Biomedical subjects

M Lavasa

Publications and source records attributed to M Lavasa.

8 recordsLinked to original sources

PubMed1994

A general method for plasma membrane isolation by colloidal gold density shift.

A general method for isolating plasma membranes is described. Vegetative amoebae of Dictyostelium discoideum were allowed to directly adsorb raw colloidal gold of particle diameter 10-20 nm. After quenching the gold surface, the cells were lysed and the lysates were diluted in 60% sucrose and centrifuged through a 65% sucrose cushion to selectively pellet the gold-laden membranes. Three generally applicable exogenous cell surface markers were used to follow the plasma membranes: intercalated [3H]cholesterol, octadecylrhodamine, and the adsorbed gold colloid itself. The isolates routinely contained approximately 60% of these tags, enriched approximately 15-fold with respect to protein. The recovery and degree of enrichment of contaminating markers in the plasma membrane fraction were lysosomes (3% and 1-fold); mitochondria (11% and 3-fold); rough endoplasmic reticulum, as reflected by RNA (3% and 0.7-fold); and DNA (9% and 4-fold). Membrane proteins and lipids were quantitatively solubilized from the gold by detergents. We conclude that this methodology provides an approach to the isolation of plasma membranes which compares favorably to existing techniques with respect to yield, purity, and ease.

Cell Membrane↗

PubMed1993

A post-lysosomal compartment in Dictyostelium discoideum.

Fluorescein isothiocyanate (FITC)-dextran and pyranine were fed to the social amoeba, Dictyostelium discoideum. These membrane-impermeable, pH-sensitive fluorophores initially entered a approximately equal to neutral endocytic compartment. They encountered maximal acidity (pH approximately equal to 5) about 15 min after ingestion, in what appeared to be digestive vacuoles (lysosomes). The environment of the probes returned to near neutrality by 30 min. At that time, the probes accumulated in a decreasing number of vacuoles of increasing size; ultimately, there were only a small number of vacuoles per cell with diameters of up to 3 microns. The late vacuoles sedimented more rapidly than did proton pumps, acid hydrolases, and recently ingested cargo. Unlike the vacuoles harvested immediately after the cells were fed FITC-dextran, the late vacuoles were not acidified by MgATP in vitro. Egestion of ingested FITC-dextran commenced after a lag of approximately equal to 45 min. A similar lag was observed for the resurfacing of two endocytosed bilayer-intercalated fluorophores. These results suggest that, in Dictyostelium, undigested endocytic cargo accumulates in and is returned to the cell surface through a distinctive compartment of large and nearly neutral post-lysosomal vacuoles. It will be important to determine the degree to which internalized plasma membrane components follow this post-lysosomal pathway.

Animals↗

PubMed1991

Reconstitution of the association of endocytic vacuoles and acidosomes from Dictyostelium.

In the amoeba, Dictyostelium discoideum, endocytic vacuoles are acidified by proton pumps which reside not in their membranes but in an associated organelle which we call the acidosome. These two organelles can be dissociated in vitro, and we now describe conditions for their functional reassociation. Fluorescein 5-isothiocyanate-dextran was fed to amoebae to report on the pH of their endocytic vacuoles. Following homogenization, the endocytic vacuoles were dissociated from acidosomes by removing Mg2+ and cytosol and purged of their native acidity by transient exposure to nigericin. The endocytic vacuoles could then be reacidified by ATP if first preincubated under these optimized conditions: 30 degrees C for 30 min in the presence of acidosomes, a 4-fold excess of cytosol, and 5 mM Mg2+ at pH 7.4. Reacidification was observed with early but not late endocytic compartments. Mn2+ and Ca2+ were poor substitutes for Mg2+; albumin did not substitute for cytosol. Neither Ca2+, ATP, nor adenosine 5'-O-(3-thiotriphosphate) affected reconstitution appreciably; guanosine 5'-O-(3-thiotriphosphate) inhibited reacidification by 50% when present during preincubation at 0.1 mM. Warming the cytosol to 50 degrees C or exposing it to protease abolished its activity but N-ethylmaleimide did not. Molecular sieving indicated that the cytosolic factor was a macromolecule. We conclude that the specific functional association of acidosomes and endocytic vacuoles can be reconstituted in vitro with soluble proteins plus Mg2+.

Adenosine Triphosphatases↗

PubMed1991

Endosomes are acidified by association with discrete proton-pumping vacuoles in Dictyostelium.

The endocytic compartment in the amoeba Dictyostelium discoideum was labeled by feeding fluorescein 5-isothiocyanate-dextran. In homogenates containing 2 mM Mg2+, the compartments so labeled copurified with all of the vacuolar H(+)-ATPase activity in a dense peak. The fluorescence properties of the probe showed that these dense vacuoles were inherently acidic. Furthermore, after purging their residual acidity, they could be re-acidified by the addition of ATP. These data suggest that the H(+)-ATPase was structurally and functionally coupled to the endocytic space. The association of the H(+)-ATPase and endocytic compartment was reversed by the removal of either Mg2+ or traces of the cytosol. Endocytic vacuoles prepared in this way were deficient in vacuolar H(+)-ATPase activity and were not acidified upon addition of MgATP. The missing proton pumps were recovered in large buoyant vacuoles that lacked ingested fluorescein 5-isothiocyanate-dextran, acid hydrolases, and residual acidity. These vacuoles were also less susceptible than endosomes to disruption by digitonin, suggesting that their bilayers were low in sterols. These results indicate that the endocytic circuit in Dictyostelium is acidified by a discrete and separable proton-pumping organelle.

Biological Transport↗

PubMed1989

Characterization of a vacuolar proton ATPase in Dictyostelium discoideum.

