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L S Mayorga

Publications and source records attributed to L S Mayorga.

54 records · Page 3Linked to original sources
PubMed1991

Interaction of Trypanosoma cruzi with macrophages influence of temperature.

The interaction of macrophages and T. cruzi has been studied in vitro culture under three different temperatures. After 24 hours incubation at 29 degree C a large number of recognizable parasites inside macrophages is observed with evidences of cell divisions. At video microscopy they show a slow motion and a predominance of epimastigotes and some round shapes (amastigotes). This was corroborated at the light and electron microscopes. No evidence of lysis in the phagosome vacuoles was observed. At 40 degrees C, macrophages show a large number of residual bodies and phagocytic vacuoles with digested parasites. At 37 degrees C, an intermediate stage with normal and digested parasites inside macrophages is observed. No significant evidences of affected phagocytic and degradative properties of the macrophages were obtained at those temperatures. It is postulated that temperature mainly affects the parasite resistance to intracellular digestion.

Animals↗
PubMed1990

In vitro depolymerization dynamics of brain endogenous microtubules.

A subcellular fraction containing fragments of endogenous microtubules stabilized in 50% glycerol was separated by diferential centrifugation of rat brain homogenates. The pellets were suspended in glycerol-deficient media, and microtubule depolymerization was monitored by measuring the decrease of sedimentable tubulin. Concomitantly, the number and size of microtubules in the suspensions were followed via electron microscopy. Depolymerization was accompanied by a proportional decrease in the number of microtubules, whereas the average size did not change significantly. After approximately 20 min, a subpopulation of microtubules became stable and did not suffer further depolymerization. These results indicate that upon dilution some microtubules completely depolymerize, whereas others remain stable in the glycerol-deficient medium. The degree of depolymerization depended on both the volume of the resuspension media and on the final glycerol concentration. The results suggest that the depolymerization of the remaining microtubules is prevented by stabilizing factors released from depolymerizing microtubules. Tubulin dimers are not one of these factors, since depolymerization was not altered by the addition of colchicine or by changing the concentration of free tubulin in the medium.

Animals↗
PubMed1989

In vitro clustering and multiple fusion among macrophage endosomes.

Early steps of receptor-mediated endocytosis appear to require the fusion of endosomes with each other. Recently, these fusion events have been reconstituted in vitro using vesicle preparations from J774 macrophages which have internalized ligands via the mannose receptor (Diaz, R., Mayorga, L., and Stahl, P. (1988) J. Biol. Chem. 263, 6093-6100). The present studies indicate that endosomes first form clusters when incubated under fusogenic conditions. Aggregation state was determined by electron microscopy using vesicles containing ligand-coated colloidal gold of different sizes previously internalized via the mannose receptor. Aggregation required cytosol and ATP. Afterwards, the limiting membranes of the vesicles composing these aggregates undergo multiple fusion and bring about the formation of large diameter vesicles that maintained the same density as endosomes when analyzed by Percoll gradient sedimentation. These large diameter vesicles were no longer fusogenic in the fusion assay. Multiple fusion was determined morphologically by the co-localization of three different size colloidal gold vesicles inside endocytic vesicles and biochemically by the fusion-dependent formation of triple immune complexes between three endocytic ligands internalized by receptor-mediated endocytosis: anti-dinitrophenol mouse IgG and dinitrophenol-derivatized beta-glucuronidase, ligands for the mannose receptor, and aggregated rabbit anti-mouse IgG, a ligand for the macrophage Fc receptor.

Animals↗
PubMed1989

Regulatory role for GTP-binding proteins in endocytosis.

Guanosine 5'-triphosphate (GTP)-binding proteins have been implicated in the transport of newly synthesized proteins along the secretory pathway of yeast and mammalian cells. Early vesicle fusion events that follow receptor-mediated endocytosis as measured by three in vitro assays were blocked by guanosine 5'-O-(3-thiotriphosphate) and aluminum fluoride. The effect was specific for guanosine nucleotides and depended on the presence of cytosolic factors. Thus, GTP-binding proteins may also have a role in the transport of molecules along the endocytic pathway.

Biological Transport↗
PubMed1989

Vesicle fusion following receptor-mediated endocytosis requires a protein active in Golgi transport.

