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Characterization of multiple conductance states in a nicotinic acetylcholine receptor/channel preparation.Montpetit, Madeleine R. January 1986 (has links)
The acetylcholine-activated channel of vertebrate skeletal muscle, as manifested in cultured developing cells, is able to adopt more than one conductance state. This thesis reviews the evidence for such multiple conductance channels and presents single channel data suggesting that subconductances represent discrete, allosterically-activated channel conformations. Since the amplitude of subconductance openings does not depend on agonist size or valence, the possibility that subconductances occur during partial occlusion of the channel by agonist molecules was ruled out. The conductances found for each event type are as follows; M Type = 40pS, S1 = 13pS and S2 = 7pS. While agonists do not determine conductance of the AChR, different agonists have differential abilities at stabilizing the sublevel openings. The kinetic analysis of open time histograms for the various conductance states reveals that the full channel openings are made up of two distinct kinetic open states; one of which is agonist-dependent and the other agonist-independent. In general, the subconductances S1 and S2 both displayed openings with a single agonist-independent time constant. Bursting behavior in the G8 AChRs was observed when channels were activated by strong agonists.
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Alkylation treatment of the Mexican Axolotl: An approach to the induction of new mutations.Ortiz, Maria Fernanda. January 1977 (has links)
In order to obtain new mutants in the axolotl, Ambystoma mexicanum, that could be used for future genetic studies, EMS treatments have been given to adult females. Our results indicate a reduced fertility among the eggs from treated females, as well as increased lethality during embryonic development. Abnormal larvae have been obtained from these embryos. Whether we have succeeded in inducing new mutations, or whether the abnormalities are only due to the teratogenic effects of the EMS, still has to be tested when the animals mature. No external signs of haploidy have been observed among these larvae, except in one case. Tail-tip squashes of this larva revealed the normal number of nucleoli. Superovulation has not been observed among the treated females. Instead, our results suggest that EMS is suppressing ovulation. To pinpoint the effects of EMS on germ cells, treatment of various concentrations have been given to mature, spawned sperm. The sperm were used to artificially inseminate newly laid eggs. Our results show that increasing concentrations of EMS reduce the fertility of eggs inseminated with treated sperm. DNA isolated from 3H-EMS treated sperm has shown approximately 1 ethylation per 1,000 nucleotides. In spite of this, abnormalities have not been observed among embryos from EMS treated sperm, and therefore, no increased lethality has been obtained. The possibility that dominant lethal induction by EMS, in the axolotl, is due to damage of extranuclear components is discussed. Possible experiments that would bring some light to this subject are proposed.
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The human parainfluenza virus 3 fusion protein: Cloning, mapping, sequence analysis and expressionCote, Marie-Jose January 1989 (has links)
Abstract not available.
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Identification and functional mapping of the protein product of the V-rel oncogeneGarson, Kenneth January 1989 (has links)
Abstract not available.
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Studies on Halobacterium cutirubrum DNA-dependent RNA polymeraseBarua, Nrityendra Nath January 1978 (has links)
Abstract not available.
