Search PubMed⌕ Search

Biomedical subjects

M C Wilson

Publications and source records attributed to M C Wilson.

At least 37 records · Page 2Linked to original sources
PubMed1998

Transient, high levels of SNAP-25 expression in cholinergic amacrine cells during postnatal development of the mammalian retina.

In the present study, we have examined the development of cholinergic amacrine cells in the retina of the Brazilian opossum, Monodelphis domestica. An antibody directed against choline acetyltransferase (ChAT) revealed that ChAT-like immunoreactivity (ChAT-IR) was first observed at 15 days postnatal (15PN). By 25PN, ChAT-IR identified two matching populations of amacrine cells in the inner nuclear and ganglion cell layer. Bromodeoxyuridine birth-dating analysis coupled with immunolabeling with the anti-ChAT antibody revealed that the cholinergic amacrine cells are born postnatally, between 2PN and 15PN. In addition, we have examined the differentiation of the cholinergic amacrine cells by using an antibody directed against a presynaptic terminal-associated protein, synaptosomal-associated protein of 25 kDa (SNAP-25). Double-labeling analysis revealed that relatively high levels of SNAP-25-IR were selectively present in cholinergic amacrine cells prior to eye opening. However, in the mature retina, high levels of SNAP-25-IR were no longer observed in the ChAT-IR amacrine cells. These results reveal a distinct period in development, prior to eye opening, when high levels of SNAP-25-IR are selectively expressed in cholinergic amacrine cells. The specificity and time course of the high levels of SNAP-25 in cholinergic amacrine cells may be critical in mediating the transient properties of these cells during visual system development.

Animals↗
PubMed1998

Möbius syndrome with oculomotor nerve paralysis without abducens paralysis.

Möbius syndrome is typified by bilateral facial nerve palsies, usually with abducens palsies. We examined an infant with Möbius syndrome who had bifacial weakness and third nerve palsies, but intact abduction of both eyes. Lower cranial nerve involvement, leading to respiratory, swallowing, and cardiac difficulties, was also present. Pathologic examination of the brainstem showed absent or hypoplastic third, seventh, tenth, and twelfth nerve nuclei. The fourth, fifth, sixth, and eighth nerve nuclei were intact. In Möbius syndrome with ocular motor palsies, rarely the sixth nerve may be spared.

Abducens Nerve↗
PubMed1997

T-cell receptor repertoire in matched MART-1 peptide-stimulated peripheral blood lymphocytes and tumor-infiltrating lymphocytes.

Characterization of tumor-associated antigens (TAAs) recognized by CTLs makes the consideration of therapeutic strategies based on peptide stimulation of peripheral blood lymphocytes (PBLs) feasible. Several such approaches are adoptive transfer of peptide-stimulated PBLs, ex vivo peptide stimulation of dendritic cells, and direct vaccination with TAA-derived peptides. A critical component of any of these peptide-based strategies is the requirement that the patient's PBLs are able to react productively against the presented TAA. The purpose of this study, through the study of T-cell receptor (TCR) usage, was to evaluate the T-cell response in matched MART-1(27-35) peptide-stimulated PBLs and tumor-infiltrating lymphocytes (TILs). MART-1(27-35)-reactive PBL and TIL cultures were generated from three patients by in vitro stimulation with an immunodominant peptide of MART-1 (MART-1(27-35)). All cultures had a human leukocyte antigen A2-restricted, MART-1(27-35)-specific CTL response. The TCR usage of each was assessed by the DNA sequence analysis of 50 TCR beta clones obtained by rapid amplification of cDNA ends per culture. TCR analysis suggests a TCR repertoire that differed from patient to patient (8-16 subfamilies were used) and a predominant usage of a different variable beta chain (BV) by each of these MART-reactive T cells. These predominant BV rearrangements were derived from multiple clonotypes because different variable, diversity, and junctional regions were observed. However, a similar pattern of expansion was present for both PBLs and TILs; the relative usage of each prevailing BV was more marked in TILs (36, 50, and 78% of TILs versus 26, 20, and 24% of PBLs, respectively), a broader TCR repertoire was used by PBLs (P > 0.05), and similar TCR subfamily usage was noted when TIL and PBL cultures from the same patient were compared (8 of 11, 7 of 9, and 7 of 8 for patients 1, 2, and 3, respectively). Furthermore, the exact same clonotypes derived from predominant TCR subfamilies in the PBLs and TILs were present in each patient, suggesting peptide-stimulated expansion in both biological compartments. These studies suggest that there will not be a limited and predictable TCR subfamily response to a specific TAA, although reproducible patterns of PBL and TIL expansion are present from patient to patient. Additionally, identical T-cell clonotypes having the same potential for antigen-driven expansion were present in a patient's PBLs and TILs. As such, our data support the conceptualization of approaches using adoptive transfer or vaccination based on TAA-derived peptide stimulation of PBLs.

