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Biomedical subjects

N D Boyd

Publications and source records attributed to N D Boyd.

16 recordsLinked to original sources
PubMed1991

Photoaffinity labeling the substance P receptor using a derivative of substance P containing p-benzoylphenylalanine.

A novel photoreactive substance P (SP) analogue has been synthesized by solid-phase peptide synthesis methodology to incorporate the amino acid p-benzoyl-L-phenylalanine [L-Phe(pBz)] in place of the Phe8 residue of SP. [Phe8(pBz)]SP was equipotent with SP in competing for SP binding sites on rat submaxillary gland membranes and had potent sialagogic activity in vivo. In the absence of light, the 125I-labeled Bolton-Hunter conjugate of [Phe8(pBz)]SP bound in a saturable and reversible manner to an apparently homogeneous class of binding sites (Bmax = 0.2 pmol/mg of membrane protein) with an affinity KD = 0.4 nM. The binding of 125I-[Phe8(pBz)]SP was inhibited competitively by various tachykinin peptides and analogues with the appropriate specificity for SP/NK-1 receptors. Upon photolysis, up to 70% of the specifically bound 125I-[Phe8(pBz)]SP underwent covalent linkage to two polypeptides of Mr = 53,000 and 46,000, identified by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and autoradiography. Quantitative analysis of the inhibitory effects of SP and related peptides on 125I-[Phe8(pBz)]SP photoincorporation indicated that the binding sites of the two photolabeled polypeptides have the same peptide specificity, namely, that typical of NK-1-type SP receptors. In addition, the labeling of the two polypeptides was equally sensitive to inhibition by guanyl-5'-yl imidodiphosphate, a nonhydrolyzable analogue of GTP. Further information on the relationship between the two labeled SP binding sites was provided by enzymatic digestion studies: the Mr = 46,000 polypeptide contains N-linked carbohydrates and is derived most likely from the higher molecular weight species by proteolytic nicking.(ABSTRACT TRUNCATED AT 250 WORDS)

Affinity Labels↗
PubMed1991

Mercury from dental "silver" tooth fillings impairs sheep kidney function.

In humans Hg vapor is released from "silver" amalgam fillings that contain 50% Hg by weight. Previous studies show that when 12 such fillings are placed in sheep teeth, the kidneys will concentrate amalgam Hg at levels ranging from 5 to 10 micrograms Hg/g renal tissue 4-20 wk after placement. In the present study 12 occlusal fillings were placed in each of six adult female sheep under general anesthesia, using standard dental procedures. Glass ionomer occlusal fillings (12) were inserted in two control sheep. At several days before dental surgery, and at 30 and 60 days after placement of fillings, renal function was evaluated by plasma clearance of inulin and by plasma and urine electrolytes, urea, and proteins. An average plasma inulin clearance rate of 69.5 +/- 7.2 ml/min before amalgam placement was reduced to 32.3 +/- 8.1 ml/min by 30 days and remained low at 27.9 +/- 8.7 ml/min after 60 days. Inulin clearance did not change in controls. After amalgam placement urine concentration of albumin decreased from 93.0 +/- 20.5 to 30.1 +/- 15.3 mg/l and urine Na+ concentration increased steadily from 24.8 +/- 7.7 to 82.2 +/- 20.3 mmol/l at 60 days. Concentrations of K+, urea, gamma-glutamyl transpeptidase, alkaline phosphatase, and total protein did not change significantly from 0 to 60 days in urine. Plasma levels of Na+, K+, urea, and albumin remained unchanged from 0 to 60 days after amalgam. Renal histology remained normal in amalgam-treated animals.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1990

Guanine nucleotides decrease the affinity of substance P binding to its receptor.

