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Biomedical subjects

M McNamee

Publications and source records attributed to M McNamee.

14 recordsLinked to original sources
PubMed2001

Probing the effects of membrane cholesterol in the Torpedo californica acetylcholine receptor and the novel lipid-exposed mutation alpha C418W in Xenopus oocytes.

The effects of cholesterol on the ion-channel function of the Torpedo acetylcholine receptor (nAChR) and the novel lipid-exposed gain in function alpha C418W mutation have been investigated in Xenopus laevis oocytes. We found conditions to increase the cholesterol/phospholipid (C/P) molar ratio on the plasma membrane of Xenopus oocytes from 0.5 to 0.87, without significant physical damage or change in morphology to the oocytes. In addition, we developed conditions to deplete endogenous cholesterol from oocytes using a methyl-beta-cyclodextrin incubation procedure without causing membrane instability of the cells. Methyl-beta-cyclodextrin was also used to examine the reversibility of the inhibitory effect of cholesterol on AChR function. Depletion of 43% of endogenous cholesterol from oocytes (C/P = 0.3) did not show any significant change in macroscopic response of the wild type, whereas in the alpha C418W mutant the same cholesterol depletion caused a dramatic gain-in-function response of this lipid-exposed mutation in addition to the increased response caused by the mutation itself. Increasing the C/P ratio to 0.87 caused an inhibition of the macroscopic response of the Torpedo wild type of about 52%, whereas the alpha C418W mutation showed an 81% inhibition compared with the responses of control oocytes. The wild type receptor did not recover from this inhibition when the excess cholesterol was depleted to near normal C/P ratios; however, the alpha C418W mutant displayed 63% of the original current, which indicates that the inhibition of this lipid-exposed mutant was significantly reversed. The ability of the alpha C418W mutation to recover from the inhibition caused by cholesterol enrichment suggests that the interaction of cholesterol with this lipid-exposed mutation is significantly different from that of the wild type. The present data demonstrate that a single lipid-exposed position in the AChR could alter the modulatory effect of cholesterol on AChR function.

Animals↗
PubMed1997

Slow-channel transgenic mice: a model of postsynaptic organellar degeneration at the neuromuscular junction.

The slow-channel congenital myasthenic syndrome (SCCMS) is a dominantly inherited disorder of neuromuscular transmission characterized by delayed closure of the skeletal muscle acetylcholine receptor (AChR) ion channel and degeneration of the neuromuscular junction. The identification of a series of AChR subunit mutations in the SCCMS supports the hypothesis that the altered kinetics of the endplate currents in this disease are attributable to inherited abnormalities of the AChR. To investigate the role of these mutant AChR subunits in the development of the synaptic degeneration seen in the SCCMS, we have studied the properties of the AChR mutation, epsilonL269F, found in a family with SCCMS, using both in vitro and in vivo expression systems. The mutation causes a sixfold increase in the open time of AChRs expressed in vitro, similar to the phenotype of other reported mutants. Transgenic mice expressing this mutant develop a syndrome that is highly reminiscent of the SCCMS. Mice have fatigability of limb muscles, electrophysiological evidence of slow AChR ion channels, and defective neuromuscular transmission. Pathologically, the motor endplates show focal accumulation of calcium and striking ultrastructural changes, including enlargement and degeneration of the subsynaptic mitochondria and nuclei. These findings clearly demonstrate the role of this mutation in the spectrum of abnormalities associated with the SCCMS and point to the subsynaptic organelles as principal targets in this disease. These transgenic mice provide a useful model for the study of excitotoxic synaptic degeneration.

Animals↗
PubMed1996

A beta-subunit mutation in the acetylcholine receptor channel gate causes severe slow-channel syndrome.

Point mutations in the genes encoding the acetylcholine receptor (AChR) subunits have been recognized in some patients with slow-channel congenital myasthenic syndromes (CMS). Clinical, electrophysiological, and pathological differences between these patients may be due to the distinct effects of individual mutations. We report that a spontaneous mutation of the beta subunit that interrupts the leucine ring of the AChR channel gate causes an eightfold increase in channel open time and a severe CMS characterized by severe endplate myopathy and extensive remodeling of the postsynaptic membrane. The pronounced abnormalities in neuromuscular synaptic architecture and function, muscle fiber damage and weakness, resulting from a single point mutation are a dramatic example of a mutation having a dominant gain of function and of hereditary excitotoxicity.

Adult↗
PubMed1994

Mutations in the M4 domain of Torpedo californica acetylcholine receptor dramatically alter ion channel function.

