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Biomedical subjects

M Willard

Publications and source records attributed to M Willard.

At least 19 recordsLinked to original sources
PubMed1997

Duodenal perforation in a cheetah (Acinonyx jubilatus).

An 11-yr-old female cheetah (Acinonyx jubilatus) from a privately owned breeding center for endangered species was referred for evaluation with a history of vomiting and depression of 10 days' duration. After anesthetic induction with tiletamine and zolazepam and anesthetic maintenance with isoflurane, a complete diagnostic workup was performed, including hematology, serum chemistry, and radiography. The clinical diagnosis was septic suppurative inflammation and hemorrhage in the abdomen, consistent with perforation or rupture of the gastrointestinal tract. An exploratory laparotomy showed a perforated duodenal ulcer, which was resected. Subsequent endoscopy revealed no further evidence of ulceration in the upper gastrointestinal tract. Biopsy of the ulcerated tissue collected from the duodenum revealed Gastrospirillum-like organisms. Histologic examination revealed widespread infiltration of lymphocytes and plasma cells into the lamina propria and submucosa. Intraepithelial leukocytes were present along with attenuation, erosion, and ulceration of the superficial epithelium. Fourteen days after surgery, this cheetah was returned to its breeding compound, and no subsequent vomiting has been observed for 4 yr.

Acinonyx↗
PubMed1994

Adenovirus-mediated transfer of a gene encoding human apolipoprotein A-I into normal mice increases circulating high-density lipoprotein cholesterol.

BACKGROUND: In animal models of atherosclerosis, augmentation of circulating high-density lipoprotein (HDL) cholesterol exerts a protective effect against development of fatty streaks and promotes plaque regression. METHODS AND RESULTS: To investigate the potential of gene transfer to increase HDL cholesterol, a fusion gene encoding human apolipoprotein A-I (apo A-I) under the control of the human cytomegalovirus (CMV) immediate-early promoter was packaged into a recombinant adenovirus (AdCMV apo A-I). BALB/c mice infected with AdCMV apo A-I by intravenous injection accumulate immunoreactive apo A-I in serum; levels 5 days after infection averaged 168 mg/dL. A 35% increase in HDL cholesterol and a 47% increase in total cholesterol were observed in mice infected with AdCMV apo A-I compared with control viruses. Analysis of size-fractionated lipoproteins revealed that human apo A-I is incorporated into murine HDL particles. Expression of human apo A-I declined to < 10% of maximum after 12 days and mRNA encoding apo A-I, prevalent 5 days after infection, was undetectable in the livers of infected mice after 12 days. CONCLUSIONS: We conclude that adenovirus-mediated transfer of a gene encoding apo A-I produces transient elevations of circulating HDL cholesterol of a magnitude correlated with important physiological effects. These observations suggest the potential for gene-based therapeutic strategies to reduce cardiovascular risk.

Adenoviridae↗
PubMed1991

The development of a patient satisfaction questionnaire in the ambulatory setting.

Patient satisfaction is of critical interest to medical care providers. The main objective of this study was to evaluate the psychometric properties of a patient satisfaction questionnaire. A preliminary 80-item questionnaire was created, and a random sample of 268 family practice patients participated. Subjects rated items on a 4-point Likert scale (strongly disagree, disagree, agree, strongly agree). Items were subjected to a principal components varimax rotated factor analysis and five factors (60 items) were extracted, accounting for 47.5% of the variance. These factors were: satisfaction with physician, dissatisfaction with practice management, physician availability, receptionist behavior, and wait time. Alpha reliability coefficients for factors 1-5 were: .96, .93, .89, .84, and .78, respectively. All items correlated highly with total scores on the respective factors. Factor intercorrelations were all significant (P less than .001) and in the expected direction. Patients with a higher level of education were significantly less satisfied about physician availability than patients without a high school education (P less than .05). Implications of the findings are discussed.

