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Biomedical subjects

J T McLaughlin

Publications and source records attributed to J T McLaughlin.

At least 19 recordsLinked to original sources
PubMed1999

Chimeric analysis of a neuronal nicotinic acetylcholine receptor reveals amino acids conferring sensitivity to alpha-bungarotoxin.

We have investigated the molecular determinants responsible for alpha-bungarotoxin (alphaBgtx) binding to nicotinic acetylcholine receptors through chimeric analysis of two homologous alpha subunits, one highly sensitive to alphaBgtx block (alpha1) and the other, alphaBgtx-insensitive (alpha3). By replacing rat alpha3 residues 184-191 with the corresponding region from the Torpedo alpha1 subunit, we introduced a cluster of five alpha1 residues (Trp-184, Trp-187, Val-188, Tyr-189, and Thr-191) into the alpha3 subunit. Functional activity and alphaBgtx sensitivity were assessed following co-expression in Xenopus oocytes of the chimeric alpha3 subunit (alpha3/alpha1[5]) with either rat beta2 or beta4 subunits. Agonist-evoked responses of alpha3/alpha1[5]-containing receptors were blocked by alphaBgtx with nanomolar affinity (IC(50) values: 41 nM for alpha3/alpha1[5]beta2 and 19 nM for alpha3/alpha1[5]beta4). Furthermore, receptors containing the single point mutation alpha3K189Y acquire significant sensitivity to alphaBgtx block (IC(50) values: 186 nM for alpha3K189Ybeta2 and 179 nM for alpha3K189Ybeta4). Another alpha3 chimeric subunit, alpha3/alpha7[6], similar to alpha3/alpha1[5] but incorporating the corresponding residues from the alphaBgtx-sensitive alpha7 subunit, also conferred potent alphaBgtx sensitivity to chimeric receptors when co-expressed with the beta4 subunit (IC(50) value = 31 nM). Our findings demonstrate that the residues between positions 184 and 191 of the alphaBgtx-sensitive subunits alpha1 and alpha7 play a critical functional role in the interaction of alphaBgtx with nicotinic acetylcholine receptors sensitive to this toxin.

Amino Acid Sequence↗
PubMed1999

Probing the agonist domain of the nicotinic acetylcholine receptor by cysteine scanning mutagenesis reveals residues in proximity to the alpha-bungarotoxin binding site.

We have constructed a series of cysteine-substitution mutants in order to identify residues in the mouse muscle nicotinic acetylcholine receptor (AChR) that are involved in alpha-bungarotoxin (alpha-Bgtx) binding. Following transient expression in HEK 293-derived TSA-201 cells, covalent modification of the introduced cysteines with thiol-specific reagents reveals that alpha subunit residues W187, V188, F189, Y190, and P194 are solvent accessible and are in a position to contribute to the alpha-Bgtx binding site in native receptors. These results with the intact receptor are consistent with NMR studies of an alpha-Bgtx/receptor-dodecapeptide complex [Basus, V., Song., G., and Hawrot, E. (1993) Biochemistry 32, 12290-12298]. We pursued a more detailed analysis of the F189C mutant as this site varies substantially between AChRs that bind Bgtx and certain neuronal AChRs that do not. Treatment of intact cells expressing F189C with either bromoacetylcholine (BrACh) or [2-(trimethylammonium)ethyl] methane-thiosulfonate (MTSET), both methylammonium-containing thiol-modifying reagents with agonist properties, results in a marked decrease ( approximately 55-70%) in the number of alpha-Bgtx binding sites, as measured under saturating conditions. The decrease in sites appears to affect both alpha/gamma and alpha/delta sites to the same extent, as shown for alphaW187C and alphaF189C which were the two mutants examined on this issue. In contrast to the results obtained with MTSET and BrACh, modification with reagents that lack the alkylammonium entity, such as methylmethanethiosulfonate (MMTS), the negatively charged 2-sulfonatoethyl methane-thiosulfonate (MTSES), or the positively charged aminoethyl methylthiosulfonate (MTSEA), has little or no effect on the maximal binding of alpha-Bgtx to the alphaW187C, alphaV188C, or alphaF189C mutant receptors. The striking alkylammonium dependency suggests that an interaction of the tethered modifying group with the negative subsite within the agonist binding domain is primarily responsible for the observed blockade of toxin binding.

