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Biomedical subjects

A D Robertson

Publications and source records attributed to A D Robertson.

At least 19 recordsLinked to original sources
PubMed2002

Coordinate changes in Myosin heavy chain isoform gene expression are selectively associated with alterations in dilated cardiomyopathy phenotype.

BACKGROUND: The most common cause of chronic heart failure in the US is secondary or primary dilated cardiomyopathy (DCM). The DCM phenotype exhibits changes in the expression of genes that regulate contractile function and pathologic hypertrophy. However, it is unclear if any of these alterations in gene expression are disease producing or modifying. MATERIALS AND METHODS: One approach to providing evidence for cause-effect of a disease-influencing gene is to quantitatively compare changes in phenotype to changes in gene expression by employing serial measurements in a longitudinal experimental design. We investigated the quantitative relationships between changes in gene expression and phenotype n 47 patients with idiopathic DCM. In endomyocardial biopsies at baseline and 6 months later, we measured mRNA expression of genes regulating contractile function (beta-adrenergic receptors, sarcoplasmic reticulum Ca(2) + ATPase, and alpha- and beta-myosin heavy chain isoforms) or associated with pathologic hypertrophy (beta-myosin heavy chain and atrial natriuretic peptide), plus beta-adrenergic receptor protein expression. Left ventricular phenotype was assessed by radionuclide ejection fraction. RESULTS: Improvement in DCM phenotype was directly related to a coordinate increase in alpha- and a decrease in beta-myosin heavy chain mRNA expression. In contrast, modification of phenotype was unrelated to changes in the expression of beta(1)- or beta(2)-adrenergic receptor mRNA or protein, or to the mRNA expression of sarcoplasmic reticulum Ca(2) + ATPase and atrial natriuretic peptide. CONCLUSION: We conclude that in human DCM, phenotypic modification is selectively associated with myosin heavy chain isoform changes. These data support the hypothesis that myosin heavy chain isoform changes contribute to disease progression in human DCM.

Antihypertensive Agents↗
PubMed2001

An iron-binding exochelin prevents restenosis due to coronary artery balloon injury in a porcine model.

Background- Vascular smooth muscle cell (VSMC) proliferation is a critical factor in the neointima formation that causes restenosis after coronary angioplasty (PTCA). Desferri-exochelin 772SM (D-EXO), a highly diffusible, lipophilic iron chelator secreted by Mycobacterium tuberculosis, inhibits proliferation of VSMCs in culture. We hypothesized that treatment with D-EXO would inhibit neointima formation in balloon-injured vessels in vivo. Methods and Results- We subjected 24 pigs to overstretch coronary artery injury with standard PTCA balloons and then administered intramural injections of either D-EXO (n=14) or vehicle (n=10) through an Infiltrator catheter. Treatments were randomized, and the investigators were blinded with regard to treatment group until data analysis was completed. One month later, we euthanized the pigs, excised the injured coronary segments, made multiple sections of each segment, and identified the site of maximal neointima formation. An injury score based on the degree of disruption of the internal or external elastic lamina or media was assigned. D-EXO reduced stenosis index (neointima area divided by the area within the internal elastic lamina), adjusted for injury score, by 47%. Neointima thickness was also reduced. Conclusions- D-EXO, injected intramurally, substantially inhibited formation of neointima in a porcine vascular injury model.

Angioplasty, Balloon, Coronary↗
PubMed2001

Aspirin impairs reverse myocardial remodeling in patients with heart failure treated with beta-blockers.

