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Biomedical subjects

F D Smith

Publications and source records attributed to F D Smith.

At least 19 recordsLinked to original sources
PubMed2001

Overexpression of muscle specific kinase increases the transcription and aggregation of acetylcholine receptors in Xenopus embryos.

Muscle specific kinase (MuSK) mediates agrin-induced acetylcholine receptor (AChR) aggregation on muscle membrane at the neuromuscular junction (NMJ). To examine whether MuSK enhances NMJ formation during embryonic development in vivo, the level of expression of MuSK was manipulated in Xenopus embryos and the functional consequence at the NMJ was assessed. We found that overexpression of MuSK enhanced the formation of NMJ by increasing the aggregation of AChRs at innervated regions in developing embryos. The area of AChR aggregation increased by approximately 2-fold in MuSK injected embryos during the critical stages of NMJ formation. Interestingly, overexpression of MuSK in Xenopus embryos was found to induce the level of AChR transcript. Deletion of the Kringle domain in the MuSK construct did not attenuate the observed induction of AChR transcription and aggregation. Taken together, our findings provide the first demonstration that increased level of MuSK expression in vivo significantly elevate the aggregation and transcription of AChR at the NMJ in developing Xenopus embryos.

Animals↗
PubMed1999

Association of the D2 dopamine receptor third cytoplasmic loop with spinophilin, a protein phosphatase-1-interacting protein.

Signaling through D2 class dopamine receptors is crucial to correct brain development and function, and dysfunction of this system is implicated in major neurological disorders such as Parkinson's disease and schizophrenia. To investigate potential novel mechanisms of D2 receptor regulation, the third cytoplasmic loop of the D2 dopamine receptor was used to screen a rat hippocampal yeast two-hybrid library. Spinophilin, a recently characterized F-actin and protein phosphatase-1-binding protein with a single PDZ domain was identified as a protein that specifically associates with this region of D2 receptors. A direct interaction between spinophilin and the D2 receptor was confirmed in vitro using recombinant fusion proteins. The portion of spinophilin responsible for interacting with the D2 third cytoplasmic loop was narrowed to a region that does not include the actin-binding domain, the PDZ domain, or the coiled-coil. This region is distinct from the site of interaction with protein phosphatase-1, and both D2 receptors and protein phosphatase-1 may bind spinophilin at the same time. The interaction is not mediated via the unique 29-amino acid insert in D2long; both D2long and D2short third cytoplasmic loops interact with spinophilin in vitro and in yeast two-hybrid assays. Expression of D2 receptors containing an extracellular hemagglutinin epitope in Madin-Darby canine kidney cells results in co-localization of receptor and endogenous spinophilin as determined by immunocytochemistry using antibodies directed against spinophilin and the HA tag. We hypothesize that spinophilin is important for establishing a signaling complex for dopaminergic neurotransmission through D2 receptors by linking receptors to downstream signaling molecules and the actin cytoskeleton.

Actins↗
PubMed1999

Xenopus muscle-specific kinase: molecular cloning and prominent expression in neural tissues during early embryonic development.

A muscle-specific receptor tyrosine kinase, designated MuSK, mediates agrin-induced aggregation of acetylcholine receptors at the vertebrate neuromuscular junction. cDNAs encoding Xenopus MuSK were isolated from embryonic cDNA libraries. The full-length MuSK cDNA encodes for a polypeptide of 948 amino acids and possesses the features unique to mammalian MuSK, including four Ig-like domains, C6 box, transmembrane region and an intracellular tyrosine kinase domain. Interestingly, Xenopus MuSK also contains a kringle domain similar to that previously reported for Torpedo MuSK. The overall amino acid sequence identity of Xenopus MuSK with mammalian MuSK is approximately 65%. Northern blot analysis demonstrated the presence of three MuSK transcripts (approximately 1 kb, approximately 3 kb and approximately 7 kb) which were differentially expressed during development. The expression of the approximately 7 kb MuSK transcript remained as the predominant species in adult tissues, e.g. skeletal muscle, spleen and lung. Immunocytochemical analysis with a MuSK-specific antibody revealed that Xenopus MuSK was colocalized with AChRs at neuromuscular junctions as well as in spontaneous acetylcholine receptor hot spots of cultured muscle cells. In situ hybridization revealed prominent expression of MuSK transcripts in neural tissues and myotomal muscle during the period of neurulation and synaptogenesis. The MuSK transcript detected at abundant levels in the central nervous system (CNS) was localized to the brain, spinal cord and eye vesicles during early embryonic development. In addition, the MuSK protein in the developing eye was found to be prominently expressed during embryonic stages of 32 and 35. These findings raise an intriguing possibility that, in addition to the known function in the formation of the neuromuscular junctions, MuSK may be involved in neural development.

Agrin↗
PubMed1995

Chronic ethanol consumption differentially alters the expression of gamma-aminobutyric acidA receptor subunit mRNAs in rat cerebral cortex: competitive, quantitative reverse transcriptase-polymerase chain reaction analysis.

