Biomedical subjects
R M Graham
Publications and source records attributed to R M Graham.
Chimeras of alpha1-adrenergic receptor subtypes identify critical residues that modulate active state isomerization.
We have identified previously two amino acids, one in each of the fifth and sixth transmembrane segments of both the alpha1a-adrenergic receptor and the alpha1b-adrenergic receptor (AR), that account almost entirely for the selectivity of agonist binding by these receptor subtypes (Hwa, J., Graham, R. M., and Perez, D. M. (1995) J. Biol. Chem. 270, 23189-23195). Thus reversal of these two residues, from those found in the native receptor of one subtype to those in the other subtype, produces complementary changes in subtype selectivity of agonist binding. Here we show that mutating only one of these residues in either the alpha1b-AR or the alpha1a-AR to the corresponding residue in the other subtype (Ala204 --> Val for the alpha1b; Met292 --> Leu for the alpha1a-AR) results in chimeras that are constitutively active for signaling by both the phospholipase C and phospholipase A2 pathways. This is evident by an increased affinity for agonists, increased basal phospholipase C and phospholipase A2 activation, and increased agonist potency. Although mutation of the other residue involved in agonist binding selectivity, to the corresponding residue in the other subtype (Leu314 --> Met for the alpha1b-AR; Val185 --> Ala for the alpha1a-AR) does not alter receptor binding or signaling, per se, when combined with the corresponding constitutively activating mutations, the resulting chimeras, Ala204 --> Val/Leu314 --> Met ( alpha1b-AR) and Val185 --> Ala/Met292 --> Leu ( alpha1a-AR), display wild type ligand binding and signaling. A simple interpretation of these results is that the alpha1a- and alpha1b-ARs possess residues that critically modulate isomerization from the basal state, R, to the active state R*, and that the native receptor structures have evolved to select residues that repress active state isomerization. It is likely that the residues identified here modulate important interhelical interactions between the fifth and sixth transmembrane segments that inhibit or promote receptor signaling.
In vivo wear of Ti6A14V femoral heads: a retrieval study.
The surface characteristics of sixteen "monobloc" titanium-6% aluminum-4% vanadium (Ti6A14V) femoral components (two of the 6-Ti-28 type and 14 of the 6-Ti-32 type) retrieved after periods of 78-131 months following loosening of the femoral component, as well as two unimplanted controls, were studied. The femoral heads were examined by a combination of noncontact light profilometry, scanning electron microscopy, and energy-dispersive X-ray analysis. No consistent correlations were found between classical surface roughness parameters (average, root mean square, peak-to-valley roughness, and radius of curvature) and any clinical parameter studied (patient gender, weight, and height; primary diagnosis; implantation time; or calculated force applied on the hip joint). This extensive quantitative topographic analysis suggests that wear mechanisms in vivo are complex and that wear of titanium alloy femoral heads is partly attributed to a combination of an imperfect nature of the surface before implantation, removal of the oxide layer causing abrasion of the alloy, subsequent deformation of the bearing surface including polishing, and, to a very small degree, patient parameters.
alpha 2-Adrenoceptors take centre stage at Nashville meeting.
Explore the source record for details and available documents.
alpha 1-adrenergic receptor subtypes. Molecular structure, function, and signaling.
Explore the source record for details and available documents.
Gitelman's syndrome (Bartter's variant) maps to the thiazide-sensitive cotransporter gene locus on chromosome 16q13 in a large kindred.
A defect in distal renal tubular sodium chloride handling is thought to be responsible for the clinical phenotype of Gitelman's syndrome, a variant of Bartter's syndrome. To study the possible involvement of the renal thiazide-sensitive NaCl cotransporter gene in the syndrome, a linkage analysis study in the largest reported kindred with the syndrome was performed. A human homolog of rat thiazide-sensitive cotransporter was cloned and mapped to chromosome 16q13 by fluorescent in situ hybridization. All 17 family members in two generations were genotyped at loci in this region. There were no recombinants observed between the Gitelman's syndrome phenotype and inheritance of D16S408 alleles, yielding a lod score of 3.88 at Q = 0. By contrast, recombinants were observed between Gitelman's syndrome and the flanking markers D16S419 and D16S400, localizing the responsible gene in this family to a 15 centimorgan region on chromosome 16q. These genetic data, together with current understanding of the molecular physiology of the thiazide-sensitive cotransporter, are strong evidence that the latter is defective in this kindred with Gitelman's syndrome.
Constitutive activation of a single effector pathway: evidence for multiple activation states of a G protein-coupled receptor.
