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Biomedical subjects

E R Stimson

Publications and source records attributed to E R Stimson.

At least 37 records · Page 2Linked to original sources
PubMed1995

Local anesthetics inhibit substance P binding and evoked increases in intracellular Ca2+.

BACKGROUND: During spinal and epidural anesthesia, local anesthetics reach concentrations in cerebrospinal fluid and spinal cord tissues at which their actions may extend beyond the classic blockade of sodium channels. This study examines the effects of several clinical and experimental local anesthetics on the binding and actions of a peptide neurotransmitter, substance P, known to be important in nociceptive transmission in the dorsal horn. METHODS: The binding of radiolabeled (Bolton-Hunter modified) substance P was studied in chick brain membranes in the presence of local anesthetics. The increase in intracellular calcium [Ca2+]in evoked by substance P was measured by the fluorescent indicator fura-2 loaded in a murine cell line expressing substance P (NK1) receptors. Cells were preincubated with bupivacaine before and during the transient addition of substance P. RESULTS: Both substance P binding and Ca2+ increase were inhibited half-maximally by approximately 1 mM bupivacaine at pH 7.5, whereas tetracaine, lidocaine, and benzocaine were slightly less potent at inhibiting binding. Concentration-dependent substance P-binding studies showed that bupivacaine's inhibition was not competitive. Inhibition of substance P binding by bupivacaine increased with increasing pH, but the protonated species appears to have some inhibitory activity, and quaternary lidocaine also inhibited binding. There was no stereoselectively to the binding inhibition. CONCLUSIONS: Because millimolar concentrations of local anesthetics are within the range measured in spinal cord during intrathecal and epidural procedures, these results are consistent with a direct action of local anesthetics on tachykinin-mediated neurotransmission during regional anesthesia.

Anesthetics, Local↗
PubMed1994

Agonist-induced photoincorporation of a p-benzoylphenylalanine derivative of substance P into membrane-spanning region 2 of the Torpedo nicotinic acetylcholine receptor delta subunit.

The neuropeptide substance P acts, at micromolar concentrations, as a noncompetitive antagonist of nicotinic acetylcholine receptors (AChRs) of both neuronal and muscle subtypes. The mechanism of this inhibition has been shown to be most consistent with stabilization of a nonconducting desensitized state of the AChR, via binding to a site distinct from both the agonist site and the high affinity noncompetitive antagonist site. We have used a radioiodinated photoreactive analogue of substance P, containing the amino acid p-benzoyl-L-phenylalanine in place of the Phe8 residue of substance P, to identify the sites of interaction of substance P within the Torpedo california AChR. AChR-rich membrane suspensions were photolabeled in the absence or presence of the agonist carbamylcholine and/or nonradioactive substance P, and incorporation into AChR subunits was assessed by autoradiography after sodium dodecyl sulfate-polyacrylamide gel electrophoresis. In the absence of agonist 125I incorporation was detected in each subunit and was insensitive to substance P, whereas in the presence of carbamylcholine there was a 2-fold increase in photoincorporation into the AChR delta subunit that was inhibited by the addition of an excess of substance P. The sites of specific photoincorporation in the delta subunit were initially mapped by use of Staphylococcus aureus V8 protease to a 14-kDa fragment extending from delta Ile-192 to Glu-280. Further fragmentation of this 14-kDa fragment with trypsin and S. aureus V8 protease established that the sites of specific incorporation were restricted to the region delta Ser-253 to Glu-280, which contains the membrane-spanning region 2 that is known to form the lining of the ion channel. These results establish that in the presence of agonist at least a part of the undecapeptide substance P binds within the ion channel in the desensitized state of the AChR, and it is likely that the binding of substance P to this site is responsible for the action of substance P as a noncompetitive AChR antagonist.

Affinity Labels↗
PubMed1993

Aluminum, iron, and zinc ions promote aggregation of physiological concentrations of beta-amyloid peptide.

A major pathological feature of Alzheimer's disease (AD) is the presence of a high density of amyloid plaques in the brain tissue of patients. The plaques are predominantly composed of human beta-amyloid peptide beta A4, a 40-mer whose neurotoxicity is related to its aggregation. Certain metals have been proposed as risk factors for AD, but the mechanism by which the metals may exert their effects is unclear. Radioiodinated human beta A4 has been used to assess the effects of various metals on the aggregation of the peptide in dilute solution (10(-10) M). In physiological buffers, 10(-3) M calcium, cobalt, copper, manganese, magnesium, sodium, or potassium had no effect on the rate of beta A4 aggregation. In sharp contrast, aluminum, iron, and zinc under the same conditions strongly promoted aggregation (rate enhancement of 100-1,000-fold). The aggregation of beta A4 induced by aluminum and iron is distinguishable from that induced by zinc in terms of rate, extent, pH and temperature dependence. These results suggest that high concentrations of certain metals may play a role in the pathogenesis of AD by promoting aggregation of beta A4.

