Biomedical subjects
Publications and source records attributed to W D Phillips.
We used optical tweezers (optical trapping technology) to measure the force required to separate antigen-antibody bonds. Under competitive-binding conditions, we used the force determination to detect and measure protein antigen concentrations as small as 1 fmol/L (10(-15) mol/L).
BACKGROUND: The biochemical and biomechanical determinants of adhesion between two biological objects following a collision are complex, and may vary from one system to another. We wished to develop an assay in which all the relevant factors, including the components of the solution, the relative orientation and the relative collision velocity, are under the user's control. RESULTS: A new assay is described in which two mesoscale particles are caused to collide using two independently controlled optical tweezers (optically controlled collision, OPTCOL). This assay enables precise examination of the probability of adhesion under biologically relevant conditions. The OPTCOL assay was used to evaluate the probability of adhesion of a single erythrocyte to a single virus-coated microsphere, in the absence and presence of a sialic acidbearing inhibitor. Inhibition constants for the most effective inhibitors could not be measured using other types of assays. The best inhibitor prevented attachment 50 % of the time at a sialic acid concentration of 35 pmol l-1; it is the most potent known inhibitor of attachment of influenza virus to erythrocytes. CONCLUSIONS: OPTCOL is a versatile new bioassay for studying dynamic interactions in biochemistry. It offers an approach to investigating interactions between moving biological objects that is both quantitative and interpretable. The simplicity of the OPTCOL technique suggests broad applicability to the study of adhesion of mesoscale (1-100 microm) objects in the areas of cell biology, microbiology, medicinal chemistry, and biophysics.
Are subjects sensitive to the frequency of parts of the same whole in the same way that they are sensitive to instances of the same category? Subjects read a randomised list of words and then estimated how many words on the list had belonged to particular groups, e.g. parts of a bird, or kinds of bird. Groups of words were either parts of the same whole, e.g. feathers, beak, wing, or members of the same category, e.g. robin, sparrow, eagle. Subjects were asked, either, "How many parts of a (bird) did you see?" or "How many kinds of (bird) did you see?". Half of the subjects expected the frequency estimation task, whereas for the other half, who performed a distractor task during encoding, the task was unexpected. In Experiment 1 subjects saw either parts of kinds. Sensitivity to frequency did not differ for parts and kinds nor as a function of whether the frequency estimation test was expected. In Experiment 2 subjects saw both parts and kinds belonging to the same superordinates, e.g. parts of birds and kinds of birds, and were asked to estimate the frequency of both types of groupings for each superordinate. The results were the same as in Experiment 1. In addition, subjects were able to separate the frequency of parts and kinds of the same superordinate. The results suggest that, in frequency estimation, part-of and kind-of associations can function similarly, and that the mechanism responsible for frequency estimation is sensitive to the type of association.
Lithography can be performed with beams of neutral atoms in metastable excited states to pattern self-assembled monolayers (SAMs) of alkanethiolates on gold. An estimated exposure of a SAM of dodecanethiolate (DDT) to 15 to 20 metastable argon atoms per DDT molecule damaged the SAM sufficiently to allow penetration of an aqueous solution of ferricyanide to the surface of the gold. This solution etched the gold and transformed the patterns in the SAMs into structures of gold; these structures had edge resolution of less than 100 nanometers. Regions of SAMs as large as 2 square centimeters were patterned by exposure to a beam of metastable argon atoms. These observations suggest that this system may be useful in new forms of micro- and nanolithography.
