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M Roa

Publications and source records attributed to M Roa.

At least 19 recordsLinked to original sources
PubMed2000

Soluble NSF attachment protein receptors (SNAREs) in RBL-2H3 mast cells: functional role of syntaxin 4 in exocytosis and identification of a vesicle-associated membrane protein 8-containing secretory compartment.

Mast cells upon stimulation through high affinity IgE receptors massively release inflammatory mediators by the fusion of specialized secretory granules (related to lysosomes) with the plasma membrane. Using the RBL-2H3 rat mast cell line, we investigated whether granule secretion involves components of the soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) machinery. Several isoforms of each family of SNARE proteins were expressed. Among those, synaptosome-associated protein of 23 kDa (SNAP23) was central in SNARE complex formation. Within the syntaxin family, syntaxin 4 interacted with SNAP23 and all vesicle-associated membrane proteins (VAMPs) examined, except tetanus neurotoxin insensitive VAMP (TI-VAMP). Overexpression of syntaxin 4, but not of syntaxin 2 nor syntaxin 3, caused inhibition of FcepsilonRI-dependent exocytosis. Four VAMP proteins, i.e., VAMP2, cellubrevin, TI-VAMP, and VAMP8, were present on intracellular membrane structures, with VAMP8 residing mainly on mediator-containing secretory granules. We suggest that syntaxin 4, SNAP23, and VAMP8 may be involved in regulation of mast cell exocytosis. Furthermore, these results are the first demonstration that the nonneuronal VAMP8 isoform, originally localized on early endosomes, is present in a regulated secretory compartment.

Animals↗
PubMed1997

Involvement of the ras-like GTPase rab3d in RBL-2H3 mast cell exocytosis following stimulation via high affinity IgE receptors (Fc epsilonRI).

Aggregation of high affinity IgE receptors (Fc epsilonRI) expressed on mast cells and basophils is a potent stimulus for the release of inflammatory mediators from cytoplasmic granules. Fc epsilonRI-dependent exocytosis requires activation of protein kinase C and mobilization of calcium from intra- and extracellular stores. However, how these events ultimately regulate the membrane fusion step between cytoplasmic granules and the plasma membrane still remains unclear. In this study, we investigated the role of the small GTPases of the rab3 subfamily in the regulated exocytosis following stimulation of rat basophilic leukemia cells (RBL-2H3). Analysis using reverse-transcriptase-based PCR showed that RBL-2H3 cells expressed rab3a and rab3d isoforms, with a predominance of rab3d at the mRNA level. Investigation of the subcellular distribution using isoform-specific Abs demonstrated that the majority of rab3a was expressed in the cytosol, whereas rab3d was found predominantly in the membrane fraction. To determine whether these proteins play a role in Fc epsilonRI-triggered exocytosis, we established RBL-2H3 transfectants that overexpressed wild-type and expressed GTP-binding mutant forms (N135I) of rab3a and rab3d. Whereas expression of rab3a proteins did not significantly affect degranulation as tested by beta-hexosaminidase release, those of both wild-type and mutant rab3d proteins inhibited degranulation. Calculations of the initial fast and of the second slow release rates showed that they are both inhibited about twofold, suggesting that rab3d interferes with a rate-limiting step in Fc epsilonRI-stimulated exocytosis.

Amino Acid Sequence↗
PubMed1997

[Edwardsellia tarda septicemia in chronic lymphoid leukemia].

Edwardsiella tarda is a Gram negative bacilli. At least 300 cases have been reported in the literature. Here we report a new case of Edwardsiella tarda septicemia in an immunocompromised patient with chronic lymphocytic leukemia. Chief infections associated with this bacterium include bacterial gastroenteritis and septicemia with fatal evolution in 50% of cases. Risk factors associated with Edwardsiella tarda infections include exposure to aquatic environments and exotic animals. Although studies indicate that this bacterium is susceptible to most commonly prescribed antibiotics, it is interesting to note that in our case, Edwardsiella tarda was resistant to numerous beta-lactamins.

Bacteremia↗
PubMed1997

[A nidulans bronchial aspergillosis after treatment of low-grade lymphoma with fludarabine].

