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R M Murphy

Publications and source records attributed to R M Murphy.

At least 37 records · Page 2Linked to original sources
PubMed1996

Effect of beta-amyloid block of the fast-inactivating K+ channel on intracellular Ca2+ and excitability in a modeled neuron.

beta-Amyloid peptide (A beta), one of the primary protein components of senile plaques found in Alzheimer disease, is believed to be toxic to neurons by a mechanism that may involve loss of intracellular calcium regulation. We have previously shown that A beta blocks the fast-inactivating potassium (A) current. In this work, we show, through the use of a mathematical model, that the A beta-mediated block of the A current could result in increased intracellular calcium levels and increased membrane excitability, both of which have been observed in vitro upon acute exposure to A beta. Simulation results are compared with experimental data from the literature; the simulations quantitatively capture the observed concentration dependence of the neuronal response and the level of increase in intracellular calcium.

Action Potentials↗
PubMed1996

A strategy for designing inhibitors of beta-amyloid toxicity.

beta-Amyloid peptide is the major protein component of Alzheimer's plaques. When aggregated into amyloid fibrils, the peptide is toxic to neuronal cells. Here, an approach to the design of inhibitors of beta-amyloid toxicity is described; in this strategy, a recognition element, which interacts specifically with beta-amyloid, is combined with a disrupting element, which alters beta-amyloid aggregation pathways. The synthesis, biophysical characterization, and biological activity of such an inhibitor is reported. This prototype inhibitor is composed of residues 15-25 of beta-amyloid peptide, designed to function as the recognition element, linked to an oligolysine disrupting element. The inhibitor does not alter the apparent secondary structure of beta-amyloid nor prevent its aggregation; rather, it causes changes in aggregation kinetics and higher order structural characteristics of the aggregate. Evidence for these effects includes changes in fibril morphology and a reduction in thioflavin T fluorescence. In addition to its influence on the physical properties of beta-amyloid aggregates, the inhibitor completely blocks beta-amyloid toxicity to PC-12 cells. Together, these data suggest that this general strategy for design of beta-amyloid toxicity inhibitors is effective. Significantly, these results demonstrate that complete disruption of amyloid fibril formation is not necessary for abrogation of toxicity.

Amyloid beta-Peptides↗
PubMed1996

The human early pregnancy factor/chaperonin 10 gene family.

cDNA clones corresponding to the sequence for human early pregnancy factor were isolated from a human melanoma library and hybridized to DNA digested with four restriction enzymes obtained from twelve different subjects. Up to 20 cross hybridizing bands were observed. When hybridized to metaphase spreads from four different humans, significant signals were present in nine locations, on eight different chromosome arms. These results suggest that the early pregnancy factor gene is a member of a large gene family. The coding sequence for early pregnancy factor has a high degree of homology with the sequence for human chaperonin 10, and the gene family described here should contain the genes for both of these proteins.

Blotting, Southern↗
PubMed1996

Beta-amyloid peptide blocks the fast-inactivating K+ current in rat hippocampal neurons.

Deposition of beta-amyloid peptide (A beta) in senile plaques is a hallmark of Alzheimer disease neuropathology. Chronic exposure of neuronal cultures to synthetic A beta is directly toxic, or enhances neuronal susceptibility to excitotoxins. Exposure to A beta may cause a loss of cellular calcium homeostasis, but the mechanism by which this occurs is uncertain. In this work, the acute response of rat hippocampal neurons to applications of synthetic A beta was measured using whole-cell voltage-clamp techniques. Pulse application of A beta caused a reversible voltage-dependent decrease in membrane conductance. A beta selectively blocked the voltage-gated fast-inactivating K+ current, with an estimated KI < 10 microM. A beta also blocked the delayed rectifying current, but only at the highest concentration tested. The response was independent of aggregation state or peptide length. The dynamic response of the fast-inactivating current to a voltage jump was consistent with a model whereby A beta binds reversibly to closed channels and prevents their opening. Blockage of fast-inactivating K+ channels by A beta could lead to prolonged cell depolarization, thereby increasing Ca2+ influx.

