The magnitude of the allosteric conformational transition of aspartate transcarbamylase is altered by mutations

Global conformational transitions are of central functional importance for many enzymes and binding proteins. It is not known, however, how much variability can exist in such structural-functional linkages. We have characterized the global magnitude of the T to R conformational transition of Escherichia coli aspartate transcarbamylase (ATCase) by measuring (1) hydration changes using osmotic stress and (2) hydrodynamic changes using high-precision analytical gel chromatography. We find that specific mutations can alter the structural magnitude of the enzyme's conformational transition without abolishing allostery, suggesting that some degree of plasticity exists in the conformational component of allostery.     

Temperature perturbation of the allosteric equilibrium in trout hemoglobin

The carbon monoxide binding kinetics of the isolated trout Hb I has been investigated by flash photolysis at various temperatures, from approximately 20 degrees to 72 degrees C. The time course of recombination has been quantitatively analyzed with a simple two-state allosteric model, making use of the thermodynamic data previously obtained. These new experiments and their analysis show that a simple two-state kinetic model is adequate, in the case of trout Hb I, to describe quantitatively the time course of CO binding at all temperatures. Moreover, we show that temperature can be used to perturb the quaternary conformational equilibrium, the high affinity state of the molecule (R) being progressively more populated at higher temperatures.     

Threonine 82 in the regulatory chain is important for nucleotide affinity and for the allosteric stabilization of Escherichia coli aspartate transcarbamoylase

Changes in the kinetic properties of voltage-activated sodium currents (I(Na)) were studied in rat retinal ganglion cells during in vivo differentiation. Whole-cell recordings from cells maintained as retinal slices or whole-mounts were examined using the patch-clamp technique in the perforated patch mode. Voltage-clamp recordings revealed significant ontogenetic modifications in key properties of I(Na) and the present study described for the first time the detailed time course of such alterations. I(Na) was first expressed on embryonic day 17/18 (E17/18). Current density increased during development from an average of -81 pA/pF on E17/18 to a maximum of -747pA/pF on postnatal day 10/12 (P10/12). Simultaneously, the activation of I(Na) shifted towards more negative potentials, reflected by a shift in the potential of half-activation from -14.1 mV on E17/18 to - 37.5 mV on P10/12. No significant changes in these parameters were observed after P10/12. Steady-state inactivation shifted first towards more positive potentials, reflected by a shift in the potential of half-inactivation from -51 mV on E17/18 to -38 mV on P3/5, but shifted back towards more negative values thereafter (-44 mV in the adult). The most striking feature of I(Na) in rat RGCs was a transient slowing of I(Na) kinetics that was never described before. Time to peak and decay time constants increased between E20 and P5, resulting in slow and broad sodium currents within a developmental period that is characterized by intensive synaptogenesis in the target structures of retinal ganglion cells and maximum retinal ganglion cell death. Thereafter, time to peak and decay time constants decreased again to values found before E20, resulting in rapid sodium spikes. In conclusion, sodium currents in rat retinal ganglion cells displayed substantial electrophysiological changes during pre- and postnatal development. These changes in the sodium system had different temporal time patterns, indicating that they may play specific roles during the development of the visual system.     

Positive effects of allosteric modulators on the binding properties and the function of muscarinic acetylcholine receptors

Data are reviewed indicating that allosteric modulators can enhance the affinities of muscarinic receptors for their antagonists and agonists, that the enhancement of the affinity for agonists is relevant functionally, and that the allosterically induced conformational change also affects the interaction between the receptors and the G proteins.     

