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Biomedical subjects

J E Fox

Publications and source records attributed to J E Fox.

At least 109 records · Page 6Linked to original sources
PubMed1987

Activation of endogenous excitatory opiate pathways in canine small intestine by field stimulation and motilin.

Field stimulation of intrinsic nerves (40 V, 0.5 ms, 1-5 pps for 3-10 min) or intraarterial administration of motilin (10(-11)-10(-8 mol) caused contractions of the canine small intestine that were partially resistant to atropine and abolished by tetrodotoxin. Naloxone (10 micrograms ia or 200 micrograms/kg iv) in selective doses (10(-10)-10(-9) mol) that left responses to intra-arterial acetylcholine unaffected reduced responses to field stimulation or motilin in the ileum in the absence of atropine or residual responses in the presence of atropine. Results in the jejunum were similar except that selective doses of naloxone (10 micrograms ia or 200 micrograms/kg iv) increased the sensitivity to motilin in the absence of atropine. Higher doses of naloxone decreased these responses. In common with naloxone, tachyphylaxis to methionine-enkephalin (Met-Enk) reduced the responses to field stimulation or motilin before or after atropine but did not affect the responses to acetylcholine before or the responses to field stimulation of muscle after atropine. These findings are consistent with the hypothesis that Met-Enk may be a noncholinergic, excitatory transmitter released by field stimulation of nerves or intra-arterial motilin in the canine intestine.

Animals↗
PubMed1987

Spectrin is associated with membrane-bound actin filaments in platelets and is hydrolyzed by the Ca2+-dependent protease during platelet activation.

We recently showed that platelets contain submembranous actin filaments that are linked to glycoprotein (GP) Ib on the plasma membrane. In the present study, experiments were performed to determine whether spectrin was associated with these filaments. The membrane-bound filaments were isolated from Triton X-100 (Sigma, St Louis) lysates of unstimulated platelets by differential centrifugation. Platelet spectrin was detected immunologically by using antibodies against human brain and RBC spectrin. Immunoblots showed that platelet spectrin consisted of two polypeptides (mol wt 240,000 and 235,000) that were similar in apparent mol wt to those of the alpha and beta chains of brain spectrin but differed slightly from those of RBC spectrin (mol wt 240,000 and 220,000). Immunoprecipitation experiments identified platelet spectrin as two minor polypeptides migrating on sodium dodecyl sulfate (SDS)-polyacrylamide gels between actin-binding protein (mol wt 250,000) and the platelet polypeptide P235 (mol wt 235,000). Immunoblots of fractions isolated from Triton X-100-lysed platelets revealed that the alpha and beta chains of platelet spectrin were associated almost entirely with the actin filaments that were linked to the plasma membrane. Little spectrin was recovered in the Triton X-100-soluble fraction or with the actin filaments that were not membrane bound. During activation of platelets with thrombin or ionophore A23187, the alpha and beta chains of spectrin were hydrolyzed, generating a major degradation product of mol wt 160,000 and a minor one of mol wt 170,000. These two hydrolytic products were also generated in Triton X-100 lysates incubated in the presence of Ca2+ but were not produced when lysates were treated with leupeptin, ethylene glycol bis(beta-aminoethyl ether)-N,N,N',N'-tetraacetic acid (EGTA), or N-ethylmaleimide, known inhibitors of the Ca2+-dependent protease. These experiments show that spectrin is a previously unidentified component of the membrane-bound actin filament network and that hydrolysis of spectrin by the Ca2+-dependent protease may regulate the interactions of the filaments during platelet activation.

Adult↗
PubMed1987

Local actions of trimebutine on canine gastrointestinal tract.

The local actions of trimebutine on the circular muscle of canine gastrointestinal tract were studied after close intraarterial injection. The effects resembled those of metenkephalin at all sites. In stomach, trimebutine had no excitatory effects, but inhibited responses mediated by cholinergic post-ganglionic nerves. In small intestine, trimebutine stimulated the quiet gut by probably both neural and direct smooth muscle mechanisms, and it inhibited the field-stimulated phasic contractions. In large intestine, trimebutine had no excitatory actions and only weak inhibitory actions on the field-stimulated gut. Excitatory actions most likely seem to use the mu or delta receptors while inhibitory actions may focus on kappa opiate receptors.

Animals↗
PubMed1986

Tachykinin activation of muscarinic inhibition in canine small intestine is SPP in nature.

