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Biomedical subjects

A Cavalli

Publications and source records attributed to A Cavalli.

At least 37 records · Page 2Linked to original sources
PubMed2001

Canine parvovirus (CPV) vaccination: comparison of neutralizing antibody responses in pups after inoculation with CPV2 or CPV2b modified live virus vaccine.

Canine parvovirus type 2 (CPV2) emerged in 1978 as causative agent of a new disease of dogs. New antigenic variants (biotypes), designated CPV2a and CPV2b, became widespread during 1979 to 1980 and 1984, respectively. At the present time the original CPV2 has disappeared in the dog population and has been replaced by the two new viruses. In the present study the comparison of neutralizing antibody titers in two groups of pups (18 pups in each group) inoculated with CPV2 and CPV2b modified live virus vaccines is reported. Using the hemagglutination inhibition (HI) test, relevant differences between antibody titers, against either the homologous or the heterologous virus, were not constantly observed. Using the neutralization (Nt) test, however, the pups inoculated with CPV2 had antibody titers which were approximately 30 times higher to the homologous virus (mean, 4,732) than to the heterologous virus (CPV2b) (mean, 162). The results of these experiments support two conclusions: (i) the HI test may not always accurately evaluate the true immune status of dogs with respect to CPV, and (ii) dogs inoculated with CPV2 vaccine develop relatively low Nt antibody titers against the heterologous virus (CPV2b). These data may suggest an advantage for new vaccines, considering that most presently licensed vaccines are produced with CPV2, which no longer exists in the dog population.

Animals↗
PubMed2001

Expiratory flow limitation and orthopnea in massively obese subjects.

BACKGROUND: Morbidly obese subjects, who often complain about breathlessness when lying down, breathe at low lung volume with a reduced expiratory reserve volume (ERV). Therefore, during tidal breathing the expiratory flow reserve is decreased, promoting expiratory flow limitation (EFL), which is more likely to occur in the supine position, when the relaxation volume of the respiratory system, and hence the functional residual capacity (FRC), decrease because of the gravitational effect of the abdominal contents. PURPOSE: The aim of the study was to assess EFL and orthopnea in massively obese subjects and to evaluate whether orthopnea was associated with the development of supine EFL. METHODS: In 46 healthy obese subjects (18 men) with a mean (+/- SD) age of 44 +/- 11 years and a mean body mass index (BMI) of 51 +/- 9 kg/m(2), we assessed EFL in both the seated and the supine positions by the negative expiratory pressure method and assessed postural changes in FRC by measuring the variations in the inspiratory capacity (IC) with recumbency. Simultaneously, dyspnea was evaluated in either position using the Borg scale dyspnea index (BSDI) to determine the presence of orthopnea, which was defined as any increase of the BSDI in the supine position. RESULTS: Partial EFL was detected in 22% and 59%, respectively, of the overall population in seated and supine position. The mean increase in the supine IC amounted to 120 +/- 200 mL (4.1 +/- 6.4%), indicating a limited decrease in FRC with recumbency in these subjects. Orthopnea, although mild (mean BSDI, 1.7 +/- 1.3), was claimed by 20 subjects, and in 15 of them EFL occurred or worsened in the supine position. Orthopnea was associated with lower values of seated ERV (p < 0.05) and was marginally related to supine EFL values (p = 0.07). No significant effect of age, BMI, obstructive sleep apnea-hypopnea syndrome, FEV(1), and forced expiratory flow at 75% of vital capacity was found on either orthopnea or EFL. CONCLUSION: In morbidly obese subjects, EFL and dyspnea frequently occur with the subject in the supine position, and both supine EFL and low-seated ERV values are related to orthopnea, suggesting that dynamic pulmonary hyperinflation and intrinsic positive end-expiratory pressure may be partly responsible for orthopnea in massively obese subjects.

Adult↗
PubMed2001

Characterization of a canine parvovirus strain isolated from an adult dog.

A CPV-2b strain was detected from an adult vaccinated dog, affected with severe gastroenteritis. The faeces of the dog were positive to canine parvovirus by a hemagglutination assay and gave a CPV-2b-like pattern by a hemagglutination inhibition test using monoclonal antibodies. In vitro-cultivation of the virus was difficult and after a few passages on canine and feline cells, the presence of the virus was detectable only by an immunofluorescence assay on the feline cells, since hemagglutinating activity had disappeared. Characterization of the virus, by an indirect immunofluorescence assay with monoclonal antibodies, confirmed the antigenic CPV-2b-like pattern of the nonhemagglutinating virus.

