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Biomedical subjects

A C McLaughlin

Publications and source records attributed to A C McLaughlin.

14 recordsLinked to original sources
PubMed1992

Significance of an opiate mechanism in the adjustment of cerebrocortical oxygen consumption and blood flow during hypercapnic stress.

The role of adrenal medulla-derived enkephalins in the control of hypercapnic cerebrocortical blood flow (CBF) and oxygen consumption (CMRO2) was investigated in the ketamine anesthetized rat. Three experimental interventions were utilized: inhibition of opioid receptors with naloxone, decrease of adrenal enkephalin production with chronic adrenal medullectomy, and treatment of adrenal demedullated animals with the synthetic enkephalin analog, D-Ala2, N-Me-Phe4, Gly5-ol-enkephalin (DAGO). In intact, untreated animals hypercapnia increased CBF and CMRO2 by approximately 300 and 35%, respectively. Naloxone reduced the hypercapnic increase of CBF, and transformed the hypercapnic increase of CMRO2 into a decrease. The mid-points of the dose-response curves for (1)-naloxone and (d)-naloxone were 10 micrograms/kg and 100 micrograms/kg, respectively. Adrenal demedullation and treatment with (1)-naloxone (0.2 mg/kg) decreased the hypercapnic CBF and CMRO2 by approximately 50%. DAGO treatment of adrenal demedullated animals restored the hypercapnic CBF and CMRO2 to values similar to those found in intact animals. These observations suggest that opioid peptides (most likely adrenal medulla-derived enkephalins) play a significant role in the regulation of CMRO2 and CBF during moderate hypercapnia.

Adrenal Medulla↗
PubMed1992

In vivo 17O NMR study of rat brain during 17O2 inhalation.

In vivo 17O NMR has been used to monitor the H2(17)O concentration in rat brain during inhalation of 17O2. The results are discussed in terms of oxygen consumption in the brain and recirculation into the brain of H2(17)O produced in other organs.

Animals↗
PubMed1991

In vivo measurement of cerebral oxygen consumption and blood flow using 17O magnetic resonance imaging.

We used 17O NMR imaging techniques to measure the H2(17)O concentration in a 0.8-ml voxel in the cat brain following injection of an arterial bolus of enriched H2(17)O and during inhalation of enriched 17O2. We also measured the H2(17)O concentration in arterial blood during 17O2 inhalation. The data from the first measurement were used to calculate the blood flow in the voxel. The data from all three measurements were combined to calculate the oxygen consumption in the voxel. The values of cerebral blood flow and oxygen consumption calculated with 17O NMR techniques agree reasonably well with values calculated for a similar region of the cat brain using autoradiographic techniques.

Administration, Inhalation↗
PubMed1991

Cerebral blood flow and metabolic rate in the conscious, freely moving rat: the effects of hypercapnia, and acute ethanol administration.

We propose a simple method that can be used to measure cerebral blood flow (CBF), cerebral oxygen consumption (CMRO2), and cerebral glucose consumption (CMRglu) in the conscious, freely moving rat. The method is based on the classical Kety-Schmidt approach, and uses a chronic cannula in the confluens sinuum. We tested the method by investigating the response of CBF, CMRO2, and CMRglu to hypercapnia and used the approach to investigate the effects of acute alcohol administration. Severe hypercapnia (PaCO2 approximately 80 mmHg) increased the CBF by a factor of 3.5, decreased the CMRO2 by 30%, and had no significant effect on the CMRglu. Under normocapnic conditions moderate blood alcohol levels (100-200 mg%) caused no significant effects on CBF, CMRO2, or CMRglu, but high blood alcohol levels (250-400 mg%) decreased all three parameters by approximately 25%. Under hypercapnic conditions high blood alcohol levels had no effect on CBF, CMRO2, and CMRglu.

Alcoholic Intoxication↗
PubMed1979

Effects of ethanol on alanine metabolism in perfused mouse liver studied by 13C NMR.

Time courses of 13C labeling from alanine and ethanol in perfused mouse livers have been followed by NMR. The enrichment at specific carbons of glucose, glutamate, glutamine, aspartate, acetate, acetoacetate, beta-hydroxybutyrate, and lactate has been measured. The specific labeling of glutamate in the presence of labeled alanine and labeled or unlabeled ethanol shows that, under these conditions, alanine enters the tricarboxylic acid cycle almost exclusively through pyruvate carboxylation, whereas ethanol is the exclusive source of acetyl-CoA. In the absence of ethanol, the alanine label flows through both paths. By comparing the scrambling of 13C between C3 and C2 of glutamate it is possible to estimate the mitochondrial fumarase activity; the C6-to-C5 ratios in glucose give the additional scrambling by cytosolic fumarase activity. In addition, the C6-to-C1 and C5-to-C2 ratios in glucose show that there is about 15% flux through the pentose cycle. Finally, the C4-to-C2 ratios in glutamine and glutamate are unequal at any time (the glutamine labels reflect the label distribution in glutamate measured 1 hr earlier), providing a method for studying flow through glutamine synthetase in situ.