In reconstitution studies N-ethylmaleimide, a sulphydryl alkylating reagent, inhibits both fusion of endocytic vesicles and vesicular transport in the Golgi apparatus. We show here that the same N-ethylmaleimide-sensitive factor that catalyses the vesicle-mediated transport within Golgi stacks is also required for endocytic vesicle fusion. Thus, it is likely that a common mechanism for vesicle fusion exists for both the secretory and endocytic pathways of eukaryotic cells.

Animals↗
PubMed1989

Reconstitution of endosomal proteolysis in a cell-free system. Transfer of immune complexes internalized via Fc receptors to an endosomal proteolytic compartment.

The presence of acid proteases in the endosomal compartment of macrophages has been recently demonstrated (Diment, S., Leech, M. S., and Stahl, P. D. (1988) J. Biol. Chem. 263, 6901-6907). This proteolytic activity allows the early degradation of ligands internalized by receptor-mediated endocytosis. To study the early steps that initiate the proteolytic processing of ligands, immune complexes formed with anti-dinitrophenol monoclonal IgG and radiolabeled dinitrophenol-derivatized bovine serum albumin were bound at 4 degrees C to Fc receptors of J774 macrophages. Cells were allowed to internalize immune complexes bound to the plasma membrane for different periods of time at 37 degrees C. Vesicle preparations generated from these cells were incubated in vitro at acidic pH to allow the hydrolysis of ligands located in protease-positive compartments. Ligand hydrolysis was observed after about 5 min of internalization, suggesting that at earlier times immune complexes were located in protease-free vesicles. Upon incubation of cell lysates under conditions that support in vitro endosome-endosome fusion, early protease-free endosomes containing ligand acquire proteolytic activity. Reconstitution of fusion-dependent proteolysis required energy, ions, membrane-associated factors, and cytosol. Cytosol was inactivated by incubation with N-ethylmaleimide. The proteolytic compartment formed upon in vitro incubation colocalized with endosomes in the light region of a Percoll gradient. Reconstitution was also achieved using an endosomal preparation separated from lysosomes in a Percoll gradient. Our results indicate that a fusion step between newly formed endocytic vesicles and a light density, protease-positive compartment triggers the proteolytic processing of ligands internalized by receptor-mediated endocytosis.

Animals↗
PubMed1989

GTP gamma S stimulation of endosome fusion suggests a role for a GTP-binding protein in the priming of vesicles before fusion.

Guanosine 5'-(3-O-thio)triphosphate (GTP gamma S), a non-hydrolyzable analogue of GTP, inhibits in vitro fusion among early endocytic vesicles in the presence of high concentrations of cytosol. In this report we show that fusion is remarkably stimulated by GTP gamma S under conditions where cytosolic components are the limiting factors for the process. The amount of cytosolic factors required for maximal fusion activity is several-fold decreased by the presence of GTP gamma S. Moreover, preincubation of vesicles in the presence of cytosol and GTP gamma S allows fusion to proceed even in the absence of cytosol. Our results indicate that a GTP-binding protein facilitates the binding of cytosolic factor(s) required for endosome fusion to the endosomal membrane and stabilizes a dilution-resistant intermediate of the fusion process.

Animals↗
PubMed1988

Plasma membrane-derived vesicles containing receptor-ligand complexes are fusogenic with early endosomes in a cell-free system.

Receptor-mediated endocytosis involves the transport of receptor-ligand complexes from the cell surface to an intracellular endocytic compartment. This study shows that plasma membrane-derived vesicles containing receptor-bound ligands (e.g. aggregated anti-dinitrophenol (DNP) IgG bound to Fc receptors) fuse with early endosomes containing DNP-beta-glucuronidase in a cell-free system. Plasma membrane vesicles were generated by homogenization of cells that had been allowed to bind ligands at 4 degrees C. Fusion between vesicles containing the two probes was assessed by (i) the formation of anti-DNP IgG-DNP-beta-glucuronidase complexes and (ii) the colocalization within closed vesicles of two different sizes of colloidal gold coated with ligands. Fusion required ATP, cytosol, and KCl. The requirements were similar to those described for endosome-endosome fusion in in vitro systems. Mild trypsinization of vesicles prior to their addition to the assay inhibited fusion. When DNP-beta-glucuronidase was chased into more mature endocytic compartments, fusion was not observed. The results indicate that cell surface regions involved in receptor-mediated endocytosis are capable of fusing to early endosomes. This fusion event may constitute the first step in the transport of ligands to an intracellular endocytic compartment.

