, are unable to recognize viable trypomastigotes. The presence of LA has been used as an important element in the criterion of cure in human Chagas' disease. Using flow cytometry technology, we introduced a new and sensitive immunomethod for the detection of anti-live trypomastigote membrane-bound antibodies. On the basis of serological tests (LA and CSA detection) and parasitological assays such as hemoculture (HE), patients were classified into the following groups: chronically infected untreated patients (NT) and treated not-cured patients (TNC), with positive HE and both LA and CSA in their sera; "dissociated" HE-negative patients (DIS), in whom LA was not detected whereas CSA were present; a group of cured HE-negative patients (CUR), who were both LA and CSA negative; and, as control, a group of non-chagasic individuals (NC). Sera from these patients were assayed by incubation with live bloodstream trypomastigotes, which were subsequently exposed to fluorescein isothiocyanate-conjugated anti-human immunoglobulin G. The parasites were then fixed, run in the cytometer, and identified on basis of their size and granularity gain adjustments. On the basis of experience with the complement-mediated lysis test, a level of 20% of parasites being fluorescein isothiocyanate fluorescence positive was used as a cutoff between effective and ineffective treatments. With this criterion, our results indicated that sera from NT and TNC patients were antibody positive whereas all sera from DIS, CUR, and NC patients did not contain membrane-bound antibodies. This new approach is a tool to easily identify anti-live T. cruzi membrane-bound antibodies that can be used to monitor the efficacy of Chagas' disease treatment.
Adult↗
Hemagglutination and proteoglycan binding by the Lyme disease spirochete, Borrelia burgdorferi.
The ability of the Lyme disease spirochete to attach to host components may contribute to its ability to infect diverse tissues. We present evidence that the Lyme disease spirochete expresses a lectin activity that promotes agglutination of erythrocytes and bacterial attachment to glycosaminoglycans. Among a diverse collection of 21 strains of Lyme disease spirochete, hemagglutinating activity was easily detected in all but 3 strains, and these three strains were noninfectious. The ability to agglutinate erythrocytes was associated with the ability of the spirochete to bind to the sulfated polysaccharide dextran sulfate and to mammalian cells. Soluble dextran sulfate was a potent inhibitor of both hemagglutination and attachment to mammalian cells, while dextran had no effect on either activity, suggesting that dextran sulfate may inhibit attachment by mimicking host cell glycosaminoglycans. Consistent with this, the spirochete bound to immobilized heparin, and soluble heparin inhibited bacterial adhesion to mammalian cells. The bacterium did not bind efficiently to Vero cells treated with heparinase or heparitinase or to mutant CHO cell lines that are deficient in proteoglycan synthesis. Sulfation of glycosaminoglycans was critical for efficient bacterial recognition, as Vero cells treated with an inhibitor of sulfation, or a mutant CHO cell line that produces undersulfated heparan sulfate, did not mediate maximal spirochetal binding. Binding of the spirochete to extracellular matrix also appeared to be dependent upon this attachment pathway. These findings suggest that a glycosaminoglycan-binding activity which can be detected by hemagglutination contributes to the attachment of the Lyme disease spirochete to host cells and matrix.
Animals↗
Phosphorylation in vitro of glial fibrillary acidic protein is increased in rat hippocampus by administration of 2,5-hexanedione.
Rats were treated with 2,5-hexanedione (2,5-HD) daily for 30 days. Hippocampal microslices were then incubated with [32P]phosphate and the in vitro rate of phosphorylation and the immuno-content of glial acidic fibrillary protein (GFAP) were measured. Exposure to 2,5-HD decreased by 25% the immuno-content of GFAP and increased by 35% its rate of phosphorylation, resulting in an increase of 88% in the ratio phosphorylation rate/immuno-content for this protein.
Animals↗
A novel in situ model to study Pneumocystis carinii adhesion to lung alveolar epithelial cells.
