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Biomedical subjects

K M Buckley

Publications and source records attributed to K M Buckley.

At least 19 recordsLinked to original sources
PubMed1997

Targeting of the synaptic vesicle protein synaptobrevin in the axon of cultured hippocampal neurons: evidence for two distinct sorting steps.

Synaptic vesicles are concentrated in the distal axon, far from the site of protein synthesis. Integral membrane proteins destined for this organelle must therefore make complex targeting decisions. Short amino acid sequences have been shown to act as targeting signals directing proteins to a variety of intracellular locations. To identify synaptic vesicle targeting sequences and to follow the path that proteins travel en route to the synaptic vesicle, we have used a defective herpes virus amplicon expression system to study the targeting of a synaptobrevin-transferrin receptor (SB-TfR) chimera in cultured hippocampal neurons. Addition of the cytoplasmic domain of synaptobrevin onto human transferrin receptor was sufficient to retarget the transferrin receptor from the dendrites to presynaptic sites in the axon. At the synapse, the SB-TfR chimera did not localize to synaptic vesicles, but was instead found in an organelle with biochemical and functional characteristics of an endosome. The chimera recycled in parallel with synaptic vesicle proteins demonstrating that the nerve terminal efficiently sorts transmembrane proteins into different pathways. The synaptobrevin sequence that controls targeting to the presynaptic endosome was not localized to a single, 10- amino acid region of the molecule, indicating that this targeting signal may be encoded by a more distributed structural conformation. However, the chimera could be shifted to synaptic vesicles by deletion of amino acids 61-70 in synaptobrevin, suggesting that separate signals encode the localization of synaptobrevin to the synapse and to the synaptic vesicle.

Animals↗
PubMed1997

Identification of a somatodendritic targeting signal in the cytoplasmic domain of the transferrin receptor.

Neurons are highly polarized cells that must sort proteins synthesized in the cell body for transport into the axon or the dendrites. Given the amount of time and energy needed to deliver proteins to the distal processes, neurons must have high fidelity mechanisms that ensure proper polarized protein trafficking. Although a variety of proteins are localized either to the somatodendritic domain or to the axon (), the question of whether there are signal-dependent mechanisms that sort proteins to distinct neuronal domains is only beginning to be addressed. To determine sequence requirements for the polarized sorting of transmembrane proteins into dendrites, we expressed mutant transferrin receptors in cultured rat hippocampal neurons, using a defective herpes virus vector. Wild-type human transferrin receptor colocalized with the endogenous protein in dendritic endosomes and was strictly excluded from axons, despite overexpression. Polarized targeting was abolished by deletion of cytoplasmic amino acids 7-10, 11-14, or 19-28, but not 29-42 or 43-58. These deletions also increased the appearance of transferrin receptor on the plasma membrane, implying that endocytosis and dendritic targeting are mediated by overlapping signals and similar molecular mechanisms. In addition, we have characterized a specialized para-Golgi endosome poised to play a critical role in the polarized recycling of transmembrane proteins.

Animals↗
PubMed1997

A sampling theorem for EEG electrode configuration.

An analytical tool to help in selecting the number of electrodes required for recording electroencephalogram (EEG) signals is presented. The main assumption made is that the scalp can be modeled as a hemispherical surface. The number of sensors required to sample a surface is derived by using a mean square error (MSE) measure to approximate the continuous potential functions on the hemispherical surface. An algorithm for selecting the number of electrodes for arbitrary head geometries is also proposed. A sampling theorem is then derived with conditions on the sampling points for electrode placement.

Algorithms↗
PubMed1995

Calcium-dependent transmitter secretion from fibroblasts: modulation by synaptotagmin I.

Following endocytic uptake of acetylcholine (ACh), CHO fibroblasts exhibit Ca(2+)-dependent spontaneous quantal ACh release and depolarization-evoked ACh release, as detected by a whole-cell voltage-clamped myocyte in contact with the fibroblast. CHO fibroblasts transfected with synaptotagmin I, an integral membrane protein of synaptic vesicles, showed a reduced spontaneous quantal ACh release and an enhanced Ca(2+)-evoked ACh release, as compared with control cells. Biochemical and ultrastructural studies of endocytic activity using horseradish peroxidase as a marker further confirmed the inhibitory action of synaptotagmin I on spontaneous vesicular exocytosis and on elevated exocytosis induced by Ca2+. Through inhibition of exocytosis at the resting intracellular concentration of Ca2+ and removal of the inhibition upon depolarization-induced Ca2+ entry, synaptotagmin I could enhance the efficiency of excitation-secretion coupling.