Animals↗
PubMed2000

The regulator of G protein signaling RGS4 selectively enhances alpha 2A-adreoreceptor stimulation of the GTPase activity of Go1alpha and Gi2alpha.

Agonist-stimulated high affinity GTPase activity of fusion proteins between the alpha(2A)-adrenoreceptor and the alpha subunits of forms of the G proteins G(i1), G(i2), G(i3), and G(o1), modified to render them insensitive to the action of pertussis toxin, was measured following transient expression in COS-7 cells. Addition of a recombinant regulator of G protein signaling protein, RGS4, did not significantly affect basal GTPase activity nor agonist stimulation of the fusion proteins containing Galpha(i1) and Galpha(i3) but markedly enhanced agonist-stimulation of the proteins containing Galpha(i2) and Galpha(o1.) The effect of RGS4 on the alpha(2A)-adrenoreceptor-Galpha(o1) fusion protein was concentration-dependent with EC(50) of 30 +/- 3 nm and the potency of the receptor agonist UK14304 was reduced 3-fold by 100 nm RGS4. Equivalent reconstitution with Asn(88)-Ser RGS4 failed to enhance agonist function on the alpha(2A)-adrenoreceptor-Galpha(o1) or alpha(2A)-adrenoreceptor-Galpha(i2) fusion proteins. Enzyme kinetic analysis of the GTPase activity of the alpha(2A)-adrenoreceptor-Galpha(o1) and alpha(2A)-adrenoreceptor-Galpha(i2) fusion proteins demonstrated that RGS4 both substantially increased GTPase V(max) and significantly increased K(m) of the fusion proteins for GTP. The increase in K(m) for GTP was dependent upon RGS4 amount and is consistent with previously proposed mechanisms of RGS function. Agonist-stimulated GTPase turnover number in the presence of 100 nm RGS4 was substantially higher for alpha(2A)-adrenoreceptor-Galpha(o1) than for alpha(2A)-adrenoreceptor-Galpha(i2). These studies demonstrate that although RGS4 has been described as a generic stimulator of the GTPase activity of G(i)-family G proteins, selectivity of this interaction and quantitative variation in its function can be monitored in the presence of receptor activation of the G proteins.

Adrenergic alpha-2 Receptor Agonists↗
PubMed2000

SAR of 9-amino-1,2,3,4-tetrahydroacridine-based acetylcholinesterase inhibitors: synthesis, enzyme inhibitory activity, QSAR, and structure-based CoMFA of tacrine analogues.

In this study, we attempted to derive a comprehensive SAR picture for the class of acetylcholinesterase (AChE) inhibitors related to tacrine, a drug currently in use for the treatment of the Alzheimer's disease. To this aim, we synthesized and tested a series of 9-amino-1,2,3,4-tetrahydroacridine derivatives substituted in the positions 6 and 7 of the acridine nucleus and bearing selected groups on the 9-amino function. By means of the Hansch approach, QSAR equations were obtained, quantitatively accounting for both the detrimental steric effect of substituents in position 7 and the favorable electron-attracting effect exerted by substituents in positions 6 and 7 of the 9-amino-1,2,3,4-tetrahydroacridine derivatives. The three-dimensional (3D) properties of the inhibitors were taken into consideration by performing a CoMFA analysis on the series of AChE inhibitors made by 12 9-amino-1,2,3, 4-tetrahydroacridines and 13 11H-indeno[1,2-b]quinolin-10-ylamines previously developed in our laboratory. The alignment of the molecules to be submitted to the CoMFA procedure was carried out by taking advantage of docking models calculated for the interactions of both the unsubstituted 9-amino-1,2,3,4-tetrahydroacridine and 11H-indeno[1,2-b]quinolin-10-ylamine with the target enzyme. A highly significant CoMFA model was obtained using the steric field alone, and the features of such a 3D QSAR model were compared with the classical QSAR equations previously calculated. The two models appeared consistent, the main aspects they had in common being (a) the individuation of the strongly negative contribution of the substituents in position 7 of tacrine and (b) a tentative assignment of the hydrophobic character to the favorable effect exerted by the substituents in position 6. Finally, a new previously unreported tacrine derivative designed on the basis of both the classical and the 3D QSAR equations was synthesized and kinetically evaluated, to test the predictive ability of the QSAR models. The 6-bromo-9-amino-1,2,3,4-tetrahydroacridine was predicted to have a pIC(50) value of 7.31 by the classical QSAR model and 7.40 by the CoMFA model, while its experimental IC(50) value was equal to 0.066 (+/-0.009) microM, corresponding to a pIC(50) of 7.18, showing a reasonable agreement between predicted and observed AChE inhibition data.