Adult↗
PubMed1990

Purification of the growth-associated protein GAP-43 by reversed phase chromatography: amino acid sequence analysis and cDNA identification.

GAP-43 is a neuronal phosphoprotein. Increased synthesis and axonal transport of GAP-43 has been associated with axon growth, and altered phosphorylation of GAP-43 has been associated with changes in synaptic efficacy. Here we report a rapid and effective procedure employing reverse-phase HPLC for the purification of GAP-43 from rat brain. To characterize the protein purified by this procedure, we generated proteolytic fragments and determined their amino acid sequences. These directly determined sequences, corresponding to 56% of the GAP-43 amino acids, confirm recently reported sequences deduced from the nucleotide sequences of cDNAs. Using oligonucleotide probes constructed according to these amino acid sequences, we identified GAP-43 cDNAs in a library prepared from neonatal rat superior cervical ganglion cells. One of these cDNAs was 1.1 kB in size; it hybridized specifically with a 1.5 kB RNA from brain, but not from liver, and contained the entire coding sequence for GAP-43. This cDNA differed from recently reported cDNAs in its 3' untranslated region.

Amino Acid Sequence↗
PubMed1990

Anxiolytic and sedative properties of BW A78U, a novel anticonvulsant adenine derivative.

The anticonvulsant BW A78U, tested in a free mouse exploratory situation, reduced in a dose-dependent fashion the locomotion and the number of rearings, this sedative effect being significant up to a dose of 15 mg/kg (IP, 20 min before testing). In an unconditioned conflict test, the light/dark box choice situation, specific for anxiolytics, low doses of BW A78U increased the time spent by mice in the lit box as well as the number of transitions between the two boxes. Finally, we demonstrated that this drug was able to protect mice against pentylenetetrazole-induced convulsions. Our data show that BW A78U possesses some of the characteristic properties of the minor tranquilizers. However, since this compound binds to the benzodiazepine receptor with a very low affinity (IC50 = 13.6 microM), it can be assumed that this drug does not exert its behavioral effects through these receptors. It may interfere with other targets involving adenosine, another potent physiological regulator of neuronal excitability.

Animals↗
PubMed1990

Identification of serine 473 as a major phosphorylation site in the neurofilament polypeptide NF-L.

Neurofilaments are composed of 3 polypeptides designated NF-H, NF-M, and NF-L, all of which are subject to posttranslational phosphorylation. It has been suggested that phosphorylation of the NF-L polypeptide can influence the assembly of NF-L into filaments, but the sites at which NF-L is phosphorylated are unknown. To locate these phosphorylation sites, we have identified phosphopeptides of NF-L by labeling them with 32P both in vitro and in cultured neurons and also by observing their change in chromatographic behavior after they have been treated with phosphatase. We report here that serine 473, in the carboxy-terminal tail domain of NF-L, is a major substrate in vitro for protein kinases endogenous to a crude cytoskeleton-containing fraction. Moreover, serine 473 is a major phosphorylation site in vivo; in neurofilaments isolated from rat spinal cord, approximately 73% of serine 473 was phosphorylated, and accounted for at least one-third of the total phosphate associated with NF-L. The identification of this phosphorylation site in NF-L provides a criterion for identifying the protein kinase that phosphorylates NF-L and raises the question of its function.

Animals↗
PubMed1989

A GAP-43-like protein in cat visual cortex.

We have purified a protein that changes in relative concentration during the development of the kitten visual cortex. It resembles GAP-43 (a neuronal protein that is expressed at elevated levels during periods of development and regenerative axon growth) in the following respects: (1) it is an acidic protein (pI = 4.7) whose electrophoretic mobility on SDS-PAGE is similar to, but lower than rat GAP-43, suggesting that the cat protein is larger; (2) its electrophoretic mobility varies with the acrylamide concentration in a manner that is characteristic of GAP-43; (3) its concentration in kitten forebrain is elevated during early postnatal development; (4) the sequence of ten consecutive amino acids from a chemically generated fragment matches the expected sequence from GAP-43; and (5) its amino-acid content also matches GAP-43. We conclude that our purified protein is cat GAP-43. Immunoblots with an antibody prepared against rat GAP-43 suggested that the concentration of GAP-43 in the visual cortex declines with age.