1. The post-synaptic membranes of neurons and muscle cells are characterized by clusters of transmitter receptors, the number and type of which help to determine synaptic efficacy. Here I briefly review what is known of the mechanism of clustering of nicotinic acetylcholine receptors (AChR) at neuromuscular synapses. 2. The extracellular protein agrin is thought to be secreted by the motor nerve terminal and trigger localized clustering of AChR in the post-synaptic membrane of the skeletal muscle cell. 3. Binding of agrin to its receptor, alpha-dystroglycan, is followed by rearrangements of the muscle membrane cytoskeleton with localized replacement of dystrophin by utrophin. It remains unclear how these changes relate to the clustering of AChR. 4. In separate studies, RAPsyn/43k protein, a protein associated with the inner face of the post-synaptic membrane was shown to be able to cluster AChR and link them to the cytoskeleton when both proteins were co-transfected into fibroblasts. 5. Mutational studies on RAPsyn identified putative binding domains for AChR and for the cytoskeleton within the RAPsyn primary structure. Targeted disruption of the RAPsyn gene in mice prevented post-synaptic AChR clustering and led to neonatal lethality. Thus RAPsyn might be the final link in the pathway that leads to AChR immobilization in the post-synaptic membrane. 6. The recent observation that active forms of agrin are not restricted to cholinergic regions of the brain suggests that analogous pathways may exist for clustering other receptor types.
Five roving symposia series directed to primary care physicians addressing the complications of diabetes were designed, implemented, and evaluated. These roving symposia effectively reached physicians statewide and appeared to influence positive practice behaviors.
The 43K protein is a cytoplasmic peripheral membrane protein concentrated subsynaptically in skeletal muscle. Recombinant 43K has been shown to cause clustering of acetylcholine receptors (AChRs) in cultured cells. However, the role of 43K in vivo is disputed, because in some cases it appears only after AChRs have clustered. We therefore examined the expression and distribution of 43K and AChRs during synapse formation in embryonic mouse muscles. Messenger RNA for 43K was detected on Embryonic Day (E) 12, a day prior to the first AChR clusters. Immunofluorescence showed that both AChRs and 43K were colocalized in patches by E13, the stage at which intramuscular nerves were first detected. The AChR/43K patches were nerve associated, and more than 98% of AChR patches were accompanied by 43K. The precise colocalization of 43K and AChRs persisted through development. These results are consistent with 43K being involved in the nerve-induced clustering of AChRs during synapse formation.
Recombinant acetylcholine receptors (AChRs) expressed on the surface of cultured fibroblasts become organized into discrete membrane domains when the 43-kD postsynaptic protein (43k) is co-expressed in the same cells (Froehner, S.C., C. W. Luetje, P. B. Scotland, and J. Patrick, 1990. Neuron. 5:403-410; Phillips, W. D., M. C. Kopta, P. Blount, P. D. Gardner, J. H. Steinbach, and J. P. Merlie. 1991. Science (Wash. DC). 251:568-570). Here we show that AChRs present on the fibroblast cell surface prior to transfection of 43k are recruited into 43k-rich membrane domains. Aggregated AChRs show increased resistance to extraction with Triton X-100, suggesting a 43k-dependent linkage to the cytoskeleton. Myotubes of the mouse cell line C2 spontaneously display occasional AChR/43k-rich membrane domains that ranged in diameter up to 15 microns, but expressed many more when 43k was overexpressed following transfection of 43k cDNA. However, the membrane domains induced by recombinant 43k were predominantly small (< or = 2 microns). We were then interested in whether the cytoskeletal component, dystrophin related protein (DRP; Tinsley, J. M., D. J. Blake, A. Roche, U. Fairbrother, J. Riss, B. C. Byth, A. E. Knight, J. Kendrick-Jones, G. K. Suthers, D. R. Love, Y. H. Edwards, and K. E. Davis, 1992. Nature (Lond.). 360:591-593) contributed to the development of AChR clusters. Immunofluorescent anti-DRP staining was present at the earliest stages of AChR clustering at the neuromuscular synapse in mouse embryos and was also concentrated at the large AChR-rich domains on nontransfected C2 myotubes. Surprisingly, anti-DRP staining was concentrated mainly at the large, but not the small AChR clusters on C2 myotubes suggesting that DRP may be principally involved in permitting the growth of AChR clusters.
