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Biomedical subjects

E P Garcia

Publications and source records attributed to E P Garcia.

12 recordsLinked to original sources
PubMed2001

Kainate receptor activation induces mixed lineage kinase-mediated cellular signaling cascades via post-synaptic density protein 95.

Kainate receptor glutamate receptor 6 (GluR6) subunit-deficient and c-Jun N-terminal kinase 3 (JNK3)-null mice share similar phenotypes including resistance to kainite-induced epileptic seizures and neuronal toxicity (Yang, D. D., Kuan, C-Y., Whitmarsh, A. J., Rincon, M., Zheng, T. S., Davis, R. J., Rakis, P., and Flavell, R. (1997) Nature 389, 865-869; Mulle, C., Seiler, A., Perez-Otano, I., Dickinson-Anson, H., Castillo, P. E., Bureau, I., Maron, C., Gage, F. H., Mann, J. R., Bettler, B., and Heinemmann, S. F. (1998) Nature 392, 601-605). This suggests that JNK activation may be involved in GluR6-mediated excitotoxicity. We provide evidence that post-synaptic density protein (PSD-95) links GluR6 to JNK activation by anchoring mixed lineage kinase (MLK) 2 or MLK3, upstream activators of JNKs, to the receptor complex. Association of MLK2 and MLK3 with PSD-95 in HN33 cells and rat brain preparations is dependent upon the SH3 domain of PSD-95, and expression of GluR6 in HN33 cells activated JNKs and induced neuronal apoptosis. Deletion of the PSD-95-binding site of GluR6 reduced both JNK activation and neuronal toxicity. Co-expression of dominant negative MLK2, MLK3, or mitogen-activated kinase kinase (MKK) 4 and MKK7 also significantly attenuated JNK activation and neuronal toxicity mediated by GluR6, and co-expression of PSD-95 with a deficient Src homology 3 domain also inhibited GluR6-induced JNK activation and neuronal toxicity. Our results suggest that PSD-95 plays a critical role in GluR6-mediated JNK activation and excitotoxicity by anchoring MLK to the receptor complex.

Animals↗
PubMed1998

SAP90 binds and clusters kainate receptors causing incomplete desensitization.

The mechanism of kainate receptor targeting and clustering is still unresolved. Here, we demonstrate that members of the SAP90/PSD-95 family colocalize and associate with kainate receptors. SAP90 and SAP102 coimmunoprecipitate with both KA2 and GluR6, but only SAP97 coimmunoprecipitates with GluR6. Similar to NMDA receptors, GluR6 clustering is mediated by the interaction of its C-terminal amino acid sequence, ETMA, with the PDZ1 domain of SAP90. In contrast, the KA2 C-terminal region binds to, and is clustered by, the SH3 and GK domains of SAP90. Finally, we show that SAP90 coexpressed with GluR6 or GluR6/KA2 receptors alters receptor function by reducing desensitization. These studies suggest that the organization and electrophysiological properties of synaptic kainate receptors are modified by association with members of the SAP90/PSD-95 family.

Amino Acid Sequence↗
PubMed1996

A presynaptic inositol-5-phosphatase.

Synaptojanin is a nerve terminal protein of relative molecular mass 145,000 which appears to participate with dynamin in synaptic vesicle recycling. The central region of synaptojanin defines it as a member of the inositol-5-phosphatase family, which includes the product of the gene that is defective in the oculocerebrorenal syndrome of Lowe. Synaptojanin has 5-phosphatase activity and its amino-terminal domain is homologous with the yeast protein Sac1 (Rsd1), which is genetically implicated in phospholipid metabolism and in the function of the actin cytoskeleton. The carboxy terminus, which is of different lengths in adult and developing neurons owing to the alternative use of two termination sites, is proline-rich, consistent with the reported interaction of synaptojanin with the SH3 domains of Grb2 (refs 1, 2). Synaptojanin is the only other major brain protein besides dynamin that binds the SH3 domain of amphiphysin, a presynaptic protein with a putative function in endocytosis. Our results suggest a link between phosphoinositide metabolism and synaptic vesicle recycling.

