SNAP-25 a membrane-associated protein of the nerve terminal, is specifically cleaved by botulinurn neurotoxins serotypes A and E, which cause human and animal botulism by blocking neurotransmitter release at the neuromuscular junction. Here we show that these two metallo-endopeptidase toxins cleave SNAP-25 at two distinct carboxyl-terminal sites. Serotype A catalyses the hydrolysis of the Gln'97-Arg'98 peptide bond, while serotype E cleaves the Arg'80-Ile'8' peptide linkage. These results indicate that the carboxyl-terminal region of SNAP-25 plays a crucial role in the multi-protein complex that mediates vesicle docking and fusion at the nerve terminal.
In neurons, depolarization induces Ca2+ influx leading to fusion of synaptic vesicles docked at the active zone for neurotransmitter release. While a number of proteins have now been identified and postulated to participate in the assembly and subsequent disengagement of a vesicle docking complex for fusion, the mechanism that ultimately triggers neuroexocytosis remains elusive. Using a cell-free, lysed synaptosomal membrane preparation, we show that Ca2+ alone is sufficient to trigger secretion of glutamate and furthermore that Ca2+-signaled exocytosis is effectively blocked by antibodies and peptides to a Several lines of evidence have identified three synaptic proteins, syntaxin, VAMP, and SNAP-25, which together with synaptotagmin form the core complex postulated to coordinate regulated vesicular fusion for neurotransmitter release (see refs. 3-5). In vitro studies with solubilized and recombinant proteins have begun to define the molecular relationships between these proteins (6-9). In an initial 7S complex, SNAP-25 binds both syntaxin 1A and VAMP to increase the strength and specificity of their interaction (8, 9). Binding of a-SNAP to this complex displaces synaptotagmin from syntaxin and provides entry of N-ethylmaleimide-sensitive fusion protein (NSF) to form a 20S prefusion complex that upon ATP hydrolysis subsequently leads to its disassembly (6). These observations have led to the proposal that neurotransmission represents a specialization of general membrane trafficking in which recognition between neural-specific vesicle-and targetsoluble NSF attachment protein receptors (v-and t-SNAREs) targets synaptic vesicles to the plasma membrane and the ATP hydrolysis step mediated by NSF to drive exocytosis (10).These in vitro studies, however, have yet to resolve the full roles played by these synaptic vesicle and plasma membrane proteins in the cascade of events required for neurotransmitter release. For
The mouse mutant coloboma (Cm/+), which exhibits profound spontaneous hyperactivity and bears a deletion mutation on chromosome 2, including the gene encoding synaptosomal protein SNAP‐25, has been proposed to model aspects of attention‐deficit hyperactivity disorder. Increasing evidence suggests a crucial role for SNAP‐25 in the release of both classical neurotransmitters and neuropeptides. In the present study, we compared the release of specific neurotransmitters in vitro from synaptosomes and slices of selected brain regions from Cm/+ mice with that of +/+ mice. The release of dopamine (DA) and serotonin (5‐HT) from striatum, and of arginine vasopressin and corticotropin‐releasing factor from hypothalamus and amygdala is calcium‐dependent. Glutamate release from and content in cortical synaptosomes of Cm/+ mice are greatly reduced, which might contribute to the learning deficits in these mutants. In dorsal striatum of Cm/+ mutants, but not ventral striatum, KCI‐induced release of DA is completely blocked and that of 5‐HT is significantly attenuated, suggesting that striatal DA and 5‐HT deficiencies may be involved in hyperactivity. Further, although acetylcholine failed to induce hypothalamic corticotropin‐releasing factor release from Cm/+ slices, restraint stress increased plasma corticosterone levels in Cm/+ mice to a significantly higher level than in +/+ mice, suggesting an important role for arginine vasopressin in hypothalamic‐pituitary‐adrenal axis activation. These results suggest that reduced SNAP‐25 expression may contribute to a region‐specific and neurotransmitter‐specific deficiency in neurotransmitter release.