Acrylamide↗
PubMed1988

Distribution and phosphorylation of the growth-associated protein GAP-43 in regenerating sympathetic neurons in culture.

Sympathetic neurons regenerating in culture were studied in order to gain further insight into the intracellular distribution and phosphorylation of GAP-43, a protein that has been suggested to have a role in axonal outgrowth and neuronal plasticity (Willard et al., 1987). Superior cervical ganglion neurons from embryonic rats were highly reactive with a polyclonal antibody against the growth-associated protein GAP-43 soon after they were placed in culture on a laminin substrate. As these neurons extended neurites, the distribution of GAP-43 reactivity changed. The cell body became progressively less reactive, whereas the growth cone at the tip of the growing neurite reacted strongly. The pattern of immunofluorescence was punctate both in the growth cone and the adjacent neurite, but appeared more diffusely distributed in the cell body. The antibody reacted only with cells that had been subjected to treatment that permeabilized the plasma membrane. When antibody was supplied in the medium of growing neurons, it neither bound to the cells nor altered normal neurite initiation or elongation. Of the different types of cells in these cultures, the antibody reacted only with neurons; it did not react with Schwann cells or fibroblasts. The stimulation of protein kinase C in these cultures resulted in a 7-fold stimulation of the phosphorylation of a protein of similar electrophoretic mobility to GAP-43. These observations demonstrate that GAP-43 is neuron-specific, is present throughout the neuron but at higher levels in the growth cone, and is a major substrate of protein kinase C. The high concentration of GAP-43 in the growth cones may necessitate its increased synthesis in neurons with elongating axons. Its location and phosphorylation by kinase C suggest that it could perform a function in the growth cone that is modulated by extracellular signals, such as those used in pathfinding or in the control of axonal elongation.

Animals↗
PubMed1987

Translocations of fodrin and its binding proteins.

Fodrin, a protein related to erythrocyte spectrin, redistributes within the cell in certain situations. We compare such movements of fodrin and several fodrin binding proteins during the processes of axonal transport in neurons, and capping of surface proteins in lymphocytes. In neurons, three different populations of newly synthesized fodrin appear to be transported down the axons at different velocities corresponding to those of groups of transported proteins designated II, IV, and V. Actin, which can interact with fodrin, is transported at the velocity of group IV. Synapsin, a component of synaptic vesicles, is also reported to bind to fodrin. One population of synapsin is transported more rapidly than fodrin, at the velocity of group I: two additional populations of transported synapsin may overlap fodrin in groups II and IV. We consider possible functional associations of these different populations of fodrin and fodrin binding proteins. We note that the transport of group IV proteins resembles in certain respects the process of capping in lymphocytes, suggesting the possibility of a common mechanism. We outline one of several possible mechanisms.

Animals↗
PubMed1987

Axonal transport of synapsin I-like proteins in rabbit retinal ganglion cells.