Adenosine Triphosphate↗
PubMed1987

beta-Galactosidase from rat epididymal fluid is bound by a recognition site attached to membranes of the epididymis different from the phosphomannosyl receptor.

In order to know if the beta-galactosidase of the rat epididymal fluid, as other secreted acid hydrolases, carries a marker in its molecule, we studied the binding of this enzyme to cellular membranes of the epididymal tissue. The binding, like that mediated by the phosphomannosyl receptor, was saturable, did not require calcium, had a Kd in the nM range and was inhibited by phosphatase or metaperiodate treatment of the enzyme. However fructose 6-phosphate derivates were more effective competitive inhibitors than mannose 6-phosphate. The binding capacity of the membranes were extractable with Triton X-100 and incorporable into liposomes. Trypsin inhibited the binding capacity of Triton extracts but it did not affect the affinity of intact cellular membranes for beta-galactosidase. The results suggest that a phosphorylated carbohydrate of the enzyme is bound by a recognizing site of the cellular membranes different from the phosphomannosyl receptor.

Animals↗
PubMed1987

Rapid in vivo assay method for type G botulinal toxin.

The survival time (ST) dose-response curve for Clostridium botulinum type G toxin was determined in mice and evaluated as a rapid assay method. As it occurs with other botulinal toxin types, the results showed a linear relationship between the logarithm of the injected dose and the logarithm of ST. The slope of the ST dose-response curve for type G toxin differed significantly from those for type A or subtype Af toxins. This parameter was altered when trypsin-activated type G toxin was used. The ST dose-response curve was rather stable. This in vivo assay method could be applied for the estimation of the potency of G toxin preparations in a short time using only some few mice.

Animals↗
PubMed1982

Effect of androgens on the activity of acid hydrolases in rat epididymis.

The enzymatic activity of 6 acid hydrolases was studied in rat epididymal homogenates following castration, testosterone replacement and during postnatal growth. Acid phosphatase and N-acetyl-beta-D-glucosaminidase activity decreased after castration and increased with hormonal treatment as well as during growth. Beta-Glucuronidase and cathepsin D activity increased during the involution of the organ and decreased or did not change with hormone treatment or during sexual maturation. Arylsulphatase and deoxyribonuclease did not recover normal activity after hormonal treatment. Their activities were particularly high in epididymal and rete testis fluid of normal animals.

Acetylglucosaminidase↗
PubMed1981

Acid hydrolases in the epididymis of normal, castrated, vasectomized, cryptorchid and cryptepididymal rats.

With the aim of gaining knowledge about the lysosomal apparatus of the rat epididymis, four acid hydrolases were analysed in homogenates of the whole organ and, in other experiments, in separated segments: proximal and distal caput, corpus and cauda. The activities were similar to those in the liver, and they were 50% recovered in a cytosol of 43 000 g x 60 min. Ten days after castration all segments showed similar changes, the activities of beta-glucuronidase and cathepsin D increased above normal levels while those of DNAase and acid phosphatase were found slightly decreased. After vasectomy region I (caput and corpus) showed decreased beta-glucuronidase activity and increased acid phosphatase activity. The activity of cathepsin D increased in both regions. In cryptorchid rats (90 days) the epididymis greatly decreased in weight, the activities of acid phosphatase and DNAase slightly decreased in region II (cauda) and in region I, respectively. In the abdominal epididymis (90 days) only region II decreased in weight. DNAase activity decreased in region I while cathepsin D did so in both regions. The results showed that a) the enzymes behave quite independently from each other, suggesting the existence of a specific regulation for each of them b) there were characteristic changes in enzymatic activity for each experimental condition.

Acid Phosphatase↗
PubMedDate not supplied

The origin of some acid hydrolases of the fluid of the rat cauda epididymidis.

