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Biomedical subjects

K M Buckley

Publications and source records attributed to K M Buckley.

33 records · Page 2Linked to original sources
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↗
PubMed1992

The synaptic vesicle protein SV2 is a novel type of transmembrane transporter.

The primary function of synaptic vesicles is to store and release neurotransmitter. Synaptic vesicles are locally recycled following exocytosis and rapidly refilled with neurotransmitter from the cytoplasm by a process that depends on the electrochemical gradient generated by a proton pump. Little is known about the molecules that import neurotransmitter into synaptic vesicles. We report here that the sequence of the synaptic vesicle protein SV2 identifies this protein as a novel type of transmembrane transporter. The deduced amino acid sequence of SV2 contains two sets of six predicted transmembrane domains: the six most N-terminal transmembrane domains are highly homologous to a subfamily of transporters that includes the human glucose transporter, while the six most C-terminal domains are homologous to the plasma membrane transporters for neurotransmitters. We propose that SV2 mediates transport of neurotransmitters into synaptic vesicles.

Amino Acid Sequence↗
PubMed1991

Detection of bullous lung disease: conventional radiography vs digital storage phosphor radiography.

The detection of fine linear contours and altered aeration are requirements for the radiologic diagnosis of bullous lung disease and are severe measures of the spatial and contrast resolution of chest imaging systems. We compared plain film radiography with five postprocessing algorithms of storage phosphor digital radiography (2144 x 1744 x 10 bit matrix with 0.2-mm pixel size) in the detection of CT-proved bullous lung disease (35 patients and 25 normal control subjects). Receiver-operating-characteristic analysis of 2160 observations by six interpreters was done to evaluate the observers' performance. By analysis of variance (p less than .05), we found that the default digital algorithm and the three edge-enhancing algorithms of high and medium frequencies performed less well than plain films did, but the differences fell short of statistical significance. Gray-scale reversal was the only digital algorithm that performed significantly less well than plain films did. We conclude that any differences between digital algorithms and plain films in the detection of bullous lung disease were too small to be detected in this study. Any difference between the two methods in providing clinically important, diagnostic information is likely to be insignificant.

Algorithms↗
PubMed1988

Targeting of secretory vesicles to cytoplasmic domains in AtT-20 and PC-12 cells.

Organelles are not uniformly distributed throughout the cytoplasm but have preferred locations that vary between tissues and during development. To investigate organelle targeting to cytoplasmic domains we have taken advantage of the mouse pituitary cell line, AtT-20, which, when induced to extend long processes, accumulates dense core secretory granules at the tips of the processes. During mitosis, these secretory granules accumulate along the plane of division. Protein synthesis is not mandatory for such redistribution of secretory granules. To explore the specificity of the redistribution we have used transfected AtT-20 cells that express the immunoglobulin kappa light chain. While the endogenous hormone ACTH is found in secretory granules, the kappa chain is a marker for organelles involved in constitutive secretion. By immunofluorescence, kappa also accumulates at the tips of growing processes, and along the midline of dividing cells, suggesting that the redistribution of vesicles is not specific for dense-core secretory granules. Since there is evidence for selective organelle transport along processes in neuronal cells, the rat pheochromocytoma cell PC-12 was transfected with DNA encoding markers for regulated and constitutive secretory vesicles. Again regulated and constitutive vesicles co-distribute, even in cells grown in the presence of nerve growth factor. We suggest that at least in the cells studied here, cytoskeletal elements normally carry exocytotic organelles to the surface; when the cytoskeletal elements coalesce in an extending process, exocytotic organelles of both the constitutive and regulated pathway are transported nonselectively to the tips of the cytoskeletal elements where they accumulate.

Adrenocorticotropic Hormone↗
PubMed1987

Cloning and sequence analysis of cDNA encoding p38, a major synaptic vesicle protein.

We have isolated from a lambda gt11 rat brain cDNA library cDNA clones encoding greater than 95% of the open reading frame and untranslated regions of the mRNA for p38, the most abundant of the integral membrane proteins of the synaptic vesicle. Phage containing cDNA that encoded vesicle proteins were identified by screening fusion proteins with a polyclonal serum to rat brain synaptic vesicles. To identify phage carrying p38 sequences, fusion proteins were used to affinity purify monospecific antibodies from the original heterogeneous serum; antibodies to a 38,000-D protein were then identified by Western blotting. Inserts carrying DNA-encoding p38 sequences were subcloned into plasmid vectors and used to generate cDNA probes for Northern blot analysis. A major transcript of 2.4 kb was expressed specifically in brain and endocrine tissue but not in liver, consistent with the tissue-specific expression of the protein detected by antibody techniques. Using three overlapping clones that encoded fusion proteins, we identified and sequenced approximately 85% of the cDNA. Two additional Eco RI fragments at the 5' end of the mRNA were obtained from a fourth clone identified by screening a second lambda gt11 library with a 5' cDNA probe. The cDNA encoded an open reading frame of 298 amino acids with a 3' untranslated region of 1.4 kb. The protein shares no sequence homology with other Ca2+-binding proteins. The availability of a cDNA clone for an integral synaptic vesicle protein should facilitate studies of its function in transmitter release, its intracellular targeting, and regulation of synaptic vesicle biogenesis during development and regeneration of nerve terminals.

