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Biomedical subjects

D R Lynch

Publications and source records attributed to D R Lynch.

At least 37 records · Page 2Linked to original sources
PubMed1998

An NR2B point mutation affecting haloperidol and CP101,606 sensitivity of single recombinant N-methyl-D-aspartate receptors.

Haloperidol and ifenprodil are N-methyl-D-aspartate (NMDA) receptor (NR) antagonists with preference for the NR1/NR2B subunit combination. Previous investigations utilizing 125I-MK801 binding assays with recombinant receptors distinguished certain structural determinants on the NR2B subunit for these two drugs, with glutamate 201 being critical for haloperidol sensitivity and arginine 337 being important for ifenprodil block. Other studies, however, suggested that these two sites pharmacologically overlap. In an attempt to resolve these discrepancies, we have characterized the actions of haloperidol and CP101,606, an ifenprodil analog, on the single-channel properties of NR1/NR2B(E201R) receptors transiently expressed in Chinese hamster ovary cells, because receptors formed by NR1/NR2B(R337K) appear to be nonfunctional. Haloperidol (10 microM) inhibited wild-type NR1/NR2B channels by decreasing the frequency of channel opening, whereas CP101,606 (0.5 microM) antagonized NR1/NR2B channel activity by decreasing both the open dwell time and the frequency of channel opening. The inhibitory actions of both drugs were virtually absent in the mutant NR1/NR2B(E201R) receptors. These results suggest that glutamate 201 is critical for both haloperidol and CP101,606 inhibition, thus demonstrating common features in the action of these two antagonists.

Animals↗
PubMed1997

The NR2B-specific interactions of polyamines and protons with the N-methyl-D-aspartate receptor.

Many compounds exhibit NR2B-specific modulation of the N-methyl-D-aspartate receptor, although their mechanism(s) of action are largely unknown. Using chimeric NR2A/NR2B subunits, we have located a region of NR2B (amino acids 138-238) which regulated glycine-independent polyamine stimulation. Mutation of glutamate 201 in this region affected stimulation by polyamines in the order E201D < E201A < E201N < E201R. The relief of proton inhibition of the N-methyl-D-aspartate-induced currents mediated by these mutant receptors correlated with the reduction in glycine-independent polyamine stimulation. Electrophysiological evidence with a triple mutant of NR2A further supports the hypothesis that polyamine stimulation may be linked to the relief of tonic inhibition by protons and demonstrates the crucial role of amino acids 200 and 201 in polyamine stimulation. Polyamines and protons, therefore, share common NR2B determinants.

Amino Acid Sequence↗
PubMed1997

N-methyl-D-aspartate receptors expressed in a nonneuronal cell line mediate subunit-specific increases in free intracellular calcium.

N-methyl-D-aspartate (NMDA) receptors can mediate cell death in neurons and in non-neuronal cells that express recombinant NMDA receptors. In neurons, increases in intracellular calcium correlate with NMDA receptor-mediated death, supporting a key role for loss of cellular calcium homeostasis in excitotoxic cell death. In the present study, free intracellular calcium concentrations were examined in response to activation of recombinant NMDA receptors expressed in human embryonic kidney 293 cells. Intracellular calcium was measured in transfected cell populations by cotransfection with the calcium-sensitive, bioluminescent protein aequorin and by single cell imaging with the fluorescent calcium indicator fluo-3. Agonist application to NR1/2A or NR1/2B-transfected cells elicited robust rises in intracellular calcium. NR1/2A responses were inhibited by the noncompetitive antagonists MK-801 and dextromethorphan and were dependent on extracellular calcium but not on intracellular calcium stores. In contrast, no detectable intracellular calcium responses were observed in NR1/2C-transfected cells. These findings indicate that NMDA receptors in the absence of other neuron-specific factors can mediate increases in intracellular calcium with subunit specificity and extracellular calcium dependence.

Calcium↗
PubMed1997

Autosomal dominant transmission of Dejerine-Sottas disease (HMSN III).

Hereditary motor-sensory neuropathy type III (HMSN III) (Dejerine-Sottas disease) is a severe demyelinating neuropathy that is traditionally considered autosomal recessive. We report a father and daughter diagnosed with HMSN III by clinical, electrophysiologic, and pathologic criteria, thus showing that it may be transmitted in an autosomal dominant fashion in selected families.

Adolescent↗
PubMed1996

Interactions between ifenprodil and the NR2B subunit of the N-methyl-D-aspartate receptor.