The inhibitory effect of guanine nucleotides on the binding of 125I-Bolton-Hunter conjugated substance P (125I-BHSP) to rat submaxillary gland membranes has been confirmed and further explored. The evidence presented here indicates that this is due to a marked loss of binding affinity. In the presence of 5'-guanylyl imidodiphosphate (GppNHp) there is (1) a greater than or equal to 20-fold increase in the Kd as determined by Scatchard analysis and (2) the concentration of SP required to inhibit half of the 125I-BHSP binding (IC50) increased approximately 30-fold. Consistent with a marked decrease in affinity is an approximately 100-fold increase in the rate of dissociation of 125I-BHSP following addition of GppNHp. Complete restoration of high-affinity binding was achieved by removal of the guanine nucleotide.

Guanine Nucleotides↗
PubMed1990

Isolation and identification of a polypeptide in the Hsp 70 family that binds substance P.

During the course of an attempt to purify the substance P (SP) receptor from horse salivary glands by substance P-affinity chromatography, a polypeptide of Mr = 78,000 was isolated. The first fifteen amino acid residues at the amino terminus were determined and, unexpectedly, were found to be identical with the amino terminus of a glucose-regulated protein (GRP) of the same molecular weight, a protein that has been identified as a member of the heat shock protein family. This finding raises the intriguing possibility that SP may interact in vivo with GRPs and other members of the heat shock protein family and play a role in modulating their biological activities.

Amino Acid Sequence↗
PubMed1990

A guinea pig model of bovine pneumonic pasteurellosis.

The induction of pneumonic pasteurellosis in guinea pigs (Cavia porcellus) was examined. Specific pathogen free male guinea pigs were anesthetized and a tracheostomy performed to introduce 10(5), 10(4) or 10(3) Pasteurella haemolytica-A1 into the left principal bronchus. The surgical site was closed with tissue adhesive and staples and the animals were monitored for signs of respiratory tract infection. Within 24 hours after inoculation they became depressed, anorectic, pyretic and dyspneic. Fibrinous pleuropneumonia with prominent areas of necrosis and hemorrhage was present. Pericardial effusion was a frequent finding. There was infiltration of the pleura and alveoli with degenerate heterophils and macrophages, a hyperplastic mesothelium and fibrin exudation on the pleura and within alveoli. Hemorrhage, congestion, consolidation, edema and fibrin exudation were prominent in the hilar region of the lungs. Bacterial colonies were evident in all airways. More bacteria were recovered from infected lungs than were inoculated (p less than 0.05) indicating P. haemolytica was actively multiplying in the lungs. Hematological and clinical chemistry data were consistent with fibrinous pneumonia, however, blood cultures were positive for P. haemolytica in 61% (11/18) of animals sampled. Examination of pneumonic pasteurellosis in guinea pigs may be useful in studying pathogenetic and pathological features applicable to bovine pneumonic pasteurellosis (shipping fever pneumonia).

Animals↗
PubMed1989

Serum immunoglobulin E levels in patients with primary biliary cirrhosis.

Serum IgE levels were measured in 22 symptomatic adults with primary biliary cirrhosis (PBC) and 19 asymptomatic female adults with positive serum mitochondrial antibody tests. Control populations included 45 adult patients with chronic liver disease consisting of autoimmune chronic active hepatitis (N = 15), alcoholic liver disease (N = 15), or miscellaneous cholestatic liver disorders (N = 15), and 87 healthy adult hospital personnel. Fourteen of 22 (64%) patients with PBC had low (less than 10 KU/L) or undetectable serum IgE levels, compared to 12 of 45 (27%) control patients with liver disease (p less than 0.005) and 39 of 87 (45%) healthy control subjects (p = 0.09). Low serum IgE levels were also found in most (13 of 19 patients, 68%) asymptomatic individuals with positive serum mitochondrial antibody tests (p less than 0.005 and p less than 0.05 versus liver disease and healthy control subjects, respectively). In vitro IgE production by peripheral blood mononuclear cells after pokeweed mitogen stimulation was determined in eight patients with PBC and in eight healthy control subjects. After pokeweed mitogen stimulation, one of three, patients with PBC compared to four of four healthy control subjects with detectable levels of serum IgE, produced sufficient amounts of IgE to be detected in culture supernatants. Neither the five patients with PBC nor the four healthy control subjects with undetectable serum IgE levels produced sufficient amounts of IgE in vitro to be detected in our assay system.(ABSTRACT TRUNCATED AT 250 WORDS)

Adult↗
PubMed1989

Regulation of substance P receptor affinity by guanine nucleotide-binding proteins.