Acetylcholine↗
PubMed1995

EEG dipole localization bounds and MAP algorithms for head models with parameter uncertainties.

The Cramer-Rao bound for unbiased dipole location estimation is derived under the assumption of a general head model parameterized by deterministic and stochastic parameters. The expression thus characterizes fundamental limits on EEG dipole localization performance due to the effects of both model uncertainty and statistical measurements noise. Expressions are derived for the cases of multivariate Gaussian and gamma distribution priors, and examples are given to illustrate the derived bounds when the radii and conductivities of a four-concentric sphere head model are allowed to be random. The joint MAP estimate of location/model parameters is then examined as a means of achieving robustness to deviations from an ideal head model. Random variations in both the multiple sphere radii and the layer conductivities are shown, via the stochastic Cramer-Rao bounds and Monte Carlo simulation of the MAP estimator, to have the most impact on localization performance in high SNR regions, where finite sample effects are not the limiting factors. This corresponds most often to spatial regions that are close to the scalp electrodes.

Algorithms↗
PubMed1995

Pulmonary lymphangiomyomatosis.

Lymphangiomyomatosis is a rare disease of unknown etiology characterized by hamartomatous proliferation of smooth muscle in the pulmonary lymphatics, blood vessels and airways. The disease occurs exclusively in women of reproductive age. Although the clinical course and radiographic findings may strongly suggest lymphangiomyomatosis, definitive diagnosis is made by obtaining open-lung biopsy. The clinical course of lymphangiomyomatosis is progressive, leading to pulmonary insufficiency and death within 10 years. Treatment with hormonal manipulation and/or oophorectomy has resulted in temporary improvement or stabilization of the disease process.

Female↗
PubMed1994

Robust adaptive microphone array processing for hearing aids: realistic speech enhancement.

The problem of combining the outputs of an array of microphones as a single input for a hearing aid is investigated. Emphasis is placed on the conservative prediction of realistically achievable performance gains provided by the array over a single microphone. Performance improvement is measured as a change in the speech reception threshold (SRT) between single microphone and multimicrophone conditions. Consistent with previous work, predictions of this change in SRT using intelligibility averaged gain, [symbol: see text] are shown to be good. Consequently, this measure is used, along with changes in signal-to-noise ratios (SNRs), to evaluate array performance. The results presented include the effects of acoustic headshadow, small room reverberation, microphone placement uncertainty, and desired speaker location uncertainty. It is in this context that realistic predictions of speech enhancement provided by robust adaptive microphone array processors are discussed. Performance improvements are demonstrated relative to the "best" single microphone in the array for three types of spatial filters: Fixed, robust block processed, and robust adaptive. The performance of the robust block processed arrays is shown to be attainable with adaptive implementations. One fundamental criterion employed in robust beamformer design directly limits the amount of cancellation of the desired signal that can occur.

Adult↗
PubMed1993

Conservation of the amino acid sequence of SV2, a transmembrane transporter in synaptic vesicles and endocrine cells.

SV2 is a secretory vesicle-specific protein produced by all neurons and by endocrine cells. The deduced amino acid (aa) sequence of this protein indicates that it is a transmembrane transporter [Bajjalieh et al., Science 257 (1992) 1271-1273; Feany et al., Cell 70 (1992) 861-867; Gingrich et al., FEBS Lett. 312 (1992) 115-122]. To determine the regions of the protein that are the most highly conserved throughout evolution, and might therefore be essential for the function of SV2, we isolated a cDNA clone encoding SV2 from the elasmobranch fish, Discopyge ommata, and compared the deduced aa sequence to two isoforms from rat, SV2A and SV2B [Bajjalieh et al., Proc. Natl. Acad. Sci. USA 90 (1993) 2150-2154]. The comparison indicates that although the N-terminal cytoplasmic domain of SV2 is the most divergent region, it contains a highly conserved sequence that is predicted to be the epitope for a monoclonal antibody that crossreacts with all species and two isoforms of SV2 [Buckley and Kelly, J. Cell Biol. 100 (1985) 1284-1294; Bajjalieh et al., Proc. Natl. Acad. Sci. USA 90 (1993) 2150-2154]. The remainder of the protein is highly conserved: 62% of the aa in SV2 from D. ommata are identical to the rat SV2A sequence, and 12% are conservative substitutions. The high degree of conservation of this protein throughout evolution and across species indicates that it mediates a critical function of synaptic vesicles.