Aluminum↗
PubMed1992

Reversible in vitro growth of Alzheimer disease beta-amyloid plaques by deposition of labeled amyloid peptide.

The salient pathological feature of Alzheimer disease (AD) is the presence of a high density of amyloid plaques in the brain tissue of victims. The plaques are predominantly composed of human beta-amyloid peptide (beta A4), a 40-mer whose neurotoxicity is related to its aggregation. Radioiodinated human beta A4 is rapidly deposited in vitro from a dilute (less than 10 pM) solution onto neuritic and diffuse plaques and cerebrovascular amyloid in AD brain tissue, whereas no deposition is detectable in tissue without performed plaques. This growth of plaques by deposition of radiolabeled beta A4 to plaques is reversible, with a dissociation half-time of approximately 1 h. The fraction of grey matter occupied by plaques that bind radiolabeled beta A4 in vitro is dramatically larger in AD cortex (23 +/- 11%) than in age-matched normal controls (less than 2%). In contrast to the human peptide, rat/mouse beta A4 (differing at three positions from human beta A4) does not affect the deposition of radiolabeled human beta A4. beta A4 has no detectable interaction with tachykinin receptors in rat or human brain. The use of radioiodinated beta A4 provides an in vitro system for the quantitative evaluation of agents or conditions that may inhibit or enhance the growth or dissolution of AD plaques. This reagent also provides an extremely sensitive method for visualizing various types of amyloid deposits and a means for characterizing and locating sites of amyloid peptide binding to cells and tissues.

Aged↗
PubMed1986

Conformational properties of trans Ac-Asn-Pro-Tyr-NHMe and trans Ac-Tyr-Pro-Asn-NHMe in dimethylsulfoxide and in water determined by multinuclear n.m.r. spectroscopy.

Vicinal coupling constants between various nuclei provide backbone and side-chain conformational information for a series of asparagine- and tyrosine-containing peptides in DMSO and in H2O. By enriching Tyr of Ac-Asn-Pro-Tyr-NHMe with 15N, it has been possible to distinguish between the resonances of the two side-chain beta protons of Tyr. Analysis of the coupling constants in terms of the distributions of side-chain conformations in these peptides indicates that the addition of Asn to the Pro-Tyr sequence leads to a less random conformational distribution. When compared to the side-chain rotamer distribution of Ac-Asn-NHMe and Ac-Tyr-NHMe, particular Asn and Tyr side-chain conformations of Ac-Asn-Pro-Tyr-NHMe are stabilized in dimethylsulfoxide solution. The interaction(s) which stabilize a unique Tyr side-chain conformation of Ac-Asn-Pro-Tyr-NHMe in dimethylsulfoxide are not present in Ac-Ala-Pro-Tyr-NHMe and are unaffected by the addition of Val-Pro to the C-terminus of Asn-Pro-Tyr. In water, a preferential stabilization of one Asn side-chain conformation of Ac-Asn-Pro-Tyr-NHMe is also observed, while the Tyr side-chain rotamer distribution is similar to that of Ac-Tyr-NHMe. An interaction between the Asn side chain and the Pro-Tyr-NHMe backbone was previously shown to stabilize a beta-bend conformation at Pro-Tyr in water. Data are also presented for Ac-Tyr-Pro-Asn-NHMe, for which local interactions do not stabilize particular backbone conformations in dimethylsulfoxide or in water. The conformations of the peptides studied here are relatively insensitive to temperatures between 27 degrees and 62 degrees, both in dimethylsulfoxide and in water. The sequences Asn-Pro-Tyr and Tyr-Pro-Asn occur in ribonuclease A, and these tripeptides serve as models for the interactions involved in the folding of this protein.

Amino Acid Sequence↗
PubMed1986

Deamidation of the asparaginyl-glycyl sequence.

The deamidation of Ac-Asn-Gly-NHMe and Ac-Isn-Gly-NHMe has been studied as a model for the facile deamidation of the Asn-Gly sequence in proteins. At alkaline pH, the product in each case is an identical mixture of Ac-alpha-Asp-Gly-NHMe (approximately 22%) and Ac-beta-Asp-Gly-NHMe (approximately 78%) as determined by n.m.r. spectroscopy. Because this same ratio is obtained from both Ac-Asn-Gly-NHMe and Ac-Isn-Gly-NHMe, the postulated mechanism, that deamidation proceeds through a cyclic imide intermediate, is confirmed. Unlike peptides of aspartyl esters, cyclization does not occur under nonaqueous conditions or at low pH in aqueous solution.