A cysteine-to-phenylalanine mutation in the third transmembrane domain of the alpha 1B-adrenergic receptor constitutively activates the receptor, resulting in G protein coupling in the absence of agonist and activation of only a single effector pathway (phospholipase C but not phospholipase A2). This mutant receptor displays a higher affinity for the catecholamines, norepinephrine, and epinephrine, as well as for other phenethylamines, but not for imidazolines, a class of structurally distinct alpha agonists. Dose-response studies demonstrate a higher potency and intrinsic activity of phenethylamines for polyphosphoinositide turnover but not for arachidonic acid release. Imidazolines have wild-type potencies and intrinsic activities for both pathways. These data indicate that a single receptor subtype forms multiple conformations (i.e., exhibits induced conformational pleiotropy) for G protein interactions (high affinity states) that are specific for a particular G protein/effector pathway and that multiple binding sites exist for agonists, which promote or induce these specific interactions. Pharmacological diversity may, thus, be achieved through a single receptor by the development of compounds that induce a single activated conformer. This has major ramifications for the eventual development of signaling-specific therapeutics.
Molecular determinants of peptide and non-peptide binding to the AT1 receptor.
1. Several residues critically involved in AT1 receptor ligand-binding and activation have now been identified based on mutational and biochemical studies. 2. Asp281 and Lys199 of the rat AT1 receptor ion-pair with Arg2 and the Phe3 alpha-COOH of angiotensin II (AngII), respectively, and the Asp281/Arg2 interaction is critical for full agonist activity. 3. Agonist activity of AngII also requires an interaction of the Phe8 side chain with His256, which is achieved by docking of the alpha-COOH with Lys199. Non-peptide agonists interact with Lys199 and His256 in a similar fashion. 4. The crucial acid pharmacophores of AngII and the non-peptide antagonist, losartan, appear to occupy the same space within the receptor pocket. Binding of the tetrazole anion moiety of losartan involves multiple contacts, such as Lys199 and His256. However, this interaction does not involve a conventional salt bridge, but rather an unusual lysine-aromatic interaction. 5. Asp1 of AngII forms an ion-pair with His183, which stabilizes the receptor-bound conformation of AngII but is not critical for receptor activation. 6. These interactions and the involvement of other residues in stabilizing the wild-type receptor conformation or in receptor/G-protein coupling are considered here. 7. Despite these insights, considerable effort is still needed to elucidate how ligand binding induces receptor activation, what determines the specificity of AT1 receptor coupling to multiple G-proteins and the in vivo role of receptor down-regulation.
Identification of critical determinants of alpha 1-adrenergic receptor subtype selective agonist binding.
alpha 1-Adrenergic receptor (AR) subtypes mediate many effects of the sympathetic nervous system. The three cloned subtypes (alpha 1a-AR, alpha 1b-AR, alpha 1d-AR), although structurally similar, bind a series of ligands with different relative potencies. This is particularly true for the alpha 1a-AR, which recognizes a number of agonists and antagonists with 5-50-fold higher affinity than the alpha 1b- or alpha 1d- subtypes. Since ligands bind to receptor-residues that are located in the transmembrane spanning domains, we hypothesize that subtype differences in ligand recognition are due to differences in the binding properties of nonconserved transmembrane residues. Using site-directed mutagenesis, selected putative ligand-binding residues in the alpha 1b-AR were converted, either individually or in combination, to the corresponding residues in the alpha 1a-AR. Mutation of two such residues (of approximately 172 amino acids in the transmembrane domains) converted the agonist binding profile entirely to that of the alpha 1a-AR. Over 80% of this conversion was due to an Ala204-->Val substitution; the remainder was due to the additional substitution of Leu314-->Met. To confirm that Ala204 and Leu314 are indeed critical for agonist subtype-selectivity, the equivalent residues in the alpha 1a-AR (Val185 and Met293) were reversed of that of the alpha 1b-AR. Correspondingly, the agonist-binding profile of this double alpha 1a-AR mutant reverted to that of the alpha 1b-AR. From these data, in conjunction with macromolecular modeling of the ligand-binding pocket, a model has been developed, which indicates that the determinants of these two residues for agonist subtype-selectivity are due not only to interactions between their side chains and specific ligand moieties but also to a critical interaction between these two amino acids.
Evolutionary conservation of both the hydrophilic and hydrophobic nature of transmembrane residues.