Ifenprodil is an atypical noncompetitive modulator of the N-methyl-D-aspartate (NMDA) receptor (NR) which demonstrates a 140-fold preference for NR2B over NR2A subunits, although the molecular basis for this subunit specificity is unknown. We have made chimeric receptors by fusing the murine forms of NR2A (epsilon 1) and NR2B (epsilon 2) to localize the high affinity determinants of ifenprodil inhibition on the 2B subunit. Binding experiments with 125I-MK-801 implicated the region between amino acids 198 and 356 of NR2B for high affinity ifenprodil interaction. Site-directed mutants at Arg-337 showed that this residue is absolutely required for high affinity ifenprodil inhibition. Polyamines also modulate the NMDA receptor with a preference for NR2B subunits, and the pharmacology of these agents overlaps with ifenprodil. Although the determinants of the polyamine enhancement of iodo-MK-801 binding also localize to the NH2 terminus of NR2B, the point mutants at Arg-337 form receptors that are polyamine-stimulated at wild type levels. In addition, polyamine stimulation depends on the expression of NR1 splice variants, whereas high affinity ifenprodil inhibition is independent of NR1 isoform expression. These studies provide evidence that ifenprodil and polyamines interact at discrete sites on the NR2B subunit.

Alternative Splicing↗
PubMed1996

Characterization of glutamate binding sites in receptors assembled from transfected NMDA receptor subunits.

Previous studies in brain and recombinant NMDA receptors have observed heterogeneity in NMDA-sensitive glutamate binding site. We further characterized the glutamate site assembled from NR1a, NR2A, and NR2B NMDA receptor subunits using L-[3H]glutamate and [3H]CGP 39653 binding assays. In contrast to earlier reports, we demonstrate a unique pharmacology for the NR2A subunit alone, which has high affinity for agonists but low affinity for competitive antagonists compared with heteromeric combinations of NR1a + NR2A and NR1a + NR2B. Similar to previous reports, we find unequal antagonist affinity between heteromeric combinations of NR1a + NR2A and NR1a + NR2B. However, unlike earlier reports, we describe two binding components within each heteromeric transfection that more closely resemble data obtained for binding to brain membranes. In addition, we show Mg2+ can alter [3H]CGP 39653 binding in both the NR1a + NR2A and the NR1a + NR2B combination, thus allowing comparison of the [3H]CGP 39653-labeled site between the two heteromeric combinations. Agonist inhibition of [3H]CGP 39653 binding revealed differences between the heteromeric combinations as well as within each heteromeric combination, the latter of which more closely resembled results from brain. These results further determine components of the agonist and antagonist binding sites of the NMDA receptor as well as suggest additional possible mechanisms of heterogeneity of the glutamate site in the brain.

2-Amino-5-phosphonovalerate↗
PubMed1996

Inhibition of N-methyl-D-aspartate receptors by haloperidol: developmental and pharmacological characterization in native and recombinant receptors.

The dopaminergic antagonist haloperidol also acts at the psychotomimetic sigma receptor and has been proposed to act at N-methyl-D-aspartate (NMDA) receptors. We have investigated the basis of its action at NMDA receptors using native and recombinant receptors and as a function of brain development. Haloperidol inhibits binding of 125I-(+) MK 801 to NMDA receptors formed from NR 1a/2B with high affinity but has lower affinity at receptors formed from NR 1a/2A. No differences are noted between different NR 1 splice variants transfected with NR 2B. Differences are also seen between neonatal receptors and adult receptors; neonatal receptors are inhibited by haloperidol with an IC50 value that is 50 times lower than that of adult receptors. Other agents acting at the sigma receptor inhibit binding of 125I-(+) MK 801 to NMDA receptors, and are slightly more potent at neonatal receptors. However, the rank order of potency for sigma agents at sigma receptors does not match their rank order of potency at NMDA receptors, and the prototypic sigma ligand ditolylguanidine shows no preference between NR 1a/2A and NR 1a/2B receptors. This suggests that haloperidol exerts its effects on the NMDA receptor at a distinct site from the pharmacologically defined sigma receptor. The effects of haloperidol are also modified by spermidine noncompetitively, which differs from the effects of spermidine on ifenprodil-mediated inhibition of NMDA receptors. This suggests that haloperidol interacts with NMDA receptors at a site distinct from the polyamine or ifenprodil sites.

Animals↗
PubMed1995

Pharmacological characterization of heterodimeric NMDA receptors composed of NR 1a and 2B subunits: differences with receptors formed from NR 1a and 2A.