Substance P when injected intraarterially into the small intestine of the anaesthetized dog during phasic activity produces three concentration dependent responses of the circular muscle. At lowest doses (approximately 10(-12) moles) inhibition occurs via release of acetylcholine to a muscarinic auto-receptor. At slightly higher doses (10(-10) moles) inhibition is preceeded by excitation via release of acetylcholine to muscarinic receptors on the smooth muscle. At still higher doses (10(-9) moles) substance P excites the smooth muscle directly. The present study demonstrates that other members of the tachykinin family also produce inhibition in vivo. The potency sequence was found to be physalaemin greater than or equal to substance P = neuromedin K greater than kassinin greater than alpha neurokinin = eledoisin. Such a sequence suggests that substance P is a natural stimulant of this pathway and that the receptor is SPP-like. The C-terminal fragment, substance P8-11, was a weak agonist at this receptor, while substance P1-9 was ineffective.

Acetylcholine↗
PubMed1986

Galanin: an inhibitory neural peptide of the canine small intestine.

Galanin injected intraarterially during phasic activity of the canine small intestine in vivo produced inhibition. Fifty percent inhibition occurred at 1.5 +/- 0.5 X 10(-10) mols lasting for 0.7 min. The inhibitory response was not decreased by treatment with atropine, hexamethonium, yohimbine or naloxone, suggesting that muscarinic, nicotinic, alpha 2 adrenergic or opiate receptors were not being stimulated. Since tetrodotoxin blockade of nerves did not reduce the response and galanin at 10(-10) mols was able to eliminate the smooth muscle response to intraarterial acetylcholine, we suggest that galanin acts to inhibit smooth muscle directly. Galanin 10(-9) M added to the muscle bath also inhibited phasic activity of the canine ileum circular muscle in vitro in the presence of tetrodotoxin. These results suggest that the neural peptide galanin may be a non-adrenergic, non-cholinergic, non-opioid neurotransmitter in the canine small intestine.

Animals↗
PubMed1986

DNA analysis for ornithine transcarbamylase deficiency.

We have utilized the Southern blotting technique to analyse genomic DNA from males with ornithine transcarbamylase (OTC) deficiency and their families. Using a nearly full-length human cDNA probe, we have identified 3 patients with deletions at this locus and have characterized 4 different restriction fragment length polymorphisms that can be used as linkage markers for the OTC mutation. These polymorphisms occur at sufficiently high frequencies so as to enable us to distinguish the two X-chromosomes in approximately 80% of OTC carriers. As a direct consequence of these findings, prenatal diagnosis and carrier assessment can be offered to a large fraction of families at risk for OTC deficiency.

Chromosome Deletion↗
PubMed1986

Substance P: a potent inhibitor of the canine small intestine in vivo.

Intra-arterially administered substance P inhibited neurally activated contractions of the circular muscle of canine small intestine in vivo (lowest effective dose approximately 10(-13) mol). Excitation of intestine required higher (10(-10) mol) doses. The inhibitory effect required functioning nerves, since tetrodotoxin treatment eliminated it. However, inhibition of neurogenic contraction by substance P was unaffected by nicotinic or opiate receptor antagonists or by catecholamine depletion but was reduced by a selective substance P antagonist. Since the inhibition by substance P was also greatly reduced by treatment with atropine or pirenzepine and acetylcholine given intra-arterially produced a similar inhibitory response, stimulation of release of acetylcholine to inhibitory muscarinic receptors on nerves appeared to be the mechanism of this action. Direct smooth muscle effects were ruled out; substance P did not inhibit contractions to intra-arterial acetylcholine or those following tetrodotoxin. In vitro in ileal strips, no inhibition by substance P of any contractile response was found. We propose that the local release of substance P into the myenteric plexus produces inhibition and suggest that this constitutes a physiological function of the neuropeptide. This action may be absent in vitro.

Acetylcholine↗
PubMed1986

Identification and application of additional restriction fragment length polymorphisms at the human ornithine transcarbamylase locus.

Two additional restriction fragment length polymorphisms (RFLPs) have been identified at the human ornithine transcarbamylase (OTC) locus. Approximately 11% of women are heterozygous for an RFLP characterized by polymorphic bands at 3.7 and 3.6 kilobasepairs (kbp) observed after DNA digestion with TaqI. Twenty-nine percent of women are heterozygous for an RFLP characterized by polymorphic bands at 18.0 and 5.2 kbp observed after digestion with BamHI. Thus, in combination with the previously reported RFLPs identified using MspI, the X chromosomes in approximately 80% of women at risk for having a son with OTC deficiency are distinguishable by RFLPs at the OTC locus. Furthermore, we show that these RFLPs will be useful in families for prenatal diagnosis of OTC deficiency, carrier detection, and carrier exclusion.