The molecular mechanisms that underlie ethanol dependence appear to involve alterations in GABAA receptor function and gene expression. In rat cerebral cortex, chronic exposure to ethanol alters many functional properties of GABAA receptors, including reduction of GABAA receptor-mediated chloride uptake. These functional alterations occur without a concomitant alteration in total receptor density or affinity. Previous investigations have shown that chronic ethanol exposure elicits alterations in mRNA and polypeptide levels for several abundant GABAA receptor subunits. For example, alpha 1 and alpha 2 subunit mRNA and polypeptide levels have been shown to decrease with chronic ethanol exposure. The present study was undertaken to further investigate the effects of chronic ethanol consumption on GABAA receptor subunit mRNA levels in rat cerebral cortex by using a competitive, quantitative reverse transcriptase-polymerase chain reaction assay that incorporates subunit-specific internal standards and allows for the absolute quantification of mRNA levels. We find that chronic ethanol consumption elicits a significant increase in alpha 4 subunit mRNA levels that is equal, in absolute amount, to a decrease in alpha 1 subunit mRNA levels. There is a small (30%) increase in gamma 2S but not gamma 2L subunit mRNA levels after chronic ethanol consumption. In addition, gamma 1 subunit mRNA levels are increased by 70%, whereas alpha 5, beta 1, beta 2, beta 3, gamma 3, and delta subunit mRNA levels do not change. We also reproduced results obtained previously by Northern blot analysis showing a 40% reduction in alpha 1 mRNA levels with no change in beta 2 subunit mRNA levels after chronic ethanol consumption. These results are consistent with the hypothesis that chronic ethanol consumption alters the function of GABAA receptors by eliciting changes in receptor subunit assembly. These changes may underlie the development of ethanol dependence.

Animals↗
PubMed1994

Biolistic transformation of conidia of Botryotinia fuckeliana.

Botryotinia fuckeliana, the causal agent of grey mould, was biolistically transformed to hygromycin B resistance using a plasmid (pOHT) containing a bacterial hygromycin phosphotransferase gene fused to regulatory sequences from Aspergillus nidulans. Multiple copies of the plasmid, precipitated onto tungsten particles, were delivered into the conidia by a helium-driven gene gun. Southern analysis showed that the plasmid was integrated into the fungal genome at one single locus. After five subsequent transfers on selective medium, all transformants were mitotically stable. When propagated on non-selective medium, four out of eight transformants retained their resistance to hygromycin B. Southern analysis of the fifth generation of transformants showed that no genetic rearrangements occurred during vegetative growth of stable transformants.

Bacterial Proteins↗
PubMed1994

GABAA and NMDA receptor subunit mRNA expression in ethanol dependent rats.

Recent studies have shown that alterations in gamma-aminobutyric acid (GABAA) and N-methyl-D-aspartate (NMDA) receptor subunit mRNA levels are associated with the effects of chronic ethanol exposure as well as genetic selection for ethanol withdrawal seizure sensitivity. We have previously shown that chronic ethanol exposure in rats results in a decrease in the levels of GABAA receptor alpha 1 and alpha 2 subunit mRNAs in cerebral cortex, an increase in the levels of alpha 6 subunit mRNAs in cerebellum and no alteration in alpha 3, GAD, ribosomal RNA or polyA + RNA levels in these regions. Since chronic ethanol administration increases the expression of [3H]Ro15-4513 binding sites in cortex and cerebellum with no effect on other GABAA receptor recognition sites, we hypothesized that the expression of other subunits would be altered in these regions. In addition, since ethanol appears to interact with zolpidem-sensitive GABAA receptors in rat brain, we investigated the effect of chronic ethanol administration on these recognition sites. Chronic ethanol administration increased [3H]zolpidem binding with no effect on levels of GABAA receptor beta 2 and gamma 2 subunit mRNAs. In addition, we examined the levels of NMDAR1 receptor subunit mRNAs since chronic ethanol administration results in increased levels of [3H]MK-801 recognition sites on NMDA receptors. NMDAR1 receptor subunit mRNAs were not altered following chronic ethanol exposure in rat cortex or hippocampus. These studies underscore the specificity of ethanol interactions with these receptors and the importance of understanding the mechanisms of both GABAA and NMDA receptor regulation in elucidating the etiology of ethanol dependence.

Alcoholism↗
PubMed1993

Optimizing the biolistic process for different biological applications.

The biolistic process is still rapidly evolving. We do not anticipate further major improvements in biolistic apparatus. There will probably still be further major improvements in particles, DNA coating, and vectors, as well as significant further advances in understanding of biological determinants of cell penetration and survival. The technology has currently reached the point at which it can be readily and reliably used for a wide range of applications. Given the information presented in this chapter, new applications can be optimized fairly readily.

Animals↗
PubMed1992

Biolistic transformation of prokaryotes: factors that affect biolistic transformation of very small cells.