The effects of local hyperthermia on the catabolism of a radioiodinated chimeric monoclonal antibody

Local hyperthermia has been shown to increase the tumor uptake and tumor:normal tissue ratios of radiolabeled monoclonal antibodies (mAbs) in athymic mouse xenograft models. The current study was undertaken to determine whether this behavior was related in part to alterations in mAb catabolism by local hyperthermia. Human/mouse chimeric 81C6 mAb reactive with tenascin and a nonspecific control mAb were labeled with 125I using Iodo-Gen and given to separate groups of athymic mice bearing s.c. D-54 MG human glioma xenografts. Half of the animals were then subjected to 4-h tumor-localized hyperthermia at 41.8 degrees C, a protocol previously shown to enhance the specific tumor uptake of the mAb in this xenograft model. The tumor, serum, liver, kidney, and urine were collected from heated as well as control animals 4 and 24 h after injection of the mAb and analyzed by SDS-PAGE and trichloroacetic acid precipitation. At 24 h, a significantly higher percentage of 81C6 was present as intact mAb in the tumors harvested from heated animals compared with those from controls. Unexpectedly, intact mAb was found in the urine of mice immediately after hyperthermia, but not in unheated control animals. We conclude that local hyperthermia decreases the catabolism of the mAb in the tumor and increases the urinary excretion of the mAb through a transient increase in glomerular permeability.     

Direct effects of metabotropic glutamate receptor compounds on native and recombinant N-methyl-D-aspartate receptors

The actions of glutamate in the central nervous system are mediated through interaction with fast activating ionotropic receptors and G protein-coupled metabotropic glutamate receptors (mGluRs). Studies of these receptors have relied on the availability of agonists and antagonists selective for each receptor class. Compounds that were thought to be selective for mGluRs have been extensively used to study the role of these receptors in the brain. Their use has implicated mGluRs in a wide range of physiological and pathological processes including the modulation of N-methyl-D-aspartate (NMDA) receptors and NMDA receptor-dependent processes. We report that some of the most commonly used mGluR compounds act as antagonists on NMDA receptors at concentrations commonly used to activate or block mGluRs. In addition, several of the drugs also act as agonists at higher concentrations due at least in part to high levels of contaminant amino acids. Our results indicate that caution should be used when using these drugs to study the roles of mGluRs in various NMDA-dependent processes. The antagonist effects were dependent on the concentration of the NMDA receptor coagonists, preventing reappraisal of previously published work.     

FGF signal transduction in PC12 cells: comparison of the responses induced by endogenous and chimeric receptors

Loss of articular cartilage, which is the most important pathological lesion occurring in osteoarthritis, has been shown to be enzymatically mediated. The matrix metalloproteinases (MMPs) are a group of enzymes which have been implicated in this degradation of articular cartilage matrix. The use of pharmacological agents to inhibit this catabolic process in the joint is a potential route for therapeutic intervention. The gelatinase MMPs, MMPs-2 and 9, were purified by affinity chromatography from equine cell cultures. The ability of phenylbutazone, flunixin, betamethasone, dexamethasone, methylprednisolone acetate (MPA), hyaluronan, pentosan polysulphate and polysulphated glycosaminoglycan (PSGAG) to inhibit equine MMPs-2 and 9 were assessed by two degradation assays. Whilst some agents did have direct effects on MMP activity, these effects were only obtained at concentrations which were unlikely to be achieved for any length of time in vivo. It is improbable that any pharmacological agent, currently used in the horse, has a significant effect on gelatinase MMP activity.     

Varied effects of polyadenylic-polyuridylic acid on immune responses

The effect of the double-stranded synthetic polynucleotide, poly(A).poly(U), on the immune response of inbred mouse strains to multichain synthetic polypeptides was studied. Poly(A).poly(U) did not affect immune responses controlled by H-2 linked genes. Thus, when either (T,G)-A- -L or (Phe,G)-A--L were injected into high or low responder mice followed by administration of poly(A).poly(U) 24 h after immunization, no increase in the antibody titers was observed. In contrast, poly(A).poly(U) increased significantly the response to polyproline, which is controlled by a non H-2 linked gene, in low responder mice. However, the polyribonucleotide had no effect on the antibody titers of the SJL mice, the high responders to multichain polyproline. When poly(A).poly(U) was injected into DBA/1 mice following immunization with (Phe,G)-Pro- -L, the polynucleotide enhanced the low response to the Pro- -l region at the expense of the anti (Phe,G) response which is normally high in this mouse strain. In this case poly(A).poly(U) caused an intramolecular antigenic competition. The general conclusion of this study is that the chemical nature of the antigenic determinant plays an important role in determining the type of influence exerted by poly(A).poly(U).     