Chromosome Mapping↗
PubMed1986

Primary care physician satisfaction with case management.

Primary care case management is frequently proposed as one mechanism to reduce the overall cost of health services. This concept places a utilization control function upon primary care physicians who, under primary care case management, must approve all patient access to specialty physician services and hospital services. This utilization control function, often called the gatekeeper role, places new responsibilities on primary care physicians. In this study we surveyed primary care physician attitudes regarding their role as case managers.

Attitude of Health Personnel↗
PubMed1985

Identification of actin-binding protein as the protein linking the membrane skeleton to glycoproteins on platelet plasma membranes.

Platelets have previously been shown to contain a membrane skeleton that is composed of actin filaments, actin-binding protein, and three membrane glycoproteins (GP), GP Ib, GP Ia, and a minor glycoprotein of Mr = 250,000. The present study was designed to determine how the membrane glycoproteins were linked to actin filaments. Unstimulated platelets were lysed with Triton X-100, and the membrane skeleton was isolated on sucrose density gradients or by high-speed centrifugation. The association of the membrane glycoproteins with the actin filaments was disrupted when actin-binding protein was hydrolyzed by activity of the Ca2+-dependent protease, which was active in platelet lysates upon addition of Ca2+ in the absence of leupeptin. Similarly, activation of the Ca2+-dependent protease in intact platelets by the addition of a platelet agonist also caused the membrane glycoproteins to dissociate from the membrane skeleton. Affinity-purified actin-binding protein antibodies immunoprecipitated the membrane glycoproteins from platelet lysates in which actin filaments had been removed by DNase I-induced depolymerization and high-speed centrifugation. These results demonstrate that actin-binding protein links actin filaments of the platelet membrane skeleton to three plasma membrane glycoproteins and that filaments are released from their attachment site when actin-binding protein is hydrolyzed by the Ca2+-dependent protease within intact platelets during platelet activation.

Actins↗
PubMed1985

Identification of two proteins (actin-binding protein and P235) that are hydrolyzed by endogenous Ca2+-dependent protease during platelet aggregation.

Our previous research has shown that the Ca2+-dependent protease within platelets is activated when platelets aggregate, resulting in the production of three polypeptides (Mr = 200,000, 100,000, and 91,000). We have now shown that these three polypeptides arise from the hydrolysis of actin-binding protein. An antibody against actin-binding protein raised in rabbits was shown to be specific for actin-binding protein on immunoblots of total platelet proteins. This antibody reacted with additional polypeptides of Mr = 200,000, 100,000, and 91,000 on immunoblots of the proteins of thrombin-activated platelets. Actin-binding protein was purified from fresh, human platelet concentrate and hydrolyzed with platelet-derived Ca2+-dependent protease; hydrolysis resulted in the appearance of three polypeptides with molecular weights and isoelectric points identical to those of the three polypeptides generated within intact, aggregating platelets. Production of these polypeptides was inhibited by leupeptin and by the chelation of Ca2+. Hydrolysis of actin-binding protein was observed at micromolar Ca2+ concentrations, demonstrating that the level of Ca2+ in aggregated platelets is sufficient to account for the hydrolysis of actin-binding protein by the Ca2+-dependent protease. P235 was also purified and tested for its susceptibility to the protease. It was hydrolyzed by the Ca2+-dependent protease, and two polypeptides (Mr = 200,000 and 46,000) were produced. Antibodies against P235 raised in rabbits reacted only with P235 on immunoblots of total platelet proteins. These antibodies also reacted with polypeptides of Mr = 200,000 and 46,000 on immunoblots of thrombin-activated platelets. These data show that both actin-binding protein and P235 are cleaved during thrombin-induced platelet aggregation and suggest that the activation of the Ca2+-dependent protease may permit reorganization of the platelet cytoskeleton in aggregating platelets.

Antibody Specificity↗
PubMed1985

Hydrolysis of cytoskeletal proteins by the Ca2+-dependent protease during platelet activation.