Of the total ATPase activity in homogenates of the ameba, Dictyostelium discoideum, approximately one-third was inhibited at pH 7 by 25 microM 7-chloro-4-nitrobenzo-2-oxa-1,3-diazole (NBD-Cl). Upon isopycnic sucrose density gradient centrifugation, the bulk of the NBD-CI-sensitive ATPase activity was recovered in a major membrane fraction with a broad peak at 1.16 g/ml, well-resolved from markers for plasma membranes, mitochondria, lysosomes and contractile vacuoles. The gradient peak had a specific activity of 0.5 mumol/min per mg protein. The activity was half-inhibited by 1 microM silicotungstate, 2 microM diisothiocyanatostilbene disulfonate (DIDS), 2.5 microM dicyclohexylcarbodiimide (DCCD), 4 microM NBD-CI and 20 microM N-ethylmaleimide (NEM) but was resistant to conventional inhibitors of mitochondrial and plasma membrane ATPase. That this ATPase activity constituted a proton pump was shown by the MgATP-dependent uptake and quenching of Acridine orange fluorescence by partially purified vacuoles. The Acridine orange uptake was specifically blocked by the aforementioned inhibitors. The generation of proton electrochemical gradients was suggested by the stimulation of enzyme activity by protonophores (fatty acids) and cation exchangers (nigericin). Uncoupling stimulated the ATPase activity as much as 20-fold, revealing an unusually high impermeability of the membranes to protons. ATPase activity was also stimulated by halide ions, apparently through a parallel conductance pathway. Under a variety of sensitive test conditions, the reverse enzyme reaction (i.e., incorporation of 32Pi into ATP) was not detected. We conclude that this major H+-ATPase serves to acidify the abundant prelysosomal vacuoles found in D. discoideum (Padh et al. (1989) J. Cell Biol. 108, 865-874). The finding of a vacuolar H+-ATPase in a protist suggests the ubiquity of this enzyme among the eukaryotic kingdoms.

Dictyostelium↗

PubMed1989

Prelysosomal acidic vacuoles in Dictyostelium discoideum.

We have examined the ameba Dictyostelium discoideum for evidence of a discrete, prelysosomal, acidic receiving compartment in endocytosis. We observed in the cytoplasm abundant round vacuoles with diameters up to 2 microns that concentrated acridine orange by a process inhibited by 7-chloro-4-nitrobenzo-2-oxa-1,3-diazole (NBD-Cl). They were therefore taken to be acidic. The vacuoles were observed to fuse nearly quantitatively with primary phagosomes over 30 min and thereby to confer upon them the ability to accumulate acridine orange. The entry into lysosomes of phagocytic cargo occurred later. In the absence of phagocytosis, almost all of the acidic vacuoles rapidly accumulated fluorescent markers that had either been covalently coupled to the cell surface or fed as the soluble dextran conjugate. Therefore, these vacuoles also lie on the pathway of pinocytosis. A prominent subcellular ATPase activity inhibited by 25 microM NBD-Cl co-distributed on sucrose equilibrium density gradients with vacuoles capable of concentrating acridine orange in vitro. The peak was broad and more buoyant than that bearing lysosomal acid hydrolases, which contained only a minor amount of this ATPase. Also migrating in the buoyant peak were internalized plasma membrane markers; e.g., 3H-galactose had been covalently coupled to the surface of intact cells and allowed to enter pinosomes. We conclude that in D. discoideum an extensive prelysosomal vacuolar compartment provides the proton pumps that acidify both phagosomes and pinosomes.

Acid Phosphatase↗

PubMed1987

Neuroimmunology: modulation of the hamster immune system by photoperiod.

Groups of adult male Syrian hamsters were kept in a long photoperiod (LD 14:10) or a short photoperiod (LD 10:14). After 12 weeks, half of the animals in each light:dark cycle were immunized with an immunogenic amino acid polymer. Exposure to short photoperiod was associated with a significant reduction in testicular, accessory sex organ, splenic and brown fat weights. However, photoperiod length did not influence whole body, thymic, adrenal or kidney weights. Spleens of immunized animals in the long photoperiod were significantly heavier than those of unimmunized animals in the long photoperiod, and both were heavier than spleens from immunized or unimmuized animals in the short photoperiod. This reflected increased splenic lymphocyte and macrophage counts. However, there was no difference in antibody production between animals kept in different photoperiods. These results demonstrate that the daily photoperiod length affects both hamster reproductive competence as well as selected immune parameters (splenic weight and mononuclear cell hyperplasia) but does not alter antibody production.

Adipose Tissue, Brown↗

PubMed1987

Suppression of acute and relapsing experimental allergic encephalomyelitis with mitoxantrone.

The effect of treatment with the antineoplastic, immunomodulatory agent mitoxantrone on the course of acute and relapsing experimental allergic encephalomyelitis (EAE) in the mouse has been studied. Untreated mice immunized to produce acute EAE had an 81% incidence of clinical disease and 100% incidence of pathologic disease. Mice treated with mitoxantrone at a dose of 0.5 mg/kg daily for the 10 days following immunization did not develop any clinical signs and had minimal pathologic signs of disease. A dose of 0.25 mg/kg gave an intermediate response. Untreated mice immunized for relapsing EAE had a 100% incidence of disease with an average onset of disease on Day 148. Mice treated with mitoxantrone at a dose of 0.05 mg/kg three times weekly for 12 weeks following immunization had a 67% incidence of clinical disease with a significant delay in the average onset date to Day 279. These results indicate that mitoxantrone was highly effective in suppressing development of acute EAE. Mitoxantrone delayed the onset of relapsing EAE in mice, but did not fully inhibit the eventual expression of the disease. These studies suggest that the use of cytotoxic therapies in the treatment of autoimmune diseases may require periodic cycles of therapy to block disease expression.

Acute Disease↗