OBJECTIVES: We hypothesized that aspirin (ASA) might alter the beneficial effect of beta-blockers on left ventricular ejection fraction (LVEF) in patients with chronic heart failure. BACKGROUND: Aspirin blunts the vasodilation caused by both angiotensin-converting enzyme (ACE) inhibitors and beta-blockers in hypertensive patients and in patients with heart failure. Several studies suggest that ASA also blunts some of beneficial effects of ACE inhibitors on mortality in patients with heart failure. To our knowledge, there have been no data evaluating the possible interaction of ASA and beta-blockers on left ventricular remodeling in patients with heart failure. METHODS: We retrospectively evaluated patients entered into the Multicenter Oral Carvedilol Heart failure Assessment (MOCHA) trial, a 6-month, double-blind, randomized, placebo-controlled, multicenter, dose-response evaluation of carvedilol in patients with chronic stable symptomatic heart failure. Multivariate analysis was performed to determine if aspirin independently influenced the improvement in LVEF. RESULTS: Over all randomized patients (n = 293), LVEF improved 8.2 +/- 0.8 ejection fraction (EF) units in ASA nonusers and 4.5 +/- 0.7 EF units in ASA users (p = 0.005). In subjects randomized to treatment with carvedilol (n = 231), LVEF improved 9.5 +/- 0.9 EF units in ASA nonusers and 5.8 +/- 0.8 EF units in ASA users (p = 0.02). In subjects randomized to treatment with placebo (n = 62), LVEF improved 2.8 +/- 1.2 EF units in ASA nonusers and 0.5 +/- 1.4 EF units in ASA users (p = 0.20). Aspirin did not significantly affect the heart rate or systolic blood pressure response in either the placebo or carvedilol groups. The effect of ASA became more significant on multivariate analysis. The change in LVEF was also influenced by carvedilol dose, etiology of heart failure, baseline heart rate, EF and coumadin use. The detrimental effect of ASA on the improvement in LVEF was dose-related and was present in both placebo and carvedilol groups, although the effect was statistically significant only in the much larger carvedilol group. CONCLUSIONS: Aspirin significantly affects the changes in LVEF over time in patients with heart failure and systolic dysfunction treated with carvedilol. The specific mechanism(s) underlying this interaction are unknown and further studies are needed to provide additional understanding of the molecular basis of factors influencing reverse remodeling in patients with heart failure.

Adrenergic beta-Antagonists↗
PubMed2001

Kinetics of unfolding and folding from amide hydrogen exchange in native ubiquitin.

Amide hydrogen (NH) exchange is one of the few experimental techniques with the potential for determining the thermodynamics and kinetics of conformational motions at nearly every residue in native proteins. Quantitative interpretation of NH exchange in terms of molecular motions relies on a simple two-state kinetic model: at any given slowly exchanging NH, a closed or exchange-incompetent conformation is in equilibrium with an open or exchange-competent conformation. Previous studies have demonstrated the accuracy of this model in measuring conformational equilibria by comparing exchange data with the thermodynamics of protein unfolding. We report here a test of the accuracy of the model in determining the kinetics of conformational changes in native proteins. The kinetics of folding and unfolding for ubiquitin have been measured by conventional methods and compared with those derived from a comprehensive analysis of the pH dependence of exchange in native ubiquitin. Rate constants for folding and unfolding from these two very different types of experiments show good agreement. The simple model for NH exchange thus appears to be a robust framework for obtaining quantitative information about molecular motions in native proteins.

Deuterium↗
PubMed2001

Some thermodynamic implications for the thermostability of proteins.

An analysis of the thermodynamics of protein stability reveals a general tendency for proteins that denature at higher temperatures to have greater free energies of maximal stability. To a reasonable approximation, the temperature of maximal stability for the set of globular, water-soluble proteins surveyed by Robertson and Murphy occurs at T* approximately 283K, independent of the heat denaturation temperature, T(m). This observation indicates, at least for these proteins, that thermostability tends to be achieved through elevation of the stability curve rather than by broadening or through a horizontal shift to higher temperatures. The relationship between the free energy of maximal stability and the temperature of heat denaturation is such that an increase in maximal stability of approximately 0.008 kJ/mole/residue is, on average, associated with a 1 degrees C increase in T(m). An estimate of the energetic consequences of thermal expansion suggests that these effects may contribute significantly to the destabilization of the native state of proteins with increasing temperature.

Hot Temperature↗
PubMed2000

Insensitivity of perturbed carboxyl pK(a) values in the ovomucoid third domain to charge replacement at a neighboring residue.

A number of carboxyl groups in turkey ovomucoid third domain (OMTKY3) have low pK(a) values. A previous study suggested that neighboring amino groups were primarily responsible for the low carboxyl pK(a) values. However, the expected elevation in pK(a) values for these amino groups was not observed. In the present study, site-directed mutagenesis is used to investigate the origins of perturbed carboxyl pK(a) values in OMTKY3. Electrostatic calculations suggest that Lys 34 has large effects, 0.4-0.6 unit, on Asp 7, Glu 10, and Glu 19 which are 5-11 A away from Lys 34. Two-dimensional (1)H NMR techniques were used to determine pK(a) values of the acidic residues in OMTKY3 mutants in which Lys 34 has been replaced with threonine and glutamine. Surprisingly, the pK(a) values in the mutants are very close to those of the wild-type protein. The insensitivity of the acidic residues to replacement of Lys 34 suggests that long-range electrostatic interactions play less of a role in perturbing carboxyl pK(a) values than originally thought. We hypothesize that hydrogen bonds play a key role in perturbing some of the carboxyl ionization equilibria in OMTKY3.