Pharmacological and molecular biological evidence indicates the existence of multiple types of NMDA receptors within the CNS. We have characterized pharmacological properties of receptors assembled from the combination of NR 1a and NR 2B subunits (NR 1a/2B) expressed in transfected cells using both 125I-MK-801 binding assays and electrophysiological measures. Binding of 125I-MK-801 to cells transfected with NR 1a/2B is saturable with a KD of 440 pM. The binding is potently inhibited by ketamine, dextromethorphan, phencyclidine, and MK-801 and is stimulated by low concentrations of magnesium. These properties resemble those of native receptors and receptors produced by NR 1a/2A. However, 125I-MK-801 binding to membranes from cells transfected with NR 1a/2B is inhibited with high affinity by ifenprodil and is stimulated by spermidine, unlike receptors assembled from NR 1a/2A. NMDA-induced currents measured in cells transfected with either NR 1a/2A or NR 1a/2B have pharmacological properties that correlate well with the binding studies. Currents in cells transfected with NR 1a/2B are potentiated by spermidine and blocked with high affinity by ifenprodil, whereas currents in cells transfected with NR 1a/2A are not enhanced by spermidine and are weakly inhibited by ifenprodil. These data suggest that pharmacological heterogeneity in native NMDA receptors may be explained by combinations of different subunits.

Animals↗
PubMed1995

Transfection of N-methyl-D-aspartate receptors in a nonneuronal cell line leads to cell death.

Neurons grown in culture die when they are exposed to high concentrations (0.1-1 mM) of the neurotransmitter L-glutamate. A similar phenomenon may occur in the mammalian brain during ischemia and other injuries that cause excessive glutamate release. Activation of N-methyl-D-aspartate (NMDA) receptors and the consequent Ca2+ influx are thought to play a critical role in the process of neuronal toxicity. Events subsequent to the Ca2+ influx are not well understood. We have discovered that nonneuronal kidney cells expressing NMDA receptors after DNA transfection undergo cell death unless they are protected by drugs that block the NMDA receptor ion channel. Furthermore, transfected cells expressing a mutated NMDA receptor that conducts less Ca2+ are less vulnerable to cell death. In addition, we find that even though several active forms of NMDA receptors can be synthesized in these cells after transfection with different cloned subunits, not all receptor types are equally toxic. These experiments suggest that Ca2+ influx through NMDA channels may be toxic to nonneuronal cells and that the NMDA receptor expression may be the major neuron-specific component of excitotoxicity.

Animals↗
PubMed1995

Cerebellar atrophy in a patient with velocardiofacial syndrome.

Velocardiofacial syndrome and DiGeorge syndrome have not previously been associated with central nervous system degeneration. We report a 34 year old man who presented for neurological evaluation with cerebellar atrophy of unknown aetiology. On historical review, he had neonatal hypocalcaemia, an atrial septal defect, and a corrected cleft palate. His physical examination showed the characteristic facies of velocardiofacial syndrome as well as dysmetria and dysdiadocho-kinesia consistent with cerebellar degeneration. Molecular cytogenetic studies showed a deletion of 22q11.2. This man is the first reported patient with the association of a neurodegenerative disorder and 22q11.2 deletion syndrome.

Abnormalities, Multiple↗
PubMed1994

Secondary mechanisms in neuronal trauma.

Secondary damage in central nervous system trauma results from toxic effects of a variety of modulators that magnify the initial traumatic damage. These modulators include the excitatory transmitter glutamate, the intracellular messenger calcium, and the intercellular messenger nitric oxide. Glutamate-induced toxicity, called excitotoxicity, occurs from excess glutamate release following trauma. Passage of calcium into the cell through a specific postsynaptic glutamate receptor ion channel, the N-methyl-D-aspartate (NMDA) receptor, is crucial in activating cellular pathways leading to excitotoxic damage. The NMDA receptor requires both glutamate and glycine for activation and is blocked by many drugs that act at either of these sites. It is also blocked by agents that selectively block the ion channel of this receptor. Blocking NMDA receptors at any of these sites decreases the cellular damage mediated by glutamate in neuronal trauma and improves physiological measures associated with traumatic damage. Other strategies to limit secondary damage include blockade of other calcium channels, of calcium-activated enzymes, and of processes that mediate the effects of calcium. Calcium-activated enzymes include nitric oxide synthase and phospholipases, which cause production of free radicals. In combination with NMDA receptor blockade, these sites represent promising areas for therapeutic intervention in secondary traumatic damage.

Animals↗
PubMed1994

N-methyl-D-aspartate receptors: different subunit requirements for binding of glutamate antagonists, glycine antagonists, and channel-blocking agents.

Expression of the NR-1 subunit in Xenopus oocytes produces channels that respond to glutamate and are blocked by competitive and noncompetitive antagonists of the N-methyl-D-aspartate (NMDA) receptor. Ionic conductances through these channels are increased by coexpression with NR-2 receptor subunits. We have characterized the pharmacological properties of NMDA receptors assembled from combinations of subunits expressed in transfected cells, to determine the minimum subunit requirements for binding of competitive glutamate antagonists, glycine antagonists, and channel-blocking agents, as detected by ligand-binding experiments. Expression of NR-1a alone produced glycine antagonist binding, whereas the combination of NR-1a and NR-2A was needed to produce binding sites for glutamate antagonists and channel-blocking agents. These results suggest that functional NMDA receptors assemble from these subunits. However, differences in the pharmacological effects of NMDA and polyamines show that not all characteristics of native NMDA receptors are reproduced by this combination of subunits.