The binding of substance P (SP) to receptors in peripheral tissues as well as in the CNS is subject to regulation by guanine nucleotides. In this report, we provide direct evidence that this effect is mediated by a guanine nucleotide-binding regulatory protein (G-protein) that is required for high-affinity binding of SP to its receptor. Rat submaxillary gland membranes bind a conjugate of SP and 125I-labeled Bolton-Hunter reagent (125I-BHSP) with high affinity (KD = 1.2 +/- 0.4 X 10(-9) M) and sensitivity to guanine nucleotide inhibition. Treatment of the membranes with alkaline buffer (pH 11.5) causes a loss of the high-affinity, GTP-sensitive binding of 125I-BHSP and a parallel loss of [35S]guanosine 5'-(3-O-thio)triphosphate ([35S]GTP gamma S) binding activity. Addition of purified G-proteins from bovine brain to the alkaline-treated membranes restores high-affinity 125I-BHSP binding. Reconstitution is maximal when the G-proteins are incorporated into the alkaline-treated membranes at a 30-fold stoichiometric excess of GTP gamma S binding sites over SP binding sites. Both Go (a pertussis toxin-sensitive G-protein having a 39,000-dalton alpha-subunit) and Gi (the G-protein that mediates inhibition of adenylate cyclase) appear to be equally effective, whereas the isolated alpha-subunit of Go is without effect. The effects of added G-proteins are specifically reversed by guanine nucleotides over the same range of nucleotide concentrations that decreases high-affinity binding of 125I-BHSP to native membranes.(ABSTRACT TRUNCATED AT 250 WORDS)

Alkalies↗
PubMed1988

Dual-precipitation method evaluated for determination of high-density lipoprotein (HDL), HDL2, and HDL3 cholesterol concentrations.

We evaluated a dual-precipitation method for determining cholesterol in high-density lipoprotein (HDL) and its subfractions HDL2 and HDL3. After total HDL was isolated by precipitation of very-low-density (VLDL) and low-density (LDL) lipoproteins with polyethylene glycol (Mr 8000), HDL2 was isolated from total HDL by precipitation with dextran sulfate (Mr 15,000), leaving HDL3 in the supernate. Concentration of total HDL cholesterol after precipitation of VLDL and LDL with PEG showed significant proportional and constant biases of -3.8% and 0.04 mmol/L, respectively, when compared with a phosphotungstic acid-based comparison method, although results by the two methods were correlated highly (r = 0.99, P less than 0.001). HDL2 and HDL3 cholesterol concentrations measured with the present technique were not different from those obtained by density-gradient ultracentrifugation or by combined precipitation-ultracentrifugation.

Centrifugation, Density Gradient↗
PubMed1987

Two distinct kinetic phases of desensitization of acetylcholine receptors of clonal rat PC12 cells.