Amyloid beta-Peptides↗
PubMed1995

Influence of cellular trafficking on protein synthesis inhibition of immunotoxins directed against the transferrin receptor.

Previously, a quantitative analysis that related protein synthesis inhibition of transferrin-toxin conjugates to the cellular trafficking of transferrin was proposed (P.T. Yazdi and R. M. Murphy, Cancer Res., 54: 6387-6394, 1994). Here, this work is extended to evaluate cellular trafficking of anti-transferrin receptor antibodies and protein synthesis inhibition kinetics of immunotoxins constructed from the same antibodies and the toxin gelonin. Cellular trafficking models for two monoclonal anti-transferrin receptor antibodies (5E9 and OKT9) in HeLa cells were developed. The two mAbs had similar trafficking parameters, which differed significantly from those for transferrin. Protein synthesis inhibition kinetics for immunotoxins constructed from 5E9 or OKT9 and gelonin were measured. Analysis of the data using our previously proposed relationship between protein synthesis and cellular trafficking indicated that the relationship is also valid for these new systems. The protein synthesis inhibition constants for 5E9-gelonin and OKT9-gelonin conjugates were similar to those for the transferrin-gelonin conjugate. These results suggest that it may be possible to predict the efficacy of gelonin immunotoxins from knowledge of the trafficking of the corresponding targeting agent. A sensitivity analysis showed which cellular trafficking parameters have the greatest influence on immunotoxin efficacy and are, therefore, the most likely to be profitably manipulated.

Antibodies, Monoclonal↗
PubMed1995

Aggregation state-dependent binding of beta-amyloid peptide to protein and lipid components of rat cortical homogenates.

Beta-amyloid peptide (A beta) is the primary protein component of senile plaques in Alzheimer's disease. A beta is toxic to neuronal cell cultures, although the mechanism of neurotoxicity is unknown. Neurotoxicity has been correlated to the aggregation state of the peptide. In this work, the synthetic beta-amyloid peptide A beta(1-39) was radioiodinated and fractionated into samples containing varying degrees of aggregated material. Binding of the peptide to rat cortical homogenates (containing both lipids and membrane-associated protein) and to artificial neuronal membrane (containing only lipids) was measured. Binding increased with increasing percent aggregated peptide in the solutions. Aggregated peptide bound to both cortical homogenate and membrane, whereas monomeric peptide bound to homogenate only. These results may help discriminate among alternative mechanisms of neurotoxicity of A beta.

Alzheimer Disease↗
PubMed1995

Solvent effects on self-assembly of beta-amyloid peptide.

beta-amyloid peptide (A beta) is the primary protein component of senile plaques in Alzheimer's disease patients. Synthetic A beta spontaneously assembles into amyloid fibrils and is neurotoxic to cortical cultures. Neurotoxicity has been associated with the degree of peptide aggregation, yet the mechanism of assembly of A beta into amyloid fibrils is poorly understood. In this work, A beta was dissolved in several different solvents commonly used in neurotoxicity assays. In pure dimethylsulfoxide (DMSO), A beta had no detectable beta-sheet content; in 0.1% trifluoroacetate, the peptide contained one-third beta-sheet; and in 35% acetonitrile/0.1% trifluoroacetate, A beta was two-thirds beta-sheet, equivalent to the fibrillar peptide in physiological buffer. Stock solutions of peptide were diluted into phosphate-buffered saline, and fibril growth was followed by static and dynamic light scattering. The growth rate was substantially faster when the peptide was predissolved in 35% acetonitrile/0.1% trifluoroacetate than in 0.1% trifluoroacetate, 10% DMSO, or 100% DMSO. Differences in growth rate were attributed to changes in the secondary structure of the peptide in the stock solvent. These results suggest that formation of an intermediate with a high beta-sheet content is a controlling step in A beta self-assembly.

Alzheimer Disease↗
PubMed1994

Quantitative analysis of protein synthesis inhibition by transferrin-toxin conjugates.