Amino Acid Sequence↗
PubMed1985

Conformational analysis of cyclized dipeptide models for specific types of beta-bends by two-dimensional nuclear Overhauser spectroscopy.

The solution conformations in DMSO-d6 of the two cyclized dipeptides, cyclo(L-alanyl-L-alanyl-epsilon-aminocaproyl) and cyclo(L-alanyl-D-alanyl-epsilon-aminocaproyl), have been analyzed by means of the two-dimensional nuclear Overhauser effect (2D-NOE). The preferred conformations for the two compounds have been deduced by comparing proton-proton distances, derived from the 2D-NOE data and relaxation-time measurements, with the corresponding distances in several possible computed low-energy conformations. The predominant conformations are a type III bend and a type II bend, respectively, for the two compounds. These conclusions agree with those deduced earlier on the basis of infrared and Raman spectra and circular dichroism measurements.

Magnetic Resonance Spectroscopy↗
PubMed1984

Determination of molecular weights by differential cryoscopy on small volumes of dilute solutions of oligopeptides.

A differential cryoscope of the equilibrium type is described. It requires only 1 ml of sample for a molecular-weight determination of a solute in a solvent that freezes below room temperature. The instrument is sensitive to +/- 0.0002 degrees C, which corresponds to +/- 0.0001 mol of solute per 1000 g of water. The apparatus was evaluated by measuring the freezing point depressions of 0.015 M urea and 0.01 M alanine in water, the measured molecular weights being accurate to within +/- 5%. The molecular weights of the following oligopeptides were then measured to determine their states of aggregation in the cited solvents: Ac-Asn-Pro-Tyr-NHMe in H2O and dimethyl sulfoxide and cyclo(L-alanyl-L-alanyl-epsilon-aminocaproyl), cyclo(L-alanyl-D-alanyl-epsilon-aminocaproyl), cyclo(L-alanyl-L-analyl-omega-capryl), cyclo(L-alanyl-D-alanyl-omega-capryl), and Ac-Tyr-Pro-Asn-NHMe in dimethyl sulfoxide.

Dimethyl Sulfoxide↗
PubMed1982

Conformations of cyclo(L-alanyl-L-alanyl-epsilon-aminocaproyl) and of cyclo(L-alanyl-D-alanyl-epsilon-aminocaproyl); cyclized dipeptide models for specific types of beta-bends.

Conformational energy calculations indicate that the peptide backbones of the low-energy conformations of the cyclized dipeptide derivatives cyclo (L-alanyl-L-alanyl-epsilon-aminocaproyl) and cyclo (L-alanyl-D-alanyl-epsilon-aminocaproyl) are constrained to form beta-bends of types I + III and II, respectively. Thus, the two compounds can serve as models for the spectroscopic properties of beta-bends of these types. The coupling constants obtained from 1H n.m.r. spectra in DMSO-d6 are consistent with the dihedral angeles of the computed lowest-energy conformations. Differences in 13C chemical shifts between the two compounds can be correlated with differences in shielding by C=O groups in bends of various types. 1H and 13C chemical shifts suggest association of cyclo (L-Ala-L-Ala-Aca) but not of cyclo (L-Ala-D-Ala-Aca) in dimethylsulfoxide. The different tendencies to associate can be explained in terms of the difference in conformation. The circular dichroism spectra of the two compounds are quite different. In methanol, trifluoroethanol and water, the L-Ala-L-Ala derivative has a positive extremum near 190 nm and two negative extrema near 206 and 220 nm, whereas the L-Ala-D-Ala derivative has a positive extremum at about 203 nm and negative extrema at about 187 and 229 nm. The spectra can be used to estimate the contribution of various bend types in a related series of compounds. A normal mode analysis of the vibrations of the computed low-energy conformations was compared with solid state infrared and Raman spectra, in order to determine the predominant conformations. The bend types determined by this comparison fully agree with the predictions of the theoretical computations for both derivatives.

Chemical Phenomena↗
PubMed1982

Solution conformations of oligomers of alpha-aminoisobutyric acid.

The N-acetyl(Aib)nN'-methylamides (with n = 1, 2 and 3) and the N-acetyl-(Aib)3 methyl ester have been synthesized using an oxazolone procedure. An experimental conformational analysis of this series of oligomers has been carried out in water, DMSO-d6 and CDCl3 using n.m.r. techniques, and in chloroform using i.r. spectroscopy. Deuterium exchange rates of amide protons in DMSO-d6 and the rates of these proton chemical shifts with temperature in water, DMSO-d6 and CDCl3 indicate that the oligomeric N'-methylamides adopt conformations that have no hydrogen bonds when n = 1, one hydrogen bond when n = 2, and two hydrogen bonds when n = 3, and that Ac(Aib)3OMe has a conformation with one hydrogen bond. An analysis of the N-H stretching region of the i.r. spectra of these compounds in CHCl3 also suggests the existence of these conformational states. These data imply that the peptides adopt the 3(10)-helical and not the alpha-helical conformation in solution. This conclusion supports the hypothesis that the Aib residue has asymmetric geometry at the C alpha atom in solution, similar to that reported in the literature for the crystalline state.