Amino Acid Sequence↗
PubMed1995

v- and t-SNAREs in neuronal exocytosis: a need for additional components to define sites of release.

Synaptic vesicle recycling is a specialized form of membrane recycling which takes place in all cells between early endosomes and the plasmalemma. Synaptic vesicles exocytosis is highly regulated and occurs only at presynaptic active zones. In contrast, exocytosis of endosome-derived vesicles of the housekeeping recycling pathway takes place constitutively and throughout the cell surface. Since v- and t-SNAREs play a key role in membrane interactions leading to fusion, unique v- and t-SNAREs may be implicated in synaptic vesicle exocytosis. It was found, however, that the same v-SNAREs of the synaptobrevin family are found both on synaptic vesicles and on endosome-derived vesicles which undergo constitutive fusion. Likewise, t-SNAREs which act as plasmalemmal receptors for synaptic vesicles are not restricted to synaptic active zones. Thus, v- and t-SNAREs interactions may define which organelles can fuse with the plasmalemma, but require additional components to define properties of the exocytotic reaction which are specific for distinct classes of secretory organelles.

Animals↗
PubMed1995

rbSec1A and B colocalize with syntaxin 1 and SNAP-25 throughout the axon, but are not in a stable complex with syntaxin.

rbSec1 is a mammalian neuronal protein homologous to the yeast SEC1 gene product which is required for exocytosis. Mutations in Sec1 homologues in the nervous systems of C. elegans and D. melanogaster lead to defective neurotransmitter secretion. Biochemical studies have shown that recombinant rbSec1 binds syntaxin 1 but not SNAP-25 or synaptobrevin/VAMP, the two proteins which together with syntaxin 1 form the synaptic SNARE complex. In this study we have examined the subcellular localization of rbSec1 and the degree of interaction between rbSec1 and syntaxin 1 in situ. rbSec1, which we show here to be represented by two alternatively spliced isoforms, rbSec1A and B, has a widespread distribution in the axon and is not restricted to the nerve terminal. This distribution parallels the localization of syntaxin 1 and SNAP-25 along the entire axonal plasmalemma. rbSec1 is found in a soluble and a membrane-associated form. Although a pool of rbSec1 is present on the plasmalemma, the majority of membrane-bound rbSec1 is not associated with syntaxin 1. We also show that rbSec1 is not part of the synaptic SNARE complex or of the syntaxin 1/SNAP-25 complex we show to be present in non-synaptic regions of the axon. Thus, in spite of biochemical studies demonstrating the high affinity interaction of rbSec1 and syntaxin 1, our results indicate that rbSec1 and syntaxin 1 are not stably associated. They also suggest that the function of rbSec1, syntaxin 1, and SNAP-25 is not restricted to synaptic vesicle exocytosis at the synapse.

Alternative Splicing↗
PubMed1994

A rat brain Sec1 homologue related to Rop and UNC18 interacts with syntaxin.

Sec1 is a hydrophilic protein that plays an essential role in exocytosis from the yeast Saccharomyces cerevisiae. Two high copy suppressors of mutations in the Sec1 gene, SSO1 and SSO2, were recently identified that encode proteins of the syntaxin family. Syntaxin (a T-SNARE), together with SNAP-25 and synaptobrevin/VAMP (a T- and a V-SNARE, respectively), is thought to form the core of the docking-fusion complex in synaptic vesicle exocytosis. Proteins that exhibit similarity to Sec1 were identified in the nervous system of Drosophila melanogaster (Rop) and Caenorhabditis elegans (UNC18). Based on the amino acid sequence alignment of Sec1, Rop, and UNC18, we have used a PCR-based approach to isolate a rat brain cDNA encoding a Sec1 homologue. The cDNA hybridizes to a 3.5-kb brain-specific mRNA by Northern blot analysis and encodes a protein of 593 amino acids (rbSec1). Antibodies raised against a central portion of rbSec1 recognize a 67.5-kDa protein in total homogenates of rat brain but not of nonneuronal tissues. When incubated with a Triton X-100 brain extract, rbSec1-glutathione S-transferase (GST) fusion protein, but not GST protein alone, specifically interacts with syntaxin but not with SNAP-25 or synaptobrevin/VAMP. We conclude that the function of proteins of the Sec1 family in membrane fusion involves an interaction with a T-SNARE.