Acetylcholine↗
PubMed1999

Dexamethasone-mediated inhibition of calcium transients and ACTH release in a pituitary cell line (AtT-20).

In the corticotroph-like murine pituitary tumor cell line, AtT-20, adrenocorticotropic hormone release is triggered by corticotropin-releasing hormone and is attenuated by the synthetic adrenal steroid dexamethasone. The precise mechanisms by which dexamethasone inhibits secretion are under investigation. We examined whether dexamethasone can modulate release via regulation of calcium homeostasis. More specifically, we have evaluated the effects of dexamethasone on calcium current, intracellular calcium concentration, and adrenocorticotropic hormone release. Using perforated patch-clamp and calcium imaging with fura PE3/AM, we found that dexamethasone decreases calcium current and intracellular calcium levels. The inhibition of current by dexamethasone is not, however, altered by the calcium channel antagonists nifedipine (L-type) or omega-agatoxin IVA (P/Q-type), despite the presence of these calcium channel subtypes in AtT-20 cells and the exclusive coupling of adrenocorticotropic hormone release to the L-type channel in these cells. We also evaluated the temporal relationship between dexamethasone-mediated inhibition of secretion and calcium influx. Whereas a prolonged (2 h) incubation with dexamethasone inhibits corticotropin-induced release by approximately 40%, a rapid (10 min) incubation (a time interval sufficient for dexamethasone-mediated inhibition of calcium transients) does not inhibit release. These data suggest, therefore, that dexamethasone does, indeed, modulate calcium homeostasis in AtT-20 cells, but that this effect is not responsible for its inhibition of secretion.

Adrenocorticotropic Hormone↗
PubMed1998

Fatty acids stimulate cholecystokinin secretion via an acyl chain length-specific, Ca2+-dependent mechanism in the enteroendocrine cell line STC-1.

1. The present study has investigated whether fatty acids directly influence peptide release from enteroendocrine cells using STC-1, a mouse intestinal endocrine tumour cell line, previously shown to release cholecystokinin (CCK) in response to other physiological stimuli. 2. Fatty acids elicited a chain length- and dose-dependent stimulation of CCK secretion. Dodecanoic acid (C12) was most effective, producing up to a 5-fold increase in CCK secretion. Fatty acids with less than ten carbon atoms did not increase secretion. The chain length dependence of these effects mimics closely fatty acid-induced CCK secretion previously observed in humans in vivo. 3. Esterification of C12 abolished CCK secretion, indicating a critical role for a free carboxyl group in eliciting secretion. In contrast, modification of the methyl terminus had no effect on C12-induced secretion. The non-metabolizable C12 analogue 2-bromododecanoic acid was equally effective. 4. C12 elicited a marked increase in intracellular calcium levels (200-300 nM) in STC-1 cells which was abolished by the L-type Ca2+ channel antagonist nicardipine. In contrast, C8 produced a smaller and more transient Ca2+ response. C12-induced CCK secretion was also blocked by nicardipine. 5. These data suggest that fatty acids can interact directly with enteroendocrine cells to stimulate CCK secretion via increases in intracellular calcium mediated primarily by L-type Ca2+ channels.

Animals↗
PubMed1998

Evidence for a lipid specific effect in nutrient induced human proximal gastric relaxation.