1. The desensitization of nicotinic acetylcholine receptors on the PC12 sympathetic cell line was investigated by using a 22Na+ influx assay to measure receptor activation. 2. The rate of desensitization was dependent on temperature and at 4 degrees C two distinct kinetic phases were readily discernible: a rapid phase that was characterized by rate constants that were dependent on the chemical nature and concentration of the agonist, and a slower phase that was characterized by rate constants that were less dependent on these. 3. For acetylcholine, carbamylcholine and l-nicotine, the equilibrium desensitization parameter, Kdes, the concentration that produces half-maximal desensitization, was determined and compared with the corresponding value for Kact, the concentration that results in a half-maximal increase in the permeability response. For each agonist, the value of Kdes was found to be lower than Kact, a result to be expected if desensitization is associated with a higher-affinity state of the receptor than that associated with ion channel activation. Thus, extensive receptor desensitization can occur even at agonist concentrations that do not produce appreciable channel activation. Both activation and desensitization functions exhibited positive cooperativity so that each function occurs over a narrow range of agonist concentrations. 4. Following removal of the agonist, recovery from desensitization was reversible and occurred by two distinct kinetic phases characterized by rate constants that were independent of the chemical nature and concentration of the agonist that produced the desensitization. The relative contribution of each kinetic phase of recovery was, however, dependent on the duration of prior exposure to agonist. Following short incubation periods with agonist, most of the receptors were in a rapidly recovering state. With increasing duration of exposure, progressively more of the receptors were converted to a desensitized state that recovered more slowly. 5. The rate constants associated with the two kinetic phases of recovery were dependent on the recovery temperature. Following the initial rapid phase of desensitization, recovery at 4 degrees C was characterized by a time constant, t1/2, of 1.9 min, a value that was about 3-fold greater than that observed at 22 degrees C. The rate of recovery of the desensitized state achieved following equilibrium exposures to agonists was considerably more temperature dependent: recovery of this desensitized state was characterized at 4 degrees C by a t1/2 of 62 min that was about 37-fold greater than that at 22 degrees C.(ABSTRACT TRUNCATED AT 400 WORDS)

Animals↗
PubMed1987

Multiple actions of substance P that regulate the functional properties of acetylcholine receptors of clonal rat PC12 cells.

1. The effects of substance P (SP) on each of the kinetic components of reversible desensitization (measured at 4 degrees C) and also on irreversible deactivation (measured at 22 degrees C) of the nicotinic acetylcholine receptor on PC12 cells were examined by 22Na+ influx measurements of the functional state of the receptor. 2. In the absence of agonists, SP converts the acetylcholine receptors in a time- and concentration-dependent manner, to a state that is not responsive to agonist. Upon removal of the peptide, this effect was reversible and the kinetics of the recovery of the permeability response were analysed to provide further characterization of the non-responsive state. Following exposure of cells to SP (10 microM) for 3 or more min, recovery was by a first-order process (time constant, t1/2 = 2.1 min), the same value, within experimental error, as that observed for recovery measured after the initial rapid phase of agonist-mediated desensitization. 3. In the presence of agonist, SP caused a strong enhancement of both the rate and extent of agonist-mediated desensitization. This effect was observed even at concentrations of peptide which produced only a small extent of desensitization when incubated alone. For 500 microM-carbamylcholine, the equilibrium level of desensitization (approximately 85% loss of the permeability response) was achieved at 4 degrees C in about 20 min by a biphasic process, while in the presence of 1.0 microM-SP, complete (100%) desensitization occurred by a single rapid exponential phase characterized by a t1/2 of 20 s. 4. The concentration of carbamylcholine required to produce half-maximal desensitization at equilibrium, Kdes, was 94 microM and was reduced by 6-fold in the presence of 0.3 microM-SP. 5. A mechanistic model is presented in which the receptor is viewed as existing in a dynamic conformational equilibrium between an activatable state Rc and the initial desensitized state Rd. It is proposed that SP binds preferentially to the Rd state and thus can allosterically (1) stabilize the receptor in the absence of agonist in that state, and (2) enhance, in an even lower concentration range, both the rate and extent of agonist-mediated stabilization of the receptor in the Rd state. 6. The second, slower component of agonist-mediated desensitization is, in contrast, inhibited by SP. This desensitization step appears to involve a covalent modification of the initial desensitized state (Rd) and is dependent on Ca2+. SP may exert this inhibitory effect by limiting the access of Ca2+ to an intracellular site of action.(ABSTRACT TRUNCATED AT 400 WORDS)

Animals↗
PubMed1979

Determination of plasma octopamine and its level in renal disease.