Chemical Phenomena↗
PubMed2000

Stereoselective binding of 2,3-substituted 3-hydroxypropionic acids on an immobilised human serum albumin chiral stationary phase: stereochemical characterisation and quantitative structure-retention relationship study.

The binding characteristics of a series of 2,3-substituted 3-hydroxypropionic acids, with anti-inflammatory properties, bearing two chiral centres, were studied by HPLC upon HSA (human serum albumin)-based stationary phase. The compounds were analysed in their stereoisomeric erythro and threo forms and the chromatographic conditions for enantioseparation of the erythro and threo forms were studied on human serum albumin stationary phase. The enantiomer elution order was determined by injection of the enriched samples or by carrying out the CD spectra of each enantiomeric fraction. The absolute configuration of the single enantiomers was assigned on the basis of their CD spectra. A QSRR study was performed by subjecting the chromatographic data of the compounds to multiparameter regression analysis against various molecular descriptors to have insight into the chiral recognition mechanism. The lipophilicity appeared to be the most important parameter in determining the affinity to the protein, the compounds' capacity factors being linearly correlated to the experimental RP-HPLC partition coefficients (log k'w). The enantioselectivity factors (alpha) related to the enantiomers of the erythro and threo forms were studied taking into consideration both the physico-chemical parameters and the conformational behaviour of the compounds.

Chromatography, High Pressure Liquid↗
PubMed2000

Linking CoMFA and protein homology models of enzyme-inhibitor interactions: an application to non-steroidal aromatase inhibitors.

An approach to compare quantitatively a ligand-based (CoMFA) model and an enzyme active site model was investigated. The active site of the cytochrome P450 human aromatase was constructed by homology modeling techniques and two structurally different non-steroidal aromatase inhibitors were docked into it. A CoMFA model was then developed on a related series of non-steroidal inhibitors by correlating their inhibitory activity (expressed as -log IC50 values) versus only 11 steric descriptors (i.e. Csp3-ligand steric interaction energies). The resulting 3D-QSAR coefficients (11) and the steric field values of the aromatase active site calculated at the same points of the CoMFA lattice (i.e. eleven Csp3-protein steric interaction energies) were pair-wise compared. Specifically, when a positive coefficient was associated with a negative or low ( < 5 kcal/mol) value of the protein steric field or, alternatively, a negative coefficient was associated with a large positive value of the protein steric field we recorded as many matches. When a 3D-QSAR coefficient did not correspond to the protein steric potential in the sense described above we considered that point as a mis-matching point. In our view, in spite of several limitations, such a comparison represents a valuable criterion to evaluate quantitatively how convergent are the results from a 3D-QSAR CoMFA model and a homology-built protein 3D structure.

Amino Acid Sequence↗
PubMed2000

Acetylcholinesterase inhibitors for potential use in Alzheimer's disease: molecular modeling, synthesis and kinetic evaluation of 11H-indeno-[1,2-b]-quinolin-10-ylamine derivatives.

Continuing our work on tetracyclic tacrine analogues, we synthesized a series of acetylcholinesterase (AChE) inhibitors of 11H-indeno-[1,2-b]-quinolin-10-ylaminic structure. Selected substituents were placed in synthetically accessible positions of the tetracyclic nucleus, in order to explore the structure-activity relationships (SAR) and the mode of action of this class of anticholinesterases. A molecular modeling investigation of the binding interaction of the lead compound (1a) with the AChE active site was performed, from which it resulted that, despite the rather wide and rigid structure of 1a, there may still be the possibility to introduce some small substituent in some positions of the tetracycle. However, from the examination of the experimental IC50 values, it derived that the indenoquinoline nucleus probably represents the maximum allowable molecular size for rigid compounds binding to AChE. In fact, only a fluorine atom in position 2 maintains the AChE inhibitory potency of the parent compound, and, actually, increases the AChE-selectivity with respect to the butyrylcholinesterase inhibition. By studying the kinetics of AChE inhibition for two representative compounds of the series, it resulted that the lead compound (1a) shows an inhibition of mixed type, binding to both the active and the peripheral sites, while the more sterically hindered analogue 2n seems to interact only at the external binding site of the enzyme. This finding seems particularly important in the context of Alzheimer's disease research in the light of recent observations showing that peripheral AChE inhibitors might decrease the aggregating effects of the enzyme on the beta-amyloid peptide (betaA).