Pulmonary aspergillosis is a common complication in neutropenic patients. The most important fungus is Aspergillus fumigatus. We report a case of invasive pulmonary aspergillosis due to Aspergillus nidulans secondary to fludarabine therapy. There are few cases of pulmonary aspergillosis due to Aspergillus nidulans (one chronic necrotizing pulmonary aspergillosis, one aspergilloma). Fludarabine induces a marked decreased of CD4 lymphocyte count responsible for opportunistic infections. It is the first case of aspergillosis after fludarabine therapy and the occurrence of such infection must be considered after purine analog therapy.

Antineoplastic Agents↗
PubMed1994

The GTPase Rab3a negatively controls calcium-dependent exocytosis in neuroendocrine cells.

There is accumulating evidence that small GTPases of the rab family regulate intracellular vesicle traffic along biosynthetic and endocytotic pathways in eukaryotic cells. It has been suggested that Rab3a, which is associated with synaptic vesicles in neurons and with secretory granules in adrenal chromaffin cells, might regulate exocytosis. We report here that overexpression in PC12 cells of Rab3a mutant proteins defective in either GTP hydrolysis or in guanine nucleotide binding inhibited exocytosis, as measured by a double indirect immunofluorescence assay. Moreover, injection of the purified mutant proteins into bovine adrenal chromaffin cells also inhibited exocytosis, as monitored by membrane capacitance measurements. Finally, the electrophysiological approach showed that bovine chromaffin cells which were intracellularly injected with antisense oligonucleotides targeted to the rab3a messenger exhibited an increasing potential to respond to repetitive stimulations. In contrast, control cells showed a phenomenon of desensitization. These results provide clear evidence that Rab3a is involved in regulated exocytosis and suggest that Rab3a is a regulatory factor that prevents exocytosis from occurring unless secretion is triggered. Furthermore, it is proposed that Rab3a is involved in adaptive processes such as response habituation.

Adrenal Cortex↗
PubMed1994

The small GTP-binding protein rab6 functions in intra-Golgi transport.

Rab6 is a ubiquitous ras-like GTP-binding protein associated with the membranes of the Golgi complex (Goud, B., A. Zahraoui, A. Tavitian, and J. Saraste. 1990. Nature (Lond.). 345:553-556; Antony, C., C. Cibert, G. Géraud, A. Santa Maria, B. Maro, V. Mayau, and B. Goud. 1992. J. Cell Sci. 103: 785-796). We have transiently overexpressed in mouse L cells and human HeLa cells wild-type rab6, GTP (rab6 Q72L), and GDP (rab6 T27N) -bound mutants of rab6 and analyzed the intracellular transport of a soluble secreted form of alkaline phosphatase (SEAP) and of a plasma membrane protein, the hemagglutinin protein (HA) of influenza virus. Over-expression of wild-type rab6 and rab6 Q72L greatly reduced transport of both markers between cis/medial (alpha-mannosidase II positive) and late (sialyl-transferase positive) Golgi compartments, without affecting transport from the endoplasmic reticulum (ER) to cis/medial-Golgi or from the trans-Golgi network (TGN) to the plasma membrane. Whereas overexpression of rab6 T27N did not affect the individual steps of transport between ER and the plasma membrane, it caused an apparent delay in secretion, most likely due to the accumulation of the transport markers in late Golgi compartments. Overexpression of both rab6 Q72L and rab6 T27N altered the morphology of the Golgi apparatus as well as that of the TGN, as assessed at the immunofluorescence level with several markers. We interpret these results as indicating that rab6 controls intra-Golgi transport, either acting as an inhibitor in anterograde transport or as a positive regulator of retrograde transport.

Alkaline Phosphatase↗
PubMed1993

The small GTP-binding protein rab6p is redistributed in the cytosol by brefeldin A.

Rab6 protein belongs to the Sec4/Ypt/rab subfamily of small GTP-binding proteins involved in intracellular membrane trafficking in yeast and mammalian cells. Its localization both in medial and trans-Golgi network prompted us to study the effects of brefeldin A (BFA) on rab6p redistribution. By two techniques, indirect immunofluorescence and cell fractionation, we investigated the fate of rab6p and compared it to other Golgi or trans-Golgi network markers in BHK-21 and NIH-3T3 cells. BFA, at 5 micrograms/ml, induced redistribution of rab6p according to a biphasic process: during the first 10-15 minutes, tubulo-vesicular structures--colabelled with a bona fide medial Golgi marker called CTR 433--were observed; these structures were then replaced by punctate diffuse staining, which was stable for up to 3 hours. The 110 kDa peripheral membrane protein beta-COP was released much more rapidly from the Golgi membranes, whereas the trans-Golgi network marker TGN 38 relocated to the microtubule organizing center. The kinetics of reversion of BFA action on these antigens was also followed by immunofluorescence. Consistent with these results, rab6 antigen, originally found as 40% in the cytosolic versus 60% in the particulate (P 150,000 g) fraction, became almost entirely cytosolic; moreover, it partitioned in the aqueous phase of Triton X-114 whereas the membrane fraction was detergent-soluble. Rab6p did not become part of the coatomers after its BFA-induced release from Golgi structures. Three requirements seemed to be necessary for such a release: integrity of the microtubules, presence of energy, and a hypothetical trimeric G protein, as revealed by the respective roles of nocodazole, ATP depletion, and sensitivity to aluminium fluoride. Finally, we have shown that BFA does not prevent attachment of newly synthesized rab6p to membranes.