During platelet stimulation, the cytosolic Ca2+ concentration increases to micromolar levels. One consequence of this increase is that the Ca2+-dependent protease within platelets is activated. Activation of the Ca2+-dependent protease results in hydrolysis of actin-binding protein and P235. Actin-binding protein and P235 can both affect the organization of actin, thus activation of the Ca2+-dependent protease may provide a regulatory mechanism by which stimulus-induced changes in the organization of actin filaments could be directed. Although both actin-binding protein and P235 affect actin polymerization, stimulus-induced actin polymerization occurs before hydrolysis of actin-binding protein or P235 can be detected, thus it seems unlikely that hydrolysis of these proteins affects actin polymerization. Actin-binding protein also cross-links actin filaments into networks, a function that is lost when it is hydrolyzed by the Ca2+-dependent protease. Thus, hydrolysis of actin-binding protein may result in disruption of the actin filament networks that form early during platelet activation and permit the reorganization of filaments into the bundles present at later stages of platelet activation.

Blood Platelets↗
PubMed1985

The sodium pump in opossum vascular smooth muscle.

Ouabain-sensitive Rb+ uptake and [3H]ouabain binding were used to measure rates of Na+ pumping and the number of pump sites, respectively, in thoracic aortae from opossums. From the number of Rb+ ions pumped per site per minute, estimates of pump turnover have been made. Values obtained are comparable to those of other species (see Table 1).

Adenosine Triphosphatases↗
PubMed1985

Expression of amplified DNA sequences for ornithine transcarbamylase in HeLa cells: arginine residues may be required for mitochondrial import of enzyme precursor.

Expression of ornithine transcarbamylase (OTC), a nuclear-coded mitochondrial enzyme, was programmed in HeLa cells by the use of a strategy of gene co-amplification. HeLa cells, ordinarily devoid of OTC activity, were transfected with a plasmid containing viral regulatory elements joined with two cDNA sequences, one encoding the human OTC precursor and a second encoding a mutant mouse dihydrofolate reductase. After transfection and selection in increasing concentrations of methotrexate, several hundred copies per cell of the sequence encoding OTC were detected by blot analysis. Immunoprecipitation of extracts of radiolabeled cells with anti-OTC antiserum revealed newly synthesized mature OTC subunits. Furthermore, OTC enzymatic activity in cell extracts was comparable to that of control human liver, and mitochondrial localization of OTC was demonstrated by immunofluorescence. When we incubated transfected HeLa cells with dinitrophenol, a known inhibitor of mitochondrial import, the only form of newly synthesized OTC detected was the precursor. We estimated the rate of import of precursor by performing an inhibitor-free chase; precursor was converted to mature subunit with a half-life of less than two minutes. When a HeLa transformant was incubated with the arginine analogue canavanine, the major form of newly synthesized OTC detected was a species migrating slightly more slowly than the normal precursor; little mature-sized subunit was recovered. This indicates that substitution of the analogue for arginine in the OTC precursor interferes with mitochondrial import and processing. Thus, arginine residues in the OTC precursor--most likely the four residues contained in its NH2-terminal leader sequence--probably play an important role in mitochondrial import and/or processing.

Amino Acid Sequence↗
PubMed1985

Organization of the cytoskeleton in resting, discoid platelets: preservation of actin filaments by a modified fixation that prevents osmium damage.

This study evaluates the structural organization of the cytoskeleton within unactivated, discoid platelets. Previously, such studies have been difficult to interpret because of the ease with which platelets are stimulated, the sensitivity of actin filaments to cell extraction buffers, and the general problem of preserving actin filaments with conventional fixatives, compounded by the density of the cytoplasm in the platelet. In this study we have employed a new fixative containing lysine, which protects actin filaments against damage during fixation and thin-section processing. We used thick (0.25-micron) sections and conventional thin sections of extracted cells (fixed and lysed simultaneously by the addition of 1% Triton X-100 to the initial fixative) as well as thin sections of whole cells to examine three preparations of human platelets: discoid platelets washed by sedimentation; discoid platelets isolated by gel filtration; and circulating platelets collected by dripping blood directly from a vein into fixative. In all of these preparations, long, interwoven actin filaments were observed within the platelet and were particularly concentrated beneath the plasma membrane. These filaments appeared to be linked at irregular intervals to the membrane and to each other via short, approximately 20- to 50-nm-long cross-links of variable width. Although most filaments were outside the circumferential band of microtubules and the cisternae of the open canalicular system, individual filaments dipped down into the cytoplasm and were found between the microtubules and in association with other membranes. The ease with which single actin filaments can be seen in the dense cytoplasm of the human platelet after lysine/aldehyde fixation suggests the great potential of this new fixative for other cells.

Actin Cytoskeleton↗