The activity of N-acetyl-beta-D-glucosaminidase (NAG, 60.1 units/mg protein) and of acid phosphatase (57.7 units/mg protein) in fluid from the cauda epididymidis formed without any contribution from the testis (fluid obtained from a perfused and isolated cauda epididymidis or from an epididymis whose corresponding efferent ducts had been ligated for 40 days) was significantly higher than the activity of these enzymes in normal fluid (39.6 and 41.2 units/mg protein, respectively). Arylsulphatase activity of the locally formed fluid (11.2 units/mg protein) was lower than that of normal fluid (74.1 units/mg protein). The rete testis fluid was relatively rich in arylsulphatase since the ratio of arylsulphatase to acid phosphatase activity was 17 times higher in this fluid than in locally formed fluid. It is concluded that the activities of NAG and acid phosphatase in normal fluid from the epididymis originate in the epididymal tissue, while most of the arylsulphatase activity comes from the testis.

Acetylglucosaminidase↗
PubMedDate not supplied

Acrosome content release in streptolysin O permeabilized mouse spermatozoa.

Sperm cell plasma membrane and the outer acrosomal membrane fuse profusely during the acrosome reaction. The process is triggered by extracellular signals that elicit several intracellular events leading ultimately to membrane fusion. We have developed a streptolysin O permeabilizing protocol that selectively affects the spermatozoon plasma membrane without causing a significant loss of the acrosomal content. Most of the acrosomal acid phosphatase remained sperm-associated even after a 20 min incubation at 37 degrees C. However, the presence of 100 microM Ca2+ in the incubation buffer stimulates the release of the enzyme. The reaction was followed biochemically, measuring the acid phosphatase activity released to the medium and morphologically by the binding of fluorescein isothiocynate-conjugated peanut agglutinin and by electron microscopy. The results show that the streptolysin O permeabilized spermatozoon is a promising model for studying the complex set of events mediating and regulating the acrosome reaction.

Acid Phosphatase↗
PubMedDate not supplied

Affinity sites for beta-glucuronidase on the surface of human spermatozoa.

Glycosidases secreted by the epididymis become bound to the surface of spermatozoa during their transit through the epididymal duct. They are believed to play a role in mammalian fertilization. In the present report, we demonstrate that beta-glucuronidase binds to the surface of ejaculated human spermatozoa with high affinity and in a saturable manner. The binding is Ca(2+)-independent, inhibited by either mannose-6-phosphate, phosphomannan fragments from the yeast Hansenula holstii and alpha-mannosidase from the Dictyostelium discoideum, suggesting that phosphomannosyl receptors are involved in the recognition of the enzyme. The catalytic site of the enzyme is not involved in the binding. The localization of the beta-glucuronidase binding-sites is restricted to the surface of the sperm head. These results suggest that the spermatozoa could be the target for glycosidases present in the seminal plasma.

Alkaline Phosphatase↗
PubMedDate not supplied

Inhibition of early endosome fusion by Trypanosoma cruzi-infected macrophage cytosol.

Trypanosoma cruzi trypomastigotes survive inside macrophages by promoting fusion between the parasitophorous vacuole and mature host lysosomes upon internalization. Since trypomastigotes can evade the lytic pathway, the earliest steps of endocytosis, such as early endosome fusion, may be affected. To test this hypothesis, we used an in vitro early endosome fusion assay. Our results show that trypomastigote-infected macrophage cytosols cannot promote fusion between early endosomes, compared to mock-infected cytosols (heat-killed trypomastigotes were used in the parasite-macrophage interaction assay). GTP gamma S addition potentiates the fusogenic activity driven by trypomastigote-infected macrophage cytosol-mediated assays, unlike the biphasic fusogenic effect obtained with GTP gamma S treatment of macrophage cytosol controls. Calcium-stimulated early endosome fusogenic processes are not affected in the assays mediated by infected macrophage cytosol. We conclude that GTP-regulated factors, and not calcium-regulated elements, are involved in the inhibition of the early endosome fusogenic process by the trypomastigote-infected macrophage cytosol. This primary impediment to the progress of a normal endocytosis may be a relevant step required for the lysosomal recruitment-fusion of the host lysosomes upon trypomastigote infection and further survival of the parasite within its host.

Animals↗