Synapsin I is a neuronal phosphoprotein that is associated with the cytoplasmic surface of small, clear synaptic vesicles in neuronal synaptic terminals; it may play an important role in synaptic transmission. In vitro, it can interact with fodrin, a relative of the erythrocyte protein spectrin. We have investigated the delivery of synapsin I from its site of synthesis in neuronal cell bodies to synaptic terminals by means of the process of axonal transport. We labeled the newly synthesized proteins of rabbit retinal ganglion cells by injecting 35S-methionine into the vitreous humour, and subsequently observed the appearance of radioactive synapsin I (identified by its 2-dimensional electrophoretic mobility) in tissues containing the axons and synaptic terminals of these neurons. A portion of the newly synthesized synapsin I was axonally transported at the velocity of the most rapidly transported (group I) proteins, which comprise membrane-associated proteins and may include elements of synaptic vesicles. However, the subsequent time course of labeling of synapsin I in the axons suggests that greater than 90% of the axonally transported synapsin I may comprise 2 additional populations--one transported rapidly, the other slowly--that are released from the cell bodies only after a delay of more than 1 d. The delayed, slowly transported population moves at the velocity (approximately 6 mm/d) of groups III and IV (which include fodrin and other proteins of the membrane cytoskeleton). We consider whether such distinct populations may correspond to functionally specialized variants of synapsin I-like proteins that may be transported in association with different organelles. The electrophoretic mobility of labeled synapsin I-like proteins in the axons changed subtly with time. Additional subtle differences between labeled synapsin I-like proteins in the axons and the terminal-containing tissues suggest that certain posttranslational modifications occur specifically in the terminals.

Animals↗
PubMed1986

Localization of fodrin during fertilization and early development of sea urchins and mice.

Fodrin, a spectrin-like protein, is localized in gametes, zygotes, and embryos from sea urchins and mice. Mammalian fodrin comprises two polypeptides with molecular weights of approximately 240 kDa (alpha) and 235 kDa (beta). An antibody specific for mammalian alpha-fodrin cross-reacted with a 240-kDa polypeptide from sea urchin egg extracts. This indicates that sea urchins contain a protein of similar electrophoretic mobility and immunological properties to mammalian alpha-fodrin. When this antibody was used to stain the sea urchin gametes with indirect immunofluorescence, fodrin-specific fluorescence was localized to the acrosome of the sperm and was distributed over the entire egg near the surface in a punctate pattern similar to the distribution of polymeric actin. During sperm incorporation, the fodrin-specific fluorescence is found at the site of sperm incorporation, in the fertilization cone. After fertilization, the intensity of fodrin fluorescence increases. During mitosis and cytokinesis in sea urchins, the entire surface of the egg remains stained; the cleavage furrow also was stained but no more intensely than was the rest of the egg surface. Antibody labeling with colloidal gold followed by electron microscopy showed that fodrin was loated in the cytoplasm immediately beneath the plasma membrane. In unfertilized mouse oocytes, both actin and fodrin were stained most intensely beneath the membrane adjacent to the meiotic spindle. After insemination, the cell surfaces of the pronucleate egg and the second polar body were stained; however, the actin matrix surrounding the apposed pronuclei did not bind the fodrin antibody. During cytokinesis in the mouse, the cleavage furrow stained more intensely than did the rest of the egg cortex, and in embryos the cell borders were delineated. These results indicate that organisms as unrelated to mammals as sea urchins have fodrin-like proteins; the rearrangements of such proteins suggest that they participate in the actin-mediated events at the cell surface during fertilization and early development in both mice and sea urchins.

Actins↗
PubMed1983

Intracellular movement of fodrin.

Fodrin is an actin/calmodulin-binding protein with similarities to spectrin (erythrocytes) and TW 260/240 (brush border). It is concentrated beneath the plasma membranes of neurons and other cells. We have observed translocations of fodrin in both neurons and lymphocytes. Newly synthesized, radiolabeled fodrin moves down axons at a maximum velocity (about 50 mm/day) that is slower than the most rapidly axonally transported proteins (group I). A portion of fodrin appears to move more slowly at velocities (1-10 mm/day) resembling those of actin and myosin (group IV) and tubulin and neurofilament proteins (group V). In lymphocytes, when certain surface antigens are induced by cross-linking agents to migrate to one pole of the cell and form a cap, fodrin redistributes beneath the membrane and forms a subcap. The movements of fodrin in lymphocyte capping and in the axonal transport of group IV polypeptides have certain similarities. In both cases, the redistribution of fodrin is accompanied by concomitant redistributions of actin, myosin, and calmodulin, and both processes proceed at similar velocities. We consider the possibilities that these two processes are related, both being driven by a submembrane force-generating system comprising in part actin, myosin, and fodrin, and that fodrin serves to link various organelles or proteins to this system.