Amino Acid Sequence↗
PubMed1997

Botulinum neurotoxin types A and E require the SNARE motif in SNAP-25 for proteolysis.

Botulinum neurotoxins type A and E (BoNT/A and BoNT/E) are metalloproteases with a unique specificity for SNAP-25 (synaptosome-associated protein of 25 kDa), an essential protein component of the neuroexocytotic machinery. It has been suggested that this specificity is directed through the recognition of a nine residue sequence, termed SNARE motif, that is common to the other two SNARE proteins: VAMP (vesicle-associated membrane protein) and syntaxin, the only known substrates of the other six clostridial neurotoxins. Here we analyse the involvement of the four copies of the SNARE motif present in SNAP-25 in its interaction with BoNT/A and BoNT/E by following the kinetics of proteolysis of SNAP-25 mutants deleted of SNARE motifs. We show that a single copy of the motif is sufficient for BoNT/A and BoNT/E to recognise SNAP-25. While the copy of the motif proximal to the cleavage site is clearly involved in recognition, in its absence, other more distant copies of the motif are able to support proteolysis. Also, a non-neuronal isoform of SNAP-25, Syndet, is shown to be sensitive to BoNT/E, but not BoNT/A, whilst the SNAP-25 isoforms from Torpedo marmorata and Drosophila melanogaster were demonstrated not to be substrates of these metalloproteases.

Amino Acid Sequence↗
PubMed1997

A single residue within the homeodomain of the Brn-3 POU family transcription factors determines whether they activate or repress the SNAP-25 promoter.

The closely related POU family transcription factors Brn-3a and Brn-3b differ in their effect on a number of different neuronally expressed promoters such as that of the gene encoding the synaptic vesicle component SNAP-25. Thus Brn-3a activates these promoters whilst Brn-3b represses both their basal activity and their activation by Brn-3a. We show here that alterations of a single amino acid at position 22 in the POU-homeodomain from the isoleucine found in Brn-3b to the valine found at the equivalent position in Brn-3a converts Brn-3b from a repressor to an activator of the SNAP-25 gene promoter. The converse mutation in Brn-3a abolishes its ability to activate the SNAP-25 gene promoter and allows it to repress the basal activity of the promoter and its activation by wild type Brn-3a. This is the first time that a single amino acid change has been shown to convert an activator of a naturally occurring promoter to a repressor and vice versa. These results are discussed in terms of the critical role of position 22 in the POU homeodomain in the protein-protein interactions of POU proteins.

Biotransformation↗
PubMed1997

Tissue distribution of SNAP-23 and its subcellular localization in 3T3-L1 cells.

The SNARE hypothesis of vesicular traffic proposes that three proteins, VAMP/synaptobrevin, syntaxin, and SNAP-25, constitute a complex that docks the vesicle at the target membrane. VAMP and syntaxin isoforms have been identified outside the nervous system, and a cDNA to a SNAP-25 related protein, SNAP-23, was recently identified in human lymphocytes. Here we report the generation of isoform-specific antibodies to SNAP-23 cloned from human melanoma cells, and their use in detecting the expression and localization of the endogenous SNAP-23 protein in several tissues and cell lines. SNAP-23 was readily detected in liver, lung, kidney, and spleen, to a lesser extent in muscle and heart, and was almost undetectable in brain. The protein was also abundant in fibroblast, muscle, and fat cell lines, but relatively less enriched in neuroendocrine PC12 cells. SNAP-23 abundance did not change during differentiation of 3T3-L1 fibroblasts into adipocytes. In both, SNAP-23 was membrane-bound and below detectable levels in the cytosolic fraction. Subcellular fractionation of 3T3-L1 adipocytes revealed that the majority of the protein was associated with plasma membranes. These findings support the conclusion that a tripartite SNARE complex exists outside of the nervous system, and suggest that SNAP-23 may play a role in vesicle traffic in most cell types.

3T3 Cells↗
PubMed1997

Differential regulation of genes encoding synaptic proteins by the Oct-2 transcription factor.

In order to investigate the effect of the Oct-2 POU family transcription factor on the regulation of genes encoding synaptic proteins, we have used cell lines in which the level of Oct-2 has been greatly reduced using an antisense approach. The reduced Oct-2 level results in enhanced expression of SNAP-25 and synapsin I, indicating that the genes encoding these proteins are normally repressed by Oct-2 in neuronal cells. In contrast, no alteration was observed in the levels of the synaptic proteins, synaptophysin and synaptotagmin. Although the neuronal forms of Oct-2 can repress the synapsin I promoter in co-transfection experiments, indicating that they have a direct effect on the expression of this gene, they have no effect on the activity of the SNAP-25 promoter, indicating that the effect of Oct-2 on this gene is likely to be indirect. These effects are discussed in terms of the differential effect of Oct-2 and the related POU family transcription factor Brn-3a, on the promoters of genes encoding different synaptic proteins.