Alanine↗
PubMed1979

Rapid ATP assays in perfused mouse liver by 31P NMR.

31P NMR was used to continuously monitor ATP and inorganic phosphate levels in perfused mouse liver. Under "optimal" conditions, the time resolution of the technique was approximately 1 min. In the absence of any metabolic perturbations the ATP level remained constant for at least 2 hr and decreased by only approximately 20% in 18 hr. Both ATP and inorganic phosphate levels responded to alterations in the oxygen supply to the liver. The half-time for this response was approximately 1 min, and the response to short periods of hypoxia or ischemia was partially reversible. The addition of insulin caused only a minor decrease in the ATP level but significantly decreased the rate of response of ATP and phosphate levels to hypoxia and ischemia.

Adenine Nucleotides↗
PubMed1977

Specificity of interactions of hapten side chains with the combining site of the myeloma protein MOPC 315.

The pKa values of the three histidine residues in the Fv fragment (variable region of the heavy and light chains) of the mouse myeloma protein MOPC 315, measured by high resolution n.m.r. (nuclear magnetic resonance), are 5.9, 6.9 and 8.2. The perturbation of the pKa of one of the histidines (pKa 6.9) on the addition of hapten and the narrow linewidth of its proton resonances suggests that it is at the edge of the combining site. References to the model of the Fv fragment [Padlan, Davies, Pecht, Givol & Wright (1976) Cold Spring Harbor Symp. Quant. Biol. 41, in the press] allows assignment of the three histidine residues, histidine-102H, histidine-97L and histidine-44L. The determination of the pKa of the phosphorus group, by 31P n.m.r., of a homologous series of Dnp- and Tnp- (di- and tri-nitrophenyl) haptens has located a positively charged residue. Molecular-model studies on the conformations of these haptens show that the residue is at the edge of the site. The model suggests that the positively charged residue is either arginine-95L or lysine-52H.

Antibody Specificity↗
PubMed1977

The determination of molecular-motion parameters from proton-relaxation-enhancement measurements in a number of Gd(III) - antibody-fragment complexes. A comparative study.

Longitudinal and transverse proton relaxation rates for water in the hydration spheres of Gd(III) bound to the non-immune rabbit IgG fragments Fc (C-terminal half of heavy-chain dimer), pFc' (C-terminal quarter of heavy-chain dimer) and Fab (N-terminal half of heavy and light chain) have been measured at a number of frequencies and temperatures using pulsed nuclear magnetic resonance spectrometry. For the fragments Fc and pFc', a full computer analysis showed that the results could be fitted by parameters of similar magnitude to those found previously for IgG. In contrast to the results for the other complexes the Fab -Gd(III) complex showed no slow exchange contribution to the relaxation rates. Under these circumstances it was found possible to obtain an accurate value for the hydration number (q) from measurements of the longitudinal and transverse relaxation rates at a chosen frequency such that the product of the nuclear Larmor frequency (omega1) and the correlation time for the dipolar relaxation processes (tauc) was approximately unity. Water-proton relaxation rates were also determined for the complex of Gd(III) with the Fv fragment of the mouse myeloma protein MOPC 315. A computer analysis of the results revealed a slow exchange contribution to the rates and this gave errors in the variable parameters similar to those observed previously for IgG, Fc and pFc'. The conclusions drawn from the different systems are discussed in terms of the present state of application of the proton relaxation enhancement technique in biology.

Animals↗
PubMed1976

Difficulties in determining accurate molecular motion parameters from proton relaxation enhancement measurements as illustrated by the immunoglobulin G-Gd(III) system.

Longitudinal and traverse proton magnetic relaxation rates for water in the hydration sphere of Gd(III) bound to non-immune rabbit IgG (immunoglobulin G) have been determined over a wide range of frequencies (4-84 MHz) at constant temperature (19 degrees C) using pulsed nuclear magnetic resonance spectrometry. The rates have also been determined at temperatures between 0 and 40 degrees C for two frequencies (61 and 84 MHz). The rates were fitted to existing theory using a computer least-squares procedure. Further computer analysis was then carried out to determine the sensitivity of the best-fit error to variation in the variable parameters in the theoretical expressions used. These include the water co-ordination number (q) for which it was found large variations could occur (between approximately 2 and 8) for only small changes in the error of best-fit. It was concluded that a slow exchange contribution to the relaxation rates was important in deciding which parameters are poorly determined. A rotational correlation time (tau r) was obtained which suggested there might be considerable internal motion of the Fc region (C-terminal half of heavy-chain dimer) of the IgG molecule. However the possibility of large errors in this value prevented unequivocal conclusions being drawn.