An algorithm (HRG), developed to allow the pairwise comparisons of the aligned residues of several members of large gene families of polytopic integral membrane proteins is described. Using hydrophobicity scales, application of this algorithm allows the number and size of the membrane-spanning domains of bacteriorhodopsin, a polytopic protein whose structure has been partially determined, to be predicted with a high degree of accuracy (sensitivity 94%, specificity 82% for predicting the membrane embedded or extramembranous location of residues). As opposed to previously reported structure-prediction algorithms, delineation of putative transmembrane segments from connecting loops is also more clearly evident with the application of the HRG algorithm, even with proteins from widely divergent species. This indicates strong evolutionary pressure for the conservation of both the hydrophobic and hydrophilic character of residues in membrane-embedded regions of polytopic proteins, such as those of the G-protein-coupled receptor superfamily. These and other structural and functional implications evident from the application of the HRG algorithm are considered.
Tetrazole and carboxylate groups of angiotensin receptor antagonists bind to the same subsite by different mechanisms.
To identify specific interactions between either the tetrazole or carboxylate pharmacophores of non-peptide antagonists and the rat AT1 receptor, 6 basic residues were examined by site-directed mutagenesis. Three of the mutants (H183Q, H256Q, and H272Q) appeared to be like wild type. Lys102 and Arg167 mutants displayed reduced binding of the non-peptide antagonist losartan. Examination of their properties employing group-specific angiotensin II analogues indicated that their effects on binding were indirect. Interestingly, the affinity of losartan was not altered by a K199Q mutation, but the same mutation reduced the affinity of angiotensin II, the antagonist [Sar1,Ile8]angiotensin II, and several carboxylate analogues of losartan. An Ala199 substitution reduced the affinity of peptide analogues to a larger extent as compared to the affinity of losartan. Thus, the crucial acidic pharmacophores of angiotensin and losartan appear to occupy the same space within the receptor pocket, but the protonated amino group of Lys199 is not essential for binding the tetrazole anion. The binding of the tetrazole moiety with the AT1 receptor involves multiple contacts with residues such as Lys199 and His256 that constitute the same subsite of the ligand binding pocket. However, this interaction does not involve a conventional salt bridge, but rather an unusual lysine-aromatic interaction.
Operative procedures in patients subsequently found to be human immunodeficiency virus positive.
This study investigated the number and range of surgical procedures performed in patients subsequently found to be human immunodeficiency virus (HIV) positive. Procedures were included if they occurred during a period when the patients were likely to have been infected with HIV but unaware of their HIV status. A total of 28 operative procedures were documented in 19 (22 per cent) of 86 patients. Several operations were major and invasive. All patients had recognized risk factors for HIV infection but these had not been elicited in the surgical records. Women and patients who acquired their disease through heterosexual intercourse were more likely to have undergone surgery before a diagnosis of HIV than homosexual men. The incidence of surgical procedures in patients with HIV infection was significantly greater than in the general population of Leicestershire; however, many of these were minor and related to the management of complications associated with HIV. Surgeons are performing operations on patients who are unknowingly infected with HIV. The risk of transmission of HIV to surgeons is remote, but surgeons must be aware of risk factors for HIV infection.
Isolation of neonatal cardiomyocytes reduces the expression of the GTP-binding protein, Gh.
alpha 1-Adrenoceptors in most tissues couple with the heterotrimeric GTP-binding protein Gq, the alpha subunit of which activates the beta-isoforms of phospholipase C. However, in heart (and in liver) alpha 1-adrenoceptors have been reported to couple to a high molecular weight GTP-binding protein. Gh, which functions both as a type II transglutaminase and as a receptor coupling protein. Gh activates a phospholipase isoform distinct from phospholipase C-beta. Here we report that isolation and culture of neonatal cardiomyocytes decreased the expression of Gh without reducing the content of Gq or Gi. Gh was readily detected in extracts from intact neonatal and adult heart tissues. The expression of Gh thus appears to be a feature of intact cardiac tissue.
Neutrophil platelet-activating factor production and acetyltransferase activity in clinical acute myocardial infarction.
1. Neutrophil function was studied in 10 males presenting with acute myocardial infarction (MI) within 6 h of onset and in 10 normal males. Neutrophil production of platelet-activating factor (PAF), determined by bioassay, that of leukotriene B4 by HPLC, and the activity of an enzyme involved in the synthesis of PAF, acetyltransferase (AT), were measured before and after stimulation with opsonized zymosan and calcium ionophore, A23187. 2. The neutrophil count was significantly raised at presentation in those with MI (8.2 +/- 0.8 vs 2.8 +/- 0.3 (s.e.m.) x 10(9) cells/L, P < 0.001; t-test, 18 d.f.). Production of PAF per neutrophil in response to both stimulants was greater than normal in those with MI (zymosan: 21 +/- 4 vs 12 +/- 1 ng/10(7) cells, P < 0.05; ionophore: 174 +/- 18 vs 113 +/- 11 ng/10(7) cells, P < 0.02) despite normal leukotriene B4 production and depressed AT activity. By 7 days, the neutrophil count had significantly fallen but it remained greater than normal as did PAF production. 3. Acute MI is associated with increased potential for production of PAF by neutrophils which may be important in the pathogenesis of MI.