Pneumocystis carinii, an extracellular parasite thriving in the lungs of immunosuppressed mammals, is a major cause of death in AIDS patients in the USA. As a prelude to growth, the parasite adheres mostly to type I pneumocytes lining the alveolar spaces. The mechanism of adherence remains unknown, largely because of difficulties in isolating type I pneumocytes and maintaining them in vitro. As a first step to understand P. carinii adherence to its natural substrate, we developed an in situ method to directly study parasite binding to lung alveolar cells. We used formaldehyde-fixed paraffin-embedded sections of normal rat lung as substrate for adhesion. As in its binding to the lungs in vivo, P. carinii adhered preferentially to type I pneumocytes. Adherence was saturable, time and dose dependent, and selectively blocked by glycoconjugates, in particular bovine submaxillary mucin, fetuin, and asialofetuin, suggesting that it may be mediated by a lectin type of interaction. Further, IgG of rats with P. carinii pneumonia inhibited adherence, suggesting that it may react with parasite ligands involved in the recognition of type I cell receptors. Our results demonstrate the usefulness of the in situ model for studying the mechanisms of P. carinii adherence to alveolar cells. In addition, this method may be valuable for identifying neutralizing antibodies and drugs potentially useful for controlling the infection in vivo.
Animals↗
Mediation of Trypanosoma cruzi invasion by heparan sulfate receptors on host cells and penetrin counter-receptors on the trypanosomes.
Trypanosoma cruzi attaches and invades a large variety of mammalian cells by receptor-mediated interactions, one of them involving the binding of parasite trans-sialidase to host sialyl receptors. Three proteoglycan-deficient mutants of Chinese hamster ovary (CHO) cells were used to probe the role of host heparin and heparan sulfate glycosaminoglycans (GAG) in T. cruzi invasion. All three mutants supported adhesion and infection to a much lower extent than the parental CHO cells. One of the mutants, pgsD-677, did not express heparan sulfate while containing three- to four-fold excess chondroitin sulfate, yet the cell line was a poor substrate for T. cruzi adhesion. Proteoglycan-deficient cells obtained by inhibiting GAG synthesis in parental cells with p-nitrophenyl-beta-D-xyloside, were also poor hosts for T. cruzi invasion. Furthermore, digestion of parental cells with heparinase and heparitinase, two lyases that specifically depolymerize heparin and heparan sulfate, reduced the potential of the cells to support T. cruzi adhesion and growth. Lyases that digested chondroitin sulfate and other GAGs did not affect T. cruzi invasion. These results suggest that heparin/heparan sulfate epitopes are receptors for T. cruzi invasion. The corresponding counter-receptor on T. cruzi appears to be penetrin, a heparin-binding protein that promotes trypanosome penetration into cells. Purified penetrin caused agglutination of red blood cells, and the hemagglutination was exquisitely sensitive to heparin and heparan sulfate. However, sialic acid and sialyl compounds did not inhibit penetrin-induced hemagglutination. Recombinant penetrin competitively inhibited T. cruzi invasion of proteoglycan-containing parental cells, but not of proteoglycan-deficient mutants nor of heparitinase-treated cells. Furthermore, consistent with the sugar specificity of penetrin as a hemagglutinin, recombinant penetrin competed for trypanosome invasion of a CHO cell mutant (Lec2) that expresses heparan sulfate but not sialyl residues. Given that the release of sialic acid from the proteoglycan-deficient mutants further reduced T. cruzi invasion, as did the removal of heparan sulfate from the Lec2 mutant, and given that penetrin does not bind to sialic acid with high affinity, the results indicate that the penetrin-heparan sulfate pathway for T. cruzi invasion is distinct from the trans-sialidase-sialic acid route.
Animals↗
Attachment of Cryptosporidium parvum sporozoites to MDCK cells in vitro.