Amino Acid Sequence↗
PubMed1991

Influence of major histocompatibility complex class I, class II and TLA genes on tumor rejection.

T lymphocytes recognize antigen associated with MHC class I and/or class II gene products. Recognition of malignant cells is therefore dependent on presentation of tumor associated antigen(s) by MHC molecules. We have studied immunity to tumors that have down-regulated class I expression. These studies demonstrate a requirement for class I antigens, but suggest that additional factors may also be required for tumor-specific immunity. The MHC also encodes TLA class I antigens, whose function is unknown. Our studies suggest that these molecules function is unknown. Our studies suggest that T lymphocytes, specifically in tumor cells that do not express H-2K or H-2D moieties. Other studies are aimed at improving tumor-specific Th cell generation by producing class II+ tumor cells. The success of these experiments indicates that this approach may be a potentially useful immunotherapy.

Animals↗
PubMed1989

Embryonal carcinoma cells express Qa and Tla class I genes of the major histocompatibility complex.

The murine major histocompatibility complex encodes H-2K and H-2D transplantation antigens and other class I-like proteins called Qa and Tla molecules; the functions of the Qa/Tla molecules are not known. That they may participate in embryonic cell-cell interactions and/or play a role in immune responses against tumors has been speculated. We have studied two murine embryonal carcinoma tumors, 402AX and PCC4, that are rejected in vivo immunologically, although they do not express H-2K or H-2D antigens. Transplantation studies with these cells suggest that rejection is mediated by class-I-like major histocompatibility complex antigens. As a first step in evaluating Qa/Tla function(s), we have characterized expression of class I-like genes and proteins in 402AX and PCC4 cells. Northern (RNA) blot hybridizations, polymerase chain reaction studies, and cDNA cloning experiments demonstrate that EC lines transcribe genes allelic to the Tla region gene "37", Qa-2 region gene "Q7", and another, previously uncharacterized, class I-like gene. Immunoprecipitation studies show that the embryonal carcinoma tumor cells contain low levels of beta 2-microglobulin expressed in association with non-H-2K, non-H-2D class I-like proteins.

Amino Acid Sequence↗
PubMed1987

Allogeneic H-2 antigen expression is insufficient for tumor rejection.

Murine A strain (KkDdLd) sarcoma I (SaI) tumor cells have been transfected with a cloned H-2Kb gene. The resulting clones (SKB clones) stably express high levels of a molecule that is serologically and biochemically indistinguishable from the H-2Kb antigen. SKB clones are not susceptible to cytotoxic T lymphocyte-mediated lysis by H-2Kb-specific bulk, cloned, or H-2Kb-restricted lymphocytic choriomeningitis virus-specific effectors. Survival times of A/J and B10.A mice challenged i.p. with the H-2Kb-expressing transfectants and the parental SaI cells are similar, suggesting that the presence of an allogeneic major histocompatibility complex class I antigen on the surface of this tumor line is insufficient for tumor rejection.

Animals↗
PubMedDate not supplied

Transperitoneal conversion of the ureter after Wertheim's operation with lymphadenectomy.

The incidence of ureteral fistula occurring after Wertheim's operation with lymphadenectomy, performed at our Department since 1953, has been high. Therefore, in 1970, a new procedure (a modification of the technique proposed by Novak) was attempted: the peritoneal flaps were used to cover the pelvic wall, which was previously completely cleaned to assure total lymphadenectomy. Thus a new bed is provided for the isolated ureter, which is prevented from falling into the retroperitoneal space. One hundred and thirty-three cases were operated on according to this technique and the incidence of ureteral fistulas fell from 10 to 0.75 per cent. Intravenous urographies taken two, six and ten months after the operation showed good results. All patients in this series who underwent this operation had previously had irradiation.

Humans↗