Acetylcholinesterase↗
PubMed2000

[4-[[N-(3-chlorophenyl)carbamoyl]oxy]-2-butynyl]-trimethylammonium (McN-A-343)-related compounds. Effect of the butynyl chain inclusion into an aromatic unit on the potency for muscarinic receptors.

A series of derivatives of the known M1 selective muscarinic receptor agonist McN-A-343 (1) was designed with the aim of investigating the effects of structural variations on both the butynyl chain and the phenyl ring of 1. The butynyl chain was replaced with an aromatic spacer, and the effects of such a modification on the stereoelectronic properties of the molecules were theoretically studied and considered compatible with muscarinic receptor affinity. Substituents on the phenyl ring of 1 were selected so as to vary their electronic and hydrophobic properties. This design strategy did not produce muscarinic M1 receptor agonists more potent than the prototype 1, even if some analogues displayed functional selectivity for different muscarinic receptor subtypes. Compounds 3 and 7 were selective agonists towards muscarinic M3 receptors, while compounds 14, 16 and 18 were selective muscarinic M2 receptor agonists. The most interesting derivative was 8, a full agonist at muscarinic M3 receptors devoid of activity at both muscarinic M1 and M2 subtypes. The pharmacological profile of the series was further characterized by studying the anticholinesterase and miotic activities of some representative compounds. Compounds 3-8 turned out to be weak acetylcholinesterase inhibitors, while derivatives 4, 6, 8 and 11 were able to significantly reduce the pupillary diameter in rabbit, indicating 8 as an effective miotic agent.

(4-(m-Chlorophenylcarbamoyloxy)-2-butynyl)trimethy↗
PubMed2000

Effects of a photic input on the human cortico-motoneuron connection.

OBJECTIVES: Disease manifestations such as photic cortical reflex myoclonus or myoclonus due to intermittent light stimulation rely on a pathologic interaction between non-structured visual inputs and the corticospinal system. We wanted to assess the normal interaction, if any, between a prior photic input and the output of the cortico-motoneuron connection. METHODS: In 9 consenting healthy subjects we quantified the changes exerted by a sudden, unexpected bright light flash on (i) the motor potentials (MEPs) evoked in the right first dorsal interosseous muscle (FDI) by transcranial magnetic or electrical stimulation (TMS/TES) of the primary motor cortex, (ii) the FDI F-waves and (iii) the soleus H-wave. Separately, we measured the simple reaction times to the flash itself. All determinations were repeated twice with an interval of 2-24 months. RESULTS: When the flash preceded TMS by 55-70 ms, the MEP size was reduced, while at interstimulus intervals (ISIs) of 90-130 ms it was enlarged. Statistical significance (P<0.05) emerged at ISIs of 55, 70, 100, 105 and 120 ms. Conversely, the MEP latency was prolonged at ISIs of 55-70 ms and shortened at ISIs of 90-130 ms (P<0.05 at ISIs of 55, 110 and 130 ms). Electrical MEPs were enhanced at an ISI of 120 ms. The F-wave size showed a non-significant trend of enhancement at ISIs of 90-130 ms. The soleus H-wave showed significant enlargement at ISIs of 90-130 ms (P<0.05 at ISIs of 100 and 105 ms). The minimum reaction time was on average 120 ms. CONCLUSIONS: An unexpected photic input, to which no reaction is planned, can cause an early inhibition of the responses to TMS. We think its origin lies within the primary motor cortex, since it is not associated with changes in spinal excitability or electrical MEPs. A later facilitation persists using TES and has a temporal relationship with an enlargement of the soleus H-wave. Thus, it likely results from activation of descending (possibly reticulospinal) fibers that excite the spinal motor nucleus.

Adult↗
PubMed2000

Hemispheric asymmetries of cortico-cortical connections in human hand motor areas.