Animals↗
PubMed1997

Differential sensitivity of c-Fos expression in hippocampus and other brain regions to moderate and low doses of alcohol.

Alcohol consumption in humans is characterized by a wide range of behavioral effects and pathological consequences that suggest several neuroanatomical targets for this drug. To identify these targets we have mapped alcohol-induced changes in the expression of the c-Fos protein in the rat brain. Administration of a moderate dose of alcohol (1.5 g kg-1) led to a suppression of basal and novel environment-induced c-Fos expression in the hippocampus and simultaneous induction of this protein in regions important for the reinforcing as well as aversive properties of drugs. These include the extended amygdala (including the central nucleus of amygdala, bed nucleus of stria terminals and nucleus accumbens), regions processing sensory information (including the Edinger-Westphal nucleus and the paraventricular nucleus of the thalamus) and in stress-related areas (including the paraventricular nucleus of the hypothalamus, nucleus of the solitary tract and several neocortical areas). Repeated administration of the same dose of alcohol did not decrease alcohol-mediated suppression of c-Fos in the hippocampus, but decreased alcohol-induced expression of c-Fos in other areas. A lower dose of acute alcohol (0.5 g kg-1) reduced basal c-Fos expression in several areas of the neocortex, hippocampus and hypothalamus. However, while this low dose of alcohol was unable to counteract the environmental novelty-induced c-Fos expression in these areas, it increased c-Fos expression in the central nucleus of amygdala (an effect similar to the one observed previously for diazepam). Our data suggest that the effects of low doses of alcohol may be due to selective GABA-like effects of ethanol, whereas higher doses of ethanol involve effects on multiple neurotransmitter systems.

Animals↗
PubMed1997

Coloboma hyperactive mutant mice exhibit regional and transmitter-specific deficits in neurotransmission.

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, KCl-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.

Acetylcholine↗
PubMed1997

Efficacy of argon laser trabeculoplasty in aphakic and pseudophakic eyes.

BACKGROUND AND OBJECTIVE: This study evaluated the efficacy of argon laser trabeculoplasty (ALT) in patients with aphakia or pseudophakia. PATIENTS AND METHODS: The authors retrospectively analyzed 63 eyes of 63 patients with aphakia or pseudophakia who had uncontrolled open-angle glaucoma and were treated with 180 degrees or 360 degrees ALT. RESULTS: The mean intraocular pressure (IOP) before ALT was 25.1 mm Hg (+/- 8.1 mm Hg). Two years after ALT, the mean IOP in the successfully treated group was 15.4 mm Hg. The mean survival time (50% success rate) for all treated eyes was 23 months, and treatment was successful in 34% of eyes at 36 months. Pseudophakic eyes had a better response than aphakic eyes (P = .06), and eyes that had extracapsular surgery did better than eyes with intracapsular surgery (P = .07). CONCLUSION: ALT in aphakic or pseudophakic eyes with uncontrolled glaucoma is a safe, reasonably effective treatment that can delay the need for trabeculectomy.

Aged↗
PubMed1997

Characterization of a sustained-release delivery system for combined cytokine/peptide vaccination using a poly-N-acetyl glucosamine-based polymer matrix.

Identification of tumor-associated antigens (TAAs) and their class I MHC-restricted epitopes now allows for the rational design of peptide-based cancer vaccines. A biocompatible system capable of sustained release of biologically relevant levels of cytokine and TAA peptide could provide a more effective microenvironment for antigen presentation. Our goal was to test a sustained-release cytokine/TAA peptide-based formulation using a highly purified polysaccharide [poly-N-acetyl glucosamine (p-GlcNAc)] polymer. Granulocyte-macrophage colony-stimulating factor (GM-CSF; 100 microgram) and MART-1(27-35) peptide (128 microgram in DMSO) were formulated into p-GlcNAc. Peptide release was assayed in vitro using interleukin 2 production from previously characterized MART-1(27-35)-specific Jurkat T cells (JRT22). GM-CSF release was assayed via ELISA and proliferation of M-07e (GM-CSF-dependent) cells. Local bioavailability of MART-1(27-35) peptide for uptake and presentation by antigen-presenting cells was demonstrated for up to 6 days (>0.5 microgram/ml). More than 1.0 microgram/ml GM-CSF was concomitantly released over the same period. Biocompatibility and local tissue response to p-GlcNAc releasing murine GM-CSF was determined in C57BL/6 mice via s.c. injection using murine GM-CSF (0. 2 microgram/ml) in 200 microliter of a 2.5% polymer gel. Significant lymphocytic and eosinophilic infiltration was observed 2-7 days after injection with polymer containing murine GM-CSF. The results of our studies show that this biocompatible system is capable of a sustained concomitant release of biologically active peptide and cytokine into the local microenvironment. These findings support further studies to validate a p-GlcNAc delivery system vehicle for a cytokine/TAA peptide-based cancer vaccine.