Amides↗
PubMed2000

Correlated motions in native proteins from MS analysis of NH exchange: evidence for a manifold of unfolding reactions in ovomucoid third domain.

Native-state amide hydrogen exchange monitored by NMR spectroscopy and mass spectrometry (MS) has the potential to provide detailed residue-level information regarding correlated motions occurring on the microseconds to seconds timescale. To expand the applicability of MS to these studies, a new algorithm has been developed to interpret MS data for exchange occurring between the EX2 and EX1 kinetic limits. Re-interpretation of MS data for ovomucoid third domain reveals multiple unfolding or partial unfolding reactions.

Algorithms↗
PubMed2000

A functional R domain from cystic fibrosis transmembrane conductance regulator is predominantly unstructured in solution.

Phosphorylation of the regulatory (R) domain initiates cystic fibrosis transmembrane conductance regulator (CFTR) Cl(-) channel activity. To discover how the function of this domain is determined by its structure, we produced an R domain protein (R8) that spanned residues 708-831 of CFTR. Phosphorylated, but not unphosphorylated, R8 stimulated activity of CFTR channels lacking this domain, indicating that R8 is functional. Unexpectedly, this functional R8 was predominantly random coil, as revealed by CD and limited proteolysis. The CD spectra of both phosphorylated and nonphosphorylated R8 were similar in aqueous buffer. The folding agent trimethylamine N-oxide induced only a small increase in the helical content of nonphosphorylated R8 and even less change in the helical content of phosphorylated R8. These data, indicating that the R domain is predominantly random coil, may explain the seemingly complex way in which phosphorylation regulates CFTR channel activity.

Circular Dichroism↗
PubMed2000

Microsecond to minute dynamics revealed by EX1-type hydrogen exchange at nearly every backbone hydrogen bond in a native protein.

A previous comprehensive analysis of the pH dependence of native-state amide hydrogen (NH) exchange in turkey ovomucoid third domain (OMTKY3) yielded apparent opening and closing rate constants (k(op) and k(cl)) at 14 NH groups involved in global conformational changes. This analysis has been extended to 18 additional slowly exchanging NH groups. Quench-flow experiments were performed to monitor NH exchange in native OMTKY3 from neutral to very alkaline pH ( approximately 12) conditions. Above pH 10 the mechanism of exchange switched from one governed by a rapid equilibrium preceding the chemistry of exchange (i.e. EX2 exchange), to one where exchange was limited by the rate of opening (i.e. EX1 exchange). Kinetics of solvent exposure are now known for nearly all backbone NH groups in native OMTKY3, yielding rate constants that span five orders of magnitude, 0.004 to 200 s(-1).

Amides↗
PubMed2000

Low-dose enoximone improves exercise capacity in chronic heart failure. Enoximone Study Group.

OBJECTIVES: This study was designed to evaluate the effects of low-dose enoximone on exercise capacity. BACKGROUND: At higher doses the phosphodiesterase inhibitor, enoximone, has been shown to increase exercise capacity and decrease symptoms in heart failure patients but also to increase mortality. The effects of lower doses of enoximone on exercise capacity and adverse events have not been evaluated. METHODS: This is a prospective, double-blind, placebo-controlled, multicenter trial (nine U.S. centers) conducted in 105 patients with New York Heart Association class II to III, ischemic or nonischemic chronic heart failure (CHF). Patients were randomized to placebo or enoximone at 25 or 50 mg orally three times a day. Treadmill maximal exercise testing was done at baseline and after 4, 8 and 12 weeks of treatment, using a modified Naughton protocol. Patients were also evaluated for changes in quality of life and for increased arrhythmias by Holter monitoring. RESULTS: By the protocol-specified method of statistical analysis (the last observation carried-forward method), enoximone at 50 mg three times a day improved exercise capacity by 117 s at 12 weeks (p = 0.003). Enoximone at 25 mg three times a day also improved exercise capacity at 12 weeks by 115 s (p = 0.013). No increases in ventricular arrhythmias were noted. There were four deaths in the placebo group and 2 and 0 deaths in the enoximone 25 mg three times a day and enoximone 50 mg three times a day groups, respectively. Effects on degree of dyspnea and patient and physician assessments of clinical status favored the enoximone groups. CONCLUSIONS: Twelve weeks of treatment with low-dose enoximone improves exercise capacity in patients with CHF, without increasing adverse events.