Amino Acid Sequence↗
PubMed1993

The synaptic vesicle protein synaptotagmin promotes formation of filopodia in fibroblasts.

Neuronal filopodia are actin-rich cytoplasmic extensions that are involved in motility and recognition in growth cones and maturing axonal endings. A detailed understanding of neuronal growth will depend on clarification of the membrane fusion events occurring during filopodial extension. The synaptic vesicle protein synaptotagmin seems to be intimately involved in exocytotic membrane fusion. Here we show that fibroblast cell lines transfected with synaptotagmin form long, highly branched, actin-rich filopodial processes, with the expressed synaptotagmin being incorporated into the plasma membrane. In contrast, cell lines expressing either of two other synaptic vesicle proteins, SV2 or synaptophysin, generate only rudimentary processes, and, like neurons, sort SV2 and synaptophysin to small intracellular vesicles. As presynaptic calcium entry regulates synaptic vesicle fusion, our results indicate that synaptotagmin might link neuronal activity with synaptic growth.

Animals↗
PubMed1993

Accumulation of mRNAs encoding synaptic vesicle-specific proteins precedes neurite extension during early neuronal development.

Synaptic vesicles are essential for neuronal synaptic function. We have analyzed the temporal and spatial pattern of mRNA accumulation of two integral membrane proteins specific for synaptic vesicles (synaptophysin and SV2) and a small GTP-binding protein associated with the vesicles (rab3a), using in situ hybridization to mouse embryonic tissue sections. Our results indicate that transcription of these mRNAs is not synchronous in the embryo. Detectable levels of synaptophysin and rab3a mRNAs appear during early neurulation (embryonic day [ED] 9.5) both in the CNS and PNS, whereas SV2 mRNA is not observed before ED 10.5. We have also compared the accumulation of these synaptic vesicle protein transcripts during neuroblast proliferation and neuronal differentiation in vitro, using as a model system the embryonic carcinoma cell line P19 which can be induced to differentiate into neurons and glial cells. We observe that transcripts for all three proteins appear in neurons virtually simultaneously soon after withdrawal from the cell cycle. These data suggest that the program of differentiation in vitro is similar to that observed in vivo, but markedly accelerated. In both embryos and P19 cells, transcripts for these three proteins are detectable at a time when most of the neurons have withdrawn from the cell cycle, but prior to neurite extension and synapse formation.

Animals↗
PubMed1993

The synaptic vesicle proteins SV2, synaptotagmin and synaptophysin are sorted to separate cellular compartments in CHO fibroblasts.

We expressed the synaptic vesicle proteins SV2, synaptotagmin, and synaptophysin in CHO fibroblasts to investigate the targeting information contained by each protein. All three proteins entered different cellular compartments. Synaptotagmin was found on the plasma membrane. Both SV2 and synaptophysin were sorted to small intracellular vesicles, but synaptophysin colocalized with early endosomal markers, while SV2 did not. SV2-containing vesicles did not have the same sedimentation characteristics as authentic synaptic vesicles, even though transfected SV2 was sorted from endosomal markers. We also created cell lines expressing both SV2 and synaptotagmin, both synaptotagmin and synaptophysin, and lines expressing all three synaptic vesicle proteins. In all cases, the proteins maintained their distinct compartmentalizations, were not found in the same organelle, and did not created synaptic vesicle-like structures. These results have important implications for models of synaptic vesicle biogenesis.

Animals↗
PubMed1993

Prewhitening for intelligibility gain in hearing aid arrays.

In this article, prewhitening multimicrophone data prior to use in an optimum spatial-filter preprocessor for a monaural hearing aid is considered. Considering preprocessor capabilities, interference signal spectral content and intelligibility, it is argued that prewhitening is advantageous. This advantage is illustrated via simulation of a head worn array. Also, an effective procedure for the design of (prewhitening) digital filters is presented. The effectiveness of this filter design technique is shown by presenting several prewhitening filter design examples.

Acoustic Stimulation↗