3T3 Cells↗
PubMed1991

Occurrence of neuropeptide K-like immunoreactivity in ventral horn cells of the chicken spinal cord during development.

The possible occurrence of NPK-LI in the ventral horns of the embryonic chicken spinal cord was investigated by means of the indirect immunofluorescence method. The results showed a transient appearance of NPK-LI in cells of the lateral motor column between day 5 of incubation and hatching. After this they disappeared and in the ventral horns NPK-LI remained only in fibers. The results are discussed in terms of a possible trophic action of NPK during development.

Animals↗
PubMed1991

Characterization and developmental evolution of a high-affinity binding site for calcitonin gene-related peptide on chick skeletal muscle membrane.

Calcitonin gene-related peptide is a putative neurotransmitter of central and peripheral nervous systems which coexists with acetylcholine in motor nerve terminals and exerts multiple effects on skeletal muscle, suggesting a trophic role for this neuropeptide. Using radiolabeled calcitonin gene-related peptide as a probe in a specific binding assay, we have characterized calcitonin gene-related peptide binding sites on chick skeletal muscle membranes. Binding is time-dependent, saturable and reversible. Scatchard analyses revealed two classes of sites: high-affinity sites with a KD value of 62 pM, and low-affinity sites with a KD value of 3.3 nM. The maximal number of sites is, respectively, 22 and 155 fmol/mg protein for high- and low-affinity binding sites. Specific binding was not affected by the presence, in excess, of other neuropeptides such as salmon calcitonin or somatostatin or vasoactive intestinal polypeptide. Affinity of the binding site for calcitonin gene-related peptide was decreased in the presence of 5'-guanylyl-imidodiphosphate, suggesting a physiological coupling of calcitonin gene-related peptide receptor to a GTP binding protein. In a developmental study of chick muscle, we found the highest activity of calcitonin gene-related peptide binding sites in 11-14 day embryos, following a pattern of evolution similar to that of acetylcholine receptors (constant ratio of 12 acetylcholine receptors per calcitonin gene-related peptide binding site). However, both receptors appear differentially regulated: while the number of acetylcholine receptors increases 5-16-fold after denervation, calcitonin gene-related peptide binding sites slightly diminish in number. These results are discussed in terms of the physiological significance of calcitonin gene-related peptide binding sites on chick skeletal muscle membrane.

Aging↗
PubMed1988

Calcitonin gene-related peptide enhances the rate of desensitization of the nicotinic acetylcholine receptor in cultured mouse muscle cells.

Calcitonin gene-related peptide (CGRP) is a neuropeptide that coexists with acetylcholine in spinal cord motoneurons. The effects of CGRP on the functional properties of the nicotinic acetylcholine receptor (AcChoR) were examined by electrophysiological methods. Using the whole-cell patch-clamp technique and a mouse cell line derived from soleus muscle, we found that CGRP produces a progressive and reversible enhancement of the rapid-decay phase of AcChoR desensitization. Single-channel data further show that CGRP decreases acetylcholine-activated channel opening frequency. This decrease occurs when CGRP and acetylcholine are applied on different cell-surface areas and thus is likely mediated by a second-messenger system. CGRP is also shown to increase cAMP accumulation in this cell line. The effects of CGRP on macroscopic acetylcholine-activated currents are mimicked by external application of forskolin (10 microM) or by internal perfusion of the cell with cAMP (1 microM). In both these cases, further application of CGRP produces no additional enhancement of AcChoR desensitization. These results suggest that, on mouse muscle cells, CGRP regulates AcChoR desensitization by a mechanism that involves, at least in part, cAMP-dependent phosphorylation of the AcChoR.