BACKGROUND/AIM: The presence of lipid in the upper gut is able to modify gastrointestinal motor performance, but its influence on the relaxation of the human stomach, which is known to modify gastric emptying, remains incompletely understood. The relaxation of the proximal stomach in response to various lipid concentrations was therefore studied in healthy volunteers. Since the observed effects could be mediated through osmolality or energy sensitive pathways, the effects of equicaloric and equiosmolar non-lipid solutions were also determined. METHODS: The tone of the proximal stomach was measured during stepwise inflation of a non-compliant bag sited in the proximal stomach, both before and after a test meal was delivered intragastrically. Iso-osmolar lipid emulsions were diluted in iso-osmolar saline at concentrations of 1.25, 2.5, 5, 10, and 20%. NaCl solutions at osmolalities of 300, 600, 1200 and 2400 mmol/kg and glucose solutions of 836 and 3344 kJ/l were also given. RESULTS: All lipid meals of 2.5% or greater concentration induced a reduction in gastric tone in a non-dose-dependent manner, responses to 5% lipid (median (range) 74 (62-92)%) being similar to those to 20% lipid (80 (55-83)%; p > 0.05). No relaxation was elicited by isocaloric glucose. NaCl only consistently caused relaxation at 2400 mmol/kg. CONCLUSION: Lipid meals reduce human proximal gastric tone by a lipid specific mechanism, independently of their energy content or osmolality.

Adult↗
PubMed1996

A classic revisited: Loewald on the therapeutic action of psychoanalysis.

Versions of the following papers were presented at the panel "Psychoanalytic Classics Revisited: Hans Loewald's "On the Therapeutic Action of Psychoanalysis' " (Gerald I. Fogel, chair) at the meetings of the American Psychoanalytic Association, December 1993. As a tribute to Loewald's lifetime of achievement, and in belated recognition of his preeminent position in the field of psychoanalysis, the exchange appears here almost in its entirety, rather than as a conventional panel report.

History, 20th Century↗
PubMed1995

Covalent modification of engineered cysteines in the nicotinic acetylcholine receptor agonist-binding domain inhibits receptor activation.

We constructed and characterized a series of nicotinic receptor mutants with a cysteine substituted for one of the amino acid residues in the alpha-subunit between positions 183 and 198. This region of the receptor is known to participate in agonist binding and channel activation. The goal of this 'cysteine scanning mutagenesis' is to introduce the reactivity of a free thiol group into functionally important protein domains; modification of the introduced cysteines can then be used to probe the structure and function of the targeted region. Mutants were examined by coexpression with the beta-, gamma- and delta-subunits in Xenopus oocytes using two-microelectrode voltage clamp recording. Twelve of fourteen mutants expressed receptors with properties comparable with the wild-type, including sensitivity to reduction by dithiothreitol (DTT). This indicates that introduction of an additional cysteine within this region of the receptor did not interfere with formation of the native disulphide between alpha Cys-192 and alpha Cys-193. Only one mutation, alpha Y198C, caused dramatic changes in the EC50 for acetylcholine (ACh) and in the sensitivity to DTT. We then examined the effects of the thiol modification and found two mutants, alpha H186C and alpha V188C, that showed significant decreases in responsiveness to ACh after exposure to methylmethanethiosulphonate (MMTS). Dose-response measurements show that exposure of alpha H186C mutants to MMTS causes a shift in apparent agonist affinity without changing the peak response, and this is not reversible by DTT. In contrast, the MMTS-treated alpha V188C mutants show changes in both apparent affinity and peak response which are readily reversed by DTT. Together, our data show that these two nearby residues occupy markedly different environments relative to the contact points for ACh. They also demonstrate that cysteine-substitution mutagenesis can be successfully applied to protein domains that include functionally important disulphides.

Acetylcholine↗
PubMed1995

Touching limits in the analytic dyad.

The matter of limits and boundary violations by both parties in the analytic dyad remains an unsettled technical and ethical concern, whether touching has to do with actual physical contact or is expanded in its meaning to include its psychic equivalents. Touching, probing, and breaching of the idiosyncratic perimeters of the private self of one by the other in the dyadic intimacy are necessary components of the healing contact but pose an inevitable liability for violation, disruption, and damage. Clinical data remind the analyst of the near-physical impact of words. And the data sometimes speak for the legitimate place of restrained forms of physical contact, as nonverbal necessities of analytic communication, in critical instances in which a viable analytic engagement could not otherwise be sustained.

Adult↗
PubMed1994

Effects of gentamicin, neomycin and tobramycin on renal calcium and magnesium handling in two rat strains.