The initial attachment of Cryptosporidium parvum sporozoites to host cells in vivo may be a critical event in the pathogenesis of this infection. The molecular basis of attachment and the conditions influencing this host-parasite interaction have not been studied systematically. Therefore, we have developed a sporozoite attachment model by using paraformaldehyde-fixed Madin-Darby canine kidney (MDCK) cells. Attachment of sporozoites to fixed MDCK cells was quantitated by indirect immunofluorescence and confirmed by transmission electron microscopy. Attachment in this system was time, temperature (37 degrees C), and pH (7.2 to 7.6) dependent. Dose-response studies demonstrated that the attachment of sporozoites to fixed MDCK cells was a saturable process. Attachment was enhanced in the presence of 10 mM manganese, 1 mM calcium, and 1 to 10 mM zinc. Attachment of sporozoites to MDCK cells was inhibited in a dose-dependent manner by polyclonal anti-Cryptosporidium antisera and by purified immunoglobulin G (IgG). This model will be useful for the study of parasite and host cell molecules involved in the initial interaction of C. parvum sporozoites with their target cell.
Adhesiveness↗
Structural and functional properties of Trypanosoma trans-sialidase.
Sialic acids and sialidases play important roles in cellular interactions and modulate the recognition of pathogenic microbes by mammalian host cells. Protozoan parasites of the genus Trypanosoma express a unique sialic acid-metabolizing enzyme. This enzyme, named trans-sialidase (TS), catalyzes the transfer of sialic acids from host glycoconjugates to acceptor molecules of the parasite plasma membrane. In African trypanosomes, the agents of sleeping sickness, TS is found only in forms developing within the insect vector, and the enzyme sialylates the major surface protein. In Trypanosoma cruzi, the causative agent of Chagas' disease in Central and South America, TS is expressed both in the insect and mammalian forms of the parasite. The T. cruzi enzyme has been biochemically characterized, and the gene encoding the enzyme has been cloned. The enzyme sialylates abundant mucin-like molecules present on the surface of the parasite. Several lines of evidence suggest that TS and sialic acid acceptors on the surface of T. cruzi participate in host-parasite interactions and mediate the initial stages of the trypanosomes' invasion of host cells.
Amino Acid Sequence↗
Growth inhibition of the intestinal parasite Giardia lamblia by a dietary lectin is associated with arrest of the cell cycle.
Giardia lamblia, a cause of diarrheal disease throughout the world, is a protozoan parasite that thrives in the small intestine. It is shown here that wheat germ agglutinin (WGA), a naturally occurring lectin widely consumed in normal human diets, reversibly inhibits the growth of G. lamblia trophozoites in vitro, and reduces infection by G. muris in the adult mouse model of giardiasis. The inhibitory effect was dose related, not associated with cytotoxicity and reversed by N-acetyl-D-glucosamine in accordance with the known specificity of the lectin and in agreement with the presence of GlcNAc residues on the surface membrane of G. lamblia trophozoites. Cell cycle analysis revealed that parasites grown in the presence of WGA are arrested in the G2/M phase, providing an explanation for the lectin-induced inhibition of cell proliferation. Comparison of electrophoretic profiles by lectin blot analysis revealed both glycoprotein induction and suppression in growth-arrested organisms. Our findings raise the possibility that blocking trophozoite growth with naturally occurring dietary lectins may influence the course of giardiasis. In addition, the study of cell cycle arrest by WGA may provide a model to study the regulation of cell division in lower eukaryotes.
Animals↗
Developmentally regulated lectins in Eimeria species and their role in avian coccidiosis.