Five bacterial species were transformed using particle gun-technology. No pretreatment of cells was necessary. Physical conditions (helium pressure, target cell distance and gap distance) and biological conditions (cell growth phase, osmoticum concentration, and cell density) were optimized for biolistic transformation of Escherichia coli and these conditions were then used to successfully transform Agrobacterium tumefaciens, Erwinia amylovora, Erwinia stewartii and Pseudomonas syringae pv. syringae. Transformation rates for E. coli were 10(4) per plate per 0.8 micrograms DNA. Although transformation rates for the other species were low (less than 10(2) per plate per 0.8 micrograms DNA), successful transformation without optimization for each species tested suggests wide utility of biolistic transformation of prokaryotes. E. coli has proven to be a useful model system to determine the effects of relative humidity, particle size and particle coating on efficiency of biolistic transformation.

Agrobacterium tumefaciens↗
PubMed1991

Biolistic transformation of a procaryote, Bacillus megaterium.

We present a simple and rapid method for introducing exogenous DNA into a bacterium, Bacillus megaterium, utilizing the recently developed biolistic process. A suspension of B. megaterium was spread onto the surface of nonselective medium. Plasmid pUB110 DNA, which contains a gene that confers kanamycin resistance, was precipitated onto tungsten particles. Using a biolistic propulsion system, the coated particles were accelerated at high velocities into the B. megaterium recipient cells. Selection was done by use of an agar overlay containing 50 micrograms of kanamycin per ml. Antibiotic-resistant transformants were recovered from the medium interface after 72 h of incubation, and the recipient strain was shown to contain the delivered plasmid by agarose gel electrophoresis of isolated plasmid DNA. All strains of B. megaterium tested were successfully transformed by this method, although transformation efficiency varied among strains. Physical variables of the biolistic process and biological variables associated with the target cells were optimized, yielding greater than 10(4) transformants per treated plate. This is the first report of the biolistic transformation of a procaryote.

Bacillus megaterium↗
PubMed1983

A method to measure pain associated with hypersensitive dentin.

Pain associated with dental hypersensitivity has been difficult to assess. An electronic threshold measure was developed to increase the degree of objectivity in a subject's response to a cold thermal stimulus. The apparatus consisted of a miniature thermistor connected to a medical multichannel recorder with a hand-held event recorder. The thermistor was placed adjacent to the hypersensitive area for an accurate temperature measure of the point at which the subject first reported pain. Room temperature air was gently blown on the hypersensitive area, dropping the surface temperature evenly from intraoral temperature to room temperature. Results indicated that this method of assessment provided consistent and reproducible data. The apparatus can detect changes in sensitivity and offers an objective approach to sensitivity studies.

Dentin Sensitivity↗
PubMed1979

Technetium-99m-labeled methylene diphosphonate and hydroxyethylidine diphosphonate--biologic and clinical comparison: concise communication.

The biologic and imaging characteristics of Tc-99m MDP and Tc-99m HEDP were compared in ten patients: Tc-99m MDP exhibited lower blood activity, lower 4-hr urinary excretion, and higher normal bone-to-background ratio. Assessment of overall image quality also favored Tc-99m MDP, indicating that the normal skeleton is better visualized with this agent. The total number of lesions seen (18) was not large enough to allow critical comparison of relative lesion-detecting efficacy. However, discrepancies between the two agents were observed, suggesting additional evaluation of the relative lesion-detecting efficacy of these two bone agents.

Adult↗
PubMed1979

Z80.1 and the state of the art.

Ophthalmic standards such as ANSI Z80.1, 1979 (Recommendation for Prescription Ophthalmic Lenses) are developed by the joint effort of many disciplines from eye care practitioners to suppliers and manufacturers. The tolerances, agreed upon by consensus, are fundamentally controlled by the state of the art, that is upon the quality of product which can be reliably produced by competent personnel using standard methods of processing. Research has demonstrated that the Z80.1, 1972 standard had been set at a level above the state of the art for today's lenses. Some of the tolerances in the 1979 revision were therefore lowered so as to establish a more realistic guideline. The new standard, if diligently applied by both laboratories and dispensers, should lead, however, to an increase and not a reduction in product quality.

Eyeglasses↗
PubMed1975

The design and prescription of multifocal lenses for civil pilots.

The aging (presbyopic) pilot has difficulty with near vision which can be a serious problem in the cockpit because of the complexity and uniqueness of his visual tasks. Numerous individual studies have been reported, but the aviation industry and vision care professionals do not have available to them a set of guidelines to permit the optimum fitting of spectacles for civil aviation pilots. In this study the cockpit vision environment was studied in ten contemporary aircraft. Twenty-five pilots of seven of these aircraft types participated in a spectacle design study. Experimental spectacles were designed for each pilot and evaluated for performance. Problems of pilot acceptance of reading aids, custom designing to the geometry of a given aircraft, balancing of the visual gains from complex designs versus difficulties in use and manufacture have been analyzed. Results of this study should be of value for human factors engineering, vision testing and accurate prescription for the presbyopic pilot.

Adult↗