Animals↗
PubMed1983

Location of a protein of the fodrin-spectrin-TW260/240 family in the mouse intestinal brush border.

We have determined that a protein of the fodrin-spectrin-TW260/240 (FST) family is a component of the thin fibrils (approximately 5 nm wide, 100-200 nm long) that cross-link bundles of actin filaments to adjacent actin bundles and to the plasma membrane in the terminal web of the brush border of the intestinal epithelium. When isolated brush borders were incubated with anti-fodrin antibodies and prepared for electron microscopy by the quick-freeze, deep-etch technique, these approximately 5 nm fibrils were specifically decorated with the antibody. In addition, these cross-linking fibrils disappeared when the anti-fodrin-reactive proteins were extracted from the brush border. We conclude that FST is a component of a cross-linking system composed of approximately 5 nm fibrils that are morphologically distinct from the approximately 8 nm myosin-containing fibrils which were identified by anti-myosin decoration. In addition to linking actin bundles to adjacent actin bundles and to the plasma membrane, these FST fibrils may mediate actin-vesicle, actin-intermediate filament and vesicle-plasma membrane linkages.

Animals↗
PubMed1983

Modulations of neurofilament axonal transport during the development of rabbit retinal ganglion cells.

We have compared the polypeptides undergoing axonal transport in the retinal ganglion cells of neonatal and adult rabbits, and have observed the following: (1) Representative polypeptides of five different adult transport groups are axonally transported from the time of birth. (2) Polypeptides of group IV (a group that includes actin and myosin) are transported two-fold more rapidly in neonates than in adults. (3) Two polypeptides, M (145K) and L (73K) that are components of neurofilaments and move with the fifth, slowest group of transported proteins, are transported approximately eight-fold more rapidly in neonatal rabbits than in adults. (4) H, a third group V polypeptide, that serves to crosslink neurofilaments, was not detected in the rabbit optic nerve until 12 days after birth. We consider the possibility that the late induction of the crosslinker precipitates a cytoskeletal "phase transition" that may be responsible for the developmental alterations in apparent transport velocities, and may have additional consequences for neuronal development.

Aging↗
PubMed1983

Redistribution of fodrin (a component of the cortical cytoplasm) accompanying capping of cell surface molecules.

Fodrin, a protein composed of two polypeptides with molecular weights of 250,000 and 240,000, is concentrated in the cortical cytoplasm of neurons, and moves down the axons by the process of axonal transport. We have used immunofluorescence techniques to determine whether fodrin antigens also move in non-neuronal cells when cell surface ligands are induced to redistribute by crosslinking them. A redistribution of fodrin antigens occurred in the following instances: (i) when 3T3 cells were incubated with concanavalin A and anti-concanavalin A, surface concanavalin A receptors formed aggregates and fodrin antigens formed corresponding intracellular aggregates; (ii) when B lymphocytes were incubated with anti-Ig, the surface Ig formed caps and fodrin antigens formed intracellular subcaps; (iii) when T lymphocytes were treated with anti-H-2 followed by a secondary antibody, the H-2 antigen formed caps and fodrin formed corresponding subcaps. These observations show that fodrin antigens can move within non-neuronal cells, as well as in axons, and that their organization can be regulated by interaction between surface proteins and environmental stimuli. They also raise the possibility that fodrin, together with other proteins that form subcaps in lymphocytes (e.g., actin, myosin, and alpha-actinin) is a component of the cellular machinery responsible for the capping process. We consider whether the similarities between the movements of fodrin in lymphocyte capping and axonal transport may indicate that certain aspects of these two processes are related.

Animals↗