Acetylglucosamine↗
PubMed1996

Differential regulation of genes encoding synaptic proteins by members of the Brn-3 subfamily of POU transcription factors.

The three members of the Brn-3 subfamily of POU transcription factors have distinct effects on target gene expression. We show that the promoter of the gene encoding the presynaptic nerve terminal protein SNAP-25 resembles previously characterised target genes in being activated by Brn-3a and Brn-3c, but being repressed by Brn-3b. Unlike other target genes, however, the SNAP-25 promoter can be activated by either the N- or C-terminal activation domains of Brn-3a. In contrast to the SNAP-25 gene, the gene encoding the synaptic vesicle protein synapsin 1 is activated by all the Brn-3 factors, the first gene for which this activation pattern has been reported Interestingly, however, similar activation by all three Brn-3 factors can be observed if the SNAP-25 promoter is truncated by removal of sequences from -2200 to -288 relative to the transcriptional start site. Moreover, a region of the SNAP-25 promoter from -283 to -126 can render a heterologous promoter responsive to activation by all three Brn-3 factors. Differences in promoter structure may thus result in differences in the response to different Brn-3 factors, thus allowing these factors to produce diverse activation patterns of neuronally expressed genes, such as those encoding different synaptic proteins.

Animals↗
PubMed1996

The effects of prenatal cocaine exposure on dopaminergic challenge and receptor binding in Wistar rats.

The behavioral teratogenic effects of prenatal cocaine administration in Wistar rats were assessed in dams treated throughout gestation via oral gavage with either 0 or 80 mg/kg of cocaine. A pair-fed (PF) cohort group for the 80-mg/kg dose was used to control for an anorexic effect of cocaine. Alterations in the dopaminergic system at maturity were evaluated using pharmacological challenges with amphetamine and cocaine and by measuring D1 and D2 receptor binding in the nucleus accumbens and caudate nucleus. No significant difference among the offspring of the treatment groups was found in amphetamine-induced locomotion. A cocaine-based conditioned taste aversion was established in all offspring, but no significant effect of prenatal cocaine treatment was seen. Dopamine receptor binding was not significantly influenced by prenatal treatment, although a decreased D1 binding in the caudate nucleus of the prenatal cocaine rats approached significance.

Animals↗
PubMed1996

SNAP-25 and synaptotagmin involvement in the final Ca(2+)-dependent triggering of neurotransmitter exocytosis.

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 Ca(2+)-signaled exocytosis is effectively blocked by antibodies and peptides to SNAP-25, a key constituent of the vesicle docking complex. In addition, Ca2+ inhibits the ability of synaptotagmin, a synaptic vesicle protein proposed as a calcium sensor and triggering device, to associate with this docking complex. These results support a model in which Ca(2+)-dependent triggering of neurotransmission at central synapses acts after ATP-dependent potentiation of the docking-fusion complex for membrane fusion.

Adenosine Triphosphate↗
PubMed1996

Tyrosine phosphorylation modulates the activity of clostridial neurotoxins.

Clostridial neurotoxins' metalloprotease domain selectively cleaves proteins implicated in the process of synaptic vesicle fusion with the plasma membrane and, accordingly, blocks neurotransmitter release into the synaptic cleft. Here we investigate the potential modulation of these neurotoxins by intracellular cascades triggered by environmental signals, which in turn may alter its activity on target substrates. We report that the nonreceptor tyrosine kinase Src phosphorylates botulinum neurotoxins A, B, and E and tetanus neurotoxin. Protein tyrosine phosphorylation of serotypes A and E dramatically increases both their catalytic activity and thermal stability, while dephosphorylation reverses the effect. This suggests that the biologically significant form of the neurotoxins inside neurons is phosphorylated. Indeed, in PC12 cells in which tyrosine kinases such as Src and PYK2 are highly abundant, stimulation by membrane depolarization in presence of extracellular calcium induces rapid and selective tyrosine phosphorylation of internalized light chain, the metalloprotease domain, of botulinum toxin A. These findings provide a conceptual framework to connect intracellular signaling pathways involving tyrosine kinases, G-proteins, phosphoinositides, and calcium with the action of botulinum neurotoxins in abrogating vesicle fusion and neurosecretion.

Animals↗