The effects of social context and defensiveness on the physiological responses of repressive copers

In previous research (T.L. Newton & R.J. Contrada, 1992), social context was found to moderate exaggerated physiological reactivity among individuals identified as using a repressive coping style. In this experiment, 119 undergraduates were classified into low-anxious, high-anxious, repressor, and defensive high-anxious coping categories. All participants completed a stressful speech task under either a public or private social context condition. The experimental social context was related to physiological reactivity and self-reported affect but did not moderate reactivity among repressive copers. Additionally, reactivity among repressive copers was not attributable to high defensiveness alone. Consistent with a theory of emotional inhibition, nonspecific skin conductance responses, but not heart rate, discriminated between repressors and nonrepressors.     

The effects of caffeine withdrawal on cardiovascular and gastrointestinal responses.

The withdrawal of caffeine from 18 psychiatric inpatients with hypertension, tachycardia, or gastrointestinal complaints (GTCs) over an 8-wk period resulted in decreased blood pressure, heart rate, and GTCs and increased headache complaints. (PsycINFO Database Record (c) 2010 APA, all rights reserved)     

Molecular-weight-dependent effects of nonanticoagulant heparins on allergic airway responses

We have hypothesized that antiallergic activity of inhaled heparin is molecular weight dependent and mediated by "nonanticoagulant fractions" (NAF-heparin). Therefore, we studied comparative effects of high-, medium-, and ultralow-molecular-weight (HMW, MMW, and ULMW, respectively) NAF-heparins on acute bronchoconstrictor response (ABR) and airway hyperresponsiveness (AHR) in allergic sheep. Specific lung resistance was measured in 23 allergic sheep, before and immediately after challenge with Ascaris suum antigen, without and after pretreatment with inhaled NAF-heparins. Airway responsiveness was estimated before and 2 h postantigen as the cumulative provocating dose of carbachol in breath units, which increased specific lung resistance by 400%. NAF-heparins attenuated ABR and AHR in a molecular-weight-dependent fashion. HMW NAF-heparin (n = 8) was the least effective agent: it attenuated ABR [inhibitory dose causing 50% protection (ID50) = 4 mg/kg] but had no effect on AHR. MMW NAF-heparin (n = 8) showed intermediate efficacy (ABR ID50 = 0.8 mg/kg, AHR ID50 = 1.4 mg/kg), whereas ULMW NAF-heparin (n = 7) was the most effective agent (ABR ID50 = 0.4 mg/kg, AHR ID50 = 0.2 mg/kg). ULMW NAF-heparin was 3.5 times more potent in attenuating antigen-induced AHR when administered "after" antigen challenge and failed to inhibit the bronchoconstrictor response to carbachol and histamine. In 15 additional sheep, segmental antigen challenge caused a marked increase in histamine in bronchoalveolar lavage fluid that was not prevented by any of the inhaled NAF-heparins. These data indicate that antiallergic activity of inhaled heparin is independent of its anticoagulant action and resides in the <2,500 ULMW chains. The antiallergic activity of NAF-heparins is mediated by an unknown biological action and may have therapeutic potential.     

Retrograde effects of electroconvulsive shock on learned responses.

Indicates that electroconvulsive shock (ECS) has 3 different effects on responses learned prior to its administration. 1st, the aversive effect of ECS, demonstrable in 1 trial but usually found after repeated presentations, can produce a delay-of-punishment gradient. 2nd, ECS apparently differentially affects memory depending upon the learning-ECS interval, an effect which can be interpreted as disruption of memory consolidation. The postlearning interval during which ECS produces temporally graded memory effects is very brief, probably well under 1 min. The 3rd effect of ECS is the halting of the incubation, i.e., the development over time, of a punishment-produced conditioned emotional response. In the passive avoidance learning situation, incubation disruption and memory disruption have identical behavioral consequences in that in both cases Ss tend to repeat the previously punished response. The postpunishment time course of incubation is relatively long, with maximum response suppression occurring between 1 and 4 hr. after punishment. The ECS disruption of the long-term incubation process has frequently been misinterpreted as ECS disruption of a long-term memory consolidation process. (60 ref.) (PsycINFO Database Record (c) 2010 APA, all rights reserved)     