The developing neuromuscular junction has provided an important paradigm for studying synapse formation. An outstanding feature of neuromuscular differentiation is the aggregation of acetylcholine receptors (AChRs) at high density in the postsynaptic membrane. While AChR aggregation is generally believed to be induced by the nerve, the mechanisms underlying aggregation remain to be clarified. A 43-kD protein (43k) normally associated with the cytoplasmic aspect of AChR clusters has long been suspected of immobilizing AChRs by linking them to the cytoskeleton. In recent studies, the AChR clustering activity of 43k has, at last, been demonstrated by expressing recombinant AChR and 43k in non-muscle cells. Mutagenesis of 43k has revealed distinct domains within the primary structure which may be responsible for plasma membrane targeting and AChR binding. Other lines of study have provided clues as to how nerve-derived (extracellular) AChR-cluster inducing factors such as agrin might activate 43k-driven postsynaptic membrane specialization.
Neurotransmitter receptors are generally clustered in the postsynaptic membrane. The mechanism of clustering was analyzed with fibroblast cell lines that were stably transfected with the four subunits for fetal (alpha, beta, gamma, delta) or adult (alpha, beta, epsilon, delta) type mouse muscle nicotinic acetylcholine receptors (AChRs). Immunofluorescent staining indicated that AChRs were dispersed on the surface of these cells. When transiently transfected with an expression construct encoding a 43-kilodalton protein that is normally concentrated under the postsynaptic membrane, AChRs expressed in these cells became aggregated in large cell-surface clusters, colocalized with the 43-kilodalton protein. This suggests that 43-kilodalton protein can induce AChR clustering and that cluster induction involves direct contact between AChR and 43-kilodalton protein.
The postsynaptic membrane of the neuromuscular junction contains a myristoylated 43-kD protein (43k) that is closely associated with the cytoplasmic face of the nicotinic acetylcholine receptor (AChR)-rich plasma membrane. Previously, we described fibroblast cell lines expressing recombinant AChRs. Transfection of these cell lines with 43k was necessary and sufficient for reorganization of AChR into discrete 43k-rich plasma membrane domains (Phillips, W. D., C. Kopta, P. Blount, P. D. Gardner, J. H. Steinbach, and J. P. Merlie. 1991. Science (Wash. DC). 251:568-570). Here we demonstrate the utility of this expression system for the study of 43k function by site-directed mutagenesis. Substitution of a termination codon for Asp254 produced a truncated (28-kD) protein that associated poorly with the cell membrane. The conversion of Gly2 to Ala2, to preclude NH2-terminal myristoylation, reduced the frequency with which 43k formed plasma membrane domains by threefold, but did not eliminate the aggregation of AChRs at these domains. Since both NH2 and COOH-termini seemed important for association of 43k with the plasma membrane, a deletion mutant was constructed in which the codon Gln15 was fused in-frame to Ile255 to create a 19-kD protein. This mutated protein formed 43k-rich plasma membrane domains at wild-type frequency, but the domains failed to aggregate AChRs, suggesting that the central part of the 43k polypeptide may be involved in AChR aggregation. Our results suggest that membrane association and AChR interactions are separable functions of the 43k molecule.