Animals↗
PubMed1987

A subpopulation of embryonic telencephalic neurons survive and develop in vitro in response to factors derived from the periphery.

Denervated chick muscle contains factors that enhance neurite outgrowth in cultures of embryonic chicken spinal neurons. Chromatography of muscle extract on a column of DEAE-Sepharose yielded a fraction which retained most of the starting neurite-promoting activity. This DEAE fraction was tested for its activity on neurons from other regions of the central nervous system of 5-day-old chicken embryos. Both neurite outgrowth and survival of telencephalic neurons in vitro were greatly enhanced when the DEAE fraction was added at protein concentrations around 1 microgram/ml. When cultures were prepared from embryos later than 6 days in ovo, the effects of the DEAE fraction progressively diminished with age. Neurons from the embryonic diencephalon, mesencephalon and rhombencephalon were not responsive to the DEAE fraction, although they all developed neurites on a laminin substratum. Similar neurite-promoting activities for telencephalic neurons were found in extracts of neonatal brain, liver and heart, but not lung.

Animals↗
PubMed1986

Tetanus toxin is labeled with photoactivatable phospholipids at low pH.

The mechanism of cell penetration by tetanus toxin is unknown; it has been suggested that the toxin may penetrate into the lipid bilayer from a low-pH vesicular compartment. In this work, the interaction of tetanus toxin with liposomal model membranes has been studied by following its photoinduced cross-linking with either a nitrene or a carbene photolytically generated from corresponding light-sensitive phosphatidylcholine analogues. The toxin was labeled only at pHs lower than 5.5. The low pH acquired hydrophobicity of tetanus toxin appears to be confined to its light chain and to the 45-kDa NH2-terminal fragment of the heavy chain. Negatively charged lipids promote the interaction of this toxin with the hydrocarbon chain of phospholipids. The relevance of the present findings to the possible mechanism of nerve cell penetration by tetanus toxin is discussed.

Carbon Radioisotopes↗
PubMed1985

Interaction of tetanus toxin with lipid vesicles at low pH. Protection of specific polypeptides against proteolysis.

Two main polypeptides, Mr about 27,000 and 21,000, were protected against pepsin proteolysis when a mixture consisting of asolectin vesicles and 125I-labeled tetanus toxin was subjected to a pH drop from 7.2 to 3.0. The same result was obtained with the amino-terminal portion of the toxin (called fragment B). These polypeptides were not found to be protected in the following conditions: (i) when vesicles were omitted from the mixture; (ii) when the external pH of the vesicles was maintained at 7.2 and trypsin was used as a proteolytic agent; and (iii) when the vesicles were ruptured either before or after addition of the toxin. By specific immunoprecipitation, we identified the protected polypeptides as part of the central fragment of tetanus toxin. In addition, a 15.5-kDa polypeptide, belonging to toxin fragment C, was shown to be particularly resistant to digestion by various proteases, even in the absence of lipid vesicles. Based on these findings, we propose a model for entry of tetanus toxin into its target cells.

Electrophoresis, Polyacrylamide Gel↗
PubMed1983

Biosynthesis of peroxisomal enzymes in the methylotrophic yeast Hansenula polymorpha.

The dramatic expansion of the peroxisomal compartment known to occur in the methanol-utilizing yeast Hansenula polymorpha on transfer from glucose- to methanol-containing media was shown to be accompanied by the synthesis of at least six major polypeptides that dominate the polypeptide pattern of total cell extracts analyzed by NaDodSO(4)/polyacrylamide gel electrophoresis. Two of these polypeptides have been identified by immunochemical methods as the monomers of the peroxisomal enzymes alcohol oxidase and catalase. We have studied the biosynthesis of these two peroxisomal enzymes, both by in vitro translation and by in vivo labeling experiments. By the criterion of mobility in NaDodSO(4)/polyacrylamide gel electrophoresis, the in vitro- and in vivo-synthesized monomers were indistinguishable from each other, both in the case of alcohol oxidase and in that of catalase. Thus, neither of these peroxisomal enzymes appear to be synthesized as larger precursors. However, further analysis of in vitro-synthesized versus mature peroxisomal alcohol oxidase showed that the in vitro-synthesized form sedimented as a 5S monomer and not, like the mature peroxisomal enzyme, as a 20S octamer. Moreover, the in vitro-synthesized form was highly susceptible to trypsin digestion whereas the mature 20S octamer appeared to be resistant.

Journal Article↗