A radioenzymatic assay method for the estimation of octopamine levels in plasma was developed. Preparation of the enzyme, phenylethanolamine-N-methyl transferase, dilution of the plasma sample, preparation of a suitable blank, and the assay conditions were found to have a significant effect on the sensitivity of the assay. Plasma octopamine levels were measured in a population of 33 normal individuals ranging in age from 19 to 94 years. Significantly higher plasma octopamine levels were found in the age group 70-90 years. Excluding those individuals over the age of 70 years, no significant differences in plasma octopamine levels were found for males or females, the range of values was 0 to 0.68 ng per ml, with a mean value of 0.23 ng per ml (n = 25). Examination of plasma octopamine levels in patients with severe renal disease requiring hemoperfusion dialysis, revealed a significantly higher level of plasma octopamine in renal disease (1.9 ng per ml), and an increase in plasma octopamine during dialysis; the mean level post dialysis being 2.7 ng per ml.

Adult↗
PubMed1979

Acetylcholine and local anesthetic binding to Torpedo nicotinic postsynaptic membranes after removal of nonreceptor peptides.

After alkaline extraction, purified subsynaptic fragments isolated from Torpedo electric tissue exhibit on sodium dodecyl sulfate/polyacrylamide gel electrophoresis predominant peptides of apparent Mr 41,000, 50,000, and 65,000 (i.e., the peptides characteristic of the nicotinic receptor purified and isolated in detergent solutions). The peptide of Mr 43,000 that is also found in the isolated postsynaptic membranes is recovered in the supernatant after alkaline extraction. The alkaline-extracted membranes were functionally intact, as demonstrated by the following criteria. The kinetics of binding of [3H]acetylcholine in the presence and absence of 30 micron carbamoylcholine to occupy acetylcholine binding sites, [14C]-meproadifen [2-(diethylmethylaminoethyl)-2,2-diphenylvalerate iodide ] was bound with a dissociation constant, KD, of 0.3 +/- 0.1 micron to 0.3 +/- 0.1 site per [3H]alpha-toxin site. This binding was displaced by perhydrohistrionicotoxin. The carbamoylcholine-stimulated efflux of 22Na+ from the Torpedo vesicles were preserved after alkaline extraction. It is concluded that not only the acetylcholine binding site, but also the local anesthetic binding site, must be associated with the peptides of the cholinergic receptor itself and not that of Mr 43,000. Those peptides remaining after alkaline extraction are also sufficient for permeability control.

Acetylcholine↗
PubMed1976

Influence of age and sex on plasma zinc levels in normal and diabetic individuals.

Plasma zinc levels were measured in both healthy and diabetic individuals having an age range of 10-93 years. No significant differences in plasma zinc concentrations were found between males and females in either healthy or diabetic individuals. Up to the age of 50 years, the mean plasma zinc of normal individuals remained relatively constant at 70 +/- 32 mug/100 ml (+/- 2 SE) after which the levels decreased. This decreasing pattern was absent in diabetics, whose plasma zinc of 65 +/- 32 mug/100 ml remained constant over the entire age range. For women on oral contraceptive agents, the mean plasma zinc was 59 +/- 18 mug/100 ml, which was significantly lower than that of controls.

Adolescent↗
PubMed1975

Effect of carbon cup aging on plasma zinc determination by flameless atomic adosorption spectrometry.

Determination of zinc in blood plasma by flameless atomic absorption spectrometry is discussed, with particular reference to the protocol required for the successful use of the Varian-Techtron Carbon Rod Atomizer. Cup aging is shown to be an important factor in limiting the precision of this analytical technique and ways of minimizing the problem are described. Matrix problems have also been encountered, which precluded the use of aqueous standard curves and the method of standard additions. We propose the use of plasma in preparing standard curves, the values for which are corrected for inherent plasma zinc, as a possible solution to the problem.

Carbon↗