Angiotensin-converting enzyme inhibition and angiotensin II subtype-1 receptor blockade during the progression of left ventricular dysfunction: differential effects on myocyte contractile processes

Inhibition of the angiotensin-converting enzyme (ACE) in the setting of chronic left ventricular (LV) dysfunction has been demonstrated to have beneficial effects on survival and symptoms. However, whether ACE inhibition has direct effects on myocyte contractile processes and if these effects are mediated primarily through the AT1 angiotensin-II receptor subtype remains unclear. The present project examined the relationship between changes in LV and myocyte function and beta adrenergic receptor transduction in four groups of six dogs each: (1) Rapid Pace: LV failure induced by chronic rapid pacing (4 weeks; 216 +/- 2 bpm); (2) Rapid Pace/ACEI: concomitant ACE inhibition (ACEI: fosinopril 30 mg/kg b.i.d.) with chronic pacing; (3) Rapid Pace/AT1 Block: concomitant AT1 Ang-II receptor blockade [Irbesartan: SR 47436(BMS-186295) 30 mg/kg b.i.d.] with chronic pacing; and (4) Control: sham controls. With Rapid Pace, the LV end-diastolic volume increased by 62% and the ejection fraction decreased by 53% from control. With Rapid Pace/ACEI, the LV end-diastolic volume was reduced by 24% and the ejection fraction increased by 26% from Rapid Pace only values. Rapid Pace/AT1 Block did not improve LV geometry or function from Rapid Pace values. Myocyte contractile function decreased by 40% with Rapid Pace and increased from this value by 32% with Rapid Pace/ACEI. Rapid Pace/AT1 Block had no effect on myocyte function when compared with Rapid Pace values. With Rapid Pace/ACEI, beta receptor density and cyclic AMP production were normalized and associated with an improvement in myocyte beta adrenergic response compared with Rapid Pace only. Although Rapid Pace/AT1 also normalized beta receptor density, cyclic AMP production was unchanged and myocyte beta adrenergic response was reduced by 15% compared with Rapid Pace only. ACE inhibition with chronic rapid pacing improved LV and myocyte geometry and function, and normalized beta receptor density and cyclic AMP production. However, AT1 Ang-II receptor blockade with chronic rapid pacing failed to provide similar protective effects on LV and myocyte geometry and function. These unique findings suggest that the effects of ACE inhibition on LV geometry and myocyte contractile processes in the setting of developing LV failure are not primarily caused by modulation of AT1 Ang-II receptor activation.     

Synthesis and characterization of a stable analog of the phosphorylated form of the chemotaxis protein CheY

The bacterial chemotaxis protein CheY is activated in vivo by the covalent phosphorylation of a single aspartate residue at position 57. However, this phosphate linkage is unstable (t1/2 approximately 20 s at room temperature), thereby precluding many biochemical analyses. Here we present a synthetic scheme to prepare an analog of CheY-phosphate (Che Y-P) with chemical stability of the phosphate linkage enhanced by several orders of magnitude relative to the native protein. Starting with CheY D57C, a site-specific mutant of CheY with a unique cysteine residue in place of the aspartate at position 57, two sequential disulfide exchange reactions were performed to form the final product 'CheY D57C-SPO3' with a thiophosphate moiety covalently bonded to the protein in a disulfide linkage. Mass spectral analysis showed that the desired analog was present at 70-80% of the total protein. The disulfide linkage had a t1/2 of 8 days at 4 degrees C. Biochemical characterization of CheY D57C-SPO3 included assessment of conformational properties using tryptophan fluorescence, evaluation of metal binding properties and measurement of binding interactions with the chemotaxis proteins CheZ and FliM. Despite possessing a phosphoryl group at a nearly identical location as native CheY-phosphate, the analog was unable to emulate CheY-phosphate function, thereby supporting the idea that there are very precise geometric requirements for successful CheY activation.      

Pharmacological inhibition of protein kinases in intact cells: antagonism of beta adrenergic receptor ligand binding by H-89 reveals limitations of usefulness

The use of pharmacological inhibitors of protein kinases represents a potentially powerful tool in dissecting the regulatory features of intracellular signaling pathways. However, although the in vitro potency, selectivity, and efficacy of numerous kinase inhibitors have been characterized, little is known regarding the usefulness of these compounds as inhibitors in intact cells. In attempting to characterize the role of protein kinase A (PKA) in regulating the beta-2 adrenergic receptor (AR) in human airway cells, we observed a seemingly profound capacity of the isoquinoline H-89, a potent and widely used PKA inhibitor, to attenuate agonist-mediated desensitization of the beta-2 AR. Although additional experiments identified H-89 as an effective inhibitor of intracellular PKA, extended analysis of the compound determined the principal effect of H-89 was via its action as a beta-2 AR antagonist. Pretreatment with or the acute addition of H-89 significantly attenuated isoproterenol-stimulated cAMP accumulation. In cells pretreated with H-89 and then washed extensively, the subsequent dose-dependent response to isoproterenol suggested beta-2 AR antagonism by retained H-89. Competition binding of [125I]iodopindolol established Ki values of approximately 180 nM and 350 nM for H-89 antagonism of beta-2 AR and beta-1 AR, respectively. Additional receptor binding studies suggest selectivity of H-89 for the beta-2 AR and beta-1 AR, although a weak antagonism (Ki values of approximately 10 microM or greater) of other G protein-coupled receptors was observed. Results from additional pharmacological and biochemical analyses of various protein kinase inhibitors further established the need for careful characterization of pharmacological inhibitors when used in intact cell models.     