Animals↗
PubMed1976

Magnetic resonance study of the three-dimensional structure of creatine kinase-substrate complexes. Implications for substrate specificity and catalytic mechanism.

The paramagnetic effects of the bound manganese ion and of a covalently attached spin label on proton nuclear spin relaxation rates have been used to calculate distances for a structural model of the MnADP and creatine complexed to creatine kinase from rabbit muscle. The nucleotide and guanidino substrates are so aligned on the enzyme that the transferable phosphoryl group on one substrate is in apposition to the acceptor moiety on the second substrate. The divalent metal ion is most probably liganded to the alpha and beta phosphates of the nucleotide substrate, both in the abortive MnADP-creatine-enzyme complex and in the active MnATP-creatine-enzyme complex. The metal ion-formate distance approximately 5 A in the Mn(II)ADP-formate-creatine-enzyme complex and less than 5 A in the Co(II)ADP-formate-creatine-enzyme complex is consistent with the suggestion that the monovalent anion is binding at the site normally occupied by the transferable phosphoryl group, thus producing a complex which mimics the transition state. Although only an upper limit of the distance from Mn(II) to the guanidino substrate could be determined in the presence of formate, it could be concluded that the disposition of the guanidino substrate changes upon addition of formate, since the relative distances of the methyl and methylene group are inverted. The effect of formate and nitrate on increasing the residence time of creatine in the MnADP-creatine-enzyme complex as determined by NMR provides evidence that the complexes observed by NMR are identical with those involved in the catalytic mechanism, since a parallel effect of formate and nitrate is observed in the kinetics of the enzymatic reaction, where the dissociation constant of creatine from the abortive quaternary complex decreases in the presence of the anions as had been determined from their inhibition of the forward reaction (Milner-White, E.J., and Watts, D.C. (1971) Biochem. J. 122, 727-740). Although the guanidino substrate is not directly liganded to the divalent metal ion, the electron paramagnetic resonance spectrum of manganese in the transition state analog complexes, i.e. nitrate-ADP-guanidino substrate-enzyme, is strongly dependent on catalytic activity of the guanidino substrate. The structural differences observed by EPR among transition state analog complexes with various guanidino substrates were not reflected in distances from Mn(II) to the guanidino substrate, which were 10% and 0.3% as active as creatine. Within the experimental error of 1 A, the distances were the same. The enzyme or the enzyme-substrate complexes may be considered to exist in a number of structurally distinct conformations in equilibrium based on the EPR spectra and on the anomalous temperature-dependence of the relaxation rates of the formate proton of the transition state analog complexes...

Adenosine Diphosphate↗
PubMed1976

Interactions of the lanthanide- and hapten-binding sites in the Fv fragment from the myeloma protein MOPC 315.

1. The interactions of lanthanide metals and dinitrophenyl spin-label haptens with the Fv fragment of the mouse myeloma protein MOPC 315 were investigated by the techniques of fluorescence, e.s.r. (electron spin resonance) and high-resolution n.m.r. (nuclear magnetic resonance). 2. The protein fluorescence of Fv fragment at 340nm is quenched by the haptens (fluorescence enhancement, epsilon=0.15) and enhanced by Gd(III) (epsilon=1.14) and other lanthanides. The binding of the haptens studied here is insensitive to pH in the range 5.5-7.0 (dissociation constant KH=0.3-1.0 muM) and shows 1:1 stoicheiometry. The binding of Gd(III) also shows 1:1 stoicheiometry, but is pH-dependent; the binding constant (KM) varies from 10 muM at pH7.0 to 700 muM at pH4.8. La(III) binding is less sensitive to pH. The pH-dependences of the metal-binding constants imply that a group in the protein with pKa greater than or equal to 6.2 is involved in the binding, and probably also other groups with lower pKa values. 3. The apparent binding of the haptens is weakened about 20-fold by Gd(III), and vice versa. An equilibrium scheme involving a ternary complex with an interaction between the two binding sites is derived in Appendix I to explain the experimental results at two pH values. 4. Time-dependent fluorescence changes are observed in the presence of Gd(III) at pH5.5. A two-state kinetic scheme involving a 'slow' conformational change in the Fv fragment is derived in Appendix II to explain this time-dependence. This scheme is consistent with the antagonistic equilibrium behaviour. 5. The e.s.r. changes in the spin-label haptens on binding to Fv fragment and on the subsequent addition of lanthanides are consistent with the binding scheme for haptens and lanthanides proposed from the fluorescence studies. A difference between the limiting quenching of the e.s.r. signal from the bound haptens in the presence of saturating concentrations of Gd(III) and La(III) is attributed to dipolar interactions between bound Gd(III) and the nitroxide moiety of the bound hapten. The residual quenching with Gd(III) allows an estimate of 1.2nm to be made for the distance between the two paramagnetic centres. 6. The 270 MHz proton difference spectrum of the Fv fragment resulting from the addition of La(III) suggests that any metal-induced conformational changes are small and involve relatively few amino acid residues on the Fv fragment...

