Transitional changes in the structure of C-reactive protein create highly pro-inflammatory molecules: Therapeutic implications for cardiovascular diseases.

C-reactive protein (CRP) is the prototypic acute-phase reactant that has long been recognized almost exclusively as a marker of inflammation and predictor of cardiovascular risk. However, accumulating evidence indicates that CRP is also a direct pathogenic pro-inflammatory mediator in atherosclerosis and cardiovascular diseases. The 'CRP system' consists of at least two protein conformations with distinct pathophysiological functions. The binding of the native, pentameric CRP (pCRP) to activated cell membranes leads to a conformational change resulting in two highly pro-inflammatory isoforms, pCRP* and monomeric CRP (mCRP). The deposition of these pro-inflammatory isoforms has been shown to aggravate the localized tissue injury in a broad range of pathological conditions including atherosclerosis and thrombosis, myocardial infarction, and stroke. Here, we review recent findings on how these structural changes contribute to the inflammatory response and discuss the transitional changes in the structure of CRP as a novel therapeutic target in cardiovascular diseases and overshooting inflammation.

Role of beta-adrenergic receptor subtypes in lipolysis.

In vitro lipolysis stimulated by low (-)-isoprenaline concentrations (< or =30 nM) in epididymal white adipocytes from Sprague-Dawley rats was inhibited at least 60-80% by the specific beta1-antagonists LK 204-545 and CGP 20712A (1 microM), suggesting that at these low (10 nM) concentrations of (-)-isoprenaline lipolysis was primarily (80%) but not solely mediated via beta1-adrenergic receptors. Low concentrations (100 nM) of (-)-noradrenaline and formoterol also confirmed a role for beta1-adrenergic receptors in mediating lipolysis at low concentrations of these agonists. At higher agonist concentrations, beta3-adrenergic receptors were fully activated and were the dominant beta-adrenergic receptor subtype mediating the maximum lipolytic response, and the maximum response was not affected by the beta1-antagonists, demonstrating that the beta3-receptor is capable of inducing maximum lipolysis on its own. Studies of lipolysis induced by the relatively beta2-selective agonist formoterol in the presence of beta1-blockade (1 microM CGP 20712A) demonstrated the inability of the beta2-selective antagonist ICI 118-551 to inhibit the residual lipolysis at concentrations of ICI 118-551 < or = 1 microM. Higher concentrations of ICI 118-551 inhibited the residual formoterol-induced lipolysis competetively, but with low affinity (approximately 500-fold lower than its beta2-adrenergic receptor pA2, 7.80 +/- 0.21), suggesting that formoterol was not acting via beta2-adrenergic receptors. These data are consistent with beta1-adrenergic receptors playing an important role in lipolysis at physiological but not pharmacological concentrations of catecholamines and that beta2-adrenergic receptors play no obvious direct role in mediating beta-adrenergic receptor agonist-induced lipolysis in vitro. Finally, racemic-SR 59230A, unlike the pure (S, S)-isomer (a beta3-selective antagonist), was found to be a nonselective antagonist at the three beta-adrenergic receptor subtypes, showing that the other enantiomers have different selectivity.

LK 204-545, a highly selective beta1-adrenoceptor antagonist at human beta-adrenoceptors.

LK 204-545 ((+/-)-1-(2-(3-(2-cyano-4-(2-cyclopropyl-methoxy-ethoxy)phenoxy)-2-hydro xy-propyl-amino)-ethyl)-3-(4-hydrxy-phenyl) urea), an antagonist that possesses high beta1-/beta2-selectivity in the rat, and a range of cardio-selective and non-selective beta-adrenoceptor antagonists were examined to compare their radioligand binding affinities for human beta1-, beta2- and beta3-adrenoceptors transfected into CHO cells. LK 204-545 and CGP 20712A displayed the highest beta1-/beta2- (approximately 1800 and approximately 650, respectively) and beta1-/beta3-selectivity (approximately 17000 and approximately 2200, respectively) at human beta-adrenoceptors with LK 204-545 being approximately 2.75-fold more beta1-/beta2-selective and approximately 8-fold beta1-/beta3-selective than CGP 20712A. The high potency of LK 204-545 at transfected human beta1-adrenoceptors and in functional models of rat beta1-adrenoceptors together with its high selectivity, identify it as a useful ligand for studying beta1-adrenoceptors and suggest that it may be the preferred ligand for human beta-adrenoceptor studies.

[3H]Rilmenidine-labelled imidazoline-receptor binding sites co-localize with [3H]2-(benzofuranyl)-2-imidazoline-labelled imidazoline-receptor binding sites and monoamine oxidase-B in rabbit, but not rat, kidney.