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In vivo zygotic- and maternal-effect characterization of the Drosophila melanogaster gene, tangoScanga, Vanessa Immacolata January 2004 (has links)
This study involved the in vivo phenotypic characterization of the Drosophila melanogaster gene tango (tgo) through classification of both its zygotic-effect during CNS development and maternal-effect during early embryonic patterning. Particularly, this study involved characterization of the prd-repeat domain of the Tango protein, or its allelic equivalent, tgo 3. tgo has been previously isolated as a bHLH-PAS nuclear transcription factor required in the regulation of CNS development through its dimerization of the CNS master transcriptional regulatory gene, single-minded. Having performed an inter-allelic analysis, I grouped the CNS zygotic effects of tgo into various phenotypic classifications. The phenotypes exhibited a pleiotropy of CNS mutant effects and were classified as fused commissure, neurogenic, stalled or ambiguous nervous system defects. These phenotypes may possibly be attributed to different but partially overlapping functions of the various Tgo domains. Additionally, I analyzed the maternal-effect of tgo during precellular blastoderm development. Germline clonal analysis of the tgo3 allele revealed that tgo has an important role during early embryogenesis. Moreover, maternal tgo3 phenotypes resemble those of the more well-characterized maternal coordinate and gap segmentation genes. tgo3 germline clones were embryonic lethal and were classified as severe or intermediate, depending on the extent of deleterious effects on the resultant cuticular phenotype. Germline clones that showed a deletion of the entire anterior end, abdominal segments Al to A5 and missing the Filzkorper and spiracular opening in the posterior end were classified as severe and are comparable to the cuticular phenotypes of amorphic bicoid mutants and Kruppel ( Kr) phenotypes. Less severe deleterious effects, such as improper development of various anterior and posterior end structures or aberrant denticle band formation, were classified as intermediate tgo 3 germline clones, comparable to weaker bicoid alleles. Subsequent stainings to observe the effect on embryonic patterning through gap and pair-rule protein localization revealed alterations in spatial expression patterns. Alterations in both Kruppel and Even-skipped localization patterns in embryos mutant for maternal tgo3 suggest a role for tgo during early embryonic patterning, perhaps through the activity of its prd-repeat domain. Ectopic expression studies, employing the GAL4-UAS system, involved heat shock trials eliciting the ubiquitous expression of a truncated C-terminal Tgo protein (UAS-tgoDeltaC ) and both the ubiquitous land targeted expression of UAS- tgoDeltac. This targeted misexpression analysis revealed tgo as both a possible activator and repressor of Kr gap gene expression, in addition to affecting the protein distribution pattern of the later-acting segmentation gene, engrailed. Taken together, these germline clonal and ectopic expression studies suggest that the role of Tgo during early embryonic patterning may function through combinatorial interactions with maternal-effect and/or early-acting segmentation gene products. Moreover, this role may depend on the function(s) of the C-terminal sequences encoded by Tgo, including the prd repeat domain. Ultimately, this early embryonic role of tgo seems to be distinct from its role as a nuclear transcription factor.
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hDREF, a MEF2 sensitive regulator of DNA synthesisRowan, Andrea Leah Margaret January 2003 (has links)
The Myocyte Enhancing Factor 2 (MEF2) family of proteins have been implicated in a wide variety of cellular mechanisms including: muscle and neuronal differentiation, inhibition of apoptosis, upregulation of c jun expression, and embryonic and post-natal cardiac development. In the course of my research I have identified a novel MEF2-responsive gene referred to as h&barbelow;uman D&barbelow;NA R&barbelow;eplication Related E&barbelow;lement F&barbelow;actor (hDREF). Three putative MEF2 consensus-binding sites have been found within the two untranslated regions (UTRs) of the hDREF sequence (which lie 5' and 3' to the open reading frame). Using CHromatin I&barbelow;mmunolP&barbelow;recipitation (CHIP) assays I have shown that MEF2 proteins associate with the MEF2 binding site found within the 5'UTR of hDREF under both growth and differentiation conditions. Furthermore, mutation of the MEF2 binding sites results in a failure to repress expression, which indicated that MEF2 acts to negatively regulate hDREF expression during differentiation. Endogenous expression studies indicate that hDREF is highly expressed during growth and downregulated during differentiation. Functional studies suggest that hDREF is directly involved in regulating DNA synthesis, and that overexpression of hDREF leads to aberrant DNA replication in post-mitotic cells leading to polyploidy. Based on my research I propose that hDREF represents a novel MEF2 regulated gene, and that the primary function of hDREF is to ensure DNA synthesis with the cell cycle.