The spatial distribution of intracellular acetylcholine receptors along the length of fibres from the avian posterior latissimus dorsi muscle has been investigated during embryonic development, when distributed synaptic sites are eliminated from the muscle fibres. Cell surface AChR were irreversibly blocked with unlabelled alpha-bungarotoxin (alpha-BGT). Muscles were then fixed and ultrasonically dissociated into fibre fragments, treated with 0.5% saponin and stained with 125I-alpha-BGT. This revealed an intracellular pool of curare sensitive binding sites equivalent to about 10% of total cell AChR. The spatial distribution of this pool was studied by autoradiography. Large (longer than 2 microns) AChR-clusters (AChR-C) characteristic of neuromuscular contacts were localized on the same fibres by immunofluorescence with an anti-AChR antibody. At E11, relatively high levels of intracellular AChR were observed throughout the length of fibres. Between E11 and E18 intracellular AChR declined (19 fold) in extrajunctional parts of fibres but remained high in segments of fibre corresponding to AChR-clusters. Treatment of E14 embryos with an inhibitor of protein synthesis (cycloheximide) reduced intracellular AChR to 22 +/- 6% (mean +/- SE) of control levels, suggesting that most of the intracellular binding represented newly-synthesized AChR. Between E11 and E18 cell nuclei were found to accumulate beneath AChR-C. The mean density of nuclei in segments of fibre corresponding to AChR-C increased 5 fold between E11 and E18, but remained unchanged in extrajunctional segments. It is suggested that the elimination of excess distributed AChR-C may be due to the preferential accumulation of nuclei at a single AChR-C on each fibre accompanied by the down regulation of AChR synthesis associated with nuclei at the remaining AChR-C.
Changes in the distribution of large acetylcholine receptor clusters (AChR-Cs) on developing fast-twitch fibres of the chicken posterior latissimus dorsi (PLD) muscle have been studied during the period of loss of polyneuronal innervation using fluorescein-conjugated alpha-bungarotoxin. Embryonic muscles were ultrasonically dissociated into single fibre fragments and presumptive fast-twitch fibres were distinguished from the minority of slow-type fibres in the PLD by immunofluorescence using an antibody against slow-type myosin. Whereas mature PLD muscle fibres are focally innervated, at embryonic day 11 (E11) many fibre fragments from the PLD displayed two or more large (longer than 2 micron) AChR-Cs. Double labelling with anti-neurofilament antibody suggested that most of these AChR-Cs (82 +/- 2%) were associated with neuromuscular contacts. There was a progressive decline in the number of large (synaptic) AChR-Cs per 1000 micron of fibre, from 3.2 +/- 0.5 at E11 to 0.4 +/- 0.1 at E18. No further decline occurred between E18 and one week post-hatch. Primary generation muscle cells identified at E11 and E16 by tritiated thymidine labelling showed a decline in the number of large AChR-Cs per 1000 micron proportional to that seen in the fibre population as a whole, suggesting that distributed synaptic AChR-Cs are eliminated from individual fibres as they mature. When embryos were treated with d-tubocurarine starting at E6 the loss of distributed AChR-Cs from fast-type PLD fibres between E11 and E14 did not occur, suggesting that neuromuscular activity may play an important role in establishing the focal synaptic site AChR-C.
The development of the focal localization of large acetylcholine receptor clusters (AChR-Cs) on avian fast muscle fibres has been investigated in the triceps brachii pars humeralis (TH) muscle of the chick embryo. The mature TH muscle consists of both fast fibres, which usually receive a focal innervation at single synaptic sites, and slow fibres which receive a distributed innervation at multiple synaptic sites. Single fibre fragments dissociated from the embryonic muscle were typed using anti-myosin antibodies; fluorescently labelled alpha-bungarotoxin was used to identify large AChR-Cs which serve as synaptic markers. In contrast to the mature focal innervation, at embryonic day 11 (E11), many fast-type fibres in the TH muscle displayed large, distributed AChR-Cs (3.7 +/- 0.7 per 1000 microns fibre length; n = 6 embryos) like neighbouring slow-type fibres. By E16 distributed AChR-Cs were rare on fast type fibres (0.9 +/- 0.2 per 1000 microns fibre length). As it was possible that the frequency of fast fibres with distributed AChR-Cs declined simply as a consequence of the increase in number of secondary generation fibres, tritiated thymidine was injected at E7 in order to identify the primary generation fibres at E14. The great majority of fast fibres that were heavily labelled with thymidine at E14 appeared to possess a focal AChR-C. The results suggest that at E11 fast-type primary fibres in the TH muscle receive a distributed innervation very similar to neighbouring slow-type fibres; this subsequently evolves into the mature focal innervation following the elimination of synaptic sites between E11 and E14.