Animals↗
PubMed1975

Antibody--hapten interactions in solution.

This paper reports the initial progress in a research programme to identify and obtain the relative orientations, in solution, of the amino acid residues that constitute the combining site of the myeloma protein MOPC 315. This protein has a molecular mass of 150,000, but enzymic digestion yields the Fv fragment of molecular mass 25,000 which still has the combining site intact, as judged by the affinity for dinitrophenyl haptens. Analysis of the e.s.r. spectra of a series of dinitrophenyl spin labelled haptens has allowed the dimensions, rigidity and polarity profile of the combining site to be determined. The combining site is a cleft of overall dimensions 1.1 nm x 0.9 nm x 0.6 nm which has considerable structural rigidity. One of these spin labels has also been used to perturb the n.m.r. spectrum of the Fv and using difference spectroscopy the 270 MHz proton n.m.r. spectrum of the amino acid residues in and around the combining site has been obtained. This spectrum contains only the equivalent of about 30 aromatic and 21 aliphatic protons. Comparison of this difference spectrum with that obtained using a diamagnetic analogue suggests that any conformational changes on hapten binding are mainly localized to the combining site. By the use of (n.m.r.) difference spectroscopy the protons of the three histidine residues in the Fv are observed to titrate with pH and have pKa values of about 8.1, 6.9 and 6.1. The histidine resonances with pKa values 6.9 and 6.1 alter slightly in the presence of haptens and also appear in the spin label difference spectrum, and must therefore be in or near to the combining site. These are assigned to His 102H and His 97L. The existence of lanthanide binding sites on the Fv, necessary for the mapping studies, has been demonstrated by measurements of Gd III water relaxation rates in Fv solutions and also by the changes in the Fv tryptophan fluorescence on addition of Gd III. At pH 5.5 there is one tight binding site for the lanthanides (KD approximately 80 muM) but in the presence of hapten this is weakened 10-20 fold with a reciprocal effect on the hapten binding. Measurements of the Gd III quenching of the e.s.r. spectrum of a spin labelled hapten bound to Fv indicate that the lanthanide site is ca. 1.5 nm from the nitroxide moiety.

Amino Acids↗
PubMed1975

The binding of lanthanides to non-immune rabbit immunoglobulin G and its fragments.

The binding of Gd(III) to rabbit IgG (immunoglobulin G) and the Fab (N-terminal half of heavy and light chain), (Bab')2 (N-terminal half of heavy and light chains joined by inter-chain disulphide bond), Fc (C-terminal half of heavy-chain dimer)and pFc' (C-terminal quarter of heavy-chain dimer) fragments was demonstrated by measurements of the enhancement of the solvent-water proton relaxation rates in the appropriate Gd(III) solutions. At pH 5.5 there are six specific Gd(III)-binding sites on the IgG. These six sites can be divided into two classes; two very 'tight' sites on the Fc fragment (Kd approx. 5 muM) and two weaker sites on each Fab region (Kd approx. 140 muM). Ca(II) does not apparently compete for these metal-binding sites. The metal-binding parameters for IgG can be explained as the sum of the metal binding to the isolated Fab and Fc fragments, suggesting that there is no apparent interaction between the Fab and Fc regions in the IgG molecule. The binding of Gd(III) to Fab and Fc fragments was also monitored by measuring changes in the electron-spin-resonance spectrum of Gd(III) in the presence of each fragment and also by monitoring the effects of Gd(III) on the protein fluorescence at 340 nm (excitation 295 nm). The fluorescence of Tb(III) solutions of 545 nm (excitation 295 nm) is enhanced slightly on addition of Fab or Fc.

Animals↗