A mathematical model was developed which relates the time and concentration dependence of protein synthesis inhibition of an immunotoxin to the properties of the targeting agent and the conjugated toxin. The role of the targeting agent and that of the toxin in determining the cytotoxicity were separated in this model by describing protein synthesis inhibition as a function of a cellular trafficking variable, which is calculated from the trafficking parameters of the targeting agent, and a protein synthesis inhibition constant, which is a property of the translocation and enzymatic rate constants of the toxin. Transferrin cellular trafficking parameters were determined experimentally for HeLa and SK-MEL-2 cells. Protein synthesis inhibition of transferrin-gelonin and transferrin-CRM107 conjugates in both cell lines was measured as a function of time and concentration. Analysis of the data showed that the model was a good representation of the experimental results, and correctly explained cell line differences in sensitivity to transferrin-toxin conjugates. The translocation rate constant for transferrin-CRM107 was approximately 3000 times greater than that for transferrin-gelonin. The model may be useful in understanding the factors that influence immunotoxin efficacy and in designing more lethal immunotoxins.

Cell Membrane↗
PubMed1994

Effect of acid predissolution on fibril size and fibril flexibility of synthetic beta-amyloid peptide.

beta-amyloid peptide (A beta) is the major protein component of senile plaques and cerebrovascular amyloid deposits in Alzheimer's patients. Several researchers have demonstrated that A beta is neurotoxic in in vitro and in vivo systems. Peptide aggregation state and/or conformation might play a significant role in determining the toxicity of the peptide. The size and flexibility of fibrils formed from the synthetic peptide beta (1-39), corresponding to the first 39 residues of A beta, were determined. Samples were prepared either directly from lyophilized peptide or diluted from a 10 mg/ml stock solution in 0.1% trifluoroacetic acid (TFA). All samples had a final peptide concentration of 0.5 mg/ml, a final pH of 7.4, and a final NaCl concentration of 0.14 M. The molecular weight and linear density of the fibrils increased with increasing pre-incubation time in TFA, based on static light scattering measurements. Analysis of the angular dependence of the intensity of scattered light indicated that the fibrils were semi-flexible chains and that the fibril flexibility decreased with increasing pre-incubation time in TFA. There was a concomitant change in phase behavior from precipitation to gelation with the decrease in fibril flexibility.

Alzheimer Disease↗
PubMed1993

Light scattering analysis of fibril growth from the amino-terminal fragment beta(1-28) of beta-amyloid peptide.

beta-Amyloid protein (beta-A/4) is the major protein component of Alzheimer disease-related senile plaques and has been postulated to be a significant contributing factor in the onset and/or progression of the disease. In the senile plaque, beta-A/4 appears as bundles of amyloid fibrils. The biological activity of beta-A/4 may be related to its state of aggregation. In this work, self-assembly, fibril formation, and interfibrillary aggregation of beta(1-28), a synthetic peptide homologous with the amino-terminal fragment of beta-A/4, were investigated. The predominant form of beta(1-28) detected by size-exclusion chromatography and polyacrylamide gel electrophoresis was apparently a tetramer which does not bind Congo red. Aggregates containing cross-beta sheet structures which bind Congo red and thioflavin T were observed at concentrations of approximately 0.3 mg/ml or greater. Concentrations of 0.5-1 mg/ml were necessary for aggregation into fibrils to be detectable by classical or quasielastic light scattering. Both fibril elongation and fibril-fibril aggregation occur over the time scale investigated. The kinetics of aggregation were much faster at physiological salt concentrations than at lower ionic strength. Ionic strength also appeared to influence the morphology of the fibril aggregates. The data indicate that sample preparation method and sample history influence fibril size and number density.