OBJECTIVE: To evaluate possible functional asymmetries of the motor cortex on the hand-dominant versus the non-dominant hemisphere. METHODS: We assessed the handedness of 15 consenting volunteers using the Edinburgh Inventory. They were divided in two groups: 9 right-handers and 6 left-handers. We used single- and paired-transcranial magnetic stimulation (TMS) to measure the relaxed and active motor threshold and the ipsilateral cortico-cortical inhibition and facilitation curve for both hand motor areas. We looked for hemispheric asymmetries of variables related to the side of stimulation (dominant versus non-dominant) and to handedness. RESULTS: We found no significant intra- or intergroup hemispheric asymmetry for the relaxed and active thresholds. Among the right-handers, the cortico-cortical inhibition and facilitation curve showed an increased amount of facilitation in the dominant as compared with the non-dominant hand area. No such changes were seen among the left-handers. Both the dominant and the non-dominant hand areas of the right-handers showed more inhibition and less facilitation on the cortico-cortical inhibition and facilitation curve than the corresponding areas of left-handers. CONCLUSION: In the right-handers, paired TMS studies showed a functional asymmetry of the motor cortex between the dominant and the non-dominant hand. The left-handers did not show this lateralization. Under TMS investigation their motor cortex function appeared different from that of right-handers.

Adult↗
PubMed2000

Immunization of pups with maternally derived antibodies to canine parvovirus (CPV) using a modified-live variant (CPV-2b).

The results of vaccination trials carried out on pups with maternally derived antibodies (MDA) to canine parvovirus (CPV), using a modified-live CPV-2b variant vaccine (29-97/40 strain), are reported. The vaccine was able to overcome the obstacle of MDA, and to elicit protective immunity in 100% of the pups whose antibody titres were 1:10-1:40, 83% of the pups with titres of 1:80, 57% of the pups with titres of 1:160, and even in 60% of the pups with antibody titres of 1:320.

Animals↗
PubMed2000

Cortical excitability in cryptogenic localization-related epilepsy: interictal transcranial magnetic stimulation studies.

PURPOSE: To assess whether single-and paired-pulse transcranial magnetic stimulation (TMS) can measure the interictal brain excitability of medicated patients with cryptogenic localization related epilepsy (CLE). Changes in the balance between excitation and inhibition are the core phenomena in focal epileptogenesis. TMS can assess this balance in the primary motor cortex. METHODS: We selected 18 patients with CLE and similar clinical features in whom we located the epileptogenic area reliably, with 11 age-and sex-matched healthy controls. For both motor cortices, we determined the threshold to TMS, the duration of the cortical silent period, and the corticocortical inhibition and facilitation curve. RESULTS: TMS was safe. The more antiepileptic drugs (AEDs) taken by the patients, the higher their threshold to TMS. The silent period duration failed to show significant changes. On paired TMS, a cluster analysis identified a homogeneous subgroup of patients (n = 7) who showed a significantly defective corticocortical inhibition and excess facilitation. With respect to the epileptogenic area, the phenomenon was bilateral in four of these patients, ipsilateral in two, and contralateral in one. The phenomenon was independent of AEDs and many other clinical variables. However, this patient group had a higher seizure frequency and a higher proportion of electroencephalograms (EEGs) showing interictal generalized epileptic discharges than the rest of the patients. CONCLUSION: Paired TMS provided a valuable pathophysiologic insight into the interictal excitatory state of the cortex in CLE. This method can potentially supply useful prognostic clinical information.

Adolescent↗
PubMed2000

Antigenic analysis of canine parvovirus strains isolated in Italy.

Eighty-two canine parvovirus type 2 strains isolated in Italy from pups with severe enteritis were characterizated using four monoclonal antibodies. Sixty-eight isolates resulted CPV-2a, whereas the other fourteen were a CPV-2b variant. The diffusion of CPV-2 variants in the Italian dog population is quite similar to that reported in the United Kingdom and Australia (CPV-2a more prevalent) and different from the epidemiological conditions of the USA and other countries where CPV-2b is more widespread.

Animals↗
PubMed2000

Imidazo[2,1 -b]thiazolylmethylene- and indolylmethylene-2-indolinones: a new class of cyclin-dependent kinase inhibitors. Design, synthesis, and CDK1/cyclin B inhibition.

Compounds containing a 2-indolinone moiety linked to imidazothiazole and indole fragments were studied as cyclin-dependent kinase inhibitors. The activity of all the new derivatives was tested in vitro against CDK1/cyclinB and the selectivity towards two other kinases was determined for the most promising compounds. The binding mode of one representative compound was investigated by means of a three-dimensional model of the inhibitor-CDK1 complex. The work allowed us to identify (2-chloroindolyl)methylene-2-indolinone as a new lead of a class of CDK1/cyclinB inhibitors, whose potency can be improved by the introduction of suitable variations on the basic molecular skeleton.