1. Standard renal clearance techniques were used to compare the acute effects of gentamicin, neomycin and tobramycin on renal calcium and magnesium handling in Sprague-Dawley and Fischer 344 rats. 2. Significant hypercalciuric and hypermagnesiuric responses to all three drugs (P < 0.01) were apparent within 30 min of the onset of drug infusion. 3. The magnitude of the acute hypercalciuric and hypermagnesiuric response to the three aminoglycosides was comparable. This contrasts with their nephrotoxic action where neomycin >> gentamicin > tobramycin. The magnitude of the acute physiological responses to these drugs do not therefore reflect their nephrotoxic potential. 4. Sprague-Dawley rats were at least as responsive as Fischer rats in their acute renal responses to gentamicin. If Fischer rats are more sensitive to aminoglycoside nephrotoxicity than Sprague-Dawley rats, this is not reflected in their acute responses to gentamicin.

Animals↗
PubMed1991

Mutations affecting agonist sensitivity of the nicotinic acetylcholine receptor.

The nicotinic acetylcholine receptor (AChR) is a pentameric transmembrane protein (alpha 2 beta gamma delta) that binds the neurotransmitter acetylcholine (ACh) and transduces this binding into the opening of a cation selective channel. The agonist, competitive antagonist, and snake toxin binding functions of the AChR are associated with the alpha subunit (Kao et al., 1984; Tzartos and Changeux, 1984; Wilson et al., 1985; Kao and Karlin, 1986; Pederson et al., 1986). We used site-directed mutagenesis and expression of AChR in Xenopus oocytes to identify amino acid residues critical for ligand binding and channel activation. Several mutations in the alpha subunit sequence were constructed based on information from sequence homology and from previous biochemical (Barkas et al., 1987; Dennis et al., 1988; Middleton and Cohen, 1990) and spectroscopic (Pearce and Hawrot, 1990; Pearce et al., 1990) studies. We have identified one mutation, Tyr190 to Phe (Y190F), that had a dramatic effect on ligand binding and channel activation. These mutant channels required more than 50-fold higher concentrations of ACh for channel activation than did wild type channels. This functional change is largely accounted for by a comparable shift in the agonist binding affinity, as assessed by the ability of ACh to compete with alpha-bungarotoxin binding. Other mutations at nearby conserved positions of the alpha subunit (H186F, P194S, Y198F) produce less dramatic changes in channel properties. Our results demonstrate that ligand binding and channel gating are separable properties of the receptor protein, and that Tyr190 appears to play a specific role in the receptor site for acetylcholine.

Amino Acid Sequence↗
PubMed1991

Clinical and theoretical aspects of enactment.

Enactment as a concept can serve analytic discourse through its established meaning of an act intended strongly to influence, persuade, or force another to react. We might agree to use the term in two complementary ways: Broadly, enactment can designate all behaviors of both parties in the analytic relationship, even verbal, in consequence of the intensification of the action intent of our words created by the constraints and regressive push induced by the analytic rules and frame. Patient and analyst are vulnerable to falling back on behaviors that actualize their intentions, doing so in ways motivated by and reflecting transference hopes, fears, and compromises shaped in their developmental past. Specifically, enactment can then be defined as those regressive (defensive) interactions between the pair experienced by either as a consequence of the behavior of the other. While nominally an interpersonal perspective, this concept of enactment facilitates more balanced attention to the involvement of both parties and to the intrapsychic dynamics in both that specifically shape their interactions. A clinical vignette illustrates the analyst's contributions to enactment, especially those reflecting his reactivated conflicts and their relation to his theoretical and technical preferences.

Female↗
PubMed1989

Structural characterization of alpha-bungarotoxin-binding proteins from Aplysia californica.