Amino Acid Sequence↗
PubMed1992

Kinetics of aggregation of synthetic beta-amyloid peptide.

beta-Amyloid peptide is the major protein component of senile plaques and cerebrovascular amyloid deposits in patients with Alzheimer's disease. The peptide deposits extracellularly in the form of amyloid fibrils, in a cross-beta conformation. beta-amyloid peptide is a 39- to 43-residue segment of a normal membrane precursor protein. In this work, a peptide homologous to the first 40 amino acids of beta-amyloid peptide, beta(1-40), was synthesized and characterized. beta(1-40) exhibited a sharp change in solubility near physiological pH and gel formation at concentrations of 3 mg/ml or greater. Circular dichroism indicated that beta(1-40) contained approximately two-thirds beta-structure, but no alpha-helical character. Quasi-elastic and classical light scattering measurements showed that beta(1-40) aggregated end-to-end in solution, reaching average molecular weights greater than 4 x 10(6) after 13 days. The aggregates were best modeled as rigid rods of 5 nm diameter, similar to the diameter of amyloid fibrils purified from plaques. A mathematical model based on diffusion-limited aggregation was developed to describe the kinetics of aggregation.

Amino Acid Sequence↗
PubMed1992

Role of spinal serotonin1 receptor subtypes in thermally and mechanically elicited nociceptive reflexes.

The ability of 5-HT1A and 5-HT1B agonists to alter a spinal animal's nociceptive threshold was examined using two analgesiometric tests. In the spinal withdrawal reflex test, administration of the selective 5-HT1A agonists ipsapirone, gepirone and PAPP resulted in significant dose-dependent increases in receptive field (RF) area for withdrawal reflexes when compared to predrug baseline values, indicating an increase in nociceptive sensitivity. The average overall percent maximal increase in RF area following administration of 5-HT1A selective compounds was: 80 +/- 16% for the ventroflexion reflex, 90 +/- 6% for the dorsiflexion reflex and 87 +/- 8% for the lateral flexion reflex. Similar to the effects noted with 5-HT1A agonists, administration of 5-HT1B agonists RU24969, mCPP and TFMPP resulted in a hyperalgesic response with an overall percent maximal increase of 43 +/- 6% for the ventroflexion reflex, 51 +/- 6% for the dorsiflexion reflex and 38 +/- 9% for the lateral flexion reflex. In the tail-flick analgesiometric test, administration of the 5-HT1A agonists 8-OH-DPAT and ipsapirone and the 5-HT1B agonists RU24969 and mCPP resulted in a significant dose-dependent increase in tail-flick latencies when compared to predrug baseline values, indicating a decrease in nociceptive sensitivity to noxious thermal stimuli. No differences in magnitude of the effect of the two receptor subtypes were found, indicating that stimulation of either 5-HT1A or 5-HT1B receptors was equipotent in producing the antinociceptive tail-flick response.(ABSTRACT TRUNCATED AT 250 WORDS)

8-Hydroxy-2-(di-n-propylamino)tetralin↗
PubMed1992

Quantitative autoradiographic mapping of a novel serotonin receptor-5-HT1S.

The spinal cord distribution of a recently identified, high affinity 3H-5-HT binding site, the 5-HT1S receptor was studied employing quantitative autoradiography with microdensitometry. The laminar distribution and density of other high affinity 3H-5-HT binding sites present in rat spinal cord, 5-HT1A, 5-HT1B and 5-HT1C, was also determined. The greatest density of 5-HT1S receptors was found in the superficial dorsal horn laminae, the autonomic nuclei around the central canal and the ventral motor nuclei. Smaller, variable densities of 5-HT1S receptors were observed in other spinal cord regions. A similar spatial pattern of receptor density was observed for 5-HT1A, 5-HT1B and 5-HT1C receptors. The distribution of 5-HT1S receptors suggests functional modulation of somatosensory input to spinal cord as well as control of visceral and somatomotor activity.

8-Hydroxy-2-(di-n-propylamino)tetralin↗
PubMed1991

Determining molecular weight distributions of antigen-antibody complexes by quasi-elastic light scattering.