Amino Acid Sequence↗
PubMed1983

Morphological studies of synapses and varicosities in dissociated cell cultures of sympathetic neurons.

Neurons dissociated from the superior cervical ganglia of newborn rats can be grown under conditions which support either adrenergic or cholinergic differentiation. In both cases, the neurons form numerous morphologically specialized synaptic terminals or synapses as well as relatively unspecialized varicosities. The ultrastructure of both types of terminal was compared in mature neuronal cultures and the effects of growth conditions on terminal morphology examined. After aldehyde-osmium fixation, synapses in cultures grown under adrenergic or cholinergic conditions were characterized by asymmetrical membrane specializations comparable to type I or asymmetric synapses; bismuth iodide and ethanolic phosphotungstic acid impregnation of neuronal cultures revealed the presence of characteristic synaptic membrane specializations: a presynaptic grid of dense projections and a wide postsynaptic dense band of uniform thickness. No membrane specializations were apparent in varicosities after aldehyde-osmium fixations or with these stains. Intramembranous particle distributions were examined in freeze-fracture replicas of neurons. Aggregates of large, 10-12 nm particles were found on P-face membrane leaflets of cell bodies and large diameter processes; this distribution is the same as that of synapses in thin-sectioned preparations. These particle aggregates may represent postsynaptic membrane specializations or acetylcholine receptors. The cytoplasmic leaflet of boutons contained large, 12-14 nm particles, which appeared to be concentrated at the region of synaptic contact at putative synapses, but were diffusely distributed in varicosity membranes. Similar large particles were also seen at a much lower density in the membrane E-face. None of these ultrastructural characteristics appeared to vary with transmitter identity or growth conditions. Synaptic vesicle shape, however, did vary in glutaraldehyde-fixed cultures. At all ages examined, neurons grown on monolayers of heart cells contained predominantly round vesicles, whereas neurons grown in the virtual absence of non-neuronal cells possessed pleiomorphic synaptic vesicles. This difference in vesicle shape appeared to be correlated more closely with growth in the presence of non-neuronal cells than with the transmitter present at the time of fixation.

Acetylcholine↗
PubMed1983

Morphological studies of neurotransmitter release and membrane recycling in sympathetic nerve terminals in culture.

The morphological correlates of transmitter release from synapses and varicosities were examined in mature cultures of sympathetic neurons dissociated from neonatal rat superior cervical ganglia. The number of synaptic vesicles decreased in synapses and varicosities depolarized with 53 mM K+. The decrease in vesicle number was accompanied by striking changes in the appearance of the synaptic terminals and an increase in their mean circumference. Coated pits and membrane-bound cisternae were observed more frequently in synapses and varicosities of depolarized neurons than in terminals of resting neurons. These morphological changes were not seen when the neurons were depolarized in the presence of Co2+, consistent with the Ca2+-dependence of transmitter release from these neurons. In freeze-fracture replicas of depolarized neurons, numerous dimples were observed in the cytoplasmic leaflet of synapses and varicosities, adjacent to large 12-14 nm particles. After a period of recovery in 5 mM K+ medium, the number of synaptic vesicles and the shape of synaptic terminals returned to normal. When horseradish peroxidase (HRP) was included in the medium as an extracellular tracer during depolarization and recovery, a significant proportion of small, synaptic vesicles contained reaction product. Label was also present in coated vesicles and cisternae. Neurons which were depolarized in medium containing Co2+ or were exposed to HRP without depolarization contained few labelled synaptic vesicles. The proportion of labelled vesicles was not significantly different in synapses and varicosities, nor did it vary consistently with the transmitter identity of the neurons. These observations are consistent with the hypothesis that transmitter release occurs from varicosities as well as from synapses of postganglionic sympathetic neurons by exocytosis of the small synaptic vesicles, and that at least some new vesicles are formed from the nerve terminal membrane.

Animals↗
PubMed1983

A synaptic vesicle antigen is restricted to the junctional region of the presynaptic plasma membrane.

The plasma membrane of electric organ nerve terminals has two domains that can be distinguished by monoclonal antibodies. A library of 111 mouse monoclonal antibodies raised to nerve terminals from Torpedo californica contains 4 antibodies that bind specifically to the outside of intact synaptosomes. The distribution of the binding sites of these monoclonal antibodies on the outside of intact nerve terminals was examined by immunofluorescence and immunoelectron microscopy. The binding sites of 3 (tor23, 25, and 132) are distributed uniformly over nerve trunks and fine terminal branches. The binding site of the fourth (tor70) is restricted to synaptic junctional regions. This antibody, but not the other 3, recognizes a major component of synaptic vesicles, a proteoglycan associated with the inner surface of the vesicle membrane. The difference in the pattern of binding of these monoclonal antibodies suggests that the region of the plasma membrane containing active zones is antigenically distinguishable from other nerve terminal plasma membrane. We suggest that the antigen recognized by tor70 is externalized by exocytosis of synaptic vesicles while other plasma antigens take a different route to the surface. The unexpected observation that the vesicle antigen remains on the surface after exocytosis and is prevented from diffusion from the synaptic junctional region would be consistent with an interaction between the vesicle proteoglycan and elements of the synaptic cleft.

Animals↗