Many avian muscles contain a characteristic topographical distribution of fibre types. In order to study the role of nerves in the establishment and distribution of these fibre types, monoclonal antibodies (McAb) to the heavy chain subunit of myosin (MHC) were produced. The anti-fast McAb (2B12) bound to adult fast MHC and cross-reacted with the embryonic isoform of MHC. The anti-slow McAb (3D1) bound specifically to the heavy chain of slow myosin 2. By indirect immunofluorescence, anti-fast (2B12) stained all myotubes in the anterior latissimus dorsi and triceps and biceps muscles at stage 37 (11 days embryonic), whilst anti-slow (3D1) staining was largely restricted to the future slow fibres of these muscles. Brachial levels of the neural tube were surgically removed at stage 12 (2 days embryonic) so that muscles developed aneurally. Muscles at aneural stage 37 were smaller than normal, but the distribution of myotube types was not altered; all myotubes present still stained with anti-fast antibody while anti-slow staining was restricted to the anterior latissimus dorsi and myotubes in the deep parts of the triceps brachii pars scapularis, triceps brachii par humeralis and biceps brachii muscles (the future slow fibres of normal muscles). The results suggest that despite an overall reduction in MHC in aneural muscles, specialized fast and slow primary myotubes arise independently of the nerve in appropriate regions of the muscle.
The size and distribution of acetylcholine receptor clusters (AChR-C) on normal and aneural developing muscle fibres of the chick wing were studied by labelling AChR with fluorescent conjugates of alpha-bungarotoxin (alpha-BGT). AChR-C of a size typical of initial synaptic contacts (5 micron long) were present at 7 days incubation, shortly after the appearance of nerves, and were grouped in bands corresponding to muscle nerve branches. A regular distribution of large (approximately equal to 5 micron) AChR-C separated by 100-200 micron had developed by 10-14 days in the slow-tonic anterior latissimus dorsi and ulnimetacarpalis dorsalis muscles. The role of motor innervation in the formation of AChR-C was assessed by removing the brachial neural tube at 2 days incubation in order to prevent nerves entering the wing. Neural-tube removal prevented the appearance of the large AChR-C normally associated with the early synaptic contacts. Small AChR-C (less than 2 micron long) appeared in aneural muscles, but these were not grouped into bands characteristic of the large AChR-C in normal muscles. The results suggest that the formation of junctional AChR-C is dependent on nerves.
The embryonic precursors of the avian slow (type I and III) and fast (type II) fibers can be distinguished from each other early in muscle formation (stage 28, V. Hamburger and H. L. Hamilton, J. Morphol, 88, 49-92, 1951) on the basis of the differential sensitivity of their myosin ATPases. To test the neural dependence of fiber type differentiation, the source of motor innervation was eliminated by excision of the brachial neural tube at stages 16-18 before muscles are innervated. Removal of the brachial neural tube did not affect the number of primary myotubes in a sample muscle of the forelimb (ulnimetacarpalis dorsalis, UMD) up until stage 36. Myosin ATPase staining at a variety of pHs revealed the typical patterns of fiber types in muscles of neural-tube free embryos in stages 35-37. These muscles included the anterior latissimus dorsi, brachialis, and UMD which showed presumptive type III staining (type IIIEMB), the pronator superficialis and flexor carpi ulnaris which showed embryonic type II staining (type IIEMB), and the triceps brachii muscles which showed characteristic arrangements of both type IEMB and type IIEMB fibers. The normal patterns of type IEMB and type IIEMB myotubes were also seen in muscles containing a heterogeneous mixture of fiber types such as the biceps brachii, extensor metacarpi radialis, and adductor indicis muscles, although the intensity of acid-stable ATPase staining of the type IEMB myotubes in these muscles was lower than in innervated muscles. It is concluded that the earliest differentiation of muscle fiber types is independent of the nervous system.