Binding Sites↗
PubMed1992

Environmental education.

The need for a new profession devoted to environmental matters is asserted. The qualities of such a profession are sketched, and it is argued that new initiatives in environmental education are needed in the form of graduate, professional programs with primary emphasis on practice. An example 2-year program is presented. A fundamental requirement is scientific competence; undergraduate preparation in the sciences or engineering is mandatory. The graduate curriculum itself is built on three primary cores: environmental science and engineering, business and management, and public policy. Additionally, an environmental round table is proposed as a focal point for academic, industrial, governmental, and public discussion on environmental matters. The round table would provide oversight for the professional educational program and an affiliated research institute.

Journal Article↗
PubMed1992

Risk factors for the neurologic complications associated with aortic aneurysms.

We reviewed the incidence of neurologic complications in 200 consecutive patients with aortic aneurysm or aortic dissection. In this 2-year period, neurologic impairment developed in 18.5% of these patients, and in 10 patients neurologic dysfunction heralded aortic rupture or dissection. Those patients with abnormal neurologic examinations at presentation frequently had aneurysm rupture or dissection and a mortality rate of 54%. Patients with thoracic or thoracoabdominal aneurysms were more likely to have neurologic complications than those with abdominal aneurysms. The most common complications were focal central nervous system ischemia, followed by disorders of consciousness and peripheral nerve complications. In patients who had elective aneurysm resection, female sex, aneurysm location, and intraoperative hypotension were risk factors for focal central nervous system ischemia. We conclude that neurologic complications depend on aneurysmal location, occur at various levels of the nervous system, and frequently develop when the intraoperative mean arterial pressure falls below 55 mm Hg.

Aged↗
PubMed1990

Carboxypeptidase E (CPE): immunocytochemical localization in the rat central nervous system and pituitary gland.

Carboxypeptidase E (CPE, enkephalin convertase, carboxypeptidase H; EC 3.4.17.10) is a carboxypeptidase B-like enzyme proposed to be involved in the synthesis of a variety of peptide hormones and neurotransmitters. In the present study we have localized CPE in the rat central nervous system using specific polyclonal antisera to the purified enzyme. Immunoreactive CPE occurs in a variety of cell somas and neuronal terminals in the central nervous system. The enzyme is most concentrated in the hypothalamus, especially in the median eminence, supraoptic nucleus, paraventricular nucleus, and suprachiasmatic nucleus. CPE is also found in axon terminals in the posterior pituitary gland, melanotropes of the intermediate pituitary gland, and select cells of the anterior pituitary gland. Immunoreactivity to CPE in the hippocampus is found in the pyramidal cells and in the inner part of the molecular layer of the dentate gyrus. CPE is also found in the central nucleus of the amygdala and the bed nucleus of the stria terminalis. These results suggest that CPE distribution corresponds to that of many neuropeptides at the fine structural level, thus further implicating CPE in peptide processing.

Animals↗
PubMed1989

Optimization of the absorbed power distribution for an annular phased array hyperthermia system.

One of the systems under investigation for producing hyperthermia noninvasively for treating deep-seated tumors is the annular phased array. This device consists of two rings of eight electromagnetic apertures that are placed concentrically about the long axis of the patient and radiate energy toward the center. Previous theoretical and clinical studies have concentrated primarily on systems where the amplitude and phase of the signal applied to each aperture were the same, and these studies have shown that the system is capable of depositing power deep within the patient. Nevertheless, in many situations the system was not capable of producing desirable temperature distributions in the tumor and normal tissue. In this paper we report on a 2-dimensional theoretical investigation where an optimization routine was used to select the amplitude and phases of each of eight apertures. The optimization procedure and resulting calculations were based on CT scans of patients with tumors. The electrical and thermal properties of the different organs and tissues were taken into account. The optimization routine tried to achieve uniform absorbed power in the tumor region with zero absorbed power outside. Using the optimized amplitudes and phases, the SAR (specific absorption rate, W/kg) was calculated for the array. The results show that in general the optimization procedure was successful in that the power deposited within the tumor volume was increased with less power deposited into normal tissue when compared to the equal amplitude and phase case. This SAR data was then used as the input to a program based on the bioheat transfer equation, which calculated the temperature distribution in the patient model for an assumed set of blood perfusion rates. Depending on the location, size of the tumor, and blood perfusion rates, the improvement in the percentage of the tumor brought to therapeutic temperature varied from 0% to as much as 80%.

Humans↗