Binding Sites↗
PubMed1999

WB 4101-related compounds. 2. Role of the ethylene chain separating amine and phenoxy units on the affinity for alpha(1)-adrenoreceptor subtypes and 5-HT(1A) receptors.

WB 4101 (1)-related benzodioxanes were synthesized by replacing the ethylene chain separating the amine and the phenoxy units of 1 with a cyclopentanol moiety, a feature of 6, 7-dihydro-5-[[(cis-2-hydroxy-trans-3-phenoxycyclopentyl)amino]meth yl] -2-methylbenzo[b]thiophen-4(5H)-one that was reported to display an intriguing selectivity profile at alpha(1)-adrenoreceptors. This synthesis strategy led to 4 out of 16 possible stereoisomers, which were isolated in the case of (-)-3, (+)-3, (-)-4, and (+)-4 and whose absolute configuration was assigned using a chiral building block for the synthesis of (-)-3 starting from (+)-(2R)-2, 3-dihydro-1,4-benzodioxine-2-carboxylic acid ((+)-9) and (1S,2S, 5S)-2-amino-5-phenoxycyclopentan-1-ol ((+)-10). The aim of this project was to further investigate whether it is possible to differentiate between these compounds with respect to their affinity for alpha(1)-adrenoreceptor subtypes and the affinity for 5-HT(1A) receptors, as 1 binds with high affinity at both receptor systems. The biological profiles of reported compounds at alpha(1)-adrenoreceptor subtypes were assessed by functional experiments in isolated rat vas deferens (alpha(1A)), spleen (alpha(1B)), and aorta (alpha(1D)) and by binding assays in CHO and HeLa cells membranes expressing the human cloned alpha(1)-adrenoreceptor subtypes and 5-HT(1A) receptors, respectively. Furthermore, the functional activity of (-)-3, (+)-3, (-)-4, and (+)-4 toward 5-HT(1A) receptors was evaluated by determining the induced stimulation of [(35)S]GTPgammaS binding in cell membranes from HeLa cells transfected with human cloned 5-HT(1A) receptors. The configuration of the cyclopentane unit determined the affinity profile: a 1R configuration, as in (+)-3 and (-)-4, conferred higher affinity at alpha(1)-adrenoreceptors, whereas a 1S configuration, as in (-)-3 and (+)-4, produced higher affinity for 5-HT(1A) receptors. For the enantiomers (+)-4 and (-)-4 also a remarkable selectivity was achieved. Functionally, the stereoisomers displayed a similar alpha(1)-selectivity profile, that is alpha(1D) > alpha(1B) > alpha(1A), which is different from that exhibited by the reference compound 1. The epimers (-)-3 and (+)-4 proved to be agonists at the 5-HT(1A) receptors, with a potency comparable to that of 5-hydroxytryptamine.

Adrenergic alpha-Agonists↗
PubMed1999

Altered adenylyl cyclase responsiveness subsequent to point mutations of Asp 128 in the third transmembrane domain of the delta-opioid receptor.

delta-Opioid receptors belong to the superfamily of G protein-coupled receptors, characterized by seven putative transmembrane domains, and have been shown to interact with a host of effector systems. It has been suggested that the charge on the conserved aspartic acid residue at position 128 in transmembrane domain 3 of the delta-opioid receptor contributes to both the conformation of the receptor binding pocket and the molecular rearrangements which accompany the establishment of high-affinity states of the receptor. In light of this, we used site-directed mutagenesis to determine whether this residue participates in the transmission of signals to adenylyl cyclase, the effector with which opioid receptors have been classically associated. Substitution of this aspartic acid (D128) for the neutral amino acid alanine, or the protonated amino acids lysine and histidine, constitutively couples the receptor to adenylyl cyclase, as evidenced by a curtailed response to forskolin stimulation in transfected cells. In addition, this constitutive activity can be blocked by pretreatment of the transfected cells with pertussis toxin. Interestingly, naloxone blocks this effect in cells expressing the D128A mutant, but acts as an agonist at the D128K mutant. Our findings support the hypothesis that the interaction between agonist and receptor promotes conformational changes that may be mimicked, at least in part, by mutation of the aspartate residue at position 128. Furthermore, these changes appear to be involved not only in receptor activation, but also in the functional discrimination between agonists and antagonists.

Adenylate Cyclase Toxin↗