Avian coccidiosis caused by Eimeria species is characterized by rather specific site infections of the intestine. We used hemagglutination and hemagglutination inhibition assays on various developmental stages of Eimeria tenella and on sporozoites of Eimeria acervulina and Eimeria maxima to assay for parasite lectins. Various monosaccharides, polysaccharides, and glycoproteins were used to demonstrate differences in sugar specificity of the lectins between these species. Surface lectins were found on the primary infective stage, i.e., sporozoites, but not on merozoites or unsporulated oocysts. Also, there were differences in the specificities of the various sugar lectins among the different parasite species. Furthermore, there was a dose-dependent reduction of infection of tissue culture cells by sporozoites of E. tenella that were continuously exposed to fetuin, 1 of the specific inhibitors of the lectin. The results of our study are unique in that in 3 species of avian Eimeria all have a lectin on their sporozoites, but the lectins for each species have different sugar specificities. We hypothesize that these lectins found on the surface of the sporozoites may play a role in determining the site of infection within the intestine of the host.
Animals↗
Enzymatic characterization of beta-D-galactoside alpha 2,3-trans-sialidase from Trypanosoma cruzi.
The substrate specificity, physico-chemical, and kinetic properties of the trans-sialidase from Trypanosoma cruzi have been investigated. The enzyme demonstrates activity towards a wide range of saccharide, glycolipid, and glycoprotein acceptors which terminate with a beta-linked galactose residue, and synthesizes exclusively an alpha 2-3 sialosidic linkage. Oligosaccharides which terminate in Gal beta 1-4(Fuc alpha 1-3)GlcNAc, Gal beta 1-3(Fuc alpha 1-4)GlcNAc, or Gal alpha 1- are not acceptor-substrates. The enzyme utilizes alpha 2,3-linked sialic acid when the donor species is an oligosaccharide and can also transfer, at a low rate, sialic acid from synthetic alpha-sialosides such as p-nitrophenyl-alpha-N-acetylneuraminic acid, but NeuAc alpha 2-3Gal beta 1-4(Fuc alpha 1-3)Glc is not a donor-substrate. The trans-sialidase has an apparent pH optimum of 7.9 and a temperature optimum of 13 degrees C. The kinetic properties of the enzyme suggest that the trans-sialylation reaction may occur via a rapid equilibrium random or steady-state ordered mechanism. A method for immobilizing the enzyme is described together with examples of its use for the synthesis of oligosaccharide and glycoprotein precursors of sialyl-Lewis and sialyl-Lewis.
Amino Acid Sequence↗
Changes in the structure and catalytic activities of the bovine pituitary multicatalytic proteinase complex following dialysis.
The multicatalytic proteinase complex (proteasome) contains at least four distinct active sites catalyzing the degradation of selected chromogenic substrates (trypsin-like, chymotrypsin-like, and peptidylglutamyl peptide hydrolyzing activities) and proteins such as beta-casein. Oxidized insulin B chain was recently proposed as a model substrate for protein degradation by the multicatalytic proteinase complex (Dick, L. R., Moomaw, C. R., DeMartino, G. N., and Slaughter, C. A. (1991) Biochemistry 30, 2725-2734). We studied the dialysis-induced activation of the hydrolysis of oxidized insulin B chain by this enzyme. Removal of EDTA from purified preparations of bovine pituitary multicatalytic proteinase complex by dialysis against Tris-HCl buffers led to marked changes in the catalytic properties and structure of the enzyme. Dialysis produced a time-dependent activation of oxidized insulin B chain hydrolysis with predominant cleavage at the Glu13-Ala14 bond. A new chromogenic assay was developed for measurement of this activity. Activation was accompanied by a virtually total inactivation of the chymotrypsin-like, trypsin-like, and peptidylglutamyl peptide hydrolyzing activities. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis revealed a loss of the 24-kDa subunit and the appearance of a new band at 21 kDa. Amino-terminal amino acid analysis established that the 21-kDa band was autolytically derived from the 24-kDa subunit. Evidence for partial dissociation and/or aggregation indicated that autolysis destabilizes the complex. By altering the profile of catalytic activities of the multicatalytic proteinase complex, autolysis may serve as a mechanism for regulation of this macromolecule.
Amino Acid Sequence↗
Epitopes shared between coxsackievirus B3 (CVB3) and normal heart tissue contribute to CVB3-induced murine myocarditis.
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Animals↗