The effects of learning on the ventilatory responses to inspiratory threshold loading

Progressive threshold loading (PTL) is frequently used to assess inspiratory muscle endurance in health and disease. We and others have noted a systematic increase in endurance with the first few exposures to the task in subjects previously na?ve to PTL, which may not be related to conditioning of the muscles themselves. The purpose of this study was to investigate the mechanisms responsible for this increased endurance by examining the ventilatory responses to 3 PTL tests, each > 24 h apart, in 18 healthy subjects. During PTL, threshold pressure (Pth) was increased by approximately 10% every 2 min until task failure. Subjects were allowed to adopt any breathing pattern. Respiratory muscle strength (maximal inspiratory pressure [PImax]) was unchanged over successive tests while maximal Pth (Pthmax) during PTL increased (69 +/- 17, 77 +/- 16, and 86 +/- 11% of PImax, respectively, p < 0.05) (mean +/- SD), indicating that the increased Pthmax could not be attributed to improved respiratory muscle strength. Breathing pattern changed with successive tests, so that for comparative loads inspiratory time (TI), respiratory frequency (f ), and duty cycle (TI/Ttot) decreased. This change in breathing pattern did not alter respiratory muscle efficiency (respiratory muscle V O2/work), which was similar in each test (2.4 +/- 2.2%), but perceived effort (Borg Score), which was maximal at task failure in each test, decreased at comparative loads with successive tests. Thus, Pthmax during initial tests appeared to be limited by discomfort rather than respiratory muscle function. These findings suggest that the increased Pthmax with successive tests is a consequence of differences in the breathing pattern adopted, reflecting neuropsychological rather than respiratory muscle conditioning. Measurements from PTL should only be used to assess respiratory muscle performance after allowing time for learning.     

Temperature effects on malonyl-CoA inhibition of carnitine palmitoyltransferase I

The calmodulin-stimulated (Ca2+, Mg2+)-ATPase (calmodulin-ATPase) of the erythrocyte membrane is susceptible to oxidative stress induced by heme and non-heme iron. There is a time-and concentration-dependent inhibition of the calmodulin-ATPase activity when the erythrocyte membranes are treated with either iron or hemin. In the present study, the calmodulin-ATPase has been used as a model system to evaluate the protective effects of a vitamin E analog (U83836E) and two 21-aminosteroids (U74500A and U74389G) against calmodulin-ATPase inhibition induced by iron and hemin. The drugs, lazaroids from Upjohn, can significantly protect the enzyme against iron-induced inhibition and also causes a decrease in the formation of thiobarbituric acid reactive species, with an IC50 of 0.4 microM for the drug U83836E and 4 microM for the drug U74500A. The 21-aminosteroid U74389G does not restore iron-inhibited calmodulin-ATPase activity under similar conditions. At higher concentrations (> 100 microM) all three drugs inhibit the calmodulin-ATPase activity. None of the drugs tested can restore hemin-inhibited calmodulin-ATPase activity.     

Preresponse cues reduce the impairing effects of alcohol on the execution and suppression of responses.

The present study examined the effects of alcohol on the ability to execute and inhibit behavior in a context in which preliminary information signaled the likelihood that a response should be executed or suppressed. Social drinkers (N =12) performed a cued go/no-go task that required quick responses to go targets and suppression of responses to no-go targets. Performance was tested under 3 doses of alcohol: 0.65 g/kg, 0.45 g/kg, and 0.0 g/kg (placebo). Alcohol had no effect on inhibition and execution when cues correctly signaled these actions. By contrast, alcohol impaired inhibition and execution in a dose-dependent manner when cues incorrectly signaled actions. These findings are consistent with a resource limitation account of alcohol impairment. (PsycINFO Database Record (c) 2010 APA, all rights reserved)