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Characterization, identification and purification of a high-affinity binding site for H4-(86-100) peptide in membrane preparations of rat alveolar macrophagesBonhomme, Andreanne January 2003 (has links)
Rat alveolar macrophages express specific binding sites for C-terminal fragments of histone H4. The present study was aimed at identifying and characterizing the receptor for the C-terminal fragment 86 to 100 of histone H4, an antinociceptive peptide structurally similar to histogranin. The inhibitory effect of GTP analogs (GTP-gamma-S and Gpp(NH)p) and pertussis toxin on membrane preparations and the finding that H4-(86--100) potently (10--8 M) inhibited forskolin-stimulated cAMP levels in cultured alveolar macrophages suggest the involvement of a GiPCR. Gel electrophoresis of cross-linked bound radiolabelled ligand revealed two binding proteins, 30 kDa and 54 kDa, whose detection was increased by stimulation of macrophages with interferon gamma (IFNgamma). Affinity chromatography and SDS-PAGE gel electrophoresis revealed a major protein band identified as beta-actin trypsin digests. (Abstract shortened by UMI.)
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TC10, a mammalian Rho GTPase responsible for actin cytoskeleton reorganization and cardiac hypertrophy in the murine heartLalonde, Julie Kathleen January 2003 (has links)
Rho guanosine triphosphatases (GTPases) act as molecular switches, cycling between two conformational states: an active, GTP-bound state and an inactive, GDP-bound state to control many complex cellular events in eukaryotic cells. Many Rho GTPases, including RhoA, Cdc42 and Rac1, have been extensively characterized and are involved in actin reorganization, activation of MAPK cascades, cell cycle progression, cellular proliferation, invasion, differentiation and apoptosis. TC10 was identified and classified as a Rho GTPase over ten years ago, however the precise role of this protein, which is highly expressed in cardiac and skeletal muscle, has only recently been explored in vitro and remains unexplored in vivo. Based on the unique expression pattern of TC10, we set out to investigate the role of TC10 by generating transgenic mice over-expressing activated TC10Q75L under the control of the cardiac-specific alpha-myosin heavy chain promoter. Transgenic mice expressing high levels of TC10Q75L showed pronounced atrial enlargement, evidence of left ventricular hypertrophy and diminished cardiomyocyte membrane integrity. In vitro, transgenic primary cardiomyocytes showed marked reorganization of the actin cytoskeleton, leading to the formation of actin-containing filopodial extensions, loss of stress fibers and actin aggregation. Together, these data suggest that TC10 functions to regulate cellular signalling to the actin cytoskeleton and processes associated with cell growth, leading to cardiac hypertrophy in TC10Q75L transgenic mice.
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Cytoprotective effects of intracellular platelet activating factor acetylhydrolasesBonin, Fanny January 2003 (has links)
Platelet activating factor (PAF) is a biologically active phospholipid implicated in the developmental brain disorder Miller-Dieker Syndrome (MDS) and purported to be a primary mediator of cell death in HIV-dementia, ischemia, and epilepsy. As part of my honour's thesis, I demonstrated that PAF can elicit cell death independently of its G-protein coupled receptor (PAFR) in PC12 cells. In my M.Sc. research, I have sought to identify how PAF-mediated cell death is regulated in PC12 cells. PAF is inactivated in brain by two intracellular PAF-acetylhydrolases (PAF-AHs): PAF-AH I and PAF-AH II. PAF-AH I is a trimeric complex composed of two catalytic subunits (alpha1 and alpha2) and one regulatory subunit (beta). Mutations in the Lis1 gene, coding for the beta subunit of PAF-AH I, are the genetic determinant of MDS. However, it is not clear whether these mutations impact on PAF-AH I enzymatic activity in MDS. Furthermore, it is not known whether cytosolic PAF-AH activity regulates the kinetics of neuronal loss following pathophysiological challenge. To begin to address these questions, I sought to identify an in vitro model system suitable for study of PAF-AH activity.* (Abstract shortened by UMI.)
*This dissertation is a compound document (contains both a paper copy and a CD as part of the dissertation). The CD requires the following system requirements: QuickTime.
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