Site-directed mutagenesis was used to mutate alpha Cys418 and beta Cys447 in the M4 domain of Torpedo californica acetylcholine receptor expressed in Xenopus laevis oocytes. The M4 region is a transmembrane domain thought to be located at the lipid-protein interface. By whole-cell voltage clamp analysis, mutation of both alpha subunits to alpha Trp418 increased maximal channel activity approximately threefold, increased the desensitization rate compared with wild-type receptor, and shifted the EC50 for acetylcholine from 32 microM to 13 microM. Patch measurements of single-channel currents revealed that the alpha Trp418 increased channel open times approximately 28-fold at 13 degrees C with no effect on channel conductance. All of our measured functional changes in the alpha Trp418 mutant are consistent with a simple kinetic model of the acetylcholine receptor in which only the channel closing rate is altered by the mutation. Our results show that changes in protein structure at the putative lipid-protein interface can dramatically affect receptor function.

Acetylcholine↗
PubMed1991

Effects of pH on acetylcholine receptor function.

We have examined the effects of changing extracellular pH on the function of nicotinic acetylcholine receptors from Torpedo californica using ion flux and electrophysiological methods. Agonist-induced cation efflux from vesicles containing purified, reconstituted receptors showed a monotonic dependence on external hydrogen ion concentration with maximal fluxes at alkaline pH and no agonist-induced efflux at pH's less than approximately 5. A similar pH dependence was measured for the peak agonist-activated membrane currents measured in microelectrode voltage-clamped Xenopus oocytes induced to express Torpedo receptor through mRNA injection. Half-maximal inhibition occurred at a similar pH in both systems, in the range of pH 6.5-7.0. Single-channel currents from Torpedo ACh receptors measured in patch-clamp recordings were also reduced in amplitude at acid pH with an apparent pKa for block of less than 5. Measurements of channel kinetics had a more complicated dependence on pH. The mean channel open time determined from patch-clamp measurements was maximal at neutral pH and decreased at both acid and alkaline pH's. Thus, both channel permeability properties and channel gating properties are affected by the extracellular pH.

Animals↗
PubMed1988

The 'Do not resuscitate' order. A profile of its changing use.

The "do not resuscitate" (DNR) order has wide-ranging ethical, legal, and economic implications. We reviewed the course of 244 patients who died during two three-month periods, in 1982 and 1986. We found that 68% of patients who died had a DNR order written, including 94% with malignancy and half of patients with cardiovascular disease. Most orders (61%) were written within three days of death, with 64% written on medical-surgical floors and 34% in critical care units. Even among patients under the age of 60 years, 57% had a DNR order written by the time of death. Ninety-one percent of DNR orders were written by attending physicians, with accompanying explanatory note in 84%. Documentation showed only 14% of patients but 77% of families being consulted. In 1983 a new two-level DNR order system defined two levels of intensity: "all but cardiopulmonary resuscitation" and "comfort measures only." Equal numbers of patients received each order in the 1986 sample. No patient was transferred to the critical care units after a DNR order had been written. The prevalence of DNR orders written for patients dying of cardiovascular disease increased from 27% to 64% over the four years. We conclude, from study of deaths in this representative community hospital, that an explicit DNR order is now the rule rather than the exception, but decisions are made late and involve family far more than the patient.

Aged↗
PubMed1981

Enzymatic activity of dystrophic chicken sarcoplasmic reticulum.

We have isolated sarcoplasmic reticulum from normal and dystrophic chicken muscle, using an improved isolation procedure. Dystrophic sarcoplasmic reticulum has a reduced level of calcium-sensitive ATPase activity, phosphoenzyme formation, and steady-state calcium transport. Anion-stimulated calcium transport by dystrophic sarcoplasmic reticulum is also reduced when measured under the proper conditions, and dystrophic sarcoplasmic reticulum shows no alteration in calcium efflux rate. Active calcium phosphate loading of the normal and dystrophic sarcoplasmic reticulum preparations indicates that a reduced percentage jof the dystrophic vesicles are capable of active calcium transport. The loaded dystrophic sarcoplasmic reticulum vesicles exhibit the same relative reductions in enzymatic activity as the starting sarcoplasmic reticulum preparations. However, the enzyme activities of normal and dystrophic sarcoplasmic reticulum are similar in the presence of detergent and exogenous phospholipid. On the basis of these results, we suggest that the lipid microenvironment of the dystrophic enzyme is altered.

Animals↗
PubMedDate not supplied

How to work with an interpreter.

BACKGROUND: A trip to China provided two nursing educators an opportunity to teach Chinese nurses and, in the process, to discover principles for using an interpreter to translate from English into the students' language. CONCLUSION: The authors present 16 tips for use by instructors who teach non-English-speaking learners. The principles apply for all situations of communicating with native speakers of another language using a translator.

China↗
PubMedDate not supplied

Developing new student clinical sites: a response to downsizing of hospitals.

The settings for nursing practice are changing rapidly. Dwindling in-patient numbers and increasing out-patient care have compelled nursing programs to find student "clinical" learning experiences that reflect the changing health care delivery system. This article presents specific strategies for changing the clinical learning practicum experiences in nursing programs to respond to the changes in the health care delivery system.

Curriculum↗