Structural features of alpha-bungarotoxin-binding proteins from the marine mollusc Aplysia californica have been examined as a first step toward delineating their potential role in cholinergic neurotransmission. Protein blotting with 125I-alpha-bungarotoxin was used to identify binding proteins in membranes prepared from Aplysia muscle and nervous tissue. Binding proteins from both tissues exhibited similar physical characteristics, which distinguish them from the prototypical alpha-bungarotoxin-binding protein, the nicotinic acetylcholine receptor obtained from Torpedo californica electric organ membranes. Aplysia binding activities migrate with an apparent molecular weight of 250 kDa on sodium dodecyl sulfate (SDS) gels in the presence of reducing agents. Binding of alpha-bungarotoxin to blots of Aplysia membranes is abolished by exposure of samples to heat or to low pH but is unaffected by reduction-alkylation treatment. In contrast, the alpha-bungarotoxin-binding subunit of the acetylcholine receptor from Torpedo membranes migrates on SDS gels at 40 kDa. It retains binding activity following exposure to heat or to low pH, but binding is substantially diminished by reduction-alkylation treatments. Another distinguishing characteristic of the Aplysia binding activities is revealed by examining recovery of membrane alpha-bungarotoxin-binding on protein blots; the high recovery of Aplysia binding contrasts sharply with the low recovery of Torpedo binding activity. The high apparent molecular weight of the Aplysia alpha-bungarotoxin-binding activities, their most distinguishing feature, is similar to an alpha-bungarotoxin-binding activity recently identified in lower vertebrate brain. Covalent cross-linking with 125I-alpha-bungarotoxin demonstrates, however, that the mobility of both Aplysia binding activities is due to a multimeric protein that is unusually resistant to dissociation in SDS. The covalently radiolabeled Aplysia alpha-bungarotoxin-binding activity migrates at approximately 260 kDa on SDS gels when solubilized at room temperature. When it was boiled before electrophoresis, the mobility of the radiolabeled protein shifts to approximately 70 kDa. Resistance to dissociation in the absence of boiling may explain both the high recovery of activity on blots and the insensitivity to reductive alkylation. Conversely, dissociation of the multimeric complex upon boiling may explain the observed loss of binding activity. Our results demonstrate structural similarities and differences between Aplysia alpha-bungarotoxin-binding proteins and the Torpedo acetylcholine receptor.(ABSTRACT TRUNCATED AT 400 WORDS)

Animals↗
PubMed1988

The analyst's insights.

The patient's fresh perceptions of himself come through mutative work shared with the analyst in the focused intimacy of their interaction. In finding transferential expectancies realized, the patient can experience these as yet different, providing he has the analyst's optimal participation. In this concurrence of crucial differences he can discount and discard the old perceptions that had shaped his psychic reality, and build out of them fresh insights. The analyst's regressive lapses in his best work are a liability inherent in the compromise formations comprising his work ego, built as it is out of the needs and motives of his own transferential past. The stagnation and tensions his regressive transferences contribute to the analytic work produce vivid actualization of the intrapsychic conflicts of both, now intertwined and mutually reinforcing. The analyst's self-analysis at such times can lead to resolving insights about himself that redress the impasse and restore the analytic work both must do.

Adult↗
PubMed1987

The play of transference: some reflections on enactment in the psychoanalytic situation.

The incessant play of nonverbal activity between patient and analyst actualizes and amplifies the primary verbal data of the psychoanalytic dialogue. Both parties must inevitably register this kinesic level of communication, and react with capacities acquired in and elaborated from earliest childhood. The analyst's apperceptive (unfocused) looking, as part of his freely hovering attentiveness, utilizes these capabilities gradually to perceive and organize patterns combining the verbal and nonverbal data. It is through the recognizing and eventual understanding of these gestalts that the analyst builds up his knowledge of his patients. In these patterns can be identified: (a) conspicuous behaviors, idiosyncratic for the individual, which often yield to psychoanalytic inquiry to reveal their dynamic-historical antecedents; and (b) inconspicuous background kinesics, habitual to the individual, which ordinarily are opaque to analytic exploration, yet hold rich meaning. Observing these small behaviors in relation to verbal content provides evidence of their linkage to, and enactment of, pregenital- and genital-level conflicts over diadic and triadic object relations, even in highly structured personalities. These enactments combine elements of play, miming, and drama to constitute an experiential dimension that actualizes and externalizes the patients' inner life of conflict and relation to objects.