Normal mammary gland homeostasis requires the coordinated regulation of protein signaling networks. However, we have little prospective information on whether activation of protein signaling occurs in premalignant mammary epithelial cells, as represented by cells with cytological atypia from women who are at high risk for breast cancer. This information is critical for understanding the role of deregulated signaling pathways in the initiation of breast cancer and for developing targeted prevention and/or treatment strategies for breast cancer in the future. In this pilot and feasibility study, we examined the expression of 52 phosphorylated, total, and cleaved proteins in 31 microdissected Random Periareolar Fine Needle Aspiration (RPFNA) samples by high-throughput Reverse Phase Protein Microarray. Unsupervised hierarchical clustering analysis indicated the presence of four clusters of proteins that represent the following signaling pathways: (1) receptor tyrosine kinase/Akt/mammalian target of rapamycin (RTK/Akt/mTOR), (2) RTK/Akt/extracellular signal-regulated kinase (RTK/Akt/ERK), (3) mitochondrial apoptosis, and (4) indeterminate. Clusters 1 through 3 comprised moderately to highly expressed proteins, while Cluster 4 comprised proteins that are lowly expressed in a majority of RPFNA samples. Our exploratory study showed that the interlinked components of mitochondrial apoptosis pathway are highly expressed in all mammary epithelial cells obtained from high-risk women. In particular, the expression levels of anti-apoptotic Bcl-xL and pro-apoptotic Bad are positively correlated in both non-atypical and atypical samples (unadjusted P < 0.0001), suggesting a delicate balance between the pro-apoptotic and anti-apoptotic regulation of cell proliferation during the early steps of mammary carcinogenesis. Our feasibility study suggests that the activation of key proteins along the RTK/Akt pathway may tip this balance to cell survival. Taken together, our results demonstrate the feasibility of mapping proteomic signaling networks in limited RPFNA samples obtained from high-risk women and the promise of developing rational drug targets or preventative strategies for breast cancer in future proteomic studies with a larger cohort of high-risk women.
Intraperitoneal administration of the nontoxic silicon compound, 1-ethoxysilatrane, to the rat did not cause proliferation of hepatic mitochondria or of endoplasmic reticulum, nor did it affect mitochondrial oxidative phosphorylation. The activities of cholesterol 7 alpha-hydroxylase in hepatic microsomes and of cholesterol oxidase in mitochondria respectively were unaffected by silatrane treatment. The rate of release of bile, whose composition remained unchanged, also was not increased in silatrane-treated animals. The results indicated that the compound did not affect the pathway of cholesterol degradation. A progressive decrease in the activity of hepatic microsomal 3-hydroxy-3-methylglutaryl coenzyme A (HMGCoA) reductase was observed on administration of the compound over a period of three weeks. Consistent with this, cholesterol biosynthesis in liver as measured by incorporation of radioactive precursors, acetate and water but not mevalonate, was significantly decreased in silatrane-treated animals. However, enzyme-linked immunosorbent assay revealed that the concentration of HMGCoA reductase protein was not decreased by the treatment indicating that inactivated enzyme was also present in such microsomes. Addition of silatrane to microsomes in the assay system did not cause inhibition indicating that the inactivation is by an indirect mechanism. It is concluded that the hypocholesterolemic action of the compound rested entirely on the inhibition of cholesterol biosynthesis in vivo by inactivation of the rate-limiting enzyme HMGCoA reductase.
Periodontitis is an infectious inflammatory disease. Turmeric and aloe vera has been used in dentistry for treating various oral conditions. In recent years various therapies like host response modulation and local drug delivery have been developed to block the pathways responsible for periodontal tissue breakdown. Although much information are there on the medical uses of Aloe vera and turmeric, limited literature is available regarding its use in field of dentistry. The purpose of this study was to evaluate the efficacy of Aloe vera as an adjunct to scaling and root planing (SRP) in patients with chronic periodontitis. The study included 20 subjects each with at least two periodontal pockets with PPD (probing pocket depths) ≥ 5-6 mm. The subjects were randomly divided into two groups. Scaling and root planning was performed for both the groups. Group I received turmeric chip and Group II received an Aloe vera chip. The clinical parameters including plaque index, gingival index, probing pocket depths and relative attachment levels were recorded at baseline, 21 days and 90 days. Both group showed improvement in site specific & full month plaque scores. Improvement in plaque score was significantly greater in the aloe group compared to turmeric group at 3 months. The GI in both group showed no difference at baseline & at 21 days, but there was a significant decrease in the GI score in the Aloe group compared to turmeric group at 3 months. PPD & CAL also showed no difference in both group at baseline & at 21 days & showed significant PPD reduction & CAL at 3 months Local drug delivery of Aloe vera chip into the periodontal pocket stimulated a significant increase in pocket depth reduction and clinical attachment level gain compared to turmeric chip as an adjunct to scaling and root planning in chronic periodontitis patients
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