Effects of protein-surface interactions on protein ion signals in MALDI mass spectrometry

The influence of polymer surface-protein binding affinity on protein ion signals in matrix-assisted laser desorption/ionization (MALDI) mass spectrometry is examined. The surfaces of poly(vinylidene fluoride) and poly(ethylene terephthalate) polymer substrates are modified by pulsed rf plasma deposition of allylamine. By varying the on/off duty cycle of the pulsed rf plasma, the polymer substrate surfaces are coated with thin films having varying densities of surface amine groups. The varying surface amine density is shown to lead to systematic changes in the surface binding affinity for the 125I-radiolabeled peptides angiotensin I and porcine insulin. Unlabeled angiotensin I and porcine insulin are then deposited on the pulsed rf plasma-modified substrates and analyzed by MALDI mass spectrometry. The experimental approach involves applying the peptide to the modified polymer surface in an aqueous phosphate-buffered saline solution and allowing the peptide solution to dry completely under ambient conditions. Subsequently, the MALDI matrix alpha-cyano-4-hydroxycinnamic acid in methanol and 10% trifluoroacetic acid in water are added to the peptide-coated modified polymer surfaces. The results of these studies demonstrate that, for the sample preparation method employed, increases in the surface peptide binding affinity lead to decreases in the peptide MALDI ion signal.     

Five-year review of endometrial ablation with the SideFire laser fiber

Radiologic assessment of fracture reduction in displaced supracondylar fractures of the elbow in children is notoriously difficult. The Baumann angle is often used as a guide to the adequacy of reduction. This is based on the assumption that the Baumann angle has a constant relationship to the carrying angle in a displaced fracture. The effect of rotation on the relationship between these angles has not been studied in detail. Computed tomography (CT) studies have shown that < or = 40 degrees of rotation may still be present after closed manipulation of a supracondylar fracture. A computer programme analysing vectors was designed to study the relationship between the Baumann angle and the carrying angle with predetermined displacements of the distal fragment. The relationship between the Baumann angle and the carrying angle was thus defined and, in contrast to the literature, found to be more complex than previously appreciated. The Baumann angle was found to be an inaccurate indicator of the carrying angle when treating displaced supracondylar fractures.     

Mycophenolate mofetil: therapeutic applications in kidney transplantation and immune-mediated renal disease

The inhibition of arylamine N-acetyltransferase (NAT) activity by ibuprofen was determined in a human colon tumour (adenocarcinoma) cell line. Two assay systems were employed, one with cellular cytosols (9000 g supernatant) and the other with intact colon tumour cell suspensions. The NAT activity in a human colon tumour cell line was inhibited by ibuprofen in a dose-dependent manner in both systems, i.e. the greater the concentration of ibuprofen in the reaction, the greater the inhibition of NAT activities in both systems. The data also indicated that ibuprofen decreases the apparent Km and Vmax of NAT enzyme from human colon tumour cells in both systems examined. This report is the first demonstration to show that ibuprofen affects human colon tumour cell NAT activity.     

Critical closing pressure explains cerebral hemodynamics during the Valsalva maneuver

Immunization with a particulate fraction of blood-stage antigens was shown previously to protect mice against Plasmodium yoelii malaria. To identify antigens inducing the protective response, sera from immunized mice were used to screen a P. yoelii cDNA expression library. Sequence analysis of one 2.6-kb cDNA clone indicated that the identified gene, pypag-1, encoded a novel plasmodial antigen. Two nonoverlapping regions of pypag-1 were expressed in Escherichia coli. The first recombinant antigen, pAg-1N, contained the N-terminal 337 residues, which included a putative transmembrane domain and a region relatively rich in tryptophan residues. The second recombinant antigen, pAg-1C, contained the remaining C-terminal 211 residues, which included 31 copies of a 5-amino-acid degenerative repeat. Immunoblot studies using rabbit antiserum raised against recombinant pAg-1N showed that the native pypAg-1 protein migrated at approximately 98 kDa, considerably slower than its predicted molecular mass of 66 kDa. Immunofluorescence studies localized the expression of the native pypAg-1 protein both to the cytoplasm and at the surface of P. yoelii-infected erythrocytes. Immunization with either pAg-1N or pAg-1C induced a four- to sevenfold reduction in P. yoelii blood-stage parasitemia. As such, pypAg-1 appears to contain at least two distinct protective epitopes. To our knowledge, this is the first characterization of a protective antigen of P. yoelii that is associated with the erythrocyte membrane.