Aminoisobutyric Acids↗
PubMed1981

Spectroscopic study of the conformations of proline-containing oligopeptides in the crystalline state and in solution.

A conformational study has been carried out on a series of linear proline-containing oligopeptides (ZGP, ZGPL, ZGPLG and ZGPLGP) in both the crystalline state and in DMSO-d6 solution, using Raman and n.m.r. spectroscopy. The amide I and III bands in the Raman spectra of the crystalline forms indicate the presence of the type I beta-bend conformation in ZGPLG and ZGPLGP, but not in ZGP and ZGPL. This result is in agreement with X-ray data on these molecules. The Raman spectra of these peptides in solution indicate that more than one conformation is present, i.e. that the beta-bend structure of the solid form of ZGPLG and ZGPLGP is destabilized by DMSO-d6. 13C and 1H n.m.r. data also demonstrate the presence of more than one conformation in ZGP, ZGPL, ZGPLG and ZGPLGP in DMSO-d6 solution. These isomers differ in their conformation (cis and trans) about their Gly-Pro peptide bonds and possibly about the c alpha-C' bond of the C-terminal proline in ZGPLGP. For ZGP, ZGPL, ZGPLG and ZGPLGP, the ensemble of conformations in DMSO-d6 includes C5 and C7 hydrogen-bonded rings; in addition, ZGPLGP may contain a small percentage of a beta-bend conformation (at Pro2-Leu3) with trans peptides in both Gly-Pro moieties.

Crystallization↗
PubMed1980

Study of the state of ionization of [Leu5]-enkephalin in the crystal and in solution.

The state of ionization of [Leu5]-enkephalin in the crystal and in solution was studied with the aid of Raman, i.r., and n.m.r. techniques. The results indicate that, in the crystal, enkephalin adopts the dipolar ion form. In water near neutral pH, enkephalin also exists in the dipolar ion form whereas in DMSO-d6 it is a mixture of the dipolar ion and uncharged species. The pK's of alpha-amino and alpha-carboxyl groups are probably closer to each other in DMSO than in H2O.

Crystallization↗
PubMed1980

Conformational study of [Leu5]-enkephalin by laser Raman spectroscopy.

The Raman spectrum of [Leu5]-enkephalin in the solid state indicates the presence of a type I' beta bend, in agreement with X-ray data of Smith & Griffin (1978). In solution, this molecule takes on different conformations in different solvents. In DMSO-d6, it forms a beta-bend structure whereas in water it exists as an ensemble of conformations.

Dimethyl Sulfoxide↗
PubMed1979

Solution conformation of enkephalin. A nuclear magnetic resonance study of 13C-enriched carbonyl carbons in [Leu5]-enkephalin.

By using 13C enrichment in [Leu5]-enkephalin, it has been possible to improve the assignment of carbonyl resonances in the nuclear resonance spectrum and to remove some of the ambiguities in the derived phi and chi dihedral angles, thereby providing information about the conformation of this molecule in solution. The combined use of 13C and 1H nuclear magnetic resonance experiments leads to the conclusion that [Leu5]0enkephalin contains a type I beta bend at residues Gly3-Phe4 in dimethyl-d6 sulfoxide (Me2SO0d6) solution. Furthermore, the side chains of Tyr1, Phe4, and Leu5 exist predominantly in one conformation (tg-) in this solvent. A comparison is made between the conformation found in Me2SO-d6 and those determined by X-ray diffraction and conformational energy calculations.

Amino Acids↗
PubMed1979

Cyclized dipeptide model for a beta-bend.

A cyclic dipeptide in which L-Ala-Gly was cyclized with epsilon-aminocaproic acid has been synthesized as a model for a beta-bend. Its conformational properties have been examined by means of conformational energy calculations and nuclear magnetic resonance, infrared, Raman, and circular dichroism spectroscopy in various solvents. These calculations and experiments suggest that a type II beta-bend exists in the Ala-Glymoiety, with an NH...O = C hydrogen bond in the epsilon-aminocaproic acid portion of the molecule, and that the molecule adopts a unique conformation in solution. In contrast, an open-chain analog of this compound exists in solution as an ensemble of conformations but with a significant amount of a type II beta-bend structure in the ensemble.

Circular Dichroism↗