Physiological properties of soluble antigen-antibody (Ag-Ab) complexes depend in part on the size of the complexes. In previous work, the size distribution and structure of model Ag-Ab complexes were determined by electron microscopy. In this study, we used constrained regularization analysis of quasi-elastic light scattering data to estimate molecular weight distributions of model Ag-Ab complexes. A conformational model was necessary to determine appropriate correlations between molecular weight and diffusion coefficient, and to estimate particle structure factors. Porod-Kratky theory proved to be an adequate conformational model for these purposes. The molecular weight distributions determined by constrained regularization compared favorably with distributions obtained either by electron microscopy or by thermodynamic modeling.

Animals↗
PubMed1991

Intensive care unit patients with acquired immunodeficiency syndrome and Pneumocystis carinii pneumonia: suggested predictors of hospital outcome.

OBJECTIVE: To define our ICU experience with AIDS patients, Pneumocystis carinii pneumonia, and respiratory failure, and to delineate factors predictive of hospital survival. DESIGN: A retrospective study in which logistic regression analysis was applied to data obtained during the first 144 hrs of ICU admission. SETTING: A university hospital medical ICU associated with a national AIDS treatment center. PATIENTS: Twenty-seven male patients with AIDS, P. carinii pneumonia, and respiratory failure who desired full supportive and resuscitative care. MEASUREMENTS AND MAIN RESULTS: Of 27 patients who met study criteria, 19 (70%) were nonsurvivors and eight (30%) were survivors. The relative risk of death was 2.2 times greater in patients who exhibited the combination of pH less than 7.35 and a base deficit greater than 4 mEq/L, at any time in their ICU course, than in patients who did not (95% confidence interval = 1.01, 4.81). Furthermore, the relative risk of death was 3.7 times greater in patients who required positive end-expiratory pressure greater than 10 cm H2O after 96 hrs of ICU care than in those patients who did not (95% confidence interval = 1.09, 12.33). Indices of oxygen transfer, severity of chest radiograph abnormalities, concurrent lung infections, and most laboratory studies on hospital admission were not different between the two groups nor predictive of hospital survival. CONCLUSIONS: When dealing with AIDS/P. carinii pneumonia/ICU patients, it is not possible to distinguish who will survive to hospital discharge based on information routinely available before ICU admission. Those patients with the greatest chance of survival demonstrate a significant decrease in the required level of respiratory support within the first 4 days of ICU care. The presence of a metabolic acidemia (pH less than 7.35 and base deficit greater than 4 mEq/L), at any time during the ICU course, is a poor prognostic sign. We suggest that such objective variables should be included in the development of any new outcome predictor model for this group of ICU patients.

Acquired Immunodeficiency Syndrome↗
PubMed1990

Size and structure of antigen-antibody complexes: thermodynamic parameters.

The role of antigen-antibody (Ag-Ab) complexes in the immune response depends, in part, on the size of the complexes. Previously, we combined electron microscopy with classical and quasi-elastic light scattering to characterize the molecular weight distribution and the conformation of Ag-Ab complexes made from bovine serum albumin (BSA) and pairs of anti-BSA monoclonal antibodies at a single concentration and Ag:Ab molar ratio. In this report, the molecular weight distribution of Ag-Ab complexes was determined by classical light scattering at a single Ag:Ab ratio and over a range of concentrations, and binding of BSA to pairs of MAb was determined by radioimmunoassay at several Ag:Ab molar ratios. A thermodynamic model was developed for the equilibrium size distribution of Ag-Ab complexes formed between a pair of MAb, each with unique affinity and specificity, and an Ag containing a single epitope for each of the pair of MAb. The combined experimental data were used in conjunction with the model to determine the values of cyclization and polymerization constants. Successful determination of the parameters required data from both classical light scattering and electron microscopy. Cyclization constants were lower than those reported in other studies of Ag-Ab complexes; this may be attributable to our use of a protein Ag, as compared to a divalent hapten. In two out of three cases, cyclization constants increased with increasing number of Ab in the complex, in contrast to previous assumptions. The validity of the thermodynamic model was further shown by its ability, in combination with conformational and hydrodynamic model, to predict the hydrodynamic radius of the complexes over a wide range of experimental conditions.

Animals↗