Effects of hypertrophy on left atrial and ventricular compliance and plasma ANF levels in conscious dogs.
Alterations in left atrial (LA) and left ventricular (LV) compliance and arterial and coronary sinus atrial natriuretic factor (ANF) concentrations at baseline and in response to both volume depletion and expansion were investigated in 15 conscious dogs with aortic banding-induced LV hypertrophy (LVH) (LV/body wt increased by 64%), which also induced LAH (LA/body wt increased by 61%). With volume expansion coronary sinus ANF increased more (P < 0.05) in dogs with LVH (+427 +/- 88 pg/ml) compared with control dogs (+146 +/- 45 pg/ml). Arterial ANF levels also rose more with volume expansion in LVH. In dogs with LVH, the LV end-diastolic pressure-diameter relationship was shifted to the left with a steeper slope with volume expansion, such that at any given diastolic dimension, diastolic pressure was higher. In contrast, the pressure-dimension relationship for the LA appendage was shifted in the opposite direction during both atrial systolic and diastolic phases, with a more shallow slope in hypertrophy compared with control dogs, resulting in an augmented pressure-dimension product during volume loading in LAH. In conclusion, in dogs with LVH and LAH, enhanced ANF was revealed in the coronary sinus and systemic circulation during volume expansion, which could be due, in part, to a more compliant, but hypertrophied, LA, which responded to equivalent volume loading with an augmented pressure-dimension product.
Production, characterization, and expression of neuropeptide Y by human pheochromocytoma.
Neuropeptide Y (NPY) levels are increased in plasma and tumors of patients with pheochromocytoma. The present study was designed to evaluate plasma and tissue NPY levels simultaneously as well as to study its release and expression in patients with either adrenal or extraadrenal pheochromocytomas. Plasma NPY levels were higher (P < 0.01) in patients with adrenal tumors than in matched normal subjects and patients with extraadrenal tumors. NPY levels were also higher (P < 0.05) in adrenal than in extraadrenal tumors. Bioactive NPY (1-36) was the predominant form in plasma and tumors of patients with adrenal pheochromocytomas. In contrast, patients with extraadrenal pheochromocytomas had an abundance of NPY fragments. NPY mRNA was abundant in 11 of 13 adrenal tumors but in only 1 of 6 extraadrenal tumors. Moreover, NPY was coreleased with NE with manipulation of adrenal but not extraadrenal tumors. These findings indicate that increased NPY gene expression in adrenal pheochromocytomas accounts for the greater biosynthesis and storage of NPY in these tumors and that increased release of NPY results in elevated plasma NPY. Factors regulating NPY gene expression in pheochromocytoma and the role of NPY in the clinical manifestations of the disease remain to be elucidated.
Functional characterization of ribozymes expressed using U1 and T7 vectors for the intracellular cleavage of ANF mRNA.
Hammerhead ribozymes targeted to various GUC or GUA sites on rat atrial natriuretic factor (ANF) mRNA were developed. The catalytic activity of ribozymes to four of these sites, synthesized by transcription off synthetic oligodeoxynucleotide duplexes, was studied in detail. In vitro, ribozyme-mediated cleavage was highly Mg(2+)-dependent, and at concentrations approaching those found intracellularly, the rate but not the extent of cleavage was markedly reduced. To test for cellular activity, synthetic genes encoding the ribozymes were cloned between the initiation and termination sequences of the U1snRNA gene or between the T7RNA polymerase promoter and terminator sequences in pSP64. Both constructs had defined initiation and termination sequences to minimize transcript size and for message stability. In vitro the addition of T7 or U1 terminator sequences had variable effects on catalytic activity, presumably due to structural interactions between the ribozyme and the added sequence. The ribozyme-encoding plasmids were cotransfected with an expression plasmid containing a rat ANF cDNA into COS-1 cells using a liposome method, which provided high-level transfection efficiency. Quantitation of ANF mRNA by RNase protection showed marked decreases in ANF transcript levels with both the U1- and the T7-expressed ribozymes directed at three of the four sites on ANF mRNA. With all constructs, target accessibility, determined in vitro, was a more important determinant of intracellular ANF mRNA cleavage than catalytic activity per se. ANF mRNA cleavage was not merely due to an antisense effect, since a mutant construct that was catalytically inactive but could still bind produced less cleavage than the corresponding wild-type ribozyme construct. These findings indicate that both U1 and T7 vector systems provide efficient ribozyme expression for the intracellular cleavage of target mRNA.