The distribution and relative densities of imidazoline-receptor binding sites (I-RBS) and monoamine oxidase (MAO)-A and -B enzyme(s) in rat and rabbit kidney were compared autoradiographically using fixed nanomolar concentrations of [3H]rilmenidine and [3H]2-(benzofuranyl)-2-imidazoline ([3H]2-BFI) to label I-RBS, and [3H]RO41-1049 and [3H]RO19-6327 to label MAO-A and -B isoenzymes, respectively. In rat kidney, high densities of I-RBS labelled by [3H]rilmenidine were observed in the cortex and outer stripe (120-280 fmol/mg tissue), in contrast to low I-RBS densities labelled by [3H]2-BFI (<4 fmol/mg). A relatively high density of [3H]RO41-1049 binding to MAO-A enzyme was present in all regions of the rat kidney (160-210 fmol/mg) compared with a low density of [3H]RO19-6327 binding to MAO-B (< 25 fmol/mg). Comparison of MAO-A and -B distributions with that of [3H]rilmenidine-labelled I-RBS strongly suggests a lack of association in rat kidney. Similarly, the extremely low densities of [3H]2-BFI-labelled I2-RBS in rat kidney contrasts with the density of MAO-A, but is consistent with the low density of MAO-B. Rabbit kidney cortex and outer stripe contained high relative densities of [3H]rilmenidine-labelled I-RBS (200-215 fmol/mg) and [3H]2-BFI-labelled I2-RBS (45-60 fmol/mg) with lower densities in the inner stripe and inner medulla (< or = 100 and 30 fmol/mg respectively). A high density of MAO-A binding was observed in the inner stripe (515 fmol/mg) with lower levels in the cortex and outer stripe (100-240 fmol/mg), while high densities of MAO-B binding were observed in the cortex and outer stripe (290-450 fmol/mg) with lower levels in the inner stripe (65 fmol/mg). The correlation between the localization of [3H]rilmenidine-labelled I-RBS and [3H]RO19-6327-labelled MAO-B in rabbit kidney (r = 0.87, P = 0.057) suggest that [3H]rilmenidine may label a binding site co-existent with MAO-B, but not MAO-A (n.s.), in this tissue, but rilmenidine did not inhibit [3H]RO41-1049 or [3H]RO19-6327 binding. The distribution of [3H]2-BFI-labelled I2-RBS overlapped the combined distributions of both MAO-A and -B isoenzymes, suggesting that [3H]2-BFI may label sites on both enzymes in the rabbit, but [3H]2-BFI binding only correlated with [3H]RO19-6327 (r = 0.84, P = 0.07), not [3H]RO41-1049 binding (n.s.). Moreover, 2-BFI only inhibited [3H]RO19-6327, not [3H]RO41-1049 binding. These data are consistent with reports that I2-RBS are located on MAO-B and allosterically influence the catalytic site. The relationship of [3H]rilmenidine- and [3H]2-BFI-labelled I-RBS and the identity of non-MAO-associated [3H]rilmenidine-labelled I-RBS requires further investigation.

Computer-aided mapping of the beta-adrenoceptor. 1. Explanation for effect of para substitution on blocking activity at the beta-1-adrenoceptor.

Anomalously low affinities for the beta-1-adrenoceptor are seen for members of a series of para-substituted N-isopropylphenoxypropanolamines in which the substituent is able to conjugate with the aromatic ring. The energy of conjugation was calculated using the AM1 semiempirical molecular orbital method and appears to correlate with the loss of binding energy, and hence affinity for the receptor. This suggests that binding is associated with movement of the substituent out of the plane of the aromatic ring due to steric interference with the receptor. A previously unrecognized binding site for aromatic groups off the para position is also identified.

Evaluation of the predicted secondary structure of bacteriorhodopsin. Prediction of the bovine rhodopsin secondary structure and its sequence similarity with bacteriorhodopsin.

The secondary structure of bacteriorhodopsin (bacR) is predicted using the Chou and Fasman method in conjunction with the hydropathic index of Kyte and Doolittle. The predicted bacR structure was compared with the structure determined by Henderson et al. (1990) using electron diffraction and was found to correlate extremely well. The secondary structure of bovine rhodopsin (bovR) was then predicted using the same techniques. The proposed transmembrane regions of bovR were then examined and found to have sequence similarity with those transmembrane regions of bacR.

Aspartate aminotransferase: investigation of the active sites.

An investigation of the crystal structure of cytosolic pig-heart aspartate aminotransferase (AAT, E.C.2.6.1.1) was carried out to determine the structural requirements for ligand recognition by the active site. Structural differences were observed between the two active sites of the AAT dimer. The natural ligand, L-aspartate, was docked into both active sites using various methods. However, due to structural differences, the ligand was able to form all the necessary interactions for initial binding in only one of the active sites. The program GRID (P.J. Goodford, J. Med. Chem. 1985, 28, 849-857) was used to predict favorable binding sites for the functional groups of the aspartate ligand. These binding sites corresponded to the position of the docked aspartate ligand, indicating that substrate recognition takes place before any major conformational changes occur within the enzyme.