Adult↗
PubMed1985

Demonstration and affinity labeling of a stereoselective binding site for a benzomorphan opiate on acetylcholine receptor-rich membranes from Torpedo electroplaque.

The interaction of an optically pure benzomorphan opiate, (-)-N-allyl-N-normetazocine [(-)-ANMC], with the nicotinic acetylcholine receptor from Torpedo electroplaque was studied by using radioligand binding and affinity labeling. The binding was complex with at least two specific components having equilibrium dissociation constants of 0.3 microM and 2 microM. The affinity of the higher affinity component was decreased by carbamoylcholine but not by alpha-bungarotoxin. The effect of carbamoylcholine was not blocked by alpha-bungarotoxin. In comparison, the affinity of [3H]phencyclidine, a well-characterized ligand for a high-affinity site for noncompetitive blockers on the acetylcholine receptor, is increased by carbamoylcholine and the increase is blocked by alpha-bungarotoxin. The binding of (-)-[3H]ANMC was inhibited by a number of other benzomorphans, with (-) isomers being 4- to 5-fold more potent than (+) isomers. Phencyclidine inhibits the binding of (-)-[3H]ANMC to its high-affinity site by a mechanism that is not competitive. UV-catalyzed affinity labeling indicated that the high-affinity-binding site for (-)-[3H]ANMC is at least partially associated with the delta subunit. Tryptic degradation of the Torpedo marmorata delta chain suggested that (-)-ANMC labeled a 16,000-dalton COOH-terminal portion of the subunit. In contrast, 5-azido-[3H]trimethisoquin, a photoaffinity label of the high-affinity site for noncompetitive blockers, labels a 47,000-dalton NH2-terminal fragment of the delta subunit. These results suggest that (-)-[3H]ANMC binds to sites completely distinct from the binding sites for acetylcholine. The high-affinity-binding site for (-)-ANMC and that for phencyclidine and 5-azidotrimethisoquin are allosterically coupled but are regulated differently and are probably physically distinct.

Affinity Labels↗
PubMed1984

On antithetic and metathetic words in the analytic situation.

Freud's view of primal words emphasized their origins in the vocal acquisition of language and their similarity to primary process behavior in young children. Our English vernacular is rich in word play of reversals and antitheses, some words being truly ancient, others originating in the subversions of slang and in the child's penchant for mirror imaging and other primary process play. In the regression of analytic work the uttered word can combine with dream imagery and behavioral enactment to convey the antitheses abounding in the oral, anal, and early phallic concerns of the individuating child acquiring language.

Female↗
PubMed1984

Mechanism of phencyclidine binding to the acetylcholine receptor from Torpedo electroplaque.

The mechanism of phencyclidine binding to Torpedo acetylcholine receptor-rich membranes was investigated. The rate of [3H]phencyclidine association is 10(3)- to 10(4)-fold more rapid when phencyclidine and carbamoylcholine are added simultaneously to acetylcholine receptor-rich membranes than when phencyclidine is added to membranes previously equilibrated with carbamoylcholine or membranes in the absence of carbamoylcholine. The mechanism of binding under conditions in which the slower rate was observed was studied with thermodynamic, viscosity, and kinetic experiments. Association and dissociation rates were highly dependent on temperature with activation energies of 26-30 kcal/mole. Viscosity had no effect on the association rate but increased the dissociation rate. These studies suggest that the binding is not diffusion-controlled but rather is limited by a significant energy barrier. The association rate was determined as a function of the concentration of acetylcholine receptor-rich membranes and the concentration of phencyclidine. In the presence of carbamoylcholine, the association rate was highly dependent upon the concentration of acetylcholine receptor but virtually insensitive to the concentration of phencyclidine. In the absence of carbamoylcholine, the association rate seemed to be a hyperbolic function of both the phencyclidine and the acetylcholine receptor concentration. The minimal model capable of explaining the data is a mechanism by which phencyclidine binds to two conformations of the acetylcholine receptor, one conformation having a higher affinity and constituting a lower percentage of receptors and the other having a lower affinity and constituting a higher percentage. The data are consistent with the possibility that the high-affinity conformation is the open-channel state of the acetylcholine receptor.

Animals↗