Adolescent↗
PubMed2000

Cardiovascular and autonomic effects of omega-conotoxins MVIIA and CVID in conscious rabbits and isolated tissue assays.

1. The effects of a novel N-type voltage-operated calcium channel antagonist, omega-conotoxin CVID, were compared with omega-conotoxin MVIIA on sympathetic-evoked activation of right atria (RA), small mesenteric arteries (MA) and vasa deferentia (VD) isolated from the rat. Their effects were also compared on blood pressure and cardiovascular reflexes in conscious rabbits. 2. The pIC(50) values for MVIIA and CVID, respectively, for inhibiting sympathetic-evoked responses were equivalent in RA (8.7 and 8.7) and VD (9.0 and 8.7); however, in MA the values were 8.4 and 7.7. The cardiac to vascular (RA/MA) potency ratios, antilog (plog RA - plog MA), for MVIIA and CVID were 2 and 10. The offset rates for CVID and MVIIA were rapid, and peptide reapplication caused rapid onset of blockade, suggesting limited desensitization. 3. In the conscious rabbit, CVID and MVIIA (100 microg kg(-1) i.v.) caused a similar fall in blood pressure and a tachycardia that rapidly reached maximum. Both peptides decreased the vagal- and sympathetic-mediated components of the baroreflex, but had no effect on the vagal nasopharyngeal reflex. The orthostatic reflex to 90 degrees tilt was blocked by MVIIA with sustained postural hypotension for > or = 90 min after administration. In contrast, CVID caused postural hypotension at 30 min which recovered rapidly. 4. Neither CVID nor MVIIA (3 microg kg(-1) i.t.) significantly altered cardiovascular variables or autonomic reflexes. 5. In conclusion, CVID appears to be relatively weak at inhibiting the reflex response to tilt consistent with its weaker inhibition of rat mesenteric artery constriction to perivascular nerve stimulation. This may point to subtype N-type calcium channel selectivity.

Animals↗
PubMed2000

Chronic hypoxia, pregnancy, and endothelium-mediated relaxation in guinea pig uterine and thoracic arteries.

Vasodilation that occurs during normal pregnancy is associated with enhanced relaxation and decreased contractile response to agonists, which are in part due to increased stimulated and basal nitric oxide (NO). In preeclampsia and/or pregnancies carried at high altitude (HA), this normal vascular adjustment is reversed or diminished. We previously reported that HA exposure did not inhibit the pregnancy-associated decrease in contractile response to agonist or basal NO in guinea pig uterine arteries (UA). We therefore sought to determine whether altitude interfered with effects of pregnancy on endothelium-dependent relaxation through a reduction in stimulated NO. We examined the relaxation response to ACh in UA and bradykinin in thoracic arteries (TA) and effects of NO inhibition with 200 microM N(G)-nitro-L-arginine (L-NNA) in arterial rings isolated from nonpregnant and pregnant guinea pigs exposed throughout gestation to low altitude (LA, 1,600 m, n = 26) or HA (3,962 m, n = 22). In pregnant UA, relaxation to ACh was enhanced (P < 0.05) at both altitudes and NO inhibition diminished, but did not reverse, ACh relaxation. The effect of L-NNA on the relaxation response to ACh was less in HA than in LA animals (P = 0.0021). In nonpregnant UA, relaxation to ACh was similar in LA and HA animals. L-NNA reversed the relaxation response to ACh at HA but not at LA. In TA, relaxation to bradykinin was unaltered by pregnancy or altitude and was completely reversed by NO inhibition. These data suggest that effects of NO inhibition are diminished in UA during pregnancy at HA. Additional studies are needed to confirm whether these effects are mediated through inhibition of stimulated NO. HA exposure did not inhibit relaxation to ACh, perhaps because of stimulation of other vasodilators.

Altitude↗
PubMed1999

Three-state unfolding and self-association of maspin, a tumor-suppressing serpin.

Maspin is a tumor suppressor protein expressed by normal human mammary epithelium but not by many breast tumor cell lines. Recombinant human maspin (rMaspin) inhibits tumor cell motility, invasion, and metastasis and thus has potential value as an anti-cancer therapeutic. Maspin is a member of the serpin family and, although the molecular mechanism by which maspin acts is unknown, recent work suggests that tissue plasminogen activator is a potential target. A puzzling observation in previous cell culture studies was loss of rMaspin activity at higher protein concentrations. One hypothesis to explain these results is self-association of rMaspin at the higher concentrations, which would be consistent with the tendency of serpins to form noncovalent polymers. This hypothesis is addressed by examining the relationship between rMaspin stability and self-association. Urea denaturation of rMaspin at pH 7 and 25 degrees C and at protein concentrations ranging from 0.01 to 0.2 mg/ml has been monitored by circular dichroism and intrinsic tryptophan fluorescence. Denaturation profiles show a protein concentration dependence and indicate the presence of at least one unfolding intermediate. The results suggest that destabilization of native monomeric rMaspin leads to partial unfolding and formation of an intermediate which can self-associate.

Circular Dichroism↗
PubMed1999

Effects of carvedilol on left ventricular mass, chamber geometry, and mitral regurgitation in chronic heart failure.

We and others have previously shown that carvedilol improves left ventricular (LV) function and symptoms in chronic heart failure. This improvement in LV function has also been shown to be associated with an improvement in survival. This study evaluates the effect of carvedilol on LV mass, geometry, and degree of mitral regurgitation (MR). In 59 patients with symptomatic heart failure and LV ejection fraction <0.35, previously randomized to either treatment with carvedilol or placebo, we evaluated LV mass, geometry, and degree of MR over the time period of carvedilol treatment. LV mass decreased as early as 4 months into the treatment protocol and continued to decrease over a period of 1 year. LV geometry, defined by the length/diameter ratio, and severity of MR also improved with 4 months of therapy. Thus, compared with placebo treatment, carvedilol decreases LV mass while improving cardiac geometry and decreasing MR in patients with chronic heart failure. These changes occur in association with an improvement in LV systolic function. This process begins by 4 months of treatment and continues for 12 months.

Adolescent↗
PubMed1999

Defining protein ensembles with native-state NH exchange: kinetics of interconversion and cooperative units from combined NMR and MS analysis.

Previous studies of native-state peptide hydrogen atom (NH) exchange in turkey ovomucoid third domain (OMTKY3) yielded the thermodynamics and kinetics of unfolding and folding for the 14 slowest-exchanging peptide hydrogen atoms (NHs). Unfolding rate constants and free energies for nine of the NHs are very similar, suggesting that these NHs exchange during a single cooperative unfolding event. Electrospray ionization mass spectrometry (ESI-MS) has been used to test this hypothesis. ESI-MS data and MS peak simulations suggest that this hypothesis is incorrect: in spite of the similarity in their unfolding rate constants, only three to five of the nine residues exchange in a cooperative manner. Thus, residues with similar thermodynamics and kinetics of exchange are probably involved in multiple conformational equilibria. Overall, combined NMR and MS analysis of NH exchange provides a rich and complex picture of the ensemble properties of native proteins.

Animals↗
PubMed1999

Right ventricular phenotypic characteristics in subjects with primary pulmonary hypertension or idiopathic dilated cardiomyopathy.

BACKGROUND: Studies of animal models and human subjects with cardiomyopathies suggest that cardiac myocyte and ventricular chamber remodeling show distinct phenotypic characteristics that may be dependent on specific signaling pathways. METHODS AND RESULTS: In this study, we characterize right ventricular (RV) chamber size, end-diastolic thickness, myocardial mass, and ejection fraction (EF) in human subjects with chronic heart failure from primary pulmonary hypertension (PPH; n = 10) and idiopathic dilated cardiomyopathy (IDC; n = 10). Subjects underwent gated cardiac magnetic resonance imaging (MRI), and the RVs were phenotypically classified based on the presence or absence of hypertrophy (increased mass), systolic dysfunction (reduced EF), and degree of wall thickness (concentric v eccentric pattern of hypertrophy). Within this schema, five abnormal phenotypes could be identified. In PPH subjects, in whom the RV is subjected to the uniform insult of chronic pressure overload, four different abnormal phenotypes were identified. CONCLUSIONS: These data indicate that distinct structural/functional ventricular chamber phenotypes may be classified by MRI, and that a uniform insult can result in multiple